Interleukin-2: Old and New Approaches to Enhance Immune-Therapeutic Efficacy.

Interleukin-2 (IL-2) is a very well-known cytokine that has been studied for the past 35 years. It plays a major role in the growth and proliferation of many immune cells such NK and T cells. It is an important immunotherapy cytokine for the treatment of various diseases including cancer. Systemic delivery of IL-2 has shown clinical benefit in renal cell carcinoma and melanoma patients. However, its use has been limited by the numerous toxicities encountered with the systemic delivery. Intravenous IL-2 causes the well-known "capillary leak syndrome," or the leakage of fluid from the circulatory system to the interstitial space resulting in hypotension (low blood pressure), edema, and dyspnea that can lead to circulatory shock and eventually cardiopulmonary collapse and multiple organ failure. Due to the toxicities associated with systemic IL-2, an aerosolized delivery approach has been developed, which enables localized delivery and a higher local immune cell activation. Since proteins are absorbed via pulmonary lymphatics, after aerosol deposition in the lung, aerosol delivery provides a means to more specifically target IL-2 to the local immune system in the lungs with less systemic effects. Its benefits have extended to diseases other than cancer. Delivery of IL-2 via aerosol or as nebulized IL-2 liposomes has been previously shown to have less toxicity and higher efficacy against sarcoma lung metastases. Dogs with cancer provided a highly relevant means to determine biodistribution of aerosolized IL-2 and IL-2 liposomes. However, efficacy of single-agent IL-2 is limited. As in general, for most immune-therapies, its effect is more beneficial in the face of minimal residual disease. To overcome this limitation, combination therapies using aerosol IL-2 with adoptive transfer of T cells or NK cells have emerged.Using a human osteosarcoma (OS) mouse model, we have demonstrated the efficacy of single-agent aerosol IL-2 and combination therapy aerosol IL-2 and NK cells or aerosol IL-2 and interleukin 11 receptor alpha-directed chimeric antigen receptor-T cells (IL-11 receptor α CAR-T cells) against OS pulmonary metastases. Combination therapy resulted in a better therapeutic effect. A Phase-I trial of aerosol IL-2 was done in Europe and proved to be safe. Others and our preclinical studies provided the basis for the development of a Phase-I aerosol IL-2 trial in our institution to include younger patients with lung metastases. OS, our disease of interest, has a peak incidence in the adolescent and young adult years. Our goal is to complete this trial in the next 2 years.In this chapter, we summarize the different effects of IL-2 and cover the advantages of the aerosol delivery route for diseases of the lung with an emphasis on some of our most recent work using combination therapy aerosol IL-2 and NK cells for the treatment of OS lung metastases.

Anti-PD-1 therapy redirects macrophages from an M2 to an M1 phenotype inducing regression of OS lung metastases.

Osteosarcoma (OS) pulmonary metastasis translates into poor patient survival. The implication of PD-1-PD-L1 pathway in the context of NK cells and/or macrophages in OS is unknown. We investigated the effect of anti-PD-1 in OS lung metastasis and the role of NK cells and/or macrophages in anti-PD-1 responses. A human LM7 OS mouse model was used. Immunohistochemistry for tissues (PD-L1, caspase-3, Ki-67, NK cells, macrophages), and Western blotting for OS lung tumors (p-Stat3, p-Erk1/2) was performed. NK and macrophages were assessed using flow cytometry. NK cell and macrophage depletion were conducted using anti-asialo GM1 and clodrosome, respectively. PD-L1 expression was observed in human OS cells and OS patient lung metastases. Anti-PD1 antibody led to a significant decrease in the number of OS lung metastases, enhanced tumor apoptosis, decreased tumor cell proliferation, and p-STAT-3/p-Erk1/2 signaling blockade in OS lung tumors. NK cells and macrophages in OS lung tumors expressed PD-1 and anti-PD1 increased NK cell and macrophage tumor infiltration. Increased numbers of antitumor M1 macrophages and decreased pro-inflammatory M2 macrophages were seen. NK depletion did not affect therapeutic effect of anti-PD-1, suggesting that NK cells were not directly involved. However, macrophage depletion significantly compromised anti-PD1 efficacy, confirming their role in efficacy of anti-PD-1 against OS lung metastasis. Our findings suggest that OS lung metastases regression by anti-PD1 can be attributed to activated tumor M1 macrophages and reduced M2 macrophages. Owing to the co-relation of M1 macrophages with OS patient outcome, we provide a novel mechanism of PD-1 blockade and a basis for future clinical trials for anti-PD-1 antibodies in OS.

Crk II silencing down-regulates IGF-IR and inhibits migration and invasion of prostate cancer cells.

Crk (C10 regulator of kinase) adaptor proteins are highly expressed in many types of human cancers and often contribute to aggressive cancer phenotypes. Crk II, a member of CRK family, has been reported to regulate cell migration and metastasis in breast cancer cells. However, its role in other cancer types has not been reported. In this study, we investigated the molecular function of Crk II in prostate cancer (PCa) cells (CWR-22rv1) in vitro and using a mouse tumor model. Results showed that Crk II knockdown by shRNA-mediated silencing (Crk II-shRNA) in the PCa cells significantly inhibited both cancer cell migration and invasion in cell culture study. Crk II-shRNA cancer cells also significantly decreased colony formation in vitro, but had no significant reduction of tumor volume after 4 weeks of cancer cell xenografting in vivo when compared to the scramble control. Interestingly, Crk II-shRNA cancer cells showed a greatly reduced level of insulin-like growth factor 1 receptor (IGF-1R) and decreased signaling of the IGF-1R/PI3K/Akt axis upon IGF-1 ligand stimulation. A close interaction between Crk II and IGF-1R was demonstrated upon co-immunoprecipitation of IGF-1R with Crk II protein. Further, treatment of cells with either proteosomal degradation or protein synthesis inhibitor showed higher proportion of ubiquitin-associated IGF-1R and faster degradation of IGF-1R in Crk II-shRNA cells in comparison with that in the control cancer cells. Taken together, these data suggest that Crk II plays an important role in the regulation of IGF-1R protein stability and affects downstream of IGF-1R signaling pathways. Therefore, targeting Crk-II can block IGF-1R growth signaling and suppress cancer cell invasion and progression.

A novel therapeutic strategy to rescue the immune effector function of proteolytically inactivated cancer therapeutic antibodies.

Primary and acquired resistance to anticancer antibody immunotherapies presents significant clinical challenges. Here, we demonstrate that proteolytic inactivation of cancer-targeting antibodies is an unappreciated contributor to cancer immune evasion, and the finding presents novel opportunities for therapeutic intervention. A single peptide bond cleavage in the IgG1 hinge impairs cancer cell killing due to structural derangement of the Fc region. Hinge-cleaved trastuzumab gradually accumulated on the surfaces of HER2-expressing cancer cell lines in vitro, and was greatly accelerated when the cells were engineered to express the potent bacterial IgG-degrading proteinase (IdeS). Similar to cancer-related matrix metalloproteinases (MMP), IdeS exposes a hinge neoepitope that we have developed an antibody, mAb2095-2, to specifically target the epitope. In in vitro studies, mAb2095-2 restored the lost antibody-dependent cell-mediated cytotoxicity functionality of cell-bound single-cleaved trastuzumab (scIgG-T). In vivo, mAb2095-2 rescued the impaired Fc-dependent tumor-suppressive activity of scIgG-T in a xenograft tumor model and restored the recruitment of immune effector cells into the tumor microenvironment. More importantly, an Fc-engineered proteinase-resistant version of mAb2095-2 rescued trastuzumab antitumor efficacy in a mouse tumor model with human cancer cells secreting IdeS, whereas trastuzumab alone showed significantly reduced antitumor activity in the same model. Consistently, an Fc-engineered proteinase-resistant version of trastuzumab also greatly improved antitumor efficacy in the xenograft tumor model. Taken together, these findings point to a novel cancer therapeutic strategy to rescue proteolytic damage of antibody effector function by an Fc-engineered mAb against the hinge neoepitope and to overcome cancer evasion of antibody immunity.

Effects of anti-EGFR antibody cetuximab on androgen-independent prostate cancer cells.

AIM: Epidermal growth factor receptor (EGFR) is a novel molecular target for anticancer therapy. This study examined the effects of anti-EGFR antibody cetuximab on two human androgen-independent prostate carcinoma cell lines, Du145 and PC-3. MATERIALS AND METHODS: Cell proliferation was monitored with a trypan blue viability assay. Cell apoptosis and cell cycle profile was evaluated by flow cytometry. The expression of various signaling molecules was examined by Western immunoblotting. RESULTS: Cetuximab (100 microg/ml) caused a significant growth inhibition by inducing cell apoptosis in Du145 cells, but not in PC-3 cells. It caused EGFR down-regulation and inhibited EGFR Tyr-845 autophosphorylation in both Du145 and PC-3 cells. However, EGFR phosphorylation at Tyr-1173 and MAPK 44/42 phosphorylation were inhibited in Du145 cells, but not in PC-3 cells. Cetuximab was not able to inhibit Akt phosphorylation in either prostate cancer cell line. CONCLUSION: Du145 cells only showed a very moderate response to cetuximab whereas PC-3 cells showed resistance. Persistent activation of EGFR downstream signaling likely contributes to cell resistance to cetuximab.