CD40 Agonism in Pancreatic Ductal Adenocarcinoma: Expression, Biology, and Therapeutic Targeting

1. Introduction

Pancreatic ductal adenocarcinoma (PDAC) is the predominant subtype of pancreatic cancer and is the sixth leading cause of cancer mortality worldwide [1]. Its incidence is rising, and projections suggest that it will become the second leading cause of cancer-related death by 2030 [2]. Standard combination chemotherapy regimens (e.g., FOLFIRINOX or gemcitabine/nab-paclitaxel) provide limited long-term benefit [3,4], with 5-year survival remaining around 13% [5], reinforcing the need for new therapeutic strategies.

Immune checkpoint inhibitors (ICIs) alone have transformed care in several solid tumors [6,7,8,9,10,11,12,13,14], but have shown limited activity in biomarker-unselected PDAC [15,16,17]. This refractoriness is largely attributed to an immunosuppressive tumor microenvironment (TME) enriched for tumor-associated macrophages, myeloid-derived suppressor cells, and regulatory T cells, combined with a dense desmoplastic stroma and abnormal, poorly perfused vasculature that restricts immune trafficking and drug delivery. Together with low tumor mutational burden and sparse tumor-specific effector T cells and DCs, these features contribute to an immunologically “cold” phenotype [18,19,20,21,22]. Therapeutic strategies in PDAC, therefore, likely require both improved antigen presentation and T cell priming, as well as relief of stromal-myeloid constraints on effector function.

CD40, a member of the tumor necrosis factor receptor (TNFR) superfamily, is a central co-stimulatory receptor in adaptive immunity that links CD4⁺ T cell help to antigen-presenting cell (APC) licensing and downstream CD8⁺ T cell priming [23,24]. Its physiological ligand, CD40L (CD154), is transiently upregulated on activated CD4⁺ T cells and can also be provided in soluble form, including from platelets, enabling CD40 signaling in both immune and vascular compartments [25,26]. CD40 is primarily expressed by APCs, including dendritic cells (DCs), macrophages, and B cells [25,26,27]. Engagement by CD40L delivers a potent activation signal that promotes APC maturation, cytokine production, and enhanced cross-presentation, thereby strengthening T cell priming and downstream effector functions [23,24,25,26,27,28]. CD40 expression has also been reported on subsets of PDAC tumor cells, with heterogeneity across tumors and disease stages, suggesting context-dependent roles in tumor biology and immune interactions [29,30]. Additionally, CD40 has been detected in stromal compartments, including cancer-associated fibroblasts (CAFs) and select endothelial populations, supporting the concept that CD40-directed therapies may exert effects beyond classical APC activation [24,30,31,32,33].

Consistent with this biology, CD40 agonism in preclinical PDAC models can induce immune activation and stromal remodeling [31,32]. Several CD40 agonists are currently in clinical trials, showing signs of immune activation and generally acceptable safety profiles across various solid tumors, including PDAC. [34,35,36]. However, CD40 signaling is context-dependent: while APC-directed activation is typically immunostimulatory, tumor-intrinsic CD40 signaling has been associated with pro-survival or pro-growth programs in some settings, with potential implications for therapeutic design and patient selection [37,38,39].

In this review, we summarize CD40 expression patterns and measurement considerations, explore CD40 signaling and cell-specific roles in PDAC, and evaluate the clinical and translational progress of CD40-directed strategies. We emphasize how combination regimens and appropriate sequencing may be required to overcome PDAC immune resistance and enable more consistent therapeutic responses.

2. CD40 Expression Landscape in PDAC

In PDAC, CD40 expression is most prominently detected on immune cells but is also reported on tumor cells and stromal elements, with variable levels across compartments and disease settings. This pattern aligns with CD40’s roles in immune activation, stromal remodeling, and tumor biology.

2.1. CD40 Expression on Non-Immune PDAC Compartments

Beyond immune cells, CD40 is present across multiple non-immune compartments in PDAC, including tumor cells and stromal elements. A substantial subset of PDAC tumor cells expresses CD40 (reported at around 68% across cohorts) [29,30], but expression is markedly heterogeneous within tumors and is often higher in advanced or metastatic disease [29]. Functionally, tumor-intrinsic CD40 signaling can yield divergent outcomes, ranging from apoptosis to enhanced proliferation and survival, depending on cellular context, with NF-κB and MAPK pathways implicated as key downstream effectors [37,38,39]. Tumor-cell CD40 expression has also been reported to impair T cell activation by reducing T cell CD154 (CD40L) expression and suppressing cytokine production and proliferation, consistent with a potential contribution to immune escape [40]. This heterogeneity likely reflects differences in oncogenic programs, local cytokine milieus, and spatiotemporal variation between primary tumors and metastatic sites, where immune and stromal architecture can differ and may influence responsiveness to CD40 agonism [41,42]. In the stroma, CD40 expression on subsets of cancer-associated fibroblasts (CAFs) and endothelial cells links CD40 signaling to extracellular matrix (ECM) remodeling and vascular regulation. CAF CD40 activation can trigger cytokine and growth factor production that influence matrix metalloproteinase (MMP) activity, leading to ECM remodeling and effects on invasion that depend on CAF subtype and context [33,43,44,45]. Additionally, CD40-expressing endothelial cells participate in inflammatory vascular remodeling and angiogenic processes [25,26,39,46,47]. Crosstalk between CAFs and endothelial cells, mediated by factors like vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF), can result in hypovascularity, reduced drug delivery, and immune exclusion [48,49,50,51]. Consistent with these compartmental effects, studies show that agonistic CD40 activation can induce stromalysis via proteases produced by activated myeloid cells, degrading dense ECM and facilitating infiltration of DCs and effector T cells into tumor tissue [31,32,43]. Collectively, these observations support the concept that CD40-directed therapies may act not only through APC activation but also via tumor-intrinsic and stromal/vascular programs that shape PDAC immune resistance and treatment sensitivity.

2.2. CD40 Expression on Immune Cells Within the PDAC Tumor Microenvironment

The most robust expression of CD40 in PDAC is observed on APC populations, including DCs, macrophages (especially TAMs), monocytes, and B cells [25,26,27]. DC subsets implicated in cross-priming, including cDC1 and cDC2 populations (including CD141⁺ DCs) and LAMP3⁺ mature/regulatory DC states, depend on CD40 engagement for functional maturation and for efficient priming of cytotoxic CD8⁺ T cells [23,26,52]. However, their function in PDAC is frequently inhibited by tumor-derived cytokines and suppressive signals from myeloid populations [53,54].

TAMs, often skewed toward an M2-like phenotype, express variable levels of CD40 [55]. Engagement of CD40 has been shown to promote M1-like inflammatory reprogramming with increased antigen presentation, pro-inflammatory cytokine production (e.g., IL-12, TNF-α), and induction of matrix-degrading programs that can facilitate immune cell infiltration [43]. Within tertiary lymphoid structures (TLS), CD40⁺ B cells may contribute to local antigen presentation and support T cell priming, and, in mature TLS, can undergo antigen-driven maturation and plasma cell differentiation with local antibody production[56]. Importantly, T cells are key providers of the CD40 signal: activated CD4⁺ T cells upregulate CD40 ligand (CD40L/CD154), which licenses CD40⁺ APCs and supports broader lymphocyte activation [24,25,28]. CD40 expression has also been described on subsets of MDSCs, where CD40 engagement may modulate suppressive or effector functions in a context-dependent manner [57,58,59]. NK cells, on the other hand, express CD40 ligand and not the CD40 receptor itself. CD40L engagement on NK cells activates them, enabling them to recognize and kill CD40-expressing target T cells, including tumor cells, and is regulated by IL-12 and IFNγ [60,61]. This activates the NF-κB signaling, resulting in cytotoxic activity [62,63].

3. Biological Functions and Signaling Pathways of CD40 in PDAC

The biological consequences of CD40 engagement are compartment-dependent, shaped by the cellular source of CD40 and the local availability and form of its ligand. The key cellular and molecular mechanisms engaged by CD40 agonists in PDAC are summarized in Figure 1.

3.1. Core CD40 Signaling Pathways Shaping Immune Activation

CD40 is a TNFR family receptor that lacks intrinsic enzymatic activity and signals by recruiting TNF receptor-associated factor (TRAF) adaptor proteins after ligand-induced receptor clustering. The composition and stoichiometry of TRAFs bias downstream pathway engagement and functional output [64,65,66]. A principal consequence of CD40 ligation is activation of canonical NF-κB signaling, inducing transcriptional programs that support APC maturation and T cell priming, including cytokines (e.g., IL-6, TNF-α), adhesion molecules (e.g., ICAM-1), and costimulatory regulators within the B7 family [66,67].

Among the latter, CD40 engagement upregulates the co-stimulatory molecules CD80 (B7-1) and CD86 (B7-2) on DCs and B cells, and enhances T cell activation [23,28,68,69]. Interestingly, CD40 activation has also been shown to upregulate B7-H3 (CD276) expression on DCs, and this upregulation was functionally required for an enhanced antitumor immune response induced by CD40-activated, tumor antigen-pulsed DCs [66,70,71]. The B7-H3 checkpoint molecule is a member of the B7 family and is overexpressed in various solid malignancies, including PDAC, where it promotes tumor growth, metastasis, and treatment resistance [72,73,74]. Therefore, it has emerged as a potential target in cancer immunotherapy. Recent advances in B7-H3-targeting antibody-drug conjugates (ADCs), such as YL201 and HS-20093 (GSK5764227), provide a promising approach for antitumor therapy in early-phase clinical trials [75,76]. Whether CD40 agonist-mediated upregulation of B7-H3 on myeloid cells within the TME could enhance the efficacy of B7-H3-directed ADCs, or, conversely, serve as an immune evasion mechanism that warrants therapeutic targeting, remains an open question with important implications for rational combination strategies that have not yet been addressed.

CD40 can also engage the non-canonical NF-κB axis, contributing to more sustained programs relevant to APC function and lymphoid organization [77]. In parallel, CD40 activates mitogen-activated protein kinase (MAPK) cascades, including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38, which shape cytokine production, differentiation, and survival programs across myeloid and stromal cells [66,70,71]. CD40 engagement additionally activates the phosphoinositide 3-kinase (PI3K)/AKT pathway, supporting cellular metabolism and antigen-processing capacity in APCs and modulating survival signaling in a context-dependent manner [78]. JAK3-STAT5 signaling is generally not a primary, direct CD40 pathway but can be engaged indirectly via autocrine/paracrine cytokines induced downstream of CD40 activation, thereby tuning inflammatory polarization and preventing tolerogenic programming in APCs [79,80,81,82]. Collectively, these signaling modules determine the magnitude, durability, and qualitative balance of CD40-driven immunity, and they provide a mechanistic basis for why CD40 agonism can have distinct effects across PDAC cellular compartments.

Targeting the JAK/STAT pathway has emerged as a potential therapeutic strategy in cancer, as it is involved in inflammatory responses and progression, particularly STAT3 in pancreatic cancer [83,84,85,86,87,88,89,90]. In a randomized, double-blind, phase II trial in patients with metastatic pancreatic cancer, a subgroup with increased systemic inflammation demonstrated signs of clinical benefit after treatment with ruxolitinib, a JAK1/JAK2 inhibitor, plus capecitabine chemotherapy [91]. Encouraged by these findings, two phase III randomized, placebo-controlled trials, JANUS 1 (NCT02117479) and JANUS 2 (NCT02119663), were initiated to evaluate the efficacy of this combination therapy [92]. Patients with advanced or metastatic pancreatic cancer and increased systemic inflammation, with one prior chemotherapy regimen, received ruxolitinib plus capecitabine or a placebo plus capecitabine. Unfortunately, both trials were terminated prematurely due to futility after interim analysis of JANUS 1, as the combination therapy failed to improve survival compared to placebo. Further investigation into JAK/STAT inhibition was pursued in another phase Ib/II study, in which itacitibin, a selective JAK1 inhibitor, was combined with gemcitabine and nab-paclitaxel in patients with treatment-naïve advanced metastatic solid tumors, including PDAC [93]. Despite demonstrating some clinical activity with an overall response rate of 24%, this trial was also terminated following the disappointing results from JANUS 1 and 2.

Given that CD40 agonism can indirectly engage JAK3-STAT5 signaling [79,80,81,82], combining CD40 agonists with JAK/STAT pathway inhibitors could offer a novel therapeutic approach to further enhance the antitumor response in PDAC. While CD40 engagement promotes APC maturation and enhanced cross-presentation for T cell priming and activation, simultaneously inhibiting the JAK/STAT pathway in the tumor might reduce progression and increase chemotherapy sensitivity, thereby enhancing antitumor efficacy and increasing survival. However, JAK/STAT pathway inhibitors may also have immunosuppressive effects [94,95,96]. Therefore, a comprehensive understanding of how JAK/STAT signaling interacts with CD40-driven immune activation and the overall effect on the immune system and TME could provide valuable insights into this combination strategy. Whether JAK/STAT pathway inhibitors would enhance or impair the efficacy of CD40 agonists, and whether the toxicity profile would be acceptable when combining these two regimens, remains an open question that warrants further preclinical investigation.

3.2. The CD40-CD40L Axis In Vivo: Ligand Sources and Signaling Context

Physiologic CD40 activation is mediated by CD40L (CD154), which is transiently upregulated on activated CD4⁺ T cells and provides the canonical help signal required for APC licensing and effective cytotoxic T cell priming [23,25,26,28]. CD40L can also be supplied by other activated immune cells and is present in soluble form, most prominently derived from platelets, supporting CD40 signaling in vascular and inflammatory contexts and contributing to endothelial activation programs [25,26,39,46,47]. The biologic outcome of CD40 engagement is therefore shaped not only by CD40 expression but also by where, when, and in what form CD40L is available (cell-bound vs soluble), as well as by the inflammatory milieu that determines pathway integration (e.g., NF-κB/MAPK tone) [71,97]. In PDAC, where productive CD4⁺ T cell help is often limited and myeloid suppression is prominent, therapeutic CD40 agonism aims to bypass inadequate endogenous CD40L delivery and restore licensing signals needed for effective priming and intratumoral immune activation [23,26,69].

3.3. CD40-Driven Activation of Antigen-Presenting Cells

CD40 engagement on DCs, macrophages, and B cells induces maturation and functional activation, thereby bridging innate and adaptive immunity. Upon CD40 ligation via CD40L or agonistic antibodies, DCs upregulate MHC class I/II and enhance cross-presentation, thereby enabling priming of both CD4⁺ and CD8⁺ T cells [23,26]. CD40 activation also increases CD80/CD86 expression on DCs, ensuring delivery of the necessary secondary signals to naive T cells, promoting their proliferation and differentiation into cytotoxic and helper subsets [23,69]. In addition, CD40 activation promotes IL-12 production, supporting Th1 polarization, CD8⁺ T cell expansion, and IFN-γ-associated effector programming, all of which are critical for antitumor responses [23,26,43,98]. These changes promote robust cross-priming of CD8⁺ T cells, a process profoundly deficient in untreated PDAC [21,99,100,101]. Therapeutic CD40 agonists could restore this critical antitumor axis.

3.4. Reprogramming of Tumor-Associated Macrophages

The PDAC TME favors immunosuppressive TAM states driven by chronic inflammation, hypoxia, and stromal cues [55,102,103,104]. CD40 signaling can reprogram M2-like macrophages toward a more inflammatory, antigen-presenting phenotype, associated with increased IL-12/TNF-α production, enhanced antigen presentation, induction of matrix-degrading programs, and improved immune cell recruitment [31,32,43,105]. In preclinical PDAC models, macrophage reprogramming is a major contributor to CD40 agonist activity and is mechanistically linked to improved T cell infiltration and intratumoral activation [31,106,107].

3.5. Stromal and Vascular Remodeling Downstream of CD40 Activation

CD40 agonism can modify PDAC stromal barriers through myeloid-driven remodeling programs [32]. CD40 activation increases expression of multiple matrix metalloproteinases (MMPs) in inflammatory macrophages and can suppress profibrotic CAF activity through paracrine and contact-dependent signaling, reducing ECM deposition and facilitating immune trafficking and drug penetration [31,32,43]. Endothelial cells express CD40, whose expression is notably upregulated by pro-inflammatory stimuli such as TNF-α and IFN-γ [46,108]. The interaction between endothelial CD40 and its ligand (CD40L), derived from activated T cells, monocytes, or platelets, triggers a potent pro-inflammatory response in the endothelium [46,47,109,110]. This activation leads to the expression of adhesion molecules (e.g., E-selectin, ICAM-1, VCAM-1), the production of inflammatory cytokines (IL-1, IL-6, IL-8), chemokines, MMPs, and increased procoagulant activity [46,109,111,112,113]. In PDAC models, inflammatory remodeling and stromal loosening have been linked to increased DC and T cell infiltration, supporting a mechanistic role for CD40 in overcoming physical and cellular barriers to immunity [24,31,43,105,114]. Furthermore, CD40-CD40L interactions can induce the production of angiogenic factors, including VEGF and fibroblast growth factor, with soluble CD40L promoting endothelial cell growth [115,116,117]. However, the function of CD40 in tumor angiogenesis is complex. Some studies indicate that CD40 activation may enhance tumor neoangiogenesis by increasing VEGF levels [118,119], whereas others show reduced tumor blood vessels and slower tumor growth due to inflammatory remodeling [39,120].

3.5. Tumor-Intrinsic CD40 Signaling in PDAC

In subsets of PDAC, CD40 is expressed by malignant epithelial cells and can transmit tumor-intrinsic signals upon ligation [29,30,121]. Downstream activation of NF-κB and MAPK pathways has been linked to divergent phenotypes, ranging from apoptosis [29,122,123] to enhanced proliferation and survival [121,124,125,126], depending on cellular state and microenvironmental context [125,127,128]. Beyond effects on tumor cell fitness, tumor-cell CD40 signaling has also been reported to modulate antitumor immunity, including attenuating T cell activation through reduced T cell CD154 (CD40L) expression and impairing cytokine production and proliferation in some settings [40,121,129]. These context-dependent tumor-intrinsic programs are relevant to therapeutic CD40 agonism because the net biologic effect may reflect the balance between APC licensing and myeloid reprogramming on one hand and tumor-cell-associated signaling on the other hand [27,33,130]. Accordingly, interpretation of CD40-targeted strategies in PDAC should consider not only immune compartment engagement but also tumor-cell CD40 expression and downstream signaling competence.

4. Therapeutic Targeting of CD40 in PDAC

CD40 agonism is being pursued in PDAC to compensate for insufficient endogenous CD40L-driven APC licensing and to induce coordinated programs that are typically deficient in this disease: improved antigen presentation and T cell priming, myeloid reprogramming, and remodeling of stromal/vascular barriers that limit immune trafficking and drug delivery. Because CD40 pathway engagement can be pharmacodynamically robust yet clinically variable as monotherapy, most development has focused on rational combinations and on defining the settings and biomarkers that best capture effective pathway engagement.

4.1. CD40 Agonistic Modalities and Design Principles

Productive CD40 signaling requires receptor clustering; therefore, in vivo agonism depends on the efficiency with which an agent promotes CD40 multimerization. This is influenced by the targeted CD40 epitope, effective valency, and, particularly for agonistic monoclonal antibodies (mAbs), Fcγ receptor (FcγR)-mediated crosslinking on accessory cells [131]. These parameters (isotype/Fc engineering, FcγR engagement profile, and binding geometry) shape the magnitude and tissue distribution of immune activation and contribute to inter-agent differences in tolerability and optimal dosing schedules. Accordingly, CD40 agonists should not be treated as a uniform class: selicrelumab (RG7876/CP-870,893), SEA-CD40, mitazalimab (JNJ-64457107/ADC-1013), sotigalimab (APX005M), CDX-1140, ChiLob 7/4, and LVGN7409 are all under clinical investigation in pancreatic cancer (Table 1). While each aims to mimic CD40L-driven activation, they differ in molecular properties that can alter clustering requirements and downstream biology. Across agents, monotherapy activity in PDAC has generally been modest, reinforcing the rationale for combination strategies that provide antigen release and/or relieve downstream suppression.

4.2. Combination Approaches with Chemotherapy

Chemotherapy establishes a mechanistic foundation for CD40 activation in PDAC by boosting antigen availability through tumor cell death and antigen release, promoting immunogenic cell death signals, and altering myeloid cell composition. Additionally, CD40-induced remodeling of myeloid and stromal components may enhance stromal and vascular permeability, facilitating better access for immune cells and drugs to the tumor tissue [32,106,132]. A key goal of chemo-CD40 therapy is to reprogram TAMs, including CD163⁺ myeloid cells, to adopt inflammatory, antigen-presenting, and matrix-remodeling phenotypes, shifting the tumor microenvironment from immunosuppressive to one more supportive of effector functions. This is important because high CD163⁺ macrophage levels correlate with poorer outcomes across various cancers [99,133,134,135]. Preclinical data support a synergistic effect between chemotherapy and CD40 activation, showing improved T cell priming and longer survival in experimental models [32,106,132].

Clinically, combinations of chemotherapy and CD40 in PDAC have been examined, including gemcitabine, gemcitabine combined with nab-paclitaxel, and mFOLFIRINOX (Table 2). In a phase I trial, patients with chemotherapy-naïve advanced PDAC received selicrelumab (CP-870893) in combination with gemcitabine, resulting in an ORR of 19% (4 of 22 patients). The median PFS was 5.2 months (95% CI: 1.9-7.4), and the median OS was 8.4 months (95% CI: 5.3-11.8), with a 1-year OS rate of 28.6%. Immune activation was observed through increased inflammatory cytokines and B cell expression of costimulatory molecules [136].

In a separate phase I study, sixteen patients with resectable PDAC received neoadjuvant selicrelumab, with or without gemcitabine/nab-paclitaxel, followed by adjuvant gemcitabine/nab-paclitaxel and selicrelumab. The median DFS and OS for the neoadjuvant selicrelumab group (n=11) were 9.8 months (95% CI: 0.4-19.2) and 23.4 months (95% CI: 9.1-37.6), respectively. For the group receiving both selicrelumab and gemcitabine/nab-paclitaxel (n=5), median DFS and OS were not reached. When combining both groups, median DFS was 13.9 months (95% CI: 2.9-24.8), and median OS was 23.4 months (95% CI: 18.0-28.8). Patients treated with selicrelumab showed a T cell-enriched tumor microenvironment (82% vs. 37% in untreated tumors, P=0.004), featuring more active and proliferative T cells and reduced fibrosis. Additionally, there were fewer M2-like macrophages and an increase in mature DCs [43].

In the phase Ib/II PRINCE trial, sotigalimab (APX005M) was combined with gemcitabine plus nab-paclitaxel in patients with untreated metastatic PDAC. In the phase 1b portion, responses were observed in 14 of 24 (58%) DLT-evaluable patients [137]. In the randomized phase 2 portion (n=105), the primary endpoint of 1-year OS was met for the nivolumab/chemotherapy arm (57.7%, P=0.006 vs. 35% historical rate) but was not met for the sotigalimab/chemotherapy arm (48.1%, P=0.062) or the triple combination arm (41.3%, P=0.223). Multi-omic biomarker analyses identified distinct treatment-specific immune signature correlates. Survival was correlated with greater intratumoral CD4⁺ T cell infiltration, increased circulating differentiated CD4⁺ T cells, and increased APC numbers after sotigalimab/chemotherapy [138].

In OPTIMIZE-1, also a phase Ib/II study, CD40 agonist mitazalimab was combined with mFOLFIRINOX in previously untreated metastatic PDAC achieved a confirmed ORR of 40% (23/57 patients; updated confirmed ORR 42.1% ), with a median PFS of 7.7 (95% CI: 5.8-11.3), median OS of 14.3 months (95% CI: 10.0-21.6), and a notably long median duration of response of 12.6 months. The 1-year PFS and OS rates were 34% (95% CI: 20.8-47.7) and 59% (95% CI: 44.2-71.1), respectively [136,139]. Here, objective responders displayed intratumoral myeloid and T cell activation, and mitazalimab-induced immune activation, including the expansion of effector CD4⁺ T cells after the priming dose, correlated with improved outcomes. Furthermore, no cytokine release syndrome was reported with mitazalimab, in contrast to earlier-generation CD40 agonists, and the safety profile was consistent with mFOLFIRINOX alone, with no hepatotoxicity-related treatment discontinuations [136].

Across these early-phase studies, chemotherapy-CD40 combination strategies have demonstrated feasibility and acceptable safety, with no unexpected immune-related toxicities [136,137,140]. Observed cytokine release syndrome (grade 1-2 with selicrelumab and sotigalimab) and liver function test elevations were transient, dose-dependent, and clinically manageable. The most common grade ≥3 adverse events have been hematologic (neutropenia, anemia, thrombocytopenia), consistent with the chemotherapy backbone. These data collectively support the continued evaluation of chemotherapy and CD40 combination strategies in both metastatic disease and neoadjuvant settings, while underscoring that the chemotherapy choice, treatment sequencing, and timing of CD40 agonist administration relative to chemotherapy are likely key determinants of both efficacy and tolerability. The planned randomized, controlled, international phase III trial of mitazalimab with mFOLFIRINOX will be critical to establish whether the encouraging results from OPTIMIZE-1 translate into a survival benefit over standard chemotherapy alone [136,139].

4.3. CD40 Agonism Combined with Immune Checkpoint Inhibition

PD-1/PD-L1 and CTLA-4 blockade have limited efficacy in PDAC, in part due to inadequate baseline priming and dominant myeloid-driven suppression. CD40 agonism provides a mechanistic rationale to improve checkpoint responsiveness by licensing APCs, expanding tumor-specific T cell priming, and shifting macrophage programs away from suppressive phenotypes, thereby creating a microenvironment more permissive to checkpoint blockade [24,141,142]. Preclinical models support durable control and immune memory with CD40-ICI combinations [105,141,142]. CD40 agonist with immune checkpoint inhibition resulted in complete tumor regression in a therapy-resistant pancreatic cancer model [142]. This effect was not obtained with checkpoint inhibition alone. In another pancreatic cancer murine model, the combination of CD40 agonism and a checkpoint inhibitor has cured 63% of tumor-bearing animals and increased tumor-specific T cells in the pancreas [143]. These findings were confirmed in an orthotopic Kras/Trp53-mutant model, demonstrating that ICI and CD40 agonism combination significantly reduced the tumor burden and shifted the TME from a suppressive to an activated state, accompanied by enhanced DC migration and decreased immunosuppressive TAMs [105].

The phase 1b PRINCE trial clinically assessed sotigalimab (APX005M), a CD40 agonist, combined with gemcitabine and nab-paclitaxel, with or without the PD-1 inhibitor nivolumab, in patients with untreated metastatic PDAC. [137]. The treatment demonstrated feasibility with manageable safety and encouraging response activity (n=30; 24 DLT-evaluable). The observed cumulative ORR was 58% (14/24 DLT-evaluable patients), with a median PFS of 11.7 months (95% CI: 7.1-17.8), and median OS 20.1 months (95% CI: 10.5-not estimable). The cumulative 1-year PFS and OS rates were 44% (95% CI: 22.2-63.4) and 70% (95% CI: 46.9-84.3), respectively [137].

However, the subsequent randomized phase II portion of the trial (n=105) did not demonstrate a survival benefit for the CD40 treatment arms compared with chemotherapy alone [138]. The primary endpoint of 1-year OS was only met for patients receiving nivolumab plus chemotherapy (57.7% vs 35% historical rate, P = 0.006, n = 34), while the sotigalimab plus chemotherapy arm showed a modest but non-significant increase in the 1-year OS (48.1%, P = 0.062, n = 36). The triple combination arm with sotigalimab plus nivolumab plus chemotherapy did not meet the endpoint (41.3%, P = 0.223, n = 35). Despite no clear clinical benefit, survival after sotigalimab plus chemotherapy was correlated with greater CD4⁺ T cell infiltration in the tumor and circulating differentiated CD4⁺ T cells and APCs. Survival after nivolumab plus chemotherapy was correlated with a less immunosuppressive TME at baseline and a higher number of activated, antigen-experienced circulating T cells. Notably, no patient subset was identified for which the triple combination was more beneficial. The lack of survival benefit in this arm could potentially be explained by the observed signs of immune exhaustion [138].

CDX-1140, a fully human IgG2 CD40 agonist antibody designed to activate CD40 without requiring FcγR cross-linking, has also been utilized in combination strategies. A phase I trial (NCT03329950) evaluated CDX-1140 as monotherapy or in combination with CDX-301 (a recombinant human Fms-like tyrosine kinase 3 ligand (Flt3L)), pembrolizumab (anti-PD1), or gemcitabine/nab-paclitaxel in patients with advanced malignancies, including PDAC, and has been completed. Preliminary data were presented at conferences and indicated that CDX-1140 was well-tolerated with evidence of immune activation [144,145].

A separate randomized, open-label phase II trial (NCT04536077) evaluating CDX-1140 with or without CDX-301 in PDAC was terminated before completion. Although no peer-reviewed publication has been issued, some preliminary results have been shared [146]. In PDAC, low tissue levels of Flt3L partly contribute to the deficits in conventional DCs (cDCs). Combining systemic Flt3L with a CD40 agonist restored cDC numbers and function in both mouse models and samples from PDAC patients in clinical trials. However, when combined with Flt3L, dual therapy triggered a cDC-driven type I immune response characterized by T cell infiltration, IL-12 production, and reciprocal IFN-γ responses. cDC1s were responsible for CD8⁺ T cell expansion, and T cell-derived IFN-γ enhanced cDC1 survival. However, the combination also increased regulatory T cells via cDC2 activation, thereby dampening immunity, highlighting the complexity of DC-centered approaches and the challenges of this combinatorial strategy.

A currently active phase I study (NCT04635995) is evaluating LVGN7409, a humanized monoclonal agonistic CD40 antibody, as monotherapy and in combination with anti-PD-1 and/or a CD137 agonist in patients with advanced or metastatic solid tumors, including potentially PDAC. Preliminary monotherapy data in 12 heavily pretreated patients did not show dose-limiting toxicities, and the treatment was well-tolerated. Among 9 evaluable patients, 44% achieved stable disease [147]. The combination arms incorporating checkpoint inhibition and CD137 agonism are of mechanistic interest, since CD137 (4-1BB) co-stimulation can amplify T cell effector function and survival downstream of CD40-licensed priming [148]. Results of this trial may inform whether this approach can overcome the immune suppression and limited T cell priming characteristics of PDAC.

Another phase Ib/II trial (NCT05419479) assessed a triple therapy of sotigalimab, domvanalimab (anti-TIGIT), and zimberelimab (anti-PD-1) in metastatic PDAC, even without checkpoint blockade. However, the trial is presently suspended, and no results have been published, underscoring the difficulties and complexity involved in multi-agent immunotherapy approaches for PDAC.

Collectively, these clinical trials demonstrate that overcoming PDACs’ resistance to checkpoint blockade with CD40 agonism is more complex. Combining checkpoint blockade with CD40 agonism and chemotherapy or other regimens may be insufficient in unselected patients. Furthermore, layering the treatment regimen may paradoxically induce immune exhaustion rather than an additive benefit [138]. Understanding the drivers of immune exhaustion and finding ways to mitigate it [149] and biomarker-driven patient selection are significant determinants of treatment efficacy and improved survival outcomes.

4.4. Integration with Cancer Vaccines and Neoantigen-Directed Therapies

Vaccine strategies in PDAC, including personalized neoantigen approaches and KRAS-targeted vaccines, aim to generate de novo tumor-specific T cell responses against epitopes less constrained by tolerance [150,151,152]. CD40 agonism is central to DC maturation and antigen cross-presentation, and IL-12-associated Th1 programming, and may increase the magnitude and functional quality of vaccine-induced responses by enhancing antigen uptake, processing, and presentation [107,153]. This rationale was confirmed in a preclinical model, where CD40 and CD80/86 signaling in cDC1s was found to play a critical role in the effective antitumor immunity of a neoantigen-based therapeutic vaccine [154]. In a murine pancreatic cancer model, DC vaccination in combination with a CD40 agonist was necessary to improve survival in an advanced PDAC setting, whereas CD40 agonism alone was ineffective [107].

The phase I REACtiVe-2 evaluated DC vaccination with MesoPher (monocyte-derived DCs pulsed with an allogeneic tumor lysate) combined with CD40 agonist mitazalimab in patients with metastatic PDAC after completion of (m)FOLFIRINOX (n=16) [155]. A systemic increase in activated and vaccine-specific T cell responses was observed. In post-therapy tumor biopsies from metastatic sites, increased T cell infiltration and decreased collagen deposition were seen. Despite these immune changes, no objective radiological responses were recorded, and half of the patients (50%) had stable disease after three administrations. Notably, half of the patients in the study (50%) had progressive disease at baseline, indicating that halting the advancing tumor biology is challenging and suggesting that this treatment may not be effective for patients already experiencing disease progression at the time of study initiation. This underscores once more the importance of patient selection to ensure an optimal therapeutic response.

Another vaccination trial (NCT02600949) is exploring a personalized neoantigen peptide-based vaccine, administered alone or combined with imiquimod, pembrolizumab, and/or sotigalimab, in patients with advanced pancreatic and colorectal cancers. Although it is not a traditional vaccine, irreversible electroporation (IRE) serves as an in-situ antigen-release platform. As a non-thermal ablation method, IRE induces immunogenic cell death and releases tumor antigens [156]. A phase I trial (NCT06205849) is currently assessing the combination of surgical IRE with intratumoral mitazalimab injections in patients with locally advanced pancreatic cancer. This approach leverages the synergy between IRE-induced antigen release and CD40-driven APC licensing in a targeted manner and could inform future locoregional immunotherapy strategies for LAPC.

Overall, this data supports the idea that vaccine strategies and CD40 agonism address different bottlenecks in PDAC immunity. Vaccines supply specific antigen targets, while CD40 activation enhances APC licensing and activates costimulatory pathways necessary for effective T cell priming. Further research is needed to determine if combining these methods can overcome immune and stromal barriers, ultimately producing durable benefits across biomarker-defined patient groups.

4.5. Neoadjuvant and Window-of-Opportunity Experience

Window-of-opportunity and neoadjuvant studies offer a direct means to interrogate intratumoral pharmacodynamics and to test whether CD40 agonism can remodel the PDAC TME when immune trafficking and lymphatic drainage may be relatively more intact than in widely metastatic disease. Neoadjuvant selicrelumab has been reported to induce measurable immune activation and TME remodeling, supporting proof-of-mechanism in human PDAC, while the linkage between these pharmacodynamic effects and long-term clinical outcomes remains under active investigation [43]. These settings are also well-suited to optimize sequencing (e.g., relative timing to chemotherapy) and to validate tissue-based pharmacodynamic endpoints for subsequent trials.

4.6. Biomarkers: Prognostic Context and Predictors of Benefit

While pan-cancer analyses have linked higher CD40 expression to improved survival in some settings, this relationship is not uniform across tumor types, and the prognostic value appears limited in PDAC [30,157,158,159,160,161]. In PDAC transcriptomic analyses, CD40 mRNA does not consistently function as an independent predictor of OS, and tumor-cell CD40 expression has not shown robust prognostic value in larger cohorts [30,162].

In a large multi-cohort study using spatial characterization of tumor samples from nine different solid tumors, CD40 expression was present in 68% of pancreatic adenocarcinomas [30]. However, CD40 expression on these tumor cells was not prognostic for OS. Similarly, in a pan-cancer transcriptomic analysis, high CD40 RNA expression was observed in 42% of pancreatic cancers and was correlated with improved OS in patients treated with immune checkpoint inhibitors, but not in multivariable analysis, suggesting that CD40 may not be an independent predictive biomarker [162]. In contrast to CD40 expression on tumor cells, serum levels of both soluble CD40 (sCD40) and CD40 ligand (sCD40L) were found to have some prognostic value. High sCD40 was associated with reduced OS, particularly in patients with neoadjuvant chemotherapy, and the combination with the tumor marker Carbohydrate Antigen 19.9 (CA19-9) increased its diagnostic value [163]. Similar to sCD40, soluble CD40 ligand (sCD40L) levels were also prognostic, with high levels associated with poor survival and correlated with unresectability and distant metastasis [117]. The predictive value of sCD40L was superior to that of the tumor markers CA19-9 and Carcinoembryonic Antigen (CEA).

Instead of CD40 expression, outcome associations in PDAC more strongly track with immune composition and functional state, particularly DC infiltration and macrophage polarization [43,138,139]. Neoadjuvant and adjuvant treatment with selicrelumab increased circulating CD4+ and CD8+ T cells and elevated inflammatory cytokine levels [43]. In the tumor, reduced fibrosis and decreased M2-like macrophages were observed, while an increase in mature DCs and enrichment of T cells were observed. Furthermore, systemic inflammatory cytokine levels were elevated after treatment. In the PRINCE trial, biomarker analysis revealed that higher baseline frequencies of circulating DCs, B cells, and experienced Th1 cells were associated with improved survival in patients treated with sotigalimab and gemcitabine plus nab-paclitaxel [138]. Within the tumor, Th1, Th2, and IFN-γ response signatures, and higher frequencies of tumor-infiltrating non-proliferating conventional and regulatory CD4⁺ T cells, were associated with improved survival outcomes. In OPTIMIZE-1, baseline tumor-intrinsic gene signatures related to fibrosis and ECM remodeling were associated with improved survival after treatment with mitazalimab and mFOLFIRINOX [139]. In peripheral blood, increased activation and proliferation of circulating T cells and NK cells were observed.

Collectively, these data support prioritizing on-treatment pharmacodynamic biomarkers, such as APC activation, shifts in myeloid polarization, T cell activation and recruitment, and immune infiltration and stromal remodeling in the TME, as more plausible indicators of effective pathway engagement over baseline CD40 abundance alone.

4.7. Safety Considerations and Toxicity Management

CD40 agonists can induce systemic immune activation with cytokine-associated symptoms, transient cytopenias, and liver enzyme elevations; cytokine release syndrome is typically moderate and transient, consistent with activation of immune and vascular compartments [24,36,43,98,164,165]. Transient transaminase elevations are often detected within around 24 hours of dosing and may persist for weeks before resolving, emphasizing the need for laboratory monitoring and dosing strategies that balance potency with tolerability [36,43,98,164,165]. Safety profiles vary among agents, plausibly reflecting differences in FcγR engagement, clustering requirements, and schedule, and intravenous administration has generally been feasible in early-phase studies, while optimal regimens continue to be refined. In PDAC combination studies, including DC vaccination plus CD40 agonism in the REACtiVe-2 trial, overall tolerability has been reported as manageable, supporting further development while underscoring the need to mitigate toxicity and optimize regimens to realize therapeutic benefit [43,136,137,138,140,155].

5. Challenges and Future Directions

5.1. Limitation to Consistent Clinical Benefit

Despite compelling biology, translating CD40 agonism into durable efficacy in PDAC remains difficult. First, PDAC imposes strong baseline constraints, marked myeloid dominance, limited endogenous priming, dense desmoplasia, and abnormal vasculature, which can blunt effector trafficking and function even when APC activation is achieved [4,104]. Second, CD40 agonists are not interchangeable: differences in clustering requirements, FcγR dependence, and exposure profiles likely contribute to heterogeneous pharmacodynamics and safety across agents [131,166]. Third, antitumor activity appears highly context dependent, with outcomes shaped by disease setting (metastatic vs neoadjuvant), tumor burden, immune composition, and treatment sequencing [43,82,136,137,138]. Finally, adaptive counter-regulation, including re-emergence of suppressive myeloid programs, regulatory T cell expansion, compensatory inhibitory pathways, and stromal reconstitution, may limit durability and argue for longitudinal tissue-based interrogation in trials [130,143,146,167].

5.1. Biomarkers and Patient Selection: Beyond Baseline CD40 Abundance

Available data do not support baseline CD40 expression as a reliable prognostic or predictive biomarker in PDAC [30,162]. Future stratification is more likely to benefit from biomarkers that capture the functional state and spatial organization of the microenvironment, such as DC subset abundance and activation [143,146,168], macrophage polarization states [130], TLS features [169,170], and stromal programs [139], rather than single-marker expression. Integrating high-dimensional profiling (single-cell approaches, multiplex tissue imaging, spatial transcriptomics/proteomics, and longitudinal immune monitoring) should help define tumor-immune states permissive to CD40-driven priming and identify early pharmacodynamic signals associated with benefit [171,172,173]. In parallel, emphasis should shift toward on-treatment pharmacodynamic endpoints (APC activation signatures, myeloid reprogramming, chemokine programs linked to trafficking, and quantitative immune infiltration/stromal remodeling readouts) as practical markers of effective pathway engagement [43,82,139].

5.2. Optimization of Dosing, Scheduling, and Delivery

The therapeutic window of CD40 agonists remains a central development constraint, requiring dosing strategies that maximize intratumoral pharmacodynamics while limiting systemic inflammatory toxicity [174]. Step-up dosing, intermittent schedules, and combination-dependent dose modulation warrant systematic evaluation, ideally linked to mechanistic readouts (e.g., APC activation and cytokine programs) that can define an exposure-response relationship [136,165]. Alternative delivery approaches, including locoregional or intratumoral administration, may improve spatial specificity and reduce systemic exposure [35,175], but will require careful feasibility evaluation in PDAC and validation that local activity translates into systemic antitumor immunity [58,176].

5.3. Context-Dependent Tumor-Intrinsic CD40 Signaling

An unresolved translational issue is tumor-intrinsic CD40 signaling, which can produce divergent outcomes depending on tumor state and microenvironmental cues, with NF-κB/MAPK programs linked to either apoptosis or pro-survival/proliferative effects in different settings [29,124,125]. Given that CD40-targeted therapies may engage tumor-cell CD40 in a subset of PDACs [30,162], future work should clarify how genomic alterations, epigenetic states, and extrinsic cytokine signals shape tumor-intrinsic CD40 responses [103,177,178], and whether tumor-cell CD40 status should inform patient selection or combination strategy to avoid unintended protumor signaling [33,40].

5.4. Next-Generation CD40 Agonists and Rational Combinations

Next-generation formats, including Fc-engineered antibodies [179,180], multivalent ligand-mimetic constructs [181], bispecific/conditional agonists [33,176,182,183], and nanoparticle or other tumor-localizing delivery strategies [58,175,184], aim to enhance productive receptor clustering in the relevant immune niche while minimizing systemic activation. Mechanistically, CD40 agonism is unlikely to provide durable benefit as monotherapy and is best developed within multidimensional regimens that coordinate antigen supply with APC licensing and relieve downstream suppression [43,107,136,146]. Chemotherapy and radiotherapy remain important partners for antigen release and microenvironmental modulation [136,137,185], checkpoint blockade may be most effective when paired with demonstrable priming [105,138,142,186], and vaccine/DC/neoantigen strategies provide a direct route to increase antigen-focused T cell responses [107,155]; collectively, these combinations are likely to be particularly informative in neoadjuvant or minimal residual disease settings where tumor burden is lower and immune trafficking may be less constrained [43,107].

5.5. Standardization of CD40 Measurement and Reporting

To enable meaningful comparison across studies and support biomarker development, CD40 measurement requires greater standardization. Bulk transcriptomic readouts quantify CD40 mRNA in mixed tissue and are sensitive to immune/stromal admixture [162,187,188], while spatial profiling confirms multicellular heterogeneity and indicates that treatment exposure can shift compartmental states, altering bulk signals independent of true cell-intrinsic changes [172,189]. Protein-level assays (IHC, multiplex IF, quantitative immunofluorescence) improve compartment attribution but vary by antibody clone, platform, and scoring method and remain sensitive to sampling and intratumoral heterogeneity [121,190]. Accordingly, studies should explicitly report assay modality, scored compartment(s), scoring thresholds, specimen type and disease site, and treatment context, and where feasible, integrate bulk and spatially resolved approaches to support reproducible interpretation [172,173,189,191,192].

6. Conclusion

CD40 is a multicompartment immunotherapeutic target in PDAC, with the capacity to license antigen-presenting cells, reprogram suppressive myeloid states, and contribute to stromal and vascular remodeling, which may help relieve immune exclusion. Early clinical studies have shown clear pharmacodynamic immune engagement, but clinical benefit has remained inconsistent, and baseline CD40 expression alone appears insufficient to guide prognosis or therapeutic selection. Further progress will likely depend on selecting CD40 agonist modalities with appropriate clustering and FcγR-dependent properties, optimizing treatment sequencing to align tumor antigen availability with APC licensing and effector T-cell support, prioritizing on-treatment pharmacodynamic and spatial biomarkers over static expression measures, and improving safety through refined dosing, scheduling, and potentially tumor-localized or conditionally active agonist designs.

A particularly interesting direction is the combination of CD40 agonism with next-generation cancer vaccines, including mRNA-based, KRAS-targeted, neoantigen-directed, and dendritic cell–based platforms. In this setting, CD40 activation may provide the APC licensing and myeloid reprogramming required to convert vaccine-induced antigen recognition into more effective and durable T-cell priming within the hostile PDAC microenvironment. Although early preclinical and clinical data support this rationale, dedicated studies are needed to define optimal timing, combination partners, biomarker strategies, and patient selection. With continued refinement, CD40-directed approaches remain a biologically plausible strategy to increase the proportion of patients with PDAC who achieve meaningful and durable immune-mediated tumor control.