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

Immunotherapy has long held the promise of controlling cancer; however, attempts to translate immunotherapeutic strategies into practice have been unsuccessful prior to the 2010 FDA approval of the autologous cellular immunotherapy, sipuleucel-T, in patients with asymptomatic or minimally symptomatic metastatic castration-resistant prostate cancer (mCRPC) [1‐4]. Each dose of sipuleucel-T is manufactured by culturing an individual’s own freshly isolated peripheral blood mononuclear cells (PBMCs), including antigen presenting cells (APCs) and T cells, with a fusion protein (PA2024) composed of prostatic acid phosphatase (PAP; an antigen expressed in prostate adenocarcinoma [5, 6]) linked to granulocyte–macrophage colony-stimulating factor (GM-CSF; an APC activator [7, 8]). A course of therapy consists of three doses of sipuleucel-T administered at two-week intervals. Before infusion, each dose of sipuleucel-T is tested to ensure that it meets quality standards, including assessments of both cell viability and potency, by quantifying the number of APCs and the activation of these cells. Thus, the preparation of each sipuleucel-T dose provides an opportunity to immunologically characterize this autologous cellular immunotherapy as it develops over the course of the three doses.

Previous studies have shown low levels of basal immune response to PAP in prostate cancer patients [9‐11], which were augmented following immunization [12, 13, 16]; however, the studies were not sufficient in size to explore correlations with overall survival (OS). In three phase 3, double-blind, placebo-controlled studies in subjects with mCRPC [14‐16], treatment with sipuleucel-T prolonged OS, providing a unique opportunity to analyze how the product parameters that were assessed on each dose of sipuleucel-T, as well as peripheral immune responses that were monitored in a subset of these subjects, related to OS.

Sipuleucel-T, the first autologous cellular immunotherapy to be FDA-approved for the treatment of cancer, is manufactured from a patient’s own PBMCs obtained during leukapheresis. The mononuclear cells removed during leukapheresis constitute only a small percentage of the body’s total pool of lymphocytes [22‐24] and are rapidly replenished [25] such that median cell counts were within normal ranges 2, 10, and 22 weeks after the third leukapheresis procedure [26, 27]. The PBMCs are cultured with the recombinant PAP-GM-CSF fusion antigen (PA2024), which is processed by APCs and presented as PAP epitopes to PAP-specific T cells [18]. The proportion of cell subtypes remains constant throughout the manufacturing process. The data presented here support the proposed mechanism of action: ex vivo-activated APCs from the first sipuleucel-T infusion engage the immune system in vivo in a manner similar to a classical vaccine-mediated memory response, where the first infusion primes the immune system and subsequent infusions boost the response.

Activation of APCs, as measured by CD54 upregulation, was evident in the first dose (week 0) in PA2024-cultured cells and further increased in the second (week 2) and third (week 4) doses. The supposition that the first infusion of activated, antigen-loaded APCs primes T cells in vivo is supported by evidence of antigen-specific T-cell proliferation and IFNγ ELISPOT activity in pre-culture cells obtained at weeks 2 and 4 (but not week 0), as well as the presence of T-cell activation-associated cytokines in the second and third doses of sipuleucel-T. While APCs do not have anamnestic properties, the presence of cytokines produced by activated T cells, such as TNFα, is known to further activate APCs and induce the expression of cytokines associated with APC activation (e.g., IL-1β) [28, 29]. Thus, the prime-boost pattern that was also detected for APC activation and associated cytokines could be due to signals from antigen-specific T cells re-stimulated with antigen during preparation of the second and third doses. Of note, the fact that re-stimulation of pre-culture cells with GM-CSF failed to induce cytokines associated with activated T cells supports the premise that GM-CSF alone is not responsible for the observed antigen-specific immune responses; this is consistent with preclinical findings [30].

TH1 cytokines (e.g., IFNγ, TNFα) in the product were present at high levels in comparison with IL-4, the classical marker of TH2 cells, but the presence of TH2 cytokines IL-5 and IL-13 implies that both TH1 and TH2 cells were activated in an antigen-specific manner. This is consistent with the observation that sipuleucel-T treatment induced both cellular and humoral responses. Intriguingly, IL-17 was also produced, suggesting the activation of TH17 cells, a TH subset known to have a pivotal role in mediating autoimmune responses [31, 32]. In addition, while IL-10 was also detectable, the relative amount of this T-cell-suppressive cytokine was markedly less than that of cytokines known to drive T-cell expansion, such as IL-2, IFNγ, and TNFα.

These data demonstrate that sipuleucel-T engages the immune system early in treatment and generates robust and persistent in vivo antigen-specific cellular and humoral immunity. In T-cell proliferative antigen recall assays, a pertinent measure of immunological responsiveness, PA2024-specific responses were present in the majority of sipuleucel-T-treated subjects with the magnitude of the response sustained through at least week 26. Furthermore, the IFNγ ELISPOT responses detected in the sipuleucel-T group at week 26 are indicative of persistent PA2024-specific memory T cells [33]. Finally, the development of early IgM responses followed by isotype switching to IgG antibody production is characteristic of memory plasma cell development and suggests the engagement of T cells and the establishment of long-lasting humoral immunity.

Immune responses to PAP were detectable but were less frequent and lower in magnitude than responses to the immunizing antigen (PA2024). Several factors may contribute to these findings. First, PA2024 was tailored to be more easily taken up and effectively processed by APCs and is larger (containing more epitopes) than PAP. Furthermore, the additional epitopes in PA2024 include the linker between GM-CSF and PAP, a neoepitope that may be more immunogenic than the epitopes to PAP. Finally, it should also be noted that tumor antigen-specific effector T cells may transit out of the peripheral blood, which is used as the source of PBMCs for the manufacture of sipuleucel-T and for immune monitoring, complicating comparisons between responses. Nevertheless, the fact that anti-PAP antibodies were detected in subjects treated with sipuleucel-T when no such antibodies could be identified in the pre-treatment specimens and that PAP-specific T-cell responders were observed only in the sipuleucel-T group supports the supposition that sipuleucel stimulates antigen-specific B- and T-cell responses that target prostate cancer.

The positive correlation observed between key product parameters, such as APC activation, and OS suggests that the broad engagement of the immune system by sipuleucel-T contributes to the survival benefit associated with therapy. Moreover, patients who developed a peripheral immune response to sipuleucel-T had improved OS. The strongest relationship between prolonged OS and long-term immune response was observed with PA2024-specific antibody and IFNγ ELISPOT responses. The absence of a significant correlation between OS and T-cell proliferation may be due to the non-specific nature of the T-cell proliferation assay (i.e., it does not distinguish between biologically functional vs. non-functional effector memory cells). As such, the IFNγ ELISPOT assay, which is a direct measure of the establishment of functional antigen-specific memory T cells, may be a better indicator of relevant immune response.

Limitations of this study include the availability of immune response data for only a subset of patients, and the post hoc correlative nature of the analyses. In addition, the correlations between immune parameters and increased OS could result from healthier subjects with an improved probability of survival being more likely to generate robust immune responses. However, the persistence of the correlations between these immune markers and OS following adjustment for baseline prognostic factors argues against this hypothesis, as does the fact that humoral recall response to tetanus did not change following treatment and was not different between the control and treatment groups (data on file; Dendreon). In addition, prior studies demonstrated no differences in baseline T-cell proliferation response to influenza between patients who did or did not subsequently develop an immune response to PAP [34]. Similarly, previous work in follicular lymphoma failed to demonstrate a correlation between anti-idiotype immune response in response to immunization and baseline prognostic factors or the ability to respond to KLH [35].

In summary, the data presented here demonstrate that ex vivo APC activation with sipuleucel-T induces a robust and long-lived in vivo immune response profile characteristic of immunological memory. Both cellular and humoral responses were evident, and the response status of subjects as well as the magnitude of the immune response correlated with OS. These data are consistent with sipuleucel-T’s intended mechanism of action: to generate antigen-specific immune responses that target prostate cancer. A recent study of sipuleucel-T administered prior to radical prostatectomy demonstrating increases in T cells at the tumor interface compared to the pre-treatment biopsy [36] provides further evidence in support of this mechanism of action. Given the increasing availability of novel immunomodulatory agents, combination studies with sipuleucel-T are warranted; augmentation of the immune parameters described herein would provide a strong rationale for larger studies to demonstrate increased clinical efficacy.