Background: Live-attenuated Listeria monocytogenes (Lm) vectors are a clinically validated cancer immunotherapy platform, but translation requires reproducible, clinically realistic workflows for dose preparation and infusion. For live bacterial products, in-use stability and device compatibility can drive dose variability through adsorption, settling, and device losses. Methods: We developed and GMP-manufactured an attenuated Lm vaccine expressing human GUCY2C (Lm-GUCY2C) and performed translational characterization, including construct verification and immunogenicity readouts, and defined the administration-focused in-use stability and device compatibility. Post-thaw stability was assessed in primary cryovials and during preparation and delivery from 250 mL saline infusion bags using standard clinical devices (syringes/needles, filter-free IV tubing) and OnGuard2 closed-system components. Samples were collected over 24 h at room temperature, and viable Lm-GUCY2C were quantified by CFU recovery. Results: Lm-GUCY2C remained stable in thawed cryovials for 24 h with no significant CFU loss. High-dose infusion bags (3 × 109 CFU/bag) maintained CFU recovery through 6 h, whereas low-dose bags (3 × 108 CFU/bag) exhibited significant losses beginning at 3 h, supporting a practical in-use window of up to 2 h for low-dose preparations. OnGuard2 intravenous (i.v.) connectors did not measurably affect CFU recovery, while OnGuard2 vial adapters reduced recovery. Conclusions: This work provides an end-to-end, translationally focused characterization of a GMP-manufactured Lm cancer vaccine, including clinically actionable in-use handling constraints and device compatibility. These data define preparation and administration guardrails (notably, time-to-infusion limits for low-dose bag preparations) that can improve dose accuracy and reproducibility in clinical testing.

Abstract Background: The WNT-β-catenin-APC signaling axis is the most mutated pathway in colorectal cancer (CRC). Two of the most commonly lost proteins in early colorectal tumorigenesis are the hormone ligands (guanylin and uroguanylin) for the tumor suppressor, guanylate cyclase 2C (GUCY2C). CRC tumorigenesis typically retains expression of GUCY2C, a transmembrane receptor expressed on the apical-lumen-facing surface of intestinal epithelial cells. Previously, we demonstrated that in CRC, activated WNT/β-catenin suppresses GUCY2C hormone expression, revealing an often-overlooked paradigm in which WNT/β-catenin signaling silences gene expression to promote CRC tumorigenesis. Results: Using four CRC lines with inducible WNT/β-catenin suppression, we found that WNT/β-catenin signaling reduces phosphorylation and activation of p38, a known tumor suppressor in CRC. We show, using both pharmacological and genetic approaches, that WNT/β-catenin-mediated inactivation of p38 is responsible for the loss of GUCY2C hormone expression. Additionally, p38 inactivation downstream of WNT/β-catenin accounts for the suppression of at least two-thirds of the genes repressed by WNT/β-catenin signaling. These studies identify a previously unrecognized gene set controlled by WNT/β-catenin via suppression of p38 activation. Next, we sought to identify the link between the WNT/β-catenin pathway and p38. Indeed, WNT/β-catenin signaling suppresses p38 activity through a MAPK-dependent mechanism, and inactivation of WNT/β-catenin increases MAP2K phosphorylation, while MAP2K3 knockdown mimics the effects of eliminating p38 on guanylin expression. In that context, phosphorylation of MAP2K is reduced in CRC tumors, compared to normal tissue, further supporting a role for this mechanism in CRC tumorigenesis. Conclusions: Our findings demonstrate that the WNT/β-catenin signaling pathway induces the expression of tumor promoters while also inducing broad gene suppression. Our results reveal broad intracellular signaling changes that occur during early CRC tumorigenesis following WNT/β-catenin mutations, providing mechanistic insight into both early CRC tumorigenesis and normal intestinal homeostasis. By elucidating these mechanisms, we can identify signaling pathways to restore guanylin and uroguanylin expression, reactivate GUCY2C, and reprogram gene expression disrupted by aberrant WNT/β-catenin signaling. In turn, this approach can provide novel strategies to prevent and treat colorectal cancer. Citation Format: Andrew E. Evans, Ariana A. Entezari, Adi Caspi, Jasmine A. Alvarez, Adam E. Snook, Scott A. Waldman. Oncogenic WNT/β-catenin signaling reduces MAPK signaling to suppress broad gene expression and promote colorectal cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(8_Suppl):Abstract nr LB283.

Abstract Background: Chimeric antigen receptor T-cell (CAR-T) therapy has shown success in hematologic malignancies; however, its efficacy in solid tumors, including colorectal cancer (CRC), remains limited by poor tumor penetration, an immunosuppressive microenvironment, antigen heterogeneity, and a risk of on-target/off-tumor toxicities. CD87, the receptor for the urokinase plasminogen activator (uPAR), has emerged as a promising target, given its near-universal overexpression in CRC, and its near absence in normal tissues, except certain myeloid cells. Recent studies on the elimination of senescent myeloid cells in mice have supported the safety of CD87-directed CAR-T cells in vivo. Here, we pursued safety and efficacy studies of human CD87-directed CART (CART87) for use in CRC. We employed a Jurkat NFAT-Lucia reporter platform to functionally screen and optimize human CD87-specific CAR designs, then advanced the top candidates into CARTs for testing against a panel of CRC cell lines. Moreover, we employed bioinformatics to identify potential risks associated with targeting CD87 in humans. Methods: To assess the potential safety of targeting CD87, we analyzed public bulk and scRNAseq datasets from human and mouse tissues. Furthermore, we quantified CD87 surface expression in human blood leukocyte populations and assessed their susceptibility to CART87. To optimize the CAR87 design, we employed a Jurkat NFAT-Lucia reporter assay, which measured CAR activation upon stimulation with recombinant uPAR and CD87-expressing cells. Constructs with high tonic signaling and non-specific activation by CD87-negative cells were eliminated. We then engineered CD87-directed CAR-T cells (CART87) and assessed their activity against CD87-positive CRC cell lines compared with CD87-negative HEK293 cells and CD87 knockout (KO) cells. Results: scRNAseq analysis of bone marrow, whole blood leukocytes, and organ tissues revealed that CD87 mRNA expression is restricted to mature monocyte-lineage cells, with murine organs/tissues showing analogous results. Correspondingly, CD87 surface expression by flow cytometry was largely restricted to mature myeloid lineage cells. The Jurkat-NFAT reporter system enabled rapid comparison of single-chain variable fragments (scFv), structural and signaling domain configurations, and identified lead construct #3 (CAR87.3). CART87.3 demonstrated specific and potent cytolytic activity in vitro against CD87-positive CRC cell lines T84, SW480, LS174T, and DLD1. Conclusions: This work identifies CD87 as a safe, selectively-expressed target antigen for the development of CAR-T therapy in CRC. The in vitro results support the advancement of human CART87 towards in vivo validation. Furthermore, we describe the rational design of a CART87 construct using a high-throughput strategy that could be broadly applied in CAR design for other target antigens. Citation Format: Andrea Feci, Trevor Baybutt, Robert Carlson, Emmett Grover, Jagmohan Singh, Ross Staudt, Miao Cao, Edoardo Manca, Jasmine Alvarez, Scott A. Waldman, Adam Snook. Preclinical development and evaluation of CD87-directed CART for colorectal cancer [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 3722.

3617 Background: Guanylyl cyclase C (GUCY2C) is an intestinal tumor antigen ectopically expressed in some gastroesophageal and pancreatic cancers, highly expressed in nearly all colorectal cancers (CRC), and retained in metastases. Adenovirus serotype 5 (Ad5)-based cancer vaccines, including those targeting GUCY2C, are limited by pre-existing neutralizing antibodies (NAbs) targeting Ad5. Ad5.F35-GUCY2C-PADRE is a chimeric Ad5 vector (fiber of serotype 35) developed to overcome anti-Ad5 immunity and induce durable antitumor T-cell responses. We report initial immunogenicity, safety, and recurrence outcomes from a phase 2A dose-finding study in patients with resected GI malignancies. Methods: This open-label phase 2A study enrolled patients with resected, high-risk GI adenocarcinomas (CRC, pancreatic, gastric, and small bowel) with no evidence of disease 4-24 wks after standard adjuvant therapies. Oligometastatic stage IV CRC patients were also allowed (n=8). Patients were randomized to receive three i.m. administrations of Ad5.F35-GUCY2C-PADRE at 1×10 11 , 1×10 12 , or 5×10 12 viral particles (vp) at 4-wk intervals. Primary endpoints were safety and GUCY2C-specific T-cell responses (IFNγ ELISpot). Exploratory endpoints included the impact of NAbs on T-cell responses and RFS, and the impact of dose on immunological and clinical outcomes. Results: Forty-six patients (29 CRC) completed treatment and were evaluable. Vaccination was well tolerated with no DLTs or TRSAEs; AEs were predominantly grade 1-2 flu-like symptoms. GUCY2C-specific T-cell responses increased in frequency and magnitude in a dose-dependent manner. Nearly all patients receiving higher doses produced T-cell responses regardless of NAb status (Table), supporting Ad5.F35 overcoming pre-existing NAbs. At the 2-year follow-up, there was a dose-dependent improvement in RFS among stage II-III CRC patients (Table), and RFS was significantly improved among immune responders vs non-responders ( P < 0.05). Conclusions: Ad5.F35-GUCY2C-PADRE is safe, overcomes pre-existing Ad5 immunity, and induces robust, dose-dependent GUCY2C-specific T-cell responses. Vaccine-induced immunity is associated with improved RFS in CRC, supporting further development of the off-the-shelf Ad5.F35 platform for cancer vaccines and Ad5.F35-GUCY2C-PADRE for CRC recurrence prevention. Clinical trial information: NCT04111172 . Dose (vp) Magnitude of GUCY2C-Specific T-cell Response 1 % of Patients with a GUCY2C-Specific T-cell Response (N/N) 2 CRC 3 Recurrences (%; N/N);Median RFS 1x10 11 9 33% (5/15) 50% (4/8); 512 days 1x10 12 92 94% (15/16) 17% (1/6); not reached 5x10 12 218 100% (15/15) 0% (0/7); not reached P < 0.0001 P < 0.0001 P = 0.051 1 Median SFCs/5×10 5 PBMCs ; 2 at 4 weeks after 1 st vaccination ; 3 excluding stage IV patients .

CAR-T cell therapies are curative for advanced hematologic cancers, however that potential has yet to be realized in epithelia-derived solid tumors reflecting the limited portfolio of cancer-restricted, cell-surface targets. Desmoglein 2 (DSG2) is a desmosomal cadherin universally overexpressed on the surface of transformed epithelial cells, with normal protein expression believed to be junctionally-restricted between adjacent cells, creating a “window of opportunity” to eliminate solid tumors without toxicity. Here, we generated DSG2-directed CAR-T cells (αDSG2) that universally recognize and lyse assorted solid tumor cell lines in vitro and eliminated patient-derived and cell-derived colon, pancreatic, lung, prostate, breast, and liver tumors in vivo. Transgenic mice expressing human DSG2 experienced no toxicity following αDSG2 CAR-T cell administration. These studies reveal safe and robust antitumor activity of αDSG2 CAR-T cells and introduce a new class of junctionally-restricted antigens that can be safely and effectively targeted across solid tumor types.

Immunoglobulin (Ig) replacement therapies (IgRT), including intravenous (IVIg) and subcutaneous (SCIg), are pooled IgG preparations widely used to restore humoral immunity and to suppress pathological inflammation in autoimmune and inflammatory disorders. Despite broad clinical use, the mechanisms underlying their immunomodulatory effects remain incompletely defined. Here, we identify extracellular vesicle (EV)-associated cytokines as mediators of IVIg activity. Multiplex bead-based flow cytometry revealed that EVs isolated by size exclusion followed by ultracentrifugation from IVIg were CD63-positive but depleted of platelet-derived and HLA markers relative to EVs from unprocessed human plasma. Luminex profiling demonstrated substantial reduction of pro-inflammatory cytokines in IVIg EVs. Notably, although IVIg EVs contained abundant IFNγ, they failed to activate IFNGR/JAK/STAT1 signaling. Instead, prolonged exposure to IVIg EVs suppressed subsequent IFNγ-induced STAT1 activation. Engineered IFNγ-coated EVs (IFNγ-eEVs) recapitulated both activating and inhibitory IVIg effects indicating context-dependent signaling bias. Critically, cold ethanol precipitation, a key step in IVIg manufacturing, selectively abrogated the activating function of IFNγ-eEVs while preserving their inhibitory capacity. These findings define a previously unrecognized mechanism where IVIg processing generates EVs that bias IFNγ signaling toward suppression. EV-associated cytokine signaling therefore represent a generalizable pathway through which IVIg exerts anti-inflammatory effects across immune-mediated diseases. ​

Abstract Guanylin (GUCA2A) is a paracrine hormone expressed in the colonic epithelium that activates the transmembrane receptor guanylyl cyclase C (GUCY2C). Activated GUCY2C promotes fluid secretion, genome stability, barrier integrity, and homeostasis through cGMP signaling. In early colorectal cancer (CRC), guanylin expression is universally suppressed, silencing the GUCY2C signaling cascade and contributing to tumorigenesis. Repression of guanylin is mediated transcriptionally by aberrant Wnt/β-catenin/TCF4 signaling at a Wnt-sensitive enhancer upstream of the GUCA2A gene. Using unbiased proteomics in Wnt-inducible CRC cell lines, we sought to directly identify regulators that mediate Wnt sensitivity to guanylin expression and control its transcriptional silencing in the context of tumorigenesis. sgRNAs targeting the GUCA2A regulatory element, consisting of the promoter and enhancer, were coupled to a dCas9-APEX2 fusion protein to capture the desired DNA locus. ChIP-qPCR and ChIP-seq confirmed sgRNA effectiveness and specificity in targeting GUCA2A. APEX2-mediated proximity biotinylation was performed to label proteins associated with the targeted site. Streptavidin affinity pulldown followed by LC-MS/MS-based proteomics revealed candidate regulatory proteins differentially enriched at the GUCA2A promoter under Wnt-on and Wnt-off conditions, including the previously identified Wnt-modulated guanylin activator p38α (MAPK14). Additionally, other candidates were enriched at the promoter irrespective of Wnt status, suggesting regulation of guanylin that is either Wnt-independent or requires post-translational control downstream of Wnt signaling. Potential regulators of guanylin expression identified in this analysis are undergoing validation using a knockdown screen in combination with a GUCA2A luciferase reporter with the goal of identifying the regulatory machinery underlying guanylin loss in CRC. As GUCY2C functions as a tumor suppressor, new insights into the mechanism of guanylin transcriptional silencing will provide an opportunity to inform prevention and treatment strategies to repair the signaling pathway and oppose disease. Citation Format: Adi Caspi, Ariana A. Entezari, Jasmine R. Alvarez, Annie K. Londregan, Thomas J. Kuret, Jeffrey A. Rappaport, Adam E. Snook, Scott A. Waldman. Proteomics at the guanylin gene locus reveals Wnt/β-catenin regulatory factors underlying GUCY2C hormone silencing in colorectal cancer [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 5946.

IntroductionFDA-approved chimeric antigen receptor (CAR)-expressing T cell therapies (CARTs) have revolutionized the treatment of blood cancers. Yet none have been successful for "solid" tumors, such as colorectal cancer (CRC), the 2nd leading cause of cancer deaths. Guanylyl cyclase C (GUCY2C) has emerged as a clinical-stage target for CART and bispecific T-cell engager (BiTE) therapies in CRC. IFNγ has been canonically recognized as beneficial for the effector functions of T cells by enhancing antigen processing and HLA presentation and is essential for CART targeting of solid malignancies by inducing adhesion molecule expression for synapse stabilization.MethodsUsing in vitro co-culture systems, conditioned media experiments, cytokine screening, pharmacologic inhibition, CRISPR-Cas9 knockout, and transcriptomic analyses, we investigated mechanisms of GUCY2C antigen loss in CRC cells exposed to activated CART cells.ResultsWe identified a novel antigen loss mechanism that limits the efficacy of CART in CRC, in which IFNγ secreted by activated CART cells causes bystander cancer cells to lose GUCY2C. This previously unexplored antigen loss mechanism is mediated through IFNγ receptor, JAK, and cellular stress signaling pathways. This mechanism of antigen loss can be rescued with anti-IFNγ neutralizing antibody, the JAK inhibitor ruxolitinib, or 4-phenylbutyrate (an ER stress reliever).DiscussionWe revealed a negative effect of IFNγ that uniquely interferes with immunotherapies targeting native surface antigens, such as CART and BiTE therapies, which may be reversed by disrupting stress signaling pathways to enhance solid tumor CART and BiTE immunotherapies.

Colorectal cancer (CRC) remains a major global health challenge and a leading cause of cancer-related mortality worldwide. Central to its pathogenesis is the dysregulation of interconnected signal transduction networks that govern cellular proliferation, survival, differentiation, apoptosis, and immune modulation. Progressive disruption of these pathways facilitates malignant transformation of the colonic epithelium and underlies disease initiation and progression. In this review, we examine the foundational signaling mechanisms implicated in CRC, with an emphasis on the Wnt/β-catenin, GUCY2C, MAPK/ERK, p53, PI3K/AKT, and SMAD4/TGF-β pathways. We also highlight the genomic and epigenomic hallmarks of CRC to illustrate how genetic and epigenetic alterations contribute to diverse oncogenic processes. Furthermore, we discuss both conventional and emerging precision-based therapeutic strategies aimed at driving mutations in CRC tumorigenesis. By synthesizing advances in signaling biology, this review aims to provide a framework for understanding CRC complexity and to inform the development of more precise and effective therapies.

TPS7087 Background: Autologous CART options for patients with relapsed or refractory (R/R) T-cell lymphomas (TCL) have faced challenges such as T-cell fratricide during CART manufacture and safety concerns regarding depletion of normal T cells. To overcome these obstacles, we proposed a dual cell population CART product, which contained both autologous 4-1BB costimulated CART cells against CD5 and healthy T-cells, with both populations knocked out for CD5 (CRISPR-Cas9 CD5 short-guide RNA to delete CD5 - Senza5). In vivo experiments using the dual population product of (Senza5 CART5) demonstrated increased CART5 expansion and enhanced antitumor efficacy in TCL xenograft models compared to wild-type (WT) CART5. For clinical use, a novel 5-day manufacturing process was designed to obtain a less differentiated and less exhausted product, with enhanced in vivo expansion and fitness. Methods: A human phase I trial was designed to determine the safety, effectiveness and recommended phase 2 dose (RP2D) of Senza5 CART5 cells in participants with R/R TCL with ≥50% expression of CD5 on malignant cells, and no circulating CD5+ cells. Participants must have a suitable backup stem cell product or donor identified in the unlikely event of T-cell aplasia. Patients with prior allo HCT are currently excluded. Cohorts of patients are treated with escalating doses of Senza5 CART5 cells (3x10 6 to 1.25x10 8 ) using a Bayesian Optimal Interval design following lymphodepletion. The study will enroll and treat participants until a maximum of 9 participants are infused and evaluable for dose limiting toxicity (DLT) assessments at a given dose level, or a maximum of 30 DLT-evaluable participants from all dose levels are infused. The RP2D will be determined based on both safety and biological evidence of efficacy. Study objectives include frequency and severity of treatment-related adverse events, as well as efficacy by assessing overall and complete response rates, duration of response, progression-free and overall survival. Manufacturing feasibility will be determined by the frequency of product release failures and occurrence of dose failures (inability to meet targeted dose). Exploratory objectives will evaluate the persistence and trafficking of Senza5 CART5 cells in blood and tumor by characterizing the kinetics of the infused cells by flow cytometry and qPCR gene expression. We will perform profiling of the tumor microenvironment and measure systemic soluble cytokines before and after treatment. We will also assess the impact of CART5 on normal T cells, and the persistence of CD5KO untransduced T cells that are infused as part of the Senza5 CART5 product by multicolor flow cytometry and qPCR. The trial is sponsored by Vittoria Biotherapeutics and is registered at clinicaltrials.gov as NCT06420089. Enrollment in this trial has begun. Clinical trial information: NCT06420089 .

The GUCY2C intestinal receptor regulates fluid secretion and epithelial transformation through its hormone ligand guanylin (GUCA2A). GUCY2C supports intestinal homeostasis and has been characterized as a tumor suppressor in colorectal cancer. In early tumorigenesis, GUCY2C signaling is silenced through transcriptional repression of guanylin. Guanylin repression is mediated by Wnt/β-catenin/TCF4, a critical signaling pathway aberrantly activated in ∼90% of colorectal cancers. Specifically, Wnt signaling regulates expression at a recently characterized 2, 683-base pair locus control region (LCR) upstream of the GUCA2A gene. This regulation is conferred indirectly, and TCF4, the transcription factor effector of the Wnt cascade, does not interact with the LCR. However, many downstream TCF4 targets are transcription factors canonically implicated in tumorigenesis. Therefore, our goal is to identify factors specifically interacting with the LCR and regulating guanylin expression. Colorectal cancer cell lines expressing inducible Wnt-silencing constructs were used to toggle the expression of guanylin. Using these models, we sought to interrogate Wnt regulation at the LCR by employing C-BERST (dCas9-APEX2 biotinylation at genomic elements by restricted spatial tagging), an unbiased proteomics-based approach to profile locus-specific protein interactions. This system relies on peroxidase-mediated biotinylation of proteins by a dCas9-APEX2 fusion protein, driven to a locus of interest by a targeting gRNA. Following localized biotinylation, proteins are enriched by streptavidin pulldown and identified by mass spectrometry analysis. Assessing proteins in the presence and absence of Wnt signaling can reveal Wnt-regulated factors controlling target gene expression. By ChIP-qPCR, we have captured the GUCA2A gene locus using targeted gRNAs. Further, dCas9-APEX2 binding does not interfere with the expression of guanylin or its Wnt regulation. Finally, streptavidin-based pulldowns reveal protein interactions at the promoter that can be further characterized by mass spectrometry to elucidate the Wnt regulatory machinery at the GUCA2A locus. Identifying the mechanism of Wnt-mediated guanylin silencing, and the transcription factors controlling that regulation, will provide novel insights to inform therapeutic targeting to reconstitute the GUCY2C signaling axis to prevent colorectal cancer. Adi Caspi, Ariana A. Entezari, Jasmine R. Alvarez, Jeffrey A. Rappaport, Adam E. Snook, Scott A. Waldman. Locus-specific proteomics to define transcriptional regulation of GUCY2C hormone expression in colorectal cancer [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 1423.

The intestinal cell surface receptor GUCY2C is universally over-expressed by colorectal tumors. Loss of expression of the intestinal hormones regulating GUCY2C silences this signaling pathway which dysregulates the cell cycle and DNA damage repair contributing to intestinal tumorigenesis. Thus, reconstituting GUCY2C activity with oral hormone replacement is being explored in clinical trials as a novel chemoprevention approach for colorectal cancer (CRC). Moreover, GUCY2C is being explored in clinical trials as an immunotherapeutic target reflecting its universal over-expression by metastatic CRC tumors. Here, we explore the expression and activity of GUCY2C in tumor stem cells (SCs) to define its potential as a therapeutic target to prevent therapeutic escape and recurrence in colorectal cancer. CRC-SCs were isolated from a library of patient-derived xenografts (PDXs) and in vitro cell lines by flow cytometry using aldehyde dehydrogenase (ALDH) activity and EpCAM expression. The ALDHHIEpCAMHI CRC-SC fraction comprised ∼1-10% of bulk tumor cells, and expressed the cardinal features of cancer SCs, including non-adherent tumor sphere and colony formation in vitro and tumor formation in immunodeficient mice compared to ALDHLOEpCAMHI differentiated tumor cells. ALDHHIEpCAM+ SCs universally express GUCY2C mRNA and protein, and expression levels are positively correlated to bulk PDX tumors of origin. GUCY2C is expressed on the surface of ALDHHIEpCAM+ SCs, which respond to the GUCY2C agonist linaclotide (Linzess™) by producing and secreting cyclic (c)GMP. Unexpectedly, ALDHHIEpCAM+ SC are hyper-responsive to linaclotide, and their hormone-induced cGMP accumulation is 5- to 10-fold greater than ALDHLOWEpCAMHI differentiated tumor cells. Universal expression, with amplified signaling, in CRC-SCs suggest that GUCY2C might be utilized as a therapeutic target. Indeed, linaclotide induces a durable block of proliferation in, and expansion of, ALDHHIEpCAM+ SCs, quantified by colony formation and expression of Ki67 in vitro. Similarly, ALDHHIEpCAM+ CRC-SCs are completely eliminated by GUCY2C-targeted chimeric antigen receptor (CAR) T cells in vitro. Taken together, these observations suggest that, beyond bulk tumor cells, GUCYC may have dual utilities as a therapeutic target in CRC-SCs. Thus, amplified GUCY2C signaling could be activated by agonists, like linaclotide, to suppress SC persistence and resistance to chemotherapy and potentially shift them to a differentiated phenotype susceptible to treatment. Moreover, metastatic CRC-SCs could be eliminated by GUCY2C-targeted immunotherapies, like CAR T cells, to prevent tumor persistence and recurrence. Jess Kopenhaver, Scott A. Waldman, Adam E. Snook, Trevor Baybutt, Lara Cheslow, Ross Staudt, Jasmine Alvarez. Targeting colorectal cancer stem cells through GUCY2C expression [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 125.

Abstract Background: Collectively, cancers of the esophagus, stomach, pancreas, and colorectum (“GI cancers”) are the leading cause of cancer-related death. Despite standard of care, 30-90% of GI cancer patients experience tumor recurrence. In colorectal cancer (CRC), guanylyl cyclase C (GUCY2C), a transmembrane heat-stable enterotoxin receptor, is overexpressed in more than 95% of colorectal tumors and in subsets of gastroesophageal and pancreatic tumors. We are conducting a Phase 2A, dose-finding, single-center, open-label, randomized trial (NCT04111172) to evaluate the safety and immunogenic activity of three dose levels of the Ad5.F35-GUCY2C-PADRE vaccine in adult patients with select solid tumors (colorectal, pancreatic, or gastroesophageal) who are at high risk of relapse following definitive surgery and adjuvant therapy. To further investigate the immunogenicity of the GUCY2C-targeting vaccine across diverse patient populations and facilitate downstream laboratory-based studies—such as autologous T cell expansion, TCR sequencing and cloning, and in vivo patient-derived xenograft (PDX) models—we explored the feasibility of a protocol for the stimulation and expansion of antigen-specific T cells derived from peripheral blood mononuclear cells (PBMCs). Methods: PBMCs from healthy donors were used as a pilot to optimize this protocol and assess its efficacy. PBMCs were cultured in vitro for approximately 10 days with adjuvants, cytokine cocktails, and antigen-specific peptides. The enriched T-cell populations were then evaluated for antigen-specific targeting using flow cytometry and ELISPOT analysis. Results and Conclusion: The stimulation protocol successfully expanded both CD4+ and CD8+ effector T cells. The final T cell population was predominantly CD8+ T cells, which secreted IFNγ following CEF (CMV, EBV, Influenza), but not MOG (self-antigen myelin oligodendrocyte glycoprotein) peptide stimulation, indicating antigen-specific reactivity of the expanded T cells. Further studies, including cytotoxicity assays and antigen-presenting cell (APC)-based longitudinal expansion, are necessary to support the high-throughput application of this protocol for future preclinical and clinical investigations. Citation Format: Zhengyang Sun, Jagmohan Singh, Miao Cao, Anne Glowacki, Tingting Zhan, Babar Bashir, Scott A Waldman, Adam E Snook. An antigen-specific t cell stimulation and expansion platform for immunological investigation of the Ad5.F35-GUCY2C-PADRE vaccine in gastrointestinal cancers [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr B067.

2539 Background: CAR-T cell therapy has been curative for many patients with refractory, progressive hematologic cancers, resulting in several FDA approvals. However, this therapy has not been successful for solid cancers, reflecting the need for suitable antigen targets for each disease and solutions to immunological barriers in solid tumors. Here, we have identified the desmosomal cadherin, desmoglein 2 (DSG2), as an effective CAR-T cell therapy target in epithelia-derived solid tumors. DSG2 contributes to cell proliferation, migration, and other emerging tumor-promoting pathways, resulting in its upregulation in nearly all solid cancers and correlating with poor prognosis. Moreover, we explored CRISPR-Cas9-mediated elimination of the inhibitory receptor CD5 to enhance in vivo CAR-T cell expansion and solid tumor efficacy. Methods: DSG2-directed CAR-T cells were generated from human T cells using a scFv derived from a murine hybridoma targeting the extracellular domain of DSG2 in a 3 rd generation CAR design with CD28, 4-1BB, and CD3ζ signaling domains. CD5 elimination employed electroporation of complexed gRNA-Cas9 ribonucleoprotein (RNP). DSG2 expression was characterized in human cancers and cell lines and CAR-T cell activity was examined in vitro by cytokine production and target cell cytolysis. In vivo efficacy studies employed cancer xenografts in NSG mice treated with CAR-T cells. Safety studies employed a human DSG2 transgenic mouse treated with syngeneic murine CAR-T cells for clinical, serum biomarkers, and histopathological evaluation. Results: In vitro studies revealed recognition and lysis of solid cancer cell lines and effector cytokine production. Administration of DSG2-directed CAR-T cells eliminated metastatic cell-derived xenografts, patient-derived xenografts, and orthotopic tumors derived from various solid cancers, including colorectal, pancreatic, lung, prostate, breast, and liver. Moreover, elimination of CD5 enhanced the expansion of DSG2-directed CAR-T cells in vivo, resulting in curative efficacy at sub-therapeutic CAR-T cell doses. Safety studies revealed no toxicity in any human DSG2 transgenic mouse tissues. Conclusions: These studies reveal the robust antitumor activity of DSG2-directed CAR-T cells in solid tumors, which is enhanced by CD5 deletion, without toxicity in a human transgenic mouse model. Thus, CD5-deleted DSG2-directed CAR-T cells are a promising therapeutic approach that may be safe and effective for all solid cancers.

Background. T-cell lymphomas carry a very poor prognosis and lack effective treatments. Despite the transformative success of chimeric antigen receptor (CAR) T-cell therapies in B-cell malignancies, translation to T-cell malignancies has been impeded by target antigen overlap that causes CAR T-cell fratricide and on-target T-cell aplasia. Senza5 CART5 is an autologous, gene-edited, dual-population, rapidly manufactured (5-day) CAR T-cell (CART) product comprised of both a CD5-deleted (CRISPR-Cas9) CAR-expressing (lentivirus transduced) T cell population (CD5⁻ CAR⁺) and a CD5-deleted, non-transduced T-cell population (CD5⁻ CAR⁻). This dual population is designed to enhance both potency and safety as CD5 deletion removes a negative regulator of T-cell effector function (Patel R., Science Immunology, 2024), while the companion CD5⁻ CAR⁻ cells are intended to mitigate on-target T-cell aplasia during immune reconstitution. Methods. In this open-label, dose-finding Phase 1 trial, adults with histologically confirmed relapsed/refractory (r/r) CD5+ T-cell lymphoma after ≥ 1 prior therapy receive lymphodepletion (fludarabine 25 mg/m² + cyclophosphamide 250 mg/m² x 3 days or bendamustine 90 mg/m² x 2 days) followed by a single IV infusion of Senza5 CART5 at doses ranging from 3x106 to 1.25x108 CAR+ cells. Primary endpoints are safety and recommended Phase 2 dose (RP2D); secondary/exploratory endpoints include manufacturing feasibility, in vivo expansion, tissue trafficking, T-cell aplasia mitigation, overall response rate (ORR), and complete response (CR) rate per Lugano criteria. The Viper 101 trial is ongoing (NCT06420089). Results. As of August 5, 2025, 6 patients were manufactured and 5 patients (pts) were treated (1 at a flat dose of 1x107 CAR+ cells and 4 at a flat dose of 3x106 CAR+ cells) with 4 pts evaluable for dose limiting toxicities (safety) and 3 pts evaluable for efficacy. Median age was 65 years (range 55–75); histologies included PTCL-NOS (n=2), TFHL (n=1), AITL (n=1), and MF (n=1). Pts had a median of 3 prior lines (range 2–5), and 2 had prior autologous stem cell transplant. Manufacturing success was 100% (6/6). Treatment-related adverse events (TRAEs) included cytokine-release syndrome (CRS) (Grade 1–2) in 100% of safety-evaluable pts (4/4) that resolved with standard care; no neurotoxicities or hemophagocytic lymphohistiocytosis syndrome were reported. All pts experienced Grade 3–4 transient cytopenias and Grade 1-2 TRAEs of the skin, mouth, and gastrointestinal tract. Infections were observed in 3/4 pts . One pt treated at the 1x107 CAR+ dose experienced a Grade 5 severe AE (infection) on Day 54 while presenting with multineage cytopenias that were considered unrelated to the investigational product. No pts experienced clinical EBV or CMV infections. All safety-evaluable pts (n=4) experienced robust in vivo CART cell expansion (median peak in peripheral blood (PB): 33,664 copies/µg gDNA). On-treatment biopsies confirmed CART trafficking to disease sites, including lymph nodes, skin, and marrow. Of note, no detectable CD5+ cells (normal or malignant) were detected in these biopsies. CART cells persisted and were still detected in the PB at last follow-up (median 1,426 copies/µg gDNA). Due to the risk of on-target T-cell toxicity, immune reconstitution was also monitored. By Day 28, all evaluable pts demonstrated reconstitution of a CD5-negative, CAR-negative (CD5- CAR-) T-cell compartment (both CD4+ and CD8+ subsets). Among efficacy-evaluable pts (n=3), overall response rate (ORR) was 100% (3/3) with all three pts achieving a complete response (CR) at a median follow-up of 4.0 months (range 1.9–5.5). 2/3 pts had FDG-avid lesions on the Day 30 scan, which, upon biopsy, were negative for the original lymphoma, and became PET negative by Day 90. No patients have relapsed at last follow up. Updated clinical outcomes and correlative data for all pts treated will be presented at the meeting. Conclusions. Early results suggest that Senza5 CART5 has manageable toxicities, potent antitumor activity, robust and durable CART expansion, and T-cell reconstitution at low doses of CART cells (3x106 CAR+ cells). While follow-up is limited, these data support further development of Senza5 CART5 for r/r T-cell lymphomas. Moreover, these results underscore the potential for CD5 deletion as a platform to broadly enhance the efficacy of engineered T-cell therapies for additional indications.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with high recurrence rates even after curative resection and adjuvant chemotherapy. Although immunotherapeutic approaches, such as immune checkpoint blockade (ICB), have revolutionized the treatment of some solid tumor malignancies, this has not been the case for PDAC. Several characteristics of PDAC, including its distinctive desmoplastic tumor microenvironment (TME), intratumor heterogeneity, and poor antigenicity and immune cell infiltration, contribute to its dismal immunotherapeutic landscape. Cancer vaccines offer one approach to overcoming these barriers, particularly in the resectable or borderline resectable settings, where tumor burden is low and immunosuppression is less pronounced. Various vaccination platforms have been tested in the clinical setting, from off-the-shelf peptide-based vaccines (e.g., AMPLFIFY-201 study, where over 80% of participants exhibited T-cell and biomarker responses) to personalized neoantigen mRNA vaccine approaches (e.g., autogene cevumeran, with significant responders experiencing longer median recurrence-free survival (RFS)). The key considerations for enhancing the efficacy of vaccination include combinations with chemotherapy, radiotherapy, and/or ICBs, as well as selecting appropriate immunomodulators or adjuvants. Recent results suggest that with continued mechanistic advancement and novel therapeutic development, cancer vaccines may finally be poised for clinical success in PDAC.

Sporadic colorectal cancers (CRCs) are initiated by APC mutations inducing aberrant β-catenin/TCF signaling regulating gene transcription. Similarly, the guanylyl cyclase C (GUCY2C) tumor suppressive axis is universally silenced in CRC. While the GUCY2C receptor is retained, transcriptional silencing of its paracrine hormones guanylin and uroguanylin by β-catenin/TCF signaling is among the earliest events in colorectal transformation. Moreover, suppression of the β-catenin/TCF pathway reconstitutes hormone expression. P38 is a tumor suppressor, and its loss promotes tumorigenesis in chemical and genetic mouse models of CRC. We aim to identify mechanisms by which β-catenin/TCF signaling silences the transcription of GUCY2C hormones to enable targeted reconstitution of ligand expression to prevent CRC. Here, we employed four human CRC cell lines with conditional β-catenin/TCF signaling and healthy human and mouse intestinal organoid models to explore the role of p38 in oncogenic transcriptional signaling. Indeed, toggling oncogenic β-catenin/TCF activity regulates p38 phosphorylation and downstream signaling, suggesting that β-catenin/TCF gain-of-function represses the p38 pathway. Further, pharmacological and genetic repression of p38 activity recapitulates β-catenin/TCF gain-of-function and directly eliminates GUCY2C hormone expression. Finally, beyond GUCY2C hormones, regulation of ∼80% of a core set of β-catenin/TCF-dependent genes common to human CRC cells is controlled by p38 signaling. Here, we reveal β-catenin/TCF regulation of p38 activity and its role in mediating transcriptional regulation of gene expression underlying nuclear oncogenic signaling in CRC. Ariana A. Entezari, Adi Caspi, Jasmine R. Alvarez, Jeffrey A. Rappaport, Adam E. Snook, Scott A. Waldman. Wnt/β-catenin/TCF signaling suppresses GUCY2C hormone transcription by silencing p38 [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 1444.