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In the three patients included in a phase I clinical trial (NCT01421524), we report the immunomodulatory effects and efficacy of CC-122, a novel pleiotropic pathway modifier compound originally developed for broad diffuse large B-cell lymphoma (DLBCL). The chemical structure of CC-122 includes the glutarimide moiety that is known to modulate the immune response. The immunomodulatory agents including lenalidomide represent a promising therapeutic strategy targeting tumors in B-cell lymphoid malignancies. We observed that CC-122 might regulate the NK phenotype and its activity due to the reduced accumulation of myeloid-derived suppressor cell and eventually decrease the Tregs subsets. Finally, the activation of T cells through co-stimulatory molecule (CD28) was detected as a delayed CC-122 effect. In this context, CC-122 arises as an alternative option for DLBCL patients refractory to the traditional chemotherapeutic agents.
As it is well known, DLBCL is the most frequent subtype of B-cell lymphoma.1 Although the addition of rituximab to the cyclophosphamide, doxorubicin, vincristine, and prednisone chemotherapy-based (R-CHOP) treatments has significantly increased the 5-y overall survival rate to 58%, nearly 15% of patients with DLBCL remain in a primary refractory state.2 Patients with DLBCL who experience R-CHOP treatment failure have limited therapy options and are likely to have dismal outcomes.
In this regard, the thalidomide analog CC-122, a new immunomodulatory drug that acts as a pleiotropic pathway modifier, has demonstrated anti-lymphoma activity through its binding to cereblon (CRBN), leading to the subsequent ubiquitination of Aiolos and Ikaros. This process results in the activation of T cells mimicking interferon signaling, ultimately leading to tumor cell apoptosis.3,4 CC-122 was administered to a cohort of refractory patients with DLBCL, resulting in increased IRF7 expression in the lymph nodes, which supported T-cell activation and co-stimulation.4 As tumor immunosurveillance in patients with refractory DLBCL has shown diminished natural killer (NK) cell populations and cytotoxic activity,5 we studied the modulatory and tumor-surveillance effects of CC-122 on the immune system of three DLBCL patients. They showed significant improvement in the innate immune response during treatment, and, we hypothesize a potential switch to the adaptive immune response, making CC-122 a promising therapy capable of reshaping the antitumor immune response.
The median follow-up was 5 mo (range 4–6 mo). The patients developed a Tumor Flare Reaction (TFR) on cycle 1, between days 7 and 10 of treatment, which disappeared within a few days. The TFR was suggestive of immune activation and correlates with NK cell proliferation and a clinical response in patients with chronic lymphocytic leukemia (CLL).6 In the first response assessment at 3 mo of treatment, patients 1 and 3 achieved a partial response, according to Cheson criteria. Patient 2 had stable disease. In the follow-up, patients 1 and 2 developed progressive disease at 4 and 6 mo, respectively. Patient 3 continues to have a very good clinical response.
A remarkable immune response was obtained in all three patients. NK cell phenotype analyses of the three patients treated with CC-122 showed a slight increase at 7 d of treatment, and patient 3 showed the highest NK cell numbers. At this time, the cytotoxic NK cell (CD3−CD56+CD16+) subset exhibited an increase, achieving similar levels to those of the healthy volunteers (HV). In line with this finding, a reduction in the non-cytotoxic NK cell subset (CD3−CD562+CD16−) was observed (Fig. 1A). Interestingly, it was reported that patients with refractory DLBCL showed low numbers of the NK cytotoxic cell subset, as we observed at pre-treatment.5 To assess the functional cytotoxic assay, we performed a standard ex vivo protocol using a leukemia cell line (K562) as a tumor target. The cytotoxic ability was not detectable at baseline in patients 1 and 2; however, during CC-122 treatment, NK cell functionality was recovered in patients 1 and 3, achieving a standard lysis activity equivalent to that of the HV (Fig. 1B). The NKG2D receptor, which plays an important role in lymphoma control,7 rapidly increased after CC-122 administration (Fig. 1C). In addition, we studied other involved cytotoxic factors such as granzyme B (GrB). GrB expression increased at 7 d in the cytotoxic NK (CD3−CD56+CD16+) subset in contrast to that observed in the non-cytotoxic (CD3−CD56+CD16−) subset (Fig. 1D).
It has been reported that CC-122 led to an antitumour res-ponse in the DLBCL cell lines and in the patients with DLBCL, caused by an increase in interferon factors such as IRF7 and a co-stimulation of T cells.4 Moreover, IRF7-enhanced T cell and NK cell activity by preventing myeloid-derived suppressor cell (MDSC) accumulation, subsequently leading to metastasis suppression in animal models.8 The MDSC subset is crucial for tumor immunosurveillance,9 and it was identified in melanoma patients as CD14+ cells expressing low levels of HLA-DRlow/neg and secreting TGFβ.10 In agreement with these data, the number of MDSC subset cells decreased 4 h after treatment initiation, with a significant improvement at 7 d (Fig. 2A). We confirmed a decrease in TGFβ mRNA expression after treatment doses, supporting the MDSC role in tumor immunosurveillance (Fig. 2B). Interestingly, other immunosuppressive functions of MDSC were reported through regulatory T cells (CD4+CD25+Foxp3+) in patients with primary liver cancer and B-cell lymphoma,11,12 and there is a close interrelationship between CD4+CD25+Foxp3+ Treg cells and disease progression.13 Our patients exhibited a patent reduced number of CD4+CD25+Foxp3+ Treg cells after CC-122 monotherapy (Fig. 2C).
Our data suggest that CC-122 treatment in DLBCL patients restores the innate immune response after discontinuing therapy. However, T-cell activation analyzed by CD28 expression in the patients treated for DLBCL showed a significant increase at day 30 after initiation of treatment in the CD8+ and δγ+ cells, suggesting a delayed effect of CC-122 (Figs. 2D and E). This immunomodulator eventually repairs T-cell immunologic synapse dysfunction in patients with DLBCL. Corroborating these results, lenalidomide, an analog of thalidomide, stimulates T-cell activation through the CD28 signaling pathway.14 In addition, CD3+CD56+ NKT-like cells possess both innate and adaptive immune functions, displaying capabilities of both T and NK cells; and a low percentage of CD3+CD56+ NKT-like cells have been associated with a higher risk of death in patients with CLL.15 All the patients showed a late enhanced number of CD3+CD56+ NKT-like cells (Fig. 2F) as a delayed effect of CC-122, supporting speculation about the adaptive immunomodulation of CC-122 in these patients.
In conclusion, clinical and immune response analyses demonstrate a promising outcome in DLBCL patients. Based on our results, CC-122 anti-lymphoma activity in patients with refractory DLBCL might be explained by tumor flare at 7 d followed by immunomodulation, cytotoxic activity, T-cell activation, and a change in adaptive immune response with a subsequent positive clinical outcome. In any case, additional controlled trials are required to assess the long-term immune effects and also increase the number patients from DLBCL treated with CC-122.
The samples were collected between December, 2014 and December, 2015; their characteristics and clinical status are summarised in Table 1. All patients fulfilled the inclusion criteria to be enrolled in the clinical trial and they had good performance status, with ECOG 0–1. The median age was 68.43 ± 18.73 y, the median time from diagnosis was 67.54 ± 24.52 y, and the median numbers of previous chemotherapy lines were 3.3 ± 0.57. All the patients were refractory to the last salvage chemotherapy. Patients provided an additional informed consent to participate in this biological study. The Institutional Review Board of La Paz University Hospital approved the study. Blood samples from the patients were obtained at various times: baseline, 4 h, 7, and 30 d after the first dose. Peripheral blood mononuclear cells (PBMCs) were isolated by centrifugation in Ficoll-Histopaque Plus (Amersham Biosciences). As control subjects (n = 5), we included sex- and age-matched HV with no history of DLBCL or any other significant illness.
PBMCs were stained with indicated antibodies (CD3, CD56, CD16, CD14, HLA-DR, CD4+, CD25, CD8+, CD28, and γδ from BD Bioscience). For the intracellular staining, the cells were labeled with granzyme B (GrB) and Foxp3 antibodies (BD Bioscience) following a standard protocol. Appropriate isotype controls were used for each experiment. After staining, the cells were acquired by BD FACS Calibur flow cytometer and the collected data were analyzed using CellQuest Pro software (BD Bioscience).
NK cytotoxicity was monitored using a conventional 2 h europium-TDA release assay (Perkin-Elmer). The primary NK cells were used as effector cells. TDA-labeled K562 cell line was used as target cells at effector-to-target cell (E:T) ratios of 8:1, 4:1, and 2:1.
Total RNA was purified from isolated monocytes using the High Pure RNA isolation kit from Roche Diagnostics. Gene expression mRNA levels were analyzed by qPCR (Light-Cycler, Roche Diagnostics) using specific primers.
No potential conflicts of interest were disclosed.
The authors would like to thank Aurora Muñoz and Anibal Varela-Serrano for the technical assistance. We also thank DLBCL patients who participated in the study.
This work was supported by grant from “Fondos de Investigación Sanitaria” (FIS) and FEDER (PI14/01234, PIE15/00065) to ELC.
Conception and design: López-Collazo E and Cubillos-Zapata C; Development of methodology: Cubillos-Zapata C; Acquisition of data (provided and managed patients, provided facilities, etc.): Villaescusa T, Moreno V, Cordoba Raúl; Analysis and interpretation of data: Avendaño-Ortiz J, Hernández-Jiménez E, Cubillos-Zapata C; Writing, review, and/or revision of the manuscript: López-Collazo E, Cubillos-Zapata C; Administrative, technical, or material support: Arribas-Jimenez C, Toledano V; Study supervision: López-Collazo E and Cubillos-Zapata C.