Grant Number: 5R01CA129350
Principal Investigator: Karolina Palucka, MD, PhD
Funding Organization: National Cancer Institute
Project Start: July 11, 2008
Project End: May 31, 2012

Abstract:

Stage IV melanoma represents an unmet medical need. Five-year survival rates in patients with visceral metastases are in the range of 5%. Adoptive T cell transfer can lead to objective clinical responses in a fraction of patients, thus confirming the role for T cell immunity in the control of metastatic melanoma. However, passive immunotherapy is not expected to yield long-lasting immune memory. Active immunotherapy with dendritic cell (DCs)-based vaccines has the potential to induce tumor-specific effectors resulting in tumor rejection and memory T cells that might control tumor outgrowth. Our results from clinical trials testing first generation DC vaccines demonstrate:

• Induction of melanoma-specific CD8+ T cells in patients with stage IV melanoma vaccinated with melanoma-antigen loaded DCs.
• Induction of durable objective responses in a fraction of patients (10%) including patients who failed other therapies.

These results provide a proof-of-principle that therapeutic immunity can be elicited by ex vivo generated DC vaccines. However, we need to improve their efficacy. Two immune parameters appear linked to clinical outcome of the patients: 1) objective clinical response is associated with induction of melanoma-specific effector cells; and 2) all patients display melanoma-specific regulatory T cells (Tregs) that may counteract effector cells. Thus, we need to identify the next generation DC vaccines able to tilt the balance towards strong effector cells and to overcome Tregs. We surmise that this can be achieved via improving the DC vaccine, and most particularly the capacity of DCs to prime CD8+ T cells. According to current concepts, DC undergo an initial activation/maturation process regulated by microbial (TLR ligands) and microenvironmental signals including surrounding cells and their products. Subsequently, DC migrate to secondary lymphoid organs where the second phase of their maturation takes place in response to T cell derived signals, for example CD40 ligation. Thus, mimicking the natural response to microbes may be optimal for increasing DC vaccine immunogenicity. Our preliminary results demonstrate that LPS signaling generates potent DC for CD8+ T cell responses. Furthermore, LPS activation of DCs permits expansion/activation of γ/δ T cells, a subset of innate lymphocytes not generally activated by other types of DCs. In turn, γ/δ T cells improve cross-priming by DCs. Altogether, these results form the basis for our hypothesis:

LPS-activated IFN-DCs will improve clinical outcomes in patients with stage IV melanoma
through the activation of cytotoxic effectors cells including conventional CD8+ T cells, NK cells, NKT cells and γ/δ T cells. This diverse repertoire of cytotoxic effector cells will overcome Tregs.

Aim 1: To determine whether LPS activated IFN-DC vaccine generates the immune and clinical response in patients with stage IV melanoma. We propose a phase II single-arm two-center clinical trial in patients with stage IV melanoma who will receive IFN-DCs loaded with killed allogeneic melanoma cells and activated with LPS. The primary endpoint is the rate of confirmed objective clinical responses as measured by RECIST criteria at Week 24. Clinical response is defined as any PR or CR at Week 24. The trial will enroll up to 35 patients as per two-stage Simon design to detect 17% objective response rate with 90% power (18 patients in stage 1 and 17 patients in stage 2). Secondary clinical outcomes are: i) progression-free survival at 24, 48 and 96 weeks; and ii) overall survival at 24, 48 and 96 weeks. Secondary immune outcome is the induction of MART-1-specific CD8+ T cell immunity at week 24 (Aim 2). We will analyze whether there is a correlation between the immune and clinical outcomes. Tertiary immune outcomes include: i) the ability of elicited CD8+ T cells to proliferate and to kill melanoma cells in vitro (Aim 2); ii) induction of MART-1 specific CD4+ T cell immunity (Aim 3); and iii) MART-1 specific IL-10 secretion as a measure of inducible Tregs; and the frequency of CD4+CD25high T cells by multicolor flow cytometry as a measure of naturally occurring Tregs (Aim 3). We will also analyze innate lymphocytes NK cells, NKT cells and γ/δ T cells (Aim 4).

Aim 2: To determine whether LPS activated IFN-DC vaccine generates MART-1-specific CD8+ T cell immunity. This will be assessed using peptide libraries covering the whole sequence of MART-1 protein and EPIMAX. The frequency of MART-1 peptide-specific circulating effector CD8+ T cells will be measured by flow cytometry (IFN-γ) and confirmed by tetramer. We will analyze a correlation between the induction of MART-1-specific CD8+ T cells and clinical outcomes. We will also analyze CTL function of elicited CD8+ T cells and their longevity in longitudinal analysis.

Aim 3: To determine the type and breadth of CD4+ T cell responses elicited by vaccination with LPS activated IFN-DCs. Specific epitopes generating secretion of IFN-γ or IL-10 will be determined by EPIMAX. Frequency will be assessed by intracellular IFN-γ /IL-10 and confirmed by tetramers. We will measure the frequency of CD25high, FoxP3high and CTLA-4high T cells. Because CD4+ T cell immunity appears essential for generation of CD8+ T cell memory and can regulate the effector function of CD8+ T cells (Tregs), we will analyze the correlation between elicited melanoma-specific CD4+ and CD8+ T cells.

Aim 4: To determine whether vaccination with LPS activated IFN-DCs leads to expansion and activation of innate cytotoxic effector cells. We will measure the expansion of NK cells, NKT cells and γ/δ T cells in vivo in vaccinated patients by flow cytometry using specific markers. While the role of these innate lymphocytes in the direct control of melanoma growth would need to be established, we propose herein to exploit their potentially beneficial effect on the DC vaccine. Therefore, we will analyze correlation between the secondary endpoint, i.e., the frequency of MART-1-specific CD8+ T cells, the induction of which is a measure of DC vaccine efficacy, and activation of innate immune effectors.