Springer Science and Business Media LLC

Despite the significant efforts to enhance immune reactivity against malignancies the clinical effect of anti-tumor vaccines and cancer immunotherapy is still below expectations. Understanding of the possible causes of such poor clinical outcome has become very important for improvement of the existing cancer treatment modalities. In particular, the critical role of HLA class I antigens in the success of T cell based immunotherapy has led to a growing interest in investigating the expression and function of these molecules in metastatic cancer progression and, especially in response to immunotherapy. In this report, we illustrate that two types of metastatic lesions are commonly generated in response to immunotherapy according to the pattern of HLA class I expression. We found that metastatic lesions, that progress after immunotherapy have low level of HLA class I antigens, while the regressing lesions demonstrate significant upregulation of these molecules. Presumably, immunotherapy changes tumor microenvironment and creates an additional immune selection pressure on tumor cells. As a result, two subtypes of metastatic lesions arise from pre-existing malignant cells: (a) regressors, with upregulated HLA class I expression after therapy, and (b) progressors with resistance to immunotherapy and with low level of HLA class I. Tumor cells with reversible defects (soft lesions) respond to therapy by upregulation of HLA class I expression and regress, while tumor cells with structural irreversible defects (hard lesions) demonstrate resistance to immunostimulation, fail to upregulate HLA class I antigens and eventually progress. These two types of metastases appear independently of type of the immunotherapy used, either non-specific immunomodulators (cytokines or BCG) or autologous tumor vaccination. Similarly, we also detected two types of metastatic colonies in a mouse fibrosarcoma model after in vitro treatment with IFN-γ. One type of metastases characterized by upregulation of all MHC class I antigens and another type with partial IFN-γ resistance, namely with lack of expression of Ld-MHC class I molecule. Our observations may shed new light on the understanding of the mechanisms of tumor escape and might have implications for improvement of the efficacy of cancer immunotherapy.

Therapeutic targeting of melanoma antigens frequently focuses on the melanocyte differentiation or cancer-testis families. Antigen-loss variants can often result, as these antigens are not critical for tumor cell survival. Exploration of functionally relevant targets has been limited. The melanoma inhibitor of apoptosis protein (ML-IAP; livin) is overexpressed in melanoma, contributing to disease progression and treatment resistance. Improved understanding of the significance of ML-IAP immune responses in patients has possible therapeutic applications. We found ML-IAP frequently expressed in melanoma metastases by immunohistochemistry. To assess spontaneous immunity to ML-IAP, an overlapping peptide library representing full-length protein was utilized to screen cellular responses in stage I–IV patients and healthy controls by ELISPOT. A broad array of CD4+ and CD8+ cellular responses against ML-IAP was observed with novel class I and class II epitopes identified. Specific HLA-A*0201 epitopes were analyzed further for frequency of reactivity. The generation of specific CD4+ and cytotoxic T cells revealed potent functional capability including cytokine responsiveness to melanoma cell lines and tumor cell killing. In addition, recombinant ML-IAP protein used in an ELISA demonstrated high titer antibody responses in a subset of patients. Several melanoma patients who received CTLA-4 blockade with ipilimumab developed augmented humoral immune responses to ML-IAP as a function of treatment which was associated with beneficial clinical outcomes. High frequency immune responses in melanoma patients, associations with favorable treatment outcomes, and its essential role in melanoma pathogenesis support the development of ML-IAP as a disease marker and therapeutic target.

High-dose interleukin-2 (HD IL-2) was approved for treatment of metastatic renal cell carcinoma (mRCC) in 1992 and for metastatic melanoma (mM) in 1998, in an era predating targeted therapies and immune checkpoint inhibitors. The PROCLAIMSM registry was established to collect and analyze data for patients treated with HD IL-2 in the current era. This analysis includes 170 patients with mM and 192 patients with mRCC treated between 2005 and 2012 with survival data current as of July 27, 2015. For patients with mM, complete response (CR) was observed in 5 %, partial response (PR) in 10 %, stable disease (SD) in 22 %, and 63 % had progressive disease (PD). The median overall survival (mOS) for these patients was 19.6 months, with a median follow-up of 43.1 months. The mOS was not reached for patients achieving CR or PR, and was 33.4 months for patients with SD. For patients with mRCC, 6 % achieved CR, 9 % had PR, 22 % had SD, and 62 % had PD. The mOS was 41 months, with a median follow-up of 46.6 months. The mOS for patients who had CR and PR was not reached and was 49.6 months for patients with SD. There were no treatment-related deaths among 362 patients. The duration of mOS for patients with mM and mRCC is longer than historically reported. These data support a continued role for IL-2 in the treatment of eligible patients with mM or mRCC and warrant further evaluation of HD IL-2 in combination or sequence with other therapeutic agents.

NK cells play a role in many cancer immunotherapies. NK cell activity is tightly regulated by killer immunoglobulin-like receptor (KIR) and KIR–ligand interactions. Inhibitory KIR–ligands have been identified as HLA molecules, while activating KIR–ligands are largely unknown. Individuals that have not inherited the corresponding KIR–ligand for at least one inhibitory KIR gene are termed the “KIR–ligand missing” genotype, and they are thought to have a subset of NK cells that express inhibitory KIRs for which the corresponding KIR–ligand is missing on autologous tissue, and thus will not be inhibited through KIR–ligand recognition. In some settings where an anticancer immunotherapeutic effect is likely mediated by NK cells, individuals with a KIR–ligand missing genotype have shown improved clinical outcome compared to individuals with an “all KIR–ligands present” genotype. In addition, patients receiving hematopoietic stem cell transplants for leukemia may do better if their donor has more activating KIR genes (i.e., KIR haplotype-B). In a recent multi-institution clinical trial of patients with metastatic renal cell carcinoma receiving high-dose IL2 (HD-IL2), 25 % of patients showed a complete or partial tumor response to this therapy. We genotyped KIR and KIR–ligand genes for these patients (n = 107) and tested whether KIR/KIR–ligand genotypes correlated with patient clinical outcomes. In these analyses, we did not find any significant association of KIR/KIR–ligand genotype (either KIR–ligand missing or the presence of KIR haplotype-B) with patient outcome in response to the HD-IL2 therapy.

Theoretical analyses suggest that the cellular internalization and catabolism of bound antibodies contribute significantly to poor penetration into tumors. Here we quantitatively assess the internalization of antibodies and antibody fragments against the commonly targeted antigen carcinoembryonic antigen (CEA). Although CEA is often referred to as a non-internalizing or shed antigen, anti-CEA antibodies and antibody fragments are shown to be slowly endocytosed by LS174T cells with a half-time of 10–16 h, a time scale consistent with the metabolic turnover rate of CEA in the absence of antibody. Anti-CEA single chain variable fragments (scFvs) with significant differences in affinity, stability against protease digestion, and valency exhibit similar uptake rates of bound antibody. In contrast, one anti-CEA IgG exhibits unique binding and trafficking properties with twice as many molecules bound per cell at saturation and significantly faster cellular internalization after binding. The internalization rates measured herein can be used in simple computational models to predict the microdistribution of these antibodies in tumor spheroids.

Transitional cell carcinoma (TCC) is the commonest cancer of the bladder. Although majority of TCC can be diagnosed at an early stage and removed easily by transurethral resection of tumor (TURT), the management of this carcinoma is complicated due to frequent recurrences usually within 6 months to one-year period. An imbalance between the Th1 and Th2 immune responses has been attributed to immune dysregulation in various malignancies. The present study aims to evaluate the Th1 and Th2 balance in Peripheral Blood Mononuclear Cells of 41 TCC patients (20 recurrent and 21 non-recurrent) using flow cytometry. It also further assesses immunological and cellular factors influencing the anti-neoplastic activity of the TCC patients and in 21 normal healthy subjects in terms of their cytokine expression and various cell surface markers. The findings of the study revealed that the cell surface markers CD3+, CD4+ and CD8+ along with NK cells were found to be significantly lower in patients than healthy controls (p<0.01). The mean percent expression of CD4+ was significantly lower in patients showing recurrence (23.9±9.84) as compared to patients with non-recurrence (31.1±12.27). The percentage of CD4+T-cells (mean±SD) producing IFN-γ, IL-2 and TNF-α were statistically significantly reduced in patients (19.1±4.94, 52.3±20.86 and 12.8±4.49) as compared to healthy controls (23.3±3.67, 67.5±12.0 and 17.6±5.96 respectively), (p<0.01, 0.018, 0.001). On the contrary, the mean levels of IL-4, IL-6 and IL-10 in patients (63.8±17.01, 60.4±14.79 and 65.7±14.84 respectively) were significantly higher as compared to healthy controls (24.4±8.77, 26.5±5.28 and 20.6±3.81 respectively), (p<0.001). No statistically significant difference was observed in the cytokine expression between patients showing recurrence and non-recurrence. Patients with bladder cancer seem to develop a Th2 dominant status with a deficient type1 immune response. The lymphocyte evaluation along with cytokine measurement can provide a sensitive and valuable tool for evaluating the function of cell-mediated immunity in these patients and can also find application in therapeutic monitoring of bladder cancer patients as new targets for immunotherapy.

Cancer immunotherapy is one the most effective approaches for treating patients with tumors, as it bolsters the generation and persistence of memory T cells. In preclinical work, it has been reported that adoptively transferred CD4+ and CD8+ lymphocytes that secrete IL-17A (i.e., Th17 and Tc17 cells) regress tumors to a greater extent than IFN-γ+Th1 or Tc1 cells in vivo. Herein, we review the mechanisms underlying how infused Th17 and Tc17 cells regress established malignancies in clinically relevant mouse models of cancer. We also discuss how unique signaling cues—such as co-stimulatory molecules (ICOS and 41BB), cytokines (IL-12 and IL-23) or pharmaceutical reagents (Akt inhibitors, etc.)—can be exploited to bolster the therapeutic potential of IL-17+ lymphocytes with an emphasis on using this knowledge to improve next-generation clinical trials for patients with cancer.

Therapeutic cancer vaccines have met limited clinical success. In the setting of cancer, the immune system is either tolerized and/or has a limited tumor-specific T cell repertoire. In this study, we explore whether intratumoral (IT) vaccination with an HPV vaccine can elicit quantitative and qualitative differences in immune response as compared to intramuscular (IM) vaccination to overcome immune resistance in established tumors. We report that IT administration of an HPV-16 E7 peptide vaccine formulated with polyinosinic–polycytidylic acid [poly(I:C)] generated an enhanced antitumor effect relative to IM delivery. The elicited anti-tumor effect with IT vaccination was consistent among the vaccinated groups and across various C57BL/6 substrains. IT vaccination resulted in an increased frequency of PD-1hi TILs, which represented both vaccine-targeted and non-vaccine-targeted tumor-specific CD8+ T cells. Overall, the CD8+/Treg ratio was increased within the tumor microenvironment using IT vaccination. We also assessed transcriptional changes in several immune-related genes in the tumor microenvironment of the various treated groups, and our data suggest that IT vaccination leads to upregulation of a broad complement of immunomodulatory genes, including upregulation of interferon gamma (IFNγ) and antigen presentation and processing machine (APM) components. IT vaccine delivery is superior to traditional IM vaccination routes with the potential to improve tumor immunogenicity, which has potential clinical application in the setting of accessible lesions such as head and neck squamous cell carcinomas (HNSCCs).