Resiquimod as an immunologic adjuvant for NY-ESO-1 protein vaccination in patients with high-risk melanoma.

The Toll-like receptor (TLR) 7/8 agonist resiquimod has been used as an immune adjuvant in cancer vaccines. We evaluated the safety and immunogenicity of the cancer testis antigen NY-ESO-1 given in combination with Montanide (Seppic) with or without resiquimod in patients with high-risk melanoma. In part I of the study, patients received 100 µg of full-length NY-ESO-1 protein emulsified in 1.25 mL of Montanide (day 1) followed by topical application of 1,000 mg of 0.2% resiquimod gel on days 1 and 3 (cohort 1) versus days 1, 3, and 5 (cohort 2) of a 21-day cycle. In part II, patients were randomized to receive 100-µg NY-ESO-1 protein plus Montanide (day 1) followed by topical application of placebo gel [(arm A; n = 8) or 1,000 mg of 0.2% resiquimod gel (arm B; n = 12)] using the dosing regimen established in part I. The vaccine regimens were generally well tolerated. NY-ESO-1-specific humoral responses were induced or boosted in all patients, many of whom had high titer antibodies. In part II, 16 of 20 patients in both arms had NY-ESO-1-specific CD4⁺ T-cell responses. CD8⁺ T-cell responses were only seen in 3 of 12 patients in arm B. Patients with TLR7 SNP rs179008 had a greater likelihood of developing NY-ESO-1-specific CD8⁺ responses. In conclusion, NY-ESO-1 protein in combination with Montanide with or without topical resiquimod is safe and induces both antibody and CD4⁺ T-cell responses in the majority of patients; the small proportion of CD8⁺ T-cell responses suggests that the addition of topical resiquimod to Montanide is not sufficient to induce consistent NY-ESO-1-specific CD8⁺ T-cell responses.

MAGE-specific T cells detected directly ex-vivo correlate with complete remission in metastatic breast cancer patients after sequential immune-endocrine therapy.

Studies suggest that conventional cancer therapies given after immunotherapy (IT) can boost antitumor immunity and possibly improve response rates and progression-free survival. We report two cases of metastatic breast cancer with durable complete responses (CRs) after sequential IT and endocrine therapy. Immune analyses of these long-term disease-free breast cancer patients previously treated with imiquimod (IMQ) suggest in-situ vaccination is achieved by topical application of the TLR-7 agonist directly onto tumors. Furthermore, IT-induced antigen-specific T cells were expanded by subsequent endocrine therapy and correlated with response, persisting > 2 years. Our findings therefore suggest that the induction/boosting of polyfunctional tumor antigen-specific T in response to sequential immune endocrine therapy and detected directly ex-vivo can serve as a peripheral blood biomarker for true clinical benefit.

Preparation of tumor antigen-loaded mature dendritic cells for immunotherapy.

While clinical studies have established that antigen-loaded DC vaccines are safe and promising therapy for tumors, their clinical efficacy remains to be established. The method described below, prepared in accordance with Good Manufacturing Process (GMP) guidelines, is an optimization of the most common ex vivo preparation method for generating large numbers of DCs for clinical studies. Our method utilizes the synthetic TLR 3 agonist Polyinosinic-Polycytidylic Acid-poly-L-lysine Carboxymethylcellulose (Poly-ICLC) to stimulate the DCs. Our previous study established that Poly-ICLC is the most potent individual maturation stimulus for human DCs as assessed by an upregulation of CD83 and CD86, induction of interleukin-12 (IL-12), tumor necrosis factor (TNF), interferon gamma-induced protein 10 (IP-10), interleukmin 1 (IL-1), and type I interferons (IFN), and minimal interleukin 10 (IL-10) production. DCs are differentiated from frozen peripheral blood mononuclear cells (PBMCs) obtained by leukapheresis. PBMCs are isolated by Ficoll gradient centrifugation and frozen in aliquots. On Day 1, PBMCs are thawed and plated onto tissue culture flasks to select for monocytes which adhere to the plastic surface after 1-2 hr incubation at 37 °C in the tissue culture incubator. After incubation, the lymphocytes are washed off and the adherent monocytes are cultured for 5 days in the presence of interleukin-4 (IL-4) and granulocyte macrophage-colony stimulating factor (GM-CSF) to differentiate to immature DCs. On Day 6, immature DCs are pulsed with the keyhole limpet hemocyanin (KLH) protein which serves as a control for the quality of the vaccine and may boost the immunogenicity of the vaccine. The DCs are stimulated to mature, loaded with peptide antigens, and incubated overnight. On Day 7, the cells are washed, and frozen in 1 ml aliquots containing 4-20 x 10(6) cells using a controlled-rate freezer. Lot release testing for the batches of DCs is performed and must meet minimum specifications before they are injected into patients.

Effective CpG immunotherapy of breast carcinoma prevents but fails to eradicate established brain metastasis.

PURPOSE: Breast cancer patients with brain metastasis have a dismal prognosis. We determined the ability of immunostimulatory CpG oligodeoxynucleotides (ODN) to treat or prevent brain metastasis in a mouse model. EXPERIMENTAL DESIGN: Mice bearing orthotopic breast carcinoma with or without concurrent i.c. tumors were treated by injections of CpG ODN at the primary tumor. Immunologic memory was tested by tumor rechallenge and immune responses were assessed by flow cytometry, delayed-type hypersensitivity, and CTL assays. RESULTS: Orthotopic tumors regressed in treated mice regardless of whether concurrent i.c. disease was present. In mice bearing only orthotopic tumors, CpG ODN rendered 50% tumor-free and they rejected tumor rechallenge in breast and brain. In mice with concurrent i.c. disease, there was no difference in brain tumor growth compared with saline controls, despite regression of the primary tumor. Flow cytometry revealed that treated mice that died from i.c. disease exhibited a significant increase in brain-infiltrating T and natural killer cells relative to saline controls. CTLs from these mice were able to kill tumor in vitro and extend survival of naive mice bearing less-established brain tumors by adoptive transfer. CONCLUSIONS: The lack of survival benefit in mice with appreciable brain metastasis was not explained by a deficit in lymphocyte trafficking or function because CTLs from these mice killed tumor and inhibited microscopic brain metastasis by adoptive transfer. These results indicate that CpG ODN might be beneficial as a preventative adjuvant to initial therapy preceding brain metastasis or to inhibit progression of microscopic brain metastases.

Combinatorial antiangiogenic gene therapy by nonviral gene transfer using the sleeping beauty transposon causes tumor regression and improves survival in mice bearing intracranial human glioblastoma.

Glioblastoma is a fatal brain tumor that becomes highly vascularized by secreting proangiogenic factors and depends on continued angiogenesis to increase in size. Consequently, a successful antiangiogenic therapy should provide long-term inhibition of tumor-induced angiogenesis, suggesting long-term gene transfer as a therapeutic strategy. In this study a soluble vascular endothelial growth factor receptor (sFlt-1) and an angiostatin-endostatin fusion gene (statin-AE) were codelivered to human glioblastoma xenografts by nonviral gene transfer using the Sleeping Beauty (SB) transposon. In subcutaneously implanted xenografts, co-injection of both transgenes showed marked anti-tumor activity as demonstrated by reduction of tumor vessel density, inhibition or abolition of glioma growth, and increase in animal survival (P = 0.003). Using luciferase-stable engrafted intracranial gliomas, the anti-tumor effect of convection-enhanced delivery of plasmid DNA into the tumor was assessed by luciferase in vivo imaging. Sustained tumor regression of intracranial gliomas was achieved only when statin-AE and sFlt-1 transposons were coadministered with SB-transposase-encoding DNA to facilitate long-term expression. We show that SB can be used to increase animal survival significantly (P = 0.008) by combinatorial antiangiogenic gene transfer in an intracranial glioma model.