B7 homolog 3 in pancreatic cancer

Abstract

Despite advances in cancer treatment, pancreatic cancer (PC) remains a disease with high mortality rates and poor survival outcomes. The B7 homolog 3 (B7-H3) checkpoint molecule is overexpressed among many malignant tumors, including PC, with low or absent expression in healthy tissues. By modulating various immunological and nonimmunological molecular mechanisms, B7-H3 may influence the progression of PC. However, the impact of B7-H3 on the survival of patients with PC remains a subject of debate. Still, most available scientific data recognize this molecule as a suppressive factor to antitumor immunity in PC. Furthermore, it has been demonstrated that B7-H3 stimulates the migration, invasion, and metastasis of PC cells, and enhances resistance to chemotherapy. In preclinical models of PC, B7-H3-targeting monoclonal antibodies have exerted profound antitumor effects by increasing natural killer cell-mediated antibody-dependent cellular cytotoxicity and delivering radioisotopes and cytotoxic drugs to the tumor site. Finally, PC treatment with B7-H3-targeting antibody-drug conjugates and chimeric antigen receptor T cells is being tested in clinical studies. This review provides a comprehensive analysis of all PC-related studies in the context of B7-H3 and points to deficiencies in the current data that should be overcome by future research.

Key Words: B7 homolog 3, Pancreatic cancer, Prognosis, Signaling pathways, Immunotherapy

Core Tip: Mortality rates of pancreatic cancer (PC) indicate that we are facing a severe illness characterized by poor survival outcomes. Despite the improvements achieved in the treatment of other types of cancer, the survival of patients with PC is still disappointing. The B7 homolog 3 (B7-H3) checkpoint molecule seems to be a promising immunotherapeutic target since it plays an important role in the progression and antitumor immunity of various cancers. In this review, we analyze the results of different studies related to the role of B7-H3 in PC and discuss its potential to be used as a target in future therapy.

INTRODUCTION

Although pancreatic cancer (PC) is not the most prevalent malignancy, its high mortality rate places it as a top priority for research and testing on a global scale. Among the various malignancies, PC has the lowest 5-year survival rate (11%), along with esophageal (20%) and lung (22%) cancers, in the United States[1]. Furthermore, in the United States PC ranks as the third most common cause of cancer-related death among males and females combined, with a mortality rate of 12.7 per 100000 in men and 9.6 per 100000 in women, as reported in 2019[1,2]. Of particular concern is the increasing incidence of PC, which is accompanied by rising mortality rates[3]. Recent global data indicate that in 2022, there were 467000 PC deaths and 511000 new cases[4].

The etiopathogenesis of PC involves interplays among genetic, molecular, and environmental factors that collectively influence the onset and advancement of the disease[5,6]. To date, many risk factors have been associated with the development of PC and may be classified as non-modifiable such as age, sex, blood type, geographic region, diabetes, genetic susceptibility and family history, and modifiable including dietary factors, obesity, smoking, alcohol abuse, gut microbiota, chronic pancreatitis, and infection. In about 90% of cases, PC is diagnosed in people older than 55 years, with most being between the ages of 70-80 years.

The poor survival of PC cancer patients partially results from the late diagnosis. This tumor is often difficult to diagnose at an early stage because it frequently presents with vague and non-specific symptoms including abdominal pain, jaundice, weight loss, and digestive problems[6]. In a small subset of patients, the diagnosis is established incidentally at earlier stages, during imaging studies for other reasons[7]. In such instances, the prognosis is comparatively more favorable, with prolonged survival rates[8].

By incorporating a spectrum of treatment modalities encompassing surgery, chemotherapy, radiotherapy, and immunotherapy, the landscape of cancer management has evolved; however, PC remains an illness with poor long-term survival[9]. Yet, the pursuit of optimal outcomes persists, propelled by ongoing advancements in drug innovation. This quest has led to the emergence of novel anticancer agents with various mechanisms of action[10]. Meanwhile, scientific endeavors converge on the identification of novel potent molecular candidates for immunotherapeutic interventions. Among these, the B7 homolog 3 (B7-H3) molecule has ascended to the forefront of research focus due to its effect on the progression of many malignant tumors[11-13].

This review evaluates the role of B7-H3 in the carcinogenesis of PC and its potential to be used as a therapeutic target, based on existing literature. Herein, we also emphasize deficiencies in the current scientific data that should be overcome by future research.

OVERVIEW OF THE B7-H3 MOLECULE AND ITS FUNCTION

B7-H3, also known as CD276, is becoming, along with B7-H1 (also known as programmed death-ligand or PD-L1), one of the most explored members of the B7 family of checkpoint molecules[12-15]. Since the publication of its discovery in 2001, B7-H3 has drawn attention among scientists and has been a focus of many laboratory and clinical studies[12,14,16]. The B7-H3 protein exists in humans either as a transmembrane or soluble variant. The transmembrane variant represents a glycoprotein consisting of 316 amino acids and has a molecular weight of approximately 45–66 kDa. It is composed of an extracellular, transmembrane, and short intracellular domain[17]. The extracellular domain contains one or two pairs of immunoglobulin variable (IgV)-like and immunoglobin constant (IgC)-like domains, giving rise to two B7-H3 isoforms, 2Ig-B7-H3 and 4Ig-B7-H3, respectively[13]. Soluble B7-H3 (sB7-H3), which is produced through the alternative splicing of the intron or cleaved from the cell surface by a matrix metalloproteinase (MMP), has also been identified in human serum[17].

B7-H3 is encoded by its gene located on chromosome 15q24.1, and its mRNA is widely expressed across the majority of normal tissues. However, B7-H3 protein expression is low in physiological conditions, indicating the influence of post-transcriptional regulatory mechanisms. It has been solidly demonstrated that various microRNAs (miRNAs), such as miR-124, miR-199a, miR-29, miR-128, and miR-187, may bind to the B7-H3 3′-untranslated mRNA region and regulate its expression[18-22]. In contrast to healthy tissues, B7-H3 is overexpressed in many malignancies including colorectal cancer, gastric cancer, esophageal cancer[12], liver cancer[23], PC[24], ovarian cancer[25], breast cancer[26], prostate cancer[27], lung cancer[28], renal cell carcinoma[29], nephroblastoma, neuroblastoma, rhabdomyosarcoma, and osteosarcoma[13]. The abundant expression of B7-H3 in most human solid tumors is associated with poor prognosis, being a consequence of the activation of various B7-H3-related immunological and nonimmunological molecular mechanisms (Figure 1A)[12,14,17,30]. By modulating the activity of CD4+ T cells, CD8+ T cells, CD45RO+ T cells, γδ T cells, natural killer (NK) cells, and macrophages, this molecule impacts both innate and adaptive immunity[12,31]. Besides, the immunomodulatory role of B7-H3 is not only a significant factor in carcinogenesis but also plays an important part in the development of various autoimmune diseases[32]. Moreover, reported evidence suggests B7-H3 may influence the pathogenesis of some allergic diseases by favoring T cell differentiation to T helper (Th) 2 phenotype and the development of pathogenic Th2 cells[33,34].

Figure 1 B7 homolog 3. A: Regulation of B7 homolog 3 (B7-H3) expression and its immunological and nonimmunological tumor-promoting molecular mechanisms; B: Role of B7-H3 in T cell activation. Although some studies recognize B7-H3 as a costimulatory molecule, most available data evidence its coinhibitory role. The B7-H3 receptor(s) is still unknown. Three possible receptors have been suggested to date, namely triggering receptor expressed on myeloid cells-like transcript 2 possibly conducting costimulatory signal, and interleukin 20 receptor subunit alpha, and phospholipase A2 receptor 1 possibly conducting coinhibitory signal. Akt: Protein kinase B; APC: Antigen-presenting cell; B7-H3: B7 homolog 3; CD276: Cluster of differentiation 276, chr: Chromosome; HIF-α: Hypoxia-inducible factor 1-alpha; IFN-γ: Interferon-gamma; IgC: Immunoglobulin constant region; IgV: Immunoglobulin variable region; IL-2: Interleukin-2; IL-10: Interleukin-10; IL-13: Interleukin-13; JAK: Janus kinase; MAPK: Mitogen-activated protein kinase, MEK: MAPK kinase; miRNAs: MicroRNAs; MMPs: Matrix metalloproteinases; mTOR: Mammalian target of rapamycin; NF-κB: Nuclear factor-kappa B; PI3K: Phosphatidylinositol 3-kinase; STAT: Signal transducer and activator of transcription; VEGF: Vascular endothelial growth factor; MHC: Major histocompatibility complex; TCR: T cell receptor. This Figure was partly generated using images from Servier Medical Art, provided by Servier (https://smart.servier.com/smart_image/), licensed under a Creative Commons Attribution 4.0 international license (https://creativecommons.org/licenses/by/4.0/) (Supplementary material).

Furthermore, scientific data have revealed the substantial influence of B7-H3 on stimulating tumor cell proliferation, migration, invasion, and angiogenesis. Apart from increasing the metastatic potential, it has been shown that this molecule may increase resistance to anticancer therapy and deregulate cancer cell metabolism[14,31]. These changes result from B7-H3-mediated activation of different signaling pathways including Janus kinase/signal transducer and activator of transcription (JAK/STAT), phosphatidylinositol 3-kinase (PI3K)/protein kinase B (also known as Akt)/mammalian target of rapamycin (mTOR), extracellular signal-regulated kinase (ERK), and nuclear factor-kappa B (NF-κB)[12,14,31].

Although B7-H3 functions have been widely investigated, the unknown nature of the B7-H3 receptor is still a major obstacle to a complete understanding of the biology of this molecule[14,31]. Few candidates have been considered potential receptors for B7-H3, and these include triggering receptor expressed on myeloid cells-like transcript 2 (TLT-2), interleukin 20 receptor subunit alpha (IL20RA), and phospholipase A2 receptor 1 (PLA2R1)[31]. Despite its receptor being unidentified, many preclinical studies investigating B7-H3-targeting agents have been conducted in recent years. The promising results of such preclinical research have provided the introduction of B7-H3-based immunotherapy in clinical trials[11-13,35].

B7-H3 EXPRESSION IN PC AND ITS PROGNOSTIC VALUE

Various studies have confirmed the overexpression of B7-H3 in PC; however, its correlation with prognosis remains a subject of debate. The inconsistent relationship between B7-H3 expression and prognostic implications across PC and other malignancies could be attributed in part to the unidentified receptor(s) for B7-H3 and the intricate tumor microenvironment (TME)[36-38]. Studies carried out by Loos et al[39] and Yamato et al[40] were among the pioneering efforts to investigate the expression of B7-H3 in PC. Both research groups discovered that the B7-H3 molecule is considerably upregulated in PC cells compared to non-cancerous tissue and/or the normal pancreas. While Yamato et al[40] did not identify a significant correlation between the level of B7-H3 expression and postoperative prognosis, Loos et al[39] observed that elevated B7-H3 expression is correlated with extended postoperative survival.

Davis et al[41] also confirmed that B7-H3 was expressed in the majority of PC samples. However, these authors discovered that B7-H3 suppressed antitumor immunity, leading to unfavorable prognostic outcomes. Likewise, studies by Zhao et al[42] and Xu et al[43] identified the pronounced overexpression of B7-H3 in PC tissue compared to normal pancreatic tissue. Furthermore, in a study conducted by Xu et al[43], B7-H3 expression exhibited an association with the early tumor-node-metastasis stage. Notably, patients whose tumors concurrently expressed both B7-H3 and B7-H4 faced an elevated mortality risk compared to those with tumors that express only one of the proteins or neither, suggesting that these markers combined have potential as a prognostic factor for poor outcomes. Intriguingly, although this study identified a positive correlation between B7-H3 and B7-H4 in PC samples, it is noteworthy that B7-H3 alone did not influence overall survival time. Nonetheless, this information should be interpreted cautiously due to the limited number of patients in their study. The co-expression of B7-H3 and B7-H4 was also examined by Si et al[44], who showed that the co-deficiency of these molecules was associated with better prognosis in patients suffering from pancreatic adenocarcinoma. However, single B7-H3 or B7-H4 expression exhibited limited prognostic value for the assessment of clinical outcomes in these patients. By contrast, Inamura et al[24] found that B7-H3 was independently associated with poor disease-free survival in patients with PC, and this association was stronger in tumors at early stages.

The correlation between B7-H3 expression in PC cells and prognosis remains a subject of debate. In addition, the investigation into B7-H3 expression within the tumor stroma of PC has been relatively limited. Inamura et al[24] observed B7-H3 expression in PC cells and stromal cells, and substantial differences in stromal B7-H3 expression among cases were noted. However, the authors further evaluated B7-H3 expression in PC without considering stromal expression and found that higher tumor expression of this molecule was independently associated with lower disease-free survival. Furthermore, Seaman et al[45] detected B7-H3 both in PC cells and the tumor vasculature. Geerdes et al[46] conducted an immunohistochemical investigation in the samples of patients with PC and ampullary cancer of the pancreato-biliary subtype. First, B7-H3 was revealed to be localized primarily in the cytoplasm of tumor cells, whereas in stromal cells, it was localized mostly at the membrane. Interestingly, the patients diagnosed with ampullary cancer exhibited markedly higher levels of B7-H3 expression in tumor cells compared to those with PC (51% vs 21% of tested samples; P < 0.001). However, the percentage of patients with B7-H3 expression in tumor stroma was not different for these two types of cancer (66% of ampullary cancer vs 63% of PC; P = 0.664). Moreover, B7-H3 expression was shown to be associated with longer disease-free survival and other survival rates among the patients with ampullary cancer, whereas this correlation was not evident among the individuals with PC.

Wang et al[47] developed a prognostic model for predicting the overall survival of pancreatic ductal adenocarcinoma (PDAC) patients, utilizing immune-associated genes. The resulting risk score showed a positive correlation with the expression levels of certain immunosuppressive checkpoint molecules including B7-H3 and high-risk scores were associated with poor prognosis.

From a broader perspective, it can be deduced that B7-H3 is overexpressed in pancreatic tumors, manifesting in both cancerous cells and the adjacent stromal tissue. Nevertheless, its reliability as a predictive biomarker for disease outcomes remains unknown. Apart from the potential influence of unidentified receptor(s) of B7-H3, the divergent outcomes witnessed to date can be attributed to variations in samples utilized by distinct researchers, encompassing differences in sample size, histopathological type, and origin of the carcinoma[39-43,46,47].

ROLE OF B7-H3 IN PATHOGENESIS OF PC

B7-H3 and the immunity in PC

The past decade has been characterized by increased awareness of the importance of cancer cell interactions with constituents of the TME. Immune cells in cancer may have two opposite roles: Producing chronic inflammation which leads to tumor development and progression, and, in antitumor immunity. Inflammation and immunity represent two crucial features of pancreatic TME, but the relationship between them is complex and not completely understood[48]. It is well known, however, that long-term inflammation in chronic pancreatitis may lead to the production of cytokines and reactive oxygen species that cause DNA damage. This kind of DNA damage may lead to oncogenic mutations (such as in KRAS, p16, p53, and DPC4) resulting in cellular transformations such as acinar cell metaplasia and pancreatic intraepithelial neoplasia[49]. Interestingly, some novel studies have uncovered the role of adipokine signaling from fat cells throughout the body and elevated levels of intrapancreatic adipocytes as factors contributing to malignant alterations in PC[50].

Desmoplastic reaction, which is not present around normal pancreatic ducts was shown to be a hallmark of TME in PC and it is believed to play an active role in disease progression and aggressiveness[51]. Cancer-associated fibroblasts (CAFs) are the dominant cells of pancreatic TME and are used as immunosuppression biomarkers[48]. Other immunosuppressive cells in pancreatic TME mainly include tumor-associated macrophages, regulatory T cells (known as Tregs), and myeloid-derived suppressor cells[51]. It is evidenced that this immune-suppressive TME leads to the low infiltration of CD8+ cytotoxic lymphocytes. In PC, these cells are usually not present within the tumor core but only localized along the invasive margin of the tumor border or trapped in the surrounding fibrotic tissue[52].

The canonical two-signal model of T cell activation requires multiple interactions of the molecules on the cell surface. Initial exposure of the peptide antigen by the major histocompatibility complex (MHC) displayed on the surface of the antigen-presenting cell (APC) (signal 1) must be combined either with a sense of danger "go" signal or with an inhibitory "stop" signal (signal 2) for optimal T cell activation to occur (Figure 1B). B7 family members are essential contributors to both T cell activation [e.g., via cluster of differentiation 28 (CD28)-B7 complexes] and inhibition or tolerance [e.g., via cytotoxic T lymphocyte-associated protein 4 (CTLA-4)-B7 complexes]. Twelve members of this molecular family have been reported to date, namely CD80 (B7-1), CD86 (B7-2), PD-L1 (B7-H1), PD-L2 (B7-DC or CD273), inducible T cell costimulator ligand (ICOSL, B7-H2), CD276 (B7-H3), B7 superfamily member 1 (B7-H4, B7x, or V-set domain containing T cell activation inhibitor-1), V-domain Ig suppressor of T cell activation (VISTA, B7-H5, GI24, or PD-1H), butyrophilin-like protein 2 (BTNL2), B7-H6, B7-H7 (human endogenous retrovirus-H long terminal repeat-associating 2, HHLA2), and immunoglobulin-like domain containing receptor 2 (ILDR2)[12,53]. In this complex network, at least five B7 family members, namely ICOSL, PD-L1, PD-L2, B7-H3, and B7-H4, are expressed on professional APCs as well as on cells within nonlymphoid organs, providing new means for regulating T cell activation and tolerance in peripheral tissues[54].

B7-H3, as a member of the B7 family, shares 20%-27% amino acid identity with other members[35]. B7-H3 was initially identified as a costimulatory molecule that can activate T cells, induce CD4+ and CD8+ T cell proliferation in vitro, and stimulate interferon-gamma (IFN-γ) production[16]. Although B7-H3 expression is reported to be inducible on T cells, B cells, and NK cells by in vitro stimulation, subsequent studies could not confirm this role[55]. In several mouse cancer models, however, this costimulatory role has manifested in the preferential expansion of CD8+ cytotoxic T cells that could slow the progression of tumor growth or even lead to its complete eradication. This was shown in mastocytoma and hepatocellular carcinoma models[56,57]. These results were in accordance with findings obtained by Loos et al[39] who noticed a positive correlation between B7-H3 tumor cell expression in PC samples and CD8+ T cell infiltration. The costimulatory role of B7-H3 was more thoroughly elaborated by the work of Hashiguchi et al[58], who noted that TLT-2 could be the specific receptor by which B7-H3 exerts its stimulatory effects. By that study[58] and their other study[59], the authors inferred that tumor-associated B7-H3 directly augments CD8+ T cell effector function, possibly by ligation of TLT-2 on tumor-infiltrating CD8+ T cells at the local tumor site. This interaction, however, was not confirmed by other investigators, and works advocating costimulatory action of B7-H3 subsided in the following years, giving way to studies further emphasizing the inhibitory role of B7-H3[60].

Suh et al[61] were the first to show a contrasting role of B7-H3 as a negative regulator that preferentially affects Th1-mediated immune responses, which was enhanced by the findings of other studies[62,63]. Si et al[44] proved that the co-deficiency of B7-H3 and B7-H4 molecules is associated with high CD8+ T cell infiltration in pancreatic adenocarcinoma patients. Accordingly, the findings of Yamato et al[40] demonstrated that blocking B7-H3 encouraged the infiltration of CD8+ T cells into the tumor and induced a significant antitumor response in murine PC. Furthermore, Davis et al[41] also found that B7-H3 suppresses antitumor immunity, consequently leading to unfavorable prognostic outcomes. The authors demonstrated that B7-H3 overexpression is associated with a decreased number of OX40+ T cells. OX40 is a molecule expressed on cells activated during immune responses, and therapy with OX40 agonists has shown efficacy against PC in both mice and humans. Moreover, it was found that higher B7-H3 expression was positively correlated with the significantly increased number of CD45RO+ memory T cells, which were previously shown to be associated with decreased lytic units of NK cell activity and decreased progression-free interval and survival[41].

Another interesting feature of the pancreatic TME is a predominance of Th2 over Th1 CD4 + subtype. Th2 immune deviation has an active role in PC progression, and it was shown that the quantity of Th2 compared to Th1 cells present in the tumor stroma has a direct correlation with prognosis. Interestingly, scientists revealed that in pancreatic TME, CAFs have a pivotal role in Th2 polarization through their secretion of thymic stromal lymphopoietin which leads to myeloid dendritic cell conditioning[51]. Moreover, Th2 polarization has been evidenced to negatively affect the CD8+ T cell compartment[64]. Intriguingly, some studies identified B7-H3 as a molecule contributing to the stimulation of pathologic Th2 immune responses[33,34]. However, the potential link between B7-H3 and Th2 polarization in PC has not yet been investigated and further research is needed. Similarly, future studies should also be directed to the examination of a possible association between B7-H3 and CAF activation in PC since this relation was previously detected in some other tumors[65,66].

Finally, it remains unknown whether different roles in the modulation of the immune response are related to different, not yet identified, B7-H3 receptors, or if it is cell-, tissue-, or tumor-specific. Although the B7-H3 receptor is still unknown, it is considered that the FG loop of the IgV domain plays a critical role in immunomodulation, presumably inhibition of T cell proliferation[67]. Recently, Husain et al[68] implemented a new platform for high-throughput detection of receptor-ligand interactions and identified the IL20RA as the first binding partner for the checkpoint inhibitor B7-H3. Subsequently, PLA2R1, a member of the mannose receptor family with tumor suppressor function, was also identified as a potential receptor using the same platform[69]. There is no direct evidence, however, that favors any of the potential candidates as a B7-H3 receptor, and further research is needed.

B7-H3-related nonimmunological molecular mechanisms in carcinogenesis of PC

Apart from the immunomodulatory role, B7-H3-related promotion of tumorigenesis is also attributed to the immune-independent functions of this B7 family member. B7-H3 downstream effectors are numerous and include known members of oncogenic signaling pathways[12,13,31]. It is reasonable to expect that both immunological and nonimmunological roles of B7-H3 in tumor progression overlap because changes in the metabolism and signaling pathways could affect both cancer cells and immune cells.

It was discovered in a study by Xu et al[70] that B7-H3 expression in PC is correlated with the expression of MMP-2, which was previously recognized as an enzyme related to malignant tumor invasion and metastasis. Furthermore, Zhao et al[42] revealed that the heightened B7-H3 expression significantly contributed to tumor migration and invasiveness of PC cancer cells, underscoring an elevated level of aggressiveness. However, this study did not reveal whether B7-H3 regulates PC progression directly or through various important intracellular pathways[42]. Along with gene mutations and specific TME, several molecular pathways are involved in the development and progression of PC including KRAS, NF-κB, TP53, Wnt/β-catenin, Notch, and Hedgehog[71-73]. These pathways regulate various cellular processes, such as cell proliferation and differentiation, and can be disrupted by genetic mutations or other factors to promote cancer growth[71]. It has been evidenced that many signaling pathways are affected by B7-H3 expression[11-13].

B7-H3 and NF-κB signaling pathway in PC: Xie et al[74] evaluated the impact of sB7-H3 on the invasion and migration of four different PC cell lines (i.e. Aspc-1, Bxpc-3, Sw1990, and Panc-1). They found that sB7-H3 could amplify the invasive and migratory capabilities of the examined PC cells through the NF-κB pathway. NF-κB governs a multitude of gene expressions, including those of interleukin-8 (IL-8) and vascular endothelial growth factor (VEGF), both recognized for their roles in stimulating tumor invasion and migration through the induction of angiogenesis[75]. Similarly, it was shown that B7-H3 expressed on colorectal cancer cells stimulates angiogenesis through the upregulation of VEGF-A expression by activating NF-κB signaling[76]. Moreover, Li et al[77] indicated in their study that activation of B7-H3 by 4H7 antibody (Ab) in PC cells induces variations in the levels of downstream molecules including ERK1/2, epidermal growth factor receptor, and inhibitor of NF-κB, leading to the increased resistance to gemcitabine chemotherapy. These findings were confirmed by Zhao et al[78], who found that knockdown of B7-H3 in PC cell lines led to increased sensitivity to gemcitabine. Additionally, PC cells with B7-H3 knockdown showed diminished survivin expression and an elevated rate of apoptosis[78]. Survivin is a member of the inhibitor of apoptosis protein family that inhibits caspases and blocks cell death. This molecule has been evidenced as expressed in most cancers and associated with a poor prognosis[79].

B7-H3 and JAK2/STAT3 signaling pathway in PC: STAT3 is a cytoplasmic transcription factor that regulates cell cycle events, differentiation, and cell survival. JAK2, as an upstream activator, leads to its dimerization and translocation to the nucleus where STAT3 can induce transcription of numerous target genes[80]. Recruitment of JAK2, on the other hand, can be activated by cytokine IL-6 after binding to its receptor, which uses the glycoprotein gp130 as a common signal transducer. This modulation of STAT3 signaling by IL-6 happens in a context-dependent manner because it is well-known that activation of other signaling pathways, such as RAS/RAF/MEK/ERK, PI3K/Akt/mTOR, or NF-κB may also be regulated by IL-6[81]. Activation of STAT3 correlates with tumor growth, survival, angiogenesis, and metastatic potential. Lesina et al[82] showed that the inactivation of either IL-6 trans-signaling or STAT3 inhibits pancreatic intraepithelial neoplasia and the development of PC. Moreover, it is evidenced that B7-H3 expressed on breast cancer cells positively regulates activation of the JAK2/STAT3 pathway and its downstream anti-apoptotic molecules such as myeloid leukemia cell differentiation protein and survivin[83]. Similarly, the overexpression of B7-H3 was shown to increase resistance to apoptosis in colorectal cancer cell lines via upregulation of the JAK2/STAT3 pathway[84].

Inhibitors of STAT3 are being increasingly used in preclinical and clinical studies in oncology. Most of these inhibitors target STAT3 indirectly by blocking upstream signaling molecules including IL-6, JAK, or some growth factor receptors. In previous years, a series of small molecule inhibitors had been designed to directly target the SH2 structural domain of STAT3[85]. Recent studies on human and mouse PC cell lines have shown that the addition of IL-6/gp130/STAT3 signaling inhibitors to standard chemotherapeutic agent paclitaxel might be advantageous in treating PDAC[86,87]. Although the effects of B7-H3 and JAK2/STAT3 on PC pathogenesis have been confirmed individually and B7-H3-related induction of this signaling pathway in other cancers has been evidenced, no studies have shown a direct relationship between B7-H3 and the JAK2/STAT3 pathway in PC.

B7-H3 and mTOR signaling pathway in PC: An atypical serine/threonine kinase, mTOR, controls key cellular processes such as cell survival, growth, and proliferation[88]. It is present in two distinct complexes, mTORC1 and mTORC2, serving as their catalytic unit. The first, mTORC1, expresses a scaffold protein named regulatory-associated protein of mTOR and is sensitive to rapamycin, which directly inhibits this complex. On the other hand, it is activated by PI3K/Akt and RAS/mitogen-activated protein kinase pathways to phosphorylate downstream proteins, mainly ribosomal S6 kinase and eIF4E-binding protein, which potentiate anabolic processes such as mRNA translation, protein turnover, and lipid synthesis and regulate catabolic pathways such as autophagy. mTORC2, defined by the rapamycin-insensitive companion of mTOR, responds primarily to growth factors promoting cell proliferation, cytoskeleton remodeling, ion transport, and glucose metabolism by phosphorylation of Akt and several members of the AGC protein kinase family such as protein kinase A, protein kinase G, and protein kinase C[89,90]. Aberrant mTOR signaling is detected in many diseases including cancer, diabetes, and tuberous sclerosis complex[88,91].

The role of the mTOR pathway in PC has been investigated in many studies in recent years. Several proteins of the mTOR pathway are frequently activated in PC, and overexpression of mTOR correlates with both the prognosis and various clinicopathological features of patients with PC[92]. Recently, Liu et al[93] reported that mTORC1 upregulates B7-H3 expression via direct phosphorylation of the transcription factor Yin Yang 2 (YY2) by p70 S6 kinase. In that study, the expression profile of B7-H3 was determined and mTORC1 activity scores were calculated for more than 10000 patients’ data from The Cancer Genome Atlas database representing 34 different cancer types including PC. It was concluded that high B7-H3 expression and high mTORC1 signatures are associated with poor prognosis and less antitumor immune cell infiltration. The expression of B7-H3 was found to be regulated by mTORC1 only and not mTORC2, and YY2 was found to be the main transcriptional regulator of CD276. Moreover, in B7-H3-deficient tumors, there was increased IFN-γ production, MHC-II expression, and a strikingly elevated number of cytotoxic CD38+ CD39+ CD4+ T cells, which is known to correlate with a better clinical prognosis in many cancers. These results demonstrated that high mTORC1 activity, which is present in many human tumors, drives CD276 expression leading to the suppression of cytotoxic CD4+ T cells.

B7-H3 AS A TARGET FOR IMMUNOTHERAPY OF PC

The identification of B7-H3 as a potential target for immunotherapeutics has sparked new hope in the field of cancer immunotherapy[11,13]. The overexpression of this molecule across different types of cancers and infrequency in normal cells is a key point for the development of B7-H3-based immunotherapeutic agents[11-13]. B7-H3 is also highly expressed in PC cells, whereas its expression in normal pancreatic tissue is significantly lower or absent[42,43], making it a suitable target to provide reduction of off-target toxicity of anti-cancer drugs.

It has been shown that experimental depletion or inhibition of B7-H3 may significantly influence PC progression[42,78]. Studies in murine models have demonstrated that the inhibition of B7-H3 expression could curtail metastasis of PC[42]. Furthermore, it was revealed that blocking B7-H3 may enhance the antitumor immune response in PC[40]. Finally, it was reported that B7-H3 knockdown may increase the sensitivity of PC cells to chemotherapy[78]. All these findings suggest B7-H3 as a potential therapeutic target for the treatment of PC using a variety of modalities that were developed thanks to recent advances in molecular biology and Ab engineering. These modalities include monoclonal Abs (mAbs), bispecific Abs (bsAbs), and chimeric antigen receptor T (CAR T) cell therapy.

mAbs

mAbs are monovalent Abs produced in the laboratory by a single B cell clone and predetermined to bind to the same epitope[94]. The anticancer activity of mAbs relies on various molecular mechanisms. Primarily, these agents may stimulate antitumor immunity by activating Fc-mediated killing, comprising NK cell-Ab-dependent cellular cytotoxicity (ADCC), neutrophil-ADCC, complement-mediated cytotoxicity, complement-dependent cellular cytotoxicity, and Ab-dependent cell-mediated phagocytosis[95]. Furthermore, mAbs may express their activity through the impairment of angiogenesis, blocking of the cellular signaling pathways, and delivery of payloads to the targeted tumor site[96]. To express their therapeutic effects, mAbs can be linked to a highly potent cytotoxic drug (giving rise to Ab-drug conjugates, ADCs), protein toxins (creating immunotoxins), or a radioisotope (making Ab-radioimmunoconjugates)[13,97].

mAbs engaging cellular cytotoxicity

The ability of mAbs to induce ADCC has greatly improved cancer therapy. ADCC involves binding of the Fc portion of the Ab to Fc receptors on immune cells, such as NK cells, neutrophils, and macrophages. Once bound, these immune cells may release cytotoxic granules or induce apoptosis in the target cells, effectively eliminating them from the body[95,96]. The fully humanized mAb, enoblituzumab (MGA271; MacroGenics, Inc., Rockville, MD, United States) was the first to use ADCC as a mechanism for destroying cancer cells[98]. Dual therapy comprising the combination of enoblituzumab with pembrolizumab [programmed cell death protein 1 (PD-1)-targeting mAb], according to preliminary findings from a Phase 1/2 trial (NCT02475213)[99], was well tolerated and demonstrated early evidence of antitumor activity across several tumors[100] but not yet in PC. However, recently Lutz et al[101] reported the preclinical characterization of a novel Fc-optimized mAb termed B7-H3-SDIE as a promising therapeutic agent for PC treatment. In their research, significant tumor cell lysis was observed in both short- and long-term cytotoxicity studies following successful NK cell activation by this mAb.

Ab-radioimmunoconjugates

Delivering radioisotopes to tumors by B7-H3-specific mAbs as carriers has been identified as a promising strategy in several studies. In 2017, Kasten et al[102] published results showing that mice with ovarian tumors treated with the B7-H3-specific mAb 376.96 conjugated with ²¹²Pb (alone or in combination with carboplatin) showed 2-3 times longer survival than controls. A similar group of authors later demonstrated the efficacy of the same B7-H3-targeting radioimmunotherapy against preclinical models of PDAC. This radioimmunoconjugate reduced the survival of tumor cells in vitro and inhibited tumor growth in patient-derived xenograft (PDX) models[103]. However, there are still no clinical trials regarding the use of B7-H3-targeting radioimmunoconjugates in PC.

ADCs

ADCs combine a mAb's target specificity with the cytotoxic effects of the drugs they are delivering to the tumor. Recent advances in technology have resulted in the development of linkers with increased serum stability and payloads with increased potency and diverse modes of action. MGC018 (humanized anti-B7-H3 mAb conjugated to the cleavable linker-duocarmycin payload; MacroGenics, Inc.) has demonstrated antitumor activity in PDX models of breast, prostate, and head and neck cancers with heterogeneous expression of B7-H3[104]. In preclinical studies, MGC018 has also demonstrated efficacy against PC PDX models and in immunocompetent mouse models expressing human B7-H3, having a synergistic impact when combined with PD-1 checkpoint suppression[104,105]. The safety and preliminary antitumor activity of MGC018 in combination with lorigerlimab, a bs dual-affinity re-targeting (known as DART) molecule that targets both CTLA-4 and PD-1, is being evaluated in a Phase 1/1b clinical trial for patients with advanced solid tumors, including PC (NCT05293496)[106]. Another novel ADC, DS-7300a, is composed of a humanized anti-B7-H3 IgG1 mAb (MABX-9001a) and a payload, a potent DNA topoisomerase I inhibitor. DS-7300a, now known as ifinatamab deruxtecan (I-DXD), has been shown to induce apoptosis in cancer cells in vitro and to exert potent antitumor activity in xenograft models of various types of solid tumors in vivo[107]. A Phase 1/2, multicenter, nonrandomized, open-label, human study of DS-7300a in patients with advanced solid tumors is currently underway in the United States and Japan (NCT04145622)[108]. Preliminary results from that study indicated a good response rate and durability in patients with refractory small-cell lung cancer[109]. A Phase 2 study evaluating I-DXD in subjects with recurrent or metastatic solid tumors, including PDAC, started recently (NCT06330064)[110].

bsAbs

Several types of B7-H3-targeting bsAbs have been developed in preclinical settings. CD3xB7-H3 bs T cell engagers are a novel class of immunotherapeutic molecules that have shown promising results in cancer treatment. These molecules are designed to engage both CD3 on T cells and B7-H3 expressed on the cell surface of tumor cells. By bridging T cells with tumor cells, CD3xB7-H3 bs T cell engagers enhance the immune response against cancer and have the potential to specifically target and kill tumor cells, potentially minimizing the off-target effects commonly seen with traditional immunotherapies[111]. T cell activation and recruitment against tumor cells have been tested with bsAbs using a B7-H3 mAb scFv linked to an anti-CD3 mAb scFv[112]. The only drug that has thus far been tested in humans for advanced B7-H3-expressing tumors including PC is the humanized CD3xB7-H3 DART protein orlotamab (MGD009; MacroGenics, Inc.) (NCT02628535)[113]. Another novel bsAb with B7-H3xCD3 specificity, named CC-3, was recently characterized in preclinical settings. Lutz et al[114] showed that CC-3 generated substantial T cell reactivity against pancreatic, hepatic, and gastric cancer cell lines leading to potent target cell lysis. This agent strongly induced T cell activation, degranulation, and secretion of IL-2, IFN-γ, and perforin and effectively stimulated T cell memory subset development and proliferation. You et al[115] developed a bsAb B7-H3x4-1BB targeting human B7-H3 and mouse or human 4-1BB, a costimulatory molecule that belongs to the TNF receptor superfamily and is broadly expressed on immune cells. 4-1BB is found on activated T cells, Tregs, B cells, NK cells, dendritic cells, and nonhematopoietic cells such as activated endothelial cells. The authors tested the 4-1BB agonistic activity of this bsAb on human-4-1BB–expressing reporter cells that were co-cultured with B7-H3-expressing cell lines including a PC cell line. The activity of B7-H3x4-1BB bsAb resulted in activation of the NF-κB signaling pathway downstream of the 4-1BB receptor. Oppositely, the evaluated antitumor agent did not activate the NF-κB pathway in the presence of cells that do not express B7-H3, indicating a need for tumor antigen–mediated 4-1BB receptor clustering for its activity. This concept may be useful in reducing the off-target toxicity which was previously identified in studies involving anti-human 4-1BB Abs.

CAR T cells

CAR T cells are genetically engineered lymphocytes designed to express a receptor that recognizes and targets specific proteins in cancer cells. Apart from being able to recognize tumor cells regardless of the expression of the MHC class I antigen, CAR T cells may also directly target and kill cancer cells through the activation of their CAR receptors. This unique feature allows CAR T cells to bypass the need for costimulatory signals from APCs. Furthermore, CAR T cells can be engineered to express multiple CAR receptors, enabling them to target multiple antigens simultaneously and potentially enhancing their efficacy against heterogeneous tumors. CAR T cells targeting B7-H3 have already demonstrated significant antitumor activity in vivo against pediatric solid tumors including Ewing sarcoma, osteosarcoma, and medulloblastoma in mouse xenograft models[116]. Currently, there is one ongoing clinical trial involving CAR T cells targeting B7-H3 in PC (NCT05143151)[117], and one that will start recruiting patients soon (NCT06158139)[118].

To summarize, in preclinical settings, B7-H3-targeting mAbs have exhibited profound antitumor effects by increasing NK cell-mediated-ADCC in PC cells[101]. Furthermore, the remarkable antitumor activity of B7-H3 mAbs conjugated to radioactive isotope or cytotoxic drug has also been observed in PC xenograft models and some ADCs have already entered clinical trials[103-105]. Initial testing of a novel technology involving the design of bsAbs having B7-H3 as one of the targets has yielded promising results against PC cells and supports further evaluation[114,115]. Finally, a strategy of B7-H3 targeting by CAR T cells is currently being tested in clinical trial involving patients with PC[117].

Although the concern about the off-target toxicity of B7-H3-targeting agents is low, we must be aware that this molecule may be expressed in some tissues in physiological conditions. This issue may be overcome by finding another tumor-specific antigen and designing an immunotherapeutic agent recognizing both B7-H3 and another PC-specific antigen. This therapeutic modality was previously tested in neuroblastoma, wherein researchers created synthetic Notch (SynNotch) gated CAR T cells, GD2-B7-H3 CAR T, recognizing GD2 as the gate and B7-H3 as the target[119]. However, a similar model has not yet been developed for the treatment of PC and further research is needed.

CONCLUSION

The B7-H3 checkpoint molecule is overexpressed in both tumor and stromal cells of PC. This molecule influences the biological behavior of PC by modulating activity of the antitumor immune response, stimulating tumor cell migration, invasion, and metastasis, and enhancing resistance to chemotherapy. Although some findings indicate the role of B7-H3 as a costimulatory molecule, most available data evidences its immunosuppressive role. The results of experimental studies have confirmed that depletion or inhibition of B7-H3 can enhance antitumor immune response, preclude metastasis, and increase sensitivity to chemotherapy of PC. These findings led to the development of preclinical (Table 1) and clinical (Table 2) studies involving B7-H3-targeting agents and the first results are promising. Therefore, there is sufficient evidence that B7-H3 is a suitable candidate for future testing as a target in PC treatment. However, further research is needed to precisely determine the molecular mechanisms of B7-H3-related carcinogenesis in PC, with anticipation that such knowledge will help to explain the current controversies in the literature regarding the influence of B7-H3 on PC prognosis and enable its use as a prognostic biomarker.

Table 1 Preclinical studies involving B7 homolog 3-targeting agents in pancreatic cancer.

Ref.	Agent/drug	Study models
Lutz et al[101]	Fc-optimized mAb B7-H3-SDIE	In vitro PDAC model
Kasten et al[103]	B7-H3-specific mAb 376.96 conjugated with 212Pb	PDAC in vitro and PDX models
Scribner et al[104] and Wittwer et al[105]	MGC0181 (alone or combined with an anti-PD-1 agent)	PC PDX and immunocompetent mouse models
Lutz et al[114]	B7-H3xCD3 bsAb CC-3	In vitro hepatic, gastric, and PDAC models
You et al[115]	B7-H3x4-1BB2 bsAb	In vitro models of PC and other solid tumors

¹Also known as vobramitamab duocarmazine, an antibody-drug conjugate comprised of a humanized B7 homolog 3 monoclonal antibody conjugated via a cleavable linker to the prodrug seco-duocarmycin hydroxybenzamide azaindole.

²Costimulatory receptor that belongs to the tumor necrosis factor receptor superfamily, broadly expressed on immune cells.

mAb: Monoclonal antibody; bsAb: Bispecific antibody; PC: Pancreatic cancer; PD-1: Programmed cell death protein 1; PDAC: Pancreatic ductal adenocarcinoma; PDX: Patient-derived xenograft; B7-H3: B7 homolog 3.

Table 2 Clinical studies involving B7 homolog 3-targeting agents in pancreatic cancer.

Ref.	Agent/drug	Route of administration	Study participants	Status
NCT05293496, phase I[106]	Vobramitamab duocarmazine1 in combination with lorigerlimab2	Intravenous	Patients with relapsed or refractory, unresectable, locally advanced, or metastatic solid tumors including PC	Recruiting
NCT06330064, phase II[110]	I-DXD3	Intravenous	Patients with recurrent or metastatic solid tumors, including PDAC	Recruiting
NCT02628535, phase I[113]	Orlotamab4	Intravenous	Patients with unresectable or metastatic B7-H3-expressing neoplasms including PC	Terminated
NCT05143151, phase I/phase II[117]	CD276 CAR T cells	Intravenous	Adult patients with refractory PC	Recruiting
NCT06158139, phase I[118]	Ic9-CAR.B7-H3 T cells	Intravenous	Adult patients with refractory PDAC	Not yet recruiting

¹Also known as MGC018, an antibody-drug conjugate comprised of a humanized anti-B7 homolog 3 (B7-H3) monoclonal antibody (mAb) conjugated via a cleavable linker to the prodrug seco-duocarmycin hydroxybenzamide azaindole.

²Bispecific dual-affinity re-targeting (DART) molecule that targets both cytotoxic T lymphocyte-associated protein 4 and programmed cell death protein 1.

³B7-H3–directed antibody-drug conjugate consisting of anti-B7-H3 IgG1 mAb (MABX-9001a) and a payload, a potent DNA topoisomerase I inhibitor.

⁴CD3xB7-H3 DART protein also known as MGD009.

CAR: Chimeric antigen receptor; I-DXD: Ifinatamab deruxtecan; PC: Pancreatic cancer; PDAC: Pancreatic ductal adenocarcinoma; B7-H3: B7 homolog 3.