Antibodies to EGF Receptor Family Members Can Upregulate Tumor Immunity.

Immunologic mechanisms influence how a cancer patient responds to therapy. Monoclonal antibodies (mAbs) to the epidermal growth factor receptor are clinically approved, and a lung cancer vaccine inducing antibodies to epidermal growth factor (EGF) has some beneficial clinical effects. We tested the hypothesis that mAbs to epidermal growth factor receptor, EGF, and tumor growth factor alpha (TGF-α), in addition to any other effects, can facilitate the generation of a tumor-destructive immunologic response. Data from studies with mouse tumors showed that all 3 of these mAbs stimulated the in vitro generation of a Th1 response with tumor cells killed by spleen cells from mice with SW1 melanoma, B16 melanoma, or ID8 ovarian carcinoma. The mAb to TGF-α was most effective, and tumor lines releasing TGF-α were more sensitive than lines not releasing TGF-α. Stimulated by these findings we then performed pilot experiments in which mice with SW1 melanoma were injected with mAbs intraperitoneally or with a combination of the 2. A combination of anti-TGF-α and anti-PD-1 mAbs could cure mice with established tumor while single anti-TGF-α or anti-PD1 mAbs could not.

Ovarian carcinomas express HE4 epitopes independently of each other.

BACKGROUND: The gene encoding HE4 undergoes alternative splicing to yield multiple protein isoforms. We investigated anti-HE4 mAbs which recognize epitopes on the C (2H5 and 3D8) or N (12A2 and 14E2) terminals. METHODS: A Luminex assay was applied to determine mAb affinity. Binding of mAbs to sections from formaline fixed ovarian carcinomas was determined by immunohistology, binding to cultured ovarian carcinoma cells was tested by flow cytometry and immunocytochemistry, and HE4 secretion to patient serum or supernatants of cultured ovarian carcinoma was assayed by ELISA. RESULTS: mAb 12A2 bound to formalin-fixed sections from 18 of 19 ovarian carcinomas, while 14E2, 2H5 and 3D8 bound to sections from 14, 7 and 4 patients, respectively. The mAbs bound independently of each other, i.e. a sample bound one mAb most strongly while another sample with similar affinity strongest bound another mAb. The intensity of binding to sections did not significantly correlate with the serum level of HE4 except for mAb 14E2, but there was a significant correlation between HE4 expression and its detection in supernatants of cultured ovarian carcinomas. CONCLUSIONS: HE4 epitopes are expressed independently of each other and their expression of HE4 by cultured ovarian carcinoma cells correlates with the release of HE4 into culture supernatants. he epitope recognized by mAb 14E2 was significantly more expressed by platinum resistant tumors. IMPACT: Expression of HE4 by most ovarian carcinomas makes it an excellent biomarker.. Further studies are needed to investigate the clinical relevance of overexpression of a particular epitope.

An In Vitro Model That Predicts the Therapeutic Efficacy of Immunomodulatory Antibodies.

Immunomodulatory monoclonal antibodies (mAbs) have efficacy in patients with advanced cancer and are the focus of intensive research. However, cures are infrequent and responses vary among tumor types and among subjects with the same tumor. An in vitro test would be valuable to determine the most effective mAb combination for a given case and to evaluate novel agents. Toward this goal, we investigated the ability of various mAb combinations to generate a tumor-destructive immune response in vitro in the presence of lymphoid cells from mice with established TC1 lung carcinoma, B16 melanoma, or SW1 melanoma. The data strongly correlate (r=0.9, 0.89, and 0.91, respectively) with the therapeutic efficacy of the respective mAb combinations. Both in vivo and in vitro, tumor destruction was associated with a shift from a Th2 to a Th1 response and included a dramatic increase of long-term memory cells. A combination of mAbs to CD137/PD-1/CTLA4/CD19 was most efficacious.

Detection of the HE4 protein in urine as a biomarker for ovarian neoplasms: Clinical correlates.

OBJECTIVES: To measure HE4 levels in urine from normal donors, patients with LMP tumors and ovarian cancer patients and to correlate levels with clinical factors in ovarian cancer patients. METHODS: Archived samples from controls, patients with LMP tumors and ovarian cancer were tested using commercial assays, including serially collected serum and urine samples from women treated for stage III/IV serous ovarian cancer. RESULTS: Five of 6 patients with stage I/II and 26 of 36 stage III/IV serous ovarian cancer patients had HE4-positive urines, similar to serum samples (4 of 5 stage I/II and 26 of 34 stage III/IV) when tested at the same level of specificity (95%). Urine HE4 was more sensitive in LMP tumors: 9 of 32 urines (28%) HE4-positive versus 3 of 68 sera (4.4%, p=0.002). Mean levels of serum CA125 and HE4 decreased comparably in patients during initial treatment regardless of their primary platinum response, but mean urine HE4 levels decreased only 7% in primary platinum resistant patients while decreasing 68% in those who were sensitive. By 7months after diagnosis, urine HE4 levels were higher in primary platinum resistant patients compared to those who proved to be sensitive (p=0.051) and persisted 12months after diagnosis (p=0.014). HE4 values in urine also became positive in advance of clinical recurrence in several women while serum HE4 and serum CA-125 remained normal. CONCLUSIONS: Measuring HE4 in urine complements serum assays for the detection of ovarian cancer and may allow identification of patients at high risk for primary platinum resistance.

Curing mice with large tumors by locally delivering combinations of immunomodulatory antibodies.

PURPOSE: Immunomodulatory mAbs can treat cancer, but cures are rare except for small tumors. Our objective was to explore whether the therapeutic window increases by combining mAbs with different modes of action and injecting them into tumors. EXPERIMENTAL DESIGN: Combinations of mAbs to CD137/PD-1/CTLA-4 or CD137/PD-1/CTLA-4/CD19 were administrated intratumorally to mice with syngeneic tumors (B16 and SW1 melanoma, TC1 lung carcinoma), including tumors with a mean surface of approximately 80 mm(2). Survival and tumor growth were assessed. Immunologic responses were evaluated using flow cytometry and qRT-PCR. RESULTS: More than 50% of tumor-bearing mice had complete regression and long-term survival after tumor injection with mAbs recognizing CD137/PD-1/CTLA-4/CD19 with similar responses in three models. Intratumoral injection was more efficacious than intraperitoneal injection in causing rejection also of untreated tumors in the same mice. The three-mAb combination could also induce regression, but was less efficacious. There were few side effects, and therapy-resistant tumors were not observed. Transplanted tumor cells rapidly caused a Th2 response with increased CD19 cells. Successful therapy shifted this response to the Th1 phenotype with decreased CD19 cells and increased numbers of long-term memory CD8 effector cells and T cells making IFNγ and TNFα. CONCLUSIONS: Intratumoral injection of mAbs recognizing CD137/PD-1/CTLA-4/CD19 can eradicate established tumors and reverse a Th2 response with tumor-associated CD19 cells to Th1 immunity, whereas a combination lacking anti-CD19 is less effective. There are several human cancers for which a similar approach may provide clinical benefit.

Anti-HE4 antibodies in infertile women and women with ovarian cancer.

OBJECTIVES: To develop an assay for anti-HE4 antibodies and assess such antibodies in sera from women with increased epidemiologic risk for ovarian cancer (infertility) and patients with ovarian cancer in comparison to controls. METHODS: An ELISA was developed to measure antibodies to recombinant full length HE4 and cut-off values were determined for different levels of specificity (up to 99%). RESULTS: Infertile women more frequently had anti-HE4 antibodies than controls (23% at 98% specificity, p < 0.001) with antibodies most frequent in women with POF (31%) and ovulatory dysfunction (47%). There was also an increased frequency of anti-HE4 antibodies in patients with ovarian cancer (14% at 97% specificity, p < 0.01), but more women with certain types of infertility have anti-HE4 antibodies than women with ovarian cancer. Most patients with ovarian cancer have circulating HE4 antigen, which may interfere with detection of antibodies, while the level of HE4 antigen in sera from infertile women was not higher than in normal controls. There was a statistically significant correlation between antibodies to HE4 and antibodies to mesothelin in the same patients. CONCLUSIONS: Women with certain types of infertility, which have increased risk to develop ovarian cancer, and women with ovarian cancer more frequently than controls have antibodies to HE4, a biomarker for ovarian cancer. The antibodies may reflect a tumor-promoting Th2 type of inflammation.

Long-lasting complete regression of established mouse tumors by counteracting Th2 inflammation.

Mice with intraperitoneal ID8 ovarian carcinoma or subcutaneous SW1 melanoma were injected with monoclonal antibodies (mAbs) to CD137PD-1CTLA4 7-15 days after tumor initiation. Survival of mice with ID8 tumors tripled and >40% of mice with SW1 tumors remained healthy >150 days after last treatment and are probably cured. Therapeutic efficacy was associated with a systemic immune response with memory and antigen specificity, required CD4 cells and involved CD8 cells and NK cells to a less extent. The 3 mAb combination significantly decreased CD19 cells at tumor sites, increased IFN-γ and TNF-α producing CD4 and CD8 T cells and mature CD86 dendritic cells (DC), and it increased the ratios of effector CD4 and CD8 T cells to CD4Foxp3 regulatory T (Treg) cells and to CD11bGr-1 myeloid suppressor cells (MDSC). This is consistent with shifting the tumor microenvironment from an immunosuppressive Th2 to an immunostimulatory Th1 type and is further supported by PCR data. Adding an anti-CD19 mAb to the 3 mAb combination in the SW1 model further increased therapeutic efficacy. Data from ongoing experiments show that intratumoral injection of a combination of mAbs to CD137PD-1CTLA4CD19 can induce complete regression and dramatically prolong survival also in the TC1 carcinoma and B16 melanoma models, suggesting that the approach has general validity.

Silencing of the TGF-ß1 gene increases the immunogenicity of cells from human ovarian carcinoma.

Cells from many tumors produce transforming growth factor (TGF)-ß which facilitates their escape from control by the immune system. We previously reported that nonimmunogenic cells from either of 2 transplantable mouse tumors became effective as therapeutic tumor vaccines after lentivirus-mediated shRNA interference to "silence" the TGF-ß1 gene. We now show that cells from in vitro cultured human ovarian carcinomas (OvC) make large amounts of TGF-ß1 and that this can be prevented by "silencing" the TGF-ß1 gene. We further show that in vitro sensitization of peripheral blood mononuclear cells in the presence of either mitomycin-treated OvC cells whose TGF-ß1 gene was silenced or in vitro matured dendritic cells that had been pulsed with homogenates from OvC cells with silenced TGF-ß1 generated a stronger Th1/Tc1 immune response to the respective wild-type OvC and also to the OvC antigens mesothelin and HE4 as measured by ELIspot assays. The percentage of interferon-γ and tumor necrosis factor-α-producing CD4+ and CD8+ T cells increased while there were fewer cells expressing markers characteristic for regulatory T cells or myeloid-derived suppressor cells. Similar results were obtained when peripheral blood mononuclear cells from a patient with OvC were sensitized to dendritic cells pulsed with homogenate from autologous TGF-ß1-silenced tumor cells, and a cytolytic lymphocyte response was generated to autologous OvC cells. Our results support clinical evaluation of TGF-ß1-silenced tumor vaccines for immunotherapy of OvC.