SUPPLEMENTAL MATERIAL: CLINICAL ASPECTS OF inkt CELL BIOLOGY

Transcription

1 SUPPLEMENTAL MATERIAL: CLINICAL ASPECTS OF inkt CELL BIOLOGY Injection of α-galcer in Phase I Clinical Trials The first in-human study was a Phase I trial using intravenous injection of α-galcer at a range of doses, from 50 4,800 mg/m 2, in patients with solid tumors (1). α-galcer was administered on days one, eight, and fifteen in a four-weekly cycle, and in the absence of contraindications, the cycle of immunization continued. As previously observed in murine models of α-galcer immunization, a rapid (within 24 h) loss of inkt cells was detected, likely due to transient TCR downregulation as a result of activation (2). Giaccone et al. (1) also described elevated serum levels of several cytokines including TNF-α and GM-CSF in a number of the patients, which correlated with the frequency of inkt cells prior to vaccination. No toxicity was observed, suggesting that activation of inkt cells through intravenous injection of α-galcer is a safe, well-tolerated treatment modality. Although no partial or complete tumor responses were observed, disease was stabilized in a proportion of the patients examined (1). It is important to note, however, that the frequency of inkt cells described in the cancer patients in this study was lower than in healthy donors, an observation that has been confirmed in other studies (3). When this initial study was undertaken, the concept of inkt cell anergy had not yet been reported (4). Repeated injection of soluble [but not cell-associated (5)] α-galcer in mice, with a short interval between administrations, led to a state in which inkt cells were no longer able to respond to a subsequent ligand challenge, a phenomenon that can last for around four weeks and was shown to depend on the PD-1/PD-L1 axis (4, 6, 7). Two Phase I/II trials with intravenous α-galcer were performed in patients with chronic Hepatitis B or Hepatitis C (8, 9). In both trials, the time between doses of α-galcer was longer than in the earlier study, with four weeks between each injection. Similar to the observations of Giaccone et al. (1), infusions of α-galcer in both groups of patients were relatively well tolerated at all the doses used, with low-grade fever and flu-like symptoms, most likely as a result of the cytokine release in response to inkt cell activation. α-galcer administration was less well tolerated in the Hepatitis C study, with a number of patients discontinuing treatment. The authors suggested that the initial baseline frequencies of inkt cells in the Hepatitis C patients may have been elevated in comparison with the previous cancer and Hepatitis B cohorts, potentially leading to increased side effects (9). Both studies reported a decrease in the blood frequency of inkt cells post α-galcer injection and an increase in cytokines, particularly TNF-α and IL-6, in some of the patients. 1

2 However, only a small number of patients in both trials showed evidence of either a transient or sustained decrease in viral DNA in response to α-galcer. These data suggested that inkt cell activation had little or no effect on viral titers and may not be a viable therapeutic strategy in such settings. α-galcer-pulsed Antigen-Presenting Cells By far the largest number of clinical trials have utilized ex vivo generated or isolated antigen-presenting cells (APCs) pulsed with α-galcer. Studies in mice demonstrated that administration of α-galcer-pulsed DCs induced a longer lasting cytokine response compared with injection of free α-galcer (10). Possible reasons for the observed differences include a more effective dose of agonist being presented by the DC, presentation of α-galcer by different CD1d-positive APCs, and the different routes of administration. Indeed, α-galcerpulsed DCs injected subcutaneously in mice did not stimulate a particularly effective inkt cell response, whereas intravenous injection induced a strong cytokine response. Furthermore, presentation of α-galcer by different APCs in vivo led to very different functional responses: DCs were found to stimulate stronger inkt cell responses than B cells, whereas B cells only stimulated Th2 cytokines and could also inhibit the DC-iNKT cell activation (11). However, targeting of inkt cell agonist directly to B cells, for BCRmediated uptake, efficiently elicits inkt cell activation and antibody responses to codelivered antigens (12). The first full-scale Phase I trial using intravenous administration of autologous immature monocyte-derived DCs pulsed with -GalCer was performed in patients with metastatic tumors (13). In a pilot study, the same group vaccinated four patients with metastatic malignancies of different origins with α-galcer-pulsed monocyte-derived DCs injected twice intravenously and twice intradermally (14). Each patient received DCs/injection, which were tolerated with no serious adverse events; an increase in the frequency of inkt cells in peripheral blood following an initial transient fall in numbers immediately postimmunization was also observed. In the larger-scale study, patients received an intravenous injection of α-galcer-pulsed immature DCs, at two weekly intervals (13). A transient decrease in the frequency of inkt, NK, T, and B cells was observed along with increased serum levels of cytokines including IFN- and IL-12 and the trans-activation of both T and NK cells. The DCs were tracked, and although initially found in the lungs, they migrated 2

3 rapidly to the liver and spleen. The authors also reported a decrease in serum tumor markers in 2 out of the 12 patients enrolled, indicating a possible antitumor effect. Two subsequent Phase I trials were published in 2005, both using monocyte-derived DCs pulsed with α-galcer. In one trial, immature DCs cultured in the presence of IL-2 were injected weekly intravenously in 11 patients with advanced or recurrent non-small-cell lung cancer (15); in the second trial, DCs matured with a cocktail of inflammatory cytokines (IL- 1β, IL-6, TNF-α, and PGE-2) and pulsed with α-galcer were injected in 5 patients with myeloma, squamous cancer, or renal cell cancer (5). As previously, DCs were generally well tolerated in both trials, with up to DCs/m 2 being injected in patients with non-smallcell cancer. The trial in non-small-cell cancer patients described an initial transient drop in blood inkt cell frequency after the first injection of DCs, followed by an expansion after the second injection. In this study, cytokine release was not measured directly, but IFN-γ RNA was quantified by qrt-pcr from sorted inkt cells and was increased after the first and second injection of DCs, but undetectable after subsequent injections, suggesting that inkt cells had become anergized (15). In the trial with matured DCs (5), patients initially received unpulsed DCs to establish a baseline response, and a reduced frequency of inkt cells was reported. α-galcer-pulsed DCs were then injected one month apart, with detailed immunomonitoring carried out. Injection of α-galcer-loaded DCs led to an increase of more than 100-fold in the frequency of inkt cells in all the patients, peaking between 7 and 30 days after injection and in some cases for more than 6 months. The authors analyzed multiple serum cytokines and demonstrated a consistent increase in the levels of IL-12p40, IP-10, and MIP-1β. The activation status of antigen-specific memory CD8 + T cells, reactive to either CMV or Influenza peptides, were assessed by HLA-tetramer staining and ELISpot. Injection of α- GalCer-pulsed DCs was associated with an increase in the frequency of IFN-γproducing CMV-specific CD8 + T cells but not a concomitant increase in the frequency of influenzaspecific T cells. These data together with the elevation in IL-12 suggest that injection of α- GalCer-pulsed DCs leads to activation of APCs in vivo, and boosting of T cell responses specific for persistent antigens. In each of the three myeloma patients enrolled in this study, the authors described a decrease in the concentration of M protein in the serum or urine, suggesting a positive effect on tumor growth. The fourth published Phase I clinical trial where α-galcer-pulsed DCs were injected enrolled nine patients with head and neck cancer (16). Patients were injected twice with 1 3

4 10 8 immature APCs directly into the nasal submucosa with a one-week interval between injections. The authors described an increase in the absolute number of inkt cells in the peripheral blood in 4 out of 9 patients, whereas a slight reduction was observed in the remaining cases. There was also clear evidence for enhanced NK cell activity in 8 out of 9 patients. The cells used for injection were a mixture of APCs derived from peripheral blood mononuclear cells, and the percentage of DCs administered in each case ranged from 30 55%. A decrease in tumor diameter in one patient was observed (from 22 to 7 mm), while there was no progression in disease in a further 5 patients. A follow-up trial was conducted in 17 patients, also with head and neck cancer injected with APCs pulsed with α-galcer (17). APCs were injected directly either into the nasal or the oral submucosa and were also labeled, allowing them to be tracked after administration. More APCs were detected in the lymph nodes after administration via the oral submucosa compared with the nasal submucosa, peaking around 48 h after injection. However, a greater increase in the absolute number of inkt cells was observed in the patients via the nasal compared with the oral route. Unfortunately, there was also evidence for an increased frequency of CD4 + /CD25 + FoxP3 + regulatory T cells after injection into the oral mucosa, indicating that despite or possibly because of the presence of activated inkt cells, there may be suppressive effects of antigen administration that are not overcome by injection of α- GalCer. More recently, four more studies were published, in which patients with different cancers were injected with APCs pulsed with α-galcer either intravenously or intradermally (18 21). Motohashi et al. (18) described the results of a large Phase I-II trial with 23 patients with non-small-cell lung cancer injected with APCs generated in the presence of GM-CSF and IL- 2, as previously described (15). Of the 23 patients enrolled, 17 completed the treatment of four intravenous injections of up to cells/m 2 of α-galcer-pulsed cells. The therapeutic regime was well tolerated and led to expansion and activation of inkt cells in some of the patients. More importantly, in the patients with increased IFN-γ production, a prolongation in survival was observed: 10 patients (more than 50% of the patients who completed the trial) who displayed an increase in the frequency of IFN-γ-producing cells of more than twofold compared with baseline levels had a median survival time of nearly 32 months. This compared favorably with the non- or poorly responding patients who had a median survival time of around 10 months, indicating that activation of inkt cells in vivo can lead to a significant enhancement in patient survival. 4

5 Nicol et al. (19) conducted a Phase I trial in patients with metastatic disease of different origins, comparing different numbers of immature monocyte-derived DCs and routes of administration (intravenous and intradermal). Minor side effects were observed, and the treatments were relatively well tolerated by the patients. A number of patients described pain and tenderness and other symptoms associated with inflammation where metastasis had been diagnosed, suggesting that inkt cell activation had led to inflammatory responses to the tumors. Interestingly, tumor flares were also experienced by a number of patients in anatomical locations harboring metastases previously unknown. Such responses were more common in patients that received intravenous DCs compared with intradermal injection. Activation of inkt cells was also more pronounced when DCs were injected intravenously. Six out of 10 of the patients experienced either a minor response (indicated by a reduction in tumor mass or a decrease in tumor-associated biomarkers) or a stabilization of disease progression, an improvement compared with the progressive disease prior to treatment initiation. In a small-scale trial with four patients with non-small-cell lung cancer, GM-CSF/IL-2 generated, α-galcer-pulsed APCs were injected intravenously 7 days prior to surgery to remove the primary tumors. At the time of surgery, an increase in both inkt numbers and IFN-γ production was described, specifically amongst tumor-infiltrating lymphocytes (TILs) (20). Recently, six patients with myeloma were injected with α-galcer-pulsed DCs and treated at the same time with the immunomodulatory drug lenalidomide, which among other effects enhances T cell activation (21). The authors suggested that by administering a lower dose of lenalidomide than is usually used, together with inkt cell agonists, the two treatment regimens may work together. An initial activation-induced decrease in inkt cell numbers was observed, together with activation of NK cells, as demonstrated by increased expression of NKG2D and CD56. The authors also observed concomitant monocyte activation and an increase in eosinophil numbers in the peripheral blood. The serum levels of soluble IL-2 receptor were also increased. Clinical responses, as indicated by a decrease in the tumorassociated monoclonal Ig levels (M spike) were observed in three of the four patients that had a detectable M spike prior to treatment. Together these data suggest that activation of inkt cells has significant potential for being combined with other immune therapies for the treatment of cancers. In Vitro Activated inkt Cells and Combination Therapies 5

6 Another monotherapy approach used in the clinic is based on ex vivo expanded, adoptively transferred inkt cells sourced from the patient. Patients with non-small-cell lung cancer received inkt cells that had been activated in vitro with α-galcer and IL-2 (22). The therapy was well tolerated, increased frequencies of inkt cells were observed, and there was evidence for downstream activation of NK cells to produce elevated levels of IFN-γ. In two other clinical trials (a Phase I trial and a subsequent Phase II trial, both in patients with head and neck squamous cell carcinoma), combination approaches were assessed (23, 24). Patients received ex vivo activated inkt cells intravenously and APCs pulsed with α- GalCer into the nasal submucosa. The frequency of inkt cells was increased in many of the patients, and there was evidence for inkt cell activation. In the Phase I study, 7 of the 8 patients demonstrated either a partial response or stabilization of disease, whereas in the Phase II trial 5 of the 10 patients had evidence for tumor regression that was associated with an increased frequency of inkt cells within the tumor (TILs). Infusion of a large number of inkt cells together with suitable APCs pulsed with α-galcer is therefore an appealing approach, as it could overcome the reduced frequency of inkt cells described in cancer patients. In mice, functionally competent inkt cells have been differentiated from induced pluripotent stem cells (ipscs), and this may represent an approach to expand inkt cells for cancer therapy in humans (25). Although ipsc-derived inkt cells were shown to release IFN-γ and to suppress tumor growth in vivo, it remains to be demonstrated whether they express PLZF and retain innate-like properties. Further studies have also been performed investigating the role of inkt cells in hematopoietic stem cell transplantation (reviewed in 26). Patients without a complete reconstitution of the inkt cell compartment have an increased rate of leukemia relapse, suggesting a role for inkt cells in the control of both the graft-versus-leukemia (GVL) and graft-versus-host disease (GVHD) responses. LITERATURE CITED 1. Giaccone G, Punt CJ, Ando Y, Ruijter R, Nishi N, et al A phase I study of the natural killer T-cell ligand α-galactosylceramide (KRN7000) in patients with solid tumors. Clin. Cancer Res. 8: Crowe NY, Uldrich AP, Kyparissoudis K, Hammond KJ, Hayakawa Y, et al Glycolipid antigen drives rapid expansion and sustained cytokine production by NK T cells. J. Immunol. 171:

7 3. Shimizu K, Hidaka M, Kadowaki N, Makita N, Konishi N, et al Evaluation of the function of human invariant NKT cells from cancer patients using α-galactosylceramideloaded murine dendritic cells. J. Immunol. 177: Parekh VV, Wilson MT, Olivares-Villagomez D, Singh AK, Wu L, et al Glycolipid antigen induces long-term natural killer T cell anergy in mice. J. Clin. Invest 115: Chang DH, Osman K, Connolly J, Kukreja A, Krasovsky J, et al Sustained expansion of NKT cells and antigen-specific T cells after injection of α-galactosylceramide loaded mature dendritic cells in cancer patients. J. Exp. Med. 201: Parekh VV, Lalani S, Kim S, Halder R, Azuma M, et al PD-1/PD-L blockade prevents anergy induction and enhances the anti-tumor activities of glycolipid-activated invariant NKT cells. J. Immunol. 182: Silk J, Salio M, Reddy BG, Shepherd D, Gileadi U, et al Cutting edge: nonglycosidic CD1d lipid ligands activate human and murine invariant NKT cells. J. Immunol. 180: Veldt BJ, van der Vliet HJ, von Blomberg BM, van Vlierberghe H, Gerken G, et al Randomized placebo controlled phase I/II trial of α-galactosylceramide for the treatment of chronic hepatitis C. J. Hepatol. 47: Woltman AM, Ter Borg MJ, Binda RS, Sprengers D, von Blomberg BM, et al α- galactosylceramide in chronic hepatitis B infection: results from a randomized placebocontrolled Phase I/II trial. Antivir. Ther. 14: Fujii S, Shimizu K, Kronenberg M, Steinman RM Prolonged IFN-γ-producing NKT response induced with α-galactosylceramide-loaded DCs. Nat. Immunol. 3: Bezbradica JS, Stanic AK, Matsuki N, Bour-Jordan H, Bluestone JA, et al Distinct roles of dendritic cells and B cells in Va14Ja18 natural T cell activation in vivo. J. Immunol. 174: Barral P, Eckl-Dorna J, Harwood NE, De Santo C, Salio M, et al B cell receptormediated uptake of CD1d-restricted antigen augments antibody responses by recruiting invariant NKT cell help in vivo. Proc. Natl. Acad. Sci. USA 105: Nieda M, Okai M, Tazbirkova A, Lin H, Yamaura A, et al Therapeutic activation of Vα24 + Vβ11 + NKT cells in human subjects results in highly coordinated secondary activation of acquired and innate immunity. Blood 103:

8 14. Okai M, Nieda M, Tazbirkova A, Horley D, Kikuchi A, et al Human peripheral blood Vα24 + Vβ11 + NKT cells expand following administration of α-galactosylceramidepulsed dendritic cells. Vox Sang. 83: Ishikawa A, Motohashi S, Ishikawa E, Fuchida H, Higashino K, et al A phase I study of α-galactosylceramide (KRN7000)-pulsed dendritic cells in patients with advanced and recurrent non-small cell lung cancer. Clin. Cancer Res. 11: Uchida T, Horiguchi S, Tanaka Y, Yamamoto H, Kunii N, et al Phase I study of α- galactosylceramide-pulsed antigen presenting cells administration to the nasal submucosa in unresectable or recurrent head and neck cancer. Cancer Immunol. Immunother. 57: Kurosaki M, Horiguchi S, Yamasaki K, Uchida Y, Motohashi S, et al Migration and immunological reaction after the administration of αgalcer-pulsed antigenpresenting cells into the submucosa of patients with head and neck cancer. Cancer Immunol. Immunother. 60: Motohashi S, Nagato K, Kunii N, Yamamoto H, Yamasaki K, et al A phase I-II study of α-galactosylceramide-pulsed IL-2/GM-CSF-cultured peripheral blood mononuclear cells in patients with advanced and recurrent non-small cell lung cancer. J. Immunol. 182: Nicol AJ, Tazbirkova A, Nieda M Comparison of clinical and immunological effects of intravenous and intradermal administration of α-galactosylceramide (KRN7000)-pulsed dendritic cells. Clin. Cancer Res. 17: Nagato K, Motohashi S, Ishibashi F, Okita K, Yamasaki K, et al Accumulation of activated invariant natural killer T cells in the tumor microenvironment after α- galactosylceramide-pulsed antigen presenting cells. J. Clin. Immunol. 32: Richter J, Neparidze N, Zhang L, Nair S, Monesmith T, et al Clinical regressions and broad immune activation following combination therapy targeting human NKT cells in myeloma. Blood 121: Motohashi S, Ishikawa A, Ishikawa E, Otsuji M, Iizasa T, et al A phase I study of in vitro expanded natural killer T cells in patients with advanced and recurrent non-small cell lung cancer. Clin. Cancer Res. 12: Kunii N, Horiguchi S, Motohashi S, Yamamoto H, Ueno N, et al Combination therapy of in vitro-expanded natural killer T cells and α-galactosylceramide-pulsed 8

9 antigen-presenting cells in patients with recurrent head and neck carcinoma. Cancer Sci. 100: Yamasaki K, Horiguchi S, Kurosaki M, Kunii N, Nagato K, et al Induction of NKT cell-specific immune responses in cancer tissues after NKT cell-targeted adoptive immunotherapy. Clin. Immunol. 138: Watarai H, Fujii S, Yamada D, Rybouchkin A, Sakata S, et al Murine induced pluripotent stem cells can be derived from and differentiate into natural killer T cells. J. Clin. Investig. 120: Dellabona P, Casorati G, de Lalla C, Montagna D, Locatelli F On the use of donorderived inkt cells for adoptive immunotherapy to prevent leukemia recurrence in pediatric recipients of HLA haploidentical HSCT for hematological malignancies. Clin. Immunol. 140: