Tuyen Vu

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

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.

Antigen-Specific CD8 Lytic Phenotype Induced by Sipuleucel-T in Hormone-Sensitive or Castration-Resistant Prostate Cancer and Association with Overall Survival

Purpose: Sipuleucel-T is FDA approved for the treatment of metastatic castration-resistant prostate cancer (mCRPC) based on the IMPACT trial showing a 4.1-month benefit in median overall survival (OS) for patients receiving sipuleucel-T versus control. Although efficacy of sipuleucel-T is well established, its mechanism remains incompletely understood. Patients and Methods: Patient samples from three sipuleucel-T trials were assessed for peripheral cellular immune responses to the immunogen PA2024 and the target antigen prostatic acid phosphatase (PAP). PAP- and PA2024-specific proliferative and cytolytic responses were characterized to delineate sipuleucel-T–induced immune responses. To quantify potential cytotoxic T lymphocyte (CTL) activity, cell-surface CD107a expression on PAP- or PA2024-specific CD8+ T cells was measured in sipuleucel-T–treated patient and healthy volunteer samples. Results: Increased PA2024-specific CD4+ (P = 0.030) and CD8+ (P = 0.052) T-cell proliferation from baseline to week 6 was observed (N = 14) post–sipuleucel-T, with greater magnitude of PA2024-specific responses compared with PAP. PAP- and PA2024-CTL activity (CD107a positivity) significantly increased at weeks 6 and 26 after sipuleucel-T treatment (P < 0.0001; N = 22). At 26 weeks post–sipuleucel-T, OS correlated with the magnitude of PAP (Pearson R, 0.52; P = 0.013) or PA2024 (Pearson R, 0.67; P = 0.0006) CTL activity. Higher PA2024-CTL activity at week 26 was significantly associated with longer OS using tertile analysis (P = 0.0005; N = 22), with PA2024 responses correlating with PAP responses at week 26 (R = 0.90; P = 1.53E−08). Conclusions: This study is the first to report PAP-specific CD8+ T-cell responses elicited by sipuleucel-T treatment. Increased and persistent potential PA2024-specific CTL activity correlated with PAP-specific CTL activity and associated with improved OS following sipuleucel-T treatment. Clin Cancer Res; 24(19); 4662–71. ©2018 AACR.

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

Abstract 2542: Sipuleucel-T-induced immune response against secondary cancer antigens is associated with improved overall survival

Introduction and Objective: Tumor cell death in response to immunotherapy may lead to the release of secondary (non-targeted) cancer antigens that prime subsequent immune responses (antigen spread) 1 . This may contribute to tumor control and reveal post-treatment biomarkers of clinical outcome. Sipuleucel-T, an immunotherapy targeted towards the prostate-specific antigen, prostatic acid phosphatase, is approved in the US and EU for treatment of asymptomatic or minimally symptomatic, metastatic castration-resistant prostate cancer. We evaluated: (i) antigen spread after sipuleucel-T treatment, based on IgG antibody responses against secondary cancer antigens, and (ii) association of such IgG responses with overall survival (OS). Methods: ProtoArray®, an unbiased protein microarray platform, was used to characterize the spectrum of serum IgG responses after treatment. IgG responses against secondary antigens were confirmed using an independent platform, Luminex®, xMAP®. Pre- and post- treatment sera from two clinical trials of sipuleucel-T were analyzed: IMPACT 2 (double-blind, placebo-controlled, phase 3, NCT00065442) and ProACT (open-label, phase 2, NCT00715078). The correlations between IgG responses (≥ 2 fold elevation post-treatment) and OS were assessed in IMPACT using a Cox regression model, adjusted for baseline PSA, LDH, bone lesions, Gleason score, and prior bisphosphonate use. Results: Serum IgG responses (p -3 ) towards several secondary cancer antigens (PSA, hK2/KLK2, K-Ras, E-Ras, and LGALS8/PCTA-1) were observed after treatment with sipuleucel-T in IMPACT as well as ProACT. Across trials, the frequency of patients with IgG responses after sipuleucel-T treatment was: ≥25% for PSA, ≥33% for hK2, ≥36% for K-Ras, ≥39% for E-Ras, ≥24% for LGALS8. These IgG responses were observed as early as 2 weeks and up to 6 months after treatment with sipuleucel-T. Control arm patients in IMPACT did not show such responses (p>0.01). In the sipuleucel-T arm of IMPACT (N=140), an IgG response to PSA ≈2 weeks after treatment was positively correlated with increased OS, compared to patients with no IgG response to PSA (Cox p Conclusions: Sipuleucel-T elicited IgG responses against multiple cancer antigens. Sipuleucel-T- induced IgG response against PSA was positively correlated with improved OS. These data enhance our understanding of sipuleucel-T9s mechanism of action, and may help to identify pharmacodynamic biomarkers of clinical outcome. The methods and results presented here may also help in the characterization of antigen spread and pharmacodynamic biomarkers after treatment with other cancer immunotherapies. 1. Ribas A, et al. Trends Immunol. 24:58-61 (2003) 2. Kantoff PW, et al. N Engl J Med. 363:411-22 (2010) Citation Format: Debraj GuhaThakurta, Li-Qun Fan, Tuyen Vu, Francis Stewart, Philip Kantoff, Eric Small, Celeste Higano, Thomas Gardner, Nadeem Sheikh, James Trager. Sipuleucel-T-induced immune response against secondary cancer antigens is associated with improved overall survival. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2542. doi:10.1158/1538-7445.AM2014-2542