The JAK-STAT signaling pathway regulates cytokine and growth factor production and signaling, hence, influencing immune recovery after allogeneic HCT. We have previously reported results of our phase 2a clinical trial (NCT04339101) showing that peri-HCT administration of Itacitinib (Ita), a selective JAK1 inhibitor, in combination with tacrolimus (Tac) / sirolimus (Siro) GVHD prophylaxis in matched donor reduced intensity HCT setting, is safe and associated with 100% engraftment, a low cumulative incidence of GVHD, and promising 1-year GVHD-free relapse-free survival of 54% [Ali et al, Blood (2024) 144 (Supplement 1)]. Here, we report the pharmacokinetics/pharmacodynamics of Ita and its impact on immune reconstitution in patients enrolled in this trial.GVHD prophylaxis was Ita (200 mg/day – from day -3 pre-HCT until day +100), with Tac/Siro (target levels: 5-10ng/ml for both). Ita exposure was assessed from day -3 to day +5. T cell subsets were monitored on days +35 and +100. Serum cytokine level was measured at pre-HCT, days +14 and +28.Ita at 200 mg/day dose resulted in variable systemic exposure, with average trough concentration of 12.5 nM (IQR: 7.3-23.0) (Fig 1A) and AUC0-12 at 2145 nM*h (IQR: 1440-2926). The average trough concentrations for Tac and Siro were less variable mainly due to therapeutic drug monitoring (TDM): 8.83 ng/ml (IQR: 7.65-10.2) and 7.06 ng/ml (IQR: 5.15-8.5), respectively (Fig 1B). When stratified by Ita trough levels, patients with higher-than-median Ita concentrations (24.5 nM; IQR: 16.8–33.7) also exhibited higher Siro trough levels (Fig 1C), likely due to shared metabolic pathway involving Cytochrome P450 3A4.The absolute number of T cell subsets on day +35 averaged 61.6/µL (IQR: 30.8–131) for CD4⁺, 21.3/µL (IQR: 10.1–39.7) for CD8⁺, increasing by day +100 to 94.5/µL (IQR: 51.9–153) and 37.8/µL (IQR: 21.5–75.9), respectively. Regulatory T cells (Treg) counts were 3.91/µL (IQR: 1.95–7.54) on day +35 and 3.31/µL (IQR: 1.74–6.42) on day +100.Patients who developed grade 2–4 acute GVHD had Treg counts of 1.22/µL (IQR: 1.1–2.3) at day +35 and 5.7/µL (IQR: 2.1–7.0) at day +100 (Fig 1D). Ita trough levels and AUC demonstrated a negative correlation with Treg counts (Fig 1E). These patients also had modest elevations in GVHD biomarkers (HGF, Reg3A, TNF-R1) on day 14 but not on day 28 (Fig 1F). While Ita trough centration did not significantly correlate with GVHD biomarkers (Fig 1G), Ita AUC showed a positive correlation with HGF, IL-6, and ST2 levels.In conclusion, oral administration of Ita during the peri-HCT period showed substantial interindividual variability in drug exposure and possible interaction with Siro. Interestingly, the higher Ita levels (trough and/or AUC) were associated with lower number of Treg and higher levels of GVHD biomarkers, suggesting that further optimization of Ita dosing and TDM may lead to further improvement of HCT outcomes.

PURPOSE:The blood-brain barrier (BBB) impedes the passage of most circulating drugs into the brain. Low-intensity pulsed ultrasound with microbubbles (LIPU/MB) transiently opens the BBB, improving parenchymal drug penetration. Parenchymal drug retention following short-lived BBB opening is unknown. We investigated the effect of LIPU/MB on the concentration of carboplatin and fluorescein over time and compared the parenchymal retention of temozolomide (TMZ), carboplatin, and fluorescein in the nonsonicated brain. EXPERIMENTAL DESIGN:We analyzed four patients who underwent intraoperative LIPU/MB with intravenous administration of carboplatin and fluorescein in the NCT04528680 clinical trial. Microdialysis catheters were implanted into sonicated and nonsonicated brain regions, and drug levels were measured over 24 hours. Published microdialysis data of TMZ without LIPU/MB were used for comparison. RESULTS:LIPU/MB led to sustained elevated parenchymal drug concentrations, achieving a 3.1-fold increase in brain-to-plasma AUC for carboplatin and fluorescein (P = 0.03). In the nonsonicated brain, TMZ concentrations remained below their plasma levels, as parenchymal drug clearance mirrored plasma clearance. In contrast, BBB-impermeable drugs such as carboplatin and fluorescein exhibited delayed parenchymal clearance, resulting in higher brain than plasma drug levels over time. Parenchymal drug clearance of carboplatin and fluorescein was not affected by sonication. CONCLUSIONS:Following LIPU/MB, BBB-impermeable drugs exhibit sustained elevated parenchymal concentrations surpassing their plasma levels, highlighting the bidirectional restriction of drug passage by the BBB. Future studies are warranted to explore drug trapping and the efficacy of sustained exposure to cytotoxic drugs for the treatment of brain-infiltrating tumors.

808 Background: Erdafitinib (E) is an approved treatment in patients with mUCwith FGFR 3 GAs after progression on platinum-based chemotherapy (PBC). Enfortumab Vedotin (EV) is approved to treat patients with mUC following prior PBC and PD1/L1 inhibitors or as First-line therapy in combination with pembrolizumab. Retrospective studies suggest that the activity of EV is not compromised by FGFR 3/2 GAs. EV and erdafitinib have different mechanisms of activity and toxicities are mostly non-overlapping. Hence, there is rationale to evaluate the feasibility of combination EV and E, to overcome the difficulties of resistance and sequencing agents in mUC patients with FGFR 3/2 GAs. Methods: This is an ongoing, single arm, multicenter, Phase I, 3+3 design dose-escalation and expansion study of E+ EV combination evaluating the safety, tolerability, PK, and antitumor activity in patients with mUC harboring FGFR3/2 GAs who have progressed after platinum and/or PD1/L1 inhibitor therapies. Dose escalation phase aims to identify the maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D) of EV in combination with fixed dose of E at 8 mg/day (table). Results: As of data cutoff, 9 patients were enrolled and completed dose limiting toxicity (DLT) period (1 st cycle) in the dose-escalation phase. Six patients were enrolled at DL1 with 1 DLT (skin rash) and 3 at DL2 (no DLT). The most common all grade treatment-related adverse events (TRAEs) included hyperphosphatemia (88%), mucositis (88%), high AST (88%), hypercalcemia (75%), palmar plantar erythrodysesthesia (75%), peripheral neuropathy (75%), alopecia (63%), diarrhea (63%), hypoalbuminemia (63%) and hypomagnesemia (63%). Grade 3 TRAEs included palmar plantar erythrodysesthesia (50%), anemia (17%), rash (17%), anorexia (17%) and paronychia (17%). One patient developed grade 4 Stevens-Johnson syndrome related to EV which subsequently improved. PK data are available for all 6 patients in DL1. The average steady-state Cmin of E and MMAE was 1430 ± 639 ng/mL and 1.4 ± 0.9 ng/ml, respectively, and the average Cmax of MMAE was 3.9 ± 0.9 ng/mL at DL1. All 9 patients are evaluable with 100% best objective rate, including 8 partial responses (PRs) and 1 complete response (CR). The mOS was NR (95% 17.1 months-NR), mPFS was 7.52 months (95% CT 5.55-NR) with median follow up of 22.7 months. The mDOR is 5.49 months. The RP2D of EV is 1.25 mg/kg in combination with E at 8 mg/day. Conclusions: E+EV combination is feasible and preliminarily exhibits promising antitumor activity. Dose expansion is ongoing at RP2D dose of EV with E. Clinical trial information: NCT04963153 . Dose Level (DL) Dose Cycle Length E EV Level -1 8 mg PO QD 0.75 mg/kg IV (maximum dose 75 mg) D1,8,15 28 days Level 1 8 mg PO QD 1 mg/kg IV (maximum dose 100 mg) D1,8,15 Level 2 8 mg PO QD 1.25 mg/kg IV (maximum dose 125 mg) D1,8,15

Polyadenosine diphosphate-ribose polymerase 1 (PARP-1) and 2 (PARP-2) are key DNA repair enzymes that promote single-strand break repair via the base excision pathway. Niraparib, a PARP inhibitor, has shown clinical efficacy with the reduction of disease progression or death and progression-free survival benefit across multiple clinical trials, leading to the Food and Drug Administration (FDA) approval for the treatment of advanced and recurrent ovarian cancers. This study presents a robust and simple 5-min assay designed for the quantitation of the single agent niraparib in human plasma utilizing liquid chromatography-tandem mass spectrometry (LC-MS/MS). A 50 mu L volume of plasma was subjected to protein precipitation, followed by chromatographic separation using a Phenomenex Kinetex C18 column (2.6 mu m, 50 x 2.1 mm) and a gradient mobile phase system consisting of 0.1% formic acid in both water and acetonitrile during a 5-min run time. Mass spectrometric detection was achieved using a SCIEX 6500 + tandem mass spectrometer with electrospray positive-mode ionization. With a stable isotopic internal standard, our assay met the criteria outlined by the FDA guidance for bioanalytical method validation, demonstrating robust performance within the range from 5 to 5000 ng/mL. This assay will support future clinical studies by defining niraparib pharmacokinetics.

Non-small cell lung cancer (NSCLC) remains the leading cause of cancer-related mortality necessitating the exploration of alternate therapeutic approaches. Tumor reactive or activated-by-cytokine killers (TRACK) are PD-L1+ highly cytolytic natural killer (NK) cells derived from umbilical cord blood NK cells and engineered to express soluble IL15 (sIL15), showing promise in preclinical studies against NSCLC. We assessed safety, persistence, homing and cytotoxic activity in six patients with advanced, refractory, and progressing NSCLC who received a low dose of unmatched, allogeneic, off-the-shelf sIL15_TRACK NK cells. We evaluated NK cell presence and persistence with droplet digital (dd) PCR, flow cytometry, and immunofluorescent staining. sIL15_TRACK NK cells had peak measurements at one hour and became undetectable four hours after each in fusion. Cognate ligands to activating NK cell receptors were found in NSCLC. sIL15_TRACK NK cells were observed in a lung tumor biopsy seven days after the final infusion, confirming their sustainment and tumor-homing ability. They retained cytolytic function following isolation from the lung tumor. Three out of six patients achieved disease stabilization on repeat imaging, while the others progressed. Unmatched, allogeneic, cryopreserved, off-the-shelf sIL15_TRACK NK cells express activating receptors, home to tumor sites that express their cognate ligands, and retain cytolytic activity after infusion, underscoring their potential as a therapeutic approach in solid tumors. At low doses, the therapy was safely administered and showed preliminary evidence of activity in three of six patients with advanced and progressive NSCLC. Additional dose escalation cohorts and co-administration with atezolizumab are planned. gov NCT05334329FUNDING. Funding was provided by CytoImmune Therapeutics; CA266457; CA033572; CA210087.

Telomerase (TERT) is an enzyme involved in maintaining telomere length in diffuse large B cell lymphoma (DLBCL). Previous attempts to target TERT+ cancers faced challenges, including the delayed clinical responses and on-target/off-tumor toxicities. Here, we present a DLBCL-targeted oligonucleotide designed to deliver a synthetic TERT substrate, 6-thio-2'-deoxy-guanosine (6tdG), damaging telomeres and triggering apoptosis. In vitro, 6tdG-oligonucleotides (6tdGOs) were selectively cytotoxic to TERT+ DLBCL cells without affecting activated T cells or non-malignant TERT- cells. Repeated intravenous administration of 6tdGO, but not 6tdG nucleoside, had significant antitumor effects against xenotransplanted human DLBCL models and syngeneic Eμ-myc/15A lymphoma in mice. In immunocompetent mice, treatment with 6tdGO induced systemic, lymphoma-specific, and CD8 T cell-mediated antitumor immune responses. The abscopal effects of 6tdGO were abolished in mice lacking expression of Sting1 or Ifnar1 but not Trl9. These findings suggest that 6tdGO-induced lymphoma cell death triggered STING-mediated type-I interferon signaling, thereby promoting recruitment/activation of CD8 T cells. Importantly, the repeated 6tdGO treatments were well-tolerated in humanized hCD34/NOG mice. Except for the reduced percentage of human B cells, 6tdGO did not decrease the numbers of hematopoietic stem cells, myeloid cells, or T cells. Overall, 6tdGO offers an effective and safer strategy against aggressive TERT+ DLBCL with potential to activate T cell-based antitumor immunity.

BACKGROUND:Non-small cell lung cancer (NSCLC) remains the leading cause of cancer-related mortality, necessitating the exploration of alternate therapeutic approaches. Tumor-reactive or activated-by-cytokine killers (TRACK) are PD-L1+, highly cytolytic NK cells derived from umbilical cord blood NK cells and engineered to express soluble IL-15 (sIL15), and these cells show promise in preclinical studies against NSCLC. METHODS:We assessed safety, persistence, homing, and cytotoxic activity in 6 patients with advanced, refractory, and progressing NSCLC who received a low dose of unmatched, allogeneic, off-the-shelf sIL15_TRACK NK cells. We evaluated NK cell presence and persistence with droplet digital PCR (ddPCR), flow cytometry, and immunofluorescence staining. RESULTS:sIL15_TRACK NK cells had peak measurements at 1 hour and became undetectable 4 hours after each infusion. Cognate ligands to activating NK cell receptors were found in NSCLC. sIL15_TRACK NK cells were observed in a lung tumor biopsy 7 days after the final infusion, confirming their sustainment and tumor-homing ability. They retained cytolytic function following isolation from the lung tumor. Three of 6 patients achieved disease stabilization on repeat imaging, while the others progressed. CONCLUSION:Unmatched, allogeneic, cryopreserved, off-the-shelf sIL15_TRACK NK cells express activating receptors, home to tumor sites that express their cognate ligands, and retain cytolytic activity after infusion, underscoring their potential as a therapeutic approach in solid tumors. At low doses, the therapy was safely administered and showed preliminary evidence of activity in 3 of 6 patients with advanced and progressive NSCLC. Additional dose escalation cohorts and coadministration with atezolizumab are planned. TRIAL REGISTRATION: CLINICALTRIALS:gov NCT05334329. FUNDING:Funding was provided by CytoImmune Therapeutics and grants from the National Cancer Institute (CA266457, CA033572, and CA210087).

Ataxia-telangiectasia and Rad3-related (ATR) protein kinase is an essential regulator of the DNA damage response (DDR) at stalled and collapsed replication forks. Tuvusertib (M1774) is a selective, orally available small molecule ATR inhibitor currently in preclinical and clinical development for cancer treatment. This study presents a robust and simple 5-min assay designed for the quantification of single agent tuvusertib in human plasma utilizing liquid chromatography tandem mass spectrometry (LC-MS/MS). A 20 μL volume of plasma was subjected to protein precipitation, followed by chromatographic separation using a Phenomenex Synergi Polar-RP (4 μm, 2.1 × 50 mm) and a gradient mobile phase system consisting of 0.1% formic acid in both water and acetonitrile during a 4-min run time. Mass spectrometric detection was achieved using a SCIEX 6500+ tandem mass spectrometer with electrospray positive-mode ionization. With a stable isotopic internal standard, our assay met the criteria outlined by the Food and Drug Administration guidance for bioanalytical method validation, demonstrating robust performance within the range from 5 to 5000 ng/mL. This assay will support ongoing and future clinical studies by defining tuvusertib pharmacokinetics.

Pressurized intraperitoneal aerosolized chemotherapy (PIPAC) is a novel, minimally invasive method of delivering intraperitoneal chemotherapy with promising peritoneal disease control in ovarian cancer. This US multicenter prospective phase I trial (NCT04329494) evaluated the safety and efficacy of PIPAC cisplatin 10.5 mg/m2 and doxorubicin 2.1 mg/m2 (PIPAC-CD) every 6 weeks in ovarian cancer at three US centers. Primary endpoints were dose-limiting toxicities and adverse events. Secondary endpoints included response according to RECIST (Response Evaluation Criteria in Solid Tumors) criteria, laparoscopic peritoneal carcinomatosis index, histologic peritoneal regression grading score, progression-free survival (PFS), and overall survival (OS). In total, 15 patients were enrolled. The median prior lines of therapy was 3 (range 1–10). The PIPAC completion rate (≥2 PIPACs) was 86.7

To support a phase 1 trial in patients with lymphomas, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for tazemetostat quantitation in 20 μL of human plasma. After protein precipitation, chromatographic separation employed a Kinetex C18 column and a gradient of 0.1% formic acid in both water and acetonitrile, during a 3-min run time. Detection was achieved using a SCIEX 6500+ tandem mass spectrometer with electrospray positive-mode ionization. Validation was based on the latest Food and Drug Administration guidance. With a stable isotopic internal standard, the assay was linear within the range of 10-5000 ng/mL and proved to be accurate (91.9%-103.7%) and precise (<4.4% imprecision). Recovery varied between 93.3% and 121.1%, and matrix effect ranged from -25.5% to -4.9%. Hemolysis, lipemia, and dilution did not impact quantitation. Plasma stability was confirmed after three freeze-thaw cycles, 24 h at room temperature, and 4 months at -80°C. Incurred sample reanalysis yielded 94.4% samples within 20% difference (n = 36). External validation showed a mean bias of -11.1%. Pharmacokinetic (PK) data obtained from three patients suggested variable concentration time profiles, warranting collection of further data. The assay proved to be suitable for tazemetostat quantitation in human plasma and will support clinical studies by defining tazemetostat PKs.

BACKGROUND AND OBJECTIVE:EVEREST is a phase 3 trial in patients with renal cell cancer (RCC) at intermediate-high or very high risk of recurrence after nephrectomy who were randomized to receive adjuvant everolimus or placebo. Longer recurrence-free survival (RFS) was observed with everolimus (hazard ratio [HR] 0.85, 95% confidence interval [CI] 0.72-1.00; p = 0.051), but the nominal significance level (p = 0.044) was not reached. To contextualize these results with positive phase 3 trials of adjuvant sunitinib and pembrolizumab, we conducted a secondary analysis in a similar population of EVEREST patients with very high-risk disease and clear cell histology. METHODS:Postnephrectomy patients with any clear cell component and very high-risk disease, defined as pT3a (grade 3-4), pT3b-c (any grade), T4 (any grade), or node-positive status (N+), were identified. A Cox regression model stratified by performance status was used to compare RFS and overall survival (OS) between the treatment arms. KEY FINDINGS AND LIMITATIONS:Of 1499 patients, 717 had clear cell histology and very high-risk disease; 699 met the eligibility criteria, of whom 348 were randomized to everolimus arm, and 351 to the placebo arm. Patient characteristics were similar between the arms. Only 163/348 (47%) patients in the everolimus arm completed all treatment as planned, versus 225/351 (64%) in the placebo arm. Adjuvant everolimus resulted in a statistically significant improvement in RFS (HR 0.80; 95%CI 0.65-0.99, p = 0.041). Evidence of a survival benefit was not seen (HR 0.85; 95%CI 0.64-1.14, p = 0.3) CONCLUSIONS AND CLINICAL IMPLICATIONS: In patients with clear cell RCC at very high-risk for recurrence, adjuvant everolimus resulted in significantly improved RFS compared to placebo but resulted in a high discontinuation rate due to adverse events. Although the treatment HR for OS was consistent with RFS findings, it did not reach statistical significance. With a focus on risk stratification tools and/or biomarkers to minimize toxicity risk in those not likely to benefit, this information can help inform the design of future adjuvant trials in high-risk RCC.

Eribulin is an inhibitor of microtubule dynamics. It is not as highly protein bound as the taxanes and is less vulnerable to extrusion by P-glycoprotein in the blood–brain barrier (BBB). These features predict that eribulin could play an active role in managing brain tumors. Indeed, the small amount of published clinical data indicates eribulin may have some efficacy against breast cancer brain metastases. To better understand the potential of eribulin for treating brain tumors, we performed an intracerebral microdialysis study to determine the neuropharmacokinetics of eribulin in cancer patients undergoing tumor resection. After tumor removal, two microdialysis catheters were inserted into peritumoral brain tissue. Approximately 24 h after surgery, a single dose of eribulin 1.4 mg/m2 was administered intravenously. Dialysate samples were collected continuously for 72 h, with plasma samples collected in parallel. Eribulin concentrations were analyzed by tandem mass spectrometry. Dialysate samples from 12 intracerebral microdialysis catheters placed in 7 study participants were included in the analysis. A statistically significant difference was observed between eribulin concentrations in brain tissue where BBB was disrupted versus intact, with a difference in mean maximum concentrations on log2 scale of 3.37 (std err = 0.59, p-value = 0.005). Nonetheless, overall brain to plasma ratios of eribulin only ranged from 0.13 to 1.99

TPS595 Background: Erdafitinib and enfortumab vedotin are available treatment options in mUC patients with somatic FGFR2/3 GAs after progression on platinum-based chemotherapy and PD-1/L1 inhibitors. However, due to decline in their clinical condition, the sequential delivery of these agents is challenging. Tubulin antagonists induce a G2-M cell-cycle block, while FGFR inhibitors cause a G1 block, with studies suggesting that their combination may be additive or synergistic. Retrospective studies suggest that the activity of EV is not compromised by somatic FGFR2/3 GAs. Hence, there is rationale to evaluate the feasibility of the combination of EV and E, to overcome the difficulties of resistance and sequencing these agents in mUC patients with FGFR2/3 GAs. Methods: This is a phase Ib, single arm, multicenter study in patients with mUC harboring somatic FGFR2/3 GAs who have progressed after platinum and PD-1/L1 inhibitor therapies with enrollment of up to 30 patients. Pts are required to have predominant urothelial component, ECOG-PS 0-2, neuropathy ≤ grade 1 and no ophthalmologic conditions precluding treatment with E. FGFR2/3 GAs may be identified by tumor tissue or circulating tumor (ct)-DNA profiling. The primary objective is feasibility and establishing a recommended phase-2 dose (RP2D). Secondary objectives include objective response rate, duration of response, progression-free survival and overall survival. The dose-escalation component will enroll up to 18 pts with 3+3 design (dosing cohorts in table), followed by dose-expansion component of 12 pts. The dose limiting toxicities (DLTs) will be evaluated using a sequential Bayesian toxicity monitoring that allows a maximum DLT rate of 0.33 during the dose-expansion phase. Exploratory biomarker analyses will be performed including 1) tumor PD-L1, Nectin-4 assessment by immunohistochemistry and association with response 2) ct-DNA evaluation at baseline and progression to evaluate resistance pathways 3) pharmacokinetic studies will assess plasma levels of E and free monomethyl auristatin-E (MMAE). This is led by North American Star Consortium as part of Experimental Therapeutics. Clinical trial information: NCT04963153. [Table: see text]

Background: Targeting of methylation by mutant isocitrate dehydrogenase (IDH) has changed the therapeutic landscape of relapsed or refractory (R/R) AML, culminating in the approval of IDH inhibitors, enasidenib, ivosidenib, and olutasidenib. Concurrent RAS-signaling mutations represent a growing problem in the management of R/R IDH mutant AML, since they are associated with resistance to IDH inhibitors and other approved therapies such as venetoclax-based regimens. In preclinical studies, co-occurring TET2 or IDH2 mutations cooperate with RAS mutations to enhance RAS signaling and increase susceptibility to MEK inhibition. In mice with co-occurring IDH2 and NRAS mutations, combined IDH2 and MEK inhibition led to a greater improvement in blood counts, decreased spleen weight and a greater reduction in leukemic stem and progenitor cells. We thus hypothesize that combined MEK inhibition with cobimetinib and IDH2 inhibition with enasidenib will be safe and overcome these mechanisms of resistance, resulting in enhanced responses. Study Design and Methods: This is a multi-center, open label, single arm, phase 1b study to investigate the safety and preliminary efficacy of the combination of cobimetinib and enasidenib in patients with R/R AML with co-occurring IDH2 and RAS-pathway mutations. The study consists of 1) a dose escalation cohort to determine the maximum tolerated dose and the recommended phase 2 dose (RP2D) and 2) a dose expansion cohort to better characterize safety of the combination as well as preliminary efficacy. Key eligibility criteria include age ≥ 18 years, R/R AML, and the presence of co-occurring IDH2 and RAS-pathway mutations. Previous treatment with enasidenib is allowed. Enasidenib (100 mg) is given orally (PO) once daily (QD) for the entire cycle (28 days). Cobimetinib is given PO QD for days 1-21 of each cycle at one of 2 dose levels (DLs), DL 1 = 40 mg and DL 2 = 60 mg. The study primary endpoint is safety. In order to determine the maximum tolerated dose (MTD) for cobimetinib in combination with enasidenib, a standard 3+3 design governs enrollment and dose escalation and de-escalation in the two potential dosing cohorts. Toxicity severity is graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE) version 5.0. Secondary endpoints include overall response (CR, CRi, MLFS, and PR) by ELN 2022 response criteria, median to first response, response duration, median and 1-year OS and EFS, pharmacokinetics, and pharmacodynamics (phospho-ERK by CYTOF). Correlative objectives include determining the expression of RAS negative regulators by RNA sequencing and epigenetic regulation of RAS regulators by bisulfite sequencing and H3K4me3 and H3K27Ac ChIP-Seq. The protocol was amended to expand eligibility to include RAS mutations involving NRAS, KRAS, PTPN11, NF1, CBL, BRAF, KIT, and RIT1. As enasidenib is a moderate CYP3A inducer, the protocol was amended to also allow concomitant use of the moderate CYP3A4 inhibiting antifungal, isavuconazonium sulfate (cresemba). Two innovative changes were made to enhance rapid identification of this very rare IDH2 and RAS-mutant AML patient population. First, a central next-generation sequencing assay with a customized Rapid Panel was developed to identify and confirm patients with IDH2 and RAS-pathway mutations within 7 days. Additionally, potential clinical trial participants have recently been identified through POSEIDON (Precision Oncology Software Environment Interoperable Data Ontologies Network), which is a secure, cloud-based Oncology Insights Engine that enables investigators to visualize clinical and comprehensive genomic profiling data. To date, four patients have enrolled with three patients completing at least 1 cycle of treatment. Enrollment is open at City of Hope and the Fred Hutchinson Cancer Center (NCT05441514).

The DNA-dependent protein kinase (DNA-PK) is an abundant nuclear protein that mediates DNA double-strand break repair by nonhomologous end joining (NHEJ). As such, DNA-PK is critical for V(D)J recombination in lymphocytes and for survival in cells exposed to ionizing radiation and clastogens. Peposertib (M3814) is a small molecule DNA-PK inhibitor currently in preclinical and clinical development for cancer treatment. We have developed a high-performance liquid chromatography-mass spectrometry method for quantitating peposertib and its metabolite in 0.1 mL human plasma. After MTBE liquid-liquid extraction, chromatographic separation was achieved with a Phenomenex Synergi polar reverse phase (4 μm, 2 × 50 mm) column and a gradient of 0.1% formic acid in acetonitrile and water over an 8 min run time. Mass spectrometric detection was performed on an ABI SCIEX 4000 with electrospray, positive-mode ionization. The assay was linear from 10 to 3000 ng/mL for peposertib and 1-300 ng/mL for the metabolite and proved to be both accurate (97.3%-103.7%) and precise (<8.9%CV) fulfilling criteria from the Food and Drug Administration (FDA) guidance on bioanalytical method validation. This liquid chromatography-tandem mass spectroscopy (LC-MS/MS) assay will support several ongoing clinical studies by defining peposertib pharmacokinetics.

TPS6127 Background: Locally advanced head and neck squamous cell carcinoma (HNSCC) is commonly treated with definitive chemoradiation therapy (CRT). However, locoregional recurrence rates of approximately 50% may occur in high risk patients. As such, there is an unmet need for up-front treatment intensification in this patient population. Preclinical evidence suggests that both radiation therapy (RT) and cisplatin activate the PI3K/AKT pathway, leading to treatment resistance through multiple processes, including increased DNA repair, decreased apoptosis, and modulation of the tumor microenvironment by promoting angiogenesis, immune escape and hypoxia. Furthermore, AKT inhibitors have been shown to sensitize a subset of HNSCC models to RT and platinum chemotherapy in vitro and in vivo. However, radiosensitization using a specific AKT inhibitor has not yet been studied in humans. This phase I studywill bethe first to establish safety and preliminary efficacy of ipatasertib combined with standard of care definitive CRT for HNSCC. Methods: The primary objective of this study is to determine the maximum tolerated dose and recommended Phase 2 dose (RP2D) of ipatasertib in combination with definitive CRT in locally advanced HNSCC based on dose-limiting toxicities (DLTs). Secondary objectives include assessment of acute and late toxicities, long term swallowing function, and a preliminary assessment of efficacy. Eligible subjects must have pathologically confirmed, previously untreated, non-metastatic HNSCC that is either human papilloma virus (HPV)-negative clinical stage III-IVB, or HPV-positive clinical stage III. The study schema includes dose escalation and expansion cohorts. All subjects will receive RT for a standard 70 Gy in 7-week course, with concurrent weekly cisplatin. Two 28-day cycles of orally administered ipatasertib will be given concurrently with CRT. The four dose levels of ipatasertib range from 100-400 mg daily. Dose escalation of ipatasertib will follow a Time-to-Event Bayesian Optimal Interval (TITE-BOIN) design, with DLT window extending from the start of CRT, through 28 days after completion of RT. The expansion cohort will enroll an additional 10 subjects at the RP2D, and incorporate pharmacodynamic biopsies for each subject to evaluate whether the addition of ipatasertib to CRT will result in increased gamma-H2AX, consistent with radiosensitization, and also decreased pS6 and pPRAS40 to evaluate AKT pathway inhibition. Additional correlative studies include assessment of the pharmacokinetic profile of ipatasertib with CRT, as well as correlation of efficacy with tumor genotype, based on whole exome sequencing of pre-treatment biopsy specimens. Enrollment is currently at dose level 2 in the escalation phase, and is expected to complete accrual in 2025. Clinical trial information: NCT05172245 .

Abstract Background: huMNC2-CAR44 and huMNC2-CAR22 are autologous CAR T cell therapies under study in an ongoing 1st-in-human trial for metastatic breast cancers (NCT04020575), being performed at City of Hope. Both CARs are targeted to the tumor by an antibody, huMNC2, that recognizes a cryptic binding site on MUC1*, which is the transmembrane cleavage product of MUC1. The antibody binds to an epitope that is only unmasked when MUC1 is cleaved to MUC1* by enzymes in the tumor microenvironment. huMNC2 strongly reacts with over 90% of breast cancers. No therapeutic that targets MUC1* had ever been tested in humans before this trial. We note that neither 5E5 nor antibodies that bind to a MUC1 “heterodimer” recognize MUC1*. Eight patients have already been treated with huMNC2-CAR44. The next 8 patients will be treated with huMNC2-CAR22 to enable comparison and inform decision as to which CAR to bring forward for completion of Phase 1 and entry into Phase 2. huMNC2-CAR22 differs from huMNC2-CAR44 in that it is resistant to exhaustion. CAR22 achieves greater in vivo persistence due to Sadelain’s “1XX” mutations of Tyr to Phe in 2 of the 3 ITAMs, which prevent Tyr phosphorylation and signaling, leaving signaling through ITAM 1 alone. Trial Design: Dose escalation or de-escalation is tested in cohorts of 3 patients each using standard “3+3” dose-finding, with the starting dose of 3.3x105 CAR+ T cells/kg up to a maximum of 1.0x107 CAR+ T cells/kg. Patients receive cyclophosphamide (300 mg/m2/day) and fludarabine (30 mg/m2/day) for 3 days prior to CAR T cell infusion. Safety will be evaluated by CTCAE version 5.0 and Lee criteria. Anti-tumor activity will be assessed by imaging studies completed between 1 and 3 months after huMNC2-CAR T cell infusion for determination of response by RECIST 1.1 or by FDG PET modified PERCIST for patients with predominant bone disease. Inclusion Criteria: Patients with confirmed diagnosis of breast cancer, with documented ER, PR, and HER2 status per ASCO/CAP guidelines. Patients with MUC1* expression of at least 30% by IHC. Patients must have received standard metastatic systemic therapy per NCCN guidelines which are known to confer benefit. No maximum on number of prior treatments. Patients must have received at least 2 or 3 prior lines of chemotherapy in the metastatic setting. Exclusion Criteria: Patients requiring >15 mg of prednisone per day or immunosuppressives; patients with major organ dysfunction; Serum creatinine > 2 mg/dL; Bilirubin ≥ 1.5 mg/dL; AST/ALT ≥ 2.5 x upper limit normal; 3x upper limit for patients with known liver metastasis; significant pulmonary dysfunction; significant cardiovascular abnormalities; ANC < 1000/mm3. Primary Objectives: To determine the safety and maximally tolerated cell dose (MTD) and recommended phase 2 cell dose (RP2D) of ex vivo expanded autologous huMNC2-CAR T cells for patients with advanced MUC1* positive breast cancer using CTCAE version 5.0 and Lee criteria. Secondary: Determine duration of in vivo persistence and phenotype of adoptively transferred huMNC2-CAR T cells. Determine antitumor activity by RECIST 1.1. Determine MTD/RP2D. Contact: City of Hope Comprehensive Cancer Center Joanne Mortimer, MD 1-800-826-4673 Minerva18625@coh.org Citation Format: Cynthia Bamdad, Joanne Mortimer, Yuan Yuan, Jennifer M. Specht, Benoit Smagghe, Stephen Chi-Min Lin, Andrew Stewart, Danica Walkley, Mark Carter, Timothy Synold, Vishwas Parekh, Kevin Yi, Jac-Leen Nash, Michael Nash, Qing Liu-Michael, Stanley Hamilton, Stephen Forman. 1st-in-human CAR T targets MUC1 transmembrane cleavage product [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-19-03.