Depleted of CD19 cells by the 4 mAb combination.

Expression of surface molecules in TIL of mice receiving mAb combinations.

Expression of surface molecules in TLN and spleen of mice receiving the mAb treatments.

Figure legends for four supplementary figures.

Lymphocyte components of mice transplanted with tumor cells.

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.

We have developed an in vitro system to predict the in vivo response when treating cancer by administering monoclonal antibodies (mAbs) to checkpoint inhibitors (Dai et al., J Immunother 39(8):298-305, 2016). In this article we review our major findings could fulfill a clinical need.

Several decades ago we published some of the first papers showing that both murine and human cancers are recognized in vitro as immunologically foreign and that this is the case also in the presence of a growing tumor. The latter situation, sometimes referred to as the Hellstrom paradox, implies that the tumor is protected in vivo by a highly immunosuppressive environment. After many disappointments, the discovery that tumor-related immunosuppression can be counteracted by administrating monoclonal antibodies (mAbs) to checkpoint inhibitors such as CTLA-4, PD-1, and PD-L1 is now revolutionizing cancer therapy. Over the past several years we have applied mouse models in attempts to further improve the ability of such mAbs to cause long-term complete tumor rejection. This review is focused on that work and emphasizes that successful immunotherapy is associated with a shift from a tumor-promoting Th2 inflammation to a tumor-inhibiting Th1 response.

•Binding affinity of the 4 mAbs to the HE4 biomarker was determined.•Studied 2 mAbs bind to the C-terminal end and 2 to the N-terminal.•mAbs with similar affinity differed in their binding was confirmed in IHC and ICC.•Correlation between HE4 expressed intracellularly and detected in cultured media.

While immunomodulatory monoclonal antibodies (mAbs) have therapeutic efficacy against many tumors, few patients are cured. Attempting to improve their therapeutic efficacy we have applied the TC1 mouse lung carcinoma model and injected established subcutaneous tumors intratumorally with 3 weekly doses of various combinations of mAbs. Combinations of mAbs to CTLA4/PD1/CD137 (the 3 mAb combination) and to CTLA4/PD1/CD137/CD19 (the 4 mAb combination) were most efficacious to induce complete regression of both the injected tumor and an untreated tumor in the same mouse. Tumor cure was consistently associated with shifting a Th2 to a Th1 response in tumor-draining lymph nodes and spleen and it involved epitope specific and long-lived memory T cells as well as M1 macrophages. This shift and accompanying tumor rejection was harder to achieve as the treated tumors increased in size. Relapse of tumors which had initially regressed following treatment with immunomodulatory mAbs was associated with return of a Th2 microenvironment in tumors, tumor-draining lymph nodes and spleens rather than the emergence of immune-resistant tumor cells. While mAbs to CTLA4 plus PD-1 were therapeutically ineffective, combining the 2 of them with intraperitoneal cisplatin, 10mg/kg, induced long-term complete tumor regression in most mice with small TC1 tumors and the therapeutic efficacy against larger tumors improved by administrating cisplatin together with the 3 or 4 mAb combination.

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Infertility is a risk factor for ovarian cancer (OvCa). The goal was to determine if antibodies to selenium-binding protein 1 (SBP1), an autoantibody we identified in patients with premature ovarian failure (POF), occurs in both infertility and OvCa patients, and thus could be associated with preneoplasia. Anti-SBP1 was measured by immunoassay against recombinant SBP1, in sera from OvCa (n = 41), infertility (n = 92) and control (n = 87) patients. Infertility causes were POF, unexplained, irregular ovulation or endometriosis. The percent of anti-SBP1-positive sera was higher in POF (P = 0.02), irregular ovulation (P = 0.001), unexplained causes (P = 0.02), late (III-IV)-stage OvCa (P = 0.02) but was not significant in endometriosis, benign ovarian tumors/cysts, early stage (I-II) OvCa or uterine cancer compared to healthy controls. Anti-SBP1 was significantly higher in women with serous (P = 0.04) but not non-serous (P = 0.33) OvCa compared to controls. Also, we determined if anti-SBP1 was associated with CA125 or anti-TP53, markers often studied in OvCa. Anti-TP53 and CA125 were measured by established immunoassays. The ability of anti-SBP1 alone to discriminate infertility or OvCa from controls or when combined with anti-TP53 and CA125, to identify OvCa was evaluated by comparing the area under the curve (AUC) in ROC analysis. Anti-SBP1 alone discriminated infertility (AUC = 0.7; P = 0.001) or OvCa (AUC = 0.67; P = 0.03) from controls. The sensitivity and specificity of OvCa identification was increased by combining CA125, anti-TP53 and anti-SBP1 (AUC = 0.96). Therefore, anti-SBP1 occurs in infertile women with POF, ovulatory disturbances or unexplained infertility and in serous OvCa. This suggests an autoimmune process is associated with the development of serous OvCa.

Over the past several years remarkable therapeutic responses have been obtained with immunomodulatory monoclonal antibodies (mAbs), both in mice \cite{10,18,20,48,54,61} and human cancer patients \cite{1,3,14,28,30,39,80}. However, complete regressions and cures are infrequent and not predictable and some tumor types respond much worse than others. As an attempt to increase curability, we have investigated in mouse models the therapeutic efficacy of several mAb combinations, focusing on anti-PD-1/CTLA-4/CD137 and anti-PD-1/CTLA-4/CD137/CD19, and we have also combined mAbs with the chemotherapeutic drug cisplatin. Our data demonstrate an important contribution of anti-CD19 mAb to therapeutic efficacy, they show that intratumoral delivery of the mAbs is therapeutically more effective than systemic delivery, and that there is synergy when the mAbs are combined with cisplatin. In an attempt to improve predictability, we developed an in vitro model that may also be employed to search for novel immunomodulatory agents and combinations. This article reviews our data and discusses what is known about the underlying mechanisms.

The present invention relates to methods which utilize anti-idiotypic antibodies, or fragments thereof, for tumor immunotherapy or immunoprophylaxis. Mono clonal anti-idiotypic antibodies which recognize an idiotype present on a second antibody or on a T lym phocyte or on an immune suppressor factor which is directed against a defined tumor antigen, can be used for immunization against a tumor, for immune anti tumor activation or inhibition of suppression, or for in vitro activation of lymphocytes to be used in adoptive immunotherapy. The anti-idiotypic antibodies, or frag ments thereof, can also be used to monitor anti-antibody induction in patients undergoing passive immunization to a tumor antigen by administration of anti-tumor anti body. In another embodiment, administration of T lym phocytes which express an idiotype directed against a defined tumor antigen can be used to transfer delayed type hypersensitivity to the tumor. In another method of the invention, the induction of anti-idiotypic antibod ies in vivo by administration of anti-tumor antibody or immune cells or factors exhibiting an anti-tumor idio type can be therapeutically valuable.

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.

Culture supernatants of spleen or thymus cells from BALB/c mice bearing transplanted, syngeneic, methyl- cholanthrene-induced sarcomas suppress T lymphocyte-me- diated lysis of cells from the tumor orne by the donor of the spleen or thymus cells. On this basis, we hybridized thymus cells from mice bearing sarcoma MCA-1490 with cells from the T lymphoma BW5147. The hybrids (hybridomas) formed were tested for production of factors that could suppress T lympho- cyte-mediated lysis of MCA-1490 cells. One hybridoma, and a clone derived from it, produced factors that suppressed the lysis of MCA-1490 cells in vitro. In addition, these factors enhanced the growth of MCA-1490 in immune mice and prevented the destruction of MCA-1490 cells by immune lymphocytes in tumor neutralization (Winn) assays. In vitro lysis of cells from another MCA-induced sarcoma by immune lymphocytes was not sup- pressed. The suppressor factors did not affect the proliferative response of BALB/c lymphocytes to mitogens or the generation of a cytotoxic response to C57BL/6 alloantigens. Neither did they inhibit the generation of primary or secondary cytotoxic responses to murine leukemia virus-related antigens present on a BALB/c lymphoma line, LSTRA. Although our findings suggest that these suppressor factors are specific for MCA-1490, their specificity for antigens restricted to this tumor needs fur- ther definition.

The HE4 protein is overexpressed in ovarian carcinomas and can be detected in serum by an ELISA with sensitivity similar to CA125 and higher specificity for malignant disease. We now demonstrate that HE4 can also be detected in the urine at a specificity level of 94.4%, including 13/15 (86.6%) with stage I/II and 57/64 (89.0%) with stage III/IV disease and including 90.5% of patients with serous ovarian carcinoma. Assaying serum and urine from the same patients showed similar sensitivity. Our data indicate that measuring HE4 in urine may aid diagnosis and the monitoring of response to therapy.

Abstract Although surgical debulking plus platinum-based chemotherapy induces clinical remission in 70+ % of patients with advanced ovarian carcinoma, most of these patients relapse and novel treatments are urgently needed. Although clinical trials of various immunotherapies have not yielded significant benefit for ovarian cancer, the failures can now be better understood as an outcome of the highly immunosuppressive tumor microenvironment so that one needs to both overcome this immunosuppression and concomitantly activate tumor-destructive immunological mechanisms. One way to achieve this is to use monoclonal antibodies (mAbs) to molecules which regulate the immune response. Antibodies blocking the co-inhibitory molecules CTLA-4 or PD-1 have already demonstrated encouraging clinical activity, particularly in melanoma, and the combination of the two mAbs has improved efficacy with long-lasting remission in some patients. We have evaluated the antitumor efficacy of mAbs against multiple co-signaling molecules, both individually and in combinations, in the ID8 syngeneic mouse model of ovarian cancer. In this model, small intraperitoneal tumor nests appear about 10 days after implantation and solid tumors as well as malignant ascites develop leading to death approximately 30 days after transplantation. Intraperitoneal injections of single mAb antagonists against co-inhibitory receptors (PD-1, CTLA-4, TIM-3, and LAG-3) or agonistic mAb targeting co-stimulatory molecules (CD137, OX40, GITR, and CD40) in mice that had been transplanted 10 days earlier with ID8 cells were ineffective. However, injection of a combination of anti-CD137 and anti-PD-1 mAbs significantly prolonged survival, and addition of a mAb to CTLA-4 further enhanced the antitumor efficacy with some mice surviving tumor-free >150 days. The mAb combination greatly increased the number of tumor-associated CD8+ effector T cells in the peritoneal cavity and decreased the number of immunosuppressive Treg and MDSC cells with a prominent increase in the T effector to T suppressive cell ratios, and it rescued the function of exhausted T cells with a PD-1+TIM3+ or PD-1+TIM3– phenotype. Importantly, pretreatment of the ID8-bearing mice with cisplatin, a drug routinely used to treat ovarian carcinoma, synergized with the anti-CD137/anti-CTLA4 mAb combination for a long-lasting and complete tumor regression in 80% of the mice. Our findings, as well as results from preclinical studies and clinical trials at many centers, strengthen the concept of targeting co-stimulatory and co-inhibitory molecules by immunomodulatory mAbs for cancer therapy. A variety of such receptors is known, and their significance as therapeutic targets, individually or in combinations, merits preclinical characterization and clinical testing of the best candidates. For example, we have recently shown that a combination of mAbs to CD137+PD1+CTLA4+CD19 is superior to the anti-CD137+PD1+CTLA4 combination when tested in mouse melanoma and lung carcinoma models and causes permanent regression of melanomas with a surface area of 80-100 mm2 at the onset of treatment, and we are now evaluating this and other combinations in the ID8 model. Although we have not observed significant toxicity, caution must be exercised since CD137 agonistic antibodies have been observed to cause liver toxicity in clinical trials. Restricting the biodistribution of mAbs for treating ovarian carcinoma to the peritoneal cavity and entrapping them in nanoparticles, may favor efficacy and minimize systemic toxicity. “Translation” of the preclinical findings to clinical trials in ovarian cancer patients should be seriously considered. Citation Format: Min Dai, Ingegerd Hellstrom, Yuen Yee Yip, Huafeng Wei, Karl Erik Hellstrom. Immunomodulatory antibodies for immunotherapy of ovarian cancer [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr AS24.

Vaccination with sipuleucel-T produced IgG antibodies to secondary prostatic carcinoma antigens and prolonged survival in some patients, and assaying for antibodies may provide prognostic information and identify new vaccine targets. Additional approaches to improve T-cell responses are needed to improve the clinical efficacy. Clin Cancer Res; 21(16); 3581–3. ©2015 AACR. See related article by GuhaThakurta et al., p. 3619