David L. Urdal

Abstract PL04-01: Sipuleucel-T and the active immunotherapy of prostate cancer.

Sipuleucel-T is an FDA approved autologous cellular immunotherapy indicated for the treatment of asymptomatic or minimally symptomatic metastatic castrate resistant (hormone refractory) prostate cancer. It consists of autologous peripheral blood mononuclear cells, including antigen presenting cells (APCs), which have been activated during a defined culture period with a recombinant human protein, PAP-GM-CSF, consisting of prostatic acid phosphatase (PAP), an antigen expressed in prostate cancer tissue, linked to granulocyte-macrophage colony stimulating factor (GM-CSF), an immune cell activator. The development history will be summarized; the regulatory milestones will be described; and the lessons learned during the development of this novel approach to the treatment of prostate cancer will be discussed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr PL04-01.

Abstract 5508: B lymphocytes are activated during the course of sipuleucel-T treatment, leading to an antigen-specific humoral response

Introduction: Sipuleucel-T is an autologous cellular immunotherapy FDA approved for men with asymptomatic or minimally symptomatic metastatic castrate resistant prostate cancer (mCRPC). Previous clinical data have shown that antibodies specific for the immunizing antigen, PA2024, develop within two weeks post-end of sipuleucel-T treatment. Here, we sought to determine whether B lymphocytes were activated during the course of treatment and to evaluate their phenotypic profile in two ongoing clinical studies (NEoACT and PRoACT). Materials and Methods: Sipuleucel-T is manufactured from peripheral blood mononuclear cells (PBMCs) isolated every two weeks by leukapheresis. The PBMCs are cultured with PA2024, an antigen composed of prostatic acid phosphatase (PAP) fused to granulocyte macrophage-colony stimulating factor (GM-CSF), washed, and then infused into the patient as sipuleucel-T. Each patient receives three infusions. The activation status of B lymphocytes was evaluated by multi-color flow cytometry during the manufacture of sipuleucel-T, before and after culture at each of the three treatment times. B lymphocyte subsets were identified by their expression of CD20, IgD and CD27: CD20+IgD+CD27− naive, CD20+IgD+CD27+ mature activated, and CD20+IgD−CD27+ memory. Activation of each of these subsets was determined by their expression of CD80 and CD96. Antigen-specific B lymphocyte function, in the form of antibody production, was evaluated in the serum of sipuleucel-T-treated subjects before and after treatment. Summary: In the mature activated B lymphocyte subset, an increase in the proportion of lymphocytes expressing CD80+ and CD86+ was detected following culture with PA2024, with the increase in CD86+ mature B lymphocytes being the highest after the second infusion of sipuleucel-T. An increase in mature activated B lymphocytes directly ex vivo, pre-culture, was not observed prior to the second or third infusion. Additionally, CD80 and CD86 expression was upregulated on memory B lymphocytes in response to culture with PA2024 and following the first infusion. Furthermore, PA2024- and PAP-specific antibody responses were detected and maintained 2-22 weeks after the completion of sipuleucel-T treatment.Conclusions: These data demonstrate that B lymphocyte activation is a consequence of ex vivo culture with PA2024 and precedes an antigen-specific humoral response, as detected in the patients’ serum after treatment with sipuleucel-T. Collectively, these findings provide evidence of therapy-induced humoral immune activation in men with mCRPC treated with sipuleucel-T. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5508. doi:10.1158/1538-7445.AM2011-5508

Abstract 5509: Evidence of T lymphocyte activation following sipuleucel-T treatment

Introduction: Sipuleucel-T is an autologous cellular immunotherapy FDA approved for men with asymptomatic or minimally symptomatic metastatic castrate resistant prostate cancer (mCRPC). Previous clinical data from Phase 3 clinical trials demonstrated T lymphocyte activation occurred in response to culture with the immunizing antigen, PA2024, in vitro, and was maintained in vivo after infusion with sipuleucel-T. Data from a current clinical study (ProACT) examined the activation profile of T lymphocytes and the production of cytokines associated with activated T lymphocytes during the preparation of sipuleucel-T. Materials and Methods: Sipuleucel-T is manufactured from peripheral blood mononuclear cells (PBMCs) isolated every two weeks by leukapheresis. The PBMCs are cultured for a defined period with the PA2024 antigen, which is composed of prostatic acid phosphatase (PAP) fused to granulocyte macrophage-colony stimulating factor (GM-CSF), washed, and then infused into the patient as sipuleucel-T, with each patient receiving three infusions. T lymphocyte activation markers, CD134 (OX 40), CD137 (4-1BB), CD278 (ICOS), and CD279 (PD-1) were measured before and after culture at weeks 0 (infusion1), 2 (infusion 2) and 4 (infusion 3) using multi-color flow cytometry. Cytokines, including IFNγ, IL-4, IL-5, IL-2, and IP-10, were measured in the culture medium at each of the three treatments using the MSD cytokine detection system. Summary: In both CD4+ and CD8+ T cell subsets, we observed increases in the percent of CD134+ and CD137+ cells, but minimal change in CD278+ population, following culture with PA2024 at each infusion of sipuleucel-T. The percent of CD8+CD279+ T lymphocytes increased slightly following culture at each infusion of sipuleucel-T; CD4+CD279+ T lymphocytes increased minimally after the first infusion, but decreased thereafter. Additionally, cytokines associated with T lymphocyte activation, such as IL-2, IL-4, IL-5, IFNγ, and IP-10, were detected during culture with PA2024, with the greatest increase between the first and second infusion.Conclusions: Collectively, these data demonstrate T lymphocyte activation is a consequence of ex vivo culture with PA2024 and subsequent infusion of sipuleucel-T. The combined T lymphocyte activation and cytokine profile suggest the first infusion of sipuleucel-T primes the immune system in vivo, while the subsequent infusions boost the immune system. Furthermore, during the course of treatment with sipuleucel-T, the upregulation of activation markers demonstrate the functional differentiation of T lymphocytes for facilitating antibody production (OX 40 expression) and enhancement of costimulatory interactions with peripheral monocytes (4-1BB expression). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5509. doi:10.1158/1538-7445.AM2011-5509

Abstract 5608: Sipuleucel-T treatment results in sequential ex vivo activation of APCs and T cells during the culture step - evidence for in vivo immunological priming

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Introduction. Sipuleucel-T is an investigational autologous immunotherapy for the treatment of prostate cancer. Product potency is measured in part by antigen presenting cell (APC) expression of CD54. Previous data have shown that CD54 upregulation correlates with APC activation (Sheikh 2008). This study investigated the ex vivo activation profile of T cells from men with metastatic castrate resistant prostate cancer enrolled in a Phase 3 study (D9902B, IMPACT) evaluating the safety and efficacy of sipuleucel-T treatment. Methods. Sipuleucel-T was prepared by incubating mononuclear cells isolated from leukapheresis obtained at Wks 0, 2, and 4 with PA2024 (a recombinant fusion protein comprising prostatic acid phosphatase and GM-CSF) ex vivo for 2 days, after which washed cells were infused. 512 subjects were randomized to sipuleucel-T or placebo (2:1). As part of product characterization efforts, fresh pre- and post-culture (final product - FP) T cells were evaluated in sipuleucel-T products by flow cytometry. The culture supernatant was evaluated for the production of APC and T cell activation-associated cytokines by multiplex luminex assay. Results. APC activation as measured by CD54 upregulation increased from Wk 0 to Wk 2 (P < 0.001) and persisted at the third dose (P < 0.001, Wk 0 vs Wk 4). An analysis of the culture supernatant at each week showed increases in both APC activation-associated cytokines (IL-1α, IL-1β, IL-10, IL-12p70, IL-23, IFNγ, and TNFα) and T cell activation-associated cytokines (IL-2, IL-4, IL-5, IL-10, IL-17, IFNγ, and TNFα). CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), and CD279 (PD-1) expression increased in both CD4+ and CD8+ T cells at Wk 2, with expression diminished at Wk 4. Conclusion. Following The Wk 0 dose, sipuleucel-T products demonstrate evidence of increased APC and T cell activation markers as well as production of T cell activation-associated cytokines. These data provide strong evidence that sipuleucel-T activates APCs ex vivo and facilitates in vivo and ex vivo priming of T cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5608.

Abstract 2932: Sipuleucel-T generates robust and persistent cellular and humoral immune responses - Results from the IMPACT trial

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Introduction. Sipuleucel-T is a patient-specific autologous cell product consisting of antigen presenting cells (APCs) loaded with a recombinant fusion protein (PA2024) comprising prostatic acid phosphatase (PAP) linked to GM-CSF. Reported here are immune response data from a randomized Phase 3 trial, D9902B (IMPACT). Methods. 512 subjects with metastatic castrate resistant prostate cancer were randomized (2:1) to receive sipuleucel-T or placebo intravenously every 2 Wks x 3. Serum and peripheral blood mononuclear cells (PBMCs) were obtained at Baseline and at Wks 6, 14, and 26 and cryopreserved; all samples from a single subject were evaluated in the same assay. Humoral responses in cryopreserved subject serum were assessed by ELISA. Cellular responses were assessed by IFNγ ELISPOT and 3H-thymidine T cell proliferation assays. Results. The median CD54 upregulation ratio increased after culture at all 3 treatment weeks in the sipuleucel-T, but not the placebo, arm. The magnitude of APC activation was significantly greater at Wk 2 (10.8-fold) and Wk 4 (11.0-fold) compared to Wk 0 (both P 400) increased after treatment with sipuleucel T, rising from 2.0% at baseline to 73.9% at Wk 14. Titers for anti-PA2024 and anti-PAP increased significantly from Wk 0 to Wk 6 after treatment with sipuleucel-T (both P 10 spots) increased after treatment with sipuleucel-T. The maximal value observed was 50.8% of subjects at Wk 6 compared with 5.7% at baseline, and specific responses were present at Wk 26. There was little increase in the proportion of subjects with PAP-specific IFNγ ELISPOT responses from Wk 0 to Wk 6. 78.6% of sipuleucel-T subjects mounted anti-PA2024 proliferative responses (stimulation index [SI] > 5). T cell proliferative responses increased significantly from Wk 0 to Wk 6 (P < 0.001) and remained above Wk 0 levels at Wk 26 (SI = 61.5). The PAP-specific proliferative response rose from Wk 0 to Wk 6 (P = 0.071), and remained above Wk 0 levels at Wk 26 (SI = 30.5). Conclusion. Immunogen and PAP-specific responses were observed after treatment with sipuleucel-T but not placebo. The majority of subjects treated with sipuleucel-T had persistent cellular and humoral responses (26 Wks). In addition the humoral class switching data suggest the establishment of a memory response. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2932.