CAR T cells redirected to B7-H3 for pediatric solid tumors: Current status and future perspectives.

Despite intensive therapies, pediatric patients with relapsed or refractory solid tumors have poor outcomes and need novel treatments. Immune therapies offer an alternative to conventional treatment options but require the identification of differentially expressed antigens to direct antitumor activity to sites of disease. B7-H3 (CD276) is an immune regulatory protein that is expressed in a range of malignancies and has limited expression in normal tissues. B7-H3 is highly expressed in pediatric solid tumors including osteosarcoma, rhabdomyosarcoma, Ewing sarcoma, Wilms tumor, neuroblastoma, and many rare tumors. In this article we review B7-H3-targeted chimeric antigen receptor (B7-H3-CAR) T cell therapies for pediatric solid tumors, reporting preclinical development strategies and outlining the landscape of active pediatric clinical trials. We identify challenges to the success of CAR T cell therapy for solid tumors including localizing to and penetrating solid tumor sites, evading the hostile tumor microenvironment, supporting T cell expansion and persistence, and avoiding intrinsic tumor resistance. We highlight strategies to overcome these challenges and enhance the effect of B7-H3-CAR T cells, including advanced CAR T cell design and incorporation of combination therapies.

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.

Rapidly-manufactured CD276 CAR-T cells exhibit enhanced persistence and efficacy in pancreatic cancer.

BACKGROUND: Pancreatic cancer is one of the most lethal malignancies and the lack of treatment options makes it more deadly. Chimeric Antigen Receptor T-cell (CAR-T) immunotherapy has revolutionized cancer treatment and made great breakthroughs in treating hematological malignancies, however its success in treating solid cancers remains limited mainly due to the lack of tumor-specific antigens. On the other hand, the prolonged traditional manufacturing process poses challenges, taking 2 to 6 weeks and impacting patient outcomes. CD276 has recently emerged as a potential therapeutic target for anti-solid cancer therapy. Here, we investigated the efficacy of CD276 CAR-T and rapidly-manufactured CAR-T against pancreatic cancer. METHODS: In the present study, CD276 CAR-T was prepared by CAR structure carrying 376.96 scFv sequence, CD8 hinge and transmembrane domain, 4-1BB and CD3ζ intracellular domains. Additionally, CD276 rapidly-manufactured CAR-T (named CD276 Dash CAR-T) was innovatively developed by shortening the duration of ex vitro culture to reduce CAR-T manufacturing time. We evaluated the anti-tumor efficacy of CD276 CAR-T and further compared the functional assessment of Dash CAR-T and conventional CAR-T in vitro and in vivo by detecting the immunophenotypes, killing ability, expansion capacity and tumor-eradicating effect of CAR-T. RESULTS: We found that CD276 was strongly expressed in multiple solid cancer cell lines and that CD276 CAR-T could efficiently kill these solid cancer cells. Moreover, Dash CAR-T was successfully manufactured within 48-72 h and the functional validation was carried out subsequently. In vitro, CD276 Dash CAR-T possessed a less-differentiated phenotype and robust proliferative ability compared to conventional CAR-T. In vivo xenograft mouse model, CD276 Dash CAR-T showed enhanced anti-pancreatic cancer efficacy and T cell expansion. Besides, except for the high-dose group, the body weight of mice was maintained stable, and the state of mice was normal. CONCLUSIONS: In this study, we proved CD276 CAR-T exhibited powerful activity against pancreatic cancer cells in vitro and in vivo. More importantly, we demonstrated the manufacturing feasibility, acceptable safety and superior anti-tumor efficacy of CD276 Dash CAR-T generated with reduced time. The results of the above studies indicated that CD276 Dash CAR-T immunotherapy might be a novel and promising strategy for pancreatic cancer treatment.

A multi-institutional survey of apheresis services among institutions in the United States.

INTRODUCTION: Apheresis practices in the United States (US) have not been comprehensively characterized to date. This study aimed to address this gap by evaluating apheresis therapy through a national survey. METHODS: A multi-institutional survey was conducted between April and July 2023. The survey, comprising 54 questions, focused on institutional demographics, procedures, equipment, staffing, training, and impacts of the Coronavirus Disease 2019 (COVID-19) pandemic. Responses from 22 institutions, primarily academic medical centers, were analyzed. RESULTS: Therapeutic plasma exchange (TPE) was the most common procedure, followed by hematopoietic progenitor cell collection (HPC-A) and red blood cell exchange (RCE). CAR-T cell collections were widespread, with some institutions supporting over 30 protocols concurrently. Most sites used the Spectra Optia Apheresis System, were managed by a transfusion medicine service, and employed internal apheresis providers. Insufficient staffing levels, exacerbated by the COVID-19 pandemic, were common and most often addressed using overtime. DISCUSSION: The survey highlighted the ubiquity of TPE, expanding cellular collections and staffing challenges. The role of apheresis in supporting cellular therapy, particularly in newly developing cell and gene therapies and clinical trials, was evident. Staffing issues during the pandemic emphasized the need for innovative recruitment strategies. CONCLUSION: This nationwide survey provides the most comprehensive analysis to date of apheresis practices in large US academic centers.

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.

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.

PET Imaging in Chimeric Antigen Receptor T-Cell Trafficking.

The evolving field of chimeric antigen receptor (CAR) T-cell therapy, though promising, necessitates more comprehensive imaging methods to enhance therapeutic effectiveness and track cell trafficking in patients and ex vivo. This review examines the application of PET imaging in CAR T-cell trafficking and optimizing their therapeutic impact. The application of PET imaging using various radiotracers is promising in providing evaluation of CAR T-cell interaction within the host, thereby facilitating strategies for improved patient outcomes. As this technology progresses, further innovative strategies to streamline assessments of immunotherapeutic effectiveness are anticipated.

Investigating multilevel cognitive processing within error-free and error-prone feedback conditions in executed and observed car driving.

Accident analyses repeatedly reported the considerable contribution of run-off-road incidents to fatalities in road traffic, and despite considerable advances in assistive technologies to mitigate devastating consequences, little insight into the drivers' brain response during such accident scenarios has been gained. While various literature documents neural correlates to steering motion, the driver's mental state, and the impact of distraction and fatigue on driving performance, the cortical substrate of continuous deviations of a car from the road - i.e., how the brain represents a varying discrepancy between the intended and observed car position and subsequently assigns customized levels of corrective measures - remains unclear. Furthermore, the superposition of multiple subprocesses, such as visual and erroneous feedback processing, performance monitoring, or motor control, complicates a clear interpretation of engaged brain regions within car driving tasks. In the present study, we thus attempted to disentangle these subprocesses, employing passive and active steering conditions within both error-free and error-prone vehicle operation conditions. We recorded EEG signals of 26 participants in 13 sessions, simultaneously measuring pairs of Executors (actively steering) and Observers (strictly observing) during a car driving task. We observed common brain patterns in the Executors regardless of error-free or error-prone vehicle operation, albeit with a shift in spectral activity from motor beta to occipital alpha oscillations within erroneous conditions. Further, significant frontocentral differences between Observers and Executors, tracing back to the caudal anterior cingulate cortex, arose during active steering conditions, indicating increased levels of motor-behavioral cognitive control. Finally, we present regression results of both the steering signal and the car position, indicating that a regression of continuous deviations from the road utilizing the EEG might be feasible.

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/.

Genetic ablation of adhesion ligands mitigates rejection of allogeneic cellular immunotherapies.

Allogeneic cellular immunotherapies hold promise for broad clinical implementation but face limitations due to potential rejection of donor cells by the host immune system. Silencing of beta-2 microglobulin (B2M) expression is commonly employed to evade T cell-mediated rejection by the host, although the absence of B2M is expected to trigger missing-self responses by host natural killer (NK) cells. Here, we demonstrate that genetic deletion of the adhesion ligands CD54 and CD58 in B2M-deficient chimeric antigen receptor (CAR) T cells and multi-edited induced pluripotent stem cell (iPSC)-derived CAR NK cells reduces their susceptibility to rejection by host NK cells in vitro and in vivo. The absence of adhesion ligands limits rejection in a unidirectional manner in B2M-deficient and B2M-sufficient settings without affecting the antitumor functionality of the engineered donor cells. Thus, these data suggest that genetic ablation of adhesion ligands effectively alleviates rejection by host immune cells, facilitating the implementation of universal immunotherapy.

IL-13Rα2/TGF-ß bispecific CAR-T cells counter TGF-ß-mediated immune suppression and potentiate anti-tumor responses in glioblastoma.

BACKGROUND: Chimeric antigen receptor (CAR)-T cell therapies targeting glioblastoma (GBM)-associated antigens such as interleukin-13 receptor subunit alpha-2 (IL-13Rα2) have achieved limited clinical efficacy to date, in part due to an immunosuppressive tumor microenvironment (TME) characterized by inhibitory molecules such as transforming growth factor-beta (TGF-ß). The aim of this study was to engineer more potent GBM-targeting CAR-T cells by countering TGF-ß-mediated immune suppression in the TME. METHODS: We engineered a single-chain, bispecific CAR targeting IL-13Rα2 and TGF-ß, which programs tumor-specific T cells to convert TGF-ß from an immunosuppressant to an immunostimulant. Bispecific IL-13Rα2/TGF-ß CAR-T cells were evaluated for efficacy and safety against both patient-derived GBM xenografts and syngeneic models of murine glioma. RESULTS: Treatment with IL-13Rα2/TGF-ß CAR-T cells leads to greater T-cell infiltration and reduced suppressive myeloid cell presence in the tumor-bearing brain compared to treatment with conventional IL-13Rα2 CAR-T cells, resulting in improved survival in both patient-derived GBM xenografts and syngeneic models of murine glioma. CONCLUSION: Our findings demonstrate that by reprogramming tumor-specific T-cell responses to TGF-ß, bispecific IL-13Rα2/TGF-ß CAR-T cells resist and remodel the immunosuppressive TME to drive potent anti-tumor responses in GBM.

Toxic epidermal necrolysis-like cutaneous toxicity following chimeric antigen receptor T-cell therapy in recurrent large B-cell lymphoma.

Chimeric antigen receptor (CAR) T-cell therapy has demonstrated remarkable success in treating various B-cell malignancies, redirecting T-cell cytotoxicity toward cancer cells. Despite its efficacy, CAR-T therapy is associated with potential risks, including cytokine release syndrome (CRS) and cytopenia. We present a case of a 69-year-old man with diffuse large B-cell lymphoma treated with axicabtagene-ciloleucel CAR-T therapy, who developed a rare and severe cutaneous toxicity resembling toxic epidermal necrolysis (TEN). The patient exhibited persistent fevers, CRS, and subsequent development of a widespread erythematous macular eruption, progressing to vesiculation with bullae. Notably, allopurinol-induced TEN was considered with the patient's recent exposure to allopurinol, although the onset and minimal mucosal involvement did not align with typical presentations of allopurinol-induced cases. The cutaneous reaction, distinct from typical SJS/TEN, showed minimal mucosal involvement and coincided with the cytokine release storm, differing from allopurinol-induced TEN. Despite the absence of guidelines, the patient was managed with systemic steroids, achieving significant improvement. This case expands the spectrum of CAR-T therapy-related cutaneous toxicities, highlighting the need for early recognition of histopathology and tailored management by dermatologists. Further understanding of these reactions is crucial for optimizing the safety profile of this groundbreaking immunotherapy.

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.

Bendamustine is a safe and effective lymphodepletion agent for axicabtagene ciloleucel in patients with refractory or relapsed large B-cell lymphoma.

BACKGROUND: Fludarabine in combination with cyclophosphamide (FC) is the standard lymphodepletion regimen for CAR T-cell therapy (CAR T). A national fludarabine shortage in 2022 necessitated the exploration of alternative regimens with many centers employing single-agent bendamustine as lymphodepletion despite a lack of clinical safety and efficacy data. To fill this gap in the literature, we evaluated the safety, efficacy, and expansion kinetics of bendamustine as lymphodepletion prior to axicabtagene ciloleucel (axi-cel) therapy. METHODS: 84 consecutive patients with relapsed or refractory large B-cell lymphoma treated with axi-cel and managed with a uniform toxicity management plan at Stanford University were studied. 27 patients received alternative lymphodepletion with bendamustine while 57 received FC. RESULTS: Best complete response rates were similar (73.7% for FC and 74% for bendamustine, p=0.28) and there was no significant difference in 12-month progression-free survival or overall survival estimates (p=0.17 and p=0.62, respectively). The frequency of high-grade cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome was similar in both the cohorts. Bendamustine cohort experienced lower proportions of hematological toxicities and antibiotic use for neutropenic fever. Immune reconstitution, as measured by quantitative assessment of cellular immunity, was better in bendamustine cohort as compared with FC cohort. CAR T expansion as measured by peak expansion and area under the curve for expansion was comparable between cohorts. CONCLUSIONS: Bendamustine is a safe and effective alternative lymphodepletion conditioning for axi-cel with lower early hematological toxicity and favorable immune reconstitution.

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.

Non-industrial production of therapeutic lentiviral vectors: How to provide vectors to academic CAR-T.

Bringing effective cancer therapy in the form of chimeric antigen receptor technology to untapped markets faces numerous challenges, including a global shortage of therapeutic lentiviral or retroviral vectors on which all current clinical therapies using genetically modified T cells are based. Production of these lentiviral vectors in academic settings in principle opens the way to local production of therapeutic cells, which is the only economically viable approach to make this therapy available to patients in developing countries. The conditions for obtaining and concentrating lentiviral vectors have been optimized and described. The calcium phosphate precipitation method was found to be suitable for transfecting high cell-density cultures, a prerequisite for high titers. We describe protocols for gradually increasing production from 6-well plates to P100 plates, T-175 flasks, and 5-layer stacks while maintaining high titers, >108 transducing units. Concentration experiments using ultracentrifugation revealed the advantage of lower centrifugation speeds compared to competing protocols. The resulting batches of lentiviral vectors had a titer of 1010 infectious particles and were used to transduce primary human T lymphocytes generating chimeric antigen receptor T cells, the quality of which was checked and found potential applicability for treatment.

Clinical and Genomic Profile of Primary Cranial Neurolymphomatosis.

Primary cranial neurolymphomatosis (PCNL) is a rare subtype of primary CNS lymphoma (PCNSL) in which infiltrative lymphomatous involvement is confined to cranial nerves. Here, we report a case of PCNL with successful genomic profiling. A 57-year-old male had a lengthy prediagnostic phase spanning approximately 30 months, characterized by multiple episodes of cranial neuropathies managed by steroids. At the time of diagnosis, the patient had right-sided cranial neuropathies involving cranial nerves (CN) V, VI, and VII. Pathological findings of the right cavernous lesion biopsy were consistent with large B-cell lymphoma-infiltrating nerve fibers. The clinical course was aggressive and refractory, characterized by relentless progression with the development of cervical spinal neurolymphomatosis, cerebrospinal fluid involvement, and ependymal and intraparenchymal cerebral involvement, despite multiple lines of therapy, including chemoimmunotherapy, Bruton's tyrosine kinase inhibitor, radiation, autologous stem cell transplant, chimeric antigen receptor T-cell therapy (CAR-T), and whole-brain radiation. The patient survived for 22 months from the time of the initial diagnosis and 52 months after the first episode of cranial neuropathy. Next-generation sequencing identified mutations (MYD88, CD79b, and PIM1) that are frequently observed in PCNSL. The unusual findings included a total of 22 mutations involving PIM1, indicating a highly active aberrant somatic hypermutation and two missense CXCR4 mutations. CXCR4 mutations have never been described in PCNSL and may have implications for disease biology and therapeutic interventions. We provide a literature review to further elucidate PCNL.