Ongoing Studies Research Articles

Abstract 15011: Recent Trends in Interventional Treatment of Acute Pulmonary Embolism and Their Implications for a National Healthcare System

Introduction: Catheter-directed treatment (CDT) of acute pulmonary embolism (PE) is gaining popularity in Europe, following a trend that originated earlier in the US. For healthcare systems, reimbursement of CDT depends on their clinical benefits along with assessing the impact on hospital costs. Hypothesis-Aims: To estimate how increasing use of CDT, as reflected by prospective US data, may affect costs of PE treatment in the German hospital remuneration system (G-DRG). Methods: We analyzed data from the German nationwide inpatient sample (Federal Statistical Office), covering all hospitalizations for PE, for the years 2018-2019. For the same period, we analyzed prospective patient-level data from the multicenter US registry of the Pulmonary Embolism Response Team (PERT) Consortium. Results: During the study period, 196,167 patients were hospitalized for acute PE in Germany. Of these, 64,056 (33%) had intermediate-risk or high-risk PE, but overall ≤ 1% received CDT. Median reimbursement was є3,572 (IQR, 2,804-5,869), corresponding to є4,088 in inflation-adjusted 2022 currency ($5,678, adjusted for purchase parity power). Of 1,349 patients with intermediate- or high-risk PE included in the US registry in 2018-2019, 284 (21%) received CDT, mostly aspiration thrombectomy (63/1,349; 4.7%), ultrasound-assisted catheter thrombolysis (151; 11.2%), or non-assisted catheter thrombolysis (47; 3.5%). Median length of stay was 4.5 (2.8-8.2) days. Based on current reimbursement rates in the G-DRG, fees for PE treated with aspiration thrombectomy amount to є12,179-15,753, and for catheter-directed thrombolysis (with or without ultrasound) to є9,858-13,829. Considering, (a) the shorter length of stay; (b) the proportion of patients receiving CDT in the elevated-risk US cohort in 2018-19; and (c) that of intermediate- and high-risk PE in the German PE inpatient sample, average remuneration per patient would rise from currently є4,088 to є4,724 ($6,552). Conclusions: CDT for PE patients at elevated risk would currently lead to a 16% increase in average per-patient hospital costs in Germany. Together with clinical validation of CDT in ongoing studies, these estimates may inform decision making on reimbursement of interventional treatment options.

The Novel Checkpoint Target Lymphocyte-Activation Gene 3 Is Highly Expressed in Cutaneous Squamous Cell Carcinoma.

Lymphocyte activation-gene 3 (LAG-3) is an emerging next-generation immune checkpoint molecule. We aim to define the expression pattern of LAG-3 in cutaneous squamous cell carcinoma (cSCC) as a first step to understand the role of LAG-3 in cSCC prognosis and therapy. To define the expression pattern of LAG-3 in cSCC as a first step to understand the role of LAG-3 in cSCC prognosis and therapy. To test whether LAG-3 is expressed on cSCC infiltrating lymphocytes, we isolated CD8 + T lymphocytes from three SCC tumors using flow cytometry and performed single-cell RNA sequencing for LAG-3 and programmed cell death protein -1 (PD-1). In addition, we evaluated LAG-3 mRNA expression in formalin-fixed, paraffin-embedded tissue using NanoString technology. Single-cell RNA sequencing showed that LAG-3 is expressed more than PD-1 in CD8 + tumor infiltrating lymphocytes (50.8% vs 35.2%, respectively). Quantifying LAG-3 mRNA expression showed that compared with normal skin, LAG-3 mRNA is approximately 8 fold higher in immunocompetent associated SCC tumors and approximately 2 fold higher in transplant associated SCC tumors ( p -values <.05). In addition, LAG-3 mRNA was expressed 7.2 fold higher in T2a SCC tumors compared with normal skin ( p -value <.05). Lymphocyte activation-gene 3 is expressed on SCC infiltrating T lymphocytes at a higher percentage than PD-1. In addition, LAG-3 mRNA expression is significantly higher in SCC tumors. Ongoing studies will be performed to define its role as an immune-related biomarker and as a therapeutic target.

Renal cell carcinoma

SummaryKidney cancer accounts for 5% and 3% of all adult malignancies in men and women. Renal cell carcinoma (RCC) accounts for approximately 80% of all kidney cancer. This year’s American Society of Clinical Oncology (ASCO) Annual Meeting was held from 2–6 June 2023, in Chicago, USA. Combination therapies for advanced RCC continue to be of interest at ASCO 2023, with the presentation of updated results from some ongoing studies. There were several studies presented at ASCO looking at treatments for non clear renal cell carcinoma. Immunotherapy- based therapy regimes are now the gold standard for rare histological subtypes of RCC.

Abstract 12785: Abnormal Dynamic Remodeling of the 12-Lead Electrocardiogram is a Risk Marker for Sudden Cardiac Death

Introduction: The ECG has always been evaluated as static risk factor for sudden cardiac death (SCD). The importance of dynamic ECG remodeling has not been investigated. Hypothesis: Abnormal ECG remodeling over time is associated with increased risk of SCD. Methods: We investigated pre-SCD ECG remodeling in SCD cases from 2 ongoing population-based studies of out-of-hospital SCD in Portland, OR (discovery) and Ventura County, CA (validation). Two archived pre-SCD ECGs performed at least 1 year apart were obtained from lifetime health records. Controls were matched on geographical region, age, sex, and duration between the 2 ECG recordings. Dynamic ECG remodeling was measured as the change in a previously validated cumulative 6-variable ECG electrical risk score (ERS) between the 1st and 2nd ECG. Results: A total of 231 SCD cases (66.5±13.6 years, 61% male), and 234 controls (65.8±11.1 years, 61% male) were included in the discovery cohort, and 203 SCD cases (70.3±14.4 years, 54% male), and 203 controls (68.4±11.8 years, 54% male) in the validation cohort. The mean time between the 2 ECG recordings in SCD cases and controls was 6.0±4.0 years vs 6.2±4.5 years (discovery) and 3.7±2.6 years vs. 3.7±1.6 years (validation), respectively. In both cohorts, SCD cases compared to controls had greater dynamic ECG remodeling over time: Discovery cohort ERS change +1.06 (95% CI +0.89 to +1.24) vs. -0.05 (-0.21 to +0.11; p&lt;0.001) and validation cohort ERS change +0.87 (+0.7 to +1.04) vs. -0.11 (-0.27 to 0.05; p&lt;0.001). In a multivariate model including clinical SCD risk factors, a 1-unit increase in the ERS was an independent risk marker for SCD: Discovery cohort OR 1.89 (95% CI 1.58-2.26; p&lt;0.001), validation cohort OR 2.17 (95% CI 1.63-2.91; p&lt;0.001). Conclusions: SCD cases were observed to have abnormal dynamic ECG remodeling prior to the SCD event, not observed in matched controls. These findings warrant further investigation of dynamic ECG remodeling as a novel risk marker for SCD.

Abstract 16802: Disparities in the Prevalence of Atrial Fibrillation and Associated Risk Characteristics Among Black and White Acute Ischemic Stroke Patients Treated With Alteplase

Introduction: Earlier studies addressing racial disparity in patients with acute ischemic stroke (AIS) and atrial fibrillation included significantly lower percentage of blacks. Demographics and post-intervention risk among white vs. black patients with AIS and AF have not been reported in a community hospital setup. Methods: In this retrospective analysis, baseline demographics, procedural characteristics and one-year clinical outcomes were collected from hospital database and compared between blacks and whites who are admitted with AIS and treated with alteplase. Results: 228 and 274 black patients with AIS admitted to our hospital during 2016 to 2021 were included in the analysis. Blacks compared to whites were younger (67 ±13 vs. 74 ± 14 years) and had lower prevalence of cardioembolism (49% vs. 61%), atrial fibrillation (10% vs. 34%), coronary artery disease (17% vs. 26%), prior coronary artery disease or myocardial infarction (17% vs. 26%), depression (16% vs. 25%), dyslipidemia (53% vs. 65%), ischemic risk at admission (6.1 ± 3.6 vs. 8.2 ± 3.8), and NIHSS-obtained (11±11 vs. 14±13), but higher prevalence of small vessel disease (15% vs. 7.9%), cryptogenic stroke (20% vs. 13%), diabetes (39% vs. 25%), smokers (26% vs. 14%), drug/alcohol abuse (21% vs. 9%), prior stroke (24% vs.15%) and one year recurrent ischemic stroke (4.7 % vs. 1.3%) (p&lt;0.05 for all comparisons). Among patients with AF, blacks were of similar age compared to whites (75±13 vs. 79±14 years) and similar prevalence of cardioembolism (99% and 96%), prior myocardial infarction/coronary artery disease (26% vs. 30%) and obesity (59% vs. 48%, p=ns for all), but higher prevalence of prior stroke (48% vs. 20%), diabetes (41% vs. 22%), renal insufficiency 30% vs. 18%) and recurrent ischemic stroke (7.4% vs. 1.3%, p&lt;0.05 for all). Conclusions: Blacks patients with AIS exhibit distinct demographics and higher prevalence of recurrent ischemic events compared to whites. Despite lower prevalence, blacks with AIS have higher prevalence of comorbidities and recurrent stroke events in the presence of AF. These data provide springboard for ongoing biomarker studies at our hospital to understand the racial disparity in the underlying pathophysiology of stroke and associated outcomes in blacks.

Abstract 11635: Device and Formulation Optimization for MK2 Inhibition and Restenosis Prevention at Sites of Peripheral Angioplasty

Introduction: Peripheral angioplasty often fails due to intimal hyperplasia (IH), where contractile smooth muscle cells (SMCs) de-differentiate into proliferative, migratory, and pro-inflammatory synthetic SMCs, leading to restenosis. We have formerly shown that potentiation of an MK2 inhibitory peptide (MK2i) via delivery with poly(propyl acrylic acid) (PPAA) can block SMC phenotype switching and neointima formation in a transplant model. This work develops strategies for targeted in vivo MK2i+PPAA delivery to the site of angioplasty. Hypothesis: MK2i+PPAA can be effectively delivered into and retained within the arterial wall using either convective delivery or a coated angioplasty balloon. Methods: These studies have utilized isolated rat aortae as a model tissue. Aortae were balloon damaged and subjected to convective or coated balloon treatment. Delivery and retention of MK2i was determined with confocal microscopy and flow cytometry. MK2i activity was measured using IHC and western blots for pCREB, a downstream substrate of MK2. Results: Convective delivery studies have shown substantial MK2i delivery into the vascular wall, with pre-formed MK2i nanopolyplexes (NPs) having significantly more cell uptake than free MK2i or sequentially delivered PPAA and MK2i. Additionally, we have created a method for layered coating of MK2i+PPAA onto an angioplasty balloon that shows rapid release and successful arterial wall delivery. In efficacy studies, convective delivery of MK2i-NPs and balloon delivery of MK2i+PPAA with 50 mg/mL lactosucrose excipient significantly decreased levels of pCREB downstream of MK2 after balloon damage. Ongoing studies show that both delivery strategies block cellular proliferation in balloon damaged arteries after 7d in a flow loop bioreactor. Conclusions: Convective and coated balloon delivery show promise as strategies for on-target delivery of MK2i+PPAA to peripheral arteries after angioplasty to prevent restenosis.

Human amniotic membrane inhibits migration and invasion of muscle-invasive bladder cancer urothelial cells by downregulating the FAK/PI3K/Akt/mTOR signalling pathway

Bladder cancer is the 10th most commonly diagnosed cancer with the highest lifetime treatment costs. The human amniotic membrane (hAM) is the innermost foetal membrane that possesses a wide range of biological properties, including anti-inflammatory, antimicrobial and anticancer properties. Despite the growing number of studies, the mechanisms associated with the anticancer effects of human amniotic membrane (hAM) are poorly understood. Here, we reported that hAM preparations (homogenate and extract) inhibited the expression of the epithelial–mesenchymal transition markers N-cadherin and MMP-2 in bladder cancer urothelial cells in a dose-dependent manner, while increasing the secretion of TIMP-2. Moreover, hAM homogenate exerted its antimigratory effect by downregulating the expression of FAK and proteins involved in actin cytoskeleton reorganisation, such as cortactin and small RhoGTPases. In muscle-invasive cancer urothelial cells, hAM homogenate downregulated the PI3K/Akt/mTOR signalling pathway, the key cascade involved in promoting bladder cancer. By using normal, non-invasive papilloma and muscle-invasive cancer urothelial models, new perspectives on the anticancer effects of hAM have emerged. The results identify new sites for therapeutic intervention and are prompt encouragement for ongoing anticancer drug development studies.

Characteristics of Clinical Trials in Sepsis Registered in Clinicaltrials.Gov between September 2007 and June 2023- a Cross Sectional Analysis

Purpose: To perform an analysis of trends and characteristics of Sepsis studies registered in ClinicalTrials.gov (CTG). Methods: The clinical studies related to sepsis registered on ClinicalTrials.gov (CTG) from September 27, 2007 to June 30, 2023, were identified using Advanced Search feature in ClinicalTrials.gov. The search terms also included bloodstream infections and toxemia. Trial characteristics were assessed by relative frequency calculations. Odds ratio and 95% confidence intervals were determined for the key characteristics. Results: September 27, 2007 to June 30, 2023, a total of 3,88,228 studies were registered in CTG, Of these, around 0.68% (2,628 studies) were conducted for sepsis disease, There were 56.01% (1472 studies) interventional and 43.99% (1156) observational studies. While comparing the proportion of Interventional studies in sepsis to the overall studies registered in CTG it was found that the proportion of interventional studies in sepsis was lower (OR 0.39; 95 % CI: 0.3618 to 0.4224; P &amp;lt; 0.0001). On further assessment, the proportion of registry studies in sepsis was higher compared to overall studies registered in CTG (OR 1.73; 95 % CI: 0.3618 to 0.4224; P &amp;lt; 0.0001). In sepsis, a total of 438 studies (16.67%) received funding from the industry, while 142 studies (5.4%) were funded by the National Institute of Health (NIH) and other U.S Federal agencies (FA). Majority of the studies, 78.86% (485) were funded by all others, including individuals, universities, research organizations etc. The proportion of sepsis studies funded by the industry and the NIH and other FA were lower in comparison with the all studies registered in CTG, with the industry funding 28.91% and the NIH funding 7.36%. Of all registered studies. Out of ongoing 615 studies in sepsis, 13.82% (85) studies are funded by the industry, 8.29% (51) were funded by the NIH and other U.S Federal agencies, and 78.86% (485) studies are funded by all others. Interestingly, there was slight decrease in the proportion of ongoing studies (indicator of recent studies) funded by the industry in comparison with all registered studies for sepsis (13.82 % vs 16.67 %), however, this difference was not statistically significant (OR 0.8; 95 % CI 0.6239 to 1.0306 and P = 0.0846), However, there was a significant increase in the funding rates by NIH and FA (OR 1.58; 95 % CI 1.1350 to 2.2081 and P &amp;lt; 0.0001). Overall rates of studies funded by others remained similar between the all the sepsis studies and the ongoing sepsis studies. The proportion of early phase studies, phase 1 (OR 0.3627; 95 % CI: 0.3012 to 0.4367; P &amp;lt; 0.0001) and phase 2 (OR 0.6581; 95 % CI: 0.5800 to 0.7466; P &amp;lt; 0.0001) were significantly lower compared to all clinical studies registered in CTG. Interestingly, proportion of late- phase clinical studies, both phase 3 (OR 1.2154; 95 % CI: 1.0668 to 1.3846; P = 0.0034) and phase 4 (OR 1.1927; 95 % CI: 1.0668 to 1.3846; P = 0.0184) were significantly higher compared to all studies registered in CTG. Conclusion: There were fewer intervention trials in the context of sepsis when compared with all clinical studies registered in CTG. Additionally, funding from the industry and NIH and FA was also lower in sepsis studies compared to overall studies. Further, overtime proportion of the studies funded by industry is reduced though not statistically. Reflection of this is seen in low number of early phase studies. A detailed analysis of the studies with trends and outcomes will be discussed in the main article.

Prothrombin complex concentrate (PCC) for treatment of trauma-induced coagulopathy: systematic review and meta-analyses

BackgroundTrauma-induced coagulopathy (TIC) is common in trauma patients with major hemorrhage. Prothrombin complex concentrate (PCC) is used as a potential treatment for the correction of TIC, but the efficacy, timing, and evidence to support its use in injured patients with hemorrhage are unclear.MethodsA systematic search of published studies was performed on MEDLINE and EMBASE databases using standardized search equations. Ongoing studies were identified using clinicaltrials.gov. Studies investigating the use of PCC to treat TIC (on its own or in combination with other treatments) in adult major trauma patients were included. Studies involving pediatric patients, studies of only traumatic brain injury (TBI), and studies involving only anticoagulated patients were excluded. Primary outcomes were in-hospital mortality and venous thromboembolism (VTE). Pooled effects of PCC use were reported using random-effects model meta-analyses. Risk of bias was assessed for each study, and we used the Grading of Recommendations Assessment, Development, and Evaluation to assess the quality of evidence.ResultsAfter removing duplicates, 1745 reports were screened and nine observational studies and one randomized controlled trial (RCT) were included, with a total of 1150 patients receiving PCC. Most studies used 4-factor-PCC with a dose of 20–30U/Kg. Among observational studies, co-interventions included whole blood (n = 1), fibrinogen concentrate (n = 2), or fresh frozen plasma (n = 4). Outcomes were inconsistently reported across studies with wide variation in both measurements and time points. The eight observational studies included reported mortality with a pooled odds ratio of 0.97 [95% CI 0.56–1.69], and five reported deep venous thrombosis (DVT) with a pooled OR of 0.83 [95% CI 0.44–1.57]. When pooling the observational studies and the RCT, the OR for mortality and DVT was 0.94 [95% CI 0.60–1.45] and 1.00 [95% CI 0.64–1.55] respectively.ConclusionsAmong published studies of TIC, PCCs did not significantly reduce mortality, nor did they increase the risk of VTE. However, the potential thrombotic risk remains a concern that should be addressed in future studies. Several RCTs are currently ongoing to further explore the efficacy and safety of PCC.

Longitudinal Correlative Profiles of Responders, Nonresponders, and Those with Relapse on Treatment with Teclistamab in the Phase 1/2 MajesTEC-1 Study of Patients with Relapsed/Refractory Multiple Myeloma

Introduction: Teclistamab (tec) is the only approved B-cell maturation antigen (BCMA) × CD3 bispecific antibody with a personalized, weight-based dosing schedule for the treatment of triple-class exposed relapsed/refractory multiple myeloma (RRMM). In the phase 1/2 MajesTEC-1 study (NCT03145181/NCT04557098), tec demonstrated deep and durable responses. To better understand mechanisms of resistance and relapse in MajesTEC-1, we assessed longitudinal BCMA expression and immune profiles. Methods: Patients (N=165) with RRMM received subcutaneous tec 1.5 mg/kg weekly after step-up doses of 0.06 and 0.3 mg/kg, with the option to switch to every-other-week dosing if they achieved at least a partial response after ≥4 cycles of therapy in phase 1 or a complete response or better for ≥6 months in phase 2. Bone marrow and peripheral blood samples at baseline, on treatment, and at disease progression (PD) were analyzed by flow cytometry for BCMA expression and immune cell populations. Bone marrow aspirates were analyzed by cytometry by time of flight (CyTOF) for immune cell populations. Soluble BCMA (sBCMA) was analyzed in serum samples by electrochemiluminescence ligand binding. Results: Patients who responded to tec had a greater recovery of CD3+ T cells in the periphery and bone marrow during the first treatment cycles, which was sustained over time compared with nonresponders. Greater activation during the first cycle was observed in responders, indicated by a transient increase in CD38 on CD8+ T cells. In contrast, an exhausted T-cell phenotype in the blood and bone marrow was observed longitudinally in nonresponders vs responders, indicated by increasing and sustained levels of activation and exhaustion markers including persistence of CD38+ T cells and expression of LAG-3, PD-1 (Figure 1A), PD-1/LAG-3, and PD-1/TIM-3 on CD4+ or CD8+ T cells. Higher levels of immunosuppressive regulatory T cells (Tregs), including CD38+ Tregs, were also sustained in the periphery in nonresponders compared with responders. BCMA expression and sBCMA levels were assessed in a subset of patients who initially responded then relapsed on tec, who had matched baseline and end-of-treatment/PD samples. There were no significant changes in the frequency of BCMA+ plasma cells, BCMA receptor density, or sBCMA levels at PD relative to baseline, suggesting BCMA loss was not a mechanism of relapse in these patients. In contrast, higher proportions of peripheral CD4+ and/or CD8+ T cells expressing CD38, TIM-3, PD-1, and PD-1/TIM-3 were observed at relapse vs baseline. Analysis of bone marrow progression samples using CyTOF in patients who relapsed showed significantly lower CD28 expression on CD8+ T cells, significantly higher expression of exhaustion markers (CD38, PD-1, TIM-3, EOMES, and TOX) and perforin on CD4+ and/or CD8+ T cells, a higher frequency of CD4+ and CD8+ T cells expressing TIM-3 and co-expressing CD38/TOX, PD-1/TIM-3, and TOX/TIM-3 (Figure 1B), and significantly higher CD38 expression on T-cell receptor gamma delta T cells, at PD than at baseline. PD-1 and TIGIT expression was also significantly higher at PD than at baseline across CD25hiCD127dim and/or CD25hiCD127dimFoxP3+ Tregs in patients who relapsed. Conclusions: Patients responding to tec exhibited a differential immune profile in early cycles compared with nonresponders, with greater transient T-cell activation. In contrast, an exhausted T-cell phenotype was sustained in nonresponders during the first 2 treatment cycles. While mutations in BCMA cannot be excluded, we did not detect BCMA loss as a mechanism of relapse, but observed a dysfunctional immune phenotype at PD, with increased T-cell exhaustion and higher frequency of gamma delta T cells and immunosuppressive Tregs. These results suggest the importance of immune fitness and T-cell function in achieving and maintaining a response to tec. Ongoing studies will evaluate these correlatives in earlier treatment lines, where patients may have more favorable immune profiles.

Thymidylate Synthase Inhibition with Capped F10 Is Effective in TP53 Null Acute Myeloid Leukemia (AML)

Acute myeloid leukemia (AML) is an aggressive cancer of the myeloid linage characterized by progressive marrow failure and death. Responses to therapy are between 50 and 80% but relapse is common and salvage rates once relapsed are low. The risk of relapse is greatly influenced by specific somatic mutations. One of the highest risk mutations in AML involves loss of the tumor suppressor TP53. Patients with AML harboring loss of TP53 function have a dismal prognosis and there is a clear need for new approaches. To identify novel agents with activity against TP53 null AML, a TP53 null and WT isogenic cell line pair was generated using CRISPR/CAS9 in a genetically defined murine AML cell line. Clonal isolates were generated by serial dilution and loss of detectable TP53 was confirmed by Western blotting. A high throughput screen of currently FDA approved agents was performed on these cell lines. When agents were ranked by equal cytotoxicity against TP53 null and WT AML cells 20% (6/30) of the top 30 agents either directly targeted thymidylate synthase or its substrates via inhibition of the folate cycle. This finding suggests that thymidylate synthase (TS) is a key enzyme in TP53 null AML cells. To confirm and extend these results, an additional murine AML TP53 null/WT cell line pair was generated. Chemotherapy resistance was confirmed in the TP53 null cell lines when treated with doxorubicin (Figure 1). Capped F10 (CF10) is a second generation fluoropyrimidine oligonucleotide that is a highly potent inhibitor of TS. In contrast to doxorubicin, CF10 treatment resulted in significant cell death when dosed in the low picomolar range with minimal differences between the null and WT cell lines (Figure 1). To extend this result TP53 null cells were injected into syngeneic recipients and upon engraftment mice were treated with CF10 at 300mg/kg twice weekly or doxorubicin 3mg/kg and cytarabine at 100 mg/kg daily for 5 days. CF10 provided a significant survival benefit in the TP53 null AML in this highly aggressive model where doxorubicin and cytarabine did not (p=0.0057, Figure 2). To confirm a TP53 independent mechanism, competition assays were performed with CAS9 expressing AML cells partially infected with a vector expressing an sgRNA targeting TP53 and containing a GFP reporter. When the mixed population was treated with a titration of doxorubicin there was a highly significant increase in GFP+ cells indicating the involvement of TP53 in doxorubicin mediated cell death. In contrast when cells were treated with a titration of CF10 there was a no significant increase in GFP+ population consistent with CF10 inducing a TP53 independent mechanism of cell death. For CF10 to of use clinically there must be an acceptable therapeutic window. Fluoropyrimidine based therapy can be myelosuppressive especially in patients with low dihydropyrimidine dehydrogenase (DPD) activity. To assess the tolerability of CF10, healthy WAG/RAJ rats were treated with the DPD inhibitor ethynyl uracil at 2mg/kg to mimic DPD deficiency. Animals were then treated with repeated doses of either 141 mg/kg CF10 or the molar equivalent of 50 mg/kg 5-flurouracil (5-FU) and weight determined. In contrast to 5-FU, CF10 treated animals maintained their weight compared to controls (data not shown). Taken together these data suggest that TS is a viable target in TP53 null AML and CF10 is well tolerated and provides a survival benefit in a highly aggressive, chemotherapy resistant model. Ongoing studies will explore the mechanisms involved in CF10 induced cell death.

High Success Rates Utilizing Local Standard of Care Cytogenetic Testing for Risk Stratified Frontline Therapy for Newly Diagnosed Multiple Myeloma: Experience from the Ukmra RADAR Study

Background. Recent studies in genetically high risk (HR) multiple myeloma (MM) such as the UKMRA OPTIMUM study have reported high response rates with intensive induction and post-ASCT consolidation and maintenance strategies, providing the rationale for stratified therapy based on genetic risk. Whilst centralised testing in clinical trials offers ready uniformity and quality control, the use of local laboratories for testing within a clinical trial paves the way for genetic testing to be available for all patients, facilitating risk stratified therapy in routine care. We designed the UKMRA RADAR study to use local laboratories for cytogenetic (CGN) testing to assign treatment according to genetic risk. Methods. RADAR is an ongoing national, multi-center, risk-adapted, response-guided multi-arm, multi-stage (MAMS) phase II/III trial for patients with newly diagnosed MM eligible for ASCT. Patients receive induction with RCyBorD (lenalidomide, cyclophosphamide, bortezomib, dexamethasone), followed by high-dose melphalan and stem cell rescue. Post ASCT, HR patients are randomised to receive isatuximab or not, alongside RBorD consolidation and R maintenance whilst standard risk (SR) patients receive MRD response guided regimens. A protocol amendment (2022) included isatuximab throughout the HR pathway, removing the post-ASCT randomisation. Risk assessment, failure rates and turnaround time are overseen by a genomics working group (GWG) including clinical and specialist cytogenetic representation. Genetic risk testing is undertaken in local genetic labs on CD138 selected bone marrow (BM) plasma cells. HR is assigned based on the presence of at least two of: t(4;14), t(14;16), t(14:20), del(17p), del(1p) and gain(1q). Clone size cut-offs are 10% for IgH translocations and 20% for copy number changes. CGN results from local laboratories are centrally reviewed to assign risk in real-time. Indeterminate cases are discussed by the GWG. Repeat testing is mandated if the initial sample fails to yield a definitive risk result for risk. Subjects with no definitive result despite 2 samples are treated as SR. Results. As of 19 th June 2023, the trial has recruited 472 patients from 72 sites across the UK. Median age is 61 years, 59.3% male, 79.9% white, 11.7% other ethnicity and 8.5% missing ethnicity data. 25 local laboratories were used for CGN testing. Of those with CGN results 354/453 patients (78.1%) were assigned standard risk, 84 (18.5%) high risk and 15 (3.3%) risk category undefined. Risk stratification is ongoing for 19 patients. Of the high risk lesions gain(1q) was most prevalent (35%), followed by t(4;14)(10.6%), del(17p)(8.7%), del(1p32)(8.7%), t(14;16)(3.8%) and t(14;20)(0.8%). In 376/453 patients (83.0%), risk was assigned from a first BM sample. 77 (17.0%) patients required a second sample; the success rate of repeat samples is 71.4%, 22 (4.9%) patients were unable to be risk-stratified despite 2 samples. Figure 1 shows the success rate of first and repeat tests. The overall median turnaround time (from D1 cycle 1 until site notified of result) was 11 days (IQR 0 to 21). Sites were sent risk status prior to cycle 1 in 99 (21.9%) patients , 285 (62.9%) prior to cycle 2 and 341 (75.3%) cycle 3. Note this is calculated where treatment data is available. Conclusion. The RADAR study demonstrates that risk-stratified treatment approaches are feasible in the context of a national phase II/III clinical trial with the use of local laboratories for risk assignment. The vast majority of patients have been successfully allocated to a risk-adapted treatment pathway following a first FISH test (83.0%). Protocol v4 requires time-critical risk-stratification for addition of isatuximab to induction treatment from cycle 2 in high-risk patients, and this has proven to be achievable in almost 2/3 of patients with over ¾ by the end of cycle 2. The RADAR study also enables hospitals across the UK to access CGN testing via a network model of laboratories with standardization of testing and clinically relevant turnaround times. This model is applicable internationally in both public and privately funded healthcare systems and supports the infrastructure for accessible genetic risk stratification for all newly diagnosed MM patients. Ongoing studies within the RADAR trial are evaluating novel and clinically deliverable risk stratification protocols to inform risk stratified approaches in newly diagnosed MM.

Chimeric Antigen Receptor T Cells Targeting LILRB4, an Immunoreceptor Mediating T-Cell Suppression, Are Potently Effective in Multiple Myeloma

Significant progress has been made in the treatment of multiple myeloma (MM) however, no strategy to date has proven curative and relapse invariably occurs. We recently developed an integrated pipeline to identify biologically and therapeutically relevant targets for MM immunotherapy by using Mass-Spectrometry analyses from seven MM cell lines and RNA-seq data from 900+ patients. Starting from 4,000+ candidates, ILT3 aka LILRB4, an immunoreceptor tyrosine-based inhibition motif-containing receptor emerged as a promising candidate target: it has absent/low expression in most normal tissues and high and frequent expression in many primary patient samples we used for validation, similarly to BCMA (Di Meo et al., Cell Reports Medicine 2023). We sought to examine its functional role in MM and develop Chimeric Antigen Receptor (CAR) T-cells targeting LILRB4. Interrogating RNA-seq data from MM patients, LILRB4 expression results higher in patients with high-risk cytogenetics such as t:(14;16), t:(14;20), and t:(8;14) translocations compared to patients with no genomic abnormalities. Investigating function, we find no difference between cell proliferation of U266/KMS11/OPM2 genetically depleted of LILRB4 (KO) compared to controls by measuring cell growth in vitro for 120 hours and MTS assays. Similarly, when KO and control cells are seeded in plastic chambers containing a porous membrane and serum-free medium, both KO and control cells are attracted to the lower chambers containing FBS, suggesting that LILRB4 is also not required for MM cell migration. Despite these initial observations, RNAseq data from LILRB4 KO and overexpressing (OE) U266 MM cells identify ~3,000 differentially expressed genes compared to controls. Thus, we next investigate potential extrinsic effects on T-cell proliferation by co-culturing T-cells from healthy donors with LILRB4 KO or OE U266 MM cells (T:E ratio 1:2). T cells are previously activated with anti-CD3/CD28-coated beads and IL-2. 5 days after activation, T cells and LILRB4 KO U266 MM are seeded in the same well plate. After 48h of co-culture, T-cell proliferation is decreased (-30%) in the presence of LILRB4 U266 OE MM cells, whereas it is increased (+50%) when the co-culture includes LILRB4 U266 KO cells. CD25 expression is also reduced by 20% on T cells co-cultured with LILRB4 OE cells and increased by the same extent when T cells are co-cultured with LILRB4 KO cells, suggesting a role as a negative immune receptor. This effect seems dependent on the direct cell-cell interaction given that when T and MM cells are separated by a transwell in co-culture, no effect on the T cell proliferation is observed. Finally, we develop multiple Chimeric Antigen Receptor (CAR) constructs targeting LILRB4, by using different single-chain variable fragments (scFVs) and rearranging the variable regions of the heavy (VH) and light (VL) regions. We used lentiviral LILRB4 scFV-41BB-CD3zeta vectors. LILRB4 CAR T-cells specifically kill U266, H929 and KMS11 MM cell lines in vitro with cytotoxicity ranging from 30 to 80% depending on the construct (T:E ratio 1:10). Equal efficacy is observed in U266 cells resistant to Bortezomib and BCMA KO U266cells. No effect on LILRB4 null cells is noted. These data suggest that LILRB4 is a negative immune receptor that is active on malignant plasma cells mediating T-cell suppression, similar to previously described data in leukemia (Deng et al., Nature 2018). Our work supports the rationale development of ILT3 CAR-T cell therapy as a viable therapeutic approach for MM patients especially high-risk patients. Ongoing studies are investigating the functional role of LILRB4 in mediating the cross-talk between MM ad immune cells.

Experiences with Delayed Diagnosis for Older Adults with SCD: “Looking Back It All Makes Sense”

Background Improvements in care over the last 50 years have extended the median lifespan of people living with sickle cell disease (SCD) from 14 years to as long as 61 years in cohorts from academic centers. Older adults with SCD have decades of unique experiences; from navigating their initial SCD diagnosis to coping with complications of SCD as an older adult. The purpose of this study was to describe the impact of delayed diagnosis of SCD on older adults (age ≥ 50 years). Methods We enrolled 19 older adults with SCD from a single comprehensive sickle cell center. We conducted semi-structured interviews by phone or in person. A single interviewer asked open-ended questions about the participant's experiences living with SCD during childhood and adulthood. Participants described their experiences being diagnosed with SCD, how they navigated healthcare systems, and their strategies to manage and prevent pain crises. Interviews were audio-recorded and transcribed verbatim. We used conventional content analysis to code the transcripts. Results The mean age of participants was 58 years (range 50-71). Over half (53%) had HbSS genotype, 37% had HbSC, and 11% had HbSβ+-thalassemia. Almost half (45%) were hospitalized and 63% had an emergency room visit in the last year. Most participants (84%) were on short-acting and 37% were on long-acting opiates. The majority (58%) reported current use of hydroxyurea. In theme 1, many participants described growing up with an unknown illness prior to their SCD diagnosis. Participants discussed having pain episodes but not knowing they had SCD. Their symptoms were often dismissed as “growing pains” or “bone pain”. Participants stated how not having a diagnosis as a child was hard on their parents and they had to see various specialists or travel to academic centers to finally receive a diagnosis. Participants shared stories about how they were diagnosed with SCD (Figure 1). Participants discussed positive aspects of not knowing they had SCD. They described how their parents allowed them to do activities and pursue ambitions without limitations since they were unaware they had a life-limiting condition. In theme 2, participants described the impact of having providers with limited knowledge about SCD. They described challenges having limited access to treatments for pain before and after their diagnosis of SCD. They discussed how doctors were not as empathetic in the past as they are now. Doctors were more likely to stigmatize them as being “drug-seekers”. Some participants stated they still felt “hurt” by how they were stigmatized and remain “very afraid” to seek care in their local hospitals for fear the providers were not knowledgeable about SCD. They recalled difficulties accessing pain medications as children and young adults due to providers' reluctance to treat them. These challenges led them to develop alternative coping strategies aside from opioid medications. Many older adults used therapies such as warm compresses, heating pads, prayer, meditation, healthy eating, proper hydration, and avoiding factors like overexertion and weather extremes. Later in life, these older adults also were more inclined to seek out alternative therapies such as acupuncture, physical therapy, aquatic therapy, and biofeedback to manage their pain. Conclusion Older adults in this study lived for many years not knowing the cause of their symptoms and how best to treat their illness. While delayed diagnosis of SCD is now rare in countries with universal newborn screening, there remains many undiagnosed people with SCD around the world. This delayed diagnosis leads to unnecessary pain and suffering for patients and their families. Participants expressed their frustration with the medical community for their lack of knowledge as to the source of their pain, their dismissive nature of said pain, and their undertreatment of these symptoms. This study highlighted the need for providers to really listen to patients and their families and take their symptoms seriously. There is much to be learned from older adults about self-management of SCD and much work to be done to repair relationships that have been broken due to mistreatment of this population by healthcare providers. In ongoing studies, we partner with older adults with SCD to increase knowledge and develop management strategies, similar to those used in geriatrics, to improve care for older adults with SCD.

Targeting Distinct Epitopes of FCRL5 Using CAR-T Generated with Sequences Derived from Newly Identified FCRL5 Humanized Antibodies Resulted in Potent Anti-Myeloma Activity in Vitro and In Vivo

Despite improvement in treatment options for multiple myeloma (MM) patients, including targeted immunotherapies, MM remains incurable. Given the potential for a single CAR-T treatment to generate long-term remission and the optimal expression profile of FCLR5 (expressed on most MM with limited expression on normal cells), we generated new FCLR5 antibodies and obtained single chain variable fragment (scFv) sequences to develop FCRL5 CAR-Ts. The extracellular domain of FCRL5 has 9 Ig domain subunits (D1-9). We immunized ATX mice (Alloy Therapeutics), genetically engineered to produce humanized antibodies, with human membrane proximal D9 of FCRL5 since cell surface membrane proximal epitopes enhance membrane synapse interactions. Engaging D1-8 could also be effective for targeting FCRL5 by CAR-T, so we immunized mice with full-length FCRL5 and screened for clones binding to D1-8. Because we found FCRL5 expression on primary MM cells was universal but modest and variable, we prioritized screening the 40 antibodies we identified (23 bound to D9 and 17 bound to D1-8) for binding to MOLP-2 cells, known to endogenously express low levels of FCRL5 using flow cytometry. The thirteen best binders (highest mean fluorescence intensity; MFI) were next tested for binding to the MM.1S cell line modified to overexpress FCRL5. All 13 antibodies bound with high efficiency. We generated CAR-T (4-1BB CD3z) for the top seven clones (four D9; three D1-8 binders) and assessed cytolytic activity on MOLP-2 target cells modified to express luciferase (MOLP2-CG) and OPM2 cells modified to express luciferase and high levels of full length human FCRL5 (OPM2-CG-FCRL5). Effector (E) CAR-T cells were incubated with target (T) cells at a range of E:T ratios for 48 hours and bioluminescence (BLI) was used to determine cytolytic activity. Most clones showed high killing activity. We tested clones 977 (D1-8), 942, 947 and 980 (D9 binders) for in vivo studies due to highest performance in vitro. NSG mice were engrafted with 1x10 6 OPM2-CG-FCRL5 cells i.v. into tail veins and treated 14 days later i.v. with 2x10 6 FCRL5 CAR-T or BCMA CAR-T, the current lead CAR-T target in MM as an independent positive control. We found significant extension of survival compared to controls (Kaplan Meier, 942: P&amp;lt;.001, 947 P&amp;lt;.003, 977 P&amp;lt;.0002, 980 P&amp;lt;.008 BCMA P&amp;lt;.001) and reduced tumor burden (longitudinal BLI) in all CAR-T treated mice compared to untreated and mice treated with non-transduced T cell controls in this aggressive tumor model ( Figure 1). Co-targeting BCMA and FCRL5 may prevent antigen escape since it targets cells expressing either or both FCRL5 and BCMA. We generated a bi-targeted CAR targeting both BCMA and FCRL5 (clone D1-8 977). To confirm each scFv was active, we ran in vitro killing assays using BCMA knockout (KO)-OPM2-CG FCRL5 (BCMA-FCRL5+), OPM2-CG (BCMA+ FCLR5-) targets and found killing of each cell type by our bi-targeted CAR-T, confirming both scFvs were active. BCMA-FCRL5 CAR-T killed OPM2-CG FCRL5 and MOLP-2-CG cells (both BCMA+FCRL5+) but not BCMA KO OPM2 (BCMA-FCLR5-) cells. The best performing BCMA CAR-T in the clinic targets two BCMA epitopes- our scFv's targeting different domains of FCLR5 opens the opportunity for bi-targeting the FCLR5 protein itself. We generated an FCRL5-FCRL5 bi-targeted CAR-T using clone 977 (D1-8) and 980 (D9) and demonstrate activity of this CAR-T on OPM2-CG-FCRL5 cells; ongoing studies will determine if each scFv is active and whether in vivo activity is superior to single epitope targeting in vivo. Finally, blocking studies of the D9 antibodies showed potential for multiple epitopes within that domain which may facilitate co-targeting D9. We tested four FCRL5 CAR-T (4-1BB CD3z) predicted to target distinct D9 epitopes and observed high cytolytic activity in killing assays using OPM2-CG-FCRL5 target cells. We tested each in vivo by injecting NSG mice in with OPM2-CG and 18 days later treated with 2x10 6 FCRL5 CAR-T. As above, we observed significant extension of survival and reduced tumor burden compared to controls. Future studies will assess feasibility of D9 co-targeting. Together, our data demonstrate efficacy of FCRL5 CAR-T using novel sequences targeting FCLR5 both within and outside of D9 and support development of an FCRL5 CAR-T clinical candidate for use in relapsed/refractory MM patients.

Modeling Gilteritinib Resistance By Enforced cxcl8a Expression in Zebrafish HSPCs

In Acute Myeloid Leukemia (AML), the FMS-like tyrosine kinase III Internal Tandem Duplication (FLT3-ITD) is common and increases the risk of relapse which is only partially mitigated by tyrosine kinase inhibitor (TKI) therapy. Gilteritinib is the current mainstay for treating relapsed FLT3-ITD AML, but therapy is non-curative, and the duration of remission is transient. Mutations in the RAS pathway are known to promote gilteritinib resistance, however non-mutational mechanisms of resistance remain under-explored and could provide new avenues for combination therapy. Our previous work has associated AML cell-autonomous expression of chemokines such as CXCL8 (IL-8) as a mechanism of resistance to gilteritinib therapy. Here we enforce expression of cxcl8a ( cxcl8, ENSDARG00000104795) by hematopoietic stem and progenitor cells (HSPCs) in transgenic zebrafish embryos using a defined Runx1 enhancer element. In the absence of gilteritinib, Runx1:cxcl8(+) animals had greater numbers of HPSCs compared to Runx1:cxcl8(-) animals (4.0 vs 2.1, p = 0.04). When treated with gilteritinib at 100 nM from 48-72 hours post-fertilization (hpf), HSPC numbers were decreased in both groups; however, Runx1:cxcl8(+) animals still had more HSPCs compared to Runx1:cxcl8(-) animals (1.6 vs 0.4, p = 0.02). This suggests that cxcl8 may act as a resistance factor for gilteritinib in vivo and that understanding regulators of cxcl8 expression may provide insight into gilteritinib resistance. Reanalysis of published single cell RNA-seq data in AML patients revealed an association between expression of CXCL8 and BTK and BMX but not other Tec-family kinases such as ITK and TEC itself. Zebrafish do not have an annotated BMX gene; BTK is expressed in HSPCs and neutrophils but not the erythroid lineage. CHMFL is a specific and potent inhibitor of human BMX with an unknown spectrum of activity in zebrafish. In Runx1:cxcl8(-) Runx1:GFP zebrafish, CHMFL treatment reduced HSPC numbers, but this was not rescued in Runx1:cxcl8(+) animals (untreated vs treated, cxcl8(-): 2.9 vs 1.5, p = 0.02, untreated vs treated, cxcl8(+): 4.0 vs 1.4, p = 0.0006). This contrasts with our prior work with the non-specific Tec family kinase inhibitor, ibrutinib, where HSPC numbers were rescued in Runx1:cxcl8(+) animals. Ongoing studies are focused on understanding the targets of ibrutinib and CHMFL in the zebrafish and how these pathways may affect gilteritinib resistance.

Cryopreservation Changes the Immune Effector Cell Composition of Peripheral Blood Stem Cell Grafts: An Analysis from the DKMS and NMDP Graft Composition Study

The impact of the cellular composition of PBSC grafts on the immune reconstitution as well as graft-versus-host and graft-versus-leukemia effects after allogeneic hematopoietic cell transplantation (alloHCT) is only poorly understood. DKMS and NMDP joined forces to comprehensively characterize the cellular composition of 2,000 PBSC products using a 34-color flow cytometry panel to define immune effector cell subsets of interest and hematopoietic stem cells. In parallel, we have created a biobank of unstimulated donor PBMC and PBSC graft samples for future analyses. As part of this larger effort, we investigated the effect of cryopreservation on the cellular composition of PBSC products. Cryopreservation of PBSC grafts was used frequently during the COVID19-pandemic and facilitated more flexible timelines for transplantation. Still, one third of the US patients and 15% of the German patients continue to receive cryopreserved PBSC grafts. Ongoing studies are investigating the impact of cryopreservation on immune-effector cell function and the outcome of hematopoietic stem cell transplantation. Several observational studies have reported delayed neutrophil and platelet engraftment with cryopreserved products. More recently, less chronic GVHD was reported for patients who had received cryopreserved PBSC products. Cryopreservation affects the cellular composition and hence has the potential to change immune effector cell functions of the graft, but available data on the alterations of cell population frequencies are scarce. We analyzed the cellular composition of 20 fresh grafts and compared them to their frozen and thawed counterparts. Cryopreservation of product samples followed routine clinical procedures using a 10% DMSO containing cryo-medium and a controlled rate freezing. After thawing at 37°C, cells were washed, stained with a 34-parameter phenotyping panel, fixed and analyzed immediately on a full spectrum flow cytometer. Following spectral unmixing and manual spillover correction, proportions of immune cells were assessed using manual gating. First, we investigated the relative frequencies of 10 non-overlapping individual cell populations relative to viable CD45+ cells, including several lymphocyte populations, monocytes, granulocytes, hematopoietic stem cells (HSC) and dendritic cells. Granulocytes showed the largest decrease after cryopreservation and thawing (18.2 % vs 3.5 %; p&amp;lt;0.001). The loss of granulocytes results in an increase of relative frequencies of many other immune cell populations including monocytes (35.4 % vs 44.7 %; p=0.018), B cells (5.8 % vs 9.6 %; p=0.006), and HSC (1.0 % vs 1.5 %; p&amp;lt;0.001). We therefore also calculated changes relative to the viable non-granulocyte cells. By doing so, a significant relative increase of cells was observed for HSC (1.2 % vs 1.6 %; p&amp;lt;0.001), whereas monocyte and B cell frequencies changed only marginally. This suggests that HSC are more resilient to freezing than other cell types. When comparing the proportions of 40 other immune cell (sub-) populations relative to their parent population in the gating hierarchy, we found a profound and significant decrease of the relative frequencies for CD16+ myeloid dendritic cells (mDCs, 68 % vs 30 % of all mDCs; p&amp;lt;0.001) and granulocytic myeloid derived suppressor cells (gMDSCs, 2 % vs 1 % of all MDSCs; p&amp;lt;0.001). In an independent set of analyses performed at Roswell Park Comprehensive Cancer Center, Buffalo, comparable results were obtained with a significant increase of HSC, a significant decrease of CD16+ mDCs, and a trend of a decrease of gMDSCs. As CD16+ mDCs may have a role in the development of chronic GVHD after alloHCT, this decrease may be meaningful. In summary, we found that cryopreservation of PBSC allografts resulted in a decreased relative frequency of granulocytes, and CD16+ mDCs, as well as an increased relative frequency of HSCs. The results provide some insight into the impact of cryopreservation on PBSC graft composition and warrant further efforts to explore the potential mechanisms for the differential outcomes observed using fresh vs cryopreserved PBSCs.

Vivovec™ Surface-Engineered Lentiviral Particles Mediate In Vivo CAR T Generation with Potent and Highly Durable Activity in Non-Human Primates and Tumor-Bearing Humanized Mice

Introduction: Ex-vivo generated autologous CAR T cell therapy has transformed the treatment of hematologic malignancies, driving deep and durable responses in patients refractory to conventional therapies. However, multiple challenges including complex manufacturing, high cost, and toxic pre-conditioning regimens limits access to these therapies. To overcome these challenges, a novel lentiviral vector platform, VivoVec™, is being developed. The VivoVec™ platform comprises lentiviral particles surface-engineered with a multi-domain fusion (MDF) protein assembled from T cell activating and costimulatory ligands along with a CAR transgene payload, designed to provide an off-the-shelf solution for generation of CAR T cells in vivo. Here we have evaluated the performance of VivoVec™ particles in multiple preclinical models. Following a single VivoVec™ particle injection, CAR T cells are efficiently generated in vivo and expand in response to cognate antigen, eradicate target antigen expressing cells, and form CAR T cell memory populations, in the absence of lymphodepleting chemotherapy or exogenous supportive cytokines. Methods: Mice (NSG MHCI/II knockout): Mice were engrafted with CD19+ Nalm6 tumors and resting human PBMCs, followed by intraperitoneal administration of varying doses of VivoVec™ carrying an aCD19 CAR payload. Absolute CAR T-cell numbers in peripheral blood were quantified by flow cytometry, and Nalm6 tumor burden was assessed by bioluminescence imaging. Non-Human Primates ( Macaca nemestrina): VivoVec™ surface engineered with NHP-specific surrogate MDF and carrying a human/NHP cross-reactive aCD20 CAR payload. VivoVec™ was administered via intranodal injection to 3 animals at doses of 0.77e8/kg TU in 1 animal and 2.50 e8/kg TU in 2 animals. aCD20 CAR T expansion and B-cell depletion were continuously evaluated by flow cytometry approximately weekly over ~4 months. Body weight, body temperature, neurological exams, serum chemistry panels, and complete blood counts were assessed weekly for the duration of the study. Results: VivoVec™ cultured with resting PBMCs selectively bind T cells and some NK cells in an MDF surface engineering-dependent manner, resulting in T cell activation, transduction, and CAR expression. The resultant CAR T cells demonstrate polyfunctional activity through antigen-specific tumor cell killing of Nalm6 tumor cells, cytokine secretion, and proliferation in response to serial antigen exposure. VivoVec™ administration led to dose-dependent generation of CAR T cells in Nalm6-tumor bearing humanized NSG MHCI/II knockout mice. In vivo generated CAR T cells expanded and mediated potent and durable anti-Nalm6 tumor activity. Non-Human Primates: In the two animals receiving 2.50e8 TU/kg of particles, aCD20 CAR T cells were detected in peripheral blood by flow cytometry with an early peak expansion by day 10 (representing 25.9% or 61.3% of total T cells, respectively). In the first animal, B-cell aplasia was observed from day 7 to day 42, when a small recovering B-cell population was detectable. This was closely followed by a secondary CAR T-cell expansion peaking at day 51: the CAR T cell population represented 43.2% of T cells present in the blood and resulted in recurrent B-cell aplasia sustained through day 70. Persistent B cell aplasia in the second animal receiving the 2.50e8 TU/kg dose began at day 7 and continues to be monitored. VivoVec™ was well-tolerated with no evidence of toxicity associated within, or shortly after the period of administration. Mild CRS was observed in association with both early and late CAR T cell expansions. Conclusions: VivoVec™ with MDF surface engineering enables potent and specific generation of functional CAR T cells in vivo in multiple preclinical models, including immune competent NHPs. Importantly, the generation and expansion of CAR T cells occurs in the absence of lymphodepleting chemotherapy, or supportive exogenous cytokines, thus the MDF engineering could impart properties consistent with a natural T cell effector response followed by formation of functional memory T cells. VivoVec™ represents a novel therapeutic platform in oncology that has the potential to overcome many of the challenges associated with the current ex vivo CAR T cell approaches. Additional data from ongoing NHP studies will be provided at the time of presentation.

Kme-0584, a Highly Potent IRAK1/IRAK4/panFLT3 Inhibitor, Is a Promising Clinical Candidate for Hypomethylating Agent Plus Venetoclax Resistant AML/MDS Patients

Leukemic cells exhibit dysregulation of innate immune signaling pathways upon diagnosis, and these pathways become further activated in drug resistance. The Interleukin-1 receptor associated kinase 1 and 4 (IRAK1/IRAK4) kinase complex is part of a critical signaling node that becomes activated in these dysregulated pathways (reviewed in J Bennett and DT Starczynowski, Curr Opin Hematol 2022). IRAK4 inhibitors are currently under investigation for the treatment of Acute Myeloid Leukemia (AML) and Myelodysplastic Syndrome (MDS) and have shown encouraging, though modest responses in clinical studies. We have recently demonstrated that limited responses to IRAK4 inhibitors in the leukemic setting can be explained by a compensated upregulation and activation of IRAK1, and the need to inhibit both IRAK1 and IRAK4 to achieve maximal therapeutic efficacy (JR Bennett et. al., Blood 2023 https://doi.org/10.1182/blood.2022018718). KME-0584 is a highly potent IRAK1/IRAK4/panFLT3 inhibitor that exhibits superior potency and therapeutic efficacy vs. IRAK4 inhibitor compounds in the FLT3 WT as well as the FLT3 mutant setting. KME-0584 exhibits &amp;gt;100-fold selectivity vs. 89% of the Kinome as measured in the Reaction Biology 374 kinase panel, with an IC50 of 23, &amp;lt;1.29, and &amp;lt;0.5nM at IRAK1, IRAK4, and FLT3 respectively. Higher kinase selectivity is seen in cell-based assays vs. biochemical assays, where high potency kinase antagonist activity is also observed against PHKg1, PDGFRβ, RET, CLK1, and CLK4. While IRAK4-selective compounds fully antagonize NF-κB signaling through the TLR pathway, KME-0584 completely inhibits NF-κB signaling through both the TLR and IL1-receptor pathways, indicating complete inhibition of multiple receptor pathways converging on NF-κB requires both IRAK1 and IRAK4 antagonism. In primary patient cell lines from FLT3 wildtype (WT) patients, KME-0584 inhibits leukemia stem cell progenitor function as measured by the colony formation assay in methylcellulose with higher potency than IRAK4 inhibitor compounds that lack IRAK1 activity such as CA-4948 (Emavusertib). This activity is independent of patient mutational status. Importantly, high potency in primary patient cell lines is observed independent of the presence of U2AF1 or SF3B1 spliceosome mutations known to drive expression of the activated IRAK4 Long isoform of IRAK4 (Fig 1), implying that the IRAK1/4/panFLT3 inhibitor KME-0584 will have activity across a broader patient population than an IRAK4 inhibitor. We have previously described an IRAK1/4 gene signature that is highly expressed in patients with myelomonocytic subtypes (M4 FAB) of adult and pediatric AML and with an antecedent MDS (JR Bennett et. al., Blood 2023 https://doi.org/10.1182/blood.2022018718). The IRAK1/4 gene signature is reversed after treatment of THP1 cells for 24 hours with KME-0584. Given that monocytic-like subtypes of AML are resistant to Venetoclax plus Azacitidine (VEN/AZA) (S Pei et. al. Cancer Discovery, 2020), these data suggest that KME-0584 has the potential to be effective across a broader range of relapsed/refractory AML/MDS patients than previously examined IRAK4 inhibitors. KME-0584 exhibits superior potency and efficacy to gilteritinib in the FLT3-ITD (D835Y) xenograft model after QD oral dosing, with sufficient PK and oral bioavailability across multiple species to support QD or BID dosing in the clinic. KME-0584 does not inhibit any of the major or minor cytochrome P450 enzymes at anticipated clinical concentrations and early indication from ongoing GLP toxicology studies suggest that it could be safely administered in humans. A clinical study of KME-0584 in relapsed/refractory AML and HR-MDS is currently planned to start in 1H 2024.

Title: MLLT1 Is Required to Maintain Normal B-Lineage Hematopoiesis and Mitochondrial Function

MLLT1 (aka ENL) is an epigenetic reader protein that binds to crotonylated/acetylated lysine residues of histone 3 through its N-terminal YEATS domain. Multiple different positive and negative regulatory complexes compete for binding to the MLLT1 C-terminal domain. MLLT1 has been implicated in leukemic stem cell survival in MLL-rearranged cell lines but its role in normal hematopoiesis has not been investigated. Mllt1 is expressed in multiple bone marrow cell subsets, with high expression levels in multipotent and B-lineage progenitors. We generated conditional Mllt1 knockout ( Mllt1 del) mice and observed a B-lineage defect in bone marrow of Mllt1 deleted mice compared to control Mllt1 fl/fl mice. Mitochondrial activity has been linked to hematopoietic lineage fate decision choices. We performed seahorse mitochondrial stress test on lineage negative (stem and progenitor) bone marrow cells and found that lineage negative cells of Mllt1 del mice have lower mitochondrial activity (as a measure of oxygen consumption rate) than control Mllt1 fl/fl mice. In addition, we observed mitochondrial mass (as a quantitative measure of ratio of mtDNA gene CytB / nDNA gene ActB) to be decreased in lineage negative cells of Mllt1 del mice when compared to control Mllt1 fl/fl mice. Surface marker labeling was used to sort more defined subpopulations from bone marrow of Mllt1 del and Mllt1 fl/fl mice. We observed decreased mitochondrial mass in MPP4s and MEPs of Mllt1 del mice when compared to control Mllt1 fl/fl mice. Flow cytometry-based cell surface labeling to distinguish different bone marrow progenitors, plus mitochondria staining using MitoTracker Green (for mitochondrial mass) and MitoTracker Red CMXRos (for mitochondrial membrane potential) showed altered mitochondrial function of B-lineage progenitors in Mllt1 del mice when compared to control Mllt1 fl/fl mice. Thus, Mllt1 is essential for normal mitochondrial function, which may be necessary to maintain specific bone marrow hematopoietic subpopulations. Ongoing studies include gene expression profiling of sorted lymphoid progenitor populations and analysis of direct Mllt1 target genes that contribute to the altered phenotype in Mllt1 del mice.