Patient-derived glioblastoma stem cells are killed by CD133-specific CAR T cells but induce the T cell aging marker CD57

Abstract Adoptive transfer of primary (unmodified) or genetically engineered antigen-specific T cells has demonstrated astonishing clinical results in the treatment of infections and some malignancies. The definition of optimal targets and antigen receptors as well as the differentiation status of transferred T cells are emerging as crucial parameters for generating cell products with predictable efficacy and safety profiles. Our laboratory has demonstrated that defined subsets within the memory CD8+ T cell compartment fulfill all key characteristics of adult tissue stem cells and are essential for robust and long-term maintained responses upon adoptive transfer. We have developed clinical multi-parameter enrichment technologies to purify these memory stem cells for clinical applications. In my presentation I will report on the status of ongoing clinical trials using such purified cell products either as a primary T cell population for the treatment of infections upon allogeneic stem cell transplantation or after genetic modification with a CD19 CAR for the treatment of malignancies (collaboration with Stan Riddell, FHCC/Seattle). Infusing small numbers of T cells within a memory stem cell product can be highly effective therapeutically, but bears some risk of toxicity. Therefore, safeguards that allow selective depletion of transferred cells in the case of un-tolerable side effects may be needed to further improve adoptive immunotherapy. I will present results exploring the capacity of a truncated version of EGFR (EGFRt) co-expressed with T cells expressing a CD19-CAR. In pre-clinical mouse models we demonstrate that application of Cetuximab, which binds to EGFRt, confers selective depletion of adoptively transferred CAR-T cells in vivo. Long-term B cell aplasia, which is a main side effect of CD19-CAR T cell therapy, can be completely reverted with this strategy. Vaccination studies upon B cell recovery demonstrate full functionality of antigen-specific antibody formation. EGFRt co-expressing CD19-CAR T cells have been successfully transferred into first human patients, providing the option to test for the first time in a clinical setting whether treatment of B cell aplasia after long-term leukemia remission can be achieved by selective depletion. Disclosures Busch: STAGE cell therapeutics: Other: I was share holder of STAGE cell therapeutics, a company that was recently bought by Juno therapeutics.. Off Label Use: CD19 CAR T cells.

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IL7-IL12 Engineered Mesenchymal Stem Cells (MSCs) Improve A CAR T Cell Attack Against Colorectal Cancer Cells

Cells ◽

10.3390/cells9040873 ◽

2020 ◽

Vol 9 (4) ◽

pp. 873 ◽

Cited By ~ 3

Author(s):

Andreas A. Hombach ◽

Ulf Geumann ◽

Christine Günther ◽

Felix G. Hermann ◽

Hinrich Abken

Keyword(s):

Stem Cells ◽

T Cells ◽

Mesenchymal Stem Cells ◽

T Cell ◽

Tumor Model ◽

T Cell Response ◽

Car T Cells ◽

Car T Cell ◽

Activation Induced Cell Death ◽

Car T

Chimeric antigen receptor (CAR) redirected T cells are efficacious in the treatment of leukemia/lymphoma, however, showed less capacities in eliminating solid tumors which is thought to be partly due to the lack of cytokine support in the tumor lesion. In order to deliver supportive cytokines, we took advantage of the inherent ability of mesenchymal stem cells (MSCs) to actively migrate to tumor sites and engineered MSCs to release both IL7 and IL12 to promote homeostatic expansion and Th1 polarization. There is a mutual interaction between engineered MSCs and CAR T cells; in presence of CAR T cell released IFN-γ and TNF-α, chronic inflammatory Th2 MSCs shifted towards a Th17/Th1 pattern with IL2 and IL15 release that mutually activated CAR T cells with extended persistence, amplification, killing and protection from activation induced cell death. MSCs releasing IL7 and IL12 were superior over non-modified MSCs in supporting the CAR T cell response and improved the anti-tumor attack in a transplant tumor model. Data demonstrate the first use of genetically modified MSCs as vehicles to deliver immuno-modulatory proteins to the tumor tissue in order to improve the efficacy of CAR T cells in the treatment of solid malignancies.

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Closely related T-memory stem cells correlate with in vivo expansion of CAR.CD19-T cells and are preserved by IL-7 and IL-15

Blood ◽

10.1182/blood-2014-01-552174 ◽

2014 ◽

Vol 123 (24) ◽

pp. 3750-3759 ◽

Cited By ~ 256

Author(s):

Yang Xu ◽

Ming Zhang ◽

Carlos A. Ramos ◽

April Durett ◽

Enli Liu ◽

...

Keyword(s):

Stem Cells ◽

T Cells ◽

T Cell ◽

Cell Expansion ◽

Ex Vivo ◽

Car T Cells ◽

Car T Cell ◽

Key Points ◽

Car T

Key Points The frequency of CD8+CD45RA+CCR7+ cells, a subset closest to T-memory stem cells, correlates with CAR–T-cell expansion in lymphoma patients. IL-7 and IL-15 increase the frequency of CD8+CD45RA+CCR7+ cells during the ex vivo expansion of CAR+ T cells.

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Chimeric Antigen Receptor-Engineered T-Cells - A New Way and Era for Lymphoma Treatment

Recent Patents on Anti-Cancer Drug Discovery ◽

10.2174/1574892814666191022164641 ◽

2020 ◽

Vol 14 (4) ◽

pp. 312-323

Author(s):

Romeo G. Mihăilă

Keyword(s):

T Cells ◽

T Cell ◽

Response Rate ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Car T Cells ◽

Future Developments ◽

Car T Cell ◽

Large B Cell Lymphoma ◽

Car T

Background: Patients with refractory or relapsed diffuse large B-cell lymphoma have a poor prognosis with the current standard of care. Objective: Chimeric Antigen Receptor T-cells (CAR T-cells) are functionally reprogrammed lymphocytes, which are able to recognize and kill tumor cells. The aim of this study is to make progress in this area. Method: A mini-review was achieved using the articles published in Web of Science and PubMed in the last year and the new patents were made in this field. Results: The responses to CAR T-cell products axicabtagene ciloleucel and tisagenlecleucel are promising; the objective response rate can reach up to 83%, and the complete response rate ranges between 40 and 58%. About half of the patients may have serious side effects, such as cytokine release syndrome and neurotoxicity. Current and future developments include the improvement of CAR T-cell expansion and polyfunctionality, the combined use of CAR T-cells with a fusion protein between interferon and an anti-CD20 monoclonal antibody, with checkpoint inhibitors or small molecule sensitizers that have apoptotic-regulatory effects. Furthermore, the use of IL-12-expressing CAR T-cells, an improved technology for the production of CAR T-cells based on targeted nucleases, the widespread use of allogeneic CAR T-cells or universal CAR T-cells obtained from genetically engineered healthy donor T-cells are future developments actively considered. Conclusion: CAR T-cell therapy significantly improved the outcome of patients with relapsed or refractory diffuse large B-cell lymphoma. The advances in CAR T-cells production technology will improve the results and enable the expansion of this new immunotherapy.

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Structure of the Signal Transduction Domain in Second-Generation CAR Regulates the Input Efficiency of CAR Signals

International Journal of Molecular Sciences ◽

10.3390/ijms22052476 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2476

Author(s):

Kento Fujiwara ◽

Masaki Kitaura ◽

Ayaka Tsunei ◽

Hotaka Kusabuka ◽

Erika Ogaki ◽

...

Keyword(s):

Signal Transduction ◽

T Cells ◽

T Cell ◽

Second Generation ◽

Independent Manner ◽

Cell Functions ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

The Impact

T cells that are genetically engineered to express chimeric antigen receptor (CAR) have a strong potential to eliminate tumor cells, yet the CAR-T cells may also induce severe side effects due to an excessive immune response. Although optimization of the CAR structure is expected to improve the efficacy and toxicity of CAR-T cells, the relationship between CAR structure and CAR-T cell functions remains unclear. Here, we constructed second-generation CARs incorporating a signal transduction domain (STD) derived from CD3ζ and a 2nd STD derived from CD28, CD278, CD27, CD134, or CD137, and investigated the impact of the STD structure and signaling on CAR-T cell functions. Cytokine secretion of CAR-T cells was enhanced by 2nd STD signaling. T cells expressing CAR with CD278-STD or CD137-STD proliferated in an antigen-independent manner by their STD tonic signaling. CAR-T cells incorporating CD28-STD or CD278-STD between TMD and CD3ζ-STD showed higher cytotoxicity than first-generation CAR or second-generation CARs with other 2nd STDs. The potent cytotoxicity of these CAR-T cells was not affected by inhibiting the 2nd STD signals, but was eliminated by placing the STDs after the CD3ζ-STD. Our data highlighted that CAR activity was affected by STD structure as well as by 2nd STD signaling.

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Anti-Mesothelin CAR T cell therapy for malignant mesothelioma

Biomarker Research ◽

10.1186/s40364-021-00264-1 ◽

2021 ◽

Vol 9 (1) ◽

Author(s):

Laura Castelletti ◽

Dannel Yeo ◽

Nico van Zandwijk ◽

John E. J. Rasko

Keyword(s):

T Cells ◽

T Cell ◽

Cell Therapy ◽

Malignant Mesothelioma ◽

Oncolytic Viruses ◽

T Cell Therapy ◽

Car T Cells ◽

Car T Cell ◽

Car T Cell Therapy ◽

Car T

AbstractMalignant mesothelioma (MM) is a treatment-resistant tumor originating in the mesothelial lining of the pleura or the abdominal cavity with very limited treatment options. More effective therapeutic approaches are urgently needed to improve the poor prognosis of MM patients. Chimeric Antigen Receptor (CAR) T cell therapy has emerged as a novel potential treatment for this incurable solid tumor. The tumor-associated antigen mesothelin (MSLN) is an attractive target for cell therapy in MM, as this antigen is expressed at high levels in the diseased pleura or peritoneum in the majority of MM patients and not (or very modestly) present in healthy tissues. Clinical trials using anti-MSLN CAR T cells in MM have shown that this potential therapeutic is relatively safe. However, efficacy remains modest, likely due to the MM tumor microenvironment (TME), which creates strong immunosuppressive conditions and thus reduces anti-MSLN CAR T cell tumor infiltration, efficacy and persistence. Various approaches to overcome these challenges are reviewed here. They include local (intratumoral) delivery of anti-MSLN CAR T cells, improved CAR design and co-stimulation, and measures to avoid T cell exhaustion. Combination therapies with checkpoint inhibitors as well as oncolytic viruses are also discussed. Preclinical studies have confirmed that increased efficacy of anti-MSLN CAR T cells is within reach and offer hope that this form of cellular immunotherapy may soon improve the prognosis of MM patients.

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Designed improvement to T-cell immunotherapy by multidimensional single cell profiling

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-001877 ◽

2021 ◽

Vol 9 (3) ◽

pp. e001877

Author(s):

Irfan N Bandey ◽

Jay R T Adolacion ◽

Gabrielle Romain ◽

Melisa Martinez Paniagua ◽

Xingyue An ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Single Cell ◽

Adoptive Cell Therapy ◽

Population Heterogeneity ◽

Serial Killer ◽

Car T Cells ◽

Car T ◽

Single Cell Profiling ◽

Killer T Cells

BackgroundAdoptive cell therapy based on the infusion of chimeric antigen receptor (CAR) T cells has shown remarkable efficacy for the treatment of hematologic malignancies. The primary mechanism of action of these infused T cells is the direct killing of tumor cells expressing the cognate antigen. However, understanding why only some T cells are capable of killing, and identifying mechanisms that can improve killing has remained elusive.MethodsTo identify molecular and cellular mechanisms that can improve T-cell killing, we utilized integrated high-throughput single-cell functional profiling by microscopy, followed by robotic retrieval and transcriptional profiling.ResultsWith the aid of mathematical modeling we demonstrate that non-killer CAR T cells comprise a heterogeneous population that arise from failure in each of the discrete steps leading to the killing. Differential transcriptional single-cell profiling of killers and non-killers identified CD137 as an inducible costimulatory molecule upregulated on killer T cells. Our single-cell profiling results directly demonstrate that inducible CD137 is feature of killer (and serial killer) T cells and this marks a different subset compared with the CD107apos (degranulating) subset of CAR T cells. Ligation of the induced CD137 with CD137 ligand (CD137L) leads to younger CD19 CAR T cells with sustained killing and lower exhaustion. We genetically modified CAR T cells to co-express CD137L, in trans, and this lead to a profound improvement in anti-tumor efficacy in leukemia and refractory ovarian cancer models in mice.ConclusionsBroadly, our results illustrate that while non-killer T cells are reflective of population heterogeneity, integrated single-cell profiling can enable identification of mechanisms that can enhance the function/proliferation of killer T cells leading to direct anti-tumor benefit.

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CARTmath—A Mathematical Model of CAR-T Immunotherapy in Preclinical Studies of Hematological Cancers

Cancers ◽

10.3390/cancers13122941 ◽

2021 ◽

Vol 13 (12) ◽

pp. 2941

Author(s):

Luciana R. C. Barros ◽

Emanuelle A. Paixão ◽

Andrea M. P. Valli ◽

Gustavo T. Naozuka ◽

Artur C. Fassoni ◽

...

Keyword(s):

Mathematical Model ◽

T Cells ◽

T Cell ◽

Mouse Models ◽

In Silico ◽

Car T Cells ◽

Car T Cell ◽

Cell Immunotherapy ◽

Car T ◽

T Cell Immunotherapy

Immunotherapy has gained great momentum with chimeric antigen receptor T cell (CAR-T) therapy, in which patient’s T lymphocytes are genetically manipulated to recognize tumor-specific antigens, increasing tumor elimination efficiency. In recent years, CAR-T cell immunotherapy for hematological malignancies achieved a great response rate in patients and is a very promising therapy for several other malignancies. Each new CAR design requires a preclinical proof-of-concept experiment using immunodeficient mouse models. The absence of a functional immune system in these mice makes them simple and suitable for use as mathematical models. In this work, we develop a three-population mathematical model to describe tumor response to CAR-T cell immunotherapy in immunodeficient mouse models, encompassing interactions between a non-solid tumor and CAR-T cells (effector and long-term memory). We account for several phenomena, such as tumor-induced immunosuppression, memory pool formation, and conversion of memory into effector CAR-T cells in the presence of new tumor cells. Individual donor and tumor specificities are considered uncertainties in the model parameters. Our model is able to reproduce several CAR-T cell immunotherapy scenarios, with different CAR receptors and tumor targets reported in the literature. We found that therapy effectiveness mostly depends on specific parameters such as the differentiation of effector to memory CAR-T cells, CAR-T cytotoxic capacity, tumor growth rate, and tumor-induced immunosuppression. In summary, our model can contribute to reducing and optimizing the number of in vivo experiments with in silico tests to select specific scenarios that could be tested in experimental research. Such an in silico laboratory is an easy-to-run open-source simulator, built on a Shiny R-based platform called CARTmath. It contains the results of this manuscript as examples and documentation. The developed model together with the CARTmath platform have potential use in assessing different CAR-T cell immunotherapy protocols and its associated efficacy, becoming an accessory for in silico trials.

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Adoptive Immunotherapy beyond CAR T-Cells

Cancers ◽

10.3390/cancers13040743 ◽

2021 ◽

Vol 13 (4) ◽

pp. 743

Author(s):

Aleksei Titov ◽

Ekaterina Zmievskaya ◽

Irina Ganeeva ◽

Aygul Valiullina ◽

Alexey Petukhov ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cell ◽

Solid Tumors ◽

Adoptive Immunotherapy ◽

Γδ T Cells ◽

Car T Cells ◽

Car T Cell ◽

Cell Immunotherapy ◽

Car T

Adoptive cell immunotherapy (ACT) is a vibrant field of cancer treatment that began progressive development in the 1980s. One of the most prominent and promising examples is chimeric antigen receptor (CAR) T-cell immunotherapy for the treatment of B-cell hematologic malignancies. Despite success in the treatment of B-cell lymphomas and leukemia, CAR T-cell therapy remains mostly ineffective for solid tumors. This is due to several reasons, such as the heterogeneity of the cellular composition in solid tumors, the need for directed migration and penetration of CAR T-cells against the pressure gradient in the tumor stroma, and the immunosuppressive microenvironment. To substantially improve the clinical efficacy of ACT against solid tumors, researchers might need to look closer into recent developments in the other branches of adoptive immunotherapy, both traditional and innovative. In this review, we describe the variety of adoptive cell therapies beyond CAR T-cell technology, i.e., exploitation of alternative cell sources with a high therapeutic potential against solid tumors (e.g., CAR M-cells) or aiming to be universal allogeneic (e.g., CAR NK-cells, γδ T-cells), tumor-infiltrating lymphocytes (TILs), and transgenic T-cell receptor (TCR) T-cell immunotherapies. In addition, we discuss the strategies for selection and validation of neoantigens to achieve efficiency and safety. We provide an overview of non-conventional TCRs and CARs, and address the problem of mispairing between the cognate and transgenic TCRs. Finally, we summarize existing and emerging approaches for manufacturing of the therapeutic cell products in traditional, semi-automated and fully automated Point-of-Care (PoC) systems.

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