Induction of NK cell reactivity against acute myeloid leukemia by Fc-optimized CD276 (B7-H3) antibody.

Acute myeloid leukemia (AML) remains a therapeutic challenge despite recent therapeutic advances. Although monoclonal antibodies (mAbs) engaging natural killer (NK) cells via antibody-dependent cellular cytotoxicity (ADCC) hold promise in cancer therapy, almost none have received clinical approval for AML, so far. Recently, CD276 (B7-H3) has emerged as a promising target for AML immunotherapy, due to its high expression on leukemic blasts of AML patients. Here, we present the preclinical development of the Fc-optimized CD276 mAb 8H8_SDIE with enhanced CD16 affinity. We demonstrate that 8H8_SDIE specifically binds to CD276 on AML cell lines and primary AML cells and induces pronounced NK cell activation and degranulation as measured by CD69, CD25, and CD107a. Secretion of IFNγ, TNF, granzyme B, granulysin, and perforin, which mediate NK cell effector functions, was induced by 8H8_SDIE. A pronounced target cell-restricted lysis of AML cell lines and primary AML cells was observed in cytotoxicity assays using 8H8_SDIE. Finally, xenograft models with 8H8_SDIE did not cause off-target immune activation and effectively inhibited leukemia growth in vivo. We here present a novel attractive immunotherapeutic compound that potently induces anti-leukemic NK cell reactivity in vitro and in vivo as treatment option for AML.

Disruption of LPA-LPAR1 pathway results in lung tumor growth inhibition by downregulating B7-H3 expression in fibroblasts.

BACKGROUND: Lysophosphatidic acids (LPAs) belong to a class of bioactive lysophospholipids with multiple functions including immunomodulatory roles in tumor microenvironment (TME). LPA exerts its biological effects via its receptors that are highly expressed in fibroblasts among other cell types. As cancer-associated fibroblasts (CAFs) are a key component of the TME, it is important to understand LPA signaling and regulation of receptors in fibroblasts or CAFs and associated regulatory roles on immunomodulation-related molecules. METHODS: Cluster analysis, immunoblotting, real-time quantitative-PCR, CRISPR-Cas9 gene editing system, immunohistochemical staining, coculture model, and in vivo xenograft model were used to investigate the effects of LPA-LPAR1 on B7-H3 in tumor promotion of CAFs. RESULTS: In this study, we found that LPAR1 and CD276 (B7-H3) were generally highly expressed in fibroblasts with good expression correlation. LPA induced B7-H3 up-expression through LPAR1, and stimulated fibroblasts proliferation that could be inhibited by silencing LPAR1 or B7-H3 as well as small molecule LPAR1 antagonist (Ki16425). Using engineered fibroblasts and non-small cell lung carcinoma (NSCLC) cell lines, subsequent investigations demonstrated that CAFs promoted the proliferation of NSCLC in vitro and in vivo, and such effect could be inhibited by knocking out LPAR1 or B7-H3. CONCLUSION: The present study provided new insights for roles of LPA in CAFs, which could lead to the development of innovative therapies targeting CAFs in the TME. It is also reasonable to postulate a combinatory approach to treat malignant fibrous tumors (such as NSCLC) with LPAR1 antagonists and B7-H3 targeting therapies.

The expression and function of the B7 family in Helicobacter pylori infection and gastric carcinogenesis process.

BACKGROUND: The B7 protein family is one of the most important immune checkpoint proteins. Gastric cancer (GC) is the fourth most common cause of cancer-related mortality worldwide and shows a significant correlation with the B7 family in tumorigenesis and progression. Helicobacter pylori infection is the most important risk factor promoting the progression of gastric precancerous lesions and GC, which also affects the expression of B7 family members. We aimed to systematically summarize and review current studies on the expression and function of B7 family members during H. pylori infection in precancerous gastric lesions and GC. MATERIALS AND METHODS: PubMed was searched for the relationship between B7 family, H. pylori and gastric carcinogenesis until April 5, 2023. Different permutation and combination of the search terms, including "H. pylori," "Helicobacter pylori," "B7," "gastric cancer," and "gastric precancerous lesions," all the different names of specific B7 molecules, and the names of signaling pathways were used. Literature related to our research topic was selected and summarized. RESULTS: The B7 family participates in gastric carcinogenesis through certain immune signaling pathways by binding to their receptors and exhibiting co-inhibitory or co-stimulatory effects. Targeting the B7 family members with mAbs may be a promising therapeutic strategy for treating gastric diseases. CONCLUSIONS: A thorough understanding of the role of B7 molecules during H. pylori infection and GC progression is helpful for the treatment and prevention of GC and the prediction of H. pylori infection outcomes, providing evidence for H. pylori eradication.

Suppressive function of bone marrow-derived mesenchymal stem cell-derived exosomal microRNA-187 in prostate cancer.

Application of bone marrow-derived mesenchymal stem cell-derived exosomes (BMSC-exos) in cancer treatment has been widely studied. Here, we elaborated the function of BMSC-exos containing microRNA-187 (miR-187) in prostate cancer. Differentially expressed miRs and genes were screened with microarray analysis. The relationship between CD276 and miR-187 in prostate cancer was evaluated. Following miR-187 mimic/inhibitor or CD276 overexpression transfection, their actions in prostate cancer cell biological processes were analyzed. Prostate cancer cells were then exposed to BMSC-exos that were treated with either miR-187 mimic/inhibitor or CD276 overexpression for pinpointing the in vitro and in vivo effects of exosomal miR-187. miR-187 was poorly expressed while CD276 was significantly upregulated in prostate cancer. Additionally, restoring miR-187 inhibited the prostate cancer cell malignant properties by targeting CD276. Upregulation of miR-187 led to declines in CD276 expression and the JAK3-STAT3-Slug signaling pathway. Next, BMSC-exos carrying miR-187 contributed to repressed cell malignant features as well as limited tumorigenicity and tumor metastasis. Collectively, this study demonstrated that BMSC-derived exosomal miR-187 restrained prostate cancer by reducing CD276/JAK3-STAT3-Slug axis.

Sevoflurane impedes glioma progression via regulating circ_0000215/miR-1200/NCR3LG1 axis.

Sevoflurane has been reported to have anti-tumorigenic effects in glioma. Circ_0000215 was found to play vital functions in the pathological progressions of glioma. However, whether circ_0000215 mediates the inhibitory effects of sevoflurane on glioma cells remains unclear. In vitro assays were performed using cell counting kit-8, flow cytometry, transwell and Western blot assays. The expression levels of circ_0000215, microRNA (miR)-1200 and NCR3LG1 (Natural Killer Cell Cytotoxicity Receptor 3 Ligand 1) were detected using quantitative real-time polymerase chain reaction (qRT-PCR) and/or Western blot. The dual-luciferase reporter assay and pull-down assay were used to investigate the relationship between miR-1200 and circ_0000215 or NCR3LG1. In vivo assay was conducted using xenograft nude mice model. In vitro assays suggested that sevoflurane repressed glioma cell proliferation, metastasis and induced apoptosis as well as hindered tumor growth in vivo, which were reversed by circ_0000215 overexpression. Mechanically, circ_0000215 was confirmed to directly target miR-1200, and NCR3LG1 was a target of miR-1200 in glioma cells. Importantly, circ_0000215 could regulate NCR3LG1 expression via miR-1200. Besides that, rescue assay suggested that circ_0000215 attenuated the inhibitory effects of sevoflurane on glioma cell growth and metastasis, which were reversed by miR-1200 overexpression or NCR3LG1 knockdown. Our study revealed that sevoflurane could suppress glioma tumorigenesis by regulating circ_0000215/miR-1200/NCR3LG1 axis, suggesting a new insight into the therapeutic potential of sevoflurane in glioma treatment.

B7-H6 as an efficient target for T cell-induced cytotoxicity in haematologic malignant cells.

T cells play crucial roles in the antitumour immune response. However, their dysfunction leads to inefficient tumour eradication. New members of the B7 family have moved to the fore of cancer research because of their involvement in T cell-mediated immune escape and tumorigenesis. Recently, bispecific antibodies (Bi-Abs) have become attractive because of their ability to activate T cells to target tumours. In this study, we examined the expression of new B7 family members B7-H4, B7-H5, B7-H6, and B7-H7 in human haematological tumour cells. Furthermore, we explored whether B7-H6 is an efficient target for T cell-induced cytotoxicity in haematologic malignant cells. We determined the capability of T cells armed with the bispecific antibody anti-CD3 × anti-B7-H6 (B7-H6Bi-Ab) to target haematological tumours in K562, Thp-1, Daudi, Jurkat, and U266 cells. Compared with their T cell counterparts, B7-H6Bi-Ab-armed T cells demonstrated significant cytotoxicity induction in B7-H6+ haematological tumour cells, according to quantitative luciferase and lactate dehydrogenase assays, and their activity was accompanied by increased levels of the secreted killing mediators granzyme B and perforin. Moreover, B7-H6Bi-Ab-armed T cells produced more T cell-derived cytokines: TNF-α, IFN-γ, and IL-2. In addition, compared to the control T cells, a higher level of the activation marker CD69 was detected on the B7-H6Bi-Ab-armed T cells. Taken together, these data suggest that the antitumour effect of B7-H6Bi-Ab-armed T cells may be a promising immunotherapy for use in future haematologic treatments.

B7H3 regulates differentiation and serves as a potential biomarker and theranostic target for human glioblastoma.

B7H3 (CD276), a co-stimulator molecule of the cell surface B7 protein superfamily, is expressed on glioblastomas (GBM). Recently, B7H3 functions beyond immune costimulation have been demonstrated. However, the mechanisms underlying B7H3 function are diverse and not well understood. GBM tumors contain undifferentiated self-renewing cells, which confound therapeutic attempts. We investigated the role of B7H3 in the regulation of GBM cell differentiation and the regulatory pathways involved. Analysis of public databases (TCGA, Rembrandt, and GEO NCBI) and RNA sequencing were performed to explore the role of B7H3 in GBM. Knockdown and overexpression of B7H3, were used to verify the downstream pathway in vitro. Further studies in vivo were performed to support the new finding. Bioinformatics analysis identified a correlation between the expression of B7H3, the expression of glioma self-renewing cell (GSC)-related genes, and MYC expression. These observations were verified by RNA-sequencing analyses in primary GBM cell lines. In vitro knockdown of B7H3-induced differentiation, associated with downregulation of SMAD6 (a TGF-ß pathway inhibitor) and enhancement of SMAD1 phosphorylation-induced SMAD4 expression. Importantly, activation of the TGF-ß pathway resulted in downregulation of MYC expression. In vivo assays conducted in a human GBM cell line xenograft mouse model demonstrated that B7H3 knockdown decreased MYC expression and inhibited tumor growth. B7H3 knockdown could regulate GBM differentiation by modulating MYC expression. So, B7H3 could serve as a potential theranostic target for the treatment of patients with GBM.

Activation of the TLR2-mediated downstream signaling pathways NF-κB and MAPK is responsible for B7-H3-augmented inflammatory response during S. pneumoniae infection.

It has been reported that B7-H3, a costimulatory protein, participates in the development and progression of experimental pneumococcal meningitis by amplifying the TLR2-mediated inflammatory response. This study attempted to clarify the pathway(s) of TLR2 signaling involved in B7-H3-augmented inflammatory response during S. pneumoniae infection. Murine microglial cell line N9 cells and primary murine microglial cells were infected with S. pneumoniae alone or in combination with B7-H3. Although B7-H3 stimulation failed to further enhance S. pneumoniae-upregulated mRNA and protein expression of TLR2, it strongly augmented S. pneumoniae-induced phosphorylation of NF-κB p65, MAPK p38, and ERK1/2 in both N9 cells and primary microglial cells. Notably, B7-H3 itself did not activate NF-κB p65, MAPK p38, and ERK1/2. Furthermore, deactivation of NF-κB p65, MAPK p38, and ERK1/2 with their specific inhibitors significantly attenuated B7-H3-amplified proinflammatory cytokine and chemokine release from S. pneumoniae-infected microglial cells. Importantly, blockage of NF-κB p65, MAPK p38, or ERK1/2 in vivo substantially diminished B7-H3-augmented TNF-α levels in the brain of S. pneumoniae-infected mice. These results indicate that the activation of both NF-κB and MAPKs is predominantly responsible for B7-H3-augmented inflammatory response during S. pneumoniae infection.

B7H3 ameliorates LPS-induced acute lung injury via attenuation of neutrophil migration and infiltration.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are characterized by an excessive inflammatory response within the lungs and severely impaired gas exchange resulting from alveolar-capillary barrier disruption and pulmonary edema. The costimulatory protein B7H3 functions as both a costimulator and coinhibitor to regulate the adaptive and innate immune response, thus participating in the development of microbial sepsis and pneumococcal meningitis. However, it is unclear whether B7H3 exerts a beneficial or detrimental role during ALI. In the present study we examined the impact of B7H3 on pulmonary inflammatory response, polymorphonuclear neutrophil (PMN) influx, and lung tissue damage in a murine model of lipopolysaccharide (LPS)-induced direct ALI. Treatment with B7H3 protected mice against LPS-induced ALI, with significantly attenuated pulmonary PMN infiltration, decreased lung myeloperoxidase (MPO) activity, reduced bronchoalveolar lavage fluid (BALF) protein content, and ameliorated lung pathological changes. In addition, B7H3 significantly diminished LPS-stimulated PMN chemoattractant CXCL2 production by inhibiting NF-κB p65 phosphorylation, and substantially attenuated LPS-induced PMN chemotaxis and transendothelial migration by down-regulating CXCR2 and Mac-1 expression. These results demonstrate that B7H3 substantially ameliorates LPS-induced ALI and this protection afforded by B7H3 is predominantly associated with its inhibitory effect on pulmonary PMN migration and infiltration.

Correlation between B7-H3 expression and rheumatoid arthritis: A new polymorphism haplotype is associated with increased disease risk.

CD276 (B7-H3) is a costimulatory molecule that plays a potent role in T cell responses, however, the role of B7-H3 in autoimmune diseases has not been elucidated. We analyzed B7-H3 expression in rheumatoid arthritis (RA) for the first time and found B7-H3 was significantly up-regulated on monocytes in RA patients, while the levels of soluble B7-H3 in serum were lower than in controls (P < 0.0001). These differences correlated with clinical and laboratory disease parameters and informatory factor TNF-α. Through in vitro experiments, we demonstrated that B7-H3 promoted TNF-α secretion. In addition, a new polymorphism variant, B7-H3-T-A-C-T, was identified and shown to be associated with the incidence of RA and the decreased release of sB7-H3. These results suggest that B7-H3 may be a promising biomarker associated with the pathogenesis of RA. Notably, the new B7-H3-T-A-C-T polymorphism variant is associated with RA risk and might be associated with the release of soluble B7-H3.

[Eukaryotic expression of recombinant B7-H3Ig protein and its effects on T cells].

OBJECTIVE: To construct a recombinant eukaryotic expression vector containing the human B7-H3Ig gene and investigate the effects of B7-H3 signal on T cells. METHODS: Being constructed by overlap extension PCR, the recombinant gene fragment of B7-H3Ig was inserted into pGEZ-Term vector. The recombinant pGEZ-Term/B7-H3Ig vector was used to transfect L929 cells to purify B7-H3Ig from conditioned medium harvested from cultured L929/B7-H3Ig cells. T cells were then stimulated with agonistic anti-CD3 mAb in the presence or absence of purified B7-H3Ig, and cell proliferation and the secretion of IL-10 and IFN-γ were analyzed 72 h later. RESULTS: The concentration of B7-H3Ig was 0.559 mg/mL with a purity of more than 90% after protein G affinity chromatography. B7-H3Ig binding test showed that the putative B7-H3 receptor was presented in activated T cells and the maximum binding capacity was observed 48 h later. B7-H3Ig enhanced, in a dose-dependent manner, the proliferation and IL-10 and IFN-γ secretion of T cells stimulated with agonistic anti-CD3 mAb. CONCLUSION: L929/B7-H3Ig transfectants have been successfully constructed. B7-H3 is a co-stimulator for T cell proliferation and IL-10 and IFN-γ expressions.

Mimicking an induced self phenotype by coating lymphomas with the NKp30 ligand B7-H6 promotes NK cell cytotoxicity.

Induced self expression of the NKp30 ligand B7-H6 facilitates NK cell-mediated elimination of stressed cells. A fusion protein consisting of the ectodomain of B7-H6 and the CD20 single-chain fragment variable 7D8 was generated to mimic an induced self phenotype required for NK cell-mediated target cell elimination. B7-H6:7D8 had bifunctional properties as reflected by its ability to simultaneously bind to the CD20 Ag and to the NKp30 receptor. B7-H6:7D8 bound by CD20(+) lymphoma cells activated human NK cells and triggered degranulation. Consequently, the immunoligand B7-H6:7D8 induced killing of lymphoma-derived cell lines as well as fresh tumor cells from chronic lymphocytic leukemia or lymphoma patients. B7-H6:7D8 was active at nanomolar concentrations in a strictly Ag-specific manner and required interaction with both CD20 and NKp30. Remarkably, NK cell cytotoxicity was further augmented by concomitant activation of Fcγ receptor IIIa or NK group 2 member D. Thus, B7-H6:7D8 acted synergistically with the CD20 Ab rituximab and the immunoligand ULBP2:7D8, which was similarly designed as B7-H6:7D8 but engaging the NK group 2 member D receptor. In conclusion, to our knowledge, B7-H6:7D8 represents the first Ab-based molecule stimulating NKp30-mediated NK cell cytotoxicity for therapeutic purposes and provides proof of concept that Ag-specific NKp30 engagement may represent an innovative strategy to enhance antitumoral NK cell cytotoxicity.