Generation and Functional Verification of Hypoxia-sensitive Chimeric Antigen Receptor-T Cells.

Extensive studies have proven the promise of chimeric antigen receptor T (CAR-T) cell therapy in treating hematological malignancies. However, treating solid tumors remains challenging, as exemplified by the safety concerns that arise when CAR-T cells attack normal cells expressing the target antigens. Researchers have explored various approaches to enhance the tumor selectivity of CAR-T cell therapy. One representative strategy along this line is the construction of hypoxia-sensitive CAR-T cells, which are designed by fusing an oxygen-dependent degradation domain to the CAR moiety and are strategized to attain high CAR expression only in a hypoxic environment-the tumor microenvironment (TME). This paper presents a protocol for the generation of such CAR-T cells and their functional characterization, including methods to analyze the changes in CAR expression and killing capacity in response to different oxygen levels established by a mobile incubator chamber. The constructed CAR-T cells are anticipated to demonstrate CAR expression and cytotoxicity in an oxygen-sensitive manner, thus supporting their capability to distinguish between hypoxic TME and normoxic normal tissues for selective activation.

PET/CT in the Evaluation of CAR-T Cell Immunotherapy in Hematological Malignancies.

Chimeric antigen receptor (CAR)-T cell-based immunotherapy has emerged as a path-breaking strategy for certain hematological malignancies. Assessment of the response to CAR-T therapy using quantitative imaging techniques such as positron emission tomography/computed tomography (PET/CT) has been broadly investigated. However, the definitive role of PET/CT in CAR-T therapy remains to be established. [18F]FDG PET/CT has demonstrated high sensitivity and specificity for differentiating patients with a partial and complete response after CAR-T therapy in lymphoma. The early therapeutic response and immune-related adverse effects such as cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome can also be detected on [18F]FDG PET images. In otherwise asymptomatic lymphoma patients with partial response following CAR-T therapy, the only positive findings could be abnormal PET/CT results. In multiple myeloma, a negative [18F]FDG PET/CT after receiving B-cell maturation antigen-directed CAR-T therapy has been associated with a favorable prognosis. In leukemia, [18F]FDG PET/CT can detect extramedullary metastases and treatment responses after therapy. Hence, PET/CT is a valuable imaging tool for patients undergoing CAR-T therapy for pretreatment evaluation, monitoring treatment response, assessing safety, and guiding therapeutic strategies. Developing guidelines with standardized cutoff values for various PET parameters and tumor cell-specific tracers may improve the efficacy and safety of CAR-T therapy.

Broadening anticancer spectrum by preprocessing and treatment of T- lymphocytes expressed FcγRI and monoclonal antibodies for refractory cancers.

Background: Chimeric antigen receptor T (CAR-T) cell therapies have achieved remarkable success in the treatment of hematological tumors. However, given the distinct features of solid tumors, particularly heterogeneity, metabolic aggressiveness, and fewer immune cells in tumor microenvironment (TME), the practical utility of CAR-T cells for solid tumors remains as a challenging issue. Meanwhile, although anti-PD-1 monoclonal antibody (mAb) has shown clinical efficacy, most mAbs also show limited clinical benefits for solid tumors due mainly to the issues associated with the lack of immune cells in TME. Thus, the infiltration of targeted immunological active cells into TME could generate synergistic efficacy for mAbs. Methods: We present a combinational strategy for solid tumor treatment, which combines armored-T cells to express Fc-gamma receptor I (FcγRI) fragment on the surfaces for targeting various tumors with therapeutically useful mAbs. Choosing CD20 and HER-2 as the targets, we characterized the in vitro and in vivo efficacy and latent mechanism of the combination drug by using flow cytometry, ELISA and other methods. Results: The combination and preprocessing of armored T-cells with corresponding antibody of Rituximab and Pertuzumab exerted profound anti-tumor effects, which is demonstrated to be mediated by synergistically produced antibody-dependent cellular cytotoxicity (ADCC) effects. Meanwhile, mAb was able to carry armored-T cell by preprocessing for the infiltration to TME in cell derived xenograft (CDX) model. Conclusions: This combination strategy showed a significant increase of safety profiles from the reduction of antibody doses. More importantly, the present strategy could be a versatile tool for a broad spectrum of cancer treatment, with a simple pairing of engineered T cells and a conventional antibody.

[CAR T-cell therapy for malignant lymphoma].

Advances in understanding of the pathogenesis of B-cell lymphoma have led to development of various novel targeted therapies. Among them, CD19-targeted chimeric antigen receptor (CAR) T-cell therapies for relapsed and refractory B-cell lymphomas have shown remarkable efficacy in clinical trials, and three CAR T-cell products are now available in Japan. Real-world evidence (RWE) has shown that these products can provide comparable efficacy to clinical trials in clinical practice, where CAR T-cells were administered in patients with wider range of backgrounds. This finding will certainly broaden the role of CAR T-cell therapies in the treatment of B-cell lymphoma. However, since about half of the patients treated with CAR T-cell therapy progress thereafter, there is an urgent need for risk stratification and optimized management of refractory cases. Here, we review the results of clinical trials and RWE of CAR T-cell therapy in B-cell lymphoma.

Engineering strategies to safely drive CAR T-cells into the future.

Chimeric antigen receptor (CAR) T-cell therapy has proven a breakthrough in cancer treatment in the last decade, giving unprecedented results against hematological malignancies. All approved CAR T-cell products, as well as many being assessed in clinical trials, are generated using viral vectors to deploy the exogenous genetic material into T-cells. Viral vectors have a long-standing clinical history in gene delivery, and thus underwent iterations of optimization to improve their efficiency and safety. Nonetheless, their capacity to integrate semi-randomly into the host genome makes them potentially oncogenic via insertional mutagenesis and dysregulation of key cellular genes. Secondary cancers following CAR T-cell administration appear to be a rare adverse event. However several cases documented in the last few years put the spotlight on this issue, which might have been underestimated so far, given the relatively recent deployment of CAR T-cell therapies. Furthermore, the initial successes obtained in hematological malignancies have not yet been replicated in solid tumors. It is now clear that further enhancements are needed to allow CAR T-cells to increase long-term persistence, overcome exhaustion and cope with the immunosuppressive tumor microenvironment. To this aim, a variety of genomic engineering strategies are under evaluation, most relying on CRISPR/Cas9 or other gene editing technologies. These approaches are liable to introduce unintended, irreversible genomic alterations in the product cells. In the first part of this review, we will discuss the viral and non-viral approaches used for the generation of CAR T-cells, whereas in the second part we will focus on gene editing and non-gene editing T-cell engineering, with particular regard to advantages, limitations, and safety. Finally, we will critically analyze the different gene deployment and genomic engineering combinations, delineating strategies with a superior safety profile for the production of next-generation CAR T-cell.

Universal CAR 2.0 to overcome current limitations in CAR therapy.

Chimeric antigen receptor (CAR) T cell therapy has effectively complemented the treatment of advanced relapsed and refractory hematological cancers. The remarkable achievements of CD19- and BCMA-CAR T therapies have raised high expectations within the fields of hematology and oncology. These groundbreaking successes are propelling a collective aspiration to extend the reach of CAR therapies beyond B-lineage malignancies. Advanced CAR technologies have created a momentum to surmount the limitations of conventional CAR concepts. Most importantly, innovations that enable combinatorial targeting to address target antigen heterogeneity, using versatile adapter CAR concepts in conjunction with recent transformative next-generation CAR design, offer the promise to overcome both the bottleneck associated with CAR manufacturing and patient-individualized treatment regimens. In this comprehensive review, we delineate the fundamental prerequisites, navigate through pivotal challenges, and elucidate strategic approaches, all aimed at paving the way for the future establishment of multitargeted immunotherapies using universal CAR technologies.

In vitro CAR-T cell killing: validation of the potency assay.

For advanced therapy medicinal products, the development and validation of potency assays are required, in accordance with international guidelines, to characterise the product and obtain reliable and consistent data. Our purpose was to validate the killing assay for the evaluation of autologous anti-CD19 chimeric antigen receptor (CAR) T potency. We used CD4 + and CD8 + lymphocytes or anti-CD19 CAR-T cells as effector cells and REH (CD19 +) or MOLM-13 (CD19 -) cell lines as target cells. After co-culturing target and effector cells (1:1 ratio) for 24 h, samples were labelled with 7-AAD, anti-CD3 and anti-CD19 antibodies and the frequency of CD19 + dead cells was evaluated by flow cytometry. In order to verify the CAR-T specificity for the CD19 + target, the co-culture between CAR-T and REH or MOLM-13 at different effector-to-target ratios was scheduled. Moreover, not transduced CD4 + and CD8 + lymphocytes were tested in comparison with CAR-T from the same donor to demonstrate the assay specificity. Linearity and accuracy were evaluated, and established acceptance criteria were compiled for both parameters (r2 ≥ 0.97 for linearity and average relative error ≤ 10% for accuracy). Furthermore, the method was considered robust when performed between 23 and 25 h of co-culture, and the intra-assay, inter-assay and inter-day precision was obtained. Finally, in order to verify the inter-analyst precision, the test was executed by three different operators and the intra-class correlation coefficient was > 0.4 in both cases. In conclusion, we consider this CAR-T potency assay as validated and usable in all steps of product development and quality control.

Impact of fludarabine and treosulfan on ovarian tumor cells and mesothelin chimeric antigen receptor T cells.

In addition to their immunosuppressive effect, cytostatics conditioning prior to adoptive therapy such as chimeric antigen receptor (CAR) T cells may play a role in debulking and remodeling the tumor microenvironment. We investigated in vitro the killing efficacy and impact of treosulfan and fludarabine on ovarian cancer cells expressing mesothelin (MSLN) and effect on MSLN-targeting CAR T cells. Treosulfan and fludarabine had a synergetic effect on killing of SKOV3 and OVCAR4 cells. Sensitivity to the combination of treosulfan and fludarabine was increased when SKOV3 cells expressed MSLN and when OVCAR4 cells were tested in hypoxia, while MSLN cells surface expression by SKOV3 and OVCAR4 cells was not altered after treosulfan or fludarabine exposure. Exposure to treosulfan or fludarabine (10 µM) neither impacted MSLN-CAR T cells degranulation, cytokines production upon challenge with MSLN + OVCAR3 cells, nor induced mitochondrial defects. Combination of treosulfan and fludarabine decreased MSLN-CAR T cells anti-tumor killing in normoxia but not hypoxia. In conclusion, treosulfan and fludarabine killed MSLN + ovarian cancer cells without altering MSLN-CAR T cells functions (at low cytostatics concentration) even in hypoxic conditions, and our data support the use of treosulfan and fludarabine as conditioning drugs prior to MSLN-CAR T cell therapy.

Systemic delivery of oncolytic herpes virus using CAR-T cells enhances targeting of antitumor immuno-virotherapy.

Recent studies have indicated that combining oncolytic viruses with CAR-T cells in therapy has shown superior anti-tumor effects, representing a promising approach. Nonetheless, the localized delivery method of intratumoral injection poses challenges for treating metastatic tumors or distal tumors that are difficult to reach. To address this obstacle, we employed HSV-1-infected CAR-T cells, which systemically delivery HSV into solid tumors. The biological function of CAR-T cells remained intact after loading them with HSV for a period of three days. In both immunocompromised and immunocompetent GBM orthotopic mouse models, B7-H3 CAR-T cells effectively delivered HSV to tumor lesions, resulting in enhanced T-cell infiltration and significantly prolonged survival in mice. We also employed a bilateral subcutaneous tumor model and observed that the group receiving intratumoral virus injection exhibited a significant reduction in tumor volume on the injected side, while the group receiving intravenous infusion of CAR-T cells carrying HSV displayed suppressed tumor growth on both sides. Hence, CAR-THSV cells offer notable advantages in the systemic delivery of HSV to distant tumors. In conclusion, our findings emphasize the potential of CAR-T cells as carriers for HSV, presenting significant advantages for oncolytic virotherapy targeting distant tumors.

Engineered CD147-CAR macrophages for enhanced phagocytosis of cancers.

Chimeric antigen receptor (CAR) T cell therapy has shown promising results in hematologic malignancies, but its effectiveness in solid cancers remains challenging. Macrophages are immune cells residing within the tumor microenvironment. They can phagocytose tumor cells. Recently, CAR macrophages (CAR-M) have been a promising candidate for treating solid cancers. One of the common cancer antigens overexpressed in various types of cancer is CD147. CAR-T and NK cells targeting CD147 antigen have shown significant efficacy against hepatocellular carcinoma. Nevertheless, CAR-M targeting the CD147 molecule has not been investigated. In this study, we generated CAR targeting the CD147 molecule using the THP-1 monocytic cell line (CD147 CAR-M). The CD147 CAR-M exhibited typical macrophage characteristics, including phagocytosis of zymosan bioparticles and polarization ability toward M1 and M2 phenotypes. Furthermore, the CD147 CAR-M demonstrated enhanced anti-tumor activity against K562 and MDA-MB-231 cells without exhibiting off-target cytotoxicity against normal cells. Our research provides valuable insights into the potential of CD147 CAR-M as a promising platform for cancer immunotherapy, with applications in both hematologic malignancies and solid cancers.

Tethered IL15-IL15Rα augments antitumor activity of CD19 CAR-T cells but displays long-term toxicity in an immunocompetent lymphoma mouse model.

BACKGROUND: Adoptive cell therapy using genetically modified T cells to express chimeric antigen receptors (CAR-T) has shown encouraging results, particularly in certain blood cancers. Nevertheless, over 40% of B cell malignancy patients experience a relapse after CAR-T therapy, likely due to inadequate persistence of the modified T cells in the body. IL15, known for its pro-survival and proliferative properties, has been suggested for incorporation into the fourth generation of CAR-T cells to enhance their persistence. However, the potential systemic toxicity associated with this cytokine warrants further evaluation. METHODS: We analyzed the persistence, antitumor efficacy and potential toxicity of anti-mouse CD19 CAR-T cells which express a membrane-bound IL15-IL15Rα chimeric protein (CD19/mbIL15q CAR-T), in BALB/c mice challenged with A20 tumor cells as well as in NSG mice. RESULTS: Conventional CD19 CAR-T cells showed low persistence and poor efficacy in BALB/c mice treated with mild lymphodepletion regimens (total body irradiation (TBI) of 1 Gy). CD19/mbIL15q CAR-T exhibits prolonged persistence and enhanced in vivo efficacy, effectively eliminating established A20 B cell lymphoma. However, this CD19/mbIL15q CAR-T displays important long-term toxicities, with marked splenomegaly, weight loss, transaminase elevations, and significant inflammatory findings in some tissues. Mice survival is highly compromised after CD19/mbIL15q CAR-T cell transfer, particularly if a high TBI regimen is applied before CAR-T cell transfer. CONCLUSION: Tethered IL15-IL15Rα augments the antitumor activity of CD19 CAR-T cells but displays long-term toxicity in immunocompetent mice. Inducible systems to regulate IL15-IL15Rα expression could be considered to control this toxicity.

Off-the-shelf CAR-NK cells targeting immunogenic cell death marker ERp57 execute robust antitumor activity and have a synergistic effect with ICD inducer oxaliplatin.

BACKGROUND: Chimeric antigen receptor natural killer (CAR-NK) therapy holds great promise for treating hematologic tumors, but its efficacy in solid tumors is limited owing to the lack of suitable targets and poor infiltration of engineered NK cells. Here, we explore whether immunogenic cell death (ICD) marker ERp57 translocated from endoplasmic reticulum to cell surface after drug treatment could be used as a target for CAR-NK therapy. METHODS: To target ERp57, a VHH phage display library was used for screening ERp57-targeted nanobodies (Nbs). A candidate Nb with high binding affinity to both human and mouse ERp57 was used for constructing CAR-NK cells. Various in vitro and in vivo studies were performed to assess the antitumor efficacy of the constructed CAR-NK cells. RESULTS: We demonstrate that the translocation of ERp57 can not only be induced by low-dose oxaliplatin (OXP) treatment but also is spontaneously expressed on the surface of various types of tumor cell lines. Our results show that G6-CAR-NK92 cells can effectively kill various tumor cell lines in vitro on which ERp57 is induced or intrinsically expressed, and also exhibit potent antitumor effects in cancer cell-derived xenograft and patient-derived xenograft mouse models. Additionally, the antitumor activity of G6-CAR-NK92 cells is synergistically enhanced by the low-dose ICD-inducible drug OXP. CONCLUSION: Collectively, our findings suggest that ERp57 can be leveraged as a new tumor antigen for CAR-NK targeting, and the resultant CAR-NK cells have the potential to be applied as a broad-spectrum immune cell therapy for various cancers by combining with ICD inducer drugs.

A bibliometric and knowledge-map study on the treatment of hematological malignancies with CAR-T cells from 2012 to 2023.

Recently, CAR-T cell therapy in hematological malignancies has received extensive attention. The objective of this study is to gain a comprehensive understanding of the current research status, development trends, research hotspots, and emerging topics pertaining to CAR-T cells in the treatment of hematological malignancies. Articles pertaining to CAR-T cell therapy for hematological malignancies from the years 2012 to 2023 were obtained and assessed from the Web of Science Core Collection (WoSCC). A bibliometric approach was employed to conduct a scientific, comprehensive, and objective quantitative analysis, as well as a visual analysis, of this particular research domain. A comprehensive analysis was conducted on a corpus of 3643 articles, which were collaboratively authored by 72 countries and various research institutions. CAR-T cell research in treating hematological malignancies shows an increasing trend each year. Notably, the study identified the countries and institutions displaying the highest level of activity, the journals with the most citations and output, as well as the authors who garnered the highest frequency of citations and co-citations. Furthermore, the analysis successfully identified the research hotspots and highlighted six emerging topics within this domain. This study conducted a comprehensive exploration and analysis of the research status, development trends, research hotspots, and emerging topics about CAR-T cells in the treatment of hematological malignancies from 2012 to 2023. The findings of this study will serve as a valuable reference and guide for researchers seeking to delve deeper into this field and determine the future direction of their research.

CARTAR: a comprehensive web tool for identifying potential targets in chimeric antigen receptor therapies using TCGA and GTEx data.

Chimeric antigen receptor (CAR) therapy has emerged as a ground-breaking advancement in cancer treatment, harnessing the power of engineered human immune cells to target and eliminate cancer cells. The escalating interest and investment in CAR therapy in recent years emphasize its profound significance in clinical research, positioning it as a rapidly expanding frontier in the field of personalized cancer therapies. A crucial step in CAR therapy design is choosing the right target as it determines the therapy's effectiveness, safety and specificity against cancer cells, while sparing healthy tissues. Herein, we propose a suite of tools for the identification and analysis of potential CAR targets leveraging expression data from The Cancer Genome Atlas and Genotype-Tissue Expression Project, which are implemented in CARTAR website. These tools focus on pinpointing tumor-associated antigens, ensuring target selectivity and assessing specificity to avoid off-tumor toxicities and can be used to rationally designing dual CARs. In addition, candidate target expression can be explored in cancer cell lines using the expression data for the Cancer Cell Line Encyclopedia. To our best knowledge, CARTAR is the first website dedicated to the systematic search of suitable candidate targets for CAR therapy. CARTAR is publicly accessible at https://gmxenomica.github.io/CARTAR/.

[Challenges and strategies for improving efficacy in CAR-T therapy].

CAR-T cell therapy targeting CD19 and BCMA for relapsed or refractory hematopoietic tumors has been adopted in routine practice and has shown dramatic results. However, half of patients who achieve remission with CAR-T therapy eventually relapse, and thus efforts to improve the efficacy of CAR-T therapy are gaining momentum. Notably, studies have described innovative technologies that enable control of cell kinetics after infusion, which is not possible with conventional CAR-T therapies. In this article, we review the challenges of CAR-T cell therapy and the development of new technologies.