Severe Neurotoxicity Research Articles

Paclitaxel alters the microvascular network in the central and peripheral nervous system of rats with chemotherapy-induced painful peripheral neuropathy.

Chemotherapy-induced peripheral neurotoxicity (CIPN), with paraesthesia, numbness, dysesthesia and neuropathic pain ranks among the most common dose-limiting toxicity of several widely used anticancer drugs. Recent studies revealed the microvascular angiogenesis as a new important actor, beside peripheral neurons, in the neurotoxicity and neuropathic pain development and chronicisation. The aim of this work is to elucidate the role of vascular alterations in CIPN. We evaluated the severity of CIPN with neurophysiological, behavioural and neuropathological analysis together with the microvascular network in central and peripheral nervous systems of rats in order to correlate the features of the CIPN and the vascular abnormalities. The vascular network was quantitatively evaluated through synchrotron radiation-based X-ray phase-contrast micro-tomography imaging, measuring four specific parameters: vascular density, vessel diameter, vessel tortuosity and branching. Rats exposed to paclitaxel and affected by a severe painful sensory axonopathy showed an increased vascular density (putative sprouting angiogenesis) in the crucial districts of the central (somatosensory cortex and lumbar spinal cord) and peripheral nervous system (lumbar dorsal root ganglia). In addition, the complexity of the vascular network and the size of neo-formed vessels were significantly decreased in specific regions. On the other hand, less significant changes were observed in rats exposed to cisplatin, affected by a painless peripheral neuropathy, suggesting a specific involvement of neo-angiogenesis in the development of severe neurotoxicity and neuropathic pain. These new ground-breaking results can shed light on new pathogenetic mechanisms and potential novel therapeutic approaches for painful-CIPN.

Safe and potent anti-CD19 CAR T-cells with shRNA-IL-6 gene silencing element in patients with refractory or relapsed B-cell acute lymphoblastic leukemia.

Severe cytokine release syndrome (sCRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) have limited the widespread use of chimeric antigen receptor T (CAR T)-cell therapy. We designed a novel anti-CD19 CAR (ssCART-19) with a small hairpin RNA (shRNA) element to silence the interleukin-6 (IL-6) gene, hypothesizing it could reduce sCRS and ICANS by alleviating monocyte activation and proinflammatory cytokine release. In a post hoc analysis of two clinical trials, we compared ssCART-19 with common CAR T-cells (cCART-19) in relapsed/refractory B-cell acute lymphoblastic leukemia (r/r B-ALL). Among 87 patients, 47 received ssCART-19 and 40 received cCART-19. Grade ≥3 CRS occurred in 14.89% (7/47) of the ssCART-19 group versus 37.5% (15/40) in the cCART-19 group (p = 0.036). ICANS occurred in 4.26% (2/47) of the ssCART-19 group (all grade 1) compared to 15% (2/40) of the cCART-19 group. Patients in the ssCART-19 group showed comparable rates of treatment response (calculated with rates of complete remission and incomplete hematological recovery) were 91.49% (43/47) for ssCART-19 and 85% (34/40) for cCART-19 (p = 0.999). With a median follow-up of 21.9 months, cumulative nonrelapse mortality was 10.4% for ssCART-19 and 13.6% for cCART-19 (p = 0.33). Median overall survival was 37.17 months for ssCART-19 and 32.93 months for cCART-19 (p = 0.40). Median progression-free survival was 24.17 months for ssCART-19 and 9.33 months for cCART-19 (p = 0.23). These data support the safety and efficacy of ssCART-19 for r/r B-ALL, suggesting its potential as a promising therapy.

Cognitive Impairment from Breast Cancer Chemotherapy in the Perspective of Traditional Chinese Medicine

In recent years, the incidence of breast cancer has been on the rise globally and in China. The conventional treatment methods for breast cancer include surgery, radiotherapy, chemotherapy, endocrine therapy, targeted therapy, traditional Chinese medicine, and other biological immunotherapy. In the treatment of breast cancer, the status of chemotherapy drugs is unshakable, and the related toxic side effects have gradually attracted people's attention. Doxorubicin is an anthracycline drug, which is the cornerstone drug for adjuvant chemotherapy for breast cancer. It exerts anti-tumor effects by inserting DNA and inhibiting topoisomerase II, but it can also cause severe neurotoxicity by crossing the blood-brain barrier, clinically manifested as memory and executive function decline, etc., which has a huge impact on the patient's work and life. With the development of modern medicine, the advantages of traditional Chinese medicine in preventing and treating adverse reactions related to breast cancer and improving patients' quality of life are increasingly being recognized. Traditional Chinese medicine classifies chemotherapy-induced cognitive impairment into related diseases such as "dementia", "brain marrow depletion", and "forgetfulness" based on its main clinical symptoms, and achieves the therapeutic effect of improving chemotherapy-induced cognitive impairment through clinical differentiation and coordinating relevant therapies, providing a scientific basis for the treatment of adverse reactions related to malignant tumors by traditional Chinese medicine in radiotherapy and chemotherapy.

Novel prognostic scoring systems for severe CRS and ICANS after anti-CD19 CAR T cells in large B-cell lymphoma

Autologous anti-CD19 chimeric antigen receptor (CAR) T cells are now used in routine practice for relapsed/refractory (R/R) large B-cell lymphoma (LBCL). Severe (grade ≥ 3) cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity (ICANS) are still the most concerning acute toxicities leading to frequent intensive care unit (ICU) admission, prolonging hospitalization, and adding significant cost to treatment. We report on the incidence of CRS and ICANS and the outcomes in a large cohort of 925 patients with LBCL treated with axicabtagene ciloleucel (axi-cel) or tisagenlecleucel (tisa-cel) in France based on patient data captured through the DESCAR-T registry. CRS of any grade occurred in 778 patients (84.1%), with 74 patients (8.0%) with grade 3 CRS or higher, while ICANS of any grade occurred in 375 patients (40.5%), with 112 patients (12.1%) with grade ≥ 3 ICANS. Based on the parameters selected by multivariable analyses, two independent prognostic scoring systems (PSS) were derived, one for grade ≥ 3 CRS and one for grade ≥ 3 ICANS. CRS-PSS included bulky disease, a platelet count < 150 G/L, a C-reactive protein (CRP) level > 30 mg/L and no bridging therapy or stable or progressive disease (SD/PD) after bridging. Patients with a CRS-PSS score > 2 had significantly higher risk to develop grade ≥ 3 CRS. ICANS-PSS included female sex, low level of platelets (< 150 G/L), use of axi-cel and no bridging therapy or SD/PD after bridging. Patients with a CRS-PSS score > 2 had significantly higher risk to develop grade ≥ 3 ICANS. Both scores were externally validated in international cohorts of patients treated with tisa-cel or axi-cel.

Environmentally relevant concentrations of microplastics from agricultural mulch and cadmium negatively impact earthworms by triggering neurotoxicity and disrupting homeostasis

Recent research has highlighted the ecological risk posed by microplastics (MPs) from mulching film and heavy metals to soil organisms. However, most studies overlooked real environmental levels of MPs and heavy metals. To address this gap, pristine and aged polyethylene (PE) mulching film-derived MPs (PMPs, 500 mg/kg; AMPs, 500 mg/kg) were combined with cadmium (Cd, 0.5 mg/kg) to assess the acute toxicity to earthworms and investigate associated molecular mechanisms (oxidative stress, osmoregulation pressure, gut microbiota, and metabolic responses) at environmentally relevant concentrations. Compared to Cd alone and Cd + PMPs treatments (11.15 ± 4.19 items/g), Cd + AMPs treatment resulted in higher MPs bioaccumulation (23.73 ± 13.14 items/g), more severe tissue lesions, and increased cell membrane osmotic pressure in earthworms’ intestines. Cd + AMPs induced neurotoxicity through elevated levels of glutamate and acetylcholinesterase. Earthworm intestines (0.98 ± 0.49 to 3.33 ± 0.37 mg/kg) exhibited significantly higher Cd content than soils (0.19 ± 0.01 to 0.51 ± 0.06 mg/kg) and casts (0.15 ± 0.01 to 0.25 ± 0.05 mg/kg), indicating PE-MPs facilitated Cd transport in earthworms’ bodies. Metabolomic analysis showed Cd + AMPs exposure depleted energy and nucleotide metabolites, disrupted cell homeostasis more profoundly than Cd and Cd + PMPs treatments. Overall, co-exposure to AMPs + Cd induced more severe neurotoxicity and disruption of homeostasis in earthworm than Cd and PMPs + Cd treatments. Our study, using Cd and MPs with environmental relevance, underscores MPs’ role in amplifying Cd accumulation and toxicity in earthworms.

Incidence and Clinical Presentation of Severe Neurotoxicity from Nelarabine in Patients with T-Cell Acute Lymphoblastic Leukemia

BackgroundNelarabine is a purine analog with demonstrated efficacy in the treatment of T-cell Lymphoblastic Leukemia and Lymphoma (T-ALL/LBL). Despite its efficacy and excellent blood-brain barrier penetration, it has a significant side effect profile which is namely concerning for neurotoxicity. Reported neurotoxicity has varied from mild peripheral neuropathy to debilitating grade 4 neurologic complications including Guillain-Barre like syndrome and myelopathy. Patients and MethodsWe conducted a single centered, retrospective case series to study patients who developed severe neurotoxicity after receiving nelarabine as part of T-ALL treatment. One hundred thirty-five patients were identified. Thirteen patients were reviewed for severe neurotoxicity (defined as ≥grade 3), and of those five patients were deemed to have neurotoxicity secondary to nelarabine exposure. ResultsFive patients (4%) developed severe neurotoxicity as manifested by Guillain-Barre like syndrome or myelopathy within a timeframe of eight to fifty-eight days from last nelarabine dose. Upon diagnosis, patients received formal neurologic evaluation by our neuro-oncology specialists including imaging, cerebrospinal fluid testing, and electromyography. Patients were treated with IVIG, and steroids upon diagnosis, but the majority of neuro-deficits were irreversible. ConclusionOur study shows that nelarabine is generally well-tolerated, and the incidence of severe neurotoxicity is rare. Given the potential risk of severe neurotoxicity, we propose capped dose of nelarabine 1000 mg/day, neurological assessment before subsequent dosing, and avoidance of simultaneous IT therapy during nelarabine administration.

Negatively charged nanodiscs for the reduction of toxicity and enhanced efficacy of polymyxin B against Acinetobacter baumannii sepsis

The treatment of sepsis caused by multidrug-resistant (MDR) Gram-negative bacterial infections remains challenging. With these pathogens exhibiting resistance to carbapenems and new generation cephalosporins, the traditional antibiotic polymyxin B (PMB) has reemerged as a critical treatment option. However, its severe neurotoxicity and nephrotoxicity greatly limit the clinical application. Therefore, we designed negatively charged high-density lipoprotein (HDL) mimicking nanodiscs as a PMB delivery system, which can simultaneously reduce toxicity and enhance drug efficacy. The negative charge prevented the PMB release in physiological conditions and binding to cell membranes, significantly reducing toxicity in mammalian cells and mice. Notably, nanodisc-PMB exhibits superior efficacy than free PMB in sepsis induced by carbapenem-resistant Acinetobacter baumannii (CRAB) strains. Nanodisc-PMB shows promise as a treatment for carbapenem-resistant Gram-negative bacterial sepsis, especially caused by Acinetobacter baumannii, and the nanodiscs could be repurposed for other toxic antibiotics as an innovative delivery system. Statement of significanceMultidrug-resistant Gram-negative bacteria, notably carbapenem-resistant Acinetobacter baumannii, currently pose a substantial challenge due to the scarcity of effective treatments, rendering Polymyxins a last-resort antibiotic option. However, their therapeutic application is significantly limited by severe neurotoxic and nephrotoxic side effects. Prevailing polymyxin delivery systems focus on either reducing toxicity or enhancing bioavailability yet fail to simultaneously achieve both. In this scenario, we have developed a distinctive HDL-mimicking nanodisc for polymyxin B, which not only significantly reduces toxicity but also improves efficacy against Gram-negative bacteria, especially in sepsis caused by CRAB. This research offers an innovative drug delivery system for polymyxin B. Such advancement could notably improve the therapeutic landscape and make a significant contribution to the arsenal against these notorious pathogens.

Self-Assembled Micelles Based on Ginsenoside Rg5 for the Targeted Treatment of PTX-Resistant Tumors.

Paclitaxel (PTX) is one of the first-line drugs for prostate cancer (PC) treatment. However, the poor water solubility, inadequate specific targeting ability, multidrug resistance, and severe neurotoxicity are far from being fully resolved, despite diverse PTX formulations in the market, such as the gold-standard PTX albumin nanoparticle (Abraxane) and polymer micelles (Genexol-PM). Some studies attempting to solve the multiple problems of chemotherapy delivery fall into the trap of an extremely complicated formulation design and sacrifice druggability. To better address these issues, this study designed an efficient, toxicity-reduced paclitaxel-ginsenoside polymeric micelle (RPM). With the aid of the inherent amphiphilic molecular structure and pharmacological effects of ginsenoside Rg5, the prepared RPM enhances the water solubility and active targeting of PTX, inhibiting chemotherapy resistance in cancer cells. Moreover, the polymeric micelles demonstrated favorable anti-inflammatory and neuroprotective effects, providing ideas for the development of new clinical anti-PC preparations.

Vinorelbine administration impedes the timely progression of meiotic maturation and induces aneuploidy in mouse oocytes

Vinorelbine is a commonly used drug to treat various malignancies, such as breast cancer, non-small cell lung cancer, and metastatic pleural mesothelioma. Its side effects include severe neutropenia, local phlebitis, gastrointestinal reactions, and neurotoxicity. In view of the scarcity of research on vinorelbine's reproductive toxicity, this study evaluated the impact of vinorelbine ditartrate, a commonly used form of vinorelbine, on oocyte maturation in vitro. Our investigation revealed that vinorelbine ditartrate had no effect on oocyte meiotic resumption. However, it did reduce the rate of first polar body extrusion, suggesting that it could significantly impede the meiotic maturation of oocytes. Vinorelbine ditartrate exposure was found to disturb the regular spindle assembly and chromosome alignment, leading to the continuous activation of the spindle assembly checkpoint (SAC) and a delayed activation of the anaphase-promoting complex/cyclosome (APC/C), ultimately causing aneuploidy in oocytes. Consequently, the administration of vinorelbine is likely to result in oocyte aneuploidy, which can be helpful in providing a drug reference and fertility guidance in a clinical context.

Early prediction of severe ICANS after standard-of-care CD19 CAR T-cell therapy using gradient-boosted classification trees.

7034 Background: Severe immune effector cell-associated neurotoxicity syndrome (ICANS) has arisen as a major complication leading to morbidity, mortality, and increased resource utilization. There is an urgent need to accurately predict the development of severe ICANS after CAR T-cell therapy. Here, we demonstrate the high performance of the XGBoost (“eXtreme Gradient Boosting”) machine learning algorithm in predicting the development of severe ICANS using commonly available laboratory and vital sign data from patients with B-cell lymphomas undergoing standard-of-care CD19 CAR T-cell therapy. Methods: We included patients who received axicabtagene ciloleucel (axi-cel) or brexucabtagene autoleucel (brexu-cel) per standard of care. ICANS was graded by CTCAE 4.03 criteria (pre-12/2018) or ASTCT Consensus Grading System (post-1/2019). Predictions were generated using XGBoost, a widely used supervised machine learning algorithm that trains ensembles of decision trees to learn iteratively from prior trees and allows for very flexible modeling (e.g. non-linear effects, complex high-order interaction effects). K-fold cross-validation was used to assess calibration and overfitting. Variables assessed included age, ferritin, CRP, LDH, IL-6, fibrinogen, platelet count, and temperature at pre-infusion, day 0, and day +3 post-infusion timepoints. Results: A total of 175 patients were included with 39 receiving brexu-cel (22%) and 136 receiving axi-cel (78%). Grade ≥3 ICANS occurred in 40 patients (23.3%). XGBoost modeling of factors demonstrated that serum ferritin level on day +3 was the most important variable predictive of grade ≥3 ICANS (accuracy gain: 0.28). Other influential factors included day 0 and +3 platelet count, patient age, day +3 IL-6 level, day 0 fibrinogen level, and day 0 and +3 CRP level, in descending order of importance. The XGBoost model maintained high discrimination (ability to distinguish high-risk from low-risk patients; mean AUROC 0.74, sensitivity 0.95, specificity 0.90). Longitudinal LOESS smoothing confirmed associations between severe ICANS and elevation in serum ferritin at day 0 (OR 3.01, 95% CI 1.41 – 6.73, p = 0.005) and day +3 (OR 5.50, 95% CI 2.31-14.5, p &lt;0.001). Conclusions: A supervised machine learning model using XGBoost incorporating age, temperature and commonly accessible laboratory data including ferritin, CRP, LDH, IL-6, fibrinogen, and platelet count predicted severe ICANS prior to the development of symptoms with high discrimination (AUROC 0.74) in patients undergoing standard of care CAR T-cell therapy. This allows for the early identification of patients at highest risk for developing severe ICANS who may benefit from prophylactic interventions. Once externally validated, we plan to develop a user-friendly web application to generate individualized predictions from our model.

Manganese and Vanadium Co-Exposure Induces Severe Neurotoxicity in the Olfactory System: Relevance to Metal-Induced Parkinsonism.

Chronic environmental exposure to toxic heavy metals, which often occurs as a mixture through occupational and industrial sources, has been implicated in various neurological disorders, including Parkinsonism. Vanadium pentoxide (V2O5) typically presents along with manganese (Mn), especially in welding rods and high-capacity batteries, including electric vehicle batteries; however, the neurotoxic effects of vanadium (V) and Mn co-exposure are largely unknown. In this study, we investigated the neurotoxic impact of MnCl2, V2O5, and MnCl2-V2O5 co-exposure in an animal model. C57BL/6 mice were intranasally administered either de-ionized water (vehicle), MnCl2 (252 µg) alone, V2O5 (182 µg) alone, or a mixture of MnCl2 (252 µg) and V2O5 (182 µg) three times a week for up to one month. Following exposure, we performed behavioral, neurochemical, and histological studies. Our results revealed dramatic decreases in olfactory bulb (OB) weight and levels of tyrosine hydroxylase, dopamine, and 3,4-dihydroxyphenylacetic acid in the treatment groups compared to the control group, with the Mn/V co-treatment group producing the most significant changes. Interestingly, increased levels of α-synuclein expression were observed in the substantia nigra (SN) of treated animals. Additionally, treatment groups exhibited locomotor deficits and olfactory dysfunction, with the co-treatment group producing the most severe deficits. The treatment groups exhibited increased levels of the oxidative stress marker 4-hydroxynonenal in the striatum and SN, as well as the upregulation of the pro-apoptotic protein PKCδ and accumulation of glomerular astroglia in the OB. The co-exposure of animals to Mn/V resulted in higher levels of these metals compared to other treatment groups. Taken together, our results suggest that co-exposure to Mn/V can adversely affect the olfactory and nigral systems. These results highlight the possible role of environmental metal mixtures in the etiology of Parkinsonism.

Comparison of intermittent intravenous tacrolimus to continuous tacrolimus in adult allogeneic stem cell transplant recipients.

Introduction: Initial continuous intravenous (CIV) tacrolimus (0.03 mg/kg/day based on ideal body weight [IBW]) has been favored for graft versus host disease (GVHD) prevention in allogeneic stem cell transplant patients due to the consistent, steady-state degree of immunosuppression; however, this method poses many logistical challenges. We implemented intermittent (IIV) tacrolimus at a starting dose of 0.015 mg/kg IBW twice daily over 4 h. To our knowledge this is the first retrospective comparison of CIV to IIV tacrolimus. Objectives: The primary objective was to evaluate the safety of IIV tacrolimus in comparison to CIV with respect to nephrotoxicity and neurotoxicity. The secondary objectives were to compare the incidence of grade II-IV acute GVHD (aGVHD) and chronic GVHD (cGVHD) at day +180, outcomes including relapse and overall survival, cell engraftment, and reactivation of cytomegalovirus and Epstein-Barr virus. Methods: This retrospective, single-center review evaluated adults who received an allogeneic stem cell transplant patients between January 1, 2020, and December 31, 2022. Results: Fifty-one unique patients were eligible for evaluation - 28 in the IIV cohort and 23 in the CIV group. The number of patients who developed nephrotoxicity and neurotoxicity were comparable between groups with no significant differences noted. No severe neurotoxicity was identified in either population. Secondary objectives revealed no significant difference in GVHD incidence or survival outcomes. Conclusion: IIV tacrolimus is comparable to CIV in terms of safety while also maintaining similar outcomes at day +180. IIV is a safe and feasible alternative to CIV in adult allogeneic stem cell transplant recipients.

Primary vs. pre-emptive anti-seizure medication prophylaxis in anti-CD19 CAR T-cell therapy

IntroductionSeizures may occur in up to 30% of non-Hodgkin lymphoma patients who received anti-CD19 CAR T-cell therapy, yet the optimal anti-seizure medication (ASM) prevention strategy has not been thoroughly investigated.MethodsConsecutive patients affected by refractory non-Hodgkin lymphoma who received anti-CD19 CAR T-cells were included. Patients were selected and assessed using similar internal protocols. ASM was started either as a primary prophylaxis (PP-group) before CAR T-cells infusion or as a pre-emptive therapy (PET-group) only upon the onset of neurotoxicity development.ResultsOne hundred fifty-six patients were included (PP-group = 88, PET-group = 66). Overall, neurotoxicity and severe neurotoxicity occurred in 45 (29%) and 20 (13%) patients, respectively, equally distributed between the two groups. Five patients experienced epileptic events (PET-group = 3 [4%]; PP-group = 2 [2%]). For all the PET-group patients, seizure/status epilepticus occurred in the absence of overt CAR-T-related neurotoxicity, whereas patients in the PP-group experienced brief seizures only in the context of critical neurotoxicity with progressive severe encephalopathy. ASMs were well-tolerated by all patients, even without titration. No patients developed epilepsy or required long-term ASMs.ConclusionOur data suggest that both primary and pre-emptive anti-seizure prophylaxis are safe and effective in anti-CD19 CAR T-cell recipients. Clinical rationale suggests a possible more favourable profile of primary prophylaxis, yet no definitive conclusion of superiority between the two ASM strategies can be drawn from our study.

Bioactivity and toxicity of coumarins from African medicinal plants.

Introduction: Coumarins are naturally occuring metabolites from plants and a few micro-organisms. They have been widely used in the food and drug industry in their natural or synthetic forms. Numerous coumarins possess several biological activities such as anti-inflammatory, anti-ulcers, anti-tumour, anti-microbial, anti-coagulant. The aim of this study was to assess the bioactivity, and toxicity of coumarins from African medicinal plants. Methods: We searched online databases and search engines such as PubMed, Google Scholar and Web of Science for key terms such as coumarins, toxicity, bioavailability, bioactivity with appropriate Boolean operators. Only full-length research articles published in English between 1956 to 2023 were reviewed. Results: We recorded 22 coumarins from 15 plant species from Africa. Most of the plant species (33%) were from North Africa. These were followed by East Africa at 21%, then West, and Central Africa at 18.2% each. Most of the coumarins (21.3%) were isolated from the entire plant and the leaves (19.1%) and most of them (46.7%) had some antimicrobial activity. Five coumarins viz osthole, pseudocordatolide C & calanolide, chartreusin and esculetin had either antitumor or anticancer activity. Six coumarins had varying levels and types of toxicity ranging from inhibiting blood clotting as anticoagulants, to cytotoxic effects, causing hyperventilation, tremor, & photophobia, pulmonary haemorrhage, carcinogenic activity, severe neurotoxicity, hepato- and phototoxicity. Conclusion: Several African medicinal plants are sources of various coumarins that possess several biological activities as well as toxicities. This calls for more research into their safety and efficacy because of their wide spread applications as therapeutic agents.

Methotrexate-induced transient encephalopathy. The experience of the Research Institute of Pediatric Oncology and Hematology named after Academician of the Russian Academy of Medical Sciences L.A. Durnov at N.N. Blokhin National Medical Research Center of Oncology, Ministry of Health of Russia

Introduction. Methotrexate is one of the main chemotherapeutic agents of group antimetabolits, includes in the first line of therapy against osteosarcoma. The drug uses in dose 12 g/m2 according to the protocol EURAMOS-1. The range of methotrexate-induced complications includes renal toxity, hepatotoxicity, myelosuppression, skin and mucosal ulcerations, dyspeptic disorders. One of the formidable, but reversible complications, is methotrexate-induced transient encephalopathy (MIE), the clinical manifestations of which occur in more than 15 % of patients in the treatment of which high doses of methotrexate (HD MTX) are used.Materials and methods. At the Research Institute of Pediatric Oncology and Hematology named after Academician of the Russian Academy of Medical Sciences L.A. Durnov at N.N. Blokhin National Medical Research Center of Oncology, Ministry of Health of Russia the period from 2013 to 2023 10 cases of MIE were recorded. All patients received therapy according to the protocol EURAMOS-1. The 4 out of 10 patients had a delay in the rate of elimination of HD MTX after the course of chemotherapy. No one patient had electrolyte disturbances with using HD MTX. The median occurrence of the complication’s emerging was 7 days (from 5 to 10 from the start of therapy) and most often developed after 3 courses of methotrexate 12 g/m2 , which corresponds in total 6 doses of methotrexate. Neurological symptoms: headache, visual impairment, aphasia, convulsions were transient and resolved after an average of 24 hours from the start of the treatment.Results. All 10 patients received obligatory alkalization, massive infusion therapy, neuroprotective drugs, as well as decongestant therapy as a treatment of MIE. Subsequently, therapy with methotrexate was continued for the 9 of 10 patients.Conclusions. The standard recommendations for the treatment of MIE do not currently exist. However, the development of severe neurotoxicity does not exclude the possibility of further using of HD MTX in the treatment program

Updated Follow-up of a Phase 1 Trial of Bispecific CART19/20 Cells for Relapsed or Refractory Non-Hodgkin Lymphoma

CART19/20 was developed by engineering autologous naïve/memory T (T N/MEM) cells with a bispecific anti-CD19/CD20 chimeric antigen receptor (CAR) with the goal of mitigating antigen escape and lack of CAR T-cell persistence. We report an update of an ongoing first-in-human phase 1 clinical trial of CART19/20 for patients with relapsed/refractory non-Hodgkin lymphoma (R/R NHL) (NCT04007029). Eligible patients were ≥18 years old with R/R diffuse large B-cell lymphoma (DLBCL) or primary mediastinal B-cell lymphoma (PMBCL) after ≥2 prior lines of therapy, or with mantle-cell lymphoma (MCL), follicular lymphoma (FL), or CLL/SLL after ≥3 prior lines of therapy. Autologous leukocytes were obtained by leukapheresis and sorted for CD14-/CD25-/CD62L+ T N/MEM cells, followed by lentiviral transduction of the bispecific CD19/CD20 CAR. Lymphodepletion chemotherapy with fludarabine 30 mg/m 2/day and cyclophosphamide 500 mg/m 2/day was administered on Days −5 to −3 prior to CAR T-cell infusion. The primary endpoint was assessment of safety. Secondary endpoints were overall response rate (ORR), progression free survival (PFS), overall survival (OS), and CART19/20 transgene persistence. As of data cutoff on July 21, 2023, 11 patients had received CART19/20 cells (6 DLBCL, including 4 transformed FL; 3 primary FL; 1 PMBCL; 1 MCL). Patients were treated at two dose levels, either 50 x 10 6 ± 30% CAR+ cells (8 patients) or 200 x 10 6 ± 30% CAR+ cells (3 patients). The median age was 58 (range: 34 to 70). Patients had received a median of 3 prior lines of therapy (range: 2 - 4). None of the treated patients had received prior CAR-T cell therapy; one patient had received a prior CD19-directed therapy. All patients had stage IV disease and 9 of 11 patients received bridging therapy. The engineered CART19/20 cells were enriched with central-memory T cells (T CM: CD45RA-/CD45RO+/CD62L+) which composed a median of 30.8% (range: 5.3 - 80.1%) of the CAR+ T-cells in the infused products. There were no occurrences of CRS grade ≥ 2 or neurotoxicity of any grade. Six patients had grade 1 cytokine release syndrome (CRS) with a median onset of 8 days from infusion (range: 5 - 11 days). One patient met criteria for dose-limiting toxicity with persistent thrombocytopenia after Day+120. Ten of the 11 patients achieved an objective response (91% ORR), with 8 patients (73%) achieving complete response (CR). One patient with FL, who progressed 18 months after receiving CART19/20, received a second infusion of CART19/20 cells and achieved a second CR for 14.9 months. With a median follow-up of 32.3 months (range: 5.1 - 44.0 months), the median PFS (95% CI 2.6 months - not estimable) and median OS (95% CI 5.7 months - not estimable) have not been reached. Overall, CART19/20 demonstrated a favorable safety profile with no severe CRS or neurotoxicity of any grade. Moreover, CART19/20 showed robust activity in patients with R/R NHL with durable responses seen. By utilizing a bispecific CAR and enriching for T N/MEM cells, this phase 1 trial of CART19/20 demonstrates the possibility of improving outcomes through targeting common mechanisms of CAR-T cell resistance.

Expression of a Novel Chimeric Inhibitory Receptor Attenuates CD19 CAR T Cells: A Promising Strategy for Toxicity Mitigation

Introduction Chimeric antigen receptor (CAR) T cells targeting CD19 are highly effective in patients with refractory/relapsed acute lymphoblastic leukemia and lymphoma but often trigger severe cytokine release syndrome (sCRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). For r/rALL patients, as many as 77% develop Grade 3-5 CRS and 40% Grade 3-5 ICANS. sCRS occurs when hyperactivated CAR T cells undergo exponential expansion accompanied by the release of excessive amounts of inflammatory cytokines. This leads to a systemic pro-inflammatory state that can cause hypotension, multi-organ failure and even death. Tocilizumab is the only FDA-approved drug for CRS and despite concurrent corticosteroids, often fails to control CRS. Attempts to improve safety have been proposed, but have significant limitations, such as permanent removal of CARs. New and innovative methods to mitigate toxicities are needed. The purpose of this study was to design and test the efficacy of a novel autoregulatory element in the form of a chimeric inhibitory receptor (CIR), that when engaged by a specific CRS-associated pro-inflammatory molecule would trigger inhibitory signaling within CAR T cells. We hypothesize that as cytokine levels increase following activation, these signals can be redirected through responsiveness of the CIR, leading to an attenuation of CAR activity. In this study, we have tested a CIR construct that couples the recognition of IFNγ to the signaling domain of the TIGIT inhibitory receptor. We co-expressed this CIR with a CD19.28ζ CAR to evaluate its ability to attenuate activation and cytokine production and to assess its cytotoxic activity. Methods An anti-IFNγ scFv followed by the transmembrane and intracellular domains of TIGIT were arranged in sequence with the truncated EGFR receptor followed by the CD19.28ζ FMC63 CAR with intervening T2A cleavage domains and cloned into a retroviral backbone. Transduction efficiency was assessed by staining with biotinylated CD19-Fc protein or biotinylated EGFR. Following expansion, memory and effector cell differentiation was evaluated by staining for CD45RO, CD45RA, CCR7, CD127, and CD95. Activation following stimulation with NALM-6 tumor cells was measured by staining for CD69, CD25, CD38, PD-1, and CD107a. Cytotoxicity was evaluated using bioluminescence. To mimic cell types present during CRS, we utilized a coculture assay that consists of CAR T cells, NALM-6 tumor cells, and either macrophages or dendritic cells (DC) differentiated from autologous CD14 + monocytes. Cocultures were incubated for 48 hr. and supernatants were collected for analyses by Luminex. Results CD19 CARs co-expressing a CIR composed of an anti-IFNγ scFv coupled to TIGIT inhibitory signaling expanded during the manufacturing process as well as T cells expressing only CD19 CAR and displayed normal levels of CAR expression. The presence of the CIR did not affect the differentiation profile of memory and effector subsets. CIR-expressing CD19 CARs exhibited robust tumor cell killing of NALM-6 targets equivalent to CD19 CARs. Next, CD19 CAR/CIR cells were pretreated with IFNγ for 24 hr. and then cultured with NALM-6 cells. Exposure to IFNγ resulted in a significant abrogation in cytotoxicity, which was eventually restored with time. Activation profile analysis of CARs equipped with CIR signaling after target stimulation revealed an overall diminution of CARs expressing multiple (4 or 5) activation markers relative to CD19 CARs. Importantly, CIR signaling resulted in significant reductions in the levels of effector, stimulatory, and inflammatory mediators including IFNγ, TNFα, GM-CSF, and IL-17. CARs expressing the CIR exhibited slower killing kinetics when challenged with sequential stimulations by fresh tumor cells. However, in vivo anti-tumor cell activity was not adversely affected as CAR/CIR T cells robustly eliminated disease in leukemia-bearing NSG mice. Lastly, using an in vitro CRS assay, we sought to determine if attenuated CAR T cells would be less potent in activating myeloid cells. We observed that monocyte-derived macrophage and DC activation by CD19 CAR/CIR T cells was reduced as evidenced by decreased secretion of proinflammatory cytokines and chemokines. Conclusion These results provide a compelling proof of concept of a novel self-regulating CAR T cell design with the potential to mitigate severe toxicities in patients.

Functional Characterization and Optimization of Switchable Allogeneic Chimeric Antigen Receptor T Cells for Targeting CD19 and CD20 in B Cell Malignancies

Chimeric antigen receptor (CAR) T cell therapy has demonstrated significant efficacy in B cell malignancies. However, despite clinically manageable on-target/off-tumor toxicity when targeting antigens like CD19 and BCMA, severe cytokine release and neurotoxicity remain a clinical challenge. Additionally, patients may require immunoglobulin replacement therapy, and antigen-loss is recognized as a cause of relapse (PMID: 30275569, 31798590, 36263838, 37051246 and 37122700). To overcome these limitations, we developed a novel switchable CAR-T cell therapy that simultaneously targets both CD19 and CD20 in B cell malignancies. This innovative therapy is based on our reverse universal chimeric antigen receptor platform (RevCAR), a 2-component CAR-T technology (Figure 1; PMID: 34638268 and 32923149). The first component is a universal CAR-T cell, incorporating a CAR based on a short, non-immunogenic peptide motif derived from the human nuclear La/SSB autoantigen. The CAR itself does not recognize any human cell surface antigen. However, it is specifically targeted by an antibody-based binder in the second component, a soluble adaptor called targeting module (TM). This TM confers specificity against the desired cancer antigens of choice. Consequently, the availability of the TM effectively controls the activity of RevCAR-T cells. We have recently reported preclinical development of an allogeneic RevCAR-T cell therapy for CD123-positive hematologic malignancies (AVC-201; Ehninger et al. 2023, AACR Abstract 4095; NCT05949125). To target CD123, which is also expressed on normal hematopoietic progenitor cells, a TM with a short half-life was selected (R-TM123), enabling a rapid switch-off of the RevCAR system by TM withdrawal to avoid acute toxicity and continued aplasia. As CD19 and CD20 have a substantially more favorable safety profile and autologous conventional CAR-T cells and bispecific T cell engagers with respective target-specificities are approved in certain B cell malignancies, a half-life extended TM was developed to allow for dosing every 1-2 weeks. After an extensive screen for an optimized TM targeting CD19 and CD20, we screened multiple RevCAR variants differing in hinge, transmembrane and costimulatory domains for optimal anti-tumor potency using both preclinical in vitro and in vivo models. Profound efficacy and complete tumor control could be observed for optimized RevCAR constructs (Figure 2). While short-term in vitro cytotoxicity assays already gave a small hint towards in vivo efficacy, surprisingly, long-term in vitro rechallenge assay results did not correlate with in vivo efficacy. A summary of functional characterization data will be presented at the meeting with a focus on the lack of predictability of certain in vitro models and underlying hypotheses. Insights from these studies are now flowing into preclinical development of a novel switchable allogeneic CAR-T cell product candidate targeting CD19 and CD20 in B cell malignancies engineered to fully overcome graft-versus-host disease as well as graft rejection by host T and NK cells.

Retrospective and Observational Study on CAR-T 19 Bridge Therapies in Pediatric, Adolescents and Young Adults

INTRODUCTION CAR-T therapy has revolutionized the treatment of B-ALL. The development of CAR-T therapy is a long and delicate process where once apheresis is done, a bridge period that least until CAR-T cells are infused starts. Meanwhile, the objective will be to maintain disease control, decrease tumor burden (TB) and prevent disease progression with no severe comorbidities by the administration of a bridge chemotherapy. That will decrease the risk of severe (grade 3-4 ASTCT) Cytokine Release Syndrome (CRS) and Immune Effector Cell-Associated Neurotoxicity Syndrome (ICANS) with an improvement of event free survival. Nevertheless, there is no consensus nor standard guidelines about bridge chemotherapy type, especially in pediatric patients. In this way, the main objective of this study is to identify CAR-T outcomes in pediatric, adolescents and young adults (AYA) diagnosed of BCP-ALL based on bridge chemotherapy. MATHERIAL AND METHODS An observational, retrospective and non-interventional study of Spanish pediatric and AYA under 24 years old diagnosed with BCP-ALL treated with tisagenlecleucel that have been registered by the Spanish Hematopoietic Stem Cell Transplantation and Cellular Therapy (GETH-TC) CAR-T clinical database from years 2017 to 2022. Data have been collected from nine centers members of GETH-TC CAR-T workgroup: La Paz University Hospital, Sant Joan de Déu University Hospital, Virgen del Rocío University Hospital, Niño Jesús Pediatric University Hospital, Gregorio Marañón General University Hospital, Vall d'Hebron University Hospital, Salamanca Clinic University Hospital, Germans Trials i Pujol University Hospital and Valencia Clinic Hospital. General, pre-CAR-T, CAR-T-related and post-CAR-T variables have been analysed from GET-TC CAR-T database, highlighting those related to bridge therapy and related outcomes. Bridging chemotherapy was analyzed considering whether the patients had received high (myelosuppression ≥7 days) or low (myelosuppression ≤7 days) intensity schemes, or in contrast, immunotherapy. Fisher's Exact Test for count data and Log Rank test for Overall Survival (OS), Relapse Free Survival (RFS) and Duration of Response (DOR). Variables with p&amp;lt;0.05 value have been considered of statistical significance. RESULTS Baseline characteristics of the 103 patients are summarized in Table 1. CRS after infusion was presented in 86 (83.5%) patients. Grade 3-4 was developed in 4/29 (13.8%) with low pre infusion TB in contrast to 18/55 (32.7%) with high TB (p=0.001). Complete remission (CR) was achieved 87 (85.3%) patients. All patients (100%) with a low TB were in CR VS 78.2% in the high TB arm (p=0.015). Otherwise, 91.9% of those patients that received low intensity bridge chemotherapy or immunotherapy were in CR VS 71% of those where high intensity bridge was administered (p=0.027). With a median of 23.75 months, OS at 12 months was 62.23% (CI95% 51.71-74.89). No statistical significance was found between bridge chemotherapy groups, although it was higher in low intensity and immunotherapy group (68.33% CI95% 55.41-84.27) VS (57.89% CI95% 40.43-82.89) (p=0.11). With a median of 9.26 months, RFS at 12 months was 41.7% (CI95% 31.71-54.85). No statistical significance was found between bridge chemotherapy groups, but was higher in low intensity group and immunotherapy (51.8% CI95% 39.45-68.01) VS (34.69% CI95% 18.59-64.74) (p=0.089). With a median of 8.28 months, DOR at 12 months was 41.7% (CI95% 31.71-54.85). No statistical significance was found between bridge chemotherapy groups, with an increasing tendency in low intensity group and immunotherapy (51.8% CI95% 39.45-68.01) VS (34.69% CI95% 18.59-64.74) (p=0.089). In the multivariate analysis, grade 3-4 CRS HR 8.7 (CI95% 0.82-92) (p = 0.072) and high TB HR 4.7 (CI95% 1.1-20) (p = 0.038) were related with a decreased OS. CONCLUSIONS Receiving low intensity bridge or immunotherapy is related with high rates of CR. In the same way, in this group of patients there is tendency to achieve higher OS, RFS and DOR. This is the reason because its use should be prioritized avoiding those of high intensity bridge chemotherapy. High TB is an important risk factor related with a poor response and high grade CRS development, so it is essential to reduce TB previous CAR-T infusion in order to improve outcomes and reduce toxicities.

Azacitidine, Venetoclax and Allogeneic NK Cells in Newly Diagnosed Acute Myeloid Leukemia (ADVENT-AML): An Investigator-Initiated Multicenter Phase Ib Trial

BACKGROUND AND SIGNIFICANCE : Older/unfit patients with acute myeloid leukemia (AML) have limited treatment options. Outcomes in adverse-risk subgroups of AML are modest with CR/CRi rates of 45% to 60% and median overall survival (OS) of 5 to 12 months with current standard frontline therapy of hypomethylating agent with venetoclax (HMA-VEN).While allogeneic stem cell transplantation (allo-SCT) can significantly improve OS in AML, only 10 to 20% of older pts are able to undergo allo-SCT (Pratz et al. Blood 2019, Maiti et al. Blood 2022) Natural killer (NK) cells bridge the innate and adaptive immune system and play a major role in graft-vs-leukemia (GVL) effect responsible for the clinical benefit noted with allo-SCT. NK cell-based adoptive cellular therapies have shown good safety profiles and promising activity in AML with responses noted in 25% to 88% of patients. Furthermore, NK cells are less likely to induce graft-versus-host disease (GVHD), severe cytokine release syndrome (CRS) or immune effector cell-associated neurotoxicity syndrome (ICANS). We hypothesize that the combination of azacitidine, venetoclax and allogeneic NK cells will be safe and improve cure rates in AML compared to either Aza/Ven or NK cells alone. Improved clinical activity may be mediated through multiple synergistic mechanisms including reduction of disease burden by Aza/Ven, upregulation of epigenetically silenced NKG2D ligands, dual activation of extrinsic and intrinsic apoptotic cascades, mitochondrial priming of leukemia cells by venetoclax, and bypassing significant morbidity and mortality associated allo-SCT. Significance: This will be the first trial to evaluate 1) cellular therapy in the frontline setting in AML; 2) the synergy of venetoclax with adoptive NK cell therapy; and 3) Aza/Ven as a lymphodepletion strategy. The results will have significant impact on the development of cellular therapies for hematological and solid tumors. STUDY DESIGN AND METHODS: This investigator-initiated, multicenter, open-label, phase Ib study will evaluate the combination of azacitidine, venetoclax and ex vivo-expanded, off-the-shelf cryopreserved allogeneic NK cells in patients with AML (NCT05834244). Eligibility criteria: The dose escalation phase will enroll adults with relapsed or refractory AML or MDS/AML, and the dose expansion phase will enroll older/unfit pts with newly diagnosed adverse-risk AML. Eligible Pts will be ≥ 75 years, or ≥ 18 years with at least one protocol-defined comorbidity. Patients with favorable risk AML, poor end-organ function, needing immunosuppression, uncontrolled infection or CNS leukemia will be excluded. Trial design: The dose escalation phase will enroll a maximum of 12 patients at 2 dose levels to establish a recommended phase 2 dose (RP2D) using a BOIN design. The dose expansion will enroll up to 20 patients at the RP2D ( Fig. 1). Study treatment: Patients will receive Aza/Ven as standard. Two dose levels of NK cells including 0.5 x10 9 and 1 x10 9 flat dose on day 8 and 15 of the first 4 cycles will be evaluated. Subsequently, pts will continue Aza/Ven indefinitely. Objectives: The primary objective is to determine the safety of this novel triplet combination in terms of dose-limiting toxicities including acute GVHD, CRS or ICANS. Secondary objectives include overall response rate, CR/CRi by 4 cycles, rate of negative measurable residual disease, OS and relapse-free survival. Exploratory objectives include additional response and survival endpoints, the persistence of NK cells using SNP-NGS chimerism, kinetics of allorejection, remodeling of host immune microenvironment and impact on AML stem/progenitor cell hierarchies using single cell CyTOF to understand response/resistance mechanisms. Status: Trial approved by the FDA and institutional IRB. Activation planned for 2H 2023. Funding: Gateway for Cancer Research, MDACC PRIME Award, Chimeric Therapeutics, NCI Cancer Center Support Grant.