Subjects and treatment
Of the 737 subjects randomized in the IMPACT, D9901, and D9902A studies, 476 received sipuleucel-T and 243 received control product, both with a median cell viability of >95 % across all 3 infusions. For the subset of subjects from IMPACT who provided blood for peripheral immune response determinations (n = 160 sipuleucel-T; n = 77 control), demographics and baseline disease characteristics were balanced between study arms and between the evaluated subgroup and the overall study population (Table 1).
Product parameters and analyses of antigen-specific T cells in the product
The sipuleucel-T final product (IMPACT only) comprised (median [25th, 75th percentile]): CD3+ T cells (62.3 % [51.8, 69.1 %]), APCs (18.3 % [12.3, 26.5 %], the majority of which were CD14+), CD56+ NK cells (11.0 % [6.9, 15.3 %]), and CD19+ B cells (4.7 % [2.6, 7.9 %]). The relative proportions of these cell types did not change across the treatment weeks.
In both the IMPACT and D9901/D9902A studies, APC activation was significantly greater with sipuleucel-T relative to control at weeks 0, 2, and 4 (Fig. 1). For sipuleucel-T, median [25th, 75th percentile] APC activation increased 6.2-fold [4.7, 7.7] in the first product, compared with 10.6-fold [7.8, 13.7] in the second and 10.5-fold [7.9, 13.7] in the third products (both P < 0.001 vs. first product, all studies pooled). The median cumulative APC activation with sipuleucel-T across the three dose preparations was 26.7 [21.5, 33.6]. TNC counts and number of APCs were consistent and not significantly different between doses prepared at weeks 0, 2, and 4; the median cumulative number [25th, 75th percentile] of TNCs and APCs across the three doses, pooled over the three studies were 9.70 × 109 [6.97 × 109, 13.55 × 109], and 1.84 × 109 [1.27 × 109, 2.88 × 109].
Antigen-specific T cells
In the subset of IMPACT patients who were evaluated, significantly greater antigen-specific T cells, detected by proliferation and IFNγ ELISPOT responses against PA2024, were observed in dose preparations from the sipuleucel-T group relative to control at weeks 2 and 4, but not at week 0; the greatest values were observed at week 4 for both assays (Fig. 2). Some sipuleucel-T subjects also demonstrated responses to PAP that were greater than any of those observed in the control group and lower in magnitude than the anti-PA2024 responses.
Cytokine/chemokine production during manufacture
Cytokines produced by activated APCs were present in the culture media of sipuleucel-T, but not control, at weeks 0, 2, and 4 (Fig. 3a). Elevated levels of T-cell activation-associated cytokines were observed in the culture medium during the manufacture of the second and third doses (Fig. 3b). T-cell activation cytokines were not induced when pre-culture cells were cultured with GM-CSF alone (Fig. 3c).
Characterization of peripheral immune responses
A positive humoral or cellular immune response to PA2024 and/or PAP in any post-baseline assay was observed in 78.8 % (123/156) of sipuleucel-T subjects compared with 13.2 % (10/76) of control subjects. An immune response to PA2024 was observed in 78.2 % (122/156) of sipuleucel-T subjects versus 10.5 % (8/76) of control subjects, and a response to PAP was observed in 39.5 % (60/152) of sipuleucel-T subjects versus 5.7 % (4/70) of control subjects (Fig. 4).
Sipuleucel-T treatment generated PA2024- and/or PAP-specific humoral responses in a majority of subjects (68 %; 102/151), compared with 3 % (2/70) of control subjects. Anti-PA2024 and anti-PAP antibody titers were greater in the sipuleucel-T group compared with control at all post-baseline time points (P < 0.001), with a positive response still evident 26 weeks after initial treatment in 53.2 % (PA2024) and 17.7 % (PAP) of subjects (Supplementary Figure 2A). The magnitude of PA2024- and PAP-specific IgM antibodies peaked at week 6 and remained detectable through week 26, while anti-PA2024 and anti-PAP IgG antibodies were detected at week 6 and further increased at weeks 14 and 26 (Supplementary Figure 2B).
Sipuleucel-T treatment also elicited PA2024- and/or PAP-specific cellular responses in a majority of subjects (60 % [61/102] T-cell proliferation; 48 % [49/102] IFNγ ELISPOT), compared with a positive response in control subjects of 6 % (3/51) for T-cell proliferation and 13 % [7/52] for IFNγ ELISPOT. PA2024-specific T-cell proliferation and IFNγ ELISPOT were significantly greater with sipuleucel-T at all post-baseline time points (P < 0.05; Supplementary Figure 3), and significantly more sipuleucel-T subjects responded to each assay (Fig. 4). The magnitude of PAP-specific T-cell proliferation was not significantly different between groups (Supplementary Figure 3); however, only sipuleucel-T subjects demonstrated a positive proliferation response to PAP (Fig. 4). PAP-specific IFNγ ELISPOT results and responder frequency were not significantly different between groups at any time point (Fig. 4; Supplementary Figure 3).
Correlations of overall survival with product parameters and peripheral immune response
In sipuleucel-T treated subjects, a positive correlation was observed between OS and cumulative APC activation, APC count, and TNC count (pooled studies; Fig. 5). These correlations remained after adjusting for baseline prognostic factors (PSA and LDH) that are independently correlated with OS . For a graphical depiction of these correlations, Kaplan–Meier curves were generated for sipuleucel-T subjects with cumulative product parameters greater than versus less than or equal to the median value (Fig. 5a–c). OS was also significantly correlated with the development of at least one post-baseline peripheral immune response to PA2024 or PAP [HR = 0.47 (95 % CI: 0.29, 0.78) P = 0.003; Fig. 5d], to PA2024 [HR = 0.46 (95 % CI: 0.28, 0.76) P = 0.002; Fig. 5e], and to PAP [HR = 0.53 (95 % CI: 0.31, 0.90) P = 0.019; Fig. 5f]. The strongest correlation between OS and the development of a post-baseline immune response to PA2024 at any time point was observed with antibody responses [HR = 0.42 (95 % CI: 0.26, 0.67) P < 0.001], while the correlation with IFNγ ELISPOT at any time point approached statistical significance [HR = 0.55 (95 % CI: 0.28, 1.08) P = 0.08]; T-cell proliferation response did not significantly correlate with OS.