PD-1/PD-L1 checkpoint inhibitors in combination with olaparib display antitumor activity in ovarian cancer patient-derived three-dimensional spheroid cultures.

Immune checkpoint inhibitors (ICIs) that target programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) have shown modest activity as monotherapies for the treatment of ovarian cancer (OC). The rationale for using these therapies in combination with poly (ADP-ribose) polymerase inhibitors (PARP-Is) has been described, and their in vivo application will benefit from ex vivo platforms that aid in the prediction of patient response or resistance to therapy. This study examined the effectiveness of detecting patient-specific immune-related activity in OC using three-dimensional (3D) spheroids. Immune-related cell composition and PD-1/PD-L1 expression status were evaluated using cells dissociated from fresh OC tissue from two patients prior to and following 3D culture. The patient sample with the greatest increase in the proportion of PD-L1 + cells also possessed more activated cytotoxic T cells and mature DCs compared to the other patient sample. Upon cytokine stimulation, patient samples demonstrated increases in cytotoxic T cell activation and DC major histocompatibility complex (MHC) class-II expression. Pembrolizumab increased cytokine secretion, enhanced olaparib cytotoxicity, and reduced spheroid viability in a T cell-dependent manner. Furthermore, durvalumab and olaparib combination treatment increased cell death in a synergistic manner. This work demonstrates that immune cell activity and functional modulation can be accurately detected using our ex vivo 3D spheroid platform, and it presents evidence for their utility to demonstrate sensitivity to ICIs alone or in combination with PARP-Is in a preclinical setting.

Prospective Validation of an Ex Vivo, Patient-Derived 3D Spheroid Model for Response Predictions in Newly Diagnosed Ovarian Cancer.

Although 70-80% of newly diagnosed ovarian cancer patients respond to first-line therapy, almost all relapse and five-year survival remains below 50%. One strategy to increase five-year survival is prolonging time to relapse by improving first-line therapy response. However, no biomarker today can accurately predict individual response to therapy. In this study, we present analytical and prospective clinical validation of a new test that utilizes primary patient tissue in 3D cell culture to make patient-specific response predictions prior to initiation of treatment in the clinic. Test results were generated within seven days of tissue receipt from newly diagnosed ovarian cancer patients obtained at standard surgical debulking or laparoscopic biopsy. Patients were followed for clinical response to chemotherapy. In a study population of 44, the 32 test-predicted Responders had a clinical response rate of 100% across both adjuvant and neoadjuvant treated populations with an overall prediction accuracy of 89% (39 of 44, p < 0.0001). The test also functioned as a prognostic readout with test-predicted Responders having a significantly increased progression-free survival compared to test-predicted Non-Responders, p = 0.01. This correlative accuracy establishes the test's potential to benefit ovarian cancer patients through accurate prediction of patient-specific response before treatment.

Combined treatment of dendritoma vaccine and low-dose interleukin-2 in stage IV renal cell carcinoma patients induced clinical response: A pilot study.

Vaccination using dendritic/tumor cell hybrids represents a novel and promising cancer immunotherapy. We have developed a technology that can instantly purify the hybrids (dendritomas) from the fusion mixture of dendritic cells (DCs) and tumor cells. Our animal studies and a phase I study of stage IV melanoma patients demonstrated that dendritoma vaccination could be conducted without major toxicity and induced tumor cell-specific immunological and clinical responses. In this pilot study, ten stage IV renal cell carcinoma patients were studied. Dendritomas were made from autologous DCs and tumor cells and administered by subcutaneous injection. After initial vaccination, three escalating doses of IL-2 (3, 6, and 9 million units each) were followed within five days. This treatment regimen was tolerated well without severe adverse events directly related to the dendritoma vaccine. Most adverse events were related to IL-2 administration or pre-existing disease. Patient-specific immune responses were evaluated by flow cytometric measurement of interferon-gamma-producing T-cells before and after vaccination in response to stimulation with tumor antigens. Nine out of nine patients eligible for the analysis showed an increase of IFN-gamma-expressing CD4+ T cells after vaccination(s); while five out of eight patients eligible for the analysis showed an increase of IFN-gamma-expressing CD8+ T cells. Clinical responses were documented in 40% of the patients, three with stabilization of disease and one with a partial response documented by a reduction in tumor size. This pilot study demonstrated that dendritoma vaccines could be administered safely to patients with metastatic renal cell carcinoma, while producing both clinical and immunologic evidence of response.

Dendritoma vaccination combined with low dose interleukin-2 in metastatic melanoma patients induced immunological and clinical responses.

A pilot clinical trial using dendritomas, purified hybrids from the fusion of dendritic/tumor cells combined with a low dose of IL-2, in metastatic melanoma patients was conducted in order to determine its safety and potential immunological and clinical responses. Ten metastatic melanoma patients were enrolled into this study. Dendritoma vaccines were created by fusing dendritic cells stained with green fluorescent dye with irradiated autologous tumor cells stained with red fluorescent dye and purifying the hybrids using immediate fluorescent-activated cell sorting. Initial vaccine was given subcutaneously and followed by IL-2 in serially elevated doses from 3-9 million units/m2 for 5 days. Repeated vaccinations were administered without IL-2, at 3-month intervals for a maximum of 5 times. Immune reactions were measured by the increase of interferon-gamma (IFN-gamma) expressing T cells. Vaccine doses ranged from 250,000 to 1,000,000 dendritomas. There was no grade 2 or higher toxicity directly attributable to the vaccine. All patients experienced toxicity due to IL-2 administration (9-grade 2, 3-grade 3, 1-grade 4). Eight of nine evaluable patients demonstrated immunologic reactions by increased IFN-gamma expressing T cells. One patient developed partial response at 12 weeks after the first vaccine. Nine months later, this patient achieved a complete response. In addition, two patients had stable disease for 9 and 4 months, respectively; one patient had a mixed response. Our findings demonstrated that dendritoma vaccines with a low dose of IL-2 can be safely administered to patients with metastatic melanoma and induce immunological and clinical responses.

Inhibition or promotion of tumor growth by granulocyte-macrophage colony stimulating factor derived from engineered tumor cells is dose-dependent.

BACKGROUND: Granulocyte-macrophage colony stimulating factor (GM-CSF) has been widely investigated as an adjuvant factor for tumor immunotherapy. However, the results are controversial with antitumor effects in some studies and a tumor growth promotion effect in others. MATERIALS AND METHODS: In order to determine whether there is a dose-dependent effect of GM-CSF on tumor growth, murine GM-CSF-expressing vector was constructed and transfected into TC-1 tumor cells and various clones stably expressing different levels of GM-CSF were obtained. The growth of these clones in vivo was studied. RESULTS: Although these clones grow at a similar rate in vitro, their growth in vivo is dramatically different. Clones expressing high levels (>10,000 pg/ml) of GM-CSF grow significantly faster than the control (p <0.001); clones expressing low levels (<100 pg/ml) of GM-CSF grow significantly slower than the control (p<0.001); while clones expressing intermediate levels (1000-2000 pg/ml) of GM-SCF grow at a similar rate as the control (p >0. 05). The high levels of GM-CSF secreted by tumor cells induced granulocytosis and lymphopenia. The antitumor growth effect induced by low levels of GM-CSF is not due to the function of lymphocytes. CONCLUSION: The inhibition or promotion of tumor growth by GM-CSF secreted from tumor cells is dose-dependent.

Synergistic anti-tumor effect of glycosylphosphatidylinositol-anchored IL-2 and IL-12.

BACKGROUND: Preclinical and clinical studies have demonstrated that interleukin 2 (IL-2), interleukin 12 (IL-12), and some other cytokines, play important roles in activating host immune responses against tumor growth. However, severe side effects caused by systemic high-dose administration of these cytokines limit their clinical application. In our previous study, local high doses of IL-2 were achieved by a GPI-anchoring technology; therefore, it will be interesting to know if this technology works for other cytokines. METHODS: A fusion gene containing murine IL-12 and the glycosylphosphatidylinositol (GPI) anchor signal sequence was generated and transfected into the murine melanoma tumor cell line B16F0 either alone or together with a vector encoding GPI-anchored IL-2. The GPI-anchored cytokine expression of the selected stable clones was assayed in vitro by ELISA and their anti-tumor effects were analyzed in vivo by tumor lymphocyte infiltration and tumor growth studies. RESULTS: GPI-anchored IL-12 was successfully expressed on the cell surface as indicated by FACS analysis and IL-12 ELISA assay. The GPI-anchored IL-12 enhanced lymphocyte infiltration and significantly inhibited tumor growth. More importantly, when GPI-anchored IL-12 and GPI-anchored IL-2 were co-delivered, a synergistic anti-tumor effect was observed in both subcutaneous and intravenous tumor models. CONCLUSIONS: GPI anchorage of cytokines represents a new approach to locally deliver high doses of cytokines without the severe adverse effects normally accompanied with systematic high-dose administration of these cytokines.

Fusion protein from RGD peptide and Fc fragment of mouse immunoglobulin G inhibits angiogenesis in tumor.

Targeting tumor vasculature represents an interesting approach for the treatment of solid tumors. The alpha v beta 3 integrins have been found to be specifically associated with angiogenesis in tumors. By using bacteriophage display technology, Ruoslahti et al found that a group of peptides containing the RGD (Arg-Gly-Asp) motif have high-binding affinity to the alpha v beta 3 integrins in tumors. In this study, we designed a fusion protein containing the RGD sequence and the Fc fragment of mouse IgG in order to target the Fc portion of IgG to the tumor vasculature to elicit an antiangiogenesis immune response. In vivo angiogenesis and tumor studies demonstrated that the fusion protein (RGD/mFc) inhibited tumor angiogenesis and tumor growth and improved overall survival. This approach may generate new therapeutic agents for solid tumor treatment.

Targeting foreign major histocompatibility complex molecules to tumors by tumor cell specific single chain antibody (scFv).

Down-regulation of the major histocompatibility complex (MHC) is one of the major mechanisms that tumor cells adopted to escape immunosurveillance. Therefore, specifically coating tumor cells with foreign MHC may make tumor cells a better target for immune recognition and surveillance. In this study, we designed and generated a fusion protein, H2Kd/scPSMA, consisting of a single chain antibody against human prostate specific membrane antigen (PSMA) and the extracellular domain of mouse H-2Kd. The expression of this fusion protein in B16F0 mouse melanoma cells was confirmed by RT-PCR and fluorescent activated cell sorting (FACS). Our animal study showed that the expression of H2Kd/scPSMA in B16F0/PSMA5, a B16F0 cell line expressing human PSMA, significantly inhibited tumor growth as demonstrated in the pulmonary metastasis assay and tumor growth study and improved overall survival.

Glycoinositol phospholipid-anchored interleukin 2 but not secreted interleukin 2 inhibits melanoma tumor growth in mice.

Whereas cancer immunotherapy with interleukin (IL) 2 and/or other cytokines has proved effective in activating immune responses against tumor cells, the major obstacle with the use of these cytokines in cancer patients is their severe side effects when delivered systemically at high doses. In an effort to overcome this problem, in the present study, a fusion protein containing human IL-2 and a glycoinositol phospholipid (GPI) anchor sequence of decay accelerating factor was generated. When expressed by transfected cells, these fusion proteins were presented on the cell surface in the GPI-anchored form as demonstrated by fluorescence-activated cell sorter and ELISA analyses. This GPI-anchored IL-2 is highly functional as indicated by significantly increased T-cell infiltration in tumor masses. Immunohistochemical analysis of tumor cells isolated from experimental tumors indicated that a local high level of IL-2 was achieved by GPI-anchored IL-2. More importantly, when injected into mice i.v., the growth of these B16F0 melanoma cells that were engineered to express this fusion protein was significantly inhibited. In contrast, the inhibition of secreted IL-2 on tumor growth was not observable in this study. These studies may provide a novel approach to locally deliver high doses of cytokines for cancer immunotherapy.