Preclinical Work Research Articles

Disturbed laterality of non-rapid eye movement sleep oscillations in post-stroke human sleep: a pilot study.

Sleep is known to promote recovery post-stroke. However, there is a paucity of data profiling sleep oscillations in the post-stroke human brain. Recent rodent work showed that resurgence of physiologic spindles coupled to sleep slow oscillations (SOs) and concomitant decrease in pathological delta (δ) waves is associated with sustained motor performance gains during stroke recovery. The goal of this study was to evaluate bilaterality of non-rapid eye movement (NREM) sleep-oscillations (namely SOs, δ-waves, spindles, and their nesting) in post-stroke patients vs. healthy control subjects. We analyzed NREM-marked electroencephalography (EEG) data in hospitalized stroke-patients (n = 5) and healthy subjects (n = 3). We used a laterality index to evaluate symmetry of NREM oscillations across hemispheres. We found that stroke subjects had pronounced asymmetry in the oscillations, with a predominance of SOs, δ-waves, spindles, and nested spindles in affected hemisphere, when compared to the healthy subjects. Recent preclinical work classified SO-nested spindles as restorative post-stroke and δ-wave-nested spindles as pathological. We found that the ratio of SO-nested spindles laterality index to δ-wave-nested spindles laterality index was lower in stroke subjects. Using linear mixed models (which included random effects of concurrent pharmacologic drugs), we found large and medium effect size for δ-wave nested spindle and SO-nested spindle, respectively. Our results in this pilot study indicate that considering laterality index of NREM oscillations might be a useful metric for assessing recovery post-stroke and that factoring in pharmacologic drugs may be important when targeting sleep modulation for neurorehabilitation post-stroke.

Hematopoietic Stem Cells from Mice and Individuals with Sickle Cell Disease Display Premature Senescence and Loss of Function That Is Targetable By Senolytic Therapy

Sickle cell disease (SCD) is an inherited anemia caused by mutations in the β-globin gene, resulting in aberrant hemoglobin polymerization driving hemolysis, vasooclusion, and systemic inflammation. Allogeneic hematopoietic stem cell (HSC) transplantation is potentially curative, and novel autologous therapies involving gene-editing of mobilized HSCs are being developed. However, many SCD patients are unable to undergo autologous gene-editing due to failure to mobilize sufficient numbers of HSCs. Further, the damaging effects of SCD on the HSC pool remain uncharacterized. Therefore, we interrogated young and aged cohorts of humanized SCD mice and non-SCD controls for HSC frequency and function. Immunophenotypic HSC frequency was mildly elevated (1.3-fold, p=0.00316) in 2-month-old SCD mice, relative to controls (n=5-6/group). In contrast, HSC numbers were halved in 6 and 12-month-old SCD mice (n=8-10/group, p=0.00358 and p=0.0148, respectively). Competitive transplantation of whole bone marrow (WBM) from 2 and 6-month-old SCD mice displayed a profound loss of peripheral blood repopulation, relative to age-matched control mice. Quantitative limiting dilution transplantation of WBM from 2 and 6-month-old SCD mice revealed a 4 and 6-fold loss of functional HSCs, relative to control mice. Thus, despite elevated numbers of phenotypic HSCs in young SCD mice, the HSC pool is depleted of functional repopulating cells during SCD. To understand the loss of functional HSCs, we performed bulk RNA-sequencing of purified HSCs from 6-month-old SCD mice. We observed downregulation of 37 genes encoding ribosomal and histone proteins, suggesting impaired histone/ribosome biogenesis, a phenotype associated with senescence. Consistently, pathway enrichment analysis also revealed perturbed p53 activity and a high degree of association with published molecular signatures of senescence. To explore this further, HSCs isolated from 6-month-old SCD mice or age-matched controls were interrogated for senescence associated β-galactosidase (SA-β-gal) activity. On average 19.5% of SCD HSCs were SA-β-gal+ versus only 7.06% of HSCs isolated from controls (n=10-11/group, p=0.004). HSCs isolated from SCD mice also displayed elevated ROS, DNA damage, and expression of molecular markers of senescence (e.g. p21, BCLXL). Importantly, 23.14 ± 5.61% of Lin-CD34+CD38- bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs) isolated from pediatric individuals with SCD were SA-β-gal+. In contrast, only 4.97 ± 2.97% of HSPCs were SA-β-gal+ in age-matched, non-SCD control individuals (n=8 control and n=5 SCD individuals, p=<0.0001). Further, 47.46 ± 17.49% of Lin-CD34+CD38-CD90+CD45RA- BM cells, which are highly enriched for human HSCs, were SA-β-gal+ in young individuals with SCD, relative to only 4.41 ± 2.37% in the BM of age-matched control individuals (n=4 control and n=5 SCD individuals, p=0.0019). We also detected elevated expression of p16, p21, and BCL2 in BM HSPCs isolated from pediatric individuals with SCD, relative to controls (n=6 control and n=9 SCD individuals). In total, these data reveal that SCD pathophysiology aberrantly invokes senescence programs and damages HSC function in mice and humans. Clearance of senescent HSCs from mouse BM has previously been shown to restore aging-related HSC dysfunction. We therefore tested if clearance of senescent HSCs from the BM could restore function to the SCD HSC pool. 2-month-old SCD and control mice were treated with two weekly cycles of 75 mg/kg/day of the senolytic, ABT-263, via oral gavage. 14 days after treatment, WBM was isolated and tested for HSC function and frequency. Senolytic therapy significantly increased HSC numbers in SCD mice BM, relative to vehicle-treated controls (n=5/group). Importantly, the in vivo hematopoietic repopulating activity of WBM from ABT-263-treated SCD mice was comparable to non-SCD controls, revealing a correction of our previously observed severe transplantation defect (n=12/group). In sum, these data reveal premature onset of senescence as a previously unappreciated phenotype of the HSC pool during SCD. This work provides the first proof-of-principle that treatment of individuals with SCD using senolytics may restore function to the HSC pool, and ongoing preclinical work aims to test if senolytic therapy enhances the yield and function of SCD HSCs mobilized for autologous therapy.

Mini-Hyper-CVD Plus Venetoclax for Treatment of Older Adults with Newly Diagnosed Ph-Negative B-ALL or T-ALL

Introduction Treatment of Philadelphia chromosome (Ph)-negative acute lymphoblastic leukemia (ALL) in older patients (pts) is challenging due to poor tolerance of conventional chemotherapy and chemo-resistant disease. Pre-clinical work demonstrates that lymphoblasts are BCL2-dependent and sensitive to the BCL2 inhibitor venetoclax (VEN). We hypothesized that VEN plus dose-reduced hyper-CVD (cyclophosphamide, vincristine, dexamethasone) chemotherapy would be well tolerated and effective in older pts. We previously reported the safety and tolerability of VEN plus hyper-CVD in newly diagnosed and relapsed/refractory (R/R) pts (Jain ASH 2019), with a maximum tolerated dose of VEN 600 mg daily on days 1-21 per 28-day cycle. Preliminarily efficacy was promising in newly diagnosed pts, which prompted study extension to phase (P) 2, enrolling newly diagnosed older pts with Ph-negative (neg) B-ALL and T-ALL. Methods Pts with Ph-neg B-ALL or T-ALL were enrolled in a multi-center Phase Ib/II study (NCT03319901). In P1, both R/R pts and newly diagnosed pts ≥ 60 years (yrs) were enrolled. In P2, only newly diagnosed pts ≥ 55 yrs old or ≥ 50 yrs old with BMI ≥ 35 kg/m2 were enrolled. Pts began with VEN ramp-up (P1 over 7d; P2 over 3d) to target dose (P1: dose level [DL]1 400 mg/d; DL2 600 mg/d; P2 400 mg/d), Figure 1. Hyper-CVD, including central nervous system (CNS) prophylaxis with intrathecal chemotherapy, was then given with VEN per DL for 21/28 days per cycle, for up to 8 cycles. Following completion of hyper-CVD courses, pts received VEN plus POMP maintenance for up to 2 yrs. Pts were recommended to receive fewer than 8 cycles of hyper-CVD in the setting of toxicity or to proceed to allogeneic hematopoietic stem cell transplant (HSCT) per physician discretion. This report updates follow-up of the newly diagnosed pts enrolled in P1 and the P2 cohort. Complete remission (CR) was defined as <5% marrow blasts, no evidence of extramedullary or CNS leukemia, ANC >1.0 K/uL and platelets >100 K/uL. Measurable residual disease (MRD) was evaluated by multicolor flow cytometry (MFC) and negative defined as <0.01%. Survival outcomes are presented via Kaplan Meier. Results In total, 30 newly diagnosed pts [P1 11 (DL1=3; DL2=8); P2 18] were treated with a median age of 68 yrs (range, 57-81 yrs, n=12 [40%] ≥70 yrs); n=15 (50%) women, n=4 (13%) Hispanic, and n=1 (3%) Black. Twenty-three (77%) pts had B-ALL and 7 (23%) pts had T-ALL (6/7 early T-cell precursor). Two (6%) pts had central nervous system (CNS) involvement at diagnosis (1-CNS3, 1-CNS2). High risk genetics were present in 56% (13/23) of B-ALL pts: TP53 (n=9 associated with complex [n=3] or hypodiploid [n=5] karyotype), KMT2A (n=1), IKZF1 abnormality (n=2), and Ph-like (n=1). Eight B-ALL pts had received prior chemotherapy for multiple myeloma (n=6; n=4 auto HSCT, n=5 lenalidomide), diffuse large B-cell lymphoma (n=1, RCHOP), and breast cancer (n=1, docetaxel, carboplatin, trastuzumab). The median number of hyper-CVD cycles received was 4 (range 1-8) among the 27 pts who have completed the hyper-CVD phase of treatment (3 pts are currently receiving consolidation). Mortality within 30 days was 3% (n=1). One patient died in CR due to sepsis during cycle 4. Venetoclax did not impair count recovery: median time from C1 to C2 was 34 days (range 27-47). Dose adjustments of VEN occurred in 4 pts (15%). An MRD-neg CR was achieved in 25/30 (83%) pts. The median cycles to achieve both CR and MRD negativity by MFC was one (range: CR 1-2 cycles; MRD negativity 1-4 cycles). Remaining pts had refractory disease (n=4, 13%), or were not assessed (n=1, 3%) due to early death. Of the 25 pts who achieved CR, 11/25 (44%) received allogeneic HSCT in CR1 after a median of 200 days (range 138 to 347). With a median follow-up of 16.4 months, the estimated 12- and 24-month OS are 82.1% (95% CI 66, 98) and 74.6% (95% CI 54, 95), Figure 2; and RFS are 82.9% (95% CI 65, 100.0) and 67.8% (95% CI 44, 92), respectively. At data cut-off, 22 pts (73%) are alive. Death events (n=8, 27%) were due to early mortality (n=1, related to both ALL and infection), post HSCT complications (n=3), refractory ALL (n=1), relapsed ALL (n=1), sepsis in CR (n=1), and complications of cardiac surgery in CR (n=1). Conclusion In this ongoing study, the addition of VEN to hyper-CVD in newly diagnosed ALL was well tolerated with encouraging responses. A future amendment will incorporate blinatumomab consolidation for pts with B-ALL.

A Phase I Study of Venetoclax in Combination with Inotuzumab Ozogamicin for Relapsed or Refractory ALL in Adults

Introduction Adults with acute lymphoblastic leukemia (ALL), especially those who are older and/or have unfavorable disease biology frequently relapse. Inotuzumab ozogamicin (INO), an anti-CD22 monoclonal antibody covalently linked to calicheamicin, is approved for treatment of relapsed or refractory (R/R) CD22+ ALL. Although INO induces measurable residual disease negative (MRD-neg) complete remission (CR) in >75% of patients (pts), the duration of response (DOR) is limited (<6 months). Pre-clinical work demonstrates that malignant lymphoblasts are BCL2-dependent (Del Gaizo Moore Blood 2008). Initial results of the BCL2 inhibitor venetoclax (VEN) combination therapies for ALL are promising (Jain ASH 2019, Pullarkat Cancer Discov 2021). Due to distinct mechanisms of activity, non-overlapping toxicity, and possible synergy (Kirchhoff Blood 2021), we hypothesized that INO and VEN can be safely and effectively combined. Study Design and Eligibility This is an investigator-sponsored phase I study (NCT05016947) with a 3+3 dose escalation design. The primary objective is to determine the maximum tolerated dose (MTD) of VEN plus INO at the FDA-approved doses in adults with R/R CD22+ ALL. Secondary objectives assess efficacy (hematologic response, MRD by multi-parametric flow cytometry [MFC] threshold <0.01%, and DOR). A 14 pt expansion cohort is planned. Dose-limiting toxicity (DLT) is defined as treatment-related CTCAE v5 non-heme toxicity grade 3+, sinusoidal obstructive syndrome (SOS) any grade, or liver toxicity during Induction (Ind) Cycle (C) 1 and/or failure of hematologic recovery (ANC ≥500, Plts ≥25 K/mL) by day 42 of Ind C1 in responding pts. Eligibility: ≥18 years (yrs); ALL (≥5% blasts) or LBL (marrow involvement not required); CD22 expression on ≥20% of blasts; R/R to one or more cycles of CC or in the case of Philadelphia-chromosome (Ph)-positive ALL, failed by two or more TKIs, or refractory to/ineligible for ponatinib; ≥60 days from bone marrow transplant (BMT), and off immune suppression ≥2 weeks; adequate liver and kidney function; no history of liver disease including SOS; blast count ≤25 K/mL prior to treatment; no symptomatic central nervous system (CNS) disease. Pts with prior INO or VEN treatment for R/R ALL are excluded. Regimen Pts are treated with a VEN Ramp-Up over 3 days (DL1: 100/200/200; DL2: 100/200/400) followed by Induction C1: IV INO days 1, 8, 15 (0.8/0.5/0.5 mg/m2) and VEN daily per DL days 1-21. DEX 10 mg/m2 IV/PO is administered during Ramp-Up and Ind C1 days 1-4 (7 days total). Pts with >5% blasts after C1 receive Ind C2 without DEX. Pts with <5% blasts after Ind (1 or 2 cycles) continue with up to four 28-day consolidation cycles (INO 0.5 mg/m2 days 1, 8, 15 plus VEN per DL days 1-21). Pts receive CNS prophylaxis with intrathecal chemotherapy. Results The 2-level dose-escalation phase has been completed; we enrolled 9 pts (5 female/4 male) with Ph-negative (n=7) or Ph-positive (n=2) ALL. The median age was 45 yrs (range 25 -74). Pts had received 1 (n=5) or 2 (n=4) prior lines of therapy. DL1 enrolled 3 pts without DLT. All DL1 pts achieved an MRD-neg CR and bridged to consolidation: 1 pt XRT to involved disease site (ankle) after 3 cycles and 2 pts to BMT after 2 cycles (Table). Three pts enrolled at DL2; no DLTs were observed. All achieved an MRD-neg CR. Two pts bridged to consolidation (donor lymphocyte infusion after 3 cycles, blinatumomab after 5 cycles) while 1 pt with KMT2A-r ALL experienced early relapse with lineage switch during second cycle. A confirmatory cohort of 3 pts enrolled at DL2 without DLT. All achieved a CR (n=2, MRD-neg; n=1, MRD-pos). The pt with MRD-pos CR as best response proceeded to blinatumomab as bridge to BMT after 2 cycles with 2 pts continuing treatment (currently cycle 2). Overall, CR was achieved in 100% (9/9) of pts with 7/8 (87%) achieving MRD-negativity. With median follow-up of 195 days (95% CI 80 - NR), 2 pts have relapsed 48 (on treatment) and 177 (after BMT) days from registration while other pts remain alive in CR. Non-heme grade 3-4 AEs include febrile neutropenia (2), DIC (1), tumor lysis syndrome (1), anxiety (1), hypertension (1), and hypotension (1). There have been no SOS. There were no delays in count recovery during C1, with median time from C1 to C2 being 30 days (range 27-42). Conclusion VEN 400mg x 21 days in combination with INO at standard doses was well tolerated and declared the recommended phase 2 dose (RP2D) with a CR rate of 100%.

Elevated gonadotropin levels are associated with increased biomarker risk of Alzheimer's disease in midlife women.

In preclinical studies, menopausal elevations in pituitary gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), trigger Alzheimer's disease (AD) pathology and synaptic loss in female animals. Herein, we took a translational approach to test whether gonadotropin elevations are linked to AD pathophysiology in women. We examined 191 women ages 40-65 years, carrying risk factors for late-onset AD, including 45 premenopausal, 67 perimenopausal, and 79 postmenopausal participants with clinical, laboratory, cognitive exams, and volumetric MRI scans. Half of the cohort completed 11C-Pittsburgh Compound B (PiB) amyloid-β (Aβ) PET scans. Associations between serum FSH, LH and biomarkers were examined using voxel-based analysis, overall and stratified by menopause status. Associations with region-of-interest (ROI) hippocampal volume, plasma estradiol levels, APOE-4 status, and cognition were assessed in sensitivity analyses. FSH levels were positively associated with Aβ load in frontal cortex (multivariable adjusted P≤0.05, corrected for family wise type error, FWE), an effect that was driven by the postmenopausal group (multivariable adjusted PFWE ≤ 0.044). LH levels were also associated with Aβ load in frontal cortex, which did not survive multivariable adjustment. FSH and LH were negatively associated with gray matter volume (GMV) in frontal cortex, overall and in each menopausal group (multivariable adjusted PFWE ≤ 0.040), and FSH was marginally associated with ROI hippocampal volume (multivariable adjusted P = 0.058). Associations were independent of age, clinical confounders, menopause type, hormone therapy status, history of depression, APOE-4 status, and regional effects of estradiol. There were no significant associations with cognitive scores. Increasing serum gonadotropin levels, especially FSH, are associated with higher Aβ load and lower GMV in some AD-vulnerable regions of midlife women at risk for AD. These findings are consistent with preclinical work and provide exploratory hormonal targets for precision medicine strategies for AD risk reduction.

Extended exposure to low doses of azacitidine induces differentiation of leukemic stem cells through activation of myeloperoxidase.

Oral azacitidine (oral-Aza) treatment results in longer median overall survival (OS) (24.7 vs. 14.8 months in placebo) in patients with acute myeloid leukemia (AML) in remission after intensive chemotherapy. The dosing schedule of oral-Aza (14 days/28-day cycle) allows for low exposure of Aza for an extended duration thereby facilitating a sustained therapeutic effect. However, the underlying mechanisms supporting the clinical impact of oral-Aza in maintenance therapy remain to be fully understood. In this preclinical work, we explore the mechanistic basis of oral-Aza/extended exposure to Aza through in vitro and in vivo modeling. In cell lines, extended exposure to Aza results in sustained DNMT1 loss, leading to durable hypomethylation, and gene expression changes. In mouse models, extended exposure to Aza, preferentially targets immature leukemic cells. In leukemic stem cell (LSC) models, the extended dose of Aza induces differentiation and depletes CD34+CD38- LSC. Mechanistically, LSC differentiation is driven in part by increased myeloperoxidase (MPO) expression. Inhibition of MPO activity either by using an MPO-specific inhibitor or blocking oxidative stress, a known mechanism of MPO, partly reverses the differentiation of LSC. Overall, our preclinical work reveals novel mechanistic insights into oral-Aza and its ability to target LSC.

Abstract 385: Comparison of Rapid Implementation of Automated Head and Thorax Elevation With Active Compression Decompression Cardiopulmonary Resuscitation and an Impedance Threshold Device versus National Outcome Data: A Prospective Observational Study

Background: Pre-clinical work has shown automated head and thorax elevation with active compression decompression CPR and an impedance threshold device (AHUP-CPR) improved 24-hour neurological survival. Propensity matched studies of out-of-hospital-cardiac arrest (OHCA) patients have shown a time dependent association between survival to hospital discharge (SHD) and application of AHUP CPR.Hypothesis: Rapid application of AHUP CPR for OHCA will have a higher incidence of SHD compared to national data. Methods: This is an ongoing prospective observational study of patients from an AHUP-CPR registry of non-traumatic adult OHCA receiving AHUP CPR as part of first responder care. OHCA patients from pre-hospital sites that consistently implemented rapid AHUP CPR were included. Patient demographics, arrest characteristics, ROSC, SHD, and SHD with favorable neurological function, defined as a Cerebral Performance Category score of 1 or 2, were recorded. Data are reported as means ± SD. Combined 2021 and 2022 data from the Cardiac Arrest Registry to Enhance Survival (CARES) were used for comparison of the primary study endpoint, SHD, and secondary endpoints. Chi-square testing was used to compare outcomes. Results: Of 11 registry sites, 7 (US States AL, FL, IL, MN, OK, TN, VA) met inclusion criteria and 1457 patients from January 2020 to June 2023 were enrolled. Average age was 65 ± 16.4 years, 61.8% (901) were male, and 71% (1035) received AHUP CPR. Average time (mm:ss) from 9-1-1 call to start of CPR was 9:52 (6:47) and time to start of AHUP CPR was 12:59 (5:51). Presenting rhythms were: asystole 47.8% (696), PEA 25.8% (376), and VF 20.5% (298). For all AHUP patients, SHD was 13.6% (198) versus 9.2% (26316/286525) for CARES (p < 0.0001). SHD with favorable neurological function was higher for AHUP (10.3% (150) versus 7.3% for CARES (20889), p < 0.0001) and ROSC (29.4% (428) vs 27.0% (77301), p = 0.039). Conclusions: In an unadjusted analysis, the incidence of SHD, ROSC, and neurologically favorable SHD with rapid implementation of AHUP-CPR was higher than nationally reported data. These results support prior findings of a time sensitive association between survival and time to AHUP-CPR. Additional analyses and study are needed for confirmation.

Innate Cell Engager (ICE®) AFM13 Combined with Preactivated and Expanded (P+E) Cord Blood (CB)-Derived Natural Killer (NK) Cells for Patients with Refractory CD30-Positive Lymphomas: Final Results

CONCLUSIONS Pts with refractory Hodgkin (HL) and other CD30+ lymphomas have few effective therapies available and targeted NK cell immunotherapy is an active area of research. Transfer of non-targeted NK cells has shown limited clinical benefit as target recognition of cancer cells by NK cells constitutes a barrier. The innate cell engager AFM13 is a first-in-class CD30/CD16A bispecific antibody construct that induces selective killing of CD30+ tumor cells by engaging and activating NK cells. As a single agent, AFM13 has limited clinical activity against HL, likely due to the reduced effector function of autologous NK cells in these pts, which provides the rationale for combining AFM13 with allogeneic NK cells. Our prior preclinical work identified a promising combination of CB-derived NK cells that were first IL-12/IL-15/IL-18-preactivated followed by ex vivo expansion (P+E) with engineered K562 feeder cells expressing membrane-bound IL-21, 4-1BBL and CD48 and exogenous IL-2, and subsequently complexed with AFM13 just before infusion; these cytokine-induced memory-like NK cells presented chimeric antigen receptor (CAR)-NK-like features and increased in vitro and in vivo antitumor activity compared to either non-AFM13-precomplexed P+E NK cells or to AFM13 alone (Kerbauy et al, Clin Cancer Res 2021). Building on this work we wished to study this CB NK-cell therapy in pts with refractory CD30+ lymphomas. This investigator-initiated single-center phase I-II trial (NCT04074746) evaluated the safety and activity of P+E AFM13-precomplexed CB NK cells followed by systemic AFM13. Pts ages 15-75 with CD30+ lymphomas refractory to brentuximab vedotin were enrolled. The goals were to identify the recommended phase 2 dose (RP2D) of NK cells, estimate the overall (ORR) and complete response (CR), event-free (EFS) and overall survival (OS) rates, and study NK cell activation and persistence. Each treatment cycle consisted of fludarabine/cyclophosphamide (days −5 to −3) followed by infusion (day 0) of the AFM13-precomplexed CB NK cells, cultured for 14 days as described above, and three weekly IV infusions of AFM13 (200 mg, days 7, 14 and 21). Two cycles were planned for pts 1-19 and up to 4 cycles from patient 20 on. Response was evaluated on day 28 of each cycle. Enrollment proceeded at 3 dose levels (DL) of 10 6, 10 7 and 10 8 NK/Kg, respectively. Each patient received the same NK dose in all cycles. Between 09/20-12/22 we enrolled 42 pts (37 HL, 5 NHL), pretreated with a median 7 lines, at DL1 (N=3), DL2 (N=3) and DL3 (N=36), with 1 (N=2), 2 (N=21), 3 (N=3) or 4 cycles (N=16). All had active progressive disease at enrollment and no bridging therapy was given. The cords used for each of the 117 cycles were selected without consideration for HLA match, which was 0/6 (N=53), 1/6 (N=46), 2/6 (N=14), 3/6 (N=3) and 4/6 (N=1). The product infused consisted of >94% NK cells (expanded >2,700 fold), with only 0.02% T cells. The NK cells were 96% viable, 91% bound to AFM13, and 97.6% CD16+. The products were infused fresh. There was 1 grade 2 infusion-related reaction (IRR) in 115 infusions of AFM13-NK (0.8%) and 27 IRR infusion related reactions in 350 infusions of AFM13 (7.7%) (1 G3, 26 G2). We saw no cases of CRS, ICANS or GVHD of any grade. DL3 (10 8 NK/Kg) was established as the RP2D. The ORR and CR were 92.8% and 66.7%, respectively (94.4% and 72.2%, respectively, in 36 pts treated at the RP2D). Nine pts had a response consolidated with SCT (5 allo, 4 auto). At median follow-up of 14 (6-34) months, the EFS/OS rates are 31%/76%; median EFS/OS are 8 months/not reached. No relapses were associated with antigen loss. In the subset of pts planned for 4 cycles at RP2D, 15 of 23 pts remained event free at 6 mo (4 pts with and 11 without SCT consolidation), for 6-month EFS/OS rates of 65%/83%. Overall, 8 of the 9 pts (89%) receiving a consolidation SCT are event free at 10 to 34 months. AFM-bound CB NK cells were detected in blood from day 1 post infusion for up to 3 weeks. Donor NK levels followed similar patterns after each cycle, which argues against a sensitization after the first NK infusion. CyTOF studies showed increased expression of AFM13 and activation markers in donor NK cells. We confirmed trafficking of AFM13-bound donor NK cells to tumor sites shortly after infusion. CB-derived cytokine-induced memory-like NK cells precomplexed with AFM13 have excellent tolerability and activity for pts with heavily pretreated and refractory CD30+ lymphoma.

Research Trends and Hot Spots of Allopregnanolone Research in the Last 20 Years: A Bibliometric Analysis.

Allopregnanolone is a kind of neuroactive steroid or neurosteroid in the central nervous system that acts as an endogenenous GABAA receptor positive modulator. However, at present, no comprehensive bibliometric analysis regarding allopregnanolone research is available. In our study, we intend to analyze the research trends and hot spots related to allopregnanolone in the past 20 years. We searched for allopregnanolone related articles and reviews between 2004 and 2023 from the Web of Science Core Collection database. Then, the bibliometric analysis was conducted using VOSviewer, CiteSpace, Microsoft Excel 2019, as well as the online bibliometric analysis platform (http://bibliometric.com/). A total of 1841 eligible publications were identified. The number of annual publications and citations was generally on the rise. Among countries, the United States ranked first in overall publications, citations, international cooperation, and the number of research institutions. The University of North Carolina was the most active institution, conducting numerous preclinical and clinical work that focusing on allopregnanolone treatment for diverse psychiatric or neurologic disorders. As for authors, Dr. Frye CA, Morrow AL, and Pinna G were identified as the top three prolific scholars due to their great publications and citations. Based on the publication clusters and citation bursts analysis, the keyword co-occurrence network, the strongest citation bursts, and co-cited references analysis, the hot spots in recent years included "depression", "postpartum depression", "GABAA receptor", and so on. Allopregnanolone is still a popular area of research, and the United States leads the way in this area. Dr. Frye CA, Morrow AL, Pinna G, and their teams contributed greatly to the mechanism study and translation study of allopregnanolone. The use of allopregnanolone for the treatment of psychiatric or neurologic disorders, especially postpartum depression, is the current hot spot. However, the underlying mechanisms of anti-depression are still not clear, deserving more in-depth research.

Chimeric autoantibody receptor T cells deplete NMDA receptor-specific B cells

Anti-NMDA receptor (NMDAR) autoantibodies cause NMDAR encephalitis, the most common autoimmune encephalitis, leading to psychosis, seizures, and autonomic dysfunction. Current treatments comprise broad immunosuppression or non-selective antibody removal. We developed NMDAR-specific chimeric autoantibody receptor (NMDAR-CAAR) Tcells to selectively eliminate anti-NMDAR B cells and disease-causing autoantibodies. NMDAR-CAARs consist of an extracellular multi-subunit NMDAR autoantigen fused to intracellular 4-1BB/CD3ζ domains. NMDAR-CAAR Tcells recognize a large panel of human patient-derived autoantibodies, release effector molecules, proliferate, and selectively kill antigen-specific target cell lines even in the presence of high autoantibody concentrations. In a passive transfer mouse model, NMDAR-CAAR Tcells led to depletion of an anti-NMDAR B cell line and sustained reduction of autoantibody levels without notable off-target toxicity. Treatment of patients may reduce side effects, prevent relapses, and improve long-term prognosis. Our preclinical work paves the way for CAAR Tcell phase I/II trials in NMDAR encephalitis and further autoantibody-mediated diseases.

Preclinical motor chunking and fine motor skill learning in fixed prosthodontics: Contribution of 3D printing and satisfaction of dental students.

In fixed prosthodontics, simulators are essential to students for a progressive transition from preclinical to clinical condition. With the 3D printing technology, we developed resin bars allowing students to better visualise by motor chunking technique. Main objectives of this work were to describe this teaching methodology used in preclinic among different promotions of second, third and fourth dental years and to evaluate students' feedback. Two hundred seventy resin strips were digitally designed and printed in resin. All participants from second, third and fourth had to fulfil a User Experience Questionnaire (UEQ) after the preclinical work. The scales of this questionnaire covered the complete impression of the user experience. Both classical aspects of usability (efficiency, insight and reliability) and aspects of user experience (originality, stimulation) were measured. For the second dental years, 'Attractiveness', 'Stimulation' and 'Novelty' were considered 'Excellent'. For the third dental year, novelty average was considered as 'Excellent'. For the fourth dental year, 'novelty' was considered as 'Good'. The resin plates used in this study are original and stimulating for the students, especially for the second-year dental students who found the exercises useful for their learning. This method can also be used by creating scenarios close to the clinical situations encountered in dentistry departments (more dilapidated teeth, preparation of inlays, post and core, etc.). This 3D printed simulation model is not intended to replace the Frasaco® models but is a complement to the learning process.

Chronic intermittent hypoxia due to obstructive sleep apnea slightly alters nutritional status: a pre-clinical study.

Obstructive sleep apnea syndrome (OSAS) is associated with chronic intermittent hypoxia (cIH) that causes disturbances in glucose and lipid metabolism. Animals exposed to cIH show lower body weight and food intake, but the protein-energy metabolism has never been investigated. Here, to address the gap, we studied the impact of cIH on nutritional status in rats. A total of 24 male Wistar rats were randomized into 3 groups (n = 8): a control group (Ctrl), a cIH group (cIH) exposed to cIH (30 s 21-30 s 5% fraction of inspired oxygen, 8 h per day, for 14 days), and a pair-fed group (PF) exposed to normoxia with food intake adjusted to the intake of the cIH group rats with anorexia. Body weight and food intake were measured throughout the study. After 14 days, the rats were euthanized, the organs were collected, weighed, and the liver, intestine mucosa, and muscles were snap-frozen to measure total protein content. Food intake was decreased in the cIH group. Body weight was significantly lower in the cIH group only (-11%, p < 0.05). Thymus and liver weight as well as EDL protein content tended to be lower in the cIH group than in the Ctrl and PF groups. Jejunum and ileum mucosa protein contents were lower in the cIH group compared to the PF group. cIH causes a slight impairment of nutritional status and immunity. This pre-clinical work argues for greater consideration of malnutrition in care for OSAS patients. Further studies are warranted to devise an adequate nutritional strategy.

Effectiveness of haptic feedback devices in preclinical training of dental students—a systematic review

BackgroundAcquisition of psychomotor skills is of utmost importance for competent preclinical restorative dentistry. Recent advancements in haptic feedback technology have been incorporated into preclinical dental education to augment the conventional phantom head-based training.ObjectiveThis systematic review aims to assess the effectiveness of haptic feedback device, Simodont, in improving the skill development and learning outcomes of dental students during their preclinical training.Materials and methodsElectronic databases Web of Science, Scopus, PubMed were searched for relevant studies since inception up until March, 2023. Only English language studies that assessed the effectiveness of haptic feedback devices in preclinical dental education were included. We excluded studies that did not use Simodont as the haptic feedback device or did not involve preclinical restorative work. Study quality was assessed using the revised Cochrane risk of bias tool and ROBINS-I. The primary goal of the study is to evaluate the efficacy of Simodont as a complementary training modality for dentistry students.ResultsResults from 9 high-quality studies were analyzed and synthesized to evaluate the overall impact of haptic feedback devices on various aspects of preclinical training. The studies were conducted on 826 undergraduate dental students enrolled in various years of their training across dental colleges and universities in different parts of the world. A majority of studies showed some concerns regarding risk of bias. Haptic feedback devices added a new layer to Virtual Reality (VR) through the perception of touch and force feedback. It assisted junior dental students improve their psychomotor skills and movement skills. Instantaneous feedback on the students' performance helped enhance their self-assessment and correction, and also eliminated the subjectivity of evaluation. Data derived from virtual simulators helped stratify dental students and predict their clinical performance, providing an opportunity to tailor the learning process to meet individual diversity in students' expertise.ConclusionBased on the limited evidence available, Simodont was effective in preclinical training of dental students, offering advantages such as unlimited reproducibility, objective evaluation of preparation by computer assessment, and cost reduction. And further studies are warranted to explore the incorporation of patient's oral environment simulation for better skill training.

THU485 The Impact Of Cured, Fried Bacon On The Reemergence From Breast Cancer Dormancy

Abstract Disclosure: C.P. Schane: None. A. Nelczyk: None. C. Chen: None. H. Vidana Gamage: None. M. Kadiri: None. M.T. McHenry: None. S. Bendre: None. N. Krawczynska: None. M.A. Henn: None. D.C. Castro: None. J. Chen: None. R.I. Tejeda: None. S. Hsiao: None. N.J. Engeseth: None. J.V. Sweedler: None. M.K. Wendt: None. T. Fan: None. W.G. Helferich: None. E.R. Nelson: None. One in eight women will develop breast cancer in her lifetime, with metastasis being the leading cause of mortality. Once the cells colonize the metastatic site, they do not always immediately begin to proliferate, but remain dormant. These dormant cells reside in the tissue, until mechanisms, which remain unknown, awaken the dormant cells and they resume malignant proliferation. Due to the large and growing population of breast cancer survivors at risk of metastatic recurrence, there is an urgent need to elucidate the mechanisms of dormancy and develop therapeutics to prevent reemergence. Elevated levels of circulating cholesterol are a risk factor for breast cancer recurrence. Preclinical work has demonstrated that oxysterol cholesterol metabolites such as 27-hydroxycholesterol (27HC) can promote tumor metastasis. Here, we show that exogenous 27HC lead to a more rapid reemergence from dormancy, using the syngeneic mouse D2.0R model of mammary cancer dormancy. Since it would be expected that cooking high cholesterol foods, such as bacon, under oxidative conditions, such as pan-frying, would produce fat containing cancer-promoting cholesterol metabolites, we hypothesized that consumption of such cooked foods would also promote reemergence from dormancy. Interestingly, consumption of cured, fried bacon fat (cfBF), but not uncured fried bacon fat (ufBF) resulted in reemergence. When metastatic tissues were assessed, it was found that consumption of cfBF dramatically reduced mast cell numbers. Since mast cells (MC) are major contributors to local histamine production, we next tested whether various anti-histamines could alter dormancy. This led to the striking finding that H2 antagonists promoted the breaking of dormancy. Our ongoing work is aimed at further elucidating this novel metastatic axis: Diet - MC - H2 - Dormancy, with the ultimate goal of leveraging it to develop novel therapeutic targets or lifestyle interventions. Therefore, our results are expected to provide the foundation for dietary and medical approaches to reduce the risk of recurrence among breast cancer survivors. This work was supported by the American Institute for Cancer Research grant (713063), the National Cancer Institute (R01CA234025) and Department of Defense Breast Cancer Research Program Era of Hope Scholar Award (BC200206). Further support awarded to ATN from a National Institutes of Environment Health Sciences and Research Training in Toxicology and Environmental Health (T32) Fellowship (ES007326). Presentation: Thursday, June 15, 2023

OR06-03 Intergenerational Exposure To Endocrine-disrupting Chemicals (Edcs) Affects Dopamine-mediated Behaviors In A Sex-Specific Manner In Rats

Abstract Disclosure: E.N. Hilz: None. C. Schnurer: None. S. Bhamidipati: None. L. Thompson: None. E. Morales-Ledesma: None. A.C. Gore: None. Polychlorinated biphenyls (PCBs) are a weakly estrogenic class of ubiquitous environmental EDCs that affect a range of sex-specific neuroendocrine and reproductive outcomes. Furthermore, these effects can extend intergenerationally via epigenetic changes carried down the germline. In humans, PCBs are associated with sexually dimorphic and hormone sensitive neurobehavioral disorders including attention deficit hyperactivity disorder (ADHD) and mood disorders such as depression; however, little preclinical work has modeled these cognitive changes in rats and none has determined if they extend intergenerationally via endocrine-mediated changes to the brain dopamine system. In the current work, three generations of male and female Sprague-Dawley rats were exposed to the PCB mixture Aroclor 1221 (A1221) at different life stages: as adults; through perinatal exposure via mother's diet; or preconceptionally, as the germ cells within the exposed fetus. Adults of each generation underwent a battery of behavioral tasks to assess attentional phenotype, cognitive flexibility, and affect as measured by attentional set-shifting, conditioned orienting, and sucrose preference tests. These outcomes were correlated with both serum estradiol concentrations and dopamine cell quantity in the midbrain. Results revealed that A1221 treatment had sex- and generation-specific effects: in adult-exposed females, decreased conditioned orienting suggested a hyporesponsive attentional phenotype; in males, preconceptional exposure reduced cognitive flexibility via impaired attentional set-shifting. All rats showed a strong preference for sucrose solution; however, A1221 treatment decreased the total amount of sucrose solution consumed (indicating anhedonia) regardless of sex or timing of exposure. Dopamine cell numbers were inversely affected by A1221 between the sexes: decreased in females and increased in males, which correlates well with the cognitive changes observed. Relationships among estradiol concentration, cognitive changes, and dopamine cell quantity will be ascertained using a multilevel model that accounts for endocrine-mediated disruptions at the brain, body, and behavioral levels. These data show that the PCB mixture A1221 impairs different aspects of cognitive behavior between the sexes depending on timing of exposure, while having a broader effect of increased anhedonia, in a manner potentially mediated by hypo- or hyper-availability of midbrain dopamine and serum estradiol. Supported by RO1 ES029464. Presentation: Thursday, June 15, 2023

The promise of microneedle technologies for drug delivery.

Microneedle (MN) technologies offer the opportunity to improve patient access and target delivery of drugs and vaccines to specific tissues. When in the form of skin patches, MNs can be administered by personnel with minimal training, or could be self-administered by patients, which can improve access to medication, especially those usually requiring injection. Because MNs are small (usually sub-millimetre), they can be used for precise tissue targeting. MN patches have been extensively studied to administer vaccines and drugs in preclinical work as well as in multiple clinical trials. When formulated with biodegradable polymer, MNs can enable long-acting therapies by slowly releasing drug as the MNs biodegrade. Targeted drug delivery by hollow MNs has resulted in FDA-approved products that are able to inject vaccines to skin-resident immune cells to improve immune response and to target specific parts of the eye (e.g., suprachoroidal space) for increased efficacy and avoidance of side effects in other parts of the eye. Cosmetic products based on MN technologies are already in widespread use, mostly as anti-aging agents. With extensive research coupled with FDA-approved products, MN technology promises to continue is growth in research leading to products that can benefit patients.

The bumpy road of purinergic inhibitors to clinical application in immune-mediated diseases.

Purinergic signaling plays important roles throughout the body in the regulation of organ functions during and following the disruption of homeostasis. This is also reflected by the widespread expression of two families of purinergic receptors (P1 and P2) with numerous subtypes. In the last few decades, there has been increasing evidence that purinergic signaling plays an important role in the regulation of immune functions. Mainly, signals mediated by P2 receptors have been shown to contribute to immune system-mediated pathologies. Thus, interference with P2 receptors may be a promising strategy for the modulation of immune responses. Although only a few clinical studies have been conducted in isolated entities with limited success, preclinical work suggests that the use of P2 receptor inhibitors may bear some promise in various autoimmune diseases. Despite the association of P2 receptors with several disorders from this field, the use of P2 receptor antagonists in clinical therapy is still very scarce. In this narrative review, we briefly review the involvement of the purinergic system in immunological responses and clinical studies on the effect of purinergic inhibition on autoimmune processes. We then open the aperture a bit and show some preclinical studies demonstrating a potential effect of purinergic blockade on autoimmune events. Using suramin, a non-specific purinergic inhibitor, as an example, we further show that off-target effects could be responsible for observed effects in immunological settings, which may have interesting implications. Overall, we believe that it is worthwhile to further investigate this hitherto underexplored area.

Cavitation in an antivascular cancer therapy setting

Focused ultrasound in combination with circulating microbubbles is being widely investigated as a means to promote spatially targeted drug delivery. This approach has considerable potential in oncology for a range of therapeutic agents. At sufficiently high pressures, above those typically employed in drug delivery, tumor microvessel damage can be induced to an extent that leads to perfusion shutdown and subsequent ischemic tissue necrosis. This approach is often referred to as antivascular ultrasound (AVUS), which has been shown in preclinical work to be capable of enhancing the effects of radiation therapy, antiangiogenic therapy, chemotherapy, and immunotherapy. At present, the mechanisms of AVUS are not well established. It is important to gain a more detailed understanding of the bubble–microvessel interactions that lead to perfusion shutdown, along with the cavitation signatures associated with these behaviors to enable the rational development of effective cavitation based control methods. This talk will provide a brief overview of AVUS therapy in oncology and highlight recent efforts employing two-photon microscopy, high speed optical imaging, and acoustic emission monitoring to gain insights into bubble behavior within small channels and in vivo microvessels under AVUS exposure conditions.

Robotic Bladder Autotransplantation: Preclinical Studies in Preparation for First-in-human Bladder Transplant.

Human urinary bladder transplantation has never been performed. From a technical standpoint, challenges include the complex deep pelvic vascular anatomy, limited intraoperative visualization, and high procedural complexity. In preparation for a first-in-human clinical trial, we report preclinical studies to develop the technique of robotic retrieval and autotransplantation of vascularized composite bladder allograft. Institutional Animal Care and Use Committee, Institutional Review Board, and UNOS (United Network for Organ Sharing) approvals were obtained, and IDEAL (Idea, Development, Exploration, Assessment, Long-term Study) Reporting Guidelines were followed. Robotic vascularized composite bladder allograft recovery, back-table graft preparation, and robotic autotransplantation were performed in 3 vascularized model settings: living porcine (n=3), pulsatile human cadavers (n=2), and heart-beating brain-dead deceased research human donors (n=5). Our primary objective was to develop a reproducible technique for robotic vascularized composite bladder allograft transplantation. Technical success was defined by adequate, sustained vascularized composite bladder allograft reperfusion. Secondary objectives were intraoperative parameters, including operative time, graft ischemia time, and blood loss. Successful robotic vascularized composite bladder allograft autotransplantation was achieved in 2 porcine, 1 cadaver, and 3 brain-dead research donors. In the heart-beating research donors, console time decreased with successive surgeries, and visual inspection revealed healthy revascularized autografts with prompt, global indocyanine green immunofluorescence uptake. In 1 heart-beating donor who was hemodynamically maintained for 12 hours postoperatively, reinspection confirmed excellent maintained global vascularized composite bladder allograft vascularity and bladder mucosal integrity. To our knowledge, the first preclinical experience of bladder autotransplantation in vascularized models is reported, including robotic vascularized composite bladder allograft recovery, back-table reconstruction, and autotransplantation. This experience represents the essential preclinical work required to build toward the first-in-human trial of bladder transplantation, performed under a UNOS-approved genitourinary vascularized composite bladder allograft program (NCT No. 05462561).

A NOVEL THERAPEUTICS ACCELERATOR TO SUPPORT A STRONGER BRAIN TUMOUR TREATMENT PIPELINE

Abstract AIMS Despite several encouraging pre-clinical studies, few therapeutics make it to successful human trials. There is a need for a stronger new treatment pipeline to improve outcomes for patients with brain tumours. The Tessa Jowell Brain Cancer Mission have launched the Brain Tumour Research Novel Therapeutics Accelerator (BTR- NTA) to provide bespoke advice and support to scientists and help position their therapeutic along a realistic pathway to clinical trials METHOD The BTR-NTA programme is supported by an international committee of brain tumour, pre-clinical science, and pharmaceutical experts who meet twice yearly to review up to five novel brain tumour therapeutics. Both academic and industry applicants, at any stage of pre-clinical or clinical work, can apply and submit their work to date before meeting with the BTR-NTA committee to discuss their therapeutic and progress. The committee then provides a written feedback report with advice and recommendations on next steps. The committee will also facilitate connections with industry partners and funders where possible. RESULTS It is hoped that BTR-NTA will provide benefits for the whole brain tumour community by: increasing the number of brain tumour therapeutics reaching patients; providing applicants with constructive feedback so that avoidable pitfalls can be overcome; strengthening international relationships between academics, industry, government, and regulatory bodies; and de-risking investment into brain tumour therapeutics for funders. CONCLUSIONS The first BTR-NTA meeting will be held in November 2023, with applications opening mid-2023.