Preclinical Research Articles

Animal models to study cognitive impairment of chronic kidney disease.

Mild cognitive impairment (MCI) is common in people with chronic kidney disease (CKD), and its prevalence increases with progressive loss of kidney function. MCI is characterized by a decline in cognitive performance greater than expected for an individual age and education level but with minimal impairment of instrumental activities of daily living. Deterioration can affect one or several cognitive domains (attention, memory, executive functions, language, and perceptual motor or social cognition). Given the increasing prevalence of kidney disease, more and more people with CKD will also develop MCI causing an enormous disease burden for these individuals, their relatives, and society. However, the underlying pathomechanisms are poorly understood, and current therapies mostly aim at supporting patients in their daily lives. This illustrates the urgent need to elucidate the pathogenesis and potential therapeutic targets and test novel therapies in appropriate preclinical models. Here, we will outline the necessary criteria for experimental modeling of cognitive disorders in CKD. We discuss the use of mice, rats, and zebrafish as model systems and present valuable techniques through which kidney function and cognitive impairment can be assessed in this setting. Our objective is to enable researchers to overcome hurdles and accelerate preclinical research aimed at improving the therapy of people with CKD and MCI.

Meeting Report for the 2023 International Scientific Conference on Complex Lymphatic Anomalies

Objectives: Complex lymphatic anomalies (CLAs) are rare diseases with variable clinical manifestations caused by the abnormal development of lymphatic vessels. The Lymphatic Malformation Institute (LMI), Lymphangiomatosis & Gorham’s Disease Alliance (LGDA), and LGD Alliance Europe sponsored an international conference on CLAs so leaders in the field could discuss recent advances in research on CLAs. Methods: The conference occurred in Dallas, Texas, between September 29th and September 30th, 2023. The event had 22 distinguished speakers and 38 attendees. Additional researchers, clinicians, and patients attended the conference virtually. Results: In this article, we summarize the key takeaways from the meeting. The conference emphasized the crucial role of the patient voice in guiding research, and attendees heard stories from patients and parents of children with CLAs. The conference covered topics such as the genetic causes of CLAs, preclinical models, clinical advances, new technologies for CLA research, lymphatic imaging, and patient-centered research. Videos of all the presentations can be found on the LGDA website. Conclusions: The meeting revealed that while progress has been made, many challenges still exist surrounding the diagnosis and treatment of CLAs. Sustained research efforts are necessary to fill the gaps in knowledge and improve the care and quality of life of CLA patients.

Effect of sodium-glucose cotransporter 2 inhibitor dapagliflozin on ejection fraction reduction, myocardial and renal NF-κB expression and systemic pro-inflammatory biomarkers in models of short-term doxorubicin cardiotoxicity.

e24013 Background: Anthracycline-mediated adverse cardiovascular events are among the leading causes of morbidity and mortality in cancer patients. Sodium-glucose cotransporter 2 inhibitors (SGLT2i) exerts multiple cardiometabolic benefits in patients with/without type 2 diabetes, chronic kidney disease and heart failure with reduced and preserved ejection fraction. We hypothesized that the SGLT2i Dapagliflozin administered before and during doxorubicin therapy, could improve cardiac function and reduce pro-inflammatory pathways in preclinical models. Methods: Female C57Bl/6 mice were treated for 10 days with a saline solution or DOXO (2.17 mg/kg), DAPA (10 mg/kg) or DOXO combined to DAPA. Systemic levels of ferroptosis-related biomarkers, galectin-3, hs-CRP and pro-inflammatory chemokines (IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, IL17-α, IL-18, IFN-γ, TNF-α, G-CSF, and GM-CSF) were quantified. After treatments, immunohistochemical staining of myocardial and renal p65/NF-kB was performed. Results: DAPA prevented the reduction of radial and longitudinal strain and ejection fraction after 10 days of treatment with DOXO. A reduced myocardial expression of NLRP-3 and MyD-88 was seen in DOXO-DAPA group compared to DOXO mice. Systemic levels of IL-1β, IL-6, TNF-α, G-CSF and GM-CSF were significantly reduced after treatment with DAPA. Serum levels of galectine-3 and hs-CRP were strongly enhanced in DOXO group; contrary, their expression were reduced in DAPA-DOXO group. Troponin-T, BNP and NT-pro-BNP were strongly reduced in DOXO-DAPA group, revealing cardioprotective properties of SGLT2-i. Mice treated with DOXO and DAPA exhibited reduced myocardial and renal NF-kB expression. Conclusions: The overall picture of the study encourages the use of DAPA in primary prevention of cardiomyopathies induced by anthracyclines in cancer patients.

Phase Ib study of a high potent PI3K-mTOR dual inhibitor WX390 combined with toripalimab in patients with advanced solid tumors.

e14627 Background: WX390 is a high potent PI3K-mTOR dual inhibitor targeting pan-PI3K and mTOR. It modulates the tumor immune microenvironment by decreasing tumor Treg and myeloid-derived suppressor cells (MDSC) and improves the immune response to checkpoint inhibitors (CPI) in preclinical models. Methods: The Phase Ib study was designed to evaluate safety, pharmacokinetics (PK), and preliminary clinical activity, to determine recommended phase 2 dose (RP2D) of WX390 combined with Toripalimab in patients with advanced solid tumors. WX390 was administered orally daily at escalating doses of 0.7 mg, 0.9mg and 1.1mg, with Toripalimab at 240 mg IV every 3 weeks until confirmed progressive disease, intolerable toxicity or withdrawal of consent. Plasma PK samples were collected up to 24 hours after the first dose at C1D1, and up to 24 hours before and after the morning dose at C1D21. Results: As of December 31, 2023, total 18 patients had been treated in 3 WX390 cohorts: 0.7 mg (n = 3), 0.9 mg (n = 3), and 1.1 mg (n = 12) once daily (QD). No dose limit toxicity (DLT) was observed, and the RP2D of WX390 in combination was 1.1 mg QD, which was consistent with the single agent RP2D. The treatment related adverse events (TRAEs) (≥20%) included hyperglycemia (17/18, 94.4%), creatinine increased (12/18, 66.7%), weight loss (10/18, 55.6%), diarrhea (9/18, 50%), proteinuria (9/18, 50%), nausea (7/18, 38.9%), lymphocytopenia (7/18, 38.9%), vomiting (7/18, 38.9%), rash (7/18, 38.9%), decreased appetite (6/18, 33.3%), hypokalemia (6/18, 33%), fatigue (5/18, 27.8%), hyponatremia (4/18, 22.2%), and thrombocytopenia (4/18, 22.2%). Grade 3 TRAEs included hyperglycemia (5/18), lymphocytopenia (4/18), rash (2/18), weight loss (2/18), leukopenia (1/18), infusion-related reaction (1/18), nausea (1/18), and stomatitis (1/18). There was no TRAE leading to treatment permanent discontinuation and no grade 4/5 TRAE were observed. PK of WX390 in combination showed similar PK profile with the single agent. Among the 16 efficacy-evaluable patients with cervical (N=8), head and neck (N=5), and urothelial cancer (N=3), 5 patients (31.3%) previously received immune checkpoint inhibitors. 3 of 5 (60%) cervical cancer patients at RP2D and 2 of 5 head and neck cancer patient (40%) have achieved confirmed partial response (PR). In addition, 3 cervical patients who were treated with 0.7 or 0.9mg QD and 1of 3 urothelial carcer patients experienced tumor shrinkage (SD). The medium progression free survival (PFS) was 5.49 months (95% CI: 2.53, 6.37). Conclusions: WX390 in combination with Toripalimab had a manageable safety profile and encouraging antitumor effects in patients with advanced solid tumors. An RP2D of 1.1 mg qd of WX390 in combination was selected to be explored in further phase II study, which is ongoing in different tumor types. Clinical trial information: NCT06117566 .

A phase Ib/II study of an anti-CD137 agonist antibody ADG106 in combination with weekly paclitaxel and dose-dense doxorubicin/cyclophosphamide.

e14507 Background: ADG106 is a fully human agonistic anti-CD137 IgG4 monoclonal antibody which modulates the tumor microenvironment and has demonstrated increased efficacy when added to chemotherapy in preclinical models. We studied the combination of ADG106 with chemotherapy. Methods: We conducted a phase Ib trial in patients with advanced solid tumors to determine the recommended phase 2 dose (RP2D) of ADG106 + weekly P (paclitaxel 80mg/m2) or ddAC (doxorubicin 60mg/m2, cyclophosphamide 600mg/m2 Q2W with pegfilgrastim), followed by a phase II trial of neoadjuvant ADG106 + weekly Px12→ADG106 + ddACx4→surgery in stage I-III HER2 negative breast cancer patients. In phase Ib, one dose of single agent ADG106 was administered followed 2 weeks later by ADG106 + ddAC (Cohort 1) or ADG106 + weekly P (Cohort 2). Dose levels (DL) of ADG106 were 50mg (DL-1), 100mg (DL1), 200mg (DL2). Serial tumor biopsies were performed at baseline, after single agent ADG106 and at disease progression in phase I, and at baseline, after single agent ADG106, after cycle 1 ADG106 + P, and at surgery in phase II for translational studies. Results: 11 patients were enrolled into Phase Ib with median 3 (range 2-11) prior lines of palliative systemic therapy. ADG106 + ddAC (n=2 at DL1) was deemed intolerable with 1 patient experiencing G4 neutropenia and the other G3 infection; thus ADG106 was not added to ddAC in phase II. No DLTs were observed in all patients receiving ADG106 + P (n=6 at DL1; n=3 at DL2); ADG106 RP2D was declared at 200mg + P. In this cohort, the most common all-grade AEs were neutropenia (n=6), peripheral neuropathy (n=6) and fatigue (n=6); significant ≥G3 AEs were infection (n=2) and G3 neutropenia (n=6). 5/11 (45%) patients in phase I achieved clinical benefit (CBR; PR=2, SD=3) which is clinically meaningful as 9/11 (82%) had prior taxane exposure. 18 of the planned 30 patients have been enrolled into Phase II (ADG106+weekly P→ddACx4→surgery); 10 have completed study treatment and 5 have undergone surgery. While on ADG106 + P, 4 patients experienced G3 adverse events (AEs): neutropenia (n=3), hypophosphatemia (n=1), fever (n=1). Most common G1-2 AEs were ALT elevation (n=4), acneiform rash (n=3) and peripheral neuropathy (n=3). While on ddAC alone, all AEs were low grade except for 1 patient who had G3 pneumonitis from Pneumocystis Jiroveci Pneumonia. 26 sets of matched tumor biopsies have been collected (9 in phase I; 17 in phase II) and will be tested for a panel of 40 markers by multiplex IHC, including CD137, PD-L1, and CD3/4/8. Conclusions: ADG106 when combined with ddAC exacerbates neutropenia and is not tolerable despite prophylactic pegfilgrastim. The RP2D of ADG106 in combination with P is 200mg and is safe and tolerable; manageable G3 neutropenia was the most common AE. There are no new safety signals thus far in Phase II. Further efficacy analyses and studies on tumor immune markers are ongoing. Clinical trial information: NCT05275777 .

Pre-clinical safety and efficacy evaluation of sodium chloride nanoparticles for treating non-muscle invasive bladder cancer.

e14591 Background: Non-muscle invasive bladder cancer (NMIBC) presents a significant therapeutic challenge. Current treatment modalities such as BCG and intravesical chemotherapy given after transurethral resection of bladder tumors (TURBT), while effective, are associated with high cost burdens and are difficult for patients to tolerate due to side effects. This ultimately limits treatment regimen duration and decreases the effectiveness of the therapy which frequently leads to local recurrence. Sodium Chloride Nanoparticles (NaCl-NPs) have previously shown promise in preclinical models of intra-tumoral injection of various tumor types but has not been tested in more clinically relevant models of bladder cancer. In this study, we evaluate the safety and efficacy of NaCl-NPs as a local intravesical therapy for preventing tumor development in a preclinical murine model of bladder cancer. Methods: We conducted a dose escalation experiment via intravesical instillation of increasing doses of NaCl-NPs (300 ug/ml-2700 ug/ml, n=3 mice per group) to identify the maximum tolerated dose. Mice were treated on a weekly dosing schedule and toxicity was evaluated at day 35 as a composite of: evaluation of bladder histology with Hematoxylin and Eosin (H&E) staining by a board-certified pathologist, blood chemistry measurements, complete blood counts, as well as body weight measurement. Secondly, we assessed the efficacy of the nanoparticles through a tumor prevention experiment where MB49 bladder cancer cells were instilled in the murine bladder and shortly after treated with intravesical: 1.) Saline, 2.) Gemcitabine, or 3.) NaCl-NPs for 1 hour (n=18 per group). Mice were monitored for tumor development with 3D bladder ultrasound. In additional, overall survival (OS) was assessed. Results: The dose escalation study showed that there was no local or systemic toxicity in the NaCl-NP treated mice, even at the highest concentration (2700ug/ml). The tumor prevention study showed that mice treated with NaCl-NPs had a significant 2.55 fold reduction in tumors that developed at 4 weeks compared to saline (22% vs 56% respectively, p <0.05). This reduction was similar to that seen with intravesical gemcitabine, which is clinically, a standard of care intravesical chemotherapy. We also found an improvement in overall survival in the NaCl NP group with 70% of mice alive at 60 days compared to the other treatment groups (65% gemcitabine; 40% saline). Conclusions: Our findings suggest that NaCl-NPs could provide a safe and effective alternative therapy for preventing recurrence of NMIBC following transurethral resection of bladder tumors. This opens avenues for safer treatment protocols that can mitigate the side effects commonly seen in the management of early-stage bladder cancer.

Effect of CD22-directed CAR-T cells secreting anti-CD19 T cell engagers on control of leukemia progression compared to tandem anti-CD19/CD22 CAR-T cells.

2550 Background: Antigen-specific cancer immunotherapies, based on engineered T cells bearing chimeric antigen receptors (CARs) or the systemic administration of bispecific T cell-engagers (TCEs), have a significant impact on relapsed/refractory (R/R) B cell malignancies. However, a significant percentage of patients relapse following CAR-T or TCE therapy, with antigen loss accounting for up to one third of relapses/progressions. To avoid antigen loss after administration of single-targeted CAR-T cells and to minimize tumor escape, strategies targeting two antigens simultaneously have been developed and validated in both preclinical models and clinical trials. Nevertheless, despite lowering the risk of antigen loss, these strategies still have some limitations, mainly related to design and manufacturing challenges. Methods: In this context, we have generated the first dual-target strategy for hematological malignancies based on T cells bearing an anti-CD22 CAR and secreting an anti-CD19 T-cell engager antibody (CAR-STAb-T cells) and conducted a comprehensive preclinical study comparing its therapeutic potential with a previously validated anti-CD19/CD22 tandem CAR therapy (TanCAR-T) for B-cell acute lymphoblastic leukemia (B-ALL). Results: We have demonstrated in both short- and long-term assays, using contact and non-contact co-culture systems, that CAR-STAb-T cells efficiently redirect bystander T cells, resulting in enhanced cytotoxic activity compared to that exhibited by TanCAR-T cells at very low E:T ratios. In addition, CAR-STAb-T cells induce more potent and faster cytotoxic responses than TanCAR-T cells in both short- and long-term co-culture assays when reproducing antigen-downmodulated conditions in vitro. In vivo assays conducted in NSG mice transplanted with a B-ALL patient-derived xenograft (PDX), followed by intravenous injection of CAR-STAb-T or TanCAR-T cells under a T cell-limiting experimental setting, also showed that CAR-STAb-T cells maintained a tighter control of tumor progression than TanCAR-T cells in peripheral blood and bone marrow. Conclusions: In conclusion, we have demonstrated that the combination of a cell surface CAR and a soluble TCE recognizing different antigens may be advantageous over the use of conventional multi-targeted strategies based on cell surface-anchored receptors. Furthermore, we have proven that a small number of transduced CAR-STAb-T cells is sufficient to redirect non-transduced bystander T cells specifically and efficiently in the presence of leukemia cells, providing a significant advantage over current dual-targeted CAR-T cell therapies. CAR-STAb-T cells could therefore become an alternative to CAR-T therapies for the treatment of R/R B cell malignancies, especially in lymphodepleted patients with low T cell counts.

Targeting CCL24 in Inflammatory and Fibrotic Diseases: Rationale and Results from Three CM-101 Phase 1 Studies.

Overexpression of C-C motif chemokine ligand 24 (CCL24) is associated with inflammatory and fibrotic diseases, including primary sclerosing cholangitis (PSC), systemic sclerosis, metabolic dysfunction-associated steatotic liver disease (MASLD) and metabolic dysfunction-associated steatohepatitis (MASH). CM-101 is a humanized monoclonal antibody that neutralizes CCL24 to attenuate inflammation and fibrosis in preclinical models. Here we report the results from two Phase 1a studies investigating the safety and tolerability of intravenous (IV) and subcutaneous (SC) CM-101 in healthy participants, and in one Phase 1b study of IV and SC CM-101 in patients with MASLD without evidence of MASH. In each dose group (0.75 mg/kg, 2.5 mg/kg, 5.0 mg/kg, and 10.0 mg/kg) of the single-center, double-blind, placebo-controlled Phase 1a IV study, healthy volunteers were randomized 3:1 to receive a single IV infusion of CM-101 or placebo. In another Phase 1a, single-center, double-blind placebo-controlled study, healthy volunteers were randomized 3:1 to receive a single SC injection of CM-101 5.0 mg/kg or placebo. In the multicenter, double-blind, placebo-controlled Phase 1b MASLD study, patients with MASLD without evidence of MASH were randomized 3:1 to receive the following: cohort 1, IV CM-101 2.5 mg/kg or placebo, and cohort 2, SC CM-101 5.0 mg/kg or placebo every three weeks for 12 weeks. The primary endpoints (for all these studies) were safety, tolerability, and serum pharmacokinetic parameters of CM-101. In each study, adverse events were rare and mild to moderate. The CM-101 pharmacokinetics profile was typical of a monoclonal antibody, with a terminal half-life of approximately 19 days when given IV and approximately 17 days when given as SC injection. In patients with MASLD without evidence of MASH, CM-101 was associated with decreased serum levels of inflammatory, fibrotic, and collagen turnover biomarkers. In healthy volunteers and patients with MASLD without evidence of MASH, IV and SC CM-101 was well tolerated at doses ranging from 0.75 mg/kg to10.0 mg/kg and engaged its target (i.e., CCL24), indicating therapeutic potential in treating inflammatory and fibrotic diseases. NCT06025851, NCT06037577, and NCT06044467. Date of registration: September 2023.

Intracellular retention of TCRαβ/CD3 to generate novel allogeneic CAR-T cells targeting CLL1 for treating acute myelocytic leukemia.

e14532 Background: The prognosis of patients with R/R AML is poor and treatment remains challenging. Human C-type lectin-like molecule 1(CLL1) is a type II transmembrane glycoprotein that is highly expressed on AML but is absent on normal HSCs. Clinical studies have shown that autologous CLL-1 is a novel and promising target for the treatment of AML. But autologous CAR-T therapy has been complicated by long production time, high-cost and risks of manufacturing failure, and allogeneic CAR-T cells can overcome these hurdles. Methods: A novel non-gene-editing allogeneic CAR-T platform was developed on the base of intracellular retention of membrane proteins, and named ThisCART (This = TCR and/or HLA-I intracellular sequestered). CLL1-directed ThisCART cells are readily produced with a single lentiviral vector, encoding both a CLL1-targeting CAR and an anti-CD3 single chain antibody with the KDEL peptide fused to its C-termini. The surface expression of CAR and TCRαβ/CD3 was measured by flow cytometer. Specific tumor cytotoxicity was evaluated both in vitro and in vivo. Furthermore, the safety of donor derived ThisCART cells were tested in a xenogeneic GvHD model. Results: The manufacturing platform of CLL1 ThisCART was able to achieve over 150-folds of ex vivo CAR-T expansion in all batches, with the purity of products (CAR-positive/TCRαβ-negative) above 99%. In preclinical models, CLL1 ThisCART did not induce GvHD, and exhibited superior antitumor function compared to benchmark (WO2017091615A) and conventional CLL1 CAR-T cells. CLL1 ThisCART cells were readily produced to a quantity and quality supporting clinical investigation. Conclusions: CLL1 ThisCART cells based on intracellular retention of TCRαβ/CD3 complex demonstrated superior CLL1-dependent cytotoxicity and no xenogeneic GvHD in murine models. With streamlined single-vector-based production, enhanced CAR signaling and potent cytotoxicity and favored preclinical safety profile, CLL1 ThisCART warrant clinical investigation.

Chimeric antigen receptor T-cell therapy in childhood acute myeloid leukemia: how far are we from a clinical application?

Recurrent and/or refractory (R/R) pediatric acute myeloid leukemia (AML) remains a recalcitrant disease with poor outcomes. Cell therapy with genetically modified immune effector cells holds the promise to improve outcomes for R/R AML since it relies on cytotoxic mechanisms that are distinct from chemotherapeutic agents. While T cells expressing chimeric antigen receptors (CAR T cells) showed significant anti-AML activity in preclinical models, early phase clinical studies have demonstrated limited activity, irrespective of the targeted AML antigen. Lack of efficacy is most likely multifactorial, including: (i) a limited array of AML-specific targets and target antigen heterogeneity; (ii) the aggressive nature of R/R AML and heavy pretreatment of patients; (iii) T-cell product manufacturing, and (iv) limited expansion and persistence of the CAR T cells, which is in part driven by the immunosuppressive AML microenvironment. Here we review the results of early phase clinical studies with AML-specific CAR T cells, and avenues investigators are exploring to improve their effector function.

A phase II study of plerixafor combined with whole brain radiation therapy (WBRT) for patients with newly diagnosed glioblastoma.

2075 Background: Plerixafor inhibits the binding of stromal cell-derived factor 1 (SDF-1a / CXCL12) to its receptor on CD11b+ monocytes, inhibiting vasculogenesis and tumor recurrence in preclinical models. An early phase trial demonstrated that Plerixafor administration decreased cerebral blood volume (CBV) within the irradiated field of glioblastoma patients, and that the majority of recurrences occurred outside of the irradiated field (Thomas R. et al, Clin Cancer Res). This follow up study investigates the efficacy of Plerixafor in combination with WBRT, with the hypothesis that widening the radiation therapy (RT) field with WBRT may reduce out-of-field tumor recurrence and improve survival. Methods: This was a Phase II, open-label, non-randomized, single-arm trial.Adults between 18-75 years old with newly diagnosed high grade glioma as defined by WHO 2016 criteria and KPS ≥ 60 were eligible for enrollment. Patients underwent maximal safe resection followed by 6 weeks of concurrent radiation therapy and temozolomide (TMZ). Of note, modifications to the conventional treatment paradigm included completing 30 Gy of standard intensity-modulated RT followed by 30 Gy of WBRT, as well as completing a 4-week continuous infusion of Plerixafor dosed at 400 µg/kg/day. Patients then started adjuvant TMZ after completing Plerixafor. Primary endpoint was 6-month progression free survival (PFS6) after chemoradiation. Secondary endpoints included median overall survival (mOS), adverse events (AEs), MRI-based patterns of recurrence, and neurocognitive outcomes measured by the Trail Making Test, Controlled Oral Word Association Test, and the Hopkins Verbal Learning Test. Additionally, maximum arterial spin labeling-cerebral blood flow (ASL-CBF) values of primary lesions were compared over various time points during the study. Results: 14 of 17 enrolled patients completed the Plerixafor infusion, and all 17 patients completed WBRT. Median age was 57 years. 15 patients (88.2%) were IDH wildtype and 13 patients (76.5%) were pMGMT unmethylated. 13 patients (76.5%) underwent gross total resection, 2 (11.8%) underwent subtotal resection, and 2 (11.8%) underwent biopsy only. The PFS6 was 91.7%. The mOS was 15.11 months. 3 patients discontinued Plerixafor early due to rash (n=1), rising creatinine (n=1), or worsening confusion and agitation (n=1). Common Gr2 treatment-related AEs included alopecia (n=4), nausea (n=4), and vomiting (n=2). 1 patient had Gr3 alopecia. There were no Grade 4/5 AEs. 80% of patients scored worse on their neurocognitive assessments at 6 months compared to screening. ASL-CBF values of primary lesions appeared relatively stable throughout the study. Conclusions: The combination of Plerixafor and WBRT did not improve mOS compared to the original study using focused RT only. There was an overall decline in neurocognitive performance over the course of the study. Clinical trial information: NCT03746080 .

Intracellular retention of TCRαβ/CD3 and CD7 to generate novel allogeneic CAR-T cells targeting CD7 for treating T-cell malignancies.

e14531 Background: The treatment for patients with T-ALL/LBL who relapse after hematopoietic stem cell transplantation (HSCT) are currently restricted. CD7 is highly expressed on the surface of T-ALL/LBL T cells and CAR-T cell therapy provides a promising avenue towards remission. However, CD7-targeting immunotherapies may be compromised by T-cell fratricide. Furthermore, in addition to the long production time, high cost, and high risk of production failure, the biggest challenges of autologous CAR-T cell therapy are how to isolate a sufficient number of healthy T cells from malignant T cells. Thus, allogeneic CAR-T cells can overcome these hurdles. Methods: A novel non-gene-editing allogeneic CAR-T platform was developed on the base of intracellular retention of membrane proteins, and named ThisCART (TCR and/or HLA-I intracellular sequestered). CD7-directed ThisCART cells are readily produced with a single lentiviral vector, encoding both a CD7-targeting CAR, an anti-CD7and anti-CD3 single chain antibody with the KDEL peptide fused to its C-termini. The surface expression of CAR, TCRαβ/CD3 and CD7 was measured by flow cytometer. Specific tumor cytotoxicity was evaluated both in vitro and in vivo. Furthermore, the safety of donor derived ThisCART cells were tested in a xenogeneic GvHD model. Results: The manufacturing platform of CD7 ThisCART was able to achieve over 130-folds of ex vivo CAR-T expansion in all batches, with the purity of products (CAR-positive/CD7-negative/TCRαβ-negative) above 99%. In preclinical models, CD7 ThisCART did not induce GvHD, and exhibited superior antitumor function compared to Control T. CD7 ThisCART cells were readily produced to a quantity and quality supporting clinical investigation. Conclusions: CD7 ThisCART cells based on intracellular retention of TCRαβ/CD3 complex and CD7 demonstrated superior CD7-dependent cytotoxicity and no xenogeneic GvHD in murine models. With streamlined single-vector-based production and potent cytotoxicity and favored preclinical safety profile, CD7 ThisCART warrant clinical investigation.

Alliance A071401: Phase II trial of abemaciclib in patients with grade 2/3 meningiomas harboring somatic NF2 or CDK pathway alterations.

2001 Background: Systemic treatments are limited for patients with meningiomas that have progressed after surgery and/or radiation. Loss of NF2and CDKN2A/Bare common in higher grade meningiomas and promote meningioma progression in preclinical models. We evaluated the efficacy of abemaciclib, a cyclin-dependent kinase (CDK) 4/6 inhibitor, as part of Alliance umbrella trial A071401, a genomically driven phase II study in recurrent or progressive meningiomas. Methods: Eligible patients (pts) with grade 2/3 tumors and NF2 mutations or CDK pathway alterations were treated with abemaciclib 200 mg orally twice daily until progressive disease. Two co-primary endpoints were used: progression-free survival at 6 months (PFS6) and response rate (RR) by Macdonald criteria; the trial would be declared positive if either endpoint was met. Twenty-four evaluable pts provided >85% power to detect a PFS6 >41.5% (vs. null 15%; alpha =0.02). The threshold for promising results for PFS6 was 8+/24 pts. For RR, 24 evaluable pts provided >89% power to detect RR >20% (vs. null 2.5%; alpha = 0.021). The threshold for promising results for RR was 3+/24 pts. Results: Of 83 pts screened while the abemaciclib arm was open between September 15, 2021 and October 3, 2022, 36 eligible pts received treatment. The mean number of treatment cycles administered was 7 and median follow-up since start of treatment was 11 months. The first 24 pts that met eligibility criteria and began treatment were considered evaluable for the primary endpoint analysis. Of the 24 pts evaluated, 58% were female and the median age was 62 years. The observed PFS6 rate was 54% (13/24 pts, 95% confidence interval 33-75%), thus the study met PFS6 endpoint. No objective responses were observed. Of the 36 pts who started treatment, eight had a grade 3 and two had a grade 4 adverse event at least possibly related to treatment. Grade 3 toxicities included anemia (2), neutropenia (2), leukopenia (1), blurry vision (1), diarrhea (2), fatigue (2), ALT elevation (1), dehydration (1), hyperkalemia (1), hyponatremia (1), dizziness (1), acute kidney injury (1), and thromboembolic event (1). Grade 4 toxicities included ALT elevation (1), AST elevation (1) and vomiting (1). Conclusions: Abemaciclib was well tolerated and resulted in an improved PFS6. The overall trial endpoint was met. Abemaciclib warrants further investigation for the treatment of patients with progressive grade 2/3 meningiomas. Support: U10CA180821, U10CA180882; UG1CA189867 (NRG Oncology); Eli Lilly; https://acknowledgments.alliancefound.org . Clinical trial information: NCT02523014 .

CD4 effector T cell expansion to identify objective responses to the CD40 agonist mitazalimab in combination with modified FOLFIRINOX (mFFX) as first-line therapy for metastatic pancreatic ductal adenocarcinoma (mPDAC) in the OPTIMIZE-1 study.

2569 Background: CD40 agonists have the potential to enhance the efficacy of standard of care chemotherapy and to trigger antitumor immunity. Preclinical models indicate that the success of this approach depends on appropriate sequencing of chemotherapy with a CD40 agonist. Preliminary efficacy results from the OPTIMIZE-1 Phase II study (NCT04888312) combining mitazalimab (anti-CD40) with mFFX chemotherapy in patients with mPDAC have been previously reported. Here, we investigated immunological determinants associated with favorable outcomes with this combination therapy. Methods: Newly diagnosed, chemotherapy-naive patients with mPDAC received mitazalimab (900 µg/kg) on day 1 prior to beginning an every 2-week mFFX regimen on day 8, followed by mitazalimab 48 hours later. Tumor response was determined using RECIST v1.1 criteria. Peripheral blood was analyzed for selected cytokines and chemokines and by flow cytometry to assess changes in leukocyte subsets including B cells, monocytes, T cells, and NK cells. Flow cytometric data from evaluable patients were subjected to unsupervised hierarchical clustering to identify immune subsets. A classifier was generated from an interim dataset (n=21) using a random forest model to predict cell populations and their association with tumor response, which were then applied to the full dataset (n=47). Results: Mitazalimab triggered an expected immune response characterized by transient cytokine (IFNg and MCP-1) release and B cell depletion. Chemotherapy caused a reduction in classical monocytes and proliferating CD4+ T cells. Both mitazalimab and chemotherapy caused a transient reduction in circulating dendritic cells and NK cells. Tumor response was associated with an expansion in the frequency of effector CD4 T cells (p < 0.0001) at day 8 after receiving mitazalimab. In a blinded analysis based on the classifier, CD4 T cell expansion was linked to an early tumor response to treatment (accuracy 70%, sensitivity 68%, specificity 72%). Conclusions: Mitazalimab and mFFX differentially modulate immune responses in patients with mPDAC. Pharmacological analyses identify mitazalimab-induced expansion of CD4 effector T cells one week after first administration as a correlate of treatment outcomes. These data suggest the contribution of mitazalimab to tumor responses, and further substantiate a priming dose of mitazalimab, prior to administering chemotherapy.

Characterization and validation of a spontaneous acute and protracted oxycodone withdrawal model in male and female mice

Opioid use disorder (OUD) is a serious health problem that may lead to physical dependence, in addition to affective disorders. Preclinical models are essential for studying the neurobiology of and developing pharmacotherapies to treat these problems. Historically, chronic morphine injections have most often been used to produce opioid-dependent animals, and withdrawal signs indicative of dependence were precipitated by administering an opioid antagonist. In the present studies, we have developed and validated a model of dependence on oxycodone (a widely prescribed opioid) during spontaneous withdrawal in male and female C57BL/6J mice. Dependence was induced by chronically administering oxycodone through osmotic minipumps at different doses for 7 days. Somatic withdrawal signs were measured after 3, 6, 24, and 48 h following minipump removal. Additionally, sensitivity to mechanical, thermal, and cold stimuli, along with anxiety-like behavior, were also measured. Our results indicated that spontaneous withdrawal following discontinuation of oxycodone produced an increase in total withdrawal signs after 60 and 120 mg/kg/day regimens of oxycodone administration. These signs were reversed by the administration of clinically approved medications for OUD. In general, both female and male mice showed similar profiles of somatic signs of spontaneous withdrawal. Spontaneous withdrawal also resulted in mechanical and cold hypersensitivity lasting for 24 and 14 days, respectively, and produced anxiety-like behaviors after 2 and 3 weeks following oxycodone removal. These results help validate a new model of oxycodone dependence, including the temporally distinct emergence of somatic, hyperalgesic, and anxiety-like behaviors, potentially useful for mechanistic and translational studies of opioid dependence.

Safety and efficacy of complement factor H (CFH) inhibitor GT103 in advanced non-small cell lung cancer (NSCLC): Results of a phase Ib first in human dose escalation study.

e14588 Background: CFH is a complement regulatory protein that protects cells from alternative complement pathway activation. Neutralizing antibodies against CFH on tumor cells increase deposition of C3b, facilitate antibody-dependent-cellular phagocytosis and complement dependent cytotoxicity, inhibit tumor growth, and modulate anti-tumor immunity. GT103 is a first-in class, fully human-derived IgG3 anti-CFH monoclonal antibody that recognizes a tumor specific epitope on CFH and has shown anti-tumor activity in preclinical models. Methods: We conducted a phase Ib, first-in-human, dose escalation trial evaluating safety and preliminary efficacy of GT103 in patients (pts) with advanced NSCLC refractory to standard therapies. Pts received GT103 intravenously across six dose levels/schedules following 3+3 design. Primary objectives were to determine maximum tolerated dose (MTD), recommended phase II dose (RP2D), and pharmacokinetic profile of GT103. Secondary objectives were to assess objective response rate, progression free survival (PFS), and overall survival (OS). Here we present updated safety and efficacy results of the study. Results: 31 pts were enrolled from 6/2020 to 9/2023 (median age 63yrs (range, 23-79), 61% had adenocarcinoma, 32% had previously treated brain metastasis). MTD was not reached. Treatment related AEs (TRAEs) observed in ≥10% pts included fatigue (19%), anemia (16%), diarrhea (16%), and nausea (13%). 3 pts developed ≥ grade 3 TRAE, including acute kidney injury, decreased lymphocyte count, and anemia. The best treatment response was stable disease in 9 (29%) pts. Median(m) PFS and mOS were 6 weeks (95% CI 6.0-6.1) and 25.7 weeks (95% CI 19.1-30.6), respectively. 24-week PFS and OS were 9.7% (95% CI 2.5-22.9%) and 50.6% (95% CI 31.9-66.6%), respectively. PD-L1 expression was available in 25 pts. No statistically significant difference (p=0.33) was noted in mPFS between PD-L1 positive (TPS > 1%) vs negative (TPS ≤1%) pts. KRAS and EGFR mutation status was available for 22pts. mPFS and mOS were numerically longer in pts with KRAS positive vs negative disease. While there was no difference in mPFS, 24-weeks PFS was 0 vs 16.7% (95% CI 4.1-36.5%) EGFR MUT vs wild type disease. Biomarker analysis of complement regulatory proteins, FcGR expression and polymorphisms, and changes in sC5b-9 is underway. Conclusions: GT103 was well tolerated across all dose levels. The RP2D of 10mg/kg IV every 3 weeks was selected, although PK modeling will be performed to optimize the dosing schedule. Encouraging anti-tumor activity was seen in heavily pretreated pts with advanced NSCLC with a subset of patients experiencing stable disease lasting for longer than 6 months. Additional biomarker analysis is ongoing. A phase II trial combining GT103 with pembrolizumab in pts with advanced NSCLC is currently enrolling. Clinical trial information: NCT04314089 .

Preclinical development of ATR04-484, an auxotrophic strain of Staphylococcus epidermidis, for the topical treatment of epidermal growth factor receptor (EGFR) inhibitor-induced dermal toxicity.

e24074 Background: Agents targeting the EGFR-mediated signaling pathway are commonly used for the treatment of advanced lung, pancreatic, colorectal, and head and neck cancers. Significant dermal toxicities, which occur in up to 90% of patients treated with EGFR inhibitors (EGFRis) and other therapies inhibiting downstream signaling pathways, may be disruptive to a patient’s quality of life and adherence to therapy. Inhibition of the EGFR pathway may suppress host defenses and lead to opportunistic pathogenic colonization or infection. EGFRi-induced dermal toxicity is associated with elevated levels of Staphylococcus aureus and IL-36γ. ATR04-484 is a topical ointment containing S. epidermidis strain SE484, isolated from a healthy human volunteer that was selected for its ability to inhibit S. aureus and IL-36γ . Methods: Reconstructed human epidermis (RHE) and ex vivo pig skin were used to measure the effect of ATR04-484 on S. aureus in therapeutic and prophylactic settings with S. aureus added prior to or after ATR04-484, respectively. To evaluate the anti-inflammatory effects of ATR04-484, IL-36γ levels were measured on untreated RHE and RHE treated with erlotinib alone or in combination with ATR04-484. 104 colony-forming units (CFU) of methicillin-resistant (MRSA) or methicillin-sensitive (MSSA) S. aureus were used. Toxicology was assessed by measuring cell viability in three in vitro toxicology assays: the eye irritant potential was tested on 3D reconstituted human cornea-like epithelium (RhCE), the skin irritant potential of the strain in the ointment was assessed on 3D RHE, and the skin corrosive potential was evaluated on RHE. Results: ATR04-484 comparably inhibited growth of both MRSA and MSSA in therapeutic and prophylactic settings. In a therapeutic setting, ATR04-484 inhibited MRSA growth by 1 log (90%) compared to untreated MRSA in both RHE and pig skin. In a prophylactic setting, ATR04-484 inhibited MRSA growth by approximately 5 logs on RHE and 2 logs on pig skin. Additionally, application of ATR04-484 reduced IL-36γ to a level comparable to untreated RHE. The effect was dose-dependent; application of 109CFU/cm2 of ATR04-484 showed more potent IL-36γ reduction compared to 108 CFU/cm2. The three toxicology assays indicate that ATR04-484 does not cause irritation or corrosion. Conclusions: In these preclinical studies, ATR-04-484 showed a promising preclinical profile for the treatment of EGFRi-induced dermal toxicity by significantly reducing S. aureus growth, completely ameliorating IL-36γ levels, and showing no safety concerns.

Evaluating antitumor activity and response determinants to trastuzumab deruxtecan in pediatric solid tumors.

10049 Background: Trastuzumab deruxtecan (T-DXd) is an anti-HER2 antibody-drug conjugate (ADC) linked to a topoisomerase inhibitor FDA-approved for several indications, including the treatment of HER2+ breast and gastric cancer. Although HER2 amplification is uncommon in pediatric cancers, recent demonstration of T-DXd efficacy in HER2-low breast cancer patients prompted us to examine the potential clinical relevance of T-DXd in pediatrics by evaluating HER2 expression and activity of T-DXd in preclinical pediatric solid tumor models. Methods: Cell viability was assessed in a panel of histologically diverse pediatric solid tumor cell lines and 2 HER2-amplified adult cancer cell lines treated with T-DXd, payload (DXd), and a control IgG-ADC. HER2 protein expression in patient-derived xenograft (PDX) tumors was evaluated by immunohistochemistry (IHC) using 5 clinically validated anti-HER2 antibodies. RNAseq was performed on clinical (n=290) and PDX (n=184) tumors to determine relative expression of ERBB2. In vivo activity of T-DXd was evaluated in osteosarcoma (OS, n=10), Wilms tumor (WT, n=1) and malignant rhabdoid tumor (MRT, n=1) PDX models and a desmoplastic small round cell tumor (DSRCT) cell line xenograft model. Differences in tumor volume and time to disease progression was assessed and compared across treatments and models. Responses were correlated with HER2 expression by IHC. Results: In vitro, HER2-amplified control cell lines demonstrated a >30-fold reduction of IC50 when comparing T-DXd to ADC control. In contrast, the ADC control IC50 was nearly identical to T-DXd across all pediatric cancer cell lines, consistent with the absence of HER2 amplification in these models. We observed focal and membranous staining of HER2 by IHC in PDXs but was quite variable across antibodies tested: WT 0-17% HER2+ cases (n=6), MRT 0-40% (n=5), DSRCT 0-33% (n=24), OS 0-7% (n=30). ERBB2 gene expression was highest in DSRCT followed by WT, OS and MRT. In vivo efficacy studies demonstrated complete and partial responses in OS, WT, and DSRCT and improved disease control rates (OS: 67%, p=0.006, Mann-Whitney; WT: 100%; DSRCT: 100%). However, T-DXd and ADC control demonstrated similar activity in all tumor types with no consistent correlation between in vivo response and HER2 expression. Consistent with these preclinical studies, 4 patients with progressive DSRCT were treated with T-DXd via compassionate/off-label access with signs of clinical and radiographic responses. Conclusions: T-DXd shows significant preclinical antitumor activity across multiple pediatric solid tumors but little correlation with HER2 expression suggesting a mechanism of action similar to the clinical activity observed in HER2-low breast cancer. Xenograft efficacy studies and preliminary clinical experience with T-DXd in DSRCT patients warrant formal clinical trial investigation in this largely incurable disease.

First-in-human phase 1 study of SPH4336, a novel cyclin-dependent kinase 4/6 inhibitor, in patients with advanced solid tumors.

e15107 Background: The inhibition of cyclin-dependent kinases (CDKs) is a highly promising and attractive strategy for the development of anticancer drugs. SPH4336, a novel oral inhibitor with high selectivity and improved inhibition for CDK4/6, has demonstrated promising anti-tumor activity in preclinical studies. This was a first-in-human, multicenter, phase 1 dose-escalation and -expansion study to assess the safety, pharmacokinetics (PK) and preliminary efficacy of SPH4336 in patients with advanced solid tumors. Methods: Patients aged 18-70 years with histologically or cytologically confirmed advanced solid tumors that were refractory or intolerant to standard treatment or for which no standard treatment exists were included. Dose escalation of SPH4336 was conducted at doses of 50 mg, 100 mg, 200 mg, 300 mg, 400 mg and 600 mg once a day. Primary endpoint was to determine the maximum tolerated dose (MTD), dose-limiting toxicity (DLT) and pharmacokinetics of SPH4336. Secondary endpoints included assessment of adverse events (AEs) and efficacy. Descriptive statistics summarized safety, efficacy and PK parameters. Kaplan-Meier method was used to analyze time-to-event variables. Results: A total of 29 patients were enrolled with a median age of 55.0 years (23–66) and 72% being females. The most common tumor types were breast cancer (n=14, 48.3%) and sarcoma (n=8, 27.6%). No DLT was observed within 50 to 600 mg and MTD was not determined. Treatment-related adverse events (TRAEs) were reported in 29 (100%) patients, the most common TRAEs were white blood cell count decreased (n=21, 72.4%), diarrhea (n=20, 69.0%), neutrophil count decreased (n=19, 65.5%) and anemia (n=18, 62.1%). No grade 4 TRAEs were observed and death occurred in 1 patient (200 mg cohort) due to disease progression. Of the 27 evaluable patients, 1 patient achieved partial response lasting 169 days, and disease control rate was 59.3% (95% CI: 38.8%-77.6%). Regression of target lesion was observed in 100, 400 and 600 mg cohorts from baseline. PK assessments showed that the exposure of SPH4336 increased in a dose-dependent manner except with 600 mg dose. SPH4336 reached a steady state at 2 weeks of continuous dosing administration at 400 mg. Conclusions: This study demonstrated that SPH4336 was well tolerated at doses ranging from 50 to 600 mg dose levels. A dose of 400 mg could be considered for further clinical trials. Clinical trial information: NCT05905614 .

Phase 2 study of novel HER2-targeting, TLR7/8 immune-stimulating antibody conjugate (ISAC) BDC-1001 (trastuzumab imbotolimod) +/- pertuzumab (P) in patients (pts) with HER2-positive metastatic breast cancer (MBC) previously treated with trastuzumab deruxtecan (T-DXd).

TPS1121 Background: Therapies to manage pts with HER2+ MBC have significantly improved, but better agents are still needed. BDC-1001 (trastuzumab imbotolimod) is being studied for MBC and other HER2+ cancers. The ISAC BDC-1001 consists of a trastuzumab biosimilar conjugated to a cell membrane impermeable TLR7/8 agonist via a non-cleavable linker and is given IV every 2 wks. Designed to trigger the innate immune system, BDC-1001 causes a durable tumor-targeted adaptive immune response. Early studies show that HER2-targeted ISACs elicit potent and durable immune-mediated antitumor efficacy, and tumor regression, in a TLR- and Fc receptor-dependent manner (1). Preclinical studies with a murine surrogate of BDC-1001 (trastuzumab-T785 ISAC) show better efficacy with trastuzumab-T785 compared to trastuzumab + P. In the BDC-1001 dose-escalation trial (BBI-20201001), 6 PRs and 12 SDs ≥ 24 weeks in 8 tumor types were observed, particularly in 20mg/kg q2w dose cohorts. The most frequent drug-related AE was low grade (grade 1/2) infusion-related reactions (29%) (2). We showed the combination of a surrogate ISAC (trastuzumab-T785) + P significantly enhances efficacy in multiple HER2-expressing tumors, including those with lower HER2 expression (1). The addition of P lowered the quantity of ISAC required for anti-tumor activity in the JIMT-1 HER2 IHC2+ model. The combination of P with the ISAC significantly increased the tumor cytokine and chemokine concentration compared to monotherapy or antibody-alone, indicating enhanced tumor myeloid activation suggesting this combination may enhance the clinical activity of trastuzumab-based ISACs. Methods: The Phase 2 multicenter, open-label, randomized study (BBI-20231001) is enrolling up to 66 patients with HER2-positive (ASCO/CAP 2018) MBC previously treated with 2 or more anti-HER2 therapies including T-DXd. Patients must be > 18 years, have measurable disease, and have ECOG performance status of 0 or 1. Patients will be randomized 1:1 to receive BDC-1001 20mg/kg every 2 weeks with or without P (840mg initial dose, followed by 420mg IV q 3w). Each arm has a Simon 2 stage design. The primary objective is anti-tumor activity of BDC-1001 alone and in combination with P. Secondary objectives will evaluate safety, PK, and immunogenicity of BDC-1001 alone and in combination with P. Exploratory objectives will include PD biomarkers in tumor tissue and in peripheral blood to elucidate the MOA and biomarkers associated with BDC-1001 activity with or without P. This study began accrual in Nov 2023. 1. Ackerman S, et al. Nature Cancer. 2021. 2. Li B, et al. ASCO 2023, Abstract 2538. Clinical trial information: NCT05954143 .