The first possible receptor identified for 2IgB7-H3 was TLT-2, which was found to bind fusion protein of murine B7-H3 and human IgG1 (B7-H3Ig) in murine T cells. The interaction with TLT-2 resulted in enhanced T-cell activation, especially CD8+ T cells correspondingly enhancing production of IFN-γ and IL-2. However, it should be considered that fusion protein B7-H3-Ig and innate membrane B7-H3 are different, the same as TLT-2 expression in transfected cells and TLT-2 normally found on cells (31). Studies show contradictory results, while some claim that both 2IgB7-H3 and 4IgB7-H3 inhibit T cell activation (35), others show the contrary (31). Additionally, Leitner et al. (35) showed that TLT-2 on human T cells does not serve as a receptor for neither 2IgB7-H3 or 4IgB7-H3 even though there is some kind of receptor on T cells that interact with B7-H3. Even though Hashiguchi et al. (31) found interaction with TLT-2 and B7-H3Ig in murine system, Leitner et al. (35) and Yan et al. (36) were not successful in reproducing the results (35). The existence of TLT-2 remains controversial, and a need of better understanding is crucial to elucidate described contradictories. It should be considered that although the same methods were used, fusion protein construct, transduction efficiency and cellular background can result in different outcomes.

Using new interactome technique, Husein et al. identified another possible receptor – the interleukin 20 receptor subunit alpha (IL20RA) (37). IL20RA is a subunit of the IL20 along with IL20RB. Upon binding of its ligand, it can form heterodimer with IL20RB. IL20RA has many ligands including IL-19, IL-20, and IL-24 (38, 39). IL20RA is expressed in skin, ovary, placenta, lungs and testes (38). IL20RA is expressed in human skin, higher in adults compared to children skin (40). It is involved in many cancers by regulating signaling pathways. Overexpression of IL20RA was found in colorectal cancer (CRC) and was also associated with greater tumor diameter and poor prognosis (41). Knockdown of IL20RA in CRC cell lines downregulates Janus kinase 1 (JAK1) and signal transducer and activator of transcription 3 (STAT3) and consistently suppress tumor growth (42). On the other hand, activation of IL20RA in ovarian cancer cells, when they disseminate into peritoneal cavity, results in polarization of macrophages into anti-tumor M1 subtype (43). They also showed expression of IL20RA and IL20RB in human brain micro vessels (44). There are currently no findings connecting IL20RA and glioblastoma.

Using Conditioned Media AlphaScreen technology, IL20RA was confirmed as an interacting partner of recombinant B7-H3 protein. B7-H3 was expressed as biotinylated Avi-tagged extracellular domain using E. coli. Additionally, binding of recombinant B7-H3 on cells transiently expressing IL20RA was analyzed with immunofluorescence. Results confirm binding of B7-H3 only on cells expressing IL20RA. This is a promising finding of another B7-H3 receptor but should be confirmed with other studies (37).

Recently a new potential B7-H3 receptor has been identified adding another piece to a puzzle of dual B7-H3 functioning. Angio-associated migratory cell protein (AAMP) plays a role in cell migration and regulates angiogenesis (45). AAMP has also role in different cancers including being overexpressed in breast cancer (46) and gastrointestinal stromal tumors (47). Furthermore AAMP is associated with poor clinical outcomes in CRC patients promoting invasion and migration both in vitro and in vivo (48). Using yeast two-hybrid screening (Y2H) and mass spectrometry phosphorylation screen, 17 potential interacting partners on natural killer (NK) cells were identified. After bimolecular fluorescent complementation (BiFC) assay, 4 candidate genes were revealed: cluster of differentiation 164 (CD164), AAMP, receptor-type tyrosine-protein phosphatase alpha (PTPRA) and SLAM Family Member 7 (SLAMF7). After that endogenously binding partners were further accessed using co-immunoprecipitation (co-IP), results confirmed AAMP as the only interaction partner. Additionally, B7-H3 interaction with AAMP knockdown and control Jurkat cell lines was analyzed. The highest concentration of B7-H3 used equally inhibited the proliferation of both cell lines, while the lower concentration of B7-H3 inhibited the knockdown of Jurkat cells more, indicating that AAMP has at least a partial interaction with B7-H3 and reduces the antiproliferative effect of B7-H3 to a limited extent. To further validate the finding, they used multivariate expression of these two genes in glioblastoma patients and showed a positive correlation in expression. Also, poor prognosis is only observed for patients with high AAMP and B7-H3 expression. Tumors can glycosylate proteins and change their interaction with receptors additionally adding another level of complexity. CD164, PTPRA and SLAMF7 could not be verified further due to shorter interaction time, poorly evaluated antibodies or Y2H false positives (49).

B7-H3 is highly expressed in several cancers ( Figure 2 ). Zhang et al. reported B7-H3 expression in 93% of analyzed ovarian tumor samples (96/103), where the protein presented with membrane and cytoplasmic localization (68). In contrary, B7-H3 expression was not detected in non-neoplastic ovarian specimens. They also reported that high B7-H3 expression was positively correlated to increased recurrence and mortality. The results of their study also show association between B7-H3 expression in tumor vasculature and histological type, stage, recurrence incidence, and poor clinical outcome. MacGregor et al. used B7-H3 as a novel target to be used in combination with existing therapies to overcome immunosuppression in the TME of ovarian cancer cells (69). With immunohistochemistry (IHC), the authors showed that B7-H3 is expressed by both tumor and stromal cells in the epithelial ovarian TME. In flow cytometry, both tumor and stromal cells were positive for surface B7-H3 expression which was lower in tumor cells compared to stromal cells. Analysis of TCGA dataset showed that B7-H3 expression is positively correlated to stromal markers (fibroblast activation protein alpha (FAP) and platelet-derived growth factor receptor beta (PDGFRβ)) and negatively correlated to epithelial markers (EpCAM and E-Cadherin). Since B7-H3 was found to be broadly expressed on tumor cells, stromal cells, and antigen-presenting cells (APCs), the authors suggest that B7-H3 therapies can target multiple cell populations. However, because its expression on non-tumor cells can be induced under specific conditions, possible off-target effects and toxicities should be considered beforehand. Yonesaka et al. reported that 74% of the examined non-small cell lung cancer (NSCLC) tested positive for B7-H3 in IHC while normal lung cancer was not stained (70). They also examined responsiveness to anti-programmed cell death protein 1 (PD-1) therapy and B7-H3 expression levels and reported that patients whose tissues were not stained for B7-H3 benefited more from anti-PD-1 therapy than patients whose tissues stained positive for B7-H3. The authors suggest that besides correlated to poor survival, high B7-H3 expression may be correlated to refractoriness to anti-PD-1/programmed death-ligand 1 (PD-L1) immunotherapy by impairing CD8+ T-cell mediated tumor immunity. Figure 3A (72) shows mRNA expression of B7-H3 across different tumors versus normal tissue. In all tumors, except for three, the expression is higher in tumor versus normal tissue. Those three exceptions are cervical squamous cell carcinoma, acute myeloid leukemia, and pheochromocytoma and paraganglioma. The protein expression ( Figures 3B–D ) show that B7-H3 is highly expressed in glioblastoma also on protein level.

B7-H3 was initially thought to be a T-cell stimulating protein, but more recent studies show that it acts as a T-cell inhibitor (73). One of the first studies to investigate the role of B7-H3 showed that it increases the proliferation of CD4+ and CD8+ T cells and is crucial for interferon‐gamma (IFN‐γ) formation during T cell activation. In mouse models, it was also proved to activate tumor-specific cytotoxic T cells and enhances antitumor immunity. However, another study showed that B7-H3 plays a co-inhibitory role, inhibiting the proliferation of CD4+ and CD8+ T cells and reducing IL-2 and IFN-γ levels. It has also been confirmed to inhibit NK cell activity and promote osteoblast differentiation (74). Oh et al. investigated the role of B7-H3 in osteoclast differentiation. B7-H3 is highly expressed in osteoclasts and its inhibition leads to inhibition of osteoclastogenesis as well as increased IFN signaling, signal transducer and activator of transcription 1 (STAT1) activation and indoleamine 2,3-dioxygenase (IDO) induction as a downstream mechanism (75). Zhang et al. analyzed the role of B7-H3 in children with allergic asthma. They found that children with asthma had higher levels of B7-H3 compared to control subjects. They also showed that B7-H3 is a target of miR-29c and is regulated by Th2/Th17 cell differentiation (76).

In cancer, Sun et al. have shown that overexpression of B7-H3 leads to a complete regression of 50% of lymphomas and significantly reduces tumor growth in mice. In addition, the mice acquired systemic immunity against tumor cells and antitumor immunity was mediated by CD8+ T cells and NK cells (77). Similarly, Muo et al. investigated the effect of overexpression of B7-H3 in mouse mastrocytomas. Transfection with the gene resulted in enhanced immunogenicity, which led to tumor regression. It also induced a clonal expansion of CD8+ cytotoxic T lymphocytes (CTL) (78). In human studies, Loos et al. observed that pancreatic cancer patients with high levels of B7-H3 had a better prognosis than patients with low levels. Furthermore, the expression correlated with the number of CD8+ T cells (79). Similarly, Wu et al. analyzed the expression of B7-H3 in patients with gastric cancer. Higher expression of B7-H3 in the tissues was associated with better survival (80). In contrary, several other studies have shown that higher expression of B7-H3 in cancer is associated with poorer survival and disease progression, such as in prostate cancer and NSCLC (23, 79, 81–85). As Suh et al. have shown, B7-H3 inhibits the proliferation of T cells, preferentially downregulating type 1 cells rather than type 2 cells (86). It has also been observed that B7-H3 is associated with a reduction of IFN-γ and TNF-α. These are known to be produced by activated T cells and are toxic to tumor cells (87). B7-H3 mediates the immunosuppression via CCL2-CCR2 axis, which leads to M2 macrophage migration and differentiation as observed in ovarian cancer (88). B7-H3 has also been investigated in inflammatory diseases, such as arthritis. Yang et al. discovered that there is positive correlation between symptoms severity and B7-H3 expression on macrophages. The activity of B7-H3 is via NF-kB pathway. When mice with arthritis were treated with anti-B7-H3 antibody, the mice had weaker symptoms, and the inflammation reduced (89).

Beside the immunological role, B7-H3 has an important role in tumor metastasis, as observed in several studies. Chen et al. investigated the role of B7-H3 in invasion. Downregulation of the gene led to reduced cell adhesion to fibronectin and reduced migration and invasion in melanoma and breast cancer cells. On the other hand, it played no role in proliferation (90). Similarly, in prostate cancer cells, Yuan et al. observed that downregulation of B7-H3 had no effect on cell proliferation. Also, B7-H3 blocks the interaction of cells with fibronectin and is related to increased cell migration and invasion (85). On the other hand, Yu et al. observed that B7-H3 promotes proliferation of lung cancer cell lines and similarly promotes invasion and migration. They also analyzed the expression of important epithelial and mesenchymal markers. After siRNA transfection, the levels of N-cadherin, vimentin and Snail were reduced, while the expression of E-cadherin was increased (91). Xie et al. investigated the role of B7-H3 in clear cell renal cell carcinoma. Knockdown of the gene led to a decrease in N-cadherin, MMP9, integrin alpha5 and beta1 genes. They showed that fibronectin promotes migration and invasion, but this effect is diminished in cells that have the B7-H3 gene knocked down. Their results also suggest that B7-H3 forms a complex with exogenous fibronectin (92). Furthermore, Xie analyzed the mechanism behind the B7-H3 related invasion in pancreatic cancer cells. sB7-H3 induced metastasis and invasion of cancer cells. It first upregulated Toll-like receptor 4 (TLR4) expression and then activated nuclear factor kappa B (NF-κB) pathway. This in turn increased the expression of IL-8 and vascular endothelial growth factor (VEGF) (93). Tekle et al. published a comprehensive study on the role of B7-H3 in melanoma cells. Similar as in previous studies, silencing of B7-H3 reduced migration and invasion in vitro. The effect was also translated in vivo, as silencing reduced metastatic potential and symptoms-free survival in mice and rats. The possible mechanism behind the metastatic potential of B7-H3 was also analyzed. In cells with knockdown, metastasis-associated proteins such as MMP2, Stat3 and IL-8 were decreased, while TIMP metallopeptidase inhibitor 1 (TIMP1) and TIMP metallopeptidase inhibitor 2 (TIMP2) were increased (94). In hepatocellular carcinoma (HCC) B7-H3 promotes cell invasion by targeting epithelial-mesenchymal transition (EMT) via partially activating JAK2/STAT3/Slug signaling pathway (95). Kang et al. first examined B7-H3 expression in HCC and found positive correlation between high B7-H3 expression and HCC metastasis. Moreover, increased B7-H3 intensity level was detected in metastatic HCC compared to other non-metastatic primary HCCs. Therefore, high B7-H3 expression was correlated to aggressive and metastatic HCC consequently also with cell migration. The authors reported that B7-H3 downregulation significantly reduces cell migration and matrigel invasion and has no effect on cell proliferation or apoptosis. In their study EMT-related protein E-cadherin was upregulated, while N-cadherin and vimentin were downregulated in B7-H3 silenced cells compared to negative controls. They propose B7-H3 to be used as a marker for tumor recurrence and/or metastasis. In a different study, Li et al. showed that silencing B7-H3 significantly suppressed migration and inhibited proliferation of lung cancer cells A549 and H460 compared to negative controls (96). Moreover, they reported that knock down of B7-H3 downregulated the expression of many integrin-associated proteins. In CRC, B7-H3 also may promote EMT by decreasing expression levels of E-cadherin and β-catenin and increasing expression levels of N-cadherin and vimentin (97). Liu et al. investigated the role of B7-H3 in the migration and invasion of CRC cells (98). With in vitro wound healing and transwell assays they observed that enhanced expression of B7-H3 promotes cell migration and invasion, respectively. The related pathway identified was Jak2/Stat3 which is known to be important in cell migration, invasion and metastasis. The authors reported MMP-9 to be a downstream target of B7-H3. Overexpression of B7-H3 increased phosphorylation of Jak2 and Stat3, which resulted in increased expression of MMP-9. B7-H3 is upregulated in bladder cancer where it promotes cell migration and invasion through the phosphoinositide 3-kinases (PI3K)/protein kinase B (AKT)/STAT3 signaling pathway (99).

As observed in several studies, B7-H3 has an important role in metabolism, particularly glycolysis. Nunes-Xavier analyzed the effect of B7-H3 levels in triple-negative breast cancer cell lines on sensitivity to 22 different anticancer drugs. In cells with B7-H3 knocked out, apoptosis inhibitor gene (API-2) and everolimus, which target the PI3K/AKT/mammalian target of rapamycin (mTOR) signaling pathway, showed greater inhibition of cell viability. In cells with B7-H3 knockdown, glycolytic capacity was reduced, while cells with B7-H3 overexpression exhibited higher glycolytic activity. Similar to previously observed, overexpression of B7-H3 had a weak effect on cell proliferation (100). Shi et al. investigated the role of B7-H3 in the metabolism in CRC cells. B7-H3 promotes glucose consumption and lactate production through the expression of hexokinase 2. In addition, B7-H3 also increased chemoresistance in cancer cells via hexokinase 2 (70). Li et al. similarly analyzed the effect of B7-H3 in oral squamous cell carcinoma. Elimination of B7-H3 resulted in decreased proliferation, colony formation, migration and invasion of the cells. B7-H3 also promoted glycolysis by regulating HIF1A hypoxia inducible factor 1 subunit alpha (HIFα) via the PI3K/AKT/mTOR pathway (101). Another mechanism was proposed by Zuo et al., who analyzed the role of B7-H3 in cervical cancer. Knockdown of B7-H3 resulted in decreased cell proliferation in the HeLa cell line. They also observed that B7-H3 forms a complex with the enolase 1 (ENO1) protein, confirmed by liquid chromatography–mass spectrometry (LC-MS) and IP. ENO1 is an important enzyme in glycolysis, and when the level of B7-H3 was reduced, adenosine triphosphate (ATP) and lactate production were also reduced. In addition, cellular Myc (c-Myc) and lactate dehydrogenase A (LDHA) were also reduced (102).

B7-H3 has a significant role in cancer resistance to both, chemo- and radiotherapy. Ma et al. revealed that patients with low expression levels of B7-H3 presented with better overall survival compared to patients with high B7-H3 expression levels. It is reported that B7-H3 enhances chemoresistance of CRC cells by regulating the expression of cell division cycle 25A (CDC25A) through STAT3 signaling pathway in CRC cells (103). Moreover, overexpression of B7-H3 enhanced chemoresistance by reducing the G2/M phase arrest in a CDC25A-dependent manner. While the overexpression of B7-H3 promoted CRC cell viability and colony formation, chemotherapy-induced apoptosis was significantly decreased in B7-H3-overexpressing CRC cells in vitro and in vivo. Chemotherapy sensitivity was increased in cells with a stable knockdown of B7-H3. Ma et al. conclude that CRC cells can acquire chemoresistance through the B7-H3/CDC25A axis. In breast cancer, high expression levels of B7-H3 are correlated to poor outcome and resistance to commonly used chemotherapeutics such as paclitaxel (104). To study the role of B7-H3 in the sensitivity of metastatic breast cancer cells to paclitaxel, Liu et al. silenced B7-H3 in three breast cancer cell lines (MDA-MB-231, MDAMB-435, and MDA-MB-436). They reported that silencing B7-H3 enhanced the effect of paclitaxel chemotherapy compared to parental and control cells. Silencing B7-H3 abrogated the phosphorylation of Stat3 via inactivation of Jak2 and downregulated the direct target genes and anti-apoptotic factors induced myeloid leukemia cell differentiation protein (Mcl-1) and, to a lesser extent, survivin. On the contrary, its overexpression increased the phosphorylation of Jak2 and Stat3. In vitro results were also confirmed in vivo where the growth of B7-H3 knockdown xenografts was significantly inhibited upon paclitaxel treatment, while control tumors were only marginally affected. They concluded that Jak2/Stat3 pathway contributes to B7-H3–mediated paclitaxel resistance. Improving the sensitivity to paclitaxel by silencing B7-H3 is an important step into the clinical management of (metastatic) breast cancer. In CRC, B7-H3 enhanced the resistance to irradiation through upregulation of Kinesin family member 15 (KIF15) expression via NF-κβ which activated the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway (105). Moreover, in CRC B7-H3 has an inducible effect on the polarization of macrophages from anti-tumor M1 into pro-tumor M2 (106).

Several studies show that B7-H3 promotes angiogenesis. Wang et al. investigated the role of B7-H3 in CRC. As observed in vitro and in vivo, knocking down B7-H3 inhibits tube formation in human umbilical vein endothelial cells and leads to decreased expression of VEGFA, VEGFC, confirmed at mRNA and protein levels. The expression of VEGFA was induced via NF-κB, indicating the mechanism behind the angiogenesis (107). Purvis et al. investigated the role of B7-H3 in medulloblastoma, the most common embryonal neuroepithelial tumor. B7-H3 promoted angiogenesis by stimulating the secretion of VEGF. Afterwards, the conditioned medium of cells with B7-H3 overexpression was analyzed. Several pro-angiogenic factors were increased, including IL-6, IL-1, VEGF-D, VEGFR2, C-X-C motif chemokine ligand 11 (CXCL11), urokinase plasminogen activator surface receptor (uPAR), monocyte-chemotactic protein 3 (MCP-3), basic fibroblast growth factor (bFGF), tumor necrosis factor-alpha (TNF-α) and MMP-9, and chemokine (C-C motif) ligand 5 (CCL5 or RANTES) (108). In NSCLC Fan et al. investigated the role of B7-H3 in angiogenesis and alternative microvascular circulation, which is independent of angiogenesis, vascular mimicry. Cancer cell with B7-H3 knockdown resulted in decreased expression of E-cadherin and MMP-14, while there was no change in VEGF secretion. In the 3D model with B7-H3 knockdown, tumor growth was significantly reduced and the formation of capillary-like tubular structures was decreased. In the in vivo xenograft, B7-H3 knockdown resulted in significantly reduced tumor growth and decreased formation of vascular mimicry, while there were no changes in CD31+ endothelial vessels. Analysis of the supernatant of the cultured cells revealed no change in VEGF production between knockdown and mock cells. The proposed signaling pathway regulating vascular mimicry is via PI3K/AKT (109). Son et al. investigated the role of B7-H3 in angiogenesis. Suppression of B7-H3 resulted in decreased proliferation, increased apoptosis, and decreased migration of late endothelial progenitor cells, which are important for recovery from vascular dysfunction. However, this also led to increased tube formation and angiogenesis. This suggests that B7-H3 is necessary to maintain the cell population while blocking the promoted angiogenic differentiation (110). Lai et al. also showed that B7-H3 promotes VEGF secretion, that leads to angiogenesis (111). Seaman et al. conducted a comprehensive study on B7-H3’s role in promoting angiogenesis. Their findings revealed high expression levels of B7-H3 on both cancer cells and tumor-associated blood vessels. Leveraging this expression pattern, they developed an anti-CD276 drug conjugate, effectively targeting both tumor cells and infiltrating blood vessels. This dual-targeting approach highlights B7-H3 as a significant therapeutic target for inhibiting tumor growth and vascular support within the tumor microenvironment (66).

Several studies show that B7-H3 is associated with cancer stem cells. For example, Liu et al. have shown that B7-H3 is upregulated in breast cancer stem cells and its inhibition leads to inhibition of cancer stem cells, both in vitro and in vivo. Namely, B7-H3 binds to major vault protein (MVP) and activates MAPK/ERK kinase (MEK) and the MAPK kinase signaling pathway (112). A similar mechanism was observed in prostate cancer, where B7-H3 is overexpressed in cancer stem cells (113). B7-H3 is also overexpressed in cancer stem cells in head and neck squamous cell carcinomas and is associated with the escape of anti-tumor immunity. Anti-B7-H3 antibodies have been observed to eliminate cancer stem cells, inhibit tumor growth and lymph node metastasis in vivo. The blockade also reduces EMT (114). Xia et al. conducted an in-depth analysis of B7-H3’s impact on stemness in gastric cancer stem cells, revealing that B7-H3 enhances this stemness through the AKT/Nrf2 pathway (115). This effect is mediated by B7-H3’s regulation of glutathione (GSH) synthesis, a critical factor in cellular redox balance that promotes cancer stem cell characteristics. Importantly, they demonstrated that inhibiting B7-H3 expression significantly reduces the stemness of these cancer cells, thereby suppressing tumorigenicity.

The Human Protein Atlas (123) is a combination of transcriptomics and antibody-based proteomics aiming to map human proteins at a single cell and spatial resolution. It provides information about gene and protein expression levels in different reference and cancer samples. To contextualize the relevance of B7-H3 in brain cancers, we analyzed its expression pattern in brain cancer and compared it to the expression pattern of the well-known immune checkpoint PD-L1). Findings are presented in Figures 4 , 5 .

In Figure 4 RNA expression overview with data from TCGA is given. Expression levels of B7-H3 and PD-L1 were analyzed in 153 glioma samples and showed a median expression of 25.2 FPKM for B7-H3 and 1.2 FPKM for PD-L1. Both B7-H3 and PD-L1 have low cancer specificity.

Analyzing single cell types ( Figure 5 , top), in particular glial cells showed low expression of B7-H3 (6.2 astrocytes, 5.2 oligodendrocyte precursor cells, 1.5 oligodendrocytes, 7.2 microglial, 7.2 Muller glia, 1.9 Schwann cells) and PD-L1 (0.6 astrocytes, 0.9 oligodendrocyte precursor cells, 0.8 oligodendrocytes, 8.1 microglial, 0.7 Muller glia, 0.5 Schwann cells). When it comes to cell line categories, both B7-H3 and PD-L1 have low cancer specificity. Expression of B7-H3 and PD-L1 was analyzed in 80 brain cancer cell lines ( Figure 5 , middle) and showed a maximum average of 76.9 nTPM for B7-H3 and 8.5 nTPM for PD-L1. The more detailed analysis of expression of separate brain cancer cell lines ( Figure 5 , bottom) showed a maximum average expression of 181.7 nTPM for B7-H3 (cell line DBTRG-05MG) and a maximum average expression of 70 nTPM for PD-L1 (cell line IOMM-Lee).

Immuno surveillance refers to the processes by which host immune cells look for and recognize pathogens and altered cells such as cancer cells. Cancer cells however have developed various mechanisms to escape immune surveillance. The immunosuppression mechanisms of B7-H3 and PD-L1 are listed in Table 1 .

Monoclonal antibodies are antibodies having only monovalent affinity for a single epitope. With changing their variable region, they can be designed to target any antigen or epitope (135). Therapeutic antibodies target cell surface antigens in cancers. The first developed monoclonal antibody for cancer treatment was against CD20 protein, which is highly expressed on cancerous B cells in non-Hodgkin lymphoma but not on healthy B cells (136). Until now, the WHO International Nonproprietary Names (INN) Program has assigned INN names to about 1,000 monoclonal antibodies, 530 of which are in the field of oncology (137). Success of monoclonal antibody in cancer treatment is now evident in multiple cancers including bladder cancer, sarcoma, colorectal cancer and breast cancer to name a few, targeting different proteins including those of B7-family PD-1 and PD-L1 (138).

Therapeutic monoclonal antibodies have different mechanisms of action: antibody-dependent-cellular-phagocytosis (ADCP), antibody-dependent-cellular-cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), radionucleotide delivery, cytotoxic drug delivery, neutralization, or signal blockade (139). The effect of the therapeutic monoclonal antibody is related to the antigen profile of the cancer, as well as the ability of the antibody to be internalized, activate Fcγ-receptors on innate cells, trigger activation of complement or block receptor-mediated oncogenic signaling (139). This depends on the isotope and nature of the monoclonal antibody (i.e., specific binding site, avidity of target binding, and particular conformation) and the target protein. Immune checkpoints enhance anti-tumor immune responses by restoring exhausted T cells. From the six major immune checkpoints for cancer immunotherapy, namely cytotoxic T-lymphocyte associated protein 4 (CTLA-4), programmed cell death 1 (PDCD1), cluster of differentiation 274 (CD274), inducible T-cell costimulator (ICOS), lymphocyte-activation gene 3 (LAG3), and CD40, ipilimumab targeted against CTLA-4 was the first monoclonal antibody approved by the Food and Drug Administration (FDA) in 2011. Ipilimumab activates the immune system by targeting CTLA-4, a protein receptor that downregulates the immune system, and is used in the treatment of metastatic melanoma. Ipilimumab mechanism of action is by blocking the binding of CTLA-4 to its ligands, inhibiting CTLA-4-mediated downregulation of T cells and promoting the interaction of CD80/CD86 with CD28 (137). This activates the immune system by increasing T cell expansion and enhancing the CTL-mediated anti-tumor immune response. Moreover, the IgG1-Fc region of ipilimumab binds to FcγRIIIa, induces ADCC and complement-dependent cytotoxicity (CDC) for enhanced anti-tumor efficacy by reducing T_reg cells.

Monoclonal antibodies have also been tested against B7-H3. In case of ovarian cancer cell lines treating with monoclonal antibodies alone significantly inhibited cell growth. In combination with tyrosine inhibitor even better outcome appeared (140). Shi et al. demonstrated monoclonal antibodies to inhibit 2IgB7-H3 and 4IgB7-H3 known to be highly expressed in variety of tumors (141). This success led to testing monoclonal antibodies including monoclonal antibodies against B7-H3 in clinical trials ( Table 2 ). However, there are no clinical trials in phase 3 yet.

For NCT02475213, patients with non-small cell lung cancer, head and neck squamous cell carcinoma, urothelial cancer and melanoma, were treated with enoblituzumab (investigational anti-B7H3 antibody), together with pembrolizumab, anti-PD1 inhibitor. 133 patients were enrolled in the study and 116 got treatment-related adverse effects, which were equal or treater than grade 3 in 28.6% of patients. There was also one treatment-related death due to pneumonitis. The response in HNSCC was in 6 cases out of 18 and in NSCLC was 5 of 14. The treatment was discontinued in 13 patients (148). Both NCT00089245 and NCT01502917, include therapy based on anti-B7-H3 antibody labeled with radioactive iodine. At clinical trial NCT00089245, 37 patients received injections of radiolabelled omburtamab. 16 of patients had metastatic neuroblastoma and other had solid tumors with B7H3 expression, including medulloblastoma, ependymoma, melanoma and rhabdomyosarcoma, choroid plexus carcinoma, atypical thabdoid teratoma, chordoma, pineoblastoma and retinoblastoma. The drug was overall well-tolerated. The most common adverse effect was myelosuppression. Patients who had neuroblastoma had significant improvement in overall survival and progression-free survival. Seven survived without progressive disease for 13-17 years from treatment. Of the remaining 8, only 2 had CNA relapse and the 6 died of systemic relapse or chemotherapy-related toxicity (149). At clinical trial NCT01502917, 28 children with diffuse intrinsic pontine glioma were treated with omburtamab labeled with radioactive 124-iodine (150). There were no dose-limiting toxicities. One out of 28 patients had treatment-related transient grade 3 hemiparesis and one had grade 3 skin infection. There were no treatment-related grade 4 adverse effects or deaths. The systemic exposure was low. The radioisotope was retained in the brain for more than 8 days.

CAR-T cell therapy is one of the main approaches of effective adoptive T-cell therapy (ACT), which kills cancer cells by the therapeutic use of transferred T cells (151). CAR is a synthetic construct i.e. a bioengineered receptor that binds to target cell surface antigens (e.g. proteins, glycolipids, and carbohydrates) through a single-chain variable fragment (scFv) recognition domain. CAR-T cells technology is an ex vivo method, which genetically modifies patient derived cells for immunotherapeutic purposes. Either NK cells or T cells can be used for CAR-T. Currently, various generations of CAR-T cells have been developed, each varying in their capacity to eliminate cancer cells (152). CAR-T cells mediate major histocompatibility complex (MHC)-unrestricted tumor cell killing by allowing T cells to bind to their cell surface antigens through a scFv recognition domain (151). Then, CAR-T cells form a non-classical immune synapse (IS), which is required for their effector function. These cells mediate their anti-tumor effects through perforin and granzyme axis, the Fas and Fas ligand axis, and cytokine release for sensitizing tumor stroma. The outputs largely depend on the individual components of the receptor i.e. scFv, spacer and costimulatory domains. First generation of CAR-T cell lacked co-stimulatory molecules which made them less powerful and inconsistent (153). However, newer generations of CAR-T cells added co-stimulatory molecules and improved its efficacy. Unlike conventional T-cells, CAR-T cells can bind to an antigen irrespective of the MHC presentation. However, their limitation is binding to cell surface expressed antigens only. CAR-T cells are currently used to treat hematological malignancies; namely, 6 CAR-T cell products have been approved by the FDA (154). Research for solid tumors is limited due to immunosuppressive TME and lack of antigens (152). Additionally, research on solid tumors was halted due to two deaths caused during clinical phase I in men with prostate cancer (155).

In clinical perspective, Majzner et al. tested B7-H3 CAR T cells on models of solid pediatric tumors (osteosarcoma, medulloblastoma and Ewing sarcoma) in xenograft mice (156). The authors developed a B7-H3 CAR, based on MGA271 (Enoblituzumab), that preferentially binds tumor tissues and shows restricted recognition of normal human tissues. They showed that the B7-H3 CAR T cells mediate antitumor activity in vivo and cause regression of established solid tumors. In particular, the B7-H3 CAR-T cells eradicated the autochthonous DAOY and D425 medulloblastoma xenografts which was observed by bioluminescent imaging. The systemically administrated B7-H3 CAR-T cells also mediated regression and eradication of established osteosarcoma and Ewing sarcoma xenografts.

CAR-T technology was tested for B7-H3 in vitro and in vivo experiments. Although no clinical trials have been completed so far, many clinical trials are currently recruiting participants. CAR-T cells targeting B7-H3 shows high tumor specific killing ability in vitro and tumor suppression effect in vivo in PDX (patient derived xenograft) model in osteosarcoma (157). CAR-T cells targeting B7-H3 are also effective in inhibiting growth in vitro and in vivo of NSCLC (158), prostate cancer (159), glioblastoma (116), ovarian and triple negative breast cancer (160).

Antibody drug conjugates (ADC) are a class of drug typically composed of monoclonal antibodies covalently attached to a cytotoxic drug i.e. payload and a linker in between. ADC combine the advantages of specific targeting ability and potent killing affect to accurately and efficiently eliminate cancer cells (161). Because of the antibody specificity, ADC target only antigen-expressing cells and therefore do not cause systemic toxicity which is common in conventional chemotherapy (148). When the antibody binds to the cell surface antigen on the target cell, the ADC is internalized to form an early endosome, followed by a maturation into late endosomes and finally fused with lysosomes. Linker breakdown promotes ADC release of the cytotoxic drug by chemical- or enzyme-mediated release into lysosomes which eventually leads to cell death or apoptosis via targeting DNA or microtubules (148, 161–163). Their mechanism of action is eliciting immunogenic cell death, ADCC, ADCP and CDC effects, as well as dendritic cell activation upon interaction with cancer and immune cells (161, 162). The “bystander effect” i.e. diffusion of the payload from antigen-expressing cancer cells to adjacent cells and therefore killing these cells contribute to the cytotoxicity of ADC in heterogenic tumors (148). Binding of the ADC to its target may also interrupt its downstream function by preventing the antigen interaction with its binding partners (163). ADC induce tumor-specific adaptive immunity by increasing the infiltration of T cells into the TME. Typically, they remain stable in blood, are target-specific and release cytotoxic component in the vicinity of cells. All components of ADC need to be carefully selected, including the right molecular target since they have an impact on safety, efficiency, and delivery of drug (161). Mostly used cytotoxic molecules inhibit tubulin, are immunomodulators or they damage DNA (164). Currently there are many ADC drugs that are under development for cancer treatment (161). First ADC drug granted approval by FDA was Mylotarg^® (gemtuzumab ozogamicin) for the treatment of adults with acute myeloid leukemia (AML) in 2000 (161, 165). The first ADC for treatment of solid tumors was Ado-Trastuzumab Emtansine (Kadcyla^®) or T-DM1 for treatment of HER2+ metastatic or locally advanced breast cancer and was approved in 2013 (162). Recently two studies demonstrated efficacy of ADC targeting B7-H3 in glioblastoma (166), lung, and breast cancer cell lines and PDX models (167). Study using ADC for killing glioblastoma showed greater killing potency for glioblastoma cells with greater expression of B7-H3. They used monomethyl auristatin E which is known microtubule-disrupting agent. When coupled with fluorescent conjugate, they showed specific accumulation in tumor cells in vivo (166). ADC used for targeting B7-H3 in lung and breast cancer cell showed the same affinity as unconjugated B7-H3 antibody. This ADC cause cell cycle arrest in S phase, DNA damage and apoptosis. In this case, they used monomethyl auristatin F, which is also microtubule-disrupting agent (167). When it comes to ADC treatment, many of the patients develop resistance and experience disease progression. Therefore, the mechanisms of resistance (eg. altered target cell surface expression, gene mutations, changes in trafficking and internalization, payload resistance) need to be studied for optimal results. In addition, combining ADC with other chemo- or immune-therapeutic approaches may increase their utility in cancer treatment (163).

The immune mechanism of antibody-dependent cell-mediated cytotoxicity (ADCC) involves three components: effector cells with NK cells being the major type in vivo, antibodies and target cells opsonized by the antibodies (168). ADCC is a method of combating tumors by introducing specific antibodies that attach to target tumor cells. This attachment triggers or recruits effector immune cells to recognize these foreign antibodies and initiate cell death through non-phagocytic mechanisms. Antigens bind to the target cells through their antigen binding fragment (Fab), while the interaction with the effector cells occurs between the fragment crystallizable region (Fc) portion of the antibody (30, 168). There are three different mechanisms that occur after the activation of effector cells: cytotoxic granule release, Fas signaling, and elaboration of reactive oxygen species (ROS). From these, the main mechanism of action in ADCC is release of perforins and granzymes from effector cell granules. Activated effector cells can also upregulate the expression of Fas ligand in order to cause apoptosis in the target via Fas signaling. Although IgG, IgA, and IgE can mediate ADCC, IgG is the most used immunoglobulin subclass for cancer therapeutic antibodies. They developed humanized mouse antibody against B7-H3, which was afucosylated afterwards. Flow cytometry showed binding on IgC1 and IgC2 domain of human B7-H3. ADCC was activated against medium and high expressing breast (MDA-MB-231) and lung cancer (NCI-H322) cell lines. Afucosylated protein induced ADCC even on low expressing B7-H3 cancer cells, while non-afucosylated did not. Additionally, the afucosylated protein showed dose-dependent antitumor efficacy in severe combined immunodeficient (SCID) mouse and small effect in new generation of severely immunodeficient (NOG) mouse (169). Xu et al. developed bispecific modified protein targeting B7-H3/PD-L1 and with modified Fc region enhanced ADCC. Fusion protein blocked PD/PD-L1 pathway, activated CD8+ cells and enhanced ADCC in tumor cells with higher expression of B7-H3 (170). Another study found that B7-H3 human mouse chimera antibody induced ADCC in primary leukemia cells but not in hematopoetic cells. Furthermore, treating PDX models in combination with antibody and human NK cells significantly prolonged survival (171).

Another important way of fighting cancer is using small molecular inhibitors. These molecules target different important molecules in signaling pathways of cancers including VEGFR, human epidermal growth factor receptor (HER), RAS, JAK, mTOR and so on. Because of their small size, small molecular inhibitors can bind to a wider range of extracellular and intracellular targets when compared to antibodies. Based on target specificity, small molecule inhibitors are divided into selective and multikinase. Multikinase small molecular inhibitors repress multiple kinases in the tumor, they do not require precise detection and rely on histology. Selective small molecule inhibitors bind to a single target and inhibit its cell signaling. They can either inhibit its unusual function or reverse its regular action. Selective small molecule inhibitors are then further divided into two groups: selective small molecule kinase inhibitors and selective small molecule non-kinase inhibitors (172). Selection of a small molecule whether it is a multikinase or selective small molecule kinase is based on the number of kinases whose inhibitory activity IC₅₀ values are below 10 nM (173). Selective small molecule non-kinase inhibitors bind to targets beyond the kinome (i.e. the complete set of protein kinases encoded by the genome), thereby blocking subsequent functions to control tumors.

Protein kinase inhibitors are the main category of small molecule inhibitors. Protein kinase enzyme has important role in cell growth, proliferation and differentiation. Protein kinase catalyzes the transfer of γ-phosphate group from ATP to protein residues containing hydroxyl groups. Dysregulations of protein kinases are linked to various diseases including cancer. Because of this, protein kinases are studied as cancer therapeutic targets. The selective small molecule kinase inhibitors contain receptor-related kinase inhibitors, kinase inhibitors targeted intracellular signaling pathways, and inhibitors targeting other cytoplasmic kinases (172). As described in (174), protein kinase inhibitors are classified into six types: Type-I inhibitors bind to the active conformation of the kinase (DFG-Asp in, αC-helix in); type-I½ inhibitors bind to a DFG-Asp in inactive kinase conformation with αC-helix out; type-II inhibitors bind to a DFG-Asp out inactive conformation; type III inhibitors restrain kinase activity by binding to an allosteric site; type IV inhibitors bind outside of the cleft; type V inhibitors are bivalent molecules that span two distinct regions of the kinase domain; and type VI inhibitors that bind covalently with the kinase active site. While type I-V inhibitors are all reversible, type VI are irreversible kinase inhibitors. The tyrosine kinase inhibitor imatinib was the first small molecule targeted drug that was approved by the FDA in 2001 (174).

Small molecular inhibitors have some important advantages that can be readily used like pharmacokinetic properties, low manufacturing cost, drug storage and transportation, small size, short half-lives, patient compliance and good distribution in tissues (174, 175). Moreover, small molecule inhibitors can be taken orally, and some of them can penetrate the BBB to control intracranial lesions (172). However, they also present with disadvantages such as low response rate and drug resistance. Understanding that the receptor(s) on activated T cells interact with the FG loop of the IgV domain of B7-H3, there is potential to create a small molecule inhibitor aimed at disrupting this precise binding region. However, it is important to acknowledge that unforeseen off-target effects may occur, underscoring the need for comprehensive assessment to minimize any potential negative outcomes. To the best of our knowledge, there are currently no clinical trials targeting B7-H3 protein and none in vitro or in vivo studies regarding B7-H3.

Nanobodies are ~15kDa proteins derived from heavy-chain only antibodies produced by just few animals, such as camelids and sharks. They were first discovered by the group of Raymond Hammers in Belgium at Vrije Universiteit Brussel in 1993 in camelids (176). Camelids have two types of IgG antibodies, the conventional and heavy chain antibodies that are lacking light chains. Nanobody is a variable domain of this heavy chain antibody. These nanobodies have several advantages compared to conventional antibodies. Due to their smaller size, they exhibit better tissue penetration and faster clearance from the body. Additionally, they are more stable due to several amino acids substitution that make them more hydrophilic. They contain 3 binding domains i.e. complementarity determining regions (CDRs - CDR1, CDR2, CDR3) compared to 6 in classical antibodies, but still bind with affinities in nano/pico molar concentrations (177). The longer CDR3 of nanobodies forms a finger-like extension that binds to small hidden epitopes in the concave surface of the antigen or in the antigen gap with high affinity. Nanobodies can selectively bind multifunctional epitopes on the receptor, block disease-related signaling pathways or responses (178). Zarantonello et al. described the selection of the C1q specific nanobody C1qNb75 that is able to inhibit activation of the classical pathway (CP) of the complement system (179). The proposed mechanism of action is by occluding the IgG binding site on C1q.

Nanobodies as the single agents have not been yet developed and tested as therapeutics. However, their unique and potentially buried binding sites, small size, as well as simple expression and production, make nanobodies suitable for use in CAR-T therapy development. Li et al. developed nanobody-based CAR-T cells targeting B7-H3 for treating several different solid tumors. Nanobody against B7-H3 was retrieved from dromedary camel phage library and specific CAR-T cells were obtained first by cloning nanobody into CAR construct. Human peripheral blood mononuclear cells (PBMCs) were then isolated from healthy donors and stimulated with anti-CD3/anti-CD28 antibody-coated beads in presence of IL-2. The therapy efficiently lysed four neuroblastoma (NB) cell lines, two pancreatic cancer cell lines, triple-negative breast cancer cell lines and lung adenocarcinoma cell line. CAR-T cells were tested also in mice model of pancreatic cancer, where they had high antitumor activity (180). One of the most important components in CAR-T cell technology is the antigen-recognition domain (180). B7-H3 has two distinct epitope domains IgC and IgV; one of the clinically tested antitumor antibodies, 8H9, binds to the IgV domain. In their study, Li et al. showed that the B7-H3 CAR-T cells targeting IgC are more potent than those targeting IgV in pancreatic ductal adenocarcinoma (PDAC) and NB mouse preclinical models. They conclude that the antigen-binding epitope is crucial for the biological function of antibody-based CAR-T cells.