Humoral Immune Response against Nontargeted Tumor Antigens after Treatment with Sipuleucel-T and Its Association with Improved Clinical Outcome.

PURPOSE: Antitumor activity of cancer immunotherapies may elicit immune responses to nontargeted (secondary) tumor antigens, or antigen spread. We evaluated humoral antigen spread after treatment with sipuleucel-T, an immunotherapy for asymptomatic or minimally symptomatic metastatic castration-resistant prostate cancer (mCRPC), designed to target prostatic acid phosphatase (PAP; primary antigen). EXPERIMENTAL DESIGN: Serum samples from patients with mCRPC enrolled in the placebo-controlled phase III IMPACT study (evaluable n = 142) were used to assess humoral antigen spread after treatment with sipuleucel-T. Immunoglobulin G (IgG) responses to self-antigens (including tumor antigens) were surveyed using protein microarrays and confirmed using Luminex xMAP. IgG responses were subsequently validated in ProACT (n = 33), an independent phase II study of sipuleucel-T. Association of IgG responses with overall survival (OS) was assessed using multivariate Cox models adjusted for baseline prostate-specific antigen (PSA) and lactate dehydrogenase levels. RESULTS: In patients from IMPACT and ProACT, levels of IgG against multiple secondary antigens, including PSA, KLK2/hK2, K-Ras, E-Ras, LGALS8/PCTA-1/galectin-8, and LGALS3/galectin-3, were elevated after treatment with sipuleucel-T (P < 0.01), but not control. IgG responses (≥ 2-fold elevation posttreatment) occurred in ≥ 25% of patients, appeared by 2 weeks after sipuleucel-T treatment, and persisted for up to 6 months. IgG responses to PSA and LGALS3 were associated with improved OS in sipuleucel-T-treated patients from IMPACT (P ≤ 0.05). CONCLUSIONS: Sipuleucel-T induced humoral antigen spread in patients with mCRPC. IgG responses were associated with improved OS in IMPACT. The methods and results reported may identify pharmacodynamic biomarkers of clinical outcome after sipuleucel-T treatment, and help in clinical assessments of other cancer immunotherapies. See related commentary by Hellstrom and Hellstrom, p. 3581.

Applications of systems biology in cancer immunotherapy: from target discovery to biomarkers of clinical outcome.

Immunotherapies are coming to the forefront as a treatment paradigm in cancer with multiple US FDA approvals in recent years and a better understanding of their therapeutic mode of action. The control of tumor growth by the immune system is orchestrated by a complex array of cellular interactions and molecular pathways, both in the immune cells as well as the tumor. Although research over the past three decades has elucidated many aspects of tumor immunosurveillance, given the inherent complexity of the immune cell phenotypes and function, high-throughput molecular profiling ('omics') approaches have now become essential to support the discovery and development of new therapies. Technologies, such as DNA and protein microarrays, deep sequencing, mass spectrometry, as well as the computational methods for their analyses, are advancing the contributions of systems biology towards the development and mechanistic understanding of cancer immunotherapies. In this review, the authors illustrate this through some recently reported studies.

Sipuleucel-T immune parameters correlate with survival: an analysis of the randomized phase 3 clinical trials in men with castration-resistant prostate cancer.

PURPOSE: Sipuleucel-T, the first FDA-approved autologous cellular immunotherapy for treatment of advanced prostate cancer, is manufactured by activating peripheral blood mononuclear cells, including antigen presenting cells (APCs), with a fusion protein containing prostatic acid phosphatase. Analysis of data from three phase 3 trials was performed to immunologically characterize this therapy during the course of the three doses, and to relate the immunological responses to overall survival (OS). METHODS: Sipuleucel-T product characteristics [APC numbers, APC activation (CD54 upregulation), and total nucleated cell (TNC) numbers] were assessed in three randomized, controlled phase 3 studies (N = 737). Antigen-specific cellular and humoral responses were assessed in a subset of subjects. The relationships between these parameters and OS were assessed. RESULTS: APC activation occurred in the first dose preparation [6.2-fold, (4.65, 7.70); median (25th, 75th percentile)] and increased in the second [10.6-fold (7.83, 13.65)] and third [10.5-fold (7.89, 13.65)] dose preparations. Cytokines and chemokines associated with activated APCs were produced during the manufacture of each dose; T-cell activation-associated cytokines were detected in the second and third dose preparations. Antigen-specific T cells were detectable after administration of the first sipuleucel-T dose. Cumulative APC activation, APC number, and TNC number correlated with OS (P < 0.05). Antigen-specific immune responses were observed in 78.8 % of monitored subjects and their presence correlated with OS (P = 0.003). CONCLUSION: Sipuleucel-T broadly engages the immune system by activating APCs ex vivo and inducing long-lived immune responses in vivo. These data indicate antigen-specific immune activation as a mechanism by which sipuleucel-T prolongs OS.

Prostatic acid phosphatase expression in human tissues.

Prostate cancer is the most common cancer and the second leading cause of cancer deaths among males in most Western countries. Autologous cellular immunotherapy for the treatment of cancer seeks to induce tumor-specific immunity in the patient and is consequently dependent on a suitable target antigen and effective presentation of that antigen to the patient's immune system. Prostatic acid phosphatase (PAP) has been tested as a target antigen due to its high and apparently specific expression in the prostate. We used a variety of approaches to analyze PAP expression, including immunohistochemistry, in situ hybridization, and quantitative polymerase chain reaction. We complemented these laboratory-based techniques with an in silico analysis of reported PAP expression in human cDNA libraries. Our studies confirmed that, while PAP expression is not restricted to prostate tissues, its expression in other human tissues is approximately 1-2 orders of magnitude less than that observed in the prostate. The relative specificity of PAP expression in the prostate supports its use as a target of autologous cellular immunotherapy. The approach described here, involving the use of multiple correlates of tissue-specific expression, is warranted as a prerequisite in selecting any suitable target for immunotherapy.

Reconstitution of telomerase activity utilizing human catalytic subunit expressed in insect cells.

Telomerase is a specialized reverse transcriptase responsible for maintaining the termini of linear chromosomes. The human enzyme is a ribonucleoprotein complex minimally comprising a catalytic protein moiety (hTERT) and an RNA subunit (hTR) which acts as the template for the reverse transcriptase reaction. Here we report expression of recombinant hTERT protein in insect cells utilizing a baculovirus expression system. The recombinant hTERT protein reconstitutes telomerase activity in the presence of hTR, either when co-expressed in insect cells or when added in vitro. Reconstitution of telomerase activity using this system will facilitate further analysis of the biochemical and biophysical properties of this enzyme.