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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 mm2. 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. Clin Cancer Res; 21(5); 1127–38. ©2014 AACR.See related commentary by Dronca and Dong, p. 944

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.

Depleted of CD19 cells by the 4 mAb combination.

Figure legends for four supplementary figures.

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.

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, 10 mg/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.

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.

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.

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.

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 mm2. 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. Clin Cancer Res; 21(5); 1127–38. ©2014 AACR. See related commentary by Dronca and Dong, p. 944

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.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%).Infertile women more frequently had anti-HE4 antibodies than controls (23% at 98% specificity, p0.001) 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, p0.01), 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.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.

INTRODUCTION: There is a need to improve the diagnosis and prognosis of ovarian carcinoma, particularly the serous type of cancer. Mesothelin and HE4 are two novel biomarkers which are expressed in serous ovarian carcinoma and can be measured in serum and other body fluids, including urine, by using ELISA. The measurement of antibodies to these markers can provide additional useful information. AREAS COVERED: A literature search was performed to as recent as December 1 2010, using the Internet (e.g. PubMed) on articles concerning mesothelin and HE4 for the diagnosis of ovarian carcinoma. The authors also included up to date recent information from the research performed in their own laboratory. EXPERT OPINION: The combination of CA125 with HE4 facilitates the triaging of women with a pelvic mass and detects more stage I/II tumors than CA125, the present 'gold standard', when used alone. Assaying urine for HE4 or mesothelin may detect early ovarian carcinoma more often than assaying serum. Antibodies to mesothelin and HE4 are more frequent in women with ovarian carcinoma or with certain types of infertility than in controls. No presently available biomarker or multi-marker panel lends itself to screening large populations of symptomless women to make possible detection of high grade serous ovarian carcinoma at stage I or II. The authors anticipate, within the next 5 years, that a greater emphasis will be given to the fact that the different subtypes of ovarian carcinoma represent different types of disease. Each different type of disease will require a different diagnostic approach and more efforts will focus on high grade serous ovarian carcinoma for which the clinical need is the greatest.

Background. There has been little success in using radioimmunotherapy in patients with adenocarcinoma, partly because of the low tumor uptake of the administered monoclonal antibody (MoAb). The authors recently reported therapeutic response in advanced cancer patients who received 131 I chimeric-L6 MoAb. The L6 MoAb identifies abundant, nonshed antigen that is expressed in many human carcinomas, including carcinomas of the lung, breast, colon, and ovary. In vitro both mouse L6 (L6) and chimeric L6 (ChL6) mediate complement-dependent tumor cytolysis with human serum, and antibody-dependent tumor cell cytolysis with normal human peripheral blood mononuclear cells

L-6, a mouse IgG2a anti-adenocarcinoma monoclonal antibody (MoAb) with favorable immunopathology and mouse biokinetics, was evaluated for cancer radioimmunotherapy by pharmacokinetic studies in 10 patients with breast cancer. The effect of escalating the preinfused protein dose was studied in two patients at each level, using 50, 100, 150, 200 and 400 mg of unlabeled L-6 prior to a 10 mCi imaging dose of 131I L-6. Quantitative imaging, and blood and urine clearances were obtained. After the 50 mg preinfusion, rapid blood clearance and lung extraction of the radiopharmaceutical occurred immediately post injection. Greater preload amounts of L-6 were associated with an increase in the intercept of the slow phase of the blood clearance from 17 to 22% injected dose (ID) with 50 mg to 70 to 80% ID with 400 mg (P less than 0.01). Lung uptake of the radiopharmaceutical immediately post injection decreased from 15 to 19% ID (50 mg) to 6 to 8% ID (400 mg). Tumors were visualized only after larger L-6 preloads, but in these patients small chest tumors contained 0.6-1.2% ID (0.1% ID/g maximum). This study suggests that L-6 reactive sites that are readily available in the lung can be saturated, so that a subsequent dose of I-131 L-6 is delivered to the tumor. This approach provides a new strategy for developing an effective method for radioimmunotherapy using a MoAb that has some cross-reactivity. Quantitative imaging contributed to detection of the cross-reactivity and the strategy for overcoming it.

Assays measuring SMRP (mesothelin) and HE4 (a secreted protease) in serum and other body fluids (including urine for SMRP) are likely to be clinically useful for patients with ovarian cancer, as data indicate that they complement CA125 for diagnosis and monitoring of patients. Both markers have temporal stability, as does CA125, which may be utilized to facilitate earlier diagnosis by performing longitudinal studies on high risk subjects. Preliminary data show autoantibodies to native mesothelin in some patients with ovarian carcinoma and in some healthy women. We are presently studying their relationship to the patients' clinical state to learn whether measurements of antibody levels provide information that can aid diagnosis and monitoring of treated patients. Prospective studies are needed to establish the clinical relevance of our findings.

We report that antibodies can be spontaneously loaded in functionalized mesoporous silica (FMS) with superhigh density (0.4−0.8 mg of antibody/mg of FMS) due to their comprehensive noncovalent interaction. The superhigh loading density and noncovalent interaction between FMS and antibodies allow long-lasting local release of the immunoregulatory molecules from FMS under physiological conditions. Preliminary data indicate that FMS-anti-CTLA4 antibody injected directly into a mouse melanoma induces much greater and extended inhibition of tumor growth than the antibody given systemically. Our findings open up a novel approach for local delivery of therapeutically active proteins to tumors and, potentially, other diseases.

Background and objectives of the study: Many cancers, including ovarian cancer, over-express transforming growth factor-beta (TGF-ß), which helps them evade immune control through a variety of mechanisms. We investigated whether the efficacy of tumor cells to induce an immune response, when used intact or as part of a dendritic cell (DC)-based vaccine can be enhanced by inhibiting their production of TGFß1 via anti-sense. Methodology: The TGFß1 expression by two ovarian cancer cell lines was silenced by transfection with a non-replicating lentivirus shRNA. Autologous PBMC were co-cultivated, in the presense of IL-2, with Mitomycin-C (MMC) treated cancer cells or mature dendritic DC that had been pulsed with tumor lysates. The in vitro sensitized lymphoid cells were evaluated by ELISPOT for production of IFN-γ and by flow cytometry. Preliminary data: Quanitative ELISA showed that TGFß1 production in the two ovarian cancer lines was signficantly inhibited by transfection with shRNA. In vitro sensitization of autologous PBMC with either tumor cells where the TGFß1 gene had been silenced or with DC that had been pulsed with homogenates of tumor cells with a silencd TGFß1 gene induced a stronger IFNγ release and increased the frequency of NK cells and DC while it decreased the population of Treg cells. We are presently investigating the anti-tumor response in a mouse melanoma model after vaccination with murine bone-marrow derived DC which have benn pulsed with TGFß silenced tumor lysates. Conclusions: The production, by many tumors, of TGFß1 interferes with their ability to induce an anti-tumor response. This is true both when intact tumor cells and DC pulsed with tumor homogenate are used as immunogen. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-320.

Many monoclonal antibodies (Mabs) have been created by immunizing mice with human carcinomas. In the vast majority of cases, these Mabs define tumor-associated differentiation antigens (TADAs), which have relative but not absolute specificity for neoplastic cells (Reisfeld, 1986; Roth, 1986; Sikorska, Shuster, and Gold, 1988; Bast, 1989; K. E. Hellstrom and Hellstrom, 1989a). Although more tumor-specific Mabs would be preferable, many potential therapeutic uses of the Mabs are already available, and several have been tested clinically and given promising results.

131I-labeled monoclonal antibodies were used in therapeutic trials in two potentially useful clinical situations: disseminated melanoma (intravenously administered Fab fragments; 21 patients) and disseminated peritoneal adenocarcinomatosis (intraperitoneal injection of IgG; 5 patients). Acute toxicity observed is consistent with mild bone marrow suppression of acute radiation syndrome and the observed toxicity is dose related in a manner that conforms to the expected human response to total body irradiation. For single doses of both i.p. administered and intravenously administered 131I-labeled anti-tumor antibodies, 100 rad to red marrow, calculated by the absorbed dose fraction method (MIRD), appeared to be a threshold below which significant acute toxicity was unlikely.

Publisher Summary This chapter explains cytotoxic lymphocytes, blocking serum factors, and unblocking antibodies in cancer patients. The majority of tumors in experimental animals have tumor-specific antigens, which can be detected with a variety of techniques. Both humoral and cell-mediated immune reactions against tumors have been identified. A major part of the immunological defense against cancer is cell-mediated as is apparent also from the fact that animals (or human patients) whose cell-mediated immunity is depressed are more prone to develop cancers than are immunologically capable individuals. Some studies have indicated that murine bladder tumors may provide good animal models: bladder papillomas and carcinomas of the rat cross-react antigenically, also when they have been induced by different carcinogenic agents, and they behave similarly, therefore, to bladder carcinomas of man. Chemically induced bladder carcinomas of mice also share a common antigen. According to preliminary studies in rats, it is possible to delay the appearance of primary chemically induced bladder papillomas by immunizing the animals with bladder tumor tissue before the chemical carcinogen is applied. These studies indicate that an immunological reaction against common bladder tumor antigens may be beneficial to the host.

Publisher Summary This chapter describes the colony inhibition technique and the cytotoxic test. The colony inhibition technique has a high sensitivity and reproducibility, while the cytotoxic test has the additional advantage that it needs small amounts of material and modest skill for the final counting. Method for colony inhibition test was first worked out to study cytotoxic humoral antibodies. Test for humoral cytotoxic antibody involves trypsinizing the target cells adhering to the bottle mildly and briefly. Next step involves adding a few ml of calf serum or medium with calf serum to inactivate the trypsin. One should add 0.1 ml of the serum being tested for cytotoxic antibody to all the wells except those in one column, which will serve as controls. This serum may be diluted as desired in complete Waymouth's medium.