Efficacy Of CAR-T Cell Therapy Research Articles

Nanotechnology Promotes Genetic and Functional Modifications of Therapeutic T Cells Against Cancer.

Growing experience with engineered chimeric antigen receptor (CAR)‐T cells has revealed some of the challenges associated with developing patient‐specific therapy. The promising clinical results obtained with CAR‐T therapy nevertheless demonstrate the urgency of advancements to promote and expand its uses. There is indeed a need to devise novel methods to generate potent CARs, and to confer them and track their anti‐tumor efficacy in CAR‐T therapy. A potentially effective approach to improve the efficacy of CAR‐T cell therapy would be to exploit the benefits of nanotechnology. This report highlights the current limitations of CAR‐T immunotherapy and pinpoints potential opportunities and tremendous advantages of using nanotechnology to 1) introduce CAR transgene cassettes into primary T cells, 2) stimulate T cell expansion and persistence, 3) improve T cell trafficking, 4) stimulate the intrinsic T cell activity, 5) reprogram the immunosuppressive cellular and vascular microenvironments, and 6) monitor the therapeutic efficacy of CAR‐T cell therapy. Therefore, genetic and functional modifications promoted by nanotechnology enable the generation of robust CAR‐T cell therapy and offer precision treatments against cancer.

Inhibition of PP2A with LB-100 Enhances Efficacy of CAR-T Cell Therapy Against Glioblastoma.

Chimeric antigen receptor (CAR)-engineered T cells represent a promising modality for treating glioblastoma. Recently, we demonstrated that CAR-T cells targeting carbonic anhydrase IX (CAIX), a protein involved in HIF-1a hypoxic signaling, is a promising CAR-T cell target in an intracranial murine glioblastoma model. Anti-CAIX CAR-T cell therapy is limited by its suboptimal activation within the tumor microenvironment. LB-100, a small molecular inhibitor of protein phosphatase 2A (PP2A), has been shown to enhance T cell anti-tumor activity through activation of the mTOR signaling pathway. Herein, we investigated if a treatment strategy consisting of a combination of LB-100 and anti-CAIX CAR-T cell therapy produced a synergistic anti-tumor effect. Our studies demonstrate that LB-100 enhanced anti-CAIX CAR-T cell treatment efficacy in vitro and in vivo. Our findings demonstrate the role of LB-100 in augmenting the cytotoxic activity of anti-CAIX CAR-T cells and underscore the synergistic therapeutic potential of applying combination LB-100 and CAR-T Cell therapy to other solid tumors.

Granulocyte-Macrophage Colony-Stimulating Factor and CAR-T Technology for Solid Tumors in Experiment

Background. Cytokines are considered as important factors that enhance the efficacy of CAR-T cell therapy. Besides, they are key elements of the pathogenesis of cytokine release syndrome and neurotoxicity in applying the CAR-T technology. However, cytokine effects in the context of CAR-T therapy have not yet been properly studied. Aim. To quantitatively assess cytokine secretion using multiplex assay with co-incubation of anti-CD19 CAR-T lymphocytes with epithelial HeLa and A431 cell lines expressing CD19 on their surface. Materials & Methods. T-lymphocytes were transduced with the lentiviral vector containing anti-CD19-CAR gene. CAR expression was tested based on GFP reporter using flow cytometry. To confirm a specific CAR-T cell activation response to tumor antigen, the levels of interleukin-2, interferon-γ, and tumor necrosis factor-α were measured by means of immunoassay. Cytotoxic activity of CAR-T lymphocytes obtained was examined with their direct co-culturing with target cells. The levels of cytokines isolated prior to and after incubation of targets with CAR-T cells were compared using multiplex assay. Results. The level of some proinflammatory cytokines (interleukin-6, interleukin-1β, interferon-γ) (P < 0.01) increased. The difference in the levels of anti-inflammatory cytokines (interleukin-4, interleukin-10) was inconsiderable, and in the HeLa cell line experiment it was insignificant (p > 0.05). The concentration of granulocyte-macrophage colony-stimulating factor (GM-CSF) was many times higher after incubation with CAR-T lymphocytes (p < 0.01). Conclusion. The trial revealed multiple enhancement of GM-CSF, one of the key elements of the pathogenesis of cy tokine release syndrome and CAR-T-associated neurotoxicity. The results of further studies of GM-CSF can contribute to improving the efficacy of CAR-T therapy with considerably lower toxicity.

TMOD-18. EXPLORING THE FACTORS LEAD TO SUCCESS OF CAR T-CELL THERAPY IN GLIOBLASTOMA WITH COMPUTATIONAL MODELING AND IN VITRO DATA

Abstract Chimeric antigen receptor (CAR) T-cell therapy is an emerging targeted immunotherapy which has shown success in liquid cancers such as leukemias. CAR T-cells are also being used for the treatment of solid tumors such as glioblastoma, which is a primary brain tumor. Ongoing phase I trials have been designed to evaluate CAR T-cell dosing, scheduling, and route of administration in order to understand and improve the efficacy of CAR T-cell therapy. A better understanding of factors leading to the success of CAR T-cell immunotherapy for solid tumors will be necessary to improve outcomes for patients with solid tumors and to advance the field of CAR T-cell immuno-oncology. Here we use mathematical model to explore factors in determining a successful response to CAR T-cell therapy: proliferation, persistence, and killing capacity of CAR T-cells. Using a novel in vitro experimental apparatus, we are able to measure the density of cancer cells over several days in 15 minute interval time resolution. This highly temporally resolved data provides a unique opportunity to confidently estimate parameters of the model and to provide insights into the dynamics of CAR T-cell proliferation, persistence, and killing capacity. Furthermore we explore the relationship between these factor with CAR T-cell dose level. We will show results from experiments using patient-derived cancer cell lines as well as cancer cells engineered to express specific levels of the target antigen (IL13Rα2) to quantitatively evaluate the roles of proliferation, persistence, and killing in cells with different levels of antigen expression. We will discuss the interpretation of the model parameters and demonstrate the clinical value of this analysis through an application of CAR T-cell treatment tailored to the dynamics of an individual patient’s cancer growth rate.

Insight into mechanisms associated with cytokine release syndrome and neurotoxicity after CD19 CAR-T cell immunotherapy.

Adoptive immunotherapy with CD19-targeted chimeric antigen receptor (CAR)-T cells has been successful in producing durable remissions in some patients with relapsed or refractory B cell malignancies. Despite the efficacy of CAR-T cell therapy, significant toxicities can occur. Cytokine release syndrome (CRS) and neurotoxicity are the most common toxicities and can range from self-limited fever to life threatening organ damage and death. Understanding the mechanisms underlying these toxicities can help guide and improve outcomes. In this review we describe CRS and neurotoxicity in patients with B cell malignancies treated with CD19 CAR-T cells in pivotal trials, and also provide insight into potential mechanisms associated with these toxicities based on studies conducted in a phase 1/2 clinical trial at the Fred Hutchinson Cancer Research Center.

Efficacy of Car T-Cell Therapy in Head and Neck Cancers- A Meta-Analysis

Background: Cancer, defined by the World Health Organization (WHO) is “a large group of diseases characterized by the growth of abnormal cells beyond their usual boundaries that can then invade adjoining parts of the body and/or spread to other organs”. From extensive surgical excisions, radiotherapy, laser therapy to immunotherapies, various treatment strategies have been proposed and implemented so far but unfortunately none could improve the five year survival rate of the patients globally. Immunotherapy, being one amongst them, is a type of cancer treatment that boosts the body’s natural defenses to fight against cancer. The current concept of immunotherapy involves Chimeric antigen receptor or the CAR T-Cell therapy which involves alterations and modifications of T cells to fight cancer cells better. Until recently, the use of CAR T-cell therapy has been restricted to small clinical trials, largely in patients with advanced blood cancers and has also shown a promising window of hope in head and neck (especially oral) cancers as well. But these treatments have nevertheless captured the attention of the people because of the remarkable responses they have produced in some patients for whom all other treatments had stopped working. The current concept of immunotherapy involves the cancer vaccines making use of CAR T-cells which are the most powerful antigen presenting cells for the induction of antigen specific T cell response. This evidence based study therefore aims to highlight the clinical perspective of CAR T-Cell based immunotherapy in oral and other head and neck cancers. Aim: Assessment of efficacy of Car T-Cell Therapy in head and neck Malignancies. Research Question: Is Car T-Cell Therapy actually effective in treating head and neck cancers? Materials and Methods: Study sample included review of 70 research articles, based on scientific data bases from the English literature based COCHRANE collaboration having a definite RCT (Randomized Control Trial). The literature was studied, analyzed and assessed; comparison was made on their p (probability) values between various techniques in terms of their sensitivity and specificity. The articles were scrutinised based on the criterion for meta-analysis and finally 11 study articles were chosen for the study. Result and Conclusion: Due to its unique individual characteristics, it helps combat against the cancer cells at its very inception. Promises a complete and permanent cure for malignancies at the grass root level. It has a sensitivity and specificity of greater than 80-90% and enhances recovery rate from 40-50% to more than 90%. So on a bulls eye view, we can say that as the already existing techniques of oral cancer treatment are very superficial and not very significant, Car T-Cell therapy tends to bring about a paradigm shift in oral cancer treatment thereby giving a new ray of hope to cancer ailing patients.

Potent and Timed Epigenetic Repression of PD-1 in Human T Cells

T cell exhaustion mediated by checkpoint genes - most prominently PD-1 - is believed to play a major role in limiting the efficacy of chimeric antigen receptor (CAR) T cell therapies of hematologic malignancies. Clinically durable responses to CAR-T therapy of B cell malignancies almost invariably occur in patients who achieve a complete remission by day 28 post infusion. Accumulating data indicate that the impact of T cell exhaustion is most prominent during the early post-infusion interval. PD-1 has recently been found to function as a tumor suppressor in T cells; as such permanent disablement of PD-1 expression via genetic knockout may thus increase long term adverse events from cell therapy.We sought to develop an approach for increasing the potency and efficacy of CAR-T cell therapy of B cell and other hematologic malignancies by timed epigenetic abrogation of PD-1 expression that was limited to the critical early (21-28d) therapeutic interval. To accomplish this we engineered synthetic transcription factors coupled to a modified KRAB repressive domain that densely tiled the transcriptional regulatory regions of PD-1 at up to single-base resolution.To identify TF-repressors that were both potent and specific, we transiently transduced each engineered TF-repressors separately into primary total CD3+, CD4+, or CD8+ human T-cells. These experiments revealed striking position dependence of TF-repressor activity, indicating that spatial and rotational positioning of the repressive domain was critical for function. We identified several TF-repressors that exhibited highly potent repression (>90%), and screened these for gene selectivity using transcriptome-wide deep RNA-seq. These studies yielded a TF-repressor with both high potency (>90%) and true single-gene (PD-1) specificity on a genomic scale.Next we examined the durability of expression. Transduction of TF-repressor mRNA resulted in rapid expression and activity, with PD-1 repression observable beginning at 12 hours and peaking by 48 hours post transduction. Experiments with tagged TF-repressors revealed that substantially all translated protein had dissipated within 72 hours post transduction. Remarkably, however, potent PD-1 repression reliably persisted for >3 weeks post-transduction.Collectively, our studies show the feasibility of generating highly potent and single-gene-selective epigenome editing reagents capable of reprogramming clinically relevant genes over a defined temporal interval. This approach should be broadly applicable to other key regulators of clinical response to produce genome-targeted medicines without incurring the genotoxic consequences of double-stranded DNA breaks. DisclosuresNo relevant conflicts of interest to declare.

Chronic Lymphocytic Leukemia Cells Impair Mitochondrial Fitness in CD8+ T Cells and Impede CAR T Cell Efficacy

Chronic Lymphocytic Leukemia Cells Impair Mitochondrial Fitness in CD8+ T Cells and Impede CAR T Cell Efficacy

The acceleration of CAR-T therapy in non-Hodgkin lymphoma.

Recent advances in diffuse large B-cell lymphomas have included both identification of high-risk subtypes characterized by multiply relapsed and/or refractory disease as well as novel treatment in the form of cellular therapy. Chimeric antigen receptor (CAR)-T cell therapy is a recently developed approach to address the poor outcomes in this patient population. The CAR-T cell construct has evolved although several iterations as it transitioned from the lab to the clinic. Three major studies have evaluated the efficacy of CD19-directed CAR-T cell therapy in aggressive B-cell non-Hodgkin lymphoma; each demonstrating durable complete remissions in heavily pretreated patients. The cost of this remarkable therapy, however, includes cytokine release syndrome and neurotoxicity shortly after administration as well as delayed infectious complications due to B-cell aplasia. Future investigations are focused on the optimizing both safety and efficacy of CAR-T cell therapy.

Targeted delivery of a PD-1-blocking scFv by CAR-T cells enhances anti-tumor efficacy in vivo.

The efficacy of CAR-T cell therapy against poorly responding tumors has been enhanced by administering the cells in combination with immune checkpoint blockade inhibitors. Alternatively, the CAR construct has been engineered to co-express factors that boost CAR-T cell function in the tumor microenvironment. Here we modified CAR-T cells to secrete PD-1-blocking single-chain variable fragments (scFv). These scFv-secreting CAR-T cells work in both a paracrine and autocrine manner to improve the anti-tumor activity of CAR-T cells and bystander tumor-specific T cells in clinically relevant syngeneic and xenogeneic mouse models of PD-L1+ hematologic and solid tumors. Efficacy was similar or better to that achieved by combination therapy with CAR-T cells and a checkpoint inhibitor. This approach could improve safety as the secreted scFv remained localized to the tumor, protecting CAR-T cells from PD-1 inhibition, which could potentially avoid toxicities associated with systemic checkpoint inhibition.

319 - Apoptotic Cells Dramatically Ameliorate the Efficacy of CAR T-Cell Therapy and Prevents Cytokine Release Syndrome (CRS)

319 - Apoptotic Cells Dramatically Ameliorate the Efficacy of CAR T-Cell Therapy and Prevents Cytokine Release Syndrome (CRS)

Single-cell multiplexed cytokine profiling of CD19 CAR-T cells reveals a diverse landscape of polyfunctional antigen-specific response

BackgroundIt remains challenging to characterize the functional attributes of chimeric antigen receptor (CAR)-engineered T cell product targeting CD19 related to potency and immunotoxicity ex vivo, despite promising in vivo efficacy in patients with B cell malignancies.MethodsWe employed a single-cell, 16-plex cytokine microfluidics device and new analysis techniques to evaluate the functional profile of CD19 CAR-T cells upon antigen-specific stimulation. CAR-T cells were manufactured from human PBMCs transfected with the lentivirus encoding the CD19-BB-z transgene and expanded with anti-CD3/anti-CD28 coated beads. The enriched CAR-T cells were stimulated with anti-CAR or control IgG beads, stained with anti-CD4 RPE and anti-CD8 Alexa Fluor 647 antibodies, and incubated for 16 h in a single-cell barcode chip (SCBC). Each SCBC contains ~12,000 microchambers, covered with a glass slide that was pre-patterned with a complete copy of a 16-plex antibody array. Protein secretions from single CAR-T cells were captured and subsequently analyzed using proprietary software and new visualization methods.ResultsWe demonstrate a new method for single-cell profiling of CD19 CAR-T pre-infusion products prepared from 4 healthy donors. CAR-T single cells exhibited a marked heterogeneity of cytokine secretions and polyfunctional (2+ cytokine) subsets specific to anti-CAR bead stimulation. The breadth of responses includes anti-tumor effector (Granzyme B, IFN-γ, MIP-1α, TNF-α), stimulatory (GM-CSF, IL-2, IL-8), regulatory (IL-4, IL-13, IL-22), and inflammatory (IL-6, IL-17A) functions. Furthermore, we developed two new bioinformatics tools for more effective polyfunctional subset visualization and comparison between donors.ConclusionsSingle-cell, multiplexed, proteomic profiling of CD19 CAR-T product reveals a diverse landscape of immune effector response of CD19 CAR-T cells to antigen-specific challenge, providing a new platform for capturing CAR-T product data for correlative analysis. Additionally, such high dimensional data requires new visualization methods to further define precise polyfunctional response differences in these products. The presented biomarker capture and analysis system provides a more sensitive and comprehensive functional assessment of CAR-T pre-infusion products and may provide insights into the safety and efficacy of CAR-T cell therapy.

Different Subsets of T Cells, Memory, Effector Functions, and CAR-T Immunotherapy.

This review is focused on different subsets of T cells: CD4 and CD8, memory and effector functions, and their role in CAR-T therapy––a cellular adoptive immunotherapy with T cells expressing chimeric antigen receptor. The CAR-T cells recognize tumor antigens and induce cytotoxic activities against tumor cells. Recently, differences in T cell functions and the role of memory and effector T cells were shown to be important in CAR-T cell immunotherapy. The CD4+ subsets (Th1, Th2, Th9, Th17, Th22, Treg, and Tfh) and CD8+ memory and effector subsets differ in extra-cellular (CD25, CD45RO, CD45RA, CCR-7, L-Selectin [CD62L], etc.); intracellular markers (FOXP3); epigenetic and genetic programs; and metabolic pathways (catabolic or anabolic); and these differences can be modulated to improve CAR-T therapy. In addition, CD4+ Treg cells suppress the efficacy of CAR-T cell therapy, and different approaches to overcome this suppression are discussed in this review. Thus, next-generation CAR-T immunotherapy can be improved, based on our knowledge of T cell subsets functions, differentiation, proliferation, and signaling pathways to generate more active CAR-T cells against tumors.

Declined presentation one dose of 5-AZA-2'-Deoxycytidine enhances therapeutic efficacy of cart-cell therapy in a murine model by improving T-cell persistence

Declined presentation one dose of 5-AZA-2'-Deoxycytidine enhances therapeutic efficacy of cart-cell therapy in a murine model by improving T-cell persistence

Identifying the Factors Modulating the Efficacy of CAR-T Cell Therapy

T cells modified to express tumour-directed chimeric antigen receptors (CARs) have shown anti-tumor efficacy in early phase clinical trials. There is no consensus yet about the optimal CAR T- cell dose needed for achieve optimal anti-leukemic activity. Early phase clinical trials have shown that the level of in vivo expansion of CAR T-cells rather than CAR T-cell dose determines the efficacy of CAR-T cell therapy. Here we identify factors that modify CAR-T cell expansion and anti-tumour activity in vitro and in vivo in a ‘humanised’ mouse model using CARs targeting CD19+ B-cell leukaemia (CAR19 T-cells). Human peripheral and cord blood T-cells were genetically modified using second generation of CAR19-T cells engineered to express CD3zeta and co-stimulatory CD28 domains cloned into PiggyBac-transposon vector. Antigen-specific stimulation using autologous mononuclear cells was used to enrich and expand CAR19 T-cells. CAR19 T-cells induced cytolysis of leukaemia cells in vitro and exhibited anti-tumour activity in vivo in xenograft mouse model of leukaemia. Antigen-specific stimulation induced rapid activation and proliferation of CAR19 T-cells, however, terminal effector differentiation and activated T cell death limited CAR19-T cell expansion. We have analysed CAR19 T-cell expansion using the panel of primary CD19+leukaemia cells expressing different levels of CD19 antigen. CAR19 T-cells expansion and cytolytic function correlated with the level of CD19 expression on target cells. In addition, increasing CD19 expression in leukaemia cells using hypomethylating agent 5-aza-2′-deoxycytidine (5-AZA) promoted antigen-specific recognition of target cells by CAR19-T cells and increased their expansion. Treatment with 5-AZA also increased CAR expression on the CAR19 T-cells, however, cytotoxicity towards CAR19 T cells mediated by the agent delayed CAR19T-cell expansion. Interestingly, allogeneic stimulation of CAR19 T-cells using CD3/CD28 activation of TCR signalling or co-culture with allogeneic bone marrow stroma cells promoted CAR19 T-cell expansion, however, addition of antigen-specific target cells (CD19+leukaemia cells) attenuated allogeneic expansion indicating the antagonism between antigen-specific and allogeneic CAR19 T- cell activation.Effector to target (E:T) cell ratio appears to be a strong determinate of CAR19 T-cell expansion and cytolytic activity towards cancer cells. CAR19 T-cells used at high E:T ratios exhibit higher cytolytic activity compared to effector cells used at a low E:T. Cell division analysis demonstrats lower proliferation of CAR19 T-cells when used at a high E:T compared to those used at low E:T ratio. The repetitive antigen-specific activation of CAR19 T-cells occurring at a high density of target cells (low E:T ratio) promotes proliferation, however, also acts to induce terminal effector differentiation resulting in rapid T cell extinction due to activated T cell death. Thus the excessive proliferation and rapid extinction of CAR19 T-cells may account for the low efficacy of CAR19 T- cell therapy when given at low E:T ratios. Similar effects were observed in a stem cell reconstituted mouse model where stem cell-derived CD19+ B cells were targeted by CAR19 T-cells. A single infusion of CAR19-T cells infused at a high E:T ratio in mice with low B cell engraftment resulted in efficient B cell depletion while the same dose of CAR19 T-cells infused to mice with high B cell engraftment level(low E:T ratio) was less efficient. These findings show that rapid exhaustion of effector cells used at low E:T ratio prevents long lasting anti-tumor effects justifying the need of pre-conditioning in patients with advanced tumors.. DisclosuresNo relevant conflicts of interest to declare.

Efficacy of CAR T-cell therapy in large tumors relies upon stromal targeting by IFNγ.

Adoptive T-cell therapy using chimeric antigen receptor-modified T cells (CAR-T therapy) has shown dramatic efficacy in patients with circulating lymphoma. However, eradication of solid tumors with CAR-T therapy has not been reported yet to be efficacious. In solid tumors, stroma destruction, due to MHC-restricted cross-presentation of tumor antigens to T cells, may be essential. However, CAR-Ts recognize antigens in an MHC-independent manner on cancer cells but not stroma cells. In this report, we show how CAR-Ts can be engineered to eradicate large established tumors with provision of a suitable CD28 costimulatory signal. In an HER2-dependent tumor model, tumor rejection by HER2-specific CAR-Ts was associated with sustained influx and proliferation of the adoptively transferred T cells. Interestingly, tumor rejection did not involve natural killer cells but was associated instead with a marked increase in the level of M1 macrophages and a requirement for IFNγ receptor expression on tumor stroma cells. Our results argue that CAR-T therapy is capable of eradicating solid tumors through a combination of antigen-independent stroma destruction and antigen-specific tumor cell targeting.

Identifying the factors modulating the efficacy of car-t cell therapy

Identifying the factors modulating the efficacy of car-t cell therapy