Abstract Background: Aging is associated with a chronic low-grade inflammatory state that profoundly influences tumor development and progression, known as inflammaging. While this is due to increased myelopoiesis, the exact mechanisms leading to increased myeloid production and the effects on the gastric tumor microenvironment remain poorly understood. Methods: We conducted comprehensive analyses of young (3 months) and aged (18 months) Hdc-GFP mice, evaluating expression of the peptide trefoil factor family 2 (TFF2) and abundance of GFP+ MDSCs in the stomach, circulation and other sites. The ACKP orthotopic model and Mist1CreERT; RhoAY42CCDH1ff; Hdc-GFP mouse (GEM) model were used to test whether aging promotes gastric cancer progression and to explore the underlying mechanisms. TFF2-MSA was given by i.p. injection twice weekly for 2 weeks before ACKP tumor inoculation, to test whether pretreatment could reverse the aging-related pro-tumor effects. Results: Aging led to a significant reduction in TFF2 levels in both the stomach and circulation, as confirmed by IHC, ELISA, and qPCR. The decline in circulating TFF2 was accompanied by elevated IL-1β expression in bone marrow, peripheral blood, and gastric neutrophils, together with increased circulating IL-1β levels. Enhanced IL-1β signaling contributed to a myeloid-biased hematopoietic phenotype, leading to the increased GFP+ MDSCs in the stomach, spleen, and peripheral blood. Elevated IL-1β in the aged stomach induced fibroblast activation with a senescence-associated secretory phenotype (SASP)-like profile, including increased IL-6, CXCL1 and CXCL2 expression. In the ACKP orthotopic and model and GEM model, with tumorigenesis initiated at 3mo or 18mo, aging significantly accelerated tumor growth and shortened survival. Immunofluorescence analyses of aged ACKP tumor identified an increased subset of IL-1R1+ cancer-associated fibroblasts (CAFs) and CGRP+ sensory nerves. Functionally, pretreatment with TFF2-MSA prior to ACKP tumor implantation decreased IL-1R1+ CAF and sensory nerves, reduced MDSC accumulation and IL-1β expression, and suppressed gastric tumor growth in aged mice. Conclusion: Our findings uncover a systemic aging axis linking reduced gastric TFF2 production to myelopoiesis and thus to gastric cancer susceptibility. Aging-associated IL-1β signaling from expanded myeloid cells reprograms gastric fibroblasts toward a pro-inflammatory SASP phenotype, fostering immune suppression and tumor growth. Restoring youthful TFF2 levels may provide effective strategies to mitigate the aging-related tumor risk and other associated inflammaging syndromes. Citation Format: Shuang Li, Hualong Zheng, Jin Qian, Puran Zhang, Feijing Wu, Yi Zeng, Biyun Zheng, Juli Lin, Hiroki Kobayashi, Yosuke Ochiai, Mashahiro Hata, Arai Junya, Leah B. Zamechek, Bruce Daugherty, Seth Lederman, Timothy C. Wang. TFF2 deficiency amplifies IL-1β-driven inflammation and promotes aging-associated gastric tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6822.

Blocking CD154 (CD40L) has the potential to prolong transplanted solid organ graft survival and treat autoimmune diseases. However, first-generation anti-CD154 IgG1 monoclonal antibodies (mAbs) were associated with an increased risk of thrombosis linked to Fc binding to FcγRIIa (CD32A). Here, we describe a first-in-human, phase 1 clinical trial of TNX-1500, a novel Fc-modified IgG4 anti-CD154 mAb designed to decrease binding to FcγRIIa. Healthy volunteers (N = 26) were enrolled into single-ascending dose (3, 10, and 30 mg/kg) cohorts and received TNX-1500 intravenously. TNX-1500 was generally well tolerated. Among participants receiving TNX-1500 3, 10, and 30 mg/kg, 1 (25

OBJECTIVE:Fibromyalgia is the prototypic nociplastic chronic pain syndrome, characterized by widespread pain, nonrestorative sleep, and fatigue. We evaluated efficacy and safety of bedtime TNX-102 SL (sublingual cyclobenzaprine) 5.6 mg for treatment of fibromyalgia. METHODS:This phase 3, double-blind, multicenter, placebo-controlled trial randomized patients 1:1 to once-nightly TNX-102 SL 2.8 mg for 2 weeks, followed by 5.6 mg for 12 weeks, or to matching placebo (NCT05273749). The primary endpoint was change from baseline at week 14 in weekly average of daily diary pain intensity scores. Secondary endpoints included Patient Global Impression of Change, Fibromyalgia Impact Questionnaire (Revised) Symptoms and Function domains, Patient-Reported Outcomes Measurement Information System instruments for Sleep Disturbance and Fatigue, and daily diary sleep quality scores. RESULTS:Overall, 81.0% (n = 187/231) and 79.6% (n = 179/225) of patients receiving TNX-102 SL and placebo completed the trial, respectively. Treatment with TNX-102 SL vs placebo was associated with significantly greater reductions in the primary pain endpoint (P < .001; mean [SE], -1.8 [0.12] vs -1.2 [0.12]) and in each of the 6 secondary endpoints (P ≤ .001; all). The most common systemic treatment-emergent adverse events (TEAEs) with TNX-102 SL and placebo were COVID-19 (4.3% vs 3.1%, respectively), headache (3.0% vs 1.8%), and somnolence (3.0% vs 1.3%); the most common TEAEs overall were local administration-site reactions including oral hypoesthesia (23.4% vs 0.4%), product taste abnormal (11.3% vs 0.9%), and oral paresthesia (6.9% vs 0.9%), which were transient and self-limited. CONCLUSION:Bedtime TNX-102 SL treatment was associated with significant improvements in fibromyalgia symptoms and function and was well tolerated. CLINICAL TRIAL REGISTRATION:ClinicalTrials.gov/study/NCT05273749, first patient screened March 21, 2022.

Problem Statement/Background TNX-102 SL significantly reduced pain in 2 Phase 3 studies in fibromyalgia (FM; RELIEF and RESILIENT studies). Purpose/Aim Assess the effects of TNX-102 SL on FM symptoms and function by pooling data from the RELIEF and RESILIENT studies. Methods and Design RELIEF (NCT04172831) and RESILIENT (NCT05273749) were similarly designed, 14-week, randomized, double-blind, placebo-controlled pivotal studies of efficacy and safety of daily bedtime TNX-102 SL 5.6mg in patients with FM. The primary efficacy endpoint was mean change from baseline to Week 14 in the weekly average of daily self-reported pain numeric rating scale intensity scores. Key secondary endpoints included proportion of patients with Patient Global Impression of Change (PGIC) response (ie, “very much” or “much” improved), mean change from baseline in the Fibromyalgia Impact Questionnaire – Revised (FIQR) Symptoms and Function domain scores and in Patient Reported Outcomes Measurement Information System (PROMIS) Fatigue 8a and Sleep Disturbance 8a score, all at Week 14. Data were pooled for this analysis, and comparisons were made between TNX-102 SL and placebo. Safety data will also be presented. Results In total, 959 patients (TNX-102 SL 5.6mg, n=479; placebo, n=480) were included; 783 (81.6%) completed the studies. Mean (SD) age was 49.5 (10.3), 95.3% of patients were female, and 85.9% were White/Caucasian. For the primary endpoint, the least-squares (LS) mean difference (95% CI) in change from baseline in pain score was -0.53 (-0.75, -0.30), significantly favoring TNX-102 SL vs placebo (P<0.001). The PGIC “response” for TNX-102 SL vs placebo at Week 14 was 36% vs 25%, respectively (P<0.001). The LS mean difference (95% CI) in change from baseline to Week 14 was -5.87 (-8.14, -3.61; P<0.001) for FIQR Symptom domain score and -4.78 (-7.15, -2.42; P<0.001) for FIQR Function domain score, significantly favoring TNX-102 SL. The LS mean difference (95% CI) in change from baseline to Week 14 in PROMIS Fatigue and Sleep Disturbance scores, -2.37 (-3.41, -1.33; P<0.001) and -3.55 (-4.66, -2.44; P<0.001, respectively, also favored TNX-102 SL. Conclusion/Implications for Nursing Profession In this pooled analysis, treatment with TNX-102 SL 5.6mg significantly reduced pain scores, provided significantly greater patient-reported response rates, and was associated with significantly greater improvements in the FIQR Symptom and Function domains and PROMIS Fatigue and Sleep Disturbance scores vs placebo.Implications for future researchFindings enhance the growing body of evidence supporting the efficacy of TNX-102 SL 5.6mg in the treatment of patients with FM. These results highlight the syndromal improvements observed and the potential of TNX-102 SL to improve other symptoms such as fatigue and nonrestorative sleep that occur in the context of chronic pain. FundingTonix Pharmaceuticals Holding Corp.

Abstract Introduction: B and T lymphocyte attenuator (BTLA) is an immune checkpoint receptor essential for immune homeostasis and tolerance. Unlike PD-1 or CTLA-4, BTLA is broadly expressed on lymphoid and some myeloid cells, including T cells, B cells, dendritic cells, and macrophages. Engagement with its ligand HVEM (herpesvirus entry mediator) transmits inhibitory signals that suppress immune activation. Within tumors, BTLA contributes to immune evasion by dampening anti-tumor responses. Given its unique expression profile and regulatory function, we developed monoclonal antibodies (mAbs) targeting BTLA as an immunotherapeutic strategy to enhance anti-tumor immunity. Methods: Fully human anti-BTLA mAbs identified by hybridoma screening were reformatted as huIgG4 (S228P/L235A) and transiently expressed using Curia’s TunaCHO® platform. The S228P mutation prevents Fab-arm exchange, stabilizing mAb structure, while L235A minimizes FcγR binding and effector functions. Binding potency was determined by ELISA; kinetics by Octet® biolayer interferometry (BLI) against human and cynomolgus BTLA. BLI also assessed FcγR, FcRn (at pH 6.0 vs 7.2), and C1q binding to evaluate recycling potential and complement activation. Neutralization of HVEM binding to BTLA was quantified by modified ELISA and cell-based reporter assays. Results: Six fully human anti-BTLA mAbs and a clinical candidate comparator JS004 displayed similar ∼1 nM binding potencies (IC50) values to human and cyno BTLA by ELISA. Clone 13-F7-A exhibited the highest affinity (Kd < 1 nM) for both human and cyno BTLA among all 7 mAbs tested. Four mAbs and the comparator JS004 were high affinity binders to human and cyno BTLA (Kd 1-32 nM). All six fully human mAbs exhibited reduced FcγR binding relative to JS004. Each bound FcRn at pH 6.0 but not at 7.2, consistent with normal endosomal recycling. None bound C1q, whereas a positive-control IgG1 did bind to C1q as expected. In an ELISA-based biofunctional assay, all mAbs blocked HVEM binding to BTLA with similar IC50 values (∼1 nM). Conclusions: We generated potent, high affinity, human/cyno cross-reactive, fully human antagonistic anti-BTLA mAbs. Targeting BTLA offers promising opportunities for cancer immunotherapy and may demonstrate strong synergy when combined with other checkpoint antagonists, potentially overcoming resistance mechanisms and improving clinical outcomes. Citation Format: Bruce L. Daugherty, Hsunhui Yang, Subhra Mahapatra, Yadong Yu, Christine L. Hsieh, Brian A. Zabel, Seth Lederman. In vitro characterization of fully human antagonistic anti-BTLA monoclonal antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6550.

Daily oral cyclobenzaprine hydrochloride (HCl) has provided transient benefits in fibromyalgia, a chronic pain condition. To improve this effect, we evaluated sublingual formulations designed to drive transmucosal absorption. Two open-label studies evaluated the pharmacokinetics (PK), tolerability, and relative bioavailability of sublingual cyclobenzaprine HCl in healthy adults. In Study 1 (n = 24), three 2.8 mg sublingual formulations of cyclobenzaprine HCl containing potassium phosphate dibasic (A), sodium phosphate dibasic (B), or trisodium citrate (C) were compared to immediate release (IR) cyclobenzaprine HCl 5 mg. All sublingual formulations showed increased bioavailability (154% [A], 126% [B], and 125% [C]) and rapid absorption. Formulation A demonstrated the most favorable PK, with a ∼3 min absorption lag versus ∼37 min for oral IR, and a 783% higher dose-normalized AUC0-1. Formulation A was designated TNX-102 SL for further development. In Study 2 (n = 16), TNX-102 SL 2.8 and 5.6 mg exhibited dose proportionality and no food effect. Furthermore, this is the first report describing the active metabolite norcyclobenzaprine in clinical studies, showing an elimination half-life of ∼60 h. Oral hypoesthesia and abnormal taste were the most common adverse events. These findings support TNX-102 SL as a rapidly absorbed and efficient sublingual tablet formulation of cyclobenzaprine HCl, providing effective transmucosal delivery.

This single-center, open-label, randomized, multiple-dose, pharmacokinetic bridging study was conducted to compare systemic exposure (AUC and Cmax) of a sublingual tablet formulation of cyclobenzaprine hydrochloride (TNX-102 SL, approved as TONMYA) at steady-state with that of an approved reference product, an oral, extended-release (ER) capsule formulation, supporting development under the 505(b)(2) regulatory pathway. Healthy adults (N = 60) were randomly assigned 1:1 to receive two sublingual 2.8-mg tablets or one oral ER 30-mg capsule once daily for 20 days. At steady state, 2 sublingual 2.8 mg tablets provided lower exposure to cyclobenzaprine and norcyclobenzaprine compared with one oral 30-mg ER capsule. However, when exposures were normalized by dose, cyclobenzaprine AUC0-24 and Cmax were higher on both Day 1 and Day 20 with two sublingual tablets versus one oral ER capsule, supporting higher bioavailability of the sublingual tablet formulation. For Day 20, the dose-normalized ratios (two sublingual tablets: one oral ER capsule) for AUC0-τ ss and Cmax ss were 140% and 152%, respectively. Sublingual tablets and oral ER capsules had comparable metabolic profiles of Phase I and II metabolites in plasma over 20 days, with no unique metabolites by the sublingual route. Sublingual cyclobenzaprine HCl tablets were generally safe and well-tolerated.

Abstract Introduction: TNX-1700 is a novel recombinant fusion molecule of human Trefoil Factor-2 (TFF2) protein and human serum albumin (HSA) that is being investigated as a potential therapeutic for gastric cancer. In syngeneic mouse models of gastric and colorectal cancer, TNX-1700 functions as a CXCR4 partial agonist that activates antitumor immunity in the tumor microenvironment by modulating myeloid cell trafficking to reduce tumor-induced granulopoiesis and accumulation of immunosuppressive neutrophils. TNX-1700 is engineered to extend plasma half-life, enhance systemic exposure, and improve cancer immunotherapy. The HSA domain in TNX-1700 provides a long circulatory half-life (>14 days) and multiple ligand-binding sites. Approved albumin-linked drugs include detemir (Levemir®), liraglutide (Victoza®), and albiglutide (Eperzan®/Tanzeum®) for diabetes, and nanoparticle albumin-bound paclitaxel (nab-paclitaxel) for cancer therapy. TNX-1700 represents a next-generation application of the albumin platform in immuno-oncology. Methods: The pharmacokinetics (PK) of TNX-1700 were evaluated in non-human primates (NHP; cynomolgus macaques) and double-transgenic mice expressing human neonatal Fc receptor (FcRn) and human serum albumin (HSA). Animals received a single dose of 1 mg/kg or 3 mg/kg TNX-1700, administered intravenously (IV) to NHPs or intraperitoneally (IP) to FcRn/HSA mice. For comparison, untagged human TFF2 (molar equivalent to TNX-1700) was also administered into FcRn/HSA mice. Serial blood samples were collected over 0-35 days and analyzed using the Boster PicoKine™ Human TFF2 ELISA kit. Pharmacokinetic parameters were determined by non-compartmental analysis. Results: All animals survived without clinical signs or >10% body-weight loss. Comparable PK profiles were observed across species and doses. In cynomolgus macaques, mean terminal half-life (t½) was 7.1 days (%CV = 9.65), clearance (CL) 13.3 mL/day (%CV = 14.3), and volume of distribution (Vz) 135.2 mL (%CV = 18.3). Allometric scaling predicted in humans a t½ of 14.2 days (%CV = 12.9), CL = 105.2 mL/day (%CV = 26.4), and Vz = 2,158 mL (%CV = 34.0). Results from the humanized murine studies provided evidence that untagged human TFF2 is rapidly cleared and that fusion with HSA significantly increased the PK profile similar to that observed in NHPs and to levels supportive for clinical candidates. Conclusion: TNX-1700 exhibited dose-independent, linear pharmacokinetics with low inter-animal variability, and exposure was consistent across doses and species. Although its half-life is shorter and clearance higher than IgG-based biologics, TNX-1700 substantially extends the half-life of TFF2 and achieves durable systemic exposure, supporting its potential as a therapeutic candidate for gastric cancer. Citation Format: Mayanka Awasthi, Jennifer Cho, Nelson Martinez, Bernd Meibohm, Seth Lederman, Christopher Cooper, Sina Bavari, Bruce L. Daugherty. Pharmacokinetics of TNX-1700 in non-human primates and human FcRn/serum albumin transgenic mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7940.

Background.Long-term renal allograft acceptance has been achieved in macaques using a transient mixed hematopoetic chimerism protocol, but similar regimens have proven unsuccessful in heart allograft recipients unless a kidney transplant was performed simultaneously. Here, we test whether a modified protocol based on targeting CD154, CD2, and CD28 is sufficient to prolong heart allograft acceptance or promote the expansion of regulatory T cells. Methods.Eight macaques underwent heterotopic allo-heart transplantation from major histocompatibility complex-mismatched donors. Induction treatment for donor bone marrow transplantation (BMT) was administered after a 4-mo delay period under TNX-1500 monotherapy. The BMT induction regimen comprised 1 (group 1, G1; n = 3) or 2 (group 2, G2; n = 5) doses of total body irradiation, thymic irradiation, and antithymocyte globulin, followed by 2 (G1) or 5 (G2) weekly doses of alpha CD2 and 5 weekly treatments with alpha CD28 and TNX-1500. Results.During the delay period, 1 G1 graft was rejected and 2 (1 in each group) exhibited moderate rejection on protocol biopsy before BMT. Lymphocyte chimerism was seen in 3 of 5 G2 animals and in 1 of 2 G1 recipients. One G1 graft was rejected despite chimerism, whereas the other recipient succumbed to anti-cytomegalovirus treatment. Two G2 monkeys succumbed due to infection (cytomegalovirus, bacteremia) post-BMT and 3 due to posttransplantation lymphoproliferative disease. Conclusions.Intensive costimulation pathway blockade with alpha CD2, alpha CD154, and alpha CD28 promotes lymphocyte chimerism at the cost of high incidence of posttransplantation lymphoproliferative disease and opportunistic infections, preventing assessment of the effectiveness of the regimen to promote alloimmune tolerance.

Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) are pathologically activated neutrophils that impair immunotherapy responses. The chemokine receptor CXCR4, a central regulator of hematopoiesis, represents an attractive PMN-MDSC target. We generated a novel peptide, TFF2-MSA, as a CXCR4 partial agonist by fusing Trefoil Factor 2 (TFF2) with murine serum albumin (MSA), and compared its treatment efficacy in combination with anti-PD-1 antibody to that of the clinically available CXCR4 antagonist plerixafor (AMD3100). Murine syngeneic gastric cancer cells were subcutaneously implanted into the Hdc-GFP transgenic mice, which enabled tracking of PMN-MDSCs via histidine decarboxylase (Hdc) expression across multiple tissues. Treatments were then initiated every three days. To elucidate mechanisms, myeloid progenitors were cultured with or without TFF2-MSA in vitro. Bone marrow Lin-c-Kit+ hematopoietic progenitors from tumor-bearing Hdc-GFP (CD45.2) mice following vehicle or TFF2-MSA treatments were transferred into CD45.1 mice to validate TFF2-MSA’s effects on PMN-MDSC generation. In tumor-free mice, we compared the short-term bone marrow cell mobilization effects of TFF2-MSA and AMD3100. Additionally, we analyzed human blood and single-cell RNA sequencing (scRNA-seq) data from gastric cancer tissues for immune profiles. The combination of TFF2-MSA plus anti-PD-1 antibody showed significant efficacy in reducing/eradicating primary and metastatic tumors and extending mouse survival, in contrast to the CXCR4 antagonist AMD3100, which did not enhance anti-PD-1 response. Mechanistically, TFF2-MSA inhibited the expansion of CXCR4-high granulocyte-monocyte progenitors (GMPs) and the generation of Hdc-GFP+ PMN-MDSCs in culture, which underlies its systemic reduction of Hdc-GFP+ PMN-MDSCs in tumor, blood, and spleen and GMPs in bone marrow in vivo. In contrast, AMD3100 induced peripheral accumulation of Hdc-GFP+ PMN-MDSCs and compensatory elevation of GMP levels in bone marrow. Congenic adoptive transfer experiments confirmed that TFF2-MSA suppressed Hdc-GFP+ PMN-MDSC generation in vivo. Consistently, in tumor-free mice, TFF2-MSA did not significantly mobilize granulocytes from bone marrow, unlike AMD3100, thus better preserving bone marrow homeostasis. In gastric cancer patients, a CXCR4+LOX-1+ PMN-MDSC population expanded in both blood and tumor, exhibiting immunosuppressive potential, and associated with reduced TFF2 plasma level compared with healthy donors. The novel CXCR4 partial agonist exhibited superiority in tumor control and effective PMN-MDSC reduction, and preservation of bone marrow homeostasis compared to the CXCR4 antagonist AMD3100. CXCR4 partial agonism may represent a promising strategy for targeting PMN-MDSCs in gastric cancer patients. Jin Qian, Chenkai Ma, Quin T. Waterbury, Xiaofei Zhi, Christine S. Moon, Ruhong Tu, Hiroki Kobayashi, Feijing Wu, Biyun Zheng, Yi Zeng, Hualong Zheng, Yosuke Ochiai, Ruth A. White, David W. Harle, Jonathan S. LaBella, Leah B. Zamechek, Lucas ZhongMing Hu, Ryan H. Moy, Arnold S. Han, Bruce L. Daugherty, Seth Lederman, Timothy C. Wang. TFF-2-mediated CXCR4 partial agonism outperforms CXCR4 antagonism in reducing murine gastric cancer by suppressing PMN-MDSC generation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2245.

Pathologically activated immunosuppressive neutrophils impair cancer immunotherapy efficacy. The chemokine receptor CXCR4, a central regulator of hematopoiesis and neutrophil biology, represents an attractive target. Here, we fuse a secreted CXCR4 partial agonist, trefoil factor 2 (TFF2), to mouse serum albumin (MSA) and demonstrate that TFF2-MSA peptide synergizes with anti-PD-1 to inhibit primary tumor growth and distant metastases and prolongs survival in gastric cancer (GC) mouse models. Using histidine decarboxylase (Hdc)-GFP transgenic mice to track polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) in vivo, we find that TFF2-MSA selectively reduces the Hdc-GFP+CXCR4high immunosuppressive neutrophils, thereby boosting CD8+ T cell-mediated tumor killing with anti-PD-1. Importantly, TFF2-MSA reduces bone marrow granulopoiesis, contrasting with CXCR4 antagonism, which fails to confer therapeutic benefits. In GC patients, elevated CXCR4+LOX-1+ low-density neutrophils correlate with lower circulating TFF2 levels. Collectively, our studies introduce a strategy that utilizes CXCR4 partial agonism to restore anti-PD-1 sensitivity by targeting immunosuppressive neutrophils and granulopoiesis.