Supplementary Methods and Materials

Abstract Current immune checkpoint therapies targeting PD-1 and PD-L1 are ineffective in treating Acute Myeloid Leukemia (AML). Vasoactive Intestinal Peptide (VIP) is an immunosuppressive 28-amino acid neuropeptide overexpressed in nearly 30% AML patients, suggesting that some AML tumors may be using VIP expression as a mechanism to evade immune surveillance. We hypothesize that AML cells secrete VIP which signals through the VIP-R (receptor) on T cells to limit anti-cancer immunity. Therefore, targeting the VIP signaling with a VIP-R antagonist can reverse immunosuppression and potentiate anti-cancer immunity. Using in silico modeling, in vitro screening for potentiation of T cell activation, and proof-of-principle screening in mouse leukemia models, we identified a novel VIP-R antagonist, ANT308. ANT308 shows stronger binding affinity to VIP-R as compared to VIP and potentiates activation of human and mouse T cells with an EC50 in the range of 200-400 nM. Immunofluorescence assays show that ANT308 colocalizes with the VIP receptor, VPAC1. Co-IP and flow cytometry confirmed ANT308 binding to VPAC1. In addition, ANT308 treatment of leukemic mice induced strong T cell-mediated anti-leukemic activity. To improve the pharmacokinetics of ANT308, we have developed a novel fusion peptide combining ANT308 and Fc fragment of IgG4 named CAMV-01. In a Jurkat T-cell activation assay, CAMV-01 has an EC50 of 100-200 nM and reduces T cell exhaustion. Furthermore, CAMV-01 significantly improved survival in multiple murine leukemia models, including those that secrete VIP (C1498) and the VIP negative cell line P815. In vitro stability studies show that CAMV-01 remains stable in plasma for > 4 days, whereas the ANT308 peptide is stable for only 15 minutes. PK studies with intravenous (i.v.) or subcutaneous (s.c.) injection of 15mg/kg CAMV-01 show a half-life of 2 weeks in mice, compared to 15 minutes for the ANT308 peptide. Using Sprague-Dawley rats receiving a single i.v. or s.c injection of 5mg/kg CAMV-01, the Cmax and t1/2 were estimated to be 82 nM and 165 hours (i.v.) respectively and 0.7 nM and 235 hours (s.c.) respectively. In a P815 mouse model, s.c. administration of CAMV-01 induced a stronger tetramer-positive CD8+ T-cell response compared to ANT308. In conclusion, CAMV-01 is a novel, first-in-class immunotherapeutic targeting the VIP immune checkpoint, with a robust pharmacokinetic and pharmacodynamic profile and promising potential for the treatment of AML. Citation Format: Swapnaa Balaji, Antonio Ward, Yuou Wang, Jian-Ming Li, Srijon Sarkar, Sonia Mecorapaj, Tenzin Kalsang, Shuhua Wang, Cynthia Giver, Edmund Waller. A first in class VIP receptor antagonist as an immunotherapeutic for acute myeloid leukemia [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 2808.

Abstract Background: Acute myeloid leukemia (AML) remains one of the most lethal hematologic malignancies, especially in TP53-mutated disease where standard chemotherapy provides poor long-term survival. Immune evasion by myeloid cells in the tumor microenvironment is a major barrier to cure. We hypothesized that targeting vasoactive intestinal peptide (VIP)/VIP receptor (VPAC) signaling would overcome myeloid cell-mediated immunosuppression and enhance anti-tumor responses in AML. Methods: RNA-seq differential expression, survival analyses, and flow cytometry of AML bone marrow and PBMC were used to profile VIP/VPAC expression and myeloid versus blast compartments in TP53-mutated and non-mutated cases. TP53-wildtype and TP53 loss-of-function human and murine AML cell lines were used to measure VIP production by ELISA and macrophage phagocytosis of VIP-producing versus low-VIP cells in M1- and M2-like co-culture assays. In vivo efficacy of the long-acting VIP receptor antagonist ANT308 and its IgG4 Fc fusion ANT308-Fc3 was evaluated in C1498 and WEHI3 AML models, including VIP, VPAC1, and VPAC2 knockout hosts. Results: VIP expression was elevated in PBMC from ∼36% of AML patients compared with healthy donors and was enriched in TP53-mutated cases. High VIP expression and TP53 loss-of-function each associated with inferior survival in cBioPortal datasets. RNA-seq showed that TP53-mutated AML is characterized by increased VIP and immunoregulatory pathway signatures together with reduced apoptosis and effector CD8+ T-cell-associated genes. Secretome analysis of human leukemia cells demonstrated higher VIP secretion from TP53 loss-of-function lines than from TP53-wildtype THP-1, confirming leukemia-intrinsic VIP production. In patient samples, both CD34+ blasts and CD34- myeloid cells contained VIP+ cells, but non-blast myeloid cells accounted for most VIP+ events, indicating they are a major VIP source in the AML microenvironment. ANT308 enhanced phagocytosis of VIP-secreting WEHI3 leukemia cells by both M1- and M2-like macrophages but did not alter phagocytosis of VIP-low C1498 cells, supporting VIP as a phagocytosis checkpoint in AML. Consistent with a host contribution, VIP-, VPAC1-, or VPAC2-knockout mice bearing C1498 leukemia showed improved survival relative to wild-type mice. In vivo treatment of wild-type C1498-bearing mice with ANT308 or the long-acting Fc fusion ANT308-Fc3 eradicated established leukemia in up to 65% of animals. Conclusions: VIP/VPAC signaling defines a previously unrecognized phagocytosis checkpoint that reinforces myeloid immunosuppression in AML and is linked to TP53-mutated disease. VPAC antagonism with ANT308-Fc3 restores macrophage phagocytosis and produces durable leukemia control in preclinical models, supporting clinical development of VPAC-targeted immunotherapy for high-risk AML. Citation Format: Zihan (Clarence) Chen, Tuisha Gupta, Fanyuan Zeng, Yujie Chen, Jian Ming Li, Cynthia R. Giver, Kiranj Chaudagar, Edmund K. Waller. VIP/VPAC signaling as a phagocytosis checkpoint in TP53-mutated acute myeloid leukemia [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 2809.

Abstract Pancreatic ductal adenocarcinoma (PDAC) remains a lethal cancer, with only 13% of patients surviving 5 years. The limited efficacy of anti-PD-1/PD-L1 therapies indicates that additional immunoregulatory pathways sustain the immunosuppressive tumor microenvironment (TME) that restricts T-cell infiltration. Our recent studies (Ravindranathan Nat Com 2022) demonstrated that combined blockade of vasoactive intestinal peptide (VIP) receptor (VPAC) and PD-1 signaling eradicated PDAC in ∼50% of mice, although the underlying mechanism of VPAC inhibition remains undefined. Therefore, we hypothesized that VPAC blockade reshapes the immune microenvironment to enhance antigen presentation and T cell responses to anti-PD1 immunotherapy in PDAC.To address this, we characterized the expression of VIP and VPAC on cancer cells, immune cells, and stromal cells in the PDAC TME. We studied the impact of VPAC signaling on the immune functions of myeloid/lymphoid cells and on the architecture of the TME using PDAC patient specimens, murine models, and relevant in vitro/ex vivo systems. Tumor growth kinetics were performed to assess the anti-tumor potential of VPAC blockade.In vitro secretome analysis showed that murine MT5 PDAC cells secrete more VIP than KPC-luc, consistent with their faster tumor-growth kinetics. To assess host-intrinsic VIP effects in TME we compared tumor progression in WT and vip-knockout (vip-ko) mice and found markedly slower growth in vip-ko mice, especially in females vs. males. To explore the cellular basis of the anti-tumor response, we depleted immune cell subsets and compared tumor growth kinetics in both female and male vip-ko mice. Depletion of CD8+ or CD4+ T cells and of CSF1R+ myeloid cells accelerated tumor growth in vip-ko mice, whereas clodronate depletion of phagocytic macrophages attenuated anti-tumor responses only in females. Androgen blockade (ARB) with degarelix restored male responsiveness to VPAC inhibition, highlighting androgen-driven resistance in male against VPAC blockade. Mechanistically, VIP from CD29+PD-L1+ fibroblasts suppressed macrophage phagocytosis of PDAC cells, while VIP loss restored this activity. Strikingly, AR activation further suppressed macrophage phagocytosis selectively in males. Mechanistically, TNF-α and MIF secreted from γδ-T cells and GZMK+CD8+ T cells, respectively, enhanced anti-tumor phagocytosis in female vip-ko mice. Combined AR blockade, anti-PD-1, VPAC antagonism, and taxotere markedly extended survival in WT male mice bearing orthotopic MT5 PDAC (MST 54 vs. 18 days for untreated control).These findings show that VPAC signaling restrains TNF-α– and MIF-dependent immune crosstalk and limits phagocytic PDAC clearance. These data also reveal androgen-suppressed phagocytosis as a driver of sexual dimorphism in response to VPAC antagonism and suggest translatable approaches for PDAC treatment. Citation Format: Sonia Mecorapaj, Tenzin Passang, Fanyuan Zeng, Zihan Chen, Shuhua Wang, Tuisha Gupta, Shayna Jankowski, Jian-Ming Li, Cynthia R. Giver, Swapnaa Balaji, Kiranj Chaudagar, Edmund K. Waller. Androgen-driven sexual dimorphism reshapes immune crosstalk and limits immunotherapy response in PDAC [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 3441.

Introduction: The clinical efficacy of chimeric antigen receptor (CAR) T cell therapy remains limited in solid tumors, with immunosuppressive and hypoxic tumor microenvironments. Enhancing CAR T cell function and harnessing the endogenous immune response through novel signaling pathways may help overcome this limitation. We previously showed that targeting the PI3K signaling pathway improves CAR T cell efficacy in hematologic cancers, however, its therapeutic potential in solid tumors has yet to be fully explored. Vasoactive intestinal peptide (VIP) is a neuropeptide which exerts its function via interaction with two receptors, VPAC1 and VPAC2. VIP signaling in T cells is known to suppress T cell proliferation and effector function. Therefore, making this pathway an intriguing target for cancer immunotherapy, and pharmacological inhibition of PI3K and VPAC increased the expansion of TCMin vitro. Thus, dual targeting of PI3K and VIP signaling pathways may be a novel approach to enhance CAR T cell function against solid tumors. CAR T cells generated from VIP-knockout (VIP-KO), and VPAC-knockout (VPAC-KO) mice were expanded using PI3Kδ/γ inhibition,metabolism and in vivo anti-cancer activity were assessed. Methods: Splenocytes isolated from C57BL/6 wild-type (WT), VIP-KO, and VPAC-KO mice were activated ex vivo using concanavalin A and IL-2, followed by retroviral transduction via spinoculation with a CAR-expressing vector. During expansion, cells were cultured in the presence or absence of the PI3Kδ/γ inhibitor, Duvelisib. Transduction efficiency and T cell phenotypes—including central memory, stem-like, and exhausted subsets—were analyzed by spectral flow cytometry using surface markers. Metabolic function was assessed using a Seahorse extracellular flux analyzer. CAR T cell function was assessed using syngeneic pancreatic ductal adenocarcinoma (PDAC) tumor model. Result: VIP-KO and VPAC-KO CAR T cells have higher frequencies of CD4⁺ and CD8⁺ naïve and stem-like memory Tscm subsets compared to WT CAR T cells. Treatment with Duvelisib further enhanced these memory subsets in both VIP-KO and VPAC-KO CAR T cells. Duvelsisib also significantly reduced effector memory Tcells and reduced phenotypically-exhausted CAR T cell populations. Seahorse metabolic analysis revealed that VIP-KO and VIPKO-DUV CAR T cells had increased basal respiration and greater ATP production through mitochondrial oxidative phosphorylation compared to WT CAR T cells. This metabolic reprogramming promoted a more quiescent phenotype in VIP-KO CAR T cells, characterized by enhanced mitochondrial function and greater metabolism up-regulation upon antigen stimulation. Despite enhanced metabolic fitness and increased frequencies of memory T cell subsets in VIPKO-DUV and VPACKO-DUV CAR T cells, in vivo studies using a syngeneic PDAC mouse model demonstrated only modest tumor control and no significant improvement in overall survival of any group compared to WT CAR T cells. Co-culture impedance experiments revealed that the addition of macrophages enhanced the function of DUV-CAR T cells, suggesting that endogenous immune cells within the tumor microenvironment may be critical for full therapeutic efficacy. Conclusion: Dual targeting of PI3Kδ/γ and VIP signaling enhances CAR T cell memory phenotype and mitochondrial metabolic fitness, promoting a less exhausted and more quiescent T cell state. These in vivo results demonstrate that infusion of greater frequencies of memory CAR T cells with greater reliance on oxidative phosphorylation were insufficient to achieve durable tumor control in a solid tumor model, highlighting the critical role of the tumor microenvironment and endogenous immune cells in CAR T cell efficacy. The genetic absence of VIP/VPAC in the context of PI3K inhibitors may have a more profound effect on T cell survial than pharmacological inhibition of VPAC and PI3K. Future studies will include evaluation of CAR T cell infiltration and persistence in solid tumors, as well as profiling of immune cell populations in tumors from responders versus non-responders to better understand the cellular context that supports effective tumor control. These findings demonstrated the need for integrated strategies that consider both intrinsic T cell programming and extrinsic immune context to optimize CAR T cell therapy for solid tumors.

Mice Resistance to Leukemia via Gut Microbiota Transfer Vasoactive Intestinal Peptide (VIP) regulates adaptive immunity through receptors expressed in T lymphocytes, VPAC1andVPAC2. VIP over production in leukemia suppresses immune function, facilitating cancer progression. VIP-knockout (VIP-KO) mice show a survival advantage against leukemia, likely due to gut microbiota-mediated immune response regulation. Cohousing enables gut microbiota transfer from VIP-KO to wild-type (WT) mice via coprophagy, potentially enhancing WT mice's resistance to leukemia. This study investigates whether cohousing WT and VIP-KO mice alters WT gut microbiota and confers resistance to leukemia challenge. Eighty mice, 42VIP-KO 38WT, were cohoused across 10cages for six weeks. Blood and stool samples were collected pre- and post-cohousing for immune and microbiome analyses. Mice were injected with syngeneic CD1498leukemiacells post-cohousing and monitored for survival. Gender differences in survival outcomes were also assessed. WT mice cohoused with VIP-KO mice demonstrated significant colonization by VIP-KO-derived gut microbiota, including Ruminococcus gnavus and Lactobacillus murinus, characterized by lower gut pH. Cohousing improved WT survival rates after leukemia challenge(p<0.009), producing long-term survivors. VIP-KO mice showed the highest presence of R. gnavus after cohousing (p=0.017)Cohousing-induced microbiota transfer enhances leukemia resistance in WT mice through gut microbiota-mediated immune modulation. Survival improvements are independent of gender, underscoring the robustness of this therapeutic strategy. These findings highlight microbiota-targeted therapies, such as fecal microbiota transplantation, as promising approaches for leukemia treatment. Future studies will evaluate combined microbiota transfer and VIP antagonism therapies i.e. ANT308tooptimizeoutcomes. Wild Type cohousing had a significance of p<0.001. Knockout cohousing had a significance of p<0.009. Wild type mice had the highest median survival time at 27days. Kayla Robinson, Shayna Jankowski, Jian Ming Li. The gut microbiota from vasoactive intestinal peptide knock-out mice confers resistance to the growth of leukemia in wild-yype mice [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 5872.

Despite significant advancements in understanding the pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment (TME) through single-cell RNA sequencing (scRNAseq) and the development of novel therapeutic combinations, PDAC remains one of the deadliest cancers, with a five-year survival rate of ∼3%. Notably, survival outcomes are particularly worse in males compared to females. The limited efficacy of immune checkpoint inhibitors (ICI), such as anti-CTLA-4 and anti-PD-1/PD-L1 therapies, in PDAC has been attributed to a profoundly immunosuppressive TME driven by M2-like tumor-associated macrophages (TAM). Our translational studies demonstrated that dual blockade of PD-1 and VPAC stimulates CD8+ T cell activity and eradicates tumors in ∼50% of mice across various PDAC models. We hypothesized that a combinatorial ICI strategy is targeting TAM-driven immunosuppression in the TME and could enhance T cell-mediated anti-tumor immunity in PDAC. To test this hypothesis, we performed in vivo tumor growth and survival experiments in syngeneic PDAC models. We also conducted RNA sequencing (RNAseq) of bone marrow-derived macrophages (BMDM) polarized to M1/M2 phenotypes to investigate immune checkpoint expressions, supplemented by scRNAseq analysis of TAM from PDAC patients using public datasets. Using VIP-knockout mice, we explored the mechanisms underlying VPAC blockade-mediated tumor control. As anticipated, syngeneic PDAC tumors (e.g., KPC-luc and MT5) grew significantly slower in VIP-knockout mice compared to wildtype controls. Notably, the anti-tumor response was markedly superior in female VIP-knockout mice compared to males. To dissect the cellular basis of this sexual dimorphism, we depleted specific immune subsets, including T cells and TAM. Phagocytic TAM depletion accelerated tumor growth in females but had no effect on males, while the depletion of other immune subsets increased tumor growth in both sexes. In vitro, androgen receptor (AR) inhibition (mimicking the female phenotype) reversed the suppressive state of BMDM, marked by reduced Arg1, CD206, and PD-L1 expression. In vivo, AR inhibition or PD-L1 loss significantly enhanced the anti-tumor efficacy of VIP blockade in male mice. Mechanistically, RNAseq and Western blotting revealed increased VPAC1 expression on IC21 and BMDM cells following AR inhibition, reflecting the female-like phenotype. These findings suggest that androgen and VIP pathways mediate sexual dimorphism in TAM phagocytosis, which limits ICI efficacy in PDAC. VPAC1 and PD-L1 are identified as potential sex-specific phagocytosis checkpoints, providing promising therapeutic targets to overcome sex-based differences in PDAC immunotherapy outcomes. Zihan Chen, Tenzin Passang, Fanyuan Zeng, Yuou Wang, JianMing Li, Cynthia Giver, Kiranj Chaudagar, Edmund Waller. Sexual dimorphism in macrophage phagocytosis limits immune checkpoint inhibitor efficacy in pancreatic ductal adenocarcinoma [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 1211.

Uveal melanoma (UVM) is resistant to immune checkpoint therapy and chemotherapy, resulting in high mortality rates, primarily due to liver metastases. While vasoactive intestinal peptide (VIP) signaling has been identified as an immune checkpoint and therapeutic target in pancreatic cancer, its role in melanoma remains unexplored. This study investigated the impact of a novel VIP receptor antagonist, ANT308, on melanoma cell behavior and tumor growth. Using both murine and human UVM/cutaneous melanoma cell lines, we examined the inhibition of VIP receptor signaling and its effects on cell migration and proliferation in vitro. Mechanistically, ANT308 downregulated melanoma cell adhesion molecule (MCAM) and N-cadherin expression at both the RNA and protein levels, as demonstrated by RNA sequencing and Western blot analyses. Knockdown of the VIP receptor VPAC2 in mouse and human melanoma cells produced similar effects on cell migration, proliferation, and MCAM protein expression, further implicating VIP-VPAC2 signaling in tumor progression. In vivo studies revealed that ANT308 treatment decreased MCAM expression in intraocular primary tumors, reduced the number and size of liver metastases following intraocular or subcutaneous melanoma injection, and showed a trend toward reduced tumor volume at the primary tumor site. In conclusion, our findings indicate that VIP receptor signaling promotes liver metastasis in melanoma, and targeting this pathway with VIP receptor antagonists may represent a novel therapeutic strategy for treating metastatic UVM.

Vasoactive intestinal peptide (VIP) is a neuropeptide involved in cancer proliferation and immune suppression. The limited potency of the VIP antagonist peptide VIPhyb in T-cell activation and murine anti-leukemia models prompted the development of a more potent antagonist. We screened a combinatorial library of VIPhyb C-terminal peptide sequence variants to identify a higher-affinity VIP-receptor (VIP-R) antagonist, hypothesizing that specific amino acid substitutions could improve receptor binding and/or plasma stability. In silico screening analyses identified sequences with docking scores predicting increased binding affinity to human VIP receptors VPAC1 and VPAC2. 15 peptides were synthesized and tested for their ability to potentiate activation of purified mouse and human T cells and enhance T cell-dependent anti-leukemia responses in murine acute myeloid leukemia models. Treating C57Bl/6 mice engrafted with a C1498 leukemia cell line with daily subcutaneous injections of VIP-R antagonist peptides induced anti-leukemia responses. Strikingly, the predicted binding of the VIP-R antagonists to VIP receptors correlated positively with their ability to augment mouse T-cell proliferation and anti-leukemia activity. ANT308 and ANT195 emerged as top candidates due to high predicted VIP-R binding, low EC50 for in vitro T cell activation, and potent anti-leukemia activities. ANT308 decreased CREB phosphorylation, a downstream signaling pathway of the VIP receptor, and stimulated granzyme B and perforin expression in CD8+ T cells from AML patients. Combining in silico modeling, in vitro T cell activation properties, and in vivo anti-leukemia activity has identified promising VIP-R antagonist candidates for further development as novel immunotherapies for patients with AML having relapsed disease.

Severe fibrosis, predominantly driven by the activation of pancreatic stellate cells (PSCs), plays a crucial role in the poor prognosis associated with pancreatic ductal adenocarcinoma (PDAC). Understanding the intricate interplay between tumor cells and their microenvironment is essential for deciphering the regulatory mechanisms underlying PSC activation. This study sheds light on the critical role of tumor-derived interleukin-35 (IL-35) in modulating PSC activation, thereby unveiling a promising therapeutic target for mitigating PDAC progression. This study demonstrates that IL-35, secreted by PDAC cells, serves as a key mediator of bidirectional communication between PDAC cells and PSCs, exacerbating fibrosis. Specifically, IL-35 upregulates the expression of IGFBP2 and Tsp-1 in PDAC cells, which subsequently activates PSCs through the IGF-1R/phosphoinositide 3-kinase/Akt and transforming growth factor beta signaling pathways, respectively. This sequential activation fosters an environment conducive to tumor cell proliferation and migration, ultimately driving accelerated tumor growth. Collectively, these findings indicate that IL-35 is a promising therapeutic target whose blockade not only suppresses PSC activation and stromal fibrosis, but also enhances the efficacy of standard chemotherapies (gemcitabine and gemcitabine/nab-paclitaxel). This provides a strong rationale for its clinical development as a combinatorial strategy in PDAC treatment.

Acute myeloid leukemia (AML) remains one of the most lethal hematologic malignancies, particularly in elderly patients and those harboring TP53 mutations. Standard-of-care chemotherapies offer limited long-term survival benefits, especially in TP53-mutated patients with chemotherapy-resistant phenotypes. Recent insights into the tumor microenvironment (TME) suggest that immune evasion mechanisms mediated by myeloid cells play a critical role in leukemia progression and therapeutic failure. Therefore, our central hypothesis is to overcome myeloid cell-mediated immunosuppression for mounting anti-tumor response in acute myeloid leukemia. Bioinformatics and flow cytometry analysis were done on bone marrow and PBMC of AML patients to investigate VIP profiling in TP53-mutated vs non-mutated cases, respectively. Human and murine AML cell lines were deployed to study impact of TP53 on VIP production and phagocytosis vulnerability of VIP-producing cells, respectively. In vivo efficacy of novel long-acting VIP receptor antagonist, ANT308-IgG4 Fc fusion was studied in the murine AML model. Our flow cytometry analysis showed a higher level of VIP expression in PBMC of ~36% AML patients relative to healthy human PBMC. Survival analysis using cBioportal datasets revealed direct association of high VIP expression with poor survival in AML patients. TP53 loss-of-function mutation followed the same pattern of mortality as VIPhi AML. Differential gene expression analysis showed an enrichment of VIP and their receptor on TP53-mutated patients, but not PD-1, other immune checkpoint or anti-apoptosis proteins. Corroborating secretome analysis of human leukemia cells demonstrated a higher VIP secretion from TP53 LOF-mutated leukemia cells (HL-60 harboring homozygous deletion in TP53; Kasumi-1 with homozygous TP53-mutation, and heterozygous TP53-mutation present in CCRF-CEM) relative to control (TP53-wildtype present in THP-1). In murine VIP-secreting WEHI3 leukemic cells treatment with the VIP receptor antagonist, ANT308 enhanced phagocytosis of leukemia cells by both M1-/M2-like macrophages but did not alter macrophage phagocytosis of VIP non-secreting C1498 cells , suggesting VIP may function as a phagocytosis checkpoint in TP53-mutated AML. Additionally, flow cytometry demonstrated increased VIP expression in non-leukemic myeloid cells in AML patients. Testing the effects of VIP signaling in host cells, we also observed a significant survival benefit in VIP knock-out mice as well as VPAC1 or VPAC2-knockout mice bearing C1498 leukemia relative to VIP-wild type mice. In vivo treatment of wild-type mice harboring C1498 with ANT308 or the long-acting Fc fusion form eradicated established C1498 leukemia in up to 65% of leukemic mice.Conclusions: The VIP/VPAC axis functions as a previously unrecognized phagocytosis checkpoint in AML. TP53-mutation evades anti-tumor immune response via VIP. These findings support the evaluation of ANT308-IgG4 Fc3 fusion, alone or in rational combinations, for high-risk AML.

Introduction: Emerging evidence highlights the gut microbiome as a critical player in gastrointestinal acute graft-versus-host disease (GvHD): microbial diversity, short-chain fatty acid (SCFA) production, and epithelial integrity all influence the incidence of acute GvHD, immune recovery and GvHD outcomes. Vasoactive intestinal peptide (VIP) is an immunosuppressive neuropeptide involved in maintaining homeostasis in the gut microbiome. VIP knockout (VIP-KO) mice exhibit greater anti-tumor immunity compared to wild-type, but have severe GvHD after allogeneic bone marrow transplant (allo-BMT) with significantly reduced survival. Furthermore, VIP-KO mice have a distinct gut microbiome that has been associated with resistance to the growth of transplantable leukemia cell lines. ANT308 is a VIP-receptor antagonist designed to block signaling through VIP-receptors VPAC1 and VPAC2. We hypothesized that pharmacological inhibition of VPAC signaling would recapitulate some of the effects of VIP-KO on the gut microbiome. We compared the effects of ANT308, a high-affinity VPAC antagonist, on the murine gut microbiome and the incidence and severity of GvHD to VIP-KO recipients in allogeneic bone marrow transplant (allo-BMT) models. Methods: Wild-type C57Bl/6 mice and VIP-KO mice were cohoused to exchange microbiota via coprophagy for 4-6 weeks. In another group, wild-type C57Bl/6 mice were treated with daily subcutaneous injections of 20ug ANT308 for 4 weeks. Both groups received allo-BMT from B10.BR donor mice. Mouse weight, GvHD score, and survival were monitored. Stool samples were collected pre- and post- VIP-KO cohouse, pre- and post- ANT308 treatment, and post-transplant for shotgun sequencing and untargeted lipidomic analysis. Taxonomic and functional data were analyzed using MaAsLin2 analysis. MetaCyc pathway mapping identified metabolic and biosynthetic pathways involved in SCFA and lipid synthesis. Lipidomic profiling of fecal pellets employed untargeted mass spectrometry. Non-parametric tests were used for differential abundance analysis, and pathway shifts were visualized via heatmaps and volcano plots. In a graft-versus-leukemia model (GvL) of allo-BMT, wild-type mice cohoused with VIP-KO mice for 4-6 weeks were inoculated with C1498-luciferase and received allo-BMT from B10.BR donors. Recipient mice were monitored for weight, GvHD score, survival, and C1498-luc growth was measured by IVIS imaging. Results: Transfer of the VIP-KO microbiome to wild-type mice following cohousing with VIP KO mice was confirmed by taxonomic and functional composition from shotgun sequencing of stool samples. Pharmacological inhibition of the VIP-signaling pathway with ANT308 treatment revealed a microbiome similar to VIP-KO mice, with increased abundance of short-chain fatty acids (SCFA)-producing taxa, including Muribaculum intestinale and Lactobacillus murinus. Metabolic pathways supporting pyruvate fermentation to acetate & lactate I/II pathways were enriched and correlated with known SCFA producer taxa in ANT308-treated wild-type mice and wild-type mice with VIP-KO microbiome. Lipidomic analysis revealed enrichment of LPC 18:2 in ANT308 and VIP-KO microbiome, and apigenin-6-C-glucoside-8-C-arabinoside in ANT308-treated microbiome only. Wild-type mice with the adoptive transfer of VIP-KO microbiome had improved median survival time and showed greater leukemia (C1498-luc) control in a GvL model compared to wild-type mice, but had worse GvHD scores with more weight loss. Interestingly, wild-type mice treated pre-transplant with ANT308 reconstituted with a microbiome characterized by increased SCFA synthesis and had improved GvHD scores, less weight loss, and greater survival compared to untreated wild-type mice. Survival benefit of ANT308-reconstituted microbiome in a Conclusion: Modulation of VIP signaling represents a promising strategy to shape gut microbial metabolism in the context of allo-BMT. Our findings suggest that microbiome-derived metabolites enriched through VIP pathway inhibition may contribute to improved immune balance and transplant outcomes. Future studies will focus on defining the mechanistic roles of these metabolites and evaluating their potential as therapeutic targets to enhance graft-versus-leukemia effects while mitigating GvHD.

Anti-programmed death-1 (PD-1) therapy enhances anti-tumor immunity in many solid tumors, but is largely ineffective in acute myeloid leukemia (AML). CD47 blockade promotes phagocytic clearance of leukemic stem cells in AML, but its efficacy is limited by off-target binding to healthy cells. Vasoactive intestinal peptide (VIP), an immunosuppressive neuropeptide overexpressed in some AML, may serve as an alternative checkpoint. ANT308 is a potent VIP antagonist that promotes T-cell activation and downregulates immune checkpoint molecule expression, demonstrating single-agent anti-leukemia activity in murine leukemia models. We hypothesized that combining ANT308 with PD-1 or CD47 blockade would improve anti-leukemia immunity in mice. DBA2/J mice were subcutaneously inoculated with 5×10⁴ or 1×10⁵ P815 AML cells. One-week later, mice were treated with daily s.c. injections of ANT308 or scrambled peptide, or biweekly injections of long-acting ANT308 (ANT308 PEG, ANT308 IgG4 Fc fusion) for 2 weeks. For combination studies, ANT308 was co-administered with either anti-PD-1 (200 µg twice/week) or anti-CD47 (1.3 nmol every other day) for 2 weeks. Tumor size and body weight were measured twice/week; survival was monitored daily. On day 17, peripheral blood was analyzed by 26-color flow cytometry for T cell phenotypes. Before treatment, tumor volumes were comparable across groups (10.5 ± 1.1 mm³). In the control mice, tumors grew rapidly, leading to death or IUCAC-specific endpoint with a median survival time [MST] of 19 days. In contrast, ANT308 monotherapy achieved a 76.4% tumor volume relative reduction [TVRR] at one-week post-treatment, compared to tumor volumes at 21 days post-inoculation, and improved survival (MST 26 d), with 30% of mice tumor-free at 45 days. Twice-weekly injections of ANT308PEG (MST 29 d) and ANT308Fc (MST 51 d; 40% tumor-free survival) outperformed daily ANT308 treatment. Anti-CD47 monotherapy achieved 10% tumor free survival through 70 days (MST 27), but its combination with ANT308 reduced anti-leukemia activity (MST 27 d vs. 60 d for ANT308 alone), with only 10% leukemia-free survivors in the combination group. In contrast, single-agent ANT308 and anti-PD-1 both demonstrated significant anti-leukemia activity (50.4% and 61.9% one-week TVRR, respectively) and were more potent when combined, resulting in an 83% one-week TVRR and 40% tumor-free survival. Furthermore, flow cytometry revealed increased Ki67 and TCF1 expression on CD8⁺ T cells following anti-PD-1 and ANT308/anti-PD-1 therapy. CXCR3 was upregulated by both anti-PD-1 and the combination. CX3CR1⁺ cells were highest with ANT308 alone or in combination, whereas mice treated with anti-PD-1 exhibited lower levels than the control. Additionally, mice treated with the combination of ANT308 and anti-PD-1 had reduced levels of exhausted CD8⁺ T cells (PD-1⁺ Tim-3⁺) compared to controls. Notably, expression of VPAC2, a receptor for VIP, was induced on T cells in both the anti-PD-1 and ANT308/anti-PD-1 combination group. These findings suggest that ANT308 synergizes with PD-1 blockade to enhance AML immunity, likely by relieving VIP-receptor-mediated suppression during T-cell priming and activation, thereby enabling PD-1 blockade to maximize effector function. Long-acting ANT308 formulations further improved survival outcomes compared to daily dosing. The combination therapy also promoted the expansion of effector T cell phenotypes with chemokine receptors associated with homing to tumors (CX3CR1⁺, CXCR3⁺) and anti-tumor immunity. A strategy that targets both VIP and PD-1 pathways is a promising approach to immunotherapy in AML.

Antagonist peptides (ANTs) of vasoactive intestinal polypeptide receptors (VIP-Rs) are shown to enhance T cell activation and proliferation in vitro, as well as improving T cell-dependent anti-tumor response in acute myeloid leukemia (AML) murine models. However, peptide therapeutics often suffer from poor metabolic stability and exhibit a short half-life/fast elimination in vivo. In this study, we describe efforts to enhance the drug properties of ANTs via chemical modifications. The lead antagonist (ANT308) is derivatized with the following modifications: N-terminus acetylation, peptide stapling, and PEGylation. Acetylated ANT308 exhibits diminished T cell activation in vitro, indicating that N-terminus conservation is critical for antagonist activity. The replacement of residues 13 and 17 with cysteine to accommodate a chemical staple results in diminished survival using the modified peptide to treat mice with AML. However, the incorporation of the constraint increases survival and reduces tumor burden relative to its unstapled counterpart. Notably, PEGylation has a significant positive effect, with fewer doses of PEGylated ANT308 needed to achieve comparable overall survival and tumor burden in leukemic mice dosed with the parenteral ANT308 peptide, suggesting that polyethylene glycol (PEG) incorporation enhances longevity, and thus the antagonist activity of ANT308.

Vasoactive intestinal peptide (VIP) is a neuropeptide involved in tumor growth and immune modulating functions. Previous research indicated that a VIP antagonist (VIPhyb) enhances T-cell activation and induces T-cell-dependent anti-leukemic activity in mice. We created a combinatorial library of VIPhyb C-terminal sequence variations to develop a more potent VIP-receptor (VIP-R) antagonist, hypothesizing that specific amino acid substitutions would improve receptor binding and plasma stability. In silico screening analyses identified sequences with improved docking scores predicting increased binding affinity to human VIP receptors VPAC1 and VPAC2. Fifteen peptides were synthesized and tested for their ability to potentiate activation of purified mouse and human T cells and enhance T cell-dependent anti-leukemia responses in murine models of acute myeloid leukemia. Treating C57Bl/6 mice engrafted with a C1498 myeloid leukemia cell line with daily subcutaneous injections of VIP-R antagonist peptides induced T cell activation resulting in specific anti-leukemia responses. Strikingly, the predicted binding affinity of the VIP-R antagonists to VIP receptors correlated positively with their ability to augment mouse T-cell proliferation and anti-leukemia activity. ANT308 and ANT195 emerged as top candidates due to their high predicted VIP-R binding, low EC 50 for in vitro T cell activation, and potent anti-leukemia activities. ANT308 decreased CREB phosphorylation, a downstream signaling pathway of the VIP receptor, and stimulated granzyme B and perforin expression in CD8+ T cells from AML patients. Combining in silico modeling, in vitro T cell activation properties, and in vivo anti-leukemia activity has identified promising VIP-R antagonist candidates for further development as novel immunotherapies for AML, especially for patients with relapsed disease.

Identifying mechanisms underlying tumor growth and immune resistance is needed to treat pancreatic ductal adenocarcinoma (PDAC) effectively. The complexity of the tumor microenvironment (TME) suggests that the crosstalk between cells in the TME could drive drug resistance and relapse in PDAC. We have previously determined that vasoactive intestinal peptide (VIP) is overexpressed in PDAC and that VIP receptors expressed on T cells are a targetable pathway that sensitizes PDAC to anti-PD1 therapy. In this study, we show that pancreatic cancer cells engage in autocrine signaling of VIP through VIP-receptor 2 (VPAC2), and that high co-expression of VIP with VPAC2 leads to reduced relapse-free survival in PDAC patients. Mechanistically, we identified piwi-like RNA-mediated gene silencing2 (Piwil2) as a tumor-cell intrinsic protein downstream of VPAC2 that regulates cancer cell growth. In addition, we discovered TGFβ-1 as a potential tumor-extrinsic inhibitor of T cell function induced by VPAC2 signaling. In vivo , knock out and knockdown of VPAC2 on PDAC cells led to reduced tumor growth rate and increased sensitivity to anti-PD-1 therapy in various mouse models of PDAC that were T-cell dependent. Overall, these findings emphasize the implications of VIP/VPAC2 signaling in the PDAC tumor microenvironment and further support the rationale for developing VPAC2-specific antagonists.Significance The autocrine VIP signaling via VPAC2 promotes cancer cell growth and dampens T cell function in pancreatic ductal adenocarcinoma and thus represents a potential therapeutic target in PDAC.### Competing Interest StatementIntellectual property related to the use of peptide antagonists to vasoactive intestinal polypeptides to treat cancer is the subject of US patent applications with SR, TP, JML, and EKW listed as inventors. These patents have been licensed to Cambium Oncology, LLC. EKW are co-founders and have equity in Cambium Oncology. A conflict-of-interest management plan has been reviewed and approved by Emory University.

Introduction: AML is a prevalent hematological malignancy in adults, with a 5-year survival rate of only 32%. While approximately 32,000 AML patients are diagnosed annually in the US today, projections suggest this will rise to 36,000 by 2027. Current treatments, such as allogeneic bone marrow transplantation, have limited applicability due to high morbidity and treatment-associated mortality, and current immune check-point therapies using anti-PD1 and anti-PDL1 antibodies are ineffective in AML patients. Vasoactive intestinal peptide (VIP) is a highly conserved peptide hormone with immunosuppressive properties. VIP is frequently upregulated in AML and expressed by activated T cells as a co-inhibitory ligand that downregulates T cell anti-leukemia activity. We have developed a peptide antagonist of VIP/VIP receptor signaling, ANT-308, and demonstrated increased T cell anti-tumor activity in murine leukemia and pancreatic cancer models. Herein, we report on developing humanized anti-VIP monoclonal antibodies as a new therapeutic to treat AML. Methods: Yeast display methods were used to screen a human scFv antibody library. Multiple rounds of FACS sorting using fluorochrome-bound VIP yielded three top candidate clones with scFv fragments having high VIP binding affinity: A6, C2, and D5. The scFv fragment VL and VH sequences were cloned into the Abvec 2.0 plasmid and co-transfected into the Freestyle 293F human expression cell line to generate humanized monoclonal antibodies. Purified anti-VIP mAbs were tested for VIP binding using biotinylated VIP bound to M-280 streptavidin Dynabeads, using an anti-human FITC-conjugated secondary antibody in a flow-cytometric assay, and also using surface plasmon resonance (SPR) analysis. In vitro testing for increased T cell activation used a pool of MACS-purified T cells from three healthy donors, incubated overnight with 50 IU/ml IL-2, followed by addition of A6, C2, or D5 mAbs at 10, 50, or 100 ug/mL and activation with a sub-maximal concentration of anti-CD3/CD28/CD2. After 48 hours, T cells were analyzed for CD69 and 4-1BB activation marker expression. In an initial in vivo test for anti-leukemia activity, groups of 10 DBA/2 mice were inoculated s.c. with 1E5 P815 myeloid sarcoma cells, followed by treatment on day 7 using a single injection of 3 nmol of a pool of the 3 anti-VIP mAbs, control mAb, ANT-308, control peptide, or PBS. Mice were followed for tumor burden and survival. Results: Analysis of anti-VIP mAb binding on VIP-coated Dynabeads demonstrated near-equivalent VIP-binding for A6, C2, and D5, with MFI 3.5-fold higher than control mAb. SPR analysis also showed VIP-binding for all three mAbs, with better binding kinetics for the D5 mAb. In the in vitro activation assay, the anti-VIP mAbs increased CD69 and 4-1BB expression in CD4+ and CD8+ T cells compared to anti-CD3/CD28/CD2 activation alone. Of note, activation levels with anti-VIP antibodies were higher than those observed with ANT-308, a peptide antagonist of the VIP receptor. In the in vivo myeloid sarcoma model, all mice in the PBS, control peptide, and control antibody groups succumbed to tumor burden by 30 days post-inoculation. The single day 7, 3 nmol dose of combined anti-VIP mAbs resulted in improved survival equivalent to that achieved with 3 nmol ANT-308 (p<0.001 compared to PBS control). Moreover, treatment of P815-bearing DBA/2 mice with a single injection of the pool of anti-VIP antibodies led to initial regression of established s.c. P815 tumors, and sampling blood for cancer-specific T cells using a P815-specific tetramer showed 5-fold expansion of tetramer+ CD8+ T cells at 14 days following anti-VIP antibody administration. Conclusions: Humanized anti-VIP antibodies are an attractive alternative to peptide-based VIP-receptor antagonists due to their improved pharmacokinetics. We demonstrate the effective use of yeast-display single-chain antibody technology, serial selection for VIP binding, and expression in a producer cell line to identify and generate novel anti-VIP human antibodies. Our initial results show the three mAbs identified here bind VIP in bead-based flow cytometry and SPR assays, increase human T cell activation in vitro, and improve survival in a mouse mastocytoma model. We are continuing to characterize the mAbs and test their ability to improve the anti-tumor activity of T cells in additional pre-clinical AML models using repeated dosing strategies.