B7 Family Research Articles

B7-H3/CD276 and small-cell lung cancer: What's new?

Immunotherapy revolutionized the treatment landscape of several cancers, including small-cell lung cancer (SCLC), with a huge number of practice-changing trials, and becoming a new frontier for their management. The addition of an anti-PD-L1, atezolizumab or durvalumab, to platinum/etoposide regimen became the standard of care for first-line therapy of extensive-stage (ES)-SCLC with the 12 months median survival exceeded for the first time.Nevertheless, most patients show primary or acquired resistance to anti-PD-L1 therefore new promising therapeutic immune-targets are under clinical investigation in several solid tumors. Among these, B7-H3, also known as CD276, is a member of the B7 family overexpressed in tumor tissues, including SCLC, while showing limited expression in normal tissues becoming an attractive and promising target for cancer immunotherapy.B7-H3 plays a dual role in the immune system during the T-cell activation, acting as a T-cell costimulatory/coinhibitory immunoregulatory protein, and promoting pro-tumorigenic functions such as tumor migration, invasion, metastases, resistance, and metabolism.Immunohistochemistry, flow cytometry, and immunofluorescence were the most used methods to assess B7-H3 expression levels and validate a possible relationship between B7-H3 staining patterns and clinicopathological features in lung cancer patients.To date, there are no clinically available therapeutics/drugs targeting B7-H3 in any solid tumors. The most promising preliminary clinical results have been reported by DS7300a and HS-20093, both are antibody-drug conjugates, that are under investigation in ongoing trials for the treatment of pretreated SCLC.This review will provide an overview of B7-H3 and corresponding inhibitors and the clinical development in the management of SCLC.

Unlocking the role of the B7-H4 polymorphism in psoriasis: Insights into methotrexate treatment outcomes: A prospective cohort study.

B7-H4 is a recently discovered member of B7 family that negatively regulates T-cell immunity, specifically Th1 and Th17 cell responses. However, its role in the pathogenesis of psoriasis has yet to be determined. This study aims to investigate the effect of B7-H4 polymorphism on the efficacy of methotrexate (MTX) and its mechanism in psoriasis. Four single nucleotide polymorphisms of B7-H4 were genotyped in 310 psoriatic patients who received 12-week MTX. The protein expression of B7-H4 in platelets was characterized using immunofluorescence staining, confocal laser scanning microscopy, and flow cytometry techniques. We found that GG genotype carriers of B7-H4 rs1935780 had a lower Psoriasis Area and Severity Index (PASI) 75 response rate and higher weight (p = 0.0245) and body mass index (p = 0.0185) than AA and AG genotype carriers. Multiple regression analysis showed that the PASI score at baseline (p = 0.01) and age at disease onset (p = 0.003) were positively correlated with PASI 75 response rate, while weight (p = 0.005) and the rs1935780 genotype (p = 0.003) were negatively associated with PASI 75 response rate. B7-H4 was expressed in the platelet plasma membrane and cytoplasm. Furthermore, the expression of B7-H4 protein in platelets was lower in good responders than in non-responders and was upregulated considerably after 12-week MTX or in vitro MTX stimulation in good responders. Collectively, these results demonstrate that psoriatic patients with GG genotype of B7-H4 rs1935780 had a poorer response to MTX. Low expression of B7-H4 protein in platelets correlated with better clinical outcomes of MTX in psoriasis.

Immunomodulatory effects of nanoparticles on dendritic cells in a model of allergic contact dermatitis: importance of PD-L2 expression

Nanoparticle (NP) skin exposure is linked to an increased prevalence of allergic contact dermatitis. In our prior studies using the mouse contact hypersensitivity (CHS) model, we reported that silica 20 nm (SiO2) NPs suppressed the allergic response and titanium dioxide NPs doped with manganese (mTiO2) exacerbated it. In this work, we conducted in vitro experiments using bone marrow-derived dendritic cells (BMDCs) to study the combinatorial effect of the potent 2,4-dinitrofluorobenzene (DNFB) hapten sensitizer with SiO2 and mTiO2 NPs on BMDC cytotoxicity, cytokine secretion and phenotype using the B7 family ligands. Results show that DNFB and mTiO2 behave similarly and exhibit proinflammatory characteristics while SiO2 promotes a naive phenotype. We observe that the B7-H3 (CD276) ligand is only expressed on CD80 + (B7-1) BMDCs. Results from adoptive transfer CHS studies, combined with BMDC phenotype analysis, point to the importance of PD-L2 expression in modulating the adaptive immune response. This work identifies metrics that can be used to predict the effects of NPs on contact allergy and to guide efforts to engineer cell-based therapies to induce hapten specific immune tolerance.

Tumor cell-expressed lipolysis-stimulated lipoprotein receptor negatively regulates T-cell function.

Lipolysis-stimulated lipoprotein receptor (LSR) is known as a lipoprotein receptor. LSR is expressed in various solid tumors, including epithelial ovarian, gastric, and colon cancers. High LSR expression is significantly associated with poor prognosis, but its role in cancer has not been fully elucidated. LSR belongs to the Ig protein superfamily, which is conserved in B7 family. Here, we assessed LSR as a novel immune checkpoint molecule. We developed a novel anti-LSR antibody (#27-6 mF-18) that defects antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity activity. The #27-6 mF-18 cross-reacts with both human and mouse LSR. We found that LSR was expressed on 4T1 murine breast cancer cell line. The #27-6 mF-18 exhibited antitumor effects against the 4T1 syngeneic tumor model, a poor immunogenic model refractory to treatment with anti-PD-1 or anti-CTLA-4 antibodies. Compared with control antibody-treated mice, mice treated with #27-6 mF-18 showed significantly increased numbers of CD8+ T cells and a ratio of activated CD8+ T cells infiltrated in the tumor tissue. This antitumor effect was abrogated by CD8+ T-cell depletion through anti-CD8 antibody treatment, indicating that LSR negatively regulates tumor immunity by repressing CD8+ T cells. These findings show that LSR negatively regulates T-cell immune activity. LSR targeting could provide immune checkpoint inhibitors for cancer immunotherapy.

Expression analysis of inhibitory B7 family members in Alzheimer's disease.

Alzheimer's disease (AD) is a global health problem due to its complexity, which frequently makes the development of treatment methods extremely difficult. Therefore, new methodologies are necessary to investigate the pathophysiology of AD and to treat AD. The interaction of immune modulation and neurodegeneration has added new dimensions in current knowledge of AD etiology and offers an attractive opportunity for the discovery of novel biomarkers and therapies. Using quantitative polymerase chain reaction, we compared the expression levels of inhibitory B7 family members (B7-1, B7-2, B7-H1, B7-DC, B7-H3, B7-H4, B7-H5, B7-H7, and ILDR2), as immune regulators, in the peripheral blood of late-onset AD (LOAD) patients (n = 50) and healthy individuals (n = 50). The levels of B7-2, B7-H4, ILDR2, and B7-DC expression were significantly higher in-patient blood samples than in control blood samples. Furthermore, we discovered a substantial positive correlation between all gene expression levels. In addition, the current study indicated that ILDR2, B7-H4, B7-2, and B7-DC might serve as diagnostic biomarkers to identify LOAD patients from healthy persons. The present work provides additional evidence for the significance of inhibitory B7 family members to the etiology of LOAD.

ITC-6102RO, a novel B7-H3 antibody-drug conjugate, exhibits potent therapeutic effects against B7-H3 expressing solid tumors

BackgroundThe B7-H3 protein, encoded by the CD276 gene, is a member of the B7 family of proteins and a transmembrane glycoprotein. It is highly expressed in various solid tumors, such as lung and breast cancer, and has been associated with limited expression in normal tissues and poor clinical outcomes across different malignancies. Additionally, B7-H3 plays a crucial role in anticancer immune responses. Antibody-drug conjugates (ADCs) are a promising therapeutic modality, utilizing antibodies targeting tumor antigens to selectively and effectively deliver potent cytotoxic agents to tumors.MethodsIn this study, we demonstrate the potential of a novel B7-H3-targeting ADC, ITC-6102RO, for B7-H3-targeted therapy. ITC-6102RO was developed and conjugated with dHBD, a soluble derivative of pyrrolobenzodiazepine (PBD), using Ortho Hydroxy-Protected Aryl Sulfate (OHPAS) linkers with high biostability. We assessed the cytotoxicity and internalization of ITC-6102RO in B7-H3 overexpressing cell lines in vitro and evaluated its anticancer efficacy and mode of action in B7-H3 overexpressing cell-derived and patient-derived xenograft models in vivo.ResultsITC-6102RO inhibited cell viability in B7-H3-positive lung and breast cancer cell lines, inducing cell cycle arrest in the S phase, DNA damage, and apoptosis in vitro. The binding activity and selectivity of ITC-6102RO with B7-H3 were comparable to those of the unconjugated anti-B7-H3 antibody. Furthermore, ITC-6102RO proved effective in B7-H3-positive JIMT-1 subcutaneously xenografted mice and exhibited a potent antitumor effect on B7-H3-positive lung cancer patient-derived xenograft (PDX) models. The mode of action, including S phase arrest and DNA damage induced by dHBD, was confirmed in JIMT-1 tumor tissues.ConclusionsOur preclinical data indicate that ITC-6102RO is a promising therapeutic agent for B7-H3-targeted therapy. Moreover, we anticipate that OHPAS linkers will serve as a valuable platform for developing novel ADCs targeting a wide range of targets.

Global research trends on B7-H3 for cancer immunotherapy: A bibliometric analysis (2012-2022)

ABSTRACT Immunotherapy has revolutionized cancer treatment. B7-H3 is a promising target for cancer immunotherapy (CI). The present study aimed to utilize bibliometric methods to assess the current research status and explore future trends in the use of B7-H3 for CI. We collected publications related to B7-H3/CI from the Clarivate Web of Science Core Collection database. VOSviewer, Microsoft Excel, the bibliometrix R package, and an online platform were used to conduct qualitative and visualized analyses of the literature. A total of 555 papers were analyzed, revealing a significant increase in annual publications since 2018. The most productive countries were China and the USA, and the leading institutions were Soochow University and Sichuan University. Zang and Ferrone were the most popular authors. Among the journals, Frontiers in Immunology had the highest number of papers, whereas Clinical Cancer Research was the most influential. Historical citation analysis reveals the development of B7-H3/CI. Top-cited papers and keyword analyses were performed to highlight current hotspots in the domain. Using cluster analysis, we classified all keywords into four clusters: “immunotherapy,” “co-stimulatory molecule,” “B7 family,” and “PD-L1.” Finally, Trends analysis suggested that future research might focus on “chimeric antigen receptor,” “pathways,” and “targeting B7-H3.” This is the first bibliometric crosstalk analysis between B7-H3 and CI. Our study illustrates that the topic of B7-H3/CI is very popular and has great clinical implications and that the number of correlative publications will continue to increase. B7-H3-based CI may lead to new research trends.

B7-H7: A potential target for cancer immunotherapy.

Cancer immunotherapy enhances the body's immunity against tumors by mitigating immune escape. Compared with traditional chemotherapy, immunotherapy has the advantages of fewer drugs, a wider range of action and fewer side effects. B7-H7 (also known as HHLA2, B7y) is a member of the B7 family of costimulatory molecules that was discovered more than 20years ago. B7-H7 is mostly expressed in organs such as the breast, intestine, gallbladder and placenta and is detected predominantly in monocytes/macrophages in the immune system. Its expression is upregulated after stimulation by inflammatory factors such as lipopolysaccharide and interferon-γ. B7-H7/transmembrane and immunoglobulin domain containing 2 (TMIGD2) and killer cell immunoglobulin-like receptor, three Ig domains and long cytoplasmic tail 3 (KIR3DL3)-B7-H7 are the two currently confirmed signaling pathways for B7-H7. An increasing number of studies have demonstrated that B7-H7 is widely present in a variety of human tumor tissues, especially in programmed cell death-1 (PD-L1)-negative human tumors. B7-H7 promotes tumor progression, disrupts T-cell-mediated antitumor immunity, and inhibits immune surveillance. B7-H7 also triggers tumor immune escape and is associated with clinical stage, depth of tumor infiltration, metastasis, prognosis, and survival related to different tumor types. Multiple studies have shown that B7-H7 is a promising immunotherapeutic target. Herein, review the current literature on the expression, regulation, receptors and function of B7-H7 and its regulation/function in tumors.

B7-H3 Associates with IMPDH2 and Regulates Cancer Cell Survival

Lung cancer is one of the most common cancers worldwide, and despite improvements in treatment regimens, patient prognosis remains poor. Lung adenocarcinomas develop from the lung epithelia and understanding how specific genetic and environmental factors lead to oncogenic transformation in these cells is of great importance to define the pathways that contribute to tumorigenesis. The recent rise in the use of immunotherapy to treat different cancers has prompted the exploration of immune modulators in tumour cells that may provide new targets to manipulate this process. Of these, the B7 family of cell surface receptors, which includes PD-1, is of particular interest due to its role in modulating immune cell responses within the tumour microenvironment. B7-H3 (CD276) is one family member that is upregulated in many cancer types and suggested to contribute to tumour–immune interactions. However, the function and ligand(s) for this receptor in normal lung epithelia and the mechanisms through which the overexpression of B7-H3 regulate cancer progression in the absence of immune cell interactions remain unclear. Here, we present evidence that B7-H3 is associated with one of the key rate-limiting metabolic enzymes IMPDH2, and the localisation of this complex is altered in human lung cancer cells that express high levels of B7-H3. Mechanistically, the IMPDH2:B7-H3 complex provides a protective role in cancer cells to escape oxidative stress triggered by chemotherapy, thus leading to cell survival. We further demonstrate that the loss of B7-H3 in cancer cells has no effect on growth or migration in 2D but promotes the expansion of 3D spheroids in an IMPDH2-dependent manner. These findings provide new insights into the B7-H3 function in the metabolic homeostasis of normal and transformed lung cancer cells, and whilst this molecule remains an interesting target for immunotherapy, these findings caution against the use of anti-B7-H3 therapies in certain clinical settings.

Polymorphic KIR3DL3 expression modulates tissue-resident and innate-like T cells.

Most human killer cell immunoglobulin-like receptors (KIR) are expressed by natural killer (NK) cells and recognize HLA class I molecules as ligands. KIR3DL3 is a conserved but polymorphic inhibitory KIR recognizing a B7 family ligand, HHLA2, and is implicated for immune checkpoint targeting. The expression profile and biological function of KIR3DL3 have been somewhat elusive, so we searched extensively for KIR3DL3 transcripts, revealing highly enriched expression in γδ and CD8+ T cells rather than NK cells. These KIR3DL3-expressing cells are rare in the blood and thymus but more common in the lungs and digestive tract. High-resolution flow cytometry and single-cell transcriptomics showed that peripheral blood KIR3DL3+ T cells have an activated transitional memory phenotype and are hypofunctional. The T cell receptor (TCR) usage is biased toward genes from early rearranged TCR-α variable segments or Vδ1 chains. In addition, we show that TCR-mediated stimulation can be inhibited through KIR3DL3 ligation. Whereas we detected no impact of KIR3DL3 polymorphism on ligand binding, variants in the proximal promoter and at residue 86 can reduce expression. Together, we demonstrate that KIR3DL3 is up-regulated alongside unconventional T cell stimulation and that individuals may vary in their ability to express KIR3DL3. These results have implications for the personalized targeting of KIR3DL3/HHLA2 checkpoint inhibition.

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

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. 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. 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. 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.

Bispecific antibodies targeting CTLA-4: game-changer troopers in cancer immunotherapy.

Antibody-based cancer immunotherapy has become a powerful asset in the arsenal against malignancies. In this regard, bispecific antibodies (BsAbs) are a ground-breaking novel approach in the therapy of cancers. Recently, BsAbs have represented a significant advancement in improving clinical outcomes. BsAbs are designed to target two different antigens specifically. Over a hundred various BsAb forms currently exist, and more are constantly being manufactured. An antagonistic regulator of T cell activation is cytotoxic T lymphocyte-associated protein 4 (CTLA-4) or CD152, a second counter-receptor for the B7 family of co-stimulatory molecules was introduced in 1996 by Professor James P. Allison and colleagues. Contrary to the explosive success of dual immune checkpoint blockade for treating cancers, a major hurdle still yet persist is that immune-related adverse events (irAEs) observed by combining immune checkpoint inhibitors (ICIs) or monoclonal antibodies such as ipilimumab (anti-CTLA-4) and nivolumab (anti-PD-1). A promising strategy to overcome this hurdle is using BsAbs. This article will summarize BsAbs targeting CTLA-4, their applications in cancer immunotherapy, and relevant clinical trial advances. We will also discuss the pre-clinical rationale for using these BsAbs, and provide the current landscape of the field.

Immune-checkpoint protein VISTA in allergic, autoimmune disease and transplant rejection.

Negative checkpoint regulators (NCRs) reduce the T cell immune response against self-antigens and limit autoimmune disease development. V-domain Ig suppressor of T cell activation (VISTA), a novel immune checkpoint in the B7 family, has recently been identified as one of the NCRs. VISTA maintains T cell quiescence and peripheral tolerance. VISTA targeting has shown promising results in treating immune-related diseases, including cancer and autoimmune disease. In this review, we summarize and discuss the immunomodulatory role of VISTA, its therapeutic potential in allergic, autoimmune disease, and transplant rejection, as well as the current therapeutic antibodies, to present a new method for regulating immune responses and achieving durable tolerance for the treatment of autoimmune disease and transplantation.

B7-H3 in Pediatric Tumors: Far beyond Neuroblastoma.

B7-H3 is a 4Ig transmembrane protein that emerged as a tumor-associated antigen in neuroblastoma. It belongs to the B7 family, shows an immunoregulatory role toward NK and T cells, and, therefore, has been included in the growing family of immune checkpoints. Besides neuroblastoma, B7-H3 is expressed by many pediatric cancers including tumors of the central nervous system, sarcomas, and acute myeloid leukemia. In children, particularly those affected by solid tumors, the therapeutic protocols are aggressive and cause important life-threatening side effects. Moreover, despite the improved survival observed in the last decade, a relevant number of patients show therapy resistance and fatal relapses. Immunotherapy represents a new frontier in the cure of cancer patients and the targeting of tumor antigens or immune checkpoints blockade showed exciting results in adults. In this encouraging scenario, researchers and clinicians are exploring the possibility to use immunotherapeutics targeting B7-H3; these include mAbs and chimeric antigen receptor T-cells (CAR-T). These tools are rapidly evolving to improve the efficacy and decrease the unwanted side effects; drug-conjugated mAbs, bi-tri-specific mAbs or CAR-T, and, very recently, NK cell engagers (NKCE), tetra-specific molecules engaging a tumor-associated antigen and NK cells, have been generated. Preclinical data are promising, and clinical trials are ongoing. Hopefully, the B7-H3 targeting will provide important benefits to cancer patients.

ALAN as an omic tool to build gene signatures and to stratify patients.

1563 Background: Understanding aberrant gene behavior is critical towards devising clinical therapeutics or gene based diagnostic panels. Clustering and AI/ML have clinical utility, but they do not contextualize changes in gene behavior that result from differences in cell type, microenvironment, or prior treatments. Here, we developed the Algorithm for Linking Activity Networks (ALAN), an informatics algorithm that allows rapid functional comparisons of all detectable genes between clinically annotated patient cohorts. ALAN outputs quantify gene behavior without any additional inputs and can be applied on all forms of clinical omic data. We tested the utility of ALAN to identify the molecular features of resistance and novel precision therapeutics. Methods: We applied the ALAN algorithm in prostate tissue using whole-transcriptome sequencing (WTS) data from SU2C/PCF (n = 209, metastatic prostate cancer), TCGA (n = 493, primary prostate cancer), and GTEX (n = 245, benign prostate tissue). We conducted informatics analysis of whole-exome sequencing (WES) data from SU2C/PCF (n = 444 mPC), Fred Hutch (n = 130 mPC), and MCTP (n = 61 mPC) to identify mPC patients with amplifications in RSPO2. We scored an ALAN gene signature in a patient with single-cell RNA sequencing pre and post AR inhibition (n = 112, 83). We examined ChIP-seq data to evaluate binding of AR and Co-factors at the CD276 transcription regulatory sequences. Results: In mPCs, ALAN unbiasedly identified genes that prior studies have implicated as resistance mechanisms towards enzalutamide. In this process, ALAN nominated RSPO2, a secreted protein, as a potential resistant gene that has never been targeted in mPC. In recent studies, we found RSPO2 amplifications in 19.7-28.5% of mPC. We utilized ALAN outputs to build a novel gene signature that was exclusively enriched (p-value < 0.0001) in the transcriptome collected from single cells of a post-resistant mPC sample. We also utilized ALAN to stratify the optimal mPC patients in receiving precision therapies against B7-H3 ( CD276). B7-H3 is an immune regulatory molecule in the B7 superfamily that includes PD-L1 and CTLA-4 and has not been targeted in mPC. B7-H3 was unlike other B7 family members and exhibited robust association with AR co-factors and AR signaling. We confirmed ALAN findings as multiple B7-H3 transcriptional enhancers and promoters were bound by AR and AR-cofactors. Conclusions: ALAN is an algorithm that can be readily deployed on existing clinical omic data to build gene signatures, identify resistant genes, and risk stratify patients. ALAN nominated a novel therapeutic target, RSPO2, which is recurrently amplified in mPC patients. ALAN predicted that mPCs with high baseline levels of AR activity would be optimal candidates for B7-H3 precision therapeutics. Altogether, ALAN outputs should be considered for making clinical decisions that would benefit by comprehensive comparisons of all detectable genes.

B7x in cancer immunity and immunotherapy

B7x (also called B7-H4) is a co-inhibitory molecule of B7 family that is highly expressed in non-inflamed or cold cancers, and its aberrant expression is contributed to cancer progression and poor outcomes. B7x preferentially expresses on antigen-presenting cells (APCs) and in tumor cells, and it acts as an alternative anti-inflammatory immune checkpoint for hampering peripheral immune responses. Augmented infiltration of immunosuppressive cells, reduced proliferation and effector function of CD4+ and CD8+ T cells, and increased generation of regulatory T cells (Tregs) are outcomes of increased B7x activity in cancer. Evaluation of B7x in sera can be exploited as an effective biomarker of response in cancer patients. B7x overexpression generally occurs in programmed death-ligand 1 (PD-L1)- cancers and is involved in tumor resistance to anti-programmed death-1 (PD-1), anti-PD-L1 or anti-cytotoxic T lymphocyte associated antigen-4 (CTLA-4) therapy. Co-expression of B7x receptor with PD-1 on CD8+ T cells has made the anti-B7x a fruitful approach for reinvigoration of the functionality of exhausted T cells and is served as a complementary regimen in patients who are irresponsive to the common immune checkpoint inhibitor (ICI) therapy. An advance in the field is the development of bispecific antibodies against B7x with other regulatory molecules within tumor microenvironment (TME).

Association of B7H3 and B7H4 gene polymorphisms and protein expression with the development and prognosis of autoimmune thyroid diseases.

The prognosis of autoimmune thyroid diseases (AITDs), including Graves' disease (GD) and Hashimoto's disease (HD), varies among patients. B7-H3 and B7-H4, members of the B7 family of proteins, regulate immune response. To clarify the association of B7-H3 and B7-H4 with the pathogenesis and prognosis of AITDs, we examined the expression of the soluble and membrane form of B7-H3 and B7-H4 and genotyped single nucleotide polymorphisms (SNPs) in the B7H3 and B7H4 genes. We examined the expression of the membrane form of B7-H3 and B7-H4 by flow cytometry and their soluble forms by enzyme-linked immunosorbent assay. We genotyped SNPs in B7H3 and B7H4 in 187 GD patients, 217 HD patients, and 110 healthy volunteers using the PCR-RFLP method. The frequency of the B7H3 rs3816661 CC genotype was higher in patients with severe HD. G carriers of B7H4 rs10754339 A/G and B7H4 rs13505 T/G were more frequent in patients with AITD. A carrier of B7H4 rs10158166 A/G and C carriers of B7H4 rs3806373 C/T were more frequent in patients with intractable GD. The proportion of B7-H3+ monocytes was higher in the CC genotype of B7H3 rs3816661 C/T than in the other genotypes and was lower in patients with GD and HD than in healthy controls. The concentration of soluble B7-H4 was lower in the TG genotype of B7H4 rs13505 T/G than in the TT genotype and was higher in patients with AITD than in healthy controls. B7H3 and B7H4 are associated with AITD susceptibility and prognosis.

B7H6 silencing increases chemosensitivity to dacarbazine and suppresses cell survival and migration in cutaneous melanoma.

Cutaneous melanoma (CM) is a highly metastatic cancer whose incidence rate is heightening worldwide. B7H6, as one of the co-stimulatory ligands of the B7 family, is expressed in malignant cells, involved in tumorigenesis. This study aimed to investigate the significance of B7H6 in CM cell chemosensitivity and metastatic ability. A375 CM cells were transfected with B7H6-siRNA and treated with dacarbazine individually or combined. The MTT assay to estimate half-maximal inhibitory concentration of dacarbazine and cell viability, the apoptotic induction using Annexin V/PI, cell cycle progression via flow cytometry, and wound healing assay for determining the migration ability of cells and assessing the clonogenic potential of A375 cells were executed. Functional analyses were performed to evaluate changes in A375 cells. The results illustrated that B7H6 suppression significantly increased the chemosensitivity of A375 cells to dacarbazine. Apoptosis induction by dacarbazine was enhanced after B7H6 knockdown through modulating Caspase-3, Bax, and Bcl-2 mRNA levels. Western blotting indicated enhancement of cleaved caspase-3 protein expression in treatment groups. A375 cells were arrested at the sub-G1 and S phases when using B7H6-siRNA and dacarbazine. B7H6 suppression combined with dacarbazine restrained cell migration through suppression of matrix metalloproteinase (MMP) expression, including MMP2, MMP3, and MMP9. In addition, the clonogenic ability of A375 cells was decreased by downregulating Sox2, Nanog, and CD44 mRNA levels. A visible decrement in STAT3 protein expression was observed in the combination group. Hence, our findings revealed that B7H6 knockdown with dacarbazine could be a promising treatment approach for cutaneous melanoma.

Abstract 6656: Expression pattern and clinicopathological implication of B7 family immune checkpoints, VTCN1 and HHLA2, in non-small cell lung cancer

Abstract Background: Efforts are needed to discover novel immunotherapeutic targets in non-responders of current immunotherapies. VTCN1 (B7-H4/B7x/B7S1) and HHLA2 (B7-H7/B7y) are B7 family co-inhibitory molecules reported to be expressed in PD-L1-negative or EGFR-mutant lung adenocarcinoma (LUAD). To elucidate the clinicopathological implications of these two immune checkpoints, we checked their detailed expression patterns including intratumoral heterogeneity. Methods: VTCN1 and HHLA2 expression were evaluated in public single-cell RNA sequencing (scRNAseq) and The Cancer Genome Atlas (TCGA) dataset. Their immunohistochemistry (IHC) was performed on tissue microarrays of 413 LUAD and 382 lung squamous cell carcinoma (LUSC) specimens. Intratumoral heterogeneity was evaluated by IHC on representative sections of 59 surgically resected LUAD and 49 LUSC specimens. Results: In LUAD scRNAseq, VTCN1 and HHLA2 were predominantly expressed on epithelial cells and were not co-expressed with PD-L1. However, IHC showed their expression in both tumor and immune cells. In TCGA analysis, there was a negative correlation between VTCN1 and PD-L1 (LUAD, r = -0.094, p = 0.033; LUSC, r = -0.227, p <0.001). VTCN1 and HHLA2 showed positive correlations in both TCGA (LUAD, r = 0.211, p <0.001; LUSC, r = 0.182, p <0.001) and IHC analysis (LUAD, p <0.001; LUSC, p = 0.016). They were more frequently expressed in EGFR-mutant LUAD (52.9% vs. 40.3%, p = 0.015 for VTCN1 in EGFR-mutant vs. wildtype; 59.8% vs. 40.5%, p <0.001 for HHLA2). CD8+ T-cell infiltration was significantly higher in VTCN1- or HHLA2-positive EGFR-mutant LUAD, but there was no significant relationship in EGFR-wildtype. In 60% of cases expressing both VTCN1 and HHLA2, the two showed spatially concordant expression (15 out of 24 LUAD; 3 out of 6 LUSC). However, VTCN1 and PD-L1 showed concordant expression in 37.2% of cases expressing both (6 out of 16 LUAD; 10 out of 27 LUSC). HHLA2 expression was associated with better overall survival in LUSC, but there was no significant difference in survival according to VTCN1 expression. Conclusions: VTCN1 and HHLA2 are frequently co-expressed in PD-L1-negative NSCLC, especially EGFR-mutant LUAD rich in CD8+ T-cells and can serve as potential novel immunotherapeutic targets. Citation Format: Sojung Lim, Jaemoon Koh, Seung Geun Song, Jeemin Yim, Bogyeong Han, Young A Kim, Doo Hyun Chung, Yoon Kyung Jeon. Expression pattern and clinicopathological implication of B7 family immune checkpoints, VTCN1 and HHLA2, in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6656.

Abstract 972: VISTA expression in patients with advanced solid tumors: A potential biomarker in VISTA-101 clinical trial

Abstract V-domain Immunoglobulin Suppressor of T cell Activation (VISTA/PD-1H) is a B7 family member highly expressed on circulating and intra-tumoral myeloid cells. It is a negative checkpoint inhibitor that inhibits anti-tumor T cell response. In patients, VISTA is associated with poor overall survival in multiple tumor indications and is also a potential mediator of resistance to anti-CTLA-4 and anti-PD1 therapies. Therefore, VISTA is a unique target for cancer immunotherapy. Kineta has developed a fully human monoclonal antibody targeting VISTA, KVA12123, that is currently being evaluated in a Phase 1/2 clinical trial in cancer patients with advanced solid tumors. This trial also includes a combination arm with pembrolizumab. In order to inform which patients may be susceptible to respond to our anti-VISTA antibody, we hypothesized that the best responders should be associated with a high expression of the target in the tumor microenvironment (TME). Therefore, after assay validation of VISTA labelling by immuno-histochemistry, we analyzed a large set of tumor samples and showed that VISTA was highly expressed on tumor infiltrating immune cells. This was particularly true for patients with non-small cell lung cancers, colorectal cancers, head and neck squamous cell carcinomas, hepatocellular carcinomas, melanomas and squamous cell carcinoma of the skin, and cervical cancers as well as ovarian cancers. VISTA expression was detected mostly on CD163 positive macrophages infiltrating the tumor. These macrophages potentially promote immunosuppression present in the TME and contribute to treatment failure with current immune checkpoint inhibitors like anti-PD1/PD-L1 and CTLA-4. While previous studies reported VISTA expression on cancer cells, we were not able to confirm these results. In all tumor tissues tested, only infiltrating immune cells were labelled for VISTA. We have investigated in parallel the expression level of soluble VISTA in the serum collected from cancer patients independent of this clinical trial. Sera were screened for patients with multiple tumor types. Patients of diverse sex and age were compared to healthy donors. After validation of the ELISA assay, we showed that sera derived from cancer patients exhibit high levels of soluble VISTA, and these levels tend to correlate with age. More data are needed to confirm that high levels of soluble VISTA are associated with advanced disease. In the ongoing Phase 1/2 clinical trial, tumor tissues and serum samples will be collected from cancer patients prior to treatment with KVA12123 to inform the possible significance of these biomarkers. This work could help to better understand the response to KVA12123 in relation to the expression level of VISTA in cancer tissues as well as in the blood. Citation Format: Neda Kabi, Chen Katz, Remington Lance, Jessica Cross, Nathan Eyde, Emily Frazier, Kurt Lustig, Yulia Ovechkina, David Peckham, Shaarwari Sridhar, Carla Talbaux, Isabelle Tihista, Mei Xu, Shawn Iadonato, Thierry Guillaudeux. VISTA expression in patients with advanced solid tumors: A potential biomarker in VISTA-101 clinical trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 972.