Nicotinamide-Expanded Allogeneic Natural Killer Cells with CD38 Deletion, Expressing an Enhanced CD38 Chimeric Antigen Receptor, Target Multiple Myeloma Cells.

Natural killer (NK) cells are a vital component of cancer immune surveillance. They provide a rapid and potent immune response, including direct cytotoxicity and mobilization of the immune system, without the need for antigen processing and presentation. NK cells may also be better tolerated than T cell therapy approaches and are susceptible to various gene manipulations. Therefore, NK cells have become the focus of extensive translational research. Gamida Cell's nicotinamide (NAM) platform for cultured NK cells provides an opportunity to enhance the therapeutic potential of NK cells. CD38 is an ectoenzyme ubiquitously expressed on the surface of various hematologic cells, including multiple myeloma (MM). It has been selected as a lead target for numerous monoclonal therapeutic antibodies against MM. Monoclonal antibodies target CD38, resulting in the lysis of MM plasma cells through various antibody-mediated mechanisms such as antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity, and antibody-dependent cellular phagocytosis, significantly improving the outcomes of patients with relapsed or refractory MM. However, this therapeutic strategy has inherent limitations, such as the anti-CD38-induced depletion of CD38-expressing NK cells, thus hindering ADCC. We have developed genetically engineered NK cells tailored to treat MM, in which CD38 was knocked-out using CRISPR-Cas9 technology and an enhanced chimeric antigen receptor (CAR) targeting CD38 was introduced using mRNA electroporation. This combined genetic approach allows for an improved cytotoxic activity directed against CD38-expressing MM cells without self-inflicted NK-cell-mediated fratricide. Preliminary results show near-complete abolition of fratricide with a 24-fold reduction in self-lysis from 19% in mock-transfected and untreated NK cells to 0.8% of self-lysis in CD38 knock-out CAR NK cells. Furthermore, we have observed significant enhancements in CD38-mediated activity in vitro, resulting in increased lysis of MM target cell lines. CD38 knock-out CAR NK cells also demonstrated significantly higher levels of NK activation markers in co-cultures with both untreated and αCD38-treated MM cell lines. These NAM-cultured NK cells with the combined genetic approach of CD38 knockout and addition of CD38 CAR represent a promising immunotherapeutic tool to target MM.

Nicotinamide enhances natural killer cell function and yields remissions in patients with non-Hodgkin lymphoma.

Allogeneic natural killer (NK) cell adoptive transfer has shown the potential to induce remissions in relapsed or refractory leukemias and lymphomas, but strategies to enhance NK cell survival and function are needed to improve clinical efficacy. Here, we demonstrated that NK cells cultured ex vivo with interleukin-15 (IL-15) and nicotinamide (NAM) exhibited stable induction of l-selectin (CD62L), a lymphocyte adhesion molecule important for lymph node homing. High frequencies of CD62L were associated with elevated transcription factor forkhead box O1 (FOXO1), and NAM promoted the stability of FOXO1 by preventing proteasomal degradation. NK cells cultured with NAM exhibited metabolic changes associated with elevated glucose flux and protection against oxidative stress. NK cells incubated with NAM also displayed enhanced cytotoxicity and inflammatory cytokine production and preferentially persisted in xenogeneic adoptive transfer experiments. We also conducted a first-in-human phase 1 clinical trial testing adoptive transfer of NK cells expanded ex vivo with IL-15 and NAM (GDA-201) combined with monoclonal antibodies in patients with relapsed or refractory non-Hodgkin lymphoma (NHL) and multiple myeloma (MM) (NCT03019666). Cellular therapy with GDA-201 and rituximab was well tolerated and yielded an overall response rate of 74% in 19 patients with advanced NHL. Thirteen patients had a complete response, and 1 patient had a partial response. GDA-201 cells were detected for up to 14 days in blood, bone marrow, and tumor tissues and maintained a favorable metabolic profile. The safety and efficacy of GDA-201 in this study support further development as a cancer therapy.

Low-dose ladostigil for mild cognitive impairment: A phase 2 placebo-controlled clinical trial.

OBJECTIVE: Ladostigil reduces oxidative stress and microglial activation in aging rats. We assessed its safety and potential efficacy in a 3-year, randomized, double-blind, placebo-controlled phase 2 clinical trial in patients with mild cognitive impairment (MCI) and medial temporal lobe atrophy. METHODS: Patients 55 to 85 years of age with MCI, Clinical Dementia Rating (CDR) score of 0.5, Mini-Mental State Examination (MMSE) score >24, Wechsler Memory Scale-Revised Verbal Paired Associates I score ≤18, and Medial Temporal Lobe Atrophy Scale score >1 were stratified by APOE ε4 genotype and randomly assigned (1:1) to ladostigil 10 mg/d or placebo. Primary outcomes were safety and onset of Alzheimer disease dementia. Secondary endpoints were Neuropsychological Test Battery (NTB) composite, Disability Assessment in Dementia (DAD), and Geriatric Depression Scale (GDS) scores. Exploratory outcomes were NTB component, CDR, and MMSE scores. Biomarkers included MRI-derived whole-brain, hippocampus, and entorhinal cortex volumes. RESULTS: Two hundred ten patients from 15 sites in Austria, Germany, and Israel were randomly allocated to placebo (107 patients) or ladostigil (103 patients). After 36 months, 21 of 103 patients on placebo and 14 of 99 patients receiving ladostigil progressed to Alzheimer disease (log-rank test p = 0.162). There were no significant effects on the NTB composite, DAD, or GDS score. Whole-brain and hippocampus volumes decreased more in the placebo than in the ladostigil group (whole brain, p = 0.025, Cohen d = 0.43; hippocampus, p = 0.043, d = 0.43). Serious adverse events were reported by 28 of 107 patients treated with placebo and 26 of 103 with ladostigil. CONCLUSION: Ladostigil was safe and well tolerated but did not delay progression to dementia. Its association with reduced brain and hippocampus volume loss suggests a potential effect on atrophy. CLINICALTRIALSGOV IDENTIFIER: NCT01429623. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that for patients with MCI and medial temporal lobe atrophy, ladostigil did not significantly decrease the risk of the development of Alzheimer disease.

Circadian rhythms in cognitive functioning among patients with schizophrenia: impact on signal detection in clinical trials of potential pro-cognitive therapies.

OBJECTIVE: Cognition is affected by circadian rhythms over the course of a day. Circadian rhythms in cognitive functioning are driven by a variety of both endogenous and exogenous factors. Patients with schizophrenia are known to have disturbed circadian rhythms that can affect their cognitive functioning. We examined the impact of time of day on cognitive test scores from subjects participating in clinical trials of potential pro-cognitive therapies for schizophrenia and then explored how this diurnal variation affected signal detection. METHOD: Baseline data from 8 separate schizophrenia clinical trials using the MATRICS Consensus Cognitive Battery (MCCB) were aggregated (Total N=2032). The MCCB assessments were divided into five 2-hour time intervals based on the start-time of the assessments (varying from 8:00 am to 5:59 pm) and then analyzed for differences by time interval. Next, data from two Phase 2 schizophrenia clinical trials of potential pro-cognitive therapies were analyzed to explore the impact of this diurnal variation on placebo separation. RESULTS: Time of day exerted a significant effect on baseline composite MCCB scores (p=.002). Follow-up comparisons revealed significant differences among multiple temporal epochs. In both Phase 2 clinical trials, subjects whose cognitive functioning was assessed at consistent times of day between their baseline and endpoint visits showed a more robust treatment response as compared to subjects assessed at inconsistent times of day. CONCLUSION: Cognitive functioning ebbs and flows over the course of the day. Maintaining consistency in the time of day of cognitive test administrations between visits can help to reduce the noise introduced by circadian rhythms, thereby enhancing signal detection in clinical trials of potential pro-cognitive therapies.

Bl-1020, a new γ-aminobutyric acid-enhanced antipsychotic: results of 6-week, randomized, double-blind, controlled, efficacy and safety study.

OBJECTIVE: BL-1020 is a γ-aminobutyric acid (GABA)-enhanced antipsychotic that combines dopamine antagonism with GABA agonist activity. On the basis of animal models, we tested the hypotheses that BL-1020 would be effective in ameliorating both psychotic symptoms and cognitive impairments, with a favorable safety profile in acutely ill schizophrenia patients. METHOD: 363 hospital-based psychiatric patients in India, Romania, and United States aged 18 to 65 years and meeting criteria for DSM-IV-TR diagnosis of chronic schizophrenia were randomized double-blind to receive BL-1020 10 mg/d, BL-1020 20-30 mg/d, placebo, or risperidone (2-8 mg/d) for 6 weeks. The main outcome measures were the positive and negative syndrome scale (PANSS), brief assessment of cognition in schizophrenia, readiness for discharge questionnaire, clinical global impressions scale (CGI) , and extrapyramidal symptom rating scale. The study ran from July 2008 to June 2009. RESULTS: BL-1020 20-30 mg was significantly better than placebo on PANSS (P = .02) and CGI (P < .001) measurements, with no significant differences noted between BL-1020 20-30 mg and risperidone. There were no significant differences in the maximum change on extrapyramidal symptom rating scale between risperidone and BL-1020 20-30 mg, and both were significantly worse (P < .001) than placebo. BL-1020 20-30 mg was associated with significantly greater improvements on cognitive functioning as measured by the brief assessment of cognition in schizophrenia composite score when compared to placebo (effect size = 0.50, P = .009), risperidone (effect size = 0.43, P = .019), and BL-1020 10 mg (effect size = 0.42, P = .013) after 6 weeks. CONCLUSIONS: BL-1020 appears to be an effective antipsychotic with possible procognitive effects that will need to be further tested for short- and long-term effects. A further randomized controlled trial using the U.S. Food and Drug Administration-recommended Measurement and Treatment Research to Improve Cognition in Schizophrenia cognitive battery is ongoing. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT00567710.

An open-label tolerability study of BL-1020 antipsychotic: a novel gamma aminobutyric acid ester of perphenazine.

BACKGROUND: BL-1020, a novel gamma aminobutyric acid (GABA) ester of perphenazine, is a new oral antipsychotic with a strong affinity for dopamine and serotonin receptors. Unlike first- and second-generation antipsychotics, it has agonist activity at GABA(A). OBJECTIVE: This is the first study to examine tolerability and safety of BL-1020 in schizophrenia. METHODS: This was a phase-II, open-label, multicenter, 6-week study treating patients (n = 36) with chronic schizophrenia. Dosing started at 20 mg/d and increased over 7 days to 40 mg/d. Weekly assessments were conducted. RESULTS: All but 1 patient was titrated to 30 mg/d at day 4; on day 7, 30 were titrated to 40 mg/d. Four patients discontinued the study prematurely. There was no clinically relevant increase in vital signs, sedation, dizziness, or other central nervous system effects or electrocardiogram or laboratory abnormalities and a small increase in weight. Ten patients experienced extrapyramidal symptoms (EPS) requiring treatment with an anticholinergic; 4 patients were unable to reach maximum dose because of EPS. Extrapyramidal Symptom Rating Scale did not indicate clinically significant changes in EPS. The most common adverse event was insomnia (6 patients); other frequent adverse effects (all n = 3) were extrapyramidal disorder, headache, parkinsonism, tremor, and hyperprolactinemia. There was improvement on Positive and Negative Syndrome Scale and Clinical Global Impression of Change with 22 patients showing at least 20% decrease by end point on Positive and Negative Syndrome Scale and 31 patients showing at least minimal improvement on Clinical Global Impression of Change. CONCLUSIONS: These data suggest that 20 to 40 mg/d of BL-1020 is associated with clinically relevant improvement of psychosis with no worsening of EPS and support further testing in randomized controlled trials.

BL-1020, a novel antipsychotic candidate with GABA-enhancing effects: D2 receptor occupancy study in humans.

BL-1020 is a potentially novel antipsychotic, which comprises the typical antipsychotic perphenazine linked by an ester bound to gamma-aminobutyric acid (GABA), intending a simultaneous dopamine-2 (D(2)) receptor blockade and GABA facilitation in the brain. This positron emission tomography (PET) study, using [(11)C]raclopride, assessed the extent and duration of D(2) receptor occupancy (D(2) RO) and safety for single doses of BL-1020 in healthy male subjects. Overall, this study did not raise any safety concern. Single doses of 16-32 mg BL-1020 caused a dose dependent striatal D(2) RO. The 32 mg dose of BL-1020 resulted in an average D(2) RO of 44% at 4-6 h post dosing (pd), which declined to 33% at 24 h pd. Equimolar doses of BL-1020 and perphenazine resulted in similar D(2) RO at 24 h pd. Pharmacokinetic-pharmacodynamic analysis predicted that oral once daily administration of 32 mg BL-1020 would result in D(2) ROs ranging from 52 to 66% at a steady state.

Gamma-aminobutyric acid amides of nortriptyline and fluoxetine display improved pain suppressing activity.

The GABA amides of the antidepressants nortriptyline and fluoxetine, 1 and 2, were compared to their respective parent compounds in rodent models of pain. The amides significantly reduced early nociceptive and late inflammatory responses compared to nortriptyline or fluoxetine, where 1 exhibited overall better efficacy than 2. Amide 1 was most efficacious in lowering cytokine secretion, edema and hyperalgesia induced by formalin and lambda-carrageenan, respectively. Thus, 1 is a promising candidate for the treatment of pain.

BL-1020: a novel antipsychotic drug with GABAergic activity and low catalepsy, is efficacious in a rat model of schizophrenia.

UNLABELLED: Reduced brain gamma-amino-butyric acid (GABA) participates in the pathogenesis of schizophrenia. GABA scarcely penetrates the brain. We evaluated the pharmacological properties of BL-1020, a novel GABA ester of perphenazine. Oral BL-1020 or perphenazine were assessed in acute and subchronic schizophrenia rat models. Catalepsy, serum prolactin, receptor binding profile and cortical (PFC), hippocampal (Hip) and dopamine (DA) levels were determined. Radioactive [14C] labeled BL-1020 was used for pharmacokinetics (PK). Acute and subchronic treatment with BL-1020 antagonized amphetamine-induced hyperactivity, with significantly lower catalepsy and sedation compared to equimolar perphenazine. At the same time, BL-1020 increased DA release in the PFC and Hip. BL-1020 and perphenazine stimulated prolactin secretion equally. BL-1020 displayed strong DA and serotonin (5HT) receptor inhibition (D(2L)K(iz)=0.066 nM, D(2S)K(i)=0.062 nM, 5-HT(2A)K(i)=0.21 nM). PK data revealed that BL-1020 penetrated the brain. CONCLUSIONS: The advantages of BL-1020 for treatment of schizophrenia stem from its being a DA/5HT antagonist and a GABAergic agonist that releases cortical DA and antagonizes amphetamine-induced hyperactivity with reduced catalepsy and sedation.

A mutual prodrug ester of GABA and perphenazine exhibits antischizophrenic efficacy with diminished extrapyramidal effects.

The perphenazine and fluphenazine GABA esters 3 and 4 evaluated in rat models for antipsychotic activity displayed a significant decrease of catalepsy associated with increased prolactin blood levels. Efficacy was evaluated in the d-amphetamine-induced hyperactivity model, where perphenazine abolished hyperactivity and induced sedation and catalepsy, whereas 3 reduced hyperactivity without sedation or catalepsy. Thus, 3 (BL-1020) constitutes a prototype of novel antipsychotics possessing GABAergic activity. A phase II study is in progress.

A novel immune-based therapy for stroke induces neuroprotection and supports neurogenesis.

The ability of the central nervous system to cope with stressful conditions was shown to be dependent on proper T-cell-mediated immune response. Because the therapeutic window for neuroprotection after acute insults such as stroke is relatively narrow, we searched for a procedure that would allow the relevant T cells to be recruited rapidly. Permanent middle cerebral artery occlusion was induced in adult rats. To facilitate a rapid poststroke T cell activity, rats were treated with poly-YE using different regimens. Control and poly-YE-treated rats were assessed for functional recovery using neurological severity score and Morris water maze. Neuroprotection, neurogenesis, growth factor expression, and microglial phenotype were assessed using histological and immunofluorescence methods. Administration of poly-YE as late as 24 hours after middle cerebral artery occlusion yielded a beneficial effect manifested by better neurological performance, reduced neuronal loss, attenuation of behavioral deficits, and increased hippocampal and cortical neurogenesis. This compound affected the subacute phase by modulating microglial response and by increasing local production of insulin-like growth factor-I, known to be a key player in neuronal survival and neurogenesis. The relative wide therapeutic window, coupled with its efficacy in attenuating further degeneration and enhancing restoration, makes poly-YE a promising immune-based candidate for stroke therapy.