Effects of TLR agonists on maturation and function of 3-day dendritic cells from AML patients in complete remission

Acute myeloid leukemia (AML) is the most common acute leukemia in adults, with a poor prognosis and an overall survival (OS) rate of only 23.6% at 5 years (SEER data). Current risk-adapted treatment approaches are determined by several parameters, including cytogenetic characteristics of AML, molecular genetics, age, initial blast count, early blast clearance, and performance status. Although complete remission (CR) rates are high, the majority of patients will suffer from relapse. In the last three decades, various post-remission strategies for elimination of minimal residual disease (MRD) have been developed. The optimal consolidation therapy has not been identified, and patients are offered cytarabine-containing regimens, allogeneic or autologous hematopoietic stem cell transplantation (HSCT), maintenance therapy, and more recently IL-2 in combination with histamine dihydrochloride according to individual risk profile and accompanying morbidity [1, 2]. Allogeneic HSCT was shown to provide a potent immunological anti-leukemic effect, with the lowest rate of relapse and a relevant benefit for overall survival in certain age groups [3]. However, this approach is restricted to a subset of patients due to patient-associated morbidity and mortality, donor availability, recipient comorbidities, or age. Clinical vaccination trials with peptides derived from leukemia-associated antigens like proteinase 3 (PR1), Wilm's tumor gene product 1 (WT-1), and the receptor for hyaluronic acid-mediated motility (RHAMM or CD168) have tried to stimulate autologous anti-leukemic T cell responses and have shown promising results regarding immunogenicity and clinical efficacy [4‐8].

More recently, an active immunization study with WT-1 RNA-transfected autologous DCs showed immunogenic and anti-leukemic activity while overcoming the HLA-restricted approach of peptide vaccination in AML [9].

DCs are recognized as key regulators of the human immune system, with the ability to induce and maintain primary immune responses as well as tolerance in vitro and in vivo [10, 11]. They have been tested as cellular adjuvants for therapeutic vaccination of solid and hematological malignancies in more than 100 clinical trials since 1996 and proven feasibility and safety. Although immune responses, such as induction of tumor-specific T cells, were observed in many studies, overall clinical response rates remain low. The vast majority of DCs used for clinical trials were derived from autologous peripheral blood monocytes and differentiated with a standard maturation cocktail composed of the cytokines TNF-α, IL-1β, IL-6, and PGE₂, therefore they lack the capacity to secrete biologically active IL-12(p70) [12]. For optimal T cell activation, it is required that DCs display peptides within MHC molecules as signal 1 and costimulatory molecules as signal 2. In addition, production of IL-12(p70) as signal 3 is desired because of its leading role in promoting T helper 1 (T_H1) cell polarization and supporting the development of CD8⁺ cytotoxic T lymphocytes, thereby fostering the appropriate adaptive immune responses needed to combat minimal residual disease and control outgrowth of malignant cells in tumor patients [13, 14]. Recently, cocktails containing synthetic TLR agonists emerged as an attractive alternative for the induction of DC maturation [15‐18]. TLRs recognize pathogen-derived signals, and stimulation leads to an induction of a T_H1 immune response via IL-12(p70). Several synthetic TLR agonists could be identified so far. R848 is a low molecular weight synthetic imidazoquinoline compound which activates immune cells via the TLR7/TLR8 MyD88-dependent signaling pathway [17]. Recently, R848 was shown to trigger NF-κB activation in cells expressing murine TLR8 when combined with poly(dT) [19]. CL075 (3M-002) is a thiazoloquinolone derivative that stimulates TLR8 in human peripheral blood mononuclear cells (PBMCs) [20]. It also activates NF-κB and preferentially triggers the production of TNF-α and IL-12(p70). Polyinosine-polycytidylic acid (poly(I:C)) is a synthetic analog of double-stranded RNA (dsRNA), a molecular pattern associated with viral infection. It is composed of a strand of poly(I) annealed to a strand of poly(C) [21]. Poly(I:C) is recognized by TLR3 located mostly in endosomal membranes. It leads to induction of inflammation and long-lasting T cell immunity and matures both mouse and human DCs in a type I IFN dependent fashion [22‐24]. The rational for the choice of TLR-agonists was not only based on in vitro evidence of effective DC activation but also upon consideration of GMP availability for usage in a clinical trial.

Our present investigation was designed to study the feasibility of a clinical grade 3-day DC generation protocol from non-leukemic monocytes of intensively pretreated AML patients. Novel cocktails containing different synthetic TLR-agonists were used for maturation and were analyzed for their phenotype, costimulatory profile, cytokine secretion pattern, migratory capacity and functional capacity to stimulate NK and T cells.

We were able to show that phenotypically and functionally mature DCs can be generated from heavily pretreated patients with AML as compared to healthy controls. The four different cocktails did not translate into significant differences in the patient and donor cohort. We conclude that this approach is feasible and supports the clinical application of autologous DCs after TLR-maturation in patients with AML for the induction of potent innate and adaptive immune responses.

DC-based vaccination has been intensively investigated since the first trial in patients with B cell lymphoma in 1996 [29]. Although DC-based immunotherapies have induced immunologic responses in the majority of trials, thus far only a limited number of clinical responses have been observed. It remains unclear why some patients respond to DC-based immunotherapy and others do not, but it has been suggested that the current protocols used to generate mature DCs may not result in optimal T_H1 and NK cell responses. Also, these trials were mainly performed in late-stage immunosuppressed cancer patients as a result of extensive radiation, chemotherapy and/or large tumor burdens [30]. The unique capacity of DCs to activate and expand different arms of cell-mediated resistance, such as NK, NKT, B and T cells, each of which recognizes different alterations in cancer cells, could be ideally exploited using IL-12(p70) secreting DCs with a positive costimulatory profile.

We therefore, evaluated the generation of such DCs from monocytes of AML patients in complete remission following intensive chemotherapy. As a prerequisite, monocyte counts of AML patients were analyzed before consolidation and maintenance therapy. The mean number of leukocytes and monocytes was comparable to HCs, making the generation of mature DCs feasible from these patients.

The discovery that adjuvants can stimulate innate immunity by interacting with specialized pattern recognition receptors (PRRs), including TLRs, opened the door for the inclusion of the respective agonists in DC maturation cocktails [31]. TLRs allow the recognition of a range of pathogens and lead to de novo transcription and secretion of cytokines and chemokines, enhanced antigen presentation capacity, and superior migration to lymphoid tissues. In humans, expression of TLR1 to 6 and TLR 8 were described in monocyte-derived DCs [32‐35]. The signaling pathways associated with ligation of each of these TLRs are not identical; therefore distinct biologic responses are initiated upon receptor ligation [36]. The availability of different synthetic ligands for TLRs, like imiquimod (R837), resiquimod (R848), S-27609, CL097, CL075 (3M-002), CL087, or loxoribine, has stimulated research to use TLR agonists in maturation cocktails. For example, combinations of the TLR3 ligand polyinosinic-polycytidylic acid (poly(I:C)) and the TLR7/8 ligands R848 or CL075, supplemented with PGE₂, yielded DCs with both high migratory capacity and high IL-12(p70) production upon T cell encounter [37, 38]. TLR-mediated maturation of ex vivo- generated human DCs may thus be used to improve immunologic and clinical responses in DC vaccination of cancer patients. Furthermore, it was shown that 3-day DCs were as effective as conventional 7-day DCs in stimulating primary, antigen-specific T_H1 immune responses. This fast generation not only reduces labor, cost and time required for DC development, but also represents a model more closely resembling DC differentiation from monocytes in vivo [26].

We tested two different TLR7/8 agonists, with or without the TLR3 agonist poly(I:C), for their potency to induce mature DCs in AML patients in comparison to HCs. Phenotypically, DCs from AML patients showed high CD83, low CD14 and a slightly reduced expression of the costimulatory molecules CD80 and CD86 in combination with a similar expression intensity of CD274 and CD273. The predominance of positive costimulatory molecules over inhibitory molecules translates into potent T cell stimulation, as demonstrated by Selenko-Gebauer et al. [39]. In accordance, we also observed a potent allostimulatory capacity of TLR-matured DCs from AML patients as well as healthy controls.

Successful anticancer immunotherapy requires strong T_H1-polarizing immune responses as well as NK cell responses [40‐43]. In a recently published clinical phase I trial using RNA-transfected DCs from AML patients, a significant correlation could be seen between clinical responses and the induction of high numbers of activated NK cells [9]. Thus, DCs are required that not only express positive costimulatory markers, but also secrete high amounts of bioactive IL-12(p70). This DC-derived cytokine constitutes signal 3 of T cell activation and determines outcomes of T cell differentiation as well as NK cell activation [40, 41, 44]. Previous reports on DC generation from monocytes of AML patients in remission have used the classical cytokine cocktail containing TNF-α, IL-1β, IL-6 and PGE₂ for DC maturation [12, 45]. This protocol generates immature DCs over 5-7 days before the maturation cocktail is added for the final 48 hours. As previously described by others, DCs generated by this cocktail do not produce measurable IL-12(p70) [27, 37]. In the published literature on DC generation from AML patients there is no data regarding secretion of DC-derived cytokines nor is there information regarding DC activation of NK cells [45, 46]. This report is the first to show that a TLR- containing maturation cocktail is able to generate DCs from AML patients in only 3 days with high potential for induction of both T- and NK-cell responses. We demonstrated high secretion of DC derived IL-12(p70) which translated into strong NK cell activation, as shown by CD69 upregulation and IFN-γ secretion by the CD3^-/CD56⁺ NK cell subpopulation. The strong effects on DC phenotype and function were primarily induced by TLR 7/8 ligation as the addition of the TLR3 ligand poly(I:C) did not result in any significant changes in the expression pattern of costimulatory molecules or IL-12(p70) secretion. This finding may be explained by the low poly(I:C) concentration used in our cocktails which is up > 100 fold lower compared to other published reports [47, 48]. The significant lower concentration of poly(I:C) was chosen due to the observations that poly(I:C) at a high concentration prevents DCs from being able to express protein after loading with exogeneous RNA, while the lower concentration is still sufficient for DC activation [27]. This is of utmost importance for our planned clinical phase I/II trial, as we will use RNA-transfected DCs for active immunotherapy in AML patients.

A promising target antigen for immunotherapeutic approaches in AML is WT-1, which is generally overexpressed in the majority of AML cases [49]. Several clinical trials using active immunotherapeutic strategies have targeted WT-1, so far with promising immunological and clinical responses [8, 50]. We tested our DCs for their potential to expand WT-1-specific, CD8⁺ T cells. Comparative data was obtained against the immunodominant CMVpp65 protein and T cell responses were analyzed by multimer and ELISPOT for IFN-γ. Equally effective expansion of WT-1 - and CMVpp65- specific T cells could be observed. Our data demonstrate that TLR-containing maturation cocktail can generate DCs from AML patients with phenotypic and functional characteristics that are highly desirable for use in a clinical trial setting.

A clinical phase I/II study is currently in preparation using non-leukemic monocytes for generation of autologous DCs in AML patients in complete remission. These DCs will be transfected with RNA encoding leukemia-associated antigens for induction of leukemia-specific immune responses in vaccinated patients. Through their secretion of IL-12(p70), the DCs will favour type 1 polarization of T cells while allowing NK cell activation to be initiated at the same time. We believe that these DCs are highly suitable for application in cancer immunotherapy.

To our knowledge, this is the first report in which a TLR-containing maturation cocktail has been used for generation of young, 3-day, DCs from AML patients in remission in comparison to HC. We found that mature DCs prepared with the TLR7/8 agonists, R848 or CL075, displayed phenotypes and functions suitable for antitumor vaccine development with no significant differences between AML and HCs and among the different TLR-containing cocktails. As the manufacturing protocol is easily adaptable to GMP requirements, we propose the implementation of our protocol in a clinical vaccination trial in AML patients in complete remission for eradication of minimal residual disease.