B7-H4 as a potential target for immunotherapy for gynecologic cancers: a closer look.

A growing body of literature suggests that B7-H4 on the cell surface binds the putative B7-H4 receptor on activated CD4 + and CD8 + T cells resulting in the inhibition of effector function via cell cycle arrest, decreased proliferation, and reduced IL-2 production. Various studies have reinforced the role of B7-H4 in diminishing both CD4 + and CD8 + T-cell functionality. Sica et al. first showed that a B7-H4 immunoglobulin fusion protein (B7-H4.Ig) decreased the proliferation, production of IL-2 and IL-10, and cytolytic activity of murine CD4 + T cells 48–72 h after stimulation with anti-CD3 antibodies [ 11 ]. Exposure to B7-H4.Ig also caused a significant inhibition of cell division, exhibiting an increase in the G 0 /G 1 phase of the cell cycle while apoptosis was not affected. In addition to studies utilizing soluble B7-H4.Ig fusion proteins, B7-H4 expressed on the cell surface also functionally inhibits proliferation and cytokine production of murine T cells [ 12 ]. Similarly, Wang et al. later showed the most profound inhibition of IL-2 secretion at 16 h in the presence of B7-H4 showing decreases of 75.4%, 72.8%, and 71.5% in CD3 + , CD4 + , and CD8 + T cells, respectively [ 30 ]. Similar results have emerged using human cell cultures. Ou et al. [ 31 ] was one of first studies to show B7-H4-mediated functional inhibition of human T cells. Much like previous reports using murine T cells, immobilized B7-H4.Ig fusion protein inhibited T-cell proliferation, arrested T cells in G 0 /G 1 of the cell cycle, and induced T-cell apoptosis in activated CD4 + and CD8 + T cells after 3–5 days in the presence of B7-H4 [ 31 ]. The diminished functional capacity of human T cells in the presence of cellular B7-H4 at both endogenous and over-expressed levels of B7-H4 protein has also been assessed, confirming the findings that B7-H4 mediates functional inhibition of human T cells [ 16 ]. Disruption of the restricted regulation of B7-H4 expression is implicated in autoimmune diseases and cancer. Loss of endogenous B7-H4 expression may play a role during induction and/or promotion of auto-immune diseases including rheumatoid arthritis and diabetes [ 32 ]. For example, Wei et al. demonstrated B7-H4 expression in murine pancreatic Langerhans islet-cells by IHC, and showed that B7-H4 deficient mice had a more aggressive form of diabetes in the presence of adoptively transferred, transgenic islet-reactive T cells, while overexpression of B7-H4 in the islet decreased the progression of diabetes [ 33 ]. However, the endogenous role of B7-H4 in normal mice is not well elucidated, as B7-H4 −/− mice do not spontaneously develop autoimmunity and do mount normal T cell responses against viral infection [ 34 ]. While loss of B7-H4 expression has been studied in the context of autoimmunity, overexpression of B7-H4 is seen in various types and stages of ovarian, lung, ductal breast, renal cell, uterine, and endometrial cancers [ 15 , 35 ]. In the vast majority of breast and ovarian cancers, B7-H4 mRNA is at least 2-fold higher than mRNA found in normal tissue [ 9 ]. B7-H4 protein is detected in half of early stage and two-thirds of late stage ovarian tumor samples [ 36 ], but not in corresponding normal ovarian tissue [ 9 , 19 , 24 ]. By protein ELISA from ovarian cancer patient tissue lysates, Simon et al. showed that patients with higher stage disease had increased B7-H4 protein expression: 48% for stage I, 55% for stage II, and 67% for stages III–IV, while normal and benign tumors had very low expression [ 36 ]. Similar to correlations found in ovarian cancer lysate samples [ 36 ], B7-H4 expression in tumor tissues from breast, uterus, ovary, colon, and pancreas showed a statistically significant increase in percentage B7-H4 expressed and stage [ 21 ]. There were 54.1% overexpression in stages III–IV, and 36.6% and 43.8% overexpressed in stages I and II respectively. Although B7-H4 research has been focused on B7-H4's effects as an immune-modulatory signaling molecule, several papers indicate distinct intracellular effects such as decreased apoptosis, enhanced proliferation, and facilitated metastasis in B7-H4-expressing cells themselves. B7-H4 may protect cancer cells from apoptosis as knockdown of B7-H4 in a breast cancer cell line increased apoptosis, and overexpression of B7-H4 protected epithelial cells from anoikis [ 9 ]. Additionally, ovarian cancer cell lines transfected with B7-H4 have increased proliferation, cell adhesion, migration, and invasion in vitro [ 38 ]. An in vivo model of a B7-H4-engineered ovarian cancer cell line demonstrated increased tumor growth compared to its parental cell line in immune-deficient mice, pointing to a possible functional role of B7-H4 in tumorigenesis even in the absence of host immunity [ 9 , 38 ]. A recent study also showed that overexpression of wild-type B7-H4 in HEK293 cells enhanced proliferation by promoting G 1 /S phase transition [ 8 ]. This data was further validated by Zhang et al.'s discovery of B7-H4's functional nuclear localization sequence (NLS) where mutation of the NLS abrogated the nuclear effects of B7-H4, deeming the NLS necessary to promote G 1 /S transition, decrease apoptosis, and increase tumor growth in vivo [ 8 ]. B7-H4 deficient mice also point to B7-H4's role in metastasis. In a 4T1 metastatic breast cancer model, B7-H4 −/− mice have fewer lung nodules, enhanced survival, and decreased infiltration of immunosuppressive cells (tumor associated macrophages and Tregs) [ 39 ], suggesting that B7-H4 plays a role in helping metastasizing cancer cells escape local antitumor immune responses. B7-H4 can also exist in a soluble form in serum of diseased patients. Levels of soluble, serum B7-H4 correlate with tumor stage, poor prognosis, and pathological types [ 17 ], and this topic was covered more thoroughly in a review paper by Fauci et al. [ 40 ]. In short, B7-H4 is elevated in ovarian cancer patients when compared with healthy controls, and not significantly elevated in other types of cancer such as colon, breast, lung, and prostate [ 23 ]. In early stage patients, adding B7-H4 to CA125 diagnostic testing improved detection of ovarian cancer from 52% to 65% [ 36 ], and in combination with other serum markers, B7-H4 was useful in predicting short-term (1 year) survival [ 41 ].

Ovarian cancer is the fifth most common cause of cancer-related death among women, accounting for approximately 14,000 deaths and 22,000 new cases estimated in 2014 in the United States [ 1 ]. Most women are asymptomatic and diagnosed late in the cancer's progression, with 5-year survival rates of only 18–45% in stages III–IV [ 2 ]. Few therapies are effective and most women ultimately die of the disease [ 3 ]. The past few decades have brought targeted treatments for many cancer types. For ovarian cancer, the most effective new treatment has been bevacizumab [ 4 ], and while effective, results have been modest [ 5 ]. Given the limitations in current treatments and the correlation of endogenous immune responses with improved survival in ovarian cancer, research in targeted immunotherapy is of great interest. One of the first publications showing immunogenicity of ovarian cancer observed that patients with detectable tumor infiltrating T cells (TILs) had a 38% 5-year overall survival rate compared to 4.5% for patients who did not [ 6 ]. A recent meta-analysis reviewed 10 studies that investigated CD3 + or CD8 + T cells in ovarian cancer, and all studies showed significantly longer survival with intraepithelial TILs [ 7 ]. While these studies suggest the importance of the immune system in the control of ovarian cancer, many mechanisms of immune evasion exist. This review focuses on the expression of one inhibitory protein, B7-H4 that facilitates escape from the host T-cell response, augments tumor cell proliferation [ 8 ] and anti-apoptotic ability [ 9 ], and promotes tumorigenesis [ 8 – 10 ].

In healthy individuals, B7-H4 mRNA is detected at low levels in many tissues [ 11 , 15 ] however, B7-H4 protein expression seems to be limited due to tight translational control in peripheral tissues in both humans [ 15 , 16 ] and mice [ 13 ]. This regulation of restricted and inducible B7-H4 expression, however, does not take place in cancer. Instead, B7-H4 is overexpressed in multiple stages of cancer and various types including ovarian, uterine, and endometrial cancers [ 15 , 35 ]. Still, its pattern of surface expression remains debated in the published literature. Within the tumor, B7-H4 protein is also expressed on the cell surface of immunosuppressive tumor-associated macrophages (TAMs) [ 16 ] and tumor cells of ovarian cancer [ 24 ]. Based upon its potential, restricted expression and functional role in cancer, B7-H4 remains a high priority candidate for targeted inhibition or elimination in ovarian cancer and other types of cancer. To date, clinical trials utilizing an anti-human B7-H4 blocking antibody have not been conducted in any disease indication. Since many clinical strategies rely on antibody/scFv specificity for generation of therapies, including monoclonal antibodies, immunotoxins, radio-therapy, bi-specific antibodies, and chimeric antigen receptors, generation of a highly specific anti-human B7-H4 antibody would open the door to robust preclinical studies. Results from several studies utilizing various anti-mouse B7-H4 antibodies have seen success in rescuing T-cell function in vitro [ 11 , 13 , 22 ], augmenting T-cell responses in vivo [ 11 , 13 ], and reducing tumor burden in a murine lung cancer model [ 49 ], demonstrating a proof of concept for targeting B7-H4. However, effective anti-human B7-H4 antibodies are not yet available [ 17 ] and pre-clinical studies have not yet assessed the ability of an anti-human B7-H4 mAb to reduce human cancer burden in vivo. Novel anti-human B7-H4 scFvs that impact human tumor progression in vivo have recently been described [ 24 ] and may have implications if engineered as radio-isotopes for imaging, or as immunotoxins, nanoparticles, or CARs for therapy. While the last decade has offered insight into the functional role of B7-H4 in tumor biology and immune evasion, no anti-B7-H4 targeted therapies have been investigated clinically. Many opportunities still exist to determine if stand-alone or combinatorial therapies to directly target or overcome B7-H4-mediated hypo-function of T cells in the ovarian tumor microenvironment can provide clinical benefit in women with ovarian cancer.

Comparison of B7-H4 to other negative immune molecules is warranted. CTLA-4 is expressed on T cells and its inhibitory effect occurs in secondary lymphoid tissues, PD-L1 is on APCs and its effect is mostly in peripheral tissues during inflammatory response, yet B7-H4 likely has more of a local effect at tumor site and may be a better target for ovarian and gynecologic cancers [ 42 , 43 ]. The CTLA-4 antibody ipilimumab (Yervoy) was FDA approved for melanoma in 2011, and is currently being investigated as a single or combinatorial therapy in clinical trials (clinicaltrials.gov). Hodi et al. showed modest antitumor effects in patients with stage IV ovarian cancer patients, 1/9 patients with a regression of disease, and 3/9 had stable disease [ 44 ]. PD-L1 pathway in ovarian cancer is of interest as higher PD-L1 expression is associated with poorer prognosis [ 42 ]. A Phase I trial in 2012 by Brahmer et al. demonstrated responses in only 1/17 ovarian cancer patients and these samples were not evaluated for expression of PD-L1 [ 43 ]. Moving forward, synergistic targeting of multiple negative immune-modulatory pathways may be necessary to fight disease. Although not yet targeted clinically, the advantage of targeting B7-H4 may be several folds since B7-H4 is expressed on tumor cells and TAMs in various cancer types. Additionally, since B7-H4 plays a role in immune evasion ( Fig. 1 ) and tumorigenesis, eliminating or diminishing the effects of B7-H4 may positively affect the host immune response and negatively affect tumor cell survival simultaneously. Although no clinical studies targeting B7-H4 have been performed to date, potential methods discussed here include siRNA, immunotoxins, blocking antibodies, or T-cell based immunotherapy. Other potential ways to target B7-H4 that are not discussed in detail here are via antibody drug conjugate (ADC) and a B7-H4-specific vaccine. Oligonucleotides, short hairpin (shRNA), or small interfering (siRNA) RNA are used to decrease expression of protein in vitro ( Fig. 2 ), yet these tools are difficult to use in clinical therapy. To reduce B7-H4 expression, Kryczek et al. designed a B7-H4-specific morpholino that specifically blocked B7-H4 expression in macrophages, resulting in increased T-cell proliferation and reduced tumor volumes in mice with tumor associated antigen (TAA)-specific T cells [ 16 ]. Knockdown of B7-H4 expression in breast cancer cell lines showed increased apoptosis of tumor cells in the absence of immune cells in vitro, while knockdown in a nonsmall-cell lung cancer cell line leads to enhanced Jurkat T cell proliferation, decreased apoptosis, and cell cycle progression [ 45 ]. The current challenge is to identify and develop safe delivery methods for these molecules to pursue clinical use ( Fig. 2 ). Immunotoxins are chimeric proteins that contain a targeting moiety (such as an antibody) linked to a toxin [ 46 ]. Immunotoxins have been studied for cancer treatment as well as autoimmune disease. SS1P immunotoxin (anti-mesothelin linked to a truncated form of Pseudomonas exotoxin) has shown modest activity in Phase I clinical trials in patients with mesothelioma, ovarian and pancreatic cancers [ 47 ], and anti-Lewis Y immunotoxin has shown one complete remission in a patient with metastatic breast cancer [ 48 ]. Development of an immunotoxin with a targeting moiety against B7-H4 could be promising ( Fig. 2 ). However, as descried in the next section, finding human/ humanized antibodies against B7-H4 has been problematic. One of the best therapeutic methods to efficiently disrupt the functionality of cell surface proteins in the tumor microenvironment is the use of monoclonal antibodies (mAbs), a strategy that has shown promise targeting other negative immune modulators such as PD-L1, PD-1, and CTLA-4. Blocking the putative B7-H4 receptor on T cells from engaging with B7-H4 on the surface of tumor cells or macrophages could be achieved using an anti-B7-H4 antibody ( Fig. 2 ). Several studies to date have utilized anti-B7-H4 antibodies for in vitro and in vivo studies, many of which are described in Table 1 . Several anti-mouse B7-H4 antibodies have demonstrated rescue of T-cell function in the presence of B7-H4 in vitro. Prasad et al. showed augmented IL-2 production and increased T-cell proliferation post-T-cell activation in the presence of B7-H4 and antibody. Sica et al. developed an anti-mouse B7-H4 mAb that showed a partial neutralization of the inhibition of T-cell proliferation post-incubation with B7-H4-transfected cells [ 11 ]. Another anti-mouse B7-H4 antibody (3E8) showed reversal of B7-H4-mediated decreases in cytokine secretion post-murine T-cell activation [ 22 ]. Additionally, anti-mouse B7-H4 antibodies are able to augment T-cell responses in vivo [ 11 , 13 ] and decrease tumor burden in a syngeneic B7-H4-expressing murine lung cancer model [ 49 ]. Although not as widely available or studied as anti-mouse B7-H4 antibodies, anti-human B7-H4 antibodies have been efficacious in rescuing B7-H4-mediated functional inhibition of T cells in vitro. Xue et al. utilized an anti-human B7-H4 blocking antibody to significantly attenuate the T-cell inhibitory effects of B7-H4 expressed by human bone marrow-derived mesechymal stem cells (hBMSCs) [ 50 ]. However, in vivo analysis of the ability of an anti-B7-H4 mAb to reduce human tumor burden in a xenograft model has not been assessed. A review by He et al. [ 17 ] highlighted the fact that efficient neutralizing antibodies specific for human B7-H4 are not yet available. A recently published paper by Dangaj et al. demonstrates potential for a novel therapeutic B7-H4 by identifying and characterizing recombinant single chain variable fragments (scFv) isolated from a yeast display library [ 24 ]. These anti-B7-H4 scFvs specifically rescued T-cell function from B7-H4-mediated T-cell inhibition, as demonstrated by increased IFN-γ secretion, up-regulation of CD69 expression, and augmented T-cell proliferation in response to anti-CD3 stimulation and inhibition through recombinant, human B7-H4 protein. These scFvs also specifically abrogated B7-H4-mediated functional inhibition of HER-2 TCR-engineered T cells in vitro in the presence of B7-H4-expressing APCs pulsed with HER-2 peptide or a B7-H4-engineered tumor cell line. Delayed growth of human B7-H4 + ovarian cancer tumor was observed with intraperitoneal injections of the anti-B7-H4 scFv in mice [ 24 ]. Although promising, moving forward with clinical trials utilizing scFv alone as systemic therapy may be difficult due to problems with tumor penetration, rapid renal clearance, and lack of effector functions. However, scFvs are versatile and can be conjugated to a myriad of efficacious tools including radio-isotopes for imaging, endotoxins, nano-particles, or even T-cell signaling domains. In spite of the association between intratumoral T cell accumulation and improved overall survival in ovarian cancer [ 6 ], targeting of solid epithelial tumors such as ovarian malignancies with naturally-occurring tumor-reactive T cells has not been highly efficacious [ 51 – 54 ]. An alternative approach is to genetically modify T cells to express chimeric antigen receptors (CARs), proteins that combine antibody recognition and TCR signaling [ 55 ]. The promise of CAR T-cell therapy has been shown in the treatment of B-cell malignancies [ 56 , 57 ], but to date, hasn't been effective in ovarian cancer. The first CAR T cell trial conducted utilized T cells targeting alpha-folate receptor in ovarian cancer [ 58 ], however, no reduction in tumor burden was seen. Lack of clinical efficacy was thought to be attributed to, in part, poor CAR T cell persistence after infusion due to the lack of T-cell co-stimulatory domains in the CAR and the development of an inhibitory factor in the serum of patients that reduced CAR activity [ 58 , 59 ]. This approach has been revitalized for ovarian cancer using a CAR that harnesses the potent pro-survival signals of 4-1BB (CD137) domain to improve T-cell persistence in vivo [ 59 ], and will soon begin in a Phase I clinical trial [ 60 ]. It is feasible that failures in prior T-cell based immune therapies for ovarian cancer may also be related to the lack of understanding of the negative effects on anti-tumor T-cells of inhibitory molecules, such as B7-H4. Therefore, it is conceivable that CAR T-cell approaches directly targeting B7-H4 may be effective by simultaneously destroying B7-H4 + tumor cells/TAMs and eliminating negative-immune modulating cells from the tumor microenvironment. Although not yet tested in the clinic, an anti-B7-H4-specific CAR could be generated through combination of an anti-B7-H4 single chain variable fragment (scFv) with optimal T cell co-stimulation domains ( Fig. 3 ). Alternatively, the activity of CAR T cells directed against a tumor antigen, such as alpha folate receptor, may be bolstered by co-administration of a B7-H4 blocking antibody. Additional therapies to resist the inhibitory effects of B7-H4 may prove useful in the field of T-cell based therapy. One example is the use of bi-specific antibodies that engage anti-CD3 expressed on T-cells and a second antigen expressed on tumor cells, such as anti-B7-H4, to facilitate the redirection of T cells to B7-H4 expressed on cancer cells and TAMs ( Fig. 2 ). Exploiting B7-H4 as a target with a T-cell based therapy, or overcoming its inhibitory effect in T cell-therapy, may be a therapeutic option in ovarian and other B7-H4 + malignancies in the near future.

B7-H4 is a transmembrane protein in the B7 family identified in 2003 by three separate groups, each performing searches in Expressed Sequence Tagged (EST) databases for sequences similar to B7 family molecules [ 11 – 13 ]. In June 2003, Sica et al. identified B7-H4 from a human EST search and predicted B7-H4 to be a 282 amino acid, type I transmembrane protein that includes an extracellular domain, hydro-phobic transmembrane domain, and a very short, 2 amino acid long intracellular domain [ 11 ]. Almost simultaneously, Prasad et al. also identified B7-H4 by the name of B7S1 [ 13 ], and Zang et al. identified mouse B7-H4 with the name B7× [ 12 ]. After the initial discovery, B7-H4 (DD-O110) was again independently identified in 2004–2005 by Salceda et al., in a genomic effort to discover genes up-regulated in tumors, specifically looking for targets for breast and ovarian cancers [ 9 ]. The authors predicted the protein core to be 28.8 kDa, yet by western blot it is in the 40–80 kDa range because of glycosylation [ 9 ].