Preclinical CAR-T cell target safety, biodistribution, and tumor infiltration analysis using in situ hybridization technology.

2019 ◽  
Vol 37 (8_suppl) ◽  
pp. 112-112 ◽  
Author(s):  
Helly Pimentel ◽  
Helen Jarnagin ◽  
Hailing Zong ◽  
Courtney Todorov ◽  
Courtney M. Anderson ◽  
...  
Keyword(s):  
T Cells ◽  
In Situ Hybridization ◽  
T Cell ◽  
Mouse Lung ◽  
Xenograft Tumor ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Low Levels

112 Background: Chimeric antigen receptor (CAR) T cell therapy is highly effective in treating hematologic malignancies, and major efforts are being made to achieve similar efficacy in solid tumors. The greater potency of CAR-T cells compared to antibody therapeutics demands a more stringent CAR-T target safety assessment to avoid adverse events resulting from “on-target/off-tumor” activity. Furthermore, it is critical to track and monitor CAR+ T cells within intact tissue and tumor to understand the mechanisms underlying off-tumor toxicity and efficacy in tumor killing. Methods: We employed the RNAscope in situ hybridization (ISH) technology to assess target expression specificity and to track CAR-T cell distribution and activation in xenograft and host tissues using the RPMI-8226 xenograft mouse model. Results: For the CAR-T target candidates, Target X and Target Y, RNA ISH revealed that Target X was only expressed in the xenograft tumor and in no mouse organs, while Target Y was found to be expressed at low levels in mouse lung and liver, as well as in the xenograft tumor. Duplex RNA ISH assays with probes targeting the CAR 3’ UTR and either IFNG or GZMB allowed for highly sensitive and specific detection of CAR-T cells and their activation state in both tumor and normal tissues from vehicle, Target X CAR-T cell, or Target Y CAR-T cell treated mice. Activated Target X CAR-T cells expressing GZMB and IFNG were found only in the xenograft tumor, where Target X was expressed. In contrast, activated Target Y CAR-T cells were found almost exclusively in mouse lung and liver, with very few Target Y CAR-T cells being found in the xenograft tumor. Lastly, a multiplex ISH-IHC approach confirmed the presence of activated Target X CAR-T cells in the xenograft tumor through simultaneous detection of the Target X CAR 3’ UTR, IFNG, GZMB, and CD3. Conclusions: These data demonstrate how the RNAscope assay can be utilized for CAR-T cell efficacy and safety/toxicity assessment in preclinical models by detecting very low levels of target antigen expression in off-tumor tissues and monitoring CAR-T cell pharmacodynamics and activation in tumor models and can also be applied for assessing TCR-T cell activity in tumors.

A Failure to Start: Aborted Activation of CAR T Cells in Chronic Lymphocytic Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 681-681
Author(s):  
McKensie Collins ◽  
Weimin Kong ◽  
Inyoung Jung ◽  
Meng Wang ◽  
Stefan M Lundh ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Research Funding ◽  
Cell Dysfunction ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
T Cell Dysfunction ◽  
Low Levels

Introduction: Chronic Lymphocytic Leukemia (CLL) is a CD19+ B-cell malignancy that accounts for approximately 25% of adult leukemia diagnoses in the developed world. While conventional therapies have some efficacy, there are few curative therapeutic options and many patients ultimately progress to relapsed or refractory disease. CD19-targeting chimeric antigen receptor (CAR) T cell therapy has provided some hope, but induces complete remission in only 26% of patients. This suboptimal response rate is believed to be due to T cell dysfunction and immune-suppression by CLL cells, the mechanisms of which are poorly understood. Results: To understand the causes of CAR T cell dysfunction in CLL we investigated the defects that CLL cells induced in normal donor CD19-targeting CAR T cells. CAR T cells were repeatedly stimulated at 5-day intervals with either primary CLL cells from patients or a CD19-expressing control cell line (aAPC). Repeat stimulation of CAR T cells with aAPCs resulted in 5.36 ± .94 population doublings after three stimulations, whereas CLL cells only evoked 2.39 ± .92 population doublings. We performed phenotyping, proliferation analysis, and cytokine analysis of stimulated CAR T cells. CLL-stimulated T cells appeared un-activated, with low levels of PD-1, LAG3, and TIM3, low levels of cytokine production, and a high proportion of non-cycling cells as measured by Ki67 staining. We first hypothesized that CLL cells induce an altered epigenetic program that prevents effector function and is stabilized by successive stimulations. To test this, we stimulated CAR T cells with CLL cells or aAPCs as indicated in Fig. 1A. CLL-stimulated CAR T cells failed to proliferate or produce cytokines, but subsequent stimulation with aAPCs rescued these functions (Fig. 1B). Further, CLL-stimulated CAR T cells did not differentiate, suggesting that CLL cells do not induce stable defects but rather insufficiently activate CAR T cells (Fig. 1C). These cells also appeared un-activated as indicated by low levels of PD-1 and Ki67. We then used flow cytometry to assess expression of costimulatory and inhibitory molecules on the primary CLL samples. We found that the levels of co-stimulatory and adhesion molecules, namely CD80/CD86 and CD54/CD58 respectively were found at low frequencies, and where present were expressed at low levels. This suggested that one mechanism behind the lack of CAR T cell effector responses may be that a lack of co-stimulation prevents proper CAR T cell targeting of these cells. Towards this, we incubated CLL cells with a murine fibroblast line expressing CD40 ligand for 24 hours with IL-4 to activate the CLL cells. We found that this activation significantly increased expression of CD80, CD86, CD54, and CD58 on the CLL cells. We then used these cells to stimulate CAR T cells in our re-stimulation assay and found that CAR T cells were able to proliferate in response to these activated CLLs (Fig. 1D). In addition, CAR T cells stimulated with activated CLL cells formed more cell-to-cell conjugates than those stimulated with un-activated CLL cells. These data suggest not only that insufficient activation of CAR T cells may be responsible for the poor response rates to CAR T cell therapy in CLL, but also implicate a need for additional co-stimulation in this CAR T cell setting. Another contributing factor may be immune suppression by CLL cells. To determine if CLL cells are immune-suppressive, we used a co-culture assay to stimulate CAR T cells with aAPCs and CLL cells mixed at known ratios. Interestingly, all mixed cultures proliferated similarly, suggesting that CLL cells did not prevent T cell activation in the presence of a strong activation signal. We also found that CLL cells are responsive to IL-2, as addition of this cytokine to culture media prolongs survival of CLL cells out to 10 days depending on the dose. This suggests that CLL cells express a functional IL-2 receptor and may be taking up IL-2 from the culture media, further impairing T cell activation. In support of this, supplementing IL-2 into our CLL/CAR T cell co-cultures rescued T cell proliferative capacity. Taken together, these data suggest that T cell dysfunction in CLL is the result of insufficient activation rather than true functional defects. Disclosures June: Novartis: Research Funding; Tmunity: Other: scientific founder, for which he has founders stock but no income, Patents & Royalties. Melenhorst:National Institutes of Health: Research Funding; Parker Institute for Cancer Immunotherapy: Research Funding; Novartis: Research Funding, Speakers Bureau; IASO Biotherapeutics, Co: Consultancy; Simcere of America, Inc: Consultancy; Shanghai Unicar Therapy, Co: Consultancy; Colorado Clinical and Translational Sciences Institute: Membership on an entity's Board of Directors or advisory committees; Genentech: Speakers Bureau; Stand Up to Cancer: Research Funding; Incyte: Research Funding.

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 Fast off-rate CD229 chimeric antigen receptor T cells efficiently target multiple myeloma, spare T cells, and exhibit reduced trogocytosis

2021 ◽  
Author(s):  
Erica R. Vander Mause ◽  
Jillian M. Baker ◽  
Sabarinath V. Radhakrishnan ◽  
Patricia Davis ◽  
Jens Panse ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
Antigen Receptors ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Life Threatening ◽  
Low Levels ◽  
Anti Tumor Activity

ABSTRACTT cells expressing chimeric antigen receptors have shown remarkable therapeutic activity against different types of cancer. However, their wider use has been hampered by the potential for life-threatening toxicities due to the unintended targeting of healthy cells expressing low levels of the targeted antigen. We have now developed an affinity-tuning approach for the generation of minimally modified, low-affinity antibody variants derived from existing high-affinity antibodies. Using this approach, we engineered low affinity variants of the fully human CD229-specific antibody 2D3. Parental 2D3 originally efficiently targeted multiple myeloma cells but also healthy T cells expressing low levels of CD229. We demonstrate that CAR T cells based on a low affinity variant of 2D3 maintain the parental antibody’s anti-tumor activity, but lack its targeting of healthy T cells. In addition, variant CD229 CAR T cells show reduced trogocytosis potentially augmenting CAR T cell persistence. The fast off-rate CAR produced using our affinity tuning approach eliminates a key liability of CD229 CAR T cells and paves the way for the effective and safe treatment of patients with multiple myeloma.One sentence summaryAffinity tuning approach yields low affinity CD229 CAR binding domain maintaining the parental clone’s anti-tumor activity while eliminating killing of healthy T cells, increasing CAR T cell expansion, and decreasing trogocytosis.

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

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Karsten Eichholz ◽  
Alvason Zhenhua Li ◽  
Kurt Diem ◽  
Michael Claus Jensen ◽  
Jia Zhu ◽  
...  
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. Here, we describe a CAR RNA FISH histo-cytometry platform combined with a random reaction seed image analysis algorithm to quantitate spatial distribution and in vivo functional activity of a CAR T cell population at a single cell resolution for preclinical models. 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 of genetically engineered cells in situ with their target cells in tissues with high statistical power and thus, can serve as an important tool for preclinical assessment of CAR T cell effectiveness.

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

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.

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

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.

scholarly journals Anti-Mesothelin CAR T cell therapy for malignant mesothelioma

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.

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

Cancers ◽  
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.

scholarly journals Adoptive Immunotherapy beyond CAR T-Cells

Cancers ◽  
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.

scholarly journals Metabolic and Mitochondrial Functioning in Chimeric Antigen Receptor (CAR)—T Cells

Cancers ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1229
Author(s):  
Ali Hosseini Rad S. M. ◽  
Joshua Colin Halpin ◽  
Mojtaba Mollaei ◽  
Samuel W. J. Smith Bell ◽  
Nattiya Hirankarn ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Chimeric Antigen Receptor ◽  
Metabolic State ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T Cell Therapy ◽  
Car T

Chimeric antigen receptor (CAR) T-cell therapy has revolutionized adoptive cell therapy with impressive therapeutic outcomes of >80% complete remission (CR) rates in some haematological malignancies. Despite this, CAR T cell therapy for the treatment of solid tumours has invariably been unsuccessful in the clinic. Immunosuppressive factors and metabolic stresses in the tumour microenvironment (TME) result in the dysfunction and exhaustion of CAR T cells. A growing body of evidence demonstrates the importance of the mitochondrial and metabolic state of CAR T cells prior to infusion into patients. The different T cell subtypes utilise distinct metabolic pathways to fulfil their energy demands associated with their function. The reprogramming of CAR T cell metabolism is a viable approach to manufacture CAR T cells with superior antitumour functions and increased longevity, whilst also facilitating their adaptation to the nutrient restricted TME. This review discusses the mitochondrial and metabolic state of T cells, and describes the potential of the latest metabolic interventions to maximise CAR T cell efficacy for solid tumours.

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