Tsin Yee Tai

Blood dendritic cells generated with Flt3 ligand and CD40 ligand prime CD8+ T cells efficiently in cancer patients.

Flt3 ligand mobilizes dendritic cells (DCs) into blood, allowing generation in vivo of large numbers of DCs for immunotherapy. These immature DCs can be rapidly activated by soluble CD40 ligand (CD40L). We developed a novel overnight method using these cytokines to produce DCs for cancer immunotherapy. Flt3 ligand-mobilized DCs (FLDCs) were isolated, activated with CD40L, loaded with antigenic peptides from influenza matrix protein, hepatitis B core antigen, NY-ESO-1, MAGE-A4, and MAGE-A10, and injected into patients with resected melanoma. Three injections were given at 4-week intervals. Study end points included antigen-specific immune responses (skin reactions to peptides alone or peptide-pulsed FLDCs; circulating T-cell responses), safety, and toxicity. No patient had a measurable tumor. Six patients were entered. FLDCs were obtained, enriched, and cultured under Good Manufacturing Practice grade conditions. Overnight culture with soluble CD40L caused marked up-regulation of activation markers (CD83 and HLA-DR). These FLDCs were functional and able to stimulate antigen-specific T cells in vitro. No significant adverse events were attributable to FLDCs. Peptide-pulsed FLDCs caused strong local skin reactions up to 60 mm diameter with intense perivascular infiltration of T cells, exceeding those seen in our previous peptide-based protocols. Antigen-specific blood T-cell responses were induced, including responses to an antigen for which the patients were naive (hepatitis B core antigen) and MAGE-A10. MAGE-A10–specific T cells with a skewed T-cell receptor repertoire were detected in 1 patient in blood ex vivo and from tumor biopsies. Vaccination with FLDCs pulsed with peptides is safe and primes immune responses to cancer antigens.

Dissecting cytotoxic T cell responses towards the NY-ESO-1 protein by peptide/MHC-specific antibody fragments

NY‐ESO‐1 is a germ cell antigen aberrantly expressed by different tumor types that elicits strong humoral and cellular immune responses, representing one of the most promising candidates for vaccination of cancer patients. A detailed analysis of CD8+ T cells generated in vaccine trials using NY‐ESO‐1‐derived peptides (157–165 and 157–167) revealed that the dominant immune response was directed against a cryptic epitope (159–167) diverting the immune response from tumor recognition. Only CTL reactivity to the NY‐ESO‐1157–165 peptide appeared to be capable of lysing NY‐ESO‐1/HLA‐A0201‐expressing tumor cells. To study the process of NY‐ESO‐1 peptide presentation by tumor cells in more detail we generated a high‐affinity (KD=60 nM) antibody fragment that specifically recognizes the NY‐ESO‐1157–165 peptide/HLA‐A0201 complex. Peptide variants such as the NY‐ESO‐1157–167 peptide or the cryptic NY‐ESO‐1159–167 peptide were not recognized. The antibody fragment blocked in a dose‐dependent fashion the recognition of NY‐ESO‐1/HLA‐A2‐positive tumor cells by NY‐ESO‐1157–165 peptide‐specific CD8+ T cells. This antibody fragment is a novel reagent that binds with TCR‐like specificity to the NY‐ESO‐1157–165/HLA‐A2 complex thus distinguishing between CTL responses against immunological meaningful or cryptic NY‐ESO‐1‐derived peptides. It may therefore become a useful monitoring tool for the development of NY‐ESO‐1‐based cancer vaccines.

Exogenous Peptides Presented by Transporter Associated with Antigen Processing (TAP)-Deficient and TAP-Competent Cells: Intracellular Loading and Kinetics of Presentation

This study investigates the differential capacity of TAP-deficient T2 cells, TAP-competent EBV cells, and immature and mature dendritic cells to present peptides to preformed CTL lines. It demonstrates that presentation of exogenous peptides involves peptide uptake and loading onto newly synthesized MHC class I molecules. This mechanism was best demonstrated for low affinity peptides in the presence of irrelevant peptides competing for HLA binding sites. Under these circumstances, inhibition of protein synthesis with cycloheximide or vesicular trafficking with brefeldin A significantly reduced the presentation of low affinity peptides. This was not restored by adding exogenous β2-microglobulin to stabilize the MHC complex on the cell surface. In contrast, presentation of high affinity peptides was not sensitive to cycloheximide or brefeldin A, which suggests that different mechanisms may operate for presentation of high and low affinity peptides by TAP-competent cells. High affinity peptides can apparently compete with peptides in preloaded MHC class I molecules at the cell surface, whereas low affinity peptides require empty MHC molecules within cells. Accordingly, very high concentrations of exogenous low affinity peptides in conjunction with active MHC class I metabolism were required to allow successful presentation against a background of competing intracellular high affinity peptides in TAP-competent cells. These findings have implications for the design of peptide and protein-based vaccines.

Striking Immunodominance Hierarchy of Naturally Occurring CD8+ and CD4+ T Cell Responses to Tumor Antigen NY-ESO-1

Immunodominance has been well-demonstrated in many antiviral and antibacterial systems, but much less so in the setting of immune responses against cancer. Tumor Ag-specific CD8+ T cells keep cancer cells in check via immunosurveillance and shape tumor development through immunoediting. Because most tumor Ags are self Ags, the breadth and depth of antitumor immune responses have not been well-appreciated. To design and develop antitumor vaccines, it is important to understand the immunodominance hierarchy and its underlying mechanisms, and to identify the most immunodominant tumor Ag-specific T cells. We have comprehensively analyzed spontaneous cellular immune responses of one individual and show that multiple tumor Ags are targeted by the patient’s immune system, especially the “cancer-testis” tumor Ag NY-ESO-1. The pattern of anti-NY-ESO-1 T cell responses in this patient closely resembles the classical broad yet hierarchical antiviral immunity and was confirmed in a second subject.

A cytotoxic anti-IL-3Rα antibody targets key cells and cytokines implicated in systemic lupus erythematosus

To date, the major target of biologic therapeutics in systemic lupus erythematosus (SLE) has been the B cell, which produces pathogenic autoantibodies. Recently, targeting type I IFN, which is elaborated by plasmacytoid dendritic cells (pDCs) in response to endosomal TLR7 and TLR9 stimulation by SLE immune complexes, has shown promising results. pDCs express high levels of the IL-3Rα chain (CD123), suggesting an alternative potential targeting strategy. We have developed an anti-CD123 monoclonal antibody, CSL362, and show here that it affects key cell types and cytokines that contribute to SLE. CSL362 potently depletes pDCs via antibody-dependent cell-mediated cytotoxicity, markedly reducing TLR7, TLR9, and SLE serum-induced IFN-α production and IFN-α-upregulated gene expression. The antibody also inhibits TLR7- and TLR9-induced plasmablast expansion by reducing IFN-α and IL-6 production. These effects are more pronounced than with IFN-α blockade alone, possibly because pDC depletion reduces production of other IFN subtypes, such as type III, as well as non-IFN proinflammatory cytokines, such as IL-6. In addition, CSL362 depletes basophils and inhibits IL-3 signaling. These effects were confirmed in cells derived from a heterogeneous population of SLE donors, various IFN-dependent autoimmune diseases, and healthy controls. We also demonstrate in vivo activity of CSL362 following its s.c. administration to cynomolgus monkeys. This spectrum of effects provides a preclinical rationale for the therapeutic evaluation of CSL362 in SLE.

A pilot study of peripheral blood BDCA-1 (CD1c) positive dendritic cells pulsed with NY-ESO-1 ISCOMATRIX™ adjuvant

AIM Pilot clinical trial of NY-ESO-1 (ESO) protein in ISCOMATRIX™ adjuvant pulsed onto peripheral blood dendritic cells (PBDC), to ascertain feasibility, evaluate toxicity and assess induction of ESO-specific immune responses. PATIENTS & METHODS Eligible participants had resected cancers expressing ESO or LAGE-1 and were at high risk of relapse. PBDC were produced using CliniMACS®plus, with initial depletion of CD1c+ B cells followed by positive selection of CD1c+ PBDC. Patients received three intradermal vaccinations of ESO/IMX-pulsed PBDC at 4-week intervals. RESULTS The process was feasible and safe. No vaccine-induced immune responses were detected. Assays of immunomodulatory cells did not correlate with outcomes. One patient had a long lasting complete remission. CONCLUSION This method was feasible and safe but was minimally immunogenic.

Abstract 5619: Natural killer cell function in multiple myeloma is impaired by dexamethasone and can not be rescued by immunomodulatory drugs

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The immunomodulatory (IMiD) drug Lenalidomide (Len) is known to stimulate natural killer (NK) cells and enhance anti-tumor responses. Len plus Dexamethasone (Dex) therapy (Len-Dex) is an effective treatment for multiple myeloma (MM). As part of a prospective clinical trial of monthly cycles of Len-Dex therapy we assessed NK cell number and function in 25 MM patients. Whilst NK numbers increased from a mean of 2×105/ml (baseline) to a mean of 3.9×105/ml (cycle 6) (p=0.05), the mean NK cell-mediated cytotoxicity in chromium assays against K562 significantly decreased from 48.9% to 27.6% respectively (p=0.0028). Further analysis showed that when NK cell cytotoxicity was assessed from MM patient peripheral blood mononuclear cells (PBMC) during therapy, no enhancement of cytotoxicity could be observed when cultured in vitro with Len. We hypothesized that Dex potently suppressed the Len-mediated enhancement of NK function. In vitro, Len increased normal donor (n=6) NK cytotoxicity from 38.5% (untreated) to 53.3%. Dex severely impaired NK cytotoxicity to <10%, this failed to increase (<13%) in the presence of Len. Only the addition of high dose IL-2 (500U/ml) rescued cytotoxicity of Dex-treated NK cells (28.6%). The suppressive effect of Dex on NK cytotoxicity was not permanent as NK function returned to normal levels within 3 days of drug removal. However, these dex-‘washout’ PBMCs could no longer respond to Len with increased NK function, suggesting a permanent Dex-induced NK impairment. In cell sorting and co-culture experiments we found that Len enhancement of NK cytotoxicity was only mediated by CD4+ T cells (NK alone 76.6% vs NK+CD4 85%) and the effect of CD4+ T cells was IL-2 dependent. Furthermore IL-2 production by CD4+ T cells in response to anti-CD3/CD28 was also suppressed by Dex. Following Len-Dex therapy in patients we observed less IL-2 and IFN-γ production from T cells in response to anti-CD3/CD28 at cycle 6 compared to baseline. Therefore, Len-mediates enhanced NK function through CD4+ T cell production of IL-2. The NK impairment observed in MM patients receiving Len-Dex is due to CD4+ T cell suppression. Our findings indicate that in order to harness both the anti-tumor and immunomodulatory capacity of Len the dose and timing of Dex therapy should be carefully reconsidered. 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 5619.