Cytotoxicity Of Chimeric Antigen Receptor Research Articles

Photo-metallo-immunotherapy: Fabricating Chromium-Based Nanocomposites to Enhance CAR-T Cell Infiltration and Cytotoxicity against Solid Tumors.

The infiltration and cytotoxicity of chimeric antigen receptor (CAR)-T cells are crucial for effective elimination of solid tumors. While metallo-immunotherapy is a promising strategy that can activate the antitumor immunity, its role in promoting CAR-T cell therapy remains elusive. The first single-element nanomaterial based on chromium nanoparticles (Cr NPs) for cancer photo-metallo-immunotherapy has been reported previously. Herein, an extended study using biodegradable polydopamine as a versatile carrier for these nanoparticles, enabling synergistic CAR-T cell therapy, is reported. The results show that these nanocomposites with or without further encapsulation of the anticancer drug alpelisib can promote the CAR-T cell migration and antitumor effect. Upon irradiation with near-infrared light, they caused mild hyperthermia that can "warm" the "cold" tumor microenvironment (TME). The administration of B7-H3 CAR-T cells to NOD severe combined immunodeficiency gamma mice bearing a human hepatoma or PIK3CA-mutated breast tumor can significantly inhibit the tumor growth after the induction of tumor hyperthermia by the nanocomposites and promote the secretion of serum cytokines, including IL-2, IFN-γ, and TNF-α. The trivalent Cr3+ ions, which are the major degradation product of these nanocomposites, can increase the CXCL13 and CCL3 chemokine expressions to generate tertiary lymphoid structures (TLSs) in the tumor tissues, facilitating the CAR-T cell infiltration.

Ex vivo pleural effusion cultures to study chimeric antigen receptor T cell cytotoxicity in an immunocompetent environment

Current invitro and invivo assays used to study immunotherapeutic interventions lack human immune components that mimic the tumor microenvironment to investigate drug potency and limitations of efficacy. Herein, we describe an exvivo pleural effusion culture (ePEC) assay, using malignant pleural-effusion-derived soluble and cellular factors that differentially affected the cytotoxicity of chimeric antigen receptor (CAR) Tcells. Following identification of CAR Tcell-suppressive factors, blocking of individual factors reveals their contribution to compromising Tcell efficacy. ePEC is a human component assay that can be utilized for developing next-generation cell and antibody therapies that counteract immunosuppression.

Targeting and cytotoxicity of chimeric antigen receptor T cells grafted with PD1 extramembrane domain

BackgroundImmunosuppression induced by programmed cell death protein 1 (PD1) presents a significant constraint on the effectiveness of chimeric antigen receptor (CAR)-T therapy. The potential of combining PD1/PDL1 (Programmed cell death 1 ligand 1) axis blockade with CAR-T cell therapy is promising. However, developing a highly efficient and minimally toxic approach requires further exploration. Our attempt to devise a novel CAR structure capable of recognizing both tumor antigens and PDL1 encountered challenges since direct targeting of PDL1 resulted in systemic adverse effects.MethodsIn this research, we innovatively engineered novel CARs by grafting the PD1 domain into a conventional second-generation (2G) CAR specifically targeting CD19. These CARs exist in two distinct forms: one with PD1 extramembrane domain (EMD) directly linked to a transmembrane domain (TMD), referred to as PE CAR, and the other with PD1 EMD connected to a TMD via a CD8 hinge domain (HD), known as PE8HT CAR. To evaluate their efficacy, we conducted comprehensive assessments of their cytotoxicity, cytokine release, and potential off-target effects both in vitro and in vivo using tumor models that overexpress CD19/PDL1.ResultsThe findings of our study indicate that PE CAR demonstrates enhanced cytotoxicity and reduced cytokine release specifically towards CD19 + PDL1 + tumor cells, without off-target effects to CD19-PDL1 + tumor cells, in contrast to 2G CAR-T cells. Additionally, PE CAR showed ameliorative differentiation, exhaustion, and apoptosis phenotypes as assessed by flow cytometry, RNA-sequencing, and metabolic parameter analysis, after encountering CD19 + PDL1 + tumor cells.ConclusionOur results revealed that CAR grafted with PD1 exhibits enhanced antitumor activity with lower cytokine release and no PD1-related off-target toxicity in tumor models that overexpress CD19 and PDL1. These findings suggest that our CAR design holds the potential for effectively addressing the PD1 signal.

Engagement of an optimized lentiviral vector enhances the expression and cytotoxicity of CAR in human NK cells.

Adoptive chimeric antigen receptor (CAR)-modified T or NK cells (CAR-T/NK) have emerged as a novel form of disease treatment. Lentiviral vectors (LVs) are commonly employed to engineer NK cells for the efficient expression of CARs. This study reported the influence of single-promoter and dual-promoter LVs on the CAR expression and cytotoxicity of engineered NK cells. We constructed a third-generation NKG2D-based CAR that kills cancer cells by targeting up to eight stress-induced ligands (NKG2DLs). Our results demonstrated that the CAR exhibits both a higher expression level and a higher coexpression concordance with the GFP reporter in HEK-293T or NK92 cells by utilizing the optimized single-promoter pCDHsp rather than the original dual-promoter pCDHdp. After puromycin selection, the pCDHsp produces robust CAR expression and enhanced in vitro cytotoxicity of engineered NK cells. Therefore, infection with a single-promoter pCDHsp lentivector is recommended to prepare CAR-engineered NK cells. This research helps to optimize the production of CAR-NK cells and enhance their functional activity, to provide CAR-NK cell products with better and more uniform quality.

Romidepsin Enhances the Killing Ability of NKG2D-CAR-T Cells through Enhanced Expression of NKG2DL against Ovarian Cancer Cells

Background: Upregulating tumor cell targeting antigens and improving the cytotoxicity of chimeric antigen receptor T cell (CAR-T) are expected to facilitate better treatment efficacy for solid cancers represented by ovarian cancer. Methods: Killer cell lectin-like receptor subfamily K member 1 ligands (NKG2DL) are the target antigens for ovarian cancer. NKG2D-CAR-T cells were constructed for NKG2D ligand on the ovarian cancer cell surface. We used flow cytometry to evaluate the expression of NKG2DL on SKOV3 (a human ovarian cancer adenocarcinoma cell line). Innovatively, when combined with romidepsin to treat ovarian cancer cell SKOV3, to evaluate the killing ability of the combined strategy, we verified the cytotoxicity of CAR-T cells by lactate dehydrogenase (LDH) release test and determined the secretion of cytokines by enzyme-linked immuno sorbent assay (ELISA). Results: The results of flow cytometry showed effective activation of the NKG2D-CAR-T cells, and romidepsin treatment resulted in increased expression of NKG2DL on the surface of SKOV3. Cytotoxicity test showed that romidepsin could enhance the killing ability of NKG2D-CAR-T cells against ovarian cancer cells by regulating their NKG2DL expression (p < 0.05). The killing effects and secretion of interferon-γ (IFN-γ) increased synchronously (p < 0.05). Levels of interleukin-2 (IL-2) and Pore-forming protein (PFP) were statistically significant at a low target ratio but programmed cell death protein 1 (PD-1) remained unaffected (p ≥ 0.05). Conclusions: Enhancing the expression of tumor target antigen is a solution to improve the limited application of CAR-T cells in solid cancers.

Abstract 5568: Revitalization of affinity-tuned CAR T cells via antigen-dependent release of interleukin-12

Abstract Introduction: The application of chimeric antigen receptor (CAR) T cells in solid tumors has met many challenges, arising from the paucity of effective yet safe targets and T cell dysfunction within the immunosuppressive milieu. Clinical trials over the years have reported severe on-target off-tumor toxicities associated with CAR T cell therapies targeting tumor-associated antigens (TAAs). Here, we propose a strategy to target TAAs effectively and safely: 1) improve tumor-targeting specificity by affinity-tuning; 2) armor T cells with antigen-dependent expression of interleukin-12 (IL-12) to compensate loss of CAR T cell activity caused by affinity-tuning. In this system, IL-12 expression is tightly regulated by the nuclear factor of the activated T cells promoter (NFAT) to limit IL-12 to the environ of activated affinity-tuned CAR T cells within the tumor. We demonstrate here the feasibility of this strategy using epithelial cell adhesion molecule (EpCAM) as a model TAA. Methods: Affinity variants were generated by alanine scanning mutagenesis in the complementary-determining region 3 (CDR3) of an EpCAM antibody (UBS54). The affinity of CAR variants was determined by flow cytometry-based saturation binding assay. On-target off-tumor cytotoxicity of CAR T cells was examined against human primary normal epithelial cells. In vivo anti-tumor efficacy was evaluated in mouse models of gastric cancer with cell line-derived (SNU-638 and MKN-45) and patient-derived xenografts. Results: Substitution of alanine into the UBS54 CDR3 led to identification of a tyrosine (6th residue in heavy chain CDR3) as a hot spot for affinity tuning. Among several more subtle amino acid substitutions for Tyr-6, we found that CAR molecule with valine substituent (Y6V) possesses 10 µM affinity toward EpCAM, rendering CAR T cells to be selective to EpCAM-high tumors while being not reactive to primary normal epithelial cells in vitro. In gastric cancer mouse models, compared to UBS54 CAR T cells that mediated rapid tumor remission, Y6V CAR T cells produced mainly partial responses. To revitalize affinity-tuned CAR T cells, we further engineered CAR T cells to release IL-12 under NFAT. We found that both the activity of the NFAT promoter and the level of IL-12 release were tightly regulated by the antigen density of targets when inducible IL-12 is combined with 10 µM affinity Y6V CAR; however, such dependence is lost when 1 µM affinity UBS54 was examined. In mouse models of gastric cancer, inducible lL-12 armored Y6V CAR T cells produced enhanced anti-tumor responses without elevating systemic exposure to IL-12, evidenced by low levels of IL-12 in mouse sera (below 100 pg/ml). Conclusions: Combination of affinity-tuned CAR with inducible expression of IL-12 is a promising strategy to develop CAR T cell therapy against TAAs with the potential for reduced on-target off-tumor toxicity and tumor resistance. Citation Format: Yanping Yang, Huan Yang, Yago Alcaina, Jaclyn E. McCloskey, Janusz Puc, Alyssa Birt, Yogindra Vedvyas, Juan Gonzalez-Valdivieso, Irene M. Min, Eric von Hofe, Moonsoo M. Jin. Revitalization of affinity-tuned CAR T cells via antigen-dependent release of interleukin-12 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5568.

CXCL9-modified CAR T cells improve immune cell infiltration and antitumor efficacy.

Chimeric antigen receptor (CAR) T cells remain unsatisfactory in treating solid tumors. The frequency of tumor-infiltrating T cells is closely related to the good prognosis of patients. Augmenting T cell accumulation in the tumor microenvironment is essential for tumor clearance. To overcome insufficient immune cell infiltration, innovative CAR designs need to be developed immediately. CXCL9 plays a pivotal role in regulating T cell migration and inhibiting tumor angiogenesis. Therefore, we engineered CAR T cells expressing CXCL9 (CART-CXCL9). The addition of CXCL9 enhanced cytokine secretion and cytotoxicity of CAR T cells and endowed CAR T cells with the ability to recruit activated T cells and antiangiogenic effect. In tumor-bearing mice, CART-CXCL9 cells attracted more T cell trafficking to the tumor site and inhibited angiogenesis than conventional CAR T cells. Additionally, CART-CXCL9 cell therapy slowed tumor growth and prolonged mouse survival, displaying superior antitumor activity. Briefly, modifying CAR T cells to express CXCL9 could effectively improve CAR T cell efficacy against solid tumors.

Evaluation of CAR-T cell cytotoxicity: Real-time impedance-based analysis.

This chapter describes the most common method for evaluating cytotoxicity of chimeric antigen receptor (CAR) T cells, the xCELLigence real-time cell analysis (RTCA) platform (Agilent Technologies, Inc., Santa Clara, CA). Though there are a variety of assays used to evaluate conventional and engineered T cell cytotoxicity, the benefit of the xCELLigence platform is the depth of real-time data collected. This chapter begins by providing information on the conceptual basis underlying the xCELLigence assay, followed by a detailed protocol for the application of this assay to evaluate your own CAR-T cells, as well as specific insight and helpful tips for assay design, usage, and data analysis. Application of the information and methods discussed within this chapter will provide a greater understanding for evaluating cytotoxicity of CAR-T cells using this in vitro model system.

The Decrease of Anti-CD3 Antibody Concentration Improved the Cytotoxicity of Chimeric Antigen Receptor (CAR) T Cells in the Treatment of Chronic Lymphoblastic Leukemia (CLL)

Purpose:Chimeric antigen receptor T (CAR-T) cell therapy has demonstrated impressive responses in refractory and relapsed acute lymphoblastic leukemia (ALL) and non-hodgkin's lymphoma (NHL), however, the outcome among chronic lymphoblastic leukemia (CLL) seems to be inferior compared to other lymphoblastic malignancies, indicating that efficacy of CAR-T cell therapy may be attributed to inherent T cell defects that are characteristic of CLL which impaired their proliferative capacity and sustained persistence in vivo. Thereby, infusion of less-differentiated T cells which have the capacity to persist and engraft long-term in vivo may enhance the anti-tumor activity.Materials and methods:On day 0, cryopreserved PBMCs from healthy donors (HDs) and CLL patients were thawed and seeded on anti-CD3 antibody (0.1μg/ml vs 1μg/ml) in combination with anti-CD28 antibody (1μg/ml) coated 24-well plates. On day 3, activated T cells (ATCs) supplied with retroviral supernatant of the third-generation RV-SFG.CD19.CD28.4-1BBzeta vector were transferred into 24-well plates previously coated with retronectin. Transduction efficiencies and phenotypes of CAR-T cells were evaluated on days 7, 10 and 14 after transduction using flow cytometry analysis. On a functional level, chromium 51 (Cr-51) release assay and intracellular staining (ICS) analysis were performed to explore the altered cytotoxic capability of CAR-T cells.Results:We observed that the decrease of anti-CD3 antibody concentration (0.1μg/ml) showed no influence on viability, expansion, transduction efficiency of CAR-T cells generated from HDs or CLL patients compared to standard anti-CD3 antibody concentration (1μg/ml). Moreover, the decrease of anti-CD3 antibody (0.1μg/ml)-mediated T cell activation resulted in an enrichment of less-differentiated naïve (CD45RA +CCR7 +) and central memory (CD45RA -CCR7 +)-like T cells both among CD4 + and CD8 + CAR-T cells. Additionally, cytokines-production (TNF-α, IFN-γ) were dramatically increased evaluated with ICS analysis from HDs and CLL patients in two different concentrations (0.1μg/ml vs. 1μg/ml) . Notably, CAR-T cells derived from HDs displayed decreased cytotoxic capability while CLL patients-derived CAR-T cells demonstrated increased cytotoxicity with lower anti-CD3 antibody concentration (0.1μg/ml) in the assessment of Cr-51 release assay, indicating that the proliferative capacity and sustained persistence of CAR-T cells derived from CLL patients were obtained in vivo.Conclusion:Anti-CD3 antibody-mediated activation of T cells altered anti-tumor efficiency of CAR-T cells before the transduction of ACTs with virus vectors. Consequently, when exploring the strategies to improve the efficacy of CAR-T cells, especially among CLL patients with inherent T cell defects, improvement of the functionality of T cells has to be taken into account. [Display omitted] DisclosuresSchmitt: TolerogenixX: Current holder of individual stocks in a privately-held company; Apogenix: Research Funding; Hexal: Other: Travel grants, Research Funding; Kite Gilead: Other: Travel grants; MSD: Membership on an entity's Board of Directors or advisory committees; Novartis: Other: Travel grants, Research Funding; Bluebird Bio: Other: Travel grants.

Combining selective inhibitors of nuclear export (SINEs) with chimeric antigen receptor (CAR) Tcells for CD19‑positive malignancies.

Chimeric antigen receptor (CAR) T cells directed against CD19 (CD19.CAR T cells) have yielded impressive clinical responses in the treatment of patients with lymphoid malignancies. However, resistance and/or relapse can limit treatment outcome. Risk of tumor escape can be reduced by combining treatment strategies. Selective inhibitors of nuclear export (SINEs) directed against nuclear exportin-1 (XPO1) have demonstrated anti-tumor efficacy in several hematological malignancies. The aim of the present study was to evaluate the combination of CAR T cells with the SINE compounds eltanexor and selinexor. As expected, eltanexor and selinexor were toxic to CD19-positive malignant cells and the sensitivity of cells towards SINEs correlated with the levels of XPO1-expression in ALL cell lines. When SINEs and CAR T cells were simultaneously combined, SINEs exerted toxicity towards CAR T cells and impaired their function affecting cytotoxicity and cytokine release ability. Flow cytometry and western blot analysis revealed that eltanexor decreased the cytoplasmic concentration of the transcription factor phosphorylated-STAT3 in CAR T cells. Due to CAR T-cell toxicity, sequential use of SINEs and CAR T cells was evaluated: Cytotoxicity of CAR T cells increased significantly when target cells were pre-treated with the SINE compound eltanexor. In addition, exhaustion of CAR T cells decreased when target cells were pre-treated with eltanexor. In summary, whereas the concomitant use of SINEs and CAR T cells does not seem advisable, sequential use of SINEs and CAR T cells might improve the anti-tumor efficacy of CAR T cells.

A novel non-viral delivery method that enables efficient engineering of primary human T cells for ex vivo cell therapy applications

Background aimsNext-generation immune cell therapy products will require complex modifications using engineering technologies that can maintain high levels of cell functionality. Non-viral engineering methods have the potential to address limitations associated with viral vectors. However, while electroporation is the most widely used non-viral modality, concerns about its effects on cell functionality have led to the exploration of alternative approaches. Here the authors have examined the suitability of the Solupore non-viral delivery system for engineering primary human T cells for cell therapy applications. MethodsThe Solupore system was used to deliver messenger RNA (mRNA) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) guide RNA ribonucleoprotein (RNP) cargos to T cells, and efficiency was measured by flow cytometry. Cell perturbation was assessed by immune gene expression profiling, including an electroporation comparator. In vitro and in vivo cytotoxicity of chimeric antigen receptor (CAR) T cells generated using the Solupore system was evaluated using a real-time cellular impedance assay and a Raji-luciferase mouse tumor model, respectively. ResultsEfficient transfection was demonstrated through delivery of mRNA and CRISPR CAS9 RNP cargos individually, simultaneously and sequentially using the Solupore system while consistently maintaining high levels of cell viability. Gene expression profiling revealed minimal alteration in immune gene expression, demonstrating the low level of perturbation experienced by the cells during this transfection process. By contrast, electroporation resulted in substantial changes in immune gene expression in T cells. CAR T cells generated using the Solupore system exhibited efficient cytotoxicity against target cancer cells in vitro and in vivo. ConclusionsThe Solupore system is a non-viral means of simply, rapidly and efficiently delivering cargos to primary human immune cells with retention of high cell viability and functionality.

Activated iNKT cells enhance the anti-tumor effect of antigen specific CD8 T cells on mesothelin-expressing salivary gland cancer

BackgroundSalivary gland cancers are not sensitive to conventional radiotherapy or chemotherapy regimens. Therefore, the development of a new treatment strategy is of critical importance for improving the prognosis. We examined the expression of mesothelin molecules in salivary gland cancers and the efficacy of adoptive cell therapy based on mesothelin-specific chimeric antigen receptor transduced T cells.MethodsThe expression of mesothelin molecule was studied in salivary gland cancer samples obtained from 16 patients as well as a salivary gland cancer cell line (A-253) and five other cell lines. The activation of mesothelin-specific chimeric antigen receptor-expressing CD8 T cells after stimulation with mesothelin and the effects of invariant natural killer T cells on this activation were evaluated.ResultsMesothelin was detected in the A-253 cells and the surgical specimens except for the case of squamous cell carcinoma to various degrees. Following stimulation with mesothelin expressing cancer cells, chimeric antigen receptor T cells were dose-dependently activated; this activation was enhanced by co-culture with invariant natural killer T cells and subsequently abrogated by treatment with anti-interferon-γ antibodies. Furthermore, the cytotoxicity of chimeric antigen receptor T cells against various cancer cells was further augmented by invariant natural killer T cells.ConclusionsThe use of adoptive transfer with mesothelin-specific chimeric antigen receptor-expressing CD8 T cells against salivary gland cancers is an effective therapy and invariant natural killer T cells are expected to be used in adjuvant treatment for T cell-based immunotherapy.

Anti-GPC3-CAR T Cells Suppress the Growth of Tumor Cells in Patient-Derived Xenografts of Hepatocellular Carcinoma

The lack of a general clinic-relevant model for human cancer is a major impediment to the acceleration of novel therapeutic approaches for clinical use. We propose to establish and characterize primary human hepatocellular carcinoma (HCC) xenografts that can be used to evaluate the cytotoxicity of adoptive chimeric antigen receptor (CAR) T cells and accelerate the clinical translation of CAR T cells used in HCC. Primary HCCs were used to establish the xenografts. The morphology, immunological markers, and gene expression characteristics of xenografts were detected and compared to those of the corresponding primary tumors. CAR T cells were adoptively transplanted into patient-derived xenograft (PDX) models of HCC. The cytotoxicity of CAR T cells in vivo was evaluated. PDX1, PDX2, and PDX3 were established using primary tumors from three individual HCC patients. All three PDXs maintained original tumor characteristics in their morphology, immunological markers, and gene expression. Tumors in PDX1 grew relatively slower than that in PDX2 and PDX3. Glypican 3 (GPC3)-CAR T cells efficiently suppressed tumor growth in PDX3 and impressively eradicated tumor cells from PDX1 and PDX2, in which GPC3 proteins were highly expressed. GPC3-CAR T cells were capable of effectively eliminating tumors in PDX model of HCC. Therefore, GPC3-CAR T cell therapy is a promising candidate for HCC treatment.