James Trager

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. Clin Cancer Res; 21(16); 3619–30. ©2015 AACR. See related commentary by Hellstrom and Hellstrom, p. 3581

An overview of sipuleucel-T: Autologous cellular immunotherapy for prostate cancer

Sipuleucel-T, the first autologous active cellular immunotherapy approved by the United States Food and Drug Administration, is designed to stimulate an immune response to prostate cancer. Sipuleucel-T is manufactured by culturing a patient’s peripheral blood mononuclear cells (including antigen presenting cells) with a recombinant protein comprising a tumor-associated antigen (prostatic acid phosphatase) and granulocyte-macrophage colony stimulating factor. Treatment consists of 3 infusions at approximately 2-week intervals, resulting in a prime-boost pattern of immune activation, a robust antigen-specific cellular and humoral immune response, and, consequently, a survival benefit in subjects with asymptomatic or minimally symptomatic metastatic castrate resistant prostate cancer. Adverse events are generally mild to moderate and resolve within 2 d. Serious adverse events occur at a low rate. As the first autologous cellular immunotherapy to demonstrate a survival benefit, sipuleucel-T is a novel oncologic therapeutic that warrants the reassessment of the current prostate cancer treatment paradigm.

Role of Antigen Spread and Distinctive Characteristics of Immunotherapy in Cancer Treatment

Immunotherapy is an important breakthrough in cancer. US Food and Drug Administration-approved immunotherapies for cancer treatment (including, but not limited to, sipuleucel-T, ipilimumab, nivolumab, pembrolizumab, and atezolizumab) substantially improve overall survival across multiple malignancies. One mechanism of action of these treatments is to induce an immune response against antigen-bearing tumor cells; the resultant cell death releases secondary (nontargeted) tumor antigens. Secondary antigens prime subsequent immune responses (antigen spread). Immunotherapy-induced antigen spread has been shown in clinical studies. For example, in metastatic castration-resistant prostate cancer patients, sipuleucel-T induced early immune responses to the immunizing antigen (PA2024) and/or the target antigen (prostatic acid phosphatase). Thereafter, most patients developed increased antibody responses to numerous secondary proteins, several of which are expressed in prostate cancer with functional relevance in cancer. The ipilimumab-induced antibody profile in melanoma patients shows that antigen spread also occurs with immune checkpoint blockade. In contrast to chemotherapy, immunotherapy often does not result in short-term changes in conventional disease progression end points (eg, progression-free survival, tumor size), which may be explained, in part, by the time taken for antigen spread to occur. Thus, immune-related response criteria need to be identified to better monitor the effectiveness of immunotherapy. As immunotherapy antitumor effects take time to evolve, immunotherapy in patients with less advanced cancer may have greater clinical benefit vs those with more advanced disease. This concept is supported by prostate cancer clinical studies with sipuleucel-T, PSA-TRICOM, and ipilimumab. We discuss antigen spread with cancer immunotherapy and its implications for clinical outcomes.

Clonotypic Diversification of Intratumoral T Cells Following Sipuleucel-T Treatment in Prostate Cancer Subjects

Sipuleucel-T is an autologous cellular therapy for asymptomatic, or minimally symptomatic, metastatic castrate-resistant prostate cancer, designed to stimulate an immune response against prostate cancer. In a recent clinical trial (NCT00715104), we found that neoadjuvant sipuleucel-T increased the number of activated T cells within the tumor microenvironment. The current analysis examined whether sipuleucel-T altered adaptive T-cell responses by expanding pre-existing T cells or by recruiting new T cells to prostate tissue. Next-generation sequencing of the T-cell receptor (TCR) genes from blood or prostate tissue was used to quantitate and track T-cell clonotypes in these treated subjects with prostate cancer. At baseline, there was a significantly greater diversity of circulating TCR sequences in subjects with prostate cancer compared with healthy donors. Among healthy donors, circulating TCR sequence diversity remained unchanged over the same time interval. In contrast, sipuleucel-T treatment reduced circulating TCR sequence diversity versus baseline as measured by the Shannon index. Interestingly, sipuleucel-T treatment resulted in greater TCR sequence diversity in resected prostate tissue in sipuleucel-T-treated subjects versus tissue of nonsipuleucel-T-treated subjects with prostate cancer. Furthermore, sipuleucel-T increased TCR sequence commonality between blood and resected prostate tissue in treated versus untreated subjects with prostate cancer. The broadening of the TCR repertoire within the prostate tissue supports the hypothesis that sipuleucel-T treatment facilitates the recruitment of T cells into the prostate. Our results highlight the importance of assessing T-cell response to immunotherapy both in the periphery and in tumor tissue. Cancer Res; 76(13); 3711-8. ©2016 AACR.

Sipuleucel-T product characterization across different disease states of prostate cancer.

42 Background: Sipuleucel-T is an autologous cellular immunotherapy approved for the treatment of asymptomatic or minimally symptomatic metastatic castrate resistant prostate cancer (mCRPC). In the Phase 3 mCRPC trials, antigen presenting cell (APC) activation in sipuleucel-T correlated with overall survival. Here product characteristics of sipuleucel-T were compared in patients (pts) from trials in the neoadjuvant (n=42), asymptomatic or minimally symptomatic mCRPC (n=341), and mCRPC (n=104) disease states. METHODS Sipuleucel-T comprises 3 treatments approximately 2 wks apart. Cellular composition and APC activation (CD54 upregulation) were evaluated in all courses of sipuleucel-T, and cumulative CD54 upregulation was calculated for each patient. In some studies, cytokines were assayed from culture supernatants and T cell and B cell activation markers were assessed by flow cytometry during manufacture. RESULTS Baseline demographics were generally representative of each disease state; neoadjuvant pts were younger with less disease burden; mCRPC pts had the highest disease burden and more frequent prior chemotherapy. While neoadjuvant pts had greater levels of T cells, the patterns of APC activation were similar among all trials, with increased CD54 upregulation at the second and third treatment. The trend for cumulative fold increase in CD54 upregulation was significantly greater in earlier disease pts (neoadjuvant: 35.5, asymptomatic or minimally symptomatic mCRPC: 28.7, mCRPC: 21.8; p<0.0001 (Joncheere-Terpstra test)). During manufacture of the product, lymphocyte activation markers and cytokines consistently showed enhanced expression during the second and third treatments in all disease states. The lymphocyte activation and cytokine profiles were similar between early and later disease state pts. CONCLUSIONS While cellular composition varied with disease state, manufacture of sipuleucel-T activated APCs in both early and late disease states of prostate cancer, and generated similar T and B cell activation and cytokine production profiles consistent with immunological prime-boost. APC activation tended to be more robust in earlier disease states.

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.

3D: diversity, dynamics, differential testing – a proposed pipeline for analysis of next-generation sequencing T cell repertoire data

BackgroundCancer immunotherapy has demonstrated significant clinical activity in different cancers. T cells represent a crucial component of the adaptive immune system and are thought to mediate anti-tumoral immunity. Antigen-specific recognition by T cells is via the T cell receptor (TCR) which is unique for each T cell. Next generation sequencing (NGS) of the TCRs can be used as a platform to profile the T cell repertoire. Though there are a number of software tools available for processing repertoire data by mapping antigen receptor segments to sequencing reads and assembling the clonotypes, most of them are not designed to track and examine the dynamic nature of the TCR repertoire across multiple time points or between different biologic compartments (e.g., blood and tissue samples) in a clinical context.ResultsWe integrated different diversity measures to assess the T cell repertoire diversity and examined the robustness of the diversity indices. Among those tested, Clonality was identified for its robustness as a key metric for study design and the first choice to measure TCR repertoire diversity. To evaluate the dynamic nature of T cell clonotypes across time, we utilized several binary similarity measures (such as Baroni-Urbani and Buser overlap index), relative clonality and Morisita’s overlap index, as well as the intraclass correlation coefficient, and performed fold change analysis, which was further extended to investigate the transition of clonotypes among different biological compartments. Furthermore, the application of differential testing enabled the detection of clonotypes which were significantly changed across time. By applying the proposed “3D” analysis pipeline to the real example of prostate cancer subjects who received sipuleucel-T, an FDA-approved immunotherapy, we were able to detect changes in TCR sequence frequency and diversity thus demonstrating that sipuleucel-T treatment affected TCR repertoire in blood and in prostate tissue. We also found that the increase in common TCR sequences between tissue and blood after sipuleucel-T treatment supported the hypothesis that treatment-induced T cell migrated into the prostate tissue. In addition, a second example of prostate cancer subjects treated with Ipilimumab and granulocyte macrophage colony stimulating factor (GM-CSF) was presented in the supplementary documents to further illustrate assessing the treatment-associated change in a clinical context by the proposed workflow.ConclusionsOur paper provides guidance to study the diversity and dynamics of NGS-based TCR repertoire profiling in a clinical context to ensure consistency and reproducibility of post-analysis. This analysis pipeline will provide an initial workflow for TCR sequencing data with serial time points and for comparing T cells in multiple compartments for a clinical study.

Induction of antigen spread after sipuleucel-T treatment and its association with improved clinical outcome

An effective cancer immunotherapy that triggers immune-mediated lysis of tumor cells, may lead to the priming of T or B lymphocytes against tumor antigens distinct from the initial target/s of the therapy. This is referred to as antigen or epitope spread. Evidence of antigen spread after treatment may not only provide insights into the mechanism of action (MoA) of cancer immunotherapies, but also provide pharmacodynamic (post-treatment) biomarkers of clinical response and outcome. Sipuleucel-T, an FDA approved autologous cellular immunotherapy for the treatment of symptomatic or minimally symptomatic, metastatic castrate-resistant prostate cancer (mCRPC), is designed to elicit immune responses to the prostate-specific antigen, Prostatic Acid Phosphatase (PAP). We sought to determine if antigen spread occurs in response to sipuleucel-T treatment.

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 patients 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.

Abstract 1313: Characterization and isolation of antigen-responding T cells in Sipuleucel-T treated patients

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Sipuleucel-T is an FDA-approved autologous cellular therapy for men with asymptomatic or minimally symptomatic metastatic castrate resistant prostate (mCRPC). Sipuleucel-T is manufactured from peripheral blood mononuclear cells (PBMCs) cultured with PA2024, a fusion antigen composed of prostatic acid phosphatase (PAP) fused to granulocyte macrophage-colony stimulating factor (GM-CSF). We have demonstrated that following three infusions this therapy results in T cell antigen specific responses directed against both PA2024 and PAP in treated patients. In this study we utilized the cellular dye Carboxyfluorescein succinimidyl ester (CSFE) and flow cytometric analysis to further characterize antigen responding T cells in Sipuleucel-T treated patients. We observed that CD4+ T cells predominate the antigen-responding T cell population following treatment, confirming our previous studies. Importantly, this approach has allowed us to also visualize a previously undetected population of antigen responding CD8+ T cells. The frequency of both of these populations is enhanced following treatment relative to baseline responses and the responding T cells display increased surface expression of the prototypical T cell activation markers CD134 and CD137 relative to non-proliferating T cells. Additionally, we are exploiting the increased surface expression of these activation markers on antigen-responding T cells to isolate these cells in an HLA and epitope-independent manner. Using this methodology we have demonstrated Sipuleucel-T is capable of inducing both CD4+ and CD8+ antigen-specific T cell responses in treated patients, and that we are capable of isolating these antigen-responding T cells specifically and with minimal manipulation. Citation Format: Jeff Pufnock, Tuyen Vu, James Trager, Nadeem Sheikh. Characterization and isolation of antigen-responding T cells in Sipuleucel-T treated patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1313. doi:10.1158/1538-7445.AM2015-1313