Caroline Sola

Anti-KIR Antibody Enhancement Of Anti-Lymphoma Activity Of Natural Killer Cells As Monotherapy and In Combination With Anti-CD20 Antibodies

Natural killer (NK) cells mediate antilymphoma activity by spontaneous cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC) when triggered by rituximab, an anti-CD20 monoclonal antibody (mAb) used to treat patients with B-cell lymphomas. The balance of inhibitory and activating signals determines the magnitude of the efficacy of NK cells by spontaneous cytotoxicity. Here, using a killer-cell immunoglobulin-like receptor (KIR) transgenic murine model, we show that blockade of the interface of inhibitory KIRs with major histocompatibility complex (MHC) class I antigens on lymphoma cells by anti-KIR antibodies prevents a tolerogenic interaction and augments NK-cell spontaneous cytotoxicity. In combination with anti-CD20 mAbs, anti-KIR treatment induces enhanced NK-cell-mediated, rituximab-dependent cytotoxicity against lymphoma in vitro and in vivo in KIR transgenic and syngeneic murine lymphoma models. These results support a therapeutic strategy of combination rituximab and KIR blockade through lirilumab, illustrating the potential efficacy of combining a tumor-targeting therapy with an NK-cell agonist, thus stimulating the postrituximab antilymphoma immune response.

Genetic and antibody-mediated reprogramming of natural killer cell missing-self recognition in vivo

Natural killer (NK) cells are lymphocytes of the innate immune system able to recognize and kill tumors lacking self-MHC class I molecules. This “missing-self” recognition is mediated by the lack of engagement of MHC class I-specific inhibitory NK cell receptors that include the killer cell Ig-like receptors (KIR) in humans and Ly49 molecules in mice. A promising immunotherapeutic strategy against MHC class I+ cancer cells is to block NK cell inhibitory receptors using monoclonal antibodies (mAb). However, interactions between MHC class I molecules and their inhibitory receptors are also required for the acquisition of NK cell functional competence, a process referred as to “education.” In addition, inhibitory receptors are involved in self-tolerance on educated NK cells. Here, we developed a preclinical mouse model in which all NK cells are educated by a single transgenic inhibitory receptor, human KIR2DL3, through the engagement with its HLA-Cw3 ligand. This approach revealed that NK cells could be reprogrammed to control the development of mouse syngenic tumors in vivo. Moreover, in vivo anti-KIR mAb treatment induced the killing of HLA+ target cells without breaking self-tolerance. Finally, the long-term infusion of anti-KIR mAb neither abolished NK cell education nor tumor cell recognition. Therefore, these results strongly support the use of inhibitory receptor blockade in cancer patients.

Invasive aspergillosis in hematopoietic stem cell transplant recipients: a retrospective analysis.

Invasive aspergillosis (IA) currently is an important cause of mortality in subjects undergoing hematopoietic stem cell transplants (HSCT) and is also an important cause of opportunistic respiratory and disseminated infections in other types of immunocompromised patients. We examined the medical records of 24 cases of proven and probable invasive aspergillosis (IA) at the Hospital de Clinicas of the Federal University of Parana, Brazil, from January 1996 to October 2006. During this period occurred a mean of 2.2 cases per year or 3.0 cases per 100 HSTC transplants. There was a significant relationship between structural changes in the bone marrow transplant (BMT) Unit and the occurrence of IA cases (p=0.034, relative risk (RR) = 2.47). Approximately 83% of the patients died due to invasive fungal infection within 60 days of follow up. Some factors tended to be associated with mortality, but these associations were not significant. These included corticosteroid use, neutropenia (<100 cells/mm(3)) at diagnosis, patients that needed to change antifungal therapy because of toxicity of the initial first-line regimen and disseminated disease. These factors should be monitored in BMT units to help prevent IA. Physicians should be aware of the risk factors for developing invasive fungal infections and try to reduce or eliminate them. However, once this invasive disease begins, appropriate diagnostic and treatment measures must be implemented as soon as possible in order to prevent the high mortality rates associated with this condition.

Anti-tumoral efficacy of therapeutic human anti-KIR antibody (Lirilumab/BMS-986015/IPH2102) in a preclinical xenograft tumor model

Natural Killer cells (NK cells) are lymphocytes able to recognize and kill tumors for which the expression of Major Histocompatibility Complex (MHC) class I molecules is altered. This “missing self” recognition is mediated in humans by the lack of engagement of MHC class I i.e. Human Leucocyte Antigens (HLA) molecules with NK cell inhibitory receptors that include Killer Immunoglobulin-like Receptors (KIR). Some tumors escape NK cell immune surveillance by increasing the expression of HLA molecules on their surface. Consequently, blocking interactions between KIR and HLA molecules constitutes an interesting therapeutic strategy. The anti-KIR2DL1/2/3-specific monoclonal antibody, lirilumab/BMS-986015/IPH2102, is a human IgG4 that is being developed for treating both hematologic malignancies and solid tumors. In rodents, the MHC class I inhibitory system regulating NK cell activation is based on lectin-like family Ly49 but the KIR molecules are not expressed. The objective of this study was to develop a preclinical model to assess the efficacy of the drug candidate used in clinical trials, lirilumab. Mice expressing the human NK inhibitory KIR2DL3, on the surface of NK cells were generated on a RAG-1deficient background (KIRtgRAG mice). The human B cell lymphoma cell line, 721.221, transduced with HLA-Cw3 molecule, a ligand of KIR2DL3, was intra-venously engrafted in these mice. The expression of HLA-C by tumor cells was sufficient to allow them to escape control of NK cells, leading to mice death in around 30 days. Lirilumab treatment increased mice survival in a dose dependent manner when injected at the same time as the tumor challenge. This protective effect was NK cell-mediated and directly correlated with the duration of KIR saturation. Interestingly, lirilumab treatment also improved survival in therapeutic conditions i.e. when the antibody was injected 5 days after the tumor, also in a NK cell-dependent manner. In conclusion, this study showed efficacy of lirilumab as single agent in a HLA-Cw3-expressing tumor model and this xenogenic pre-clinical model will be an excellent tool to investigate the therapeutic benefits of combination treatments.

Treatment outcomes for Hodgkin lymphoma: First report from the Brazilian Prospective Registry

Data about Hodgkin lymphoma (HL) in developing countries are scarce and suggest the existence of substantial disparities in healthcare and outcomes in large areas of the world. In 2009, a prospective registry of HL was implemented in Brazil. Web‐based data were contributed by 20 institutions across the country participating in the Brazilian Prospective Hodgkins Lymphoma Registry. The aim of this study was to present the clinical features and outcomes of newly diagnosed patients with HL aged 13 to 90 years. Multivariate Cox regression models were used to estimate progression‐free (PFS) and overall survival (OS) by clinical factors. A total of 674 patients with classical HL were analysed, with a median follow‐up of 37 months. Median age was 30 years (13‐90). The median time from the onset of symptoms to diagnosis was 6 months (0‐60). Only 6% of patients had early favourable disease, while 65% had advanced disease. Stage IVB was present in 26% and a high‐risk International Prognostic Score in 38%. Doxorubicin, bleomycin, vinblastine, and dacarbazine was used in 93%. The median dose of radiotherapy was 36 Gy for localized disease and 32 Gy for advanced disease. The 3 year PFS in early favourable, early unfavourable, and advanced disease were 95%, 88%, and 66%, respectively. High‐risk International Prognostic Score, advanced disease, and age greater than or equal to 60 were independently associated with poorer PFS and OS; performance status greater than or equal to 2 was also associated with a poorer OS. Poor‐risk patients predominated. Radiation doses for localized disease appear higher than current recommendations. Outcomes appear inferior in developing countries than in developed countries. Delayed diagnosis is probably a major factor underlying these findings. Scattered reports from developing nations suggest that many aspects of standard care in developed countries remain unmet needs for populations living in developing countries. The present report contributes to this body of data, with a proper description of what is currently achieved in urban areas in Brazil.

Abstract 2342: NKG2A immune checkpoint blockade enhances the anti-tumor efficacy of PD1/PD-L1 inhibitors in a preclinical model

Monalizumab (IPH2201) is a novel, first-in-class humanized IgG4 targeting the immune checkpoint receptor NKG2A (Natural Killer Group 2A). NKG2A is expressed as a heterodimer with CD94 on the surface of subsets of cytotoxic lymphocytes: NK (Natural Killer) cells, γδ T cells and tumor infiltrating CD8 T lymphocytes. CD94/NKG2A is an inhibitory receptor that binds to HLA-E (Human Leukocyte Antigen-E) in humans and orthologous Qa-1b in mice. Upon ligand binding, CD94/NKG2A triggers inhibitory signaling that reduces NK and CD8 T cell responses. HLA-E is frequently up-regulated on cancer cells of many solid tumors or hematological malignancies, protecting from killing by NKG2A+ immune cells. By blocking the binding of CD94/NKG2A to HLA-E, monalizumab leads to enhancement of NK and cytotoxic T cell responses. The immune checkpoint receptor programmed cell death 1 (PD-1) is another inhibitory receptor widely upregulated by tumor-infiltrating T lymphocytes (TILs). In many tumor types immune surveillance is hampered by the expression of PD-L1, one of the ligands of PD-1. Blocking the PD-1 pathway has proven efficient as anti-tumor therapy. Nevertheless many patients remain refractory to these therapeutics. Combination treatment with PD-1 blockers and mAb to a second checkpoint receptor, CTLA-4, have proven effective only for some patients, suggesting a need for combining with other checkpoint blockers. The A20 cell line is a mouse B cell lymphoma line that expresses PD-L1, but not Qa-1b. Upon subcutaneous injection into Balb/c mice, A20 formed solid tumors in which PD-L1 expression was retained and where Qa-1b expression was induced. A20 tumor growth was controlled by NK and CD8 T cells. In spite of high expression of PD-1 on many immune infiltrating cells, and high expression of PD-L1 on tumor cells, monotherapy with anti-PD-1 or -PD-L1 mAb resulted in only moderate reduction in tumor growth. Interestingly, more than 50% of A20 tumor infiltrating NK cells and about 10% of CD8 T cells expressed CD94/NKG2A. The NKG2A+ CD8 T cell population also co-expressed PD-1. Qa-1b expression was induced not only on the surface of tumor cells but also on infiltrating immune cells in vivo. Consistent with the hypothesis that Qa-1b protects tumor cells from killing by NKG2A+ effector NK and T cells, treatment with an antibody that blocks NKG2A significantly delayed A20 tumor growth. Mice were then treated with both anti-NKG2A and anti-PD-1 or -PD-L1 in combination. These combinations resulted in significantly higher anti-tumor responses compared to monotherapies, characterized by an increased frequency of complete tumor cell regression. Together, these data indicate that blocking NKG2A in conjunction with PD-1/PD-L1 checkpoint inhibitors could provide synergistic anti-tumor efficacy and support the rationale for investigating this combination in clinical trials. Citation Format: Caroline Sola, Thomas Arnoux, Fabien Chanuc, Nicolas Fuseri, Benjamin Rossi, Laurent Gauthier, Corinne Leget, Cecile Bonnafous, Nicolai Wagtmann, Yannis Morel, Pascale Andre. NKG2A immune checkpoint blockade enhances the anti-tumor efficacy of PD1/PD-L1 inhibitors in a preclinical model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2342.

Lower socioeconomic status is independently associated with shorter survival in Hodgkin Lymphoma patients—An analysis from the Brazilian Hodgkin Lymphoma Registry

Socioeconomic status (SES) is a well‐known determinant of outcomes in cancer. The purpose of this study was to analyze the impact of the SES on the outcomes of Hodgkin lymphoma (HL) patients from the Brazilian Prospective HL Registry. SES stratification was done using an individual asset/education‐based household index. A total of 624 classical HL patients with diagnosis from January/2009 to December/2014, and treated with ABVD (doxorubicin, bleomycin, vinblastine and dacarbazine), were analyzed. The median follow‐up was 35.6 months, and 33% were classified as lower SES. The 3‐year progression‐ free survival (PFS) in higher and lower SES were 78 and 64% (p < 0.0001), respectively. The 3‐year overall survival (OS) in higher and lower SES were 94 and 82% (p < 0.0001), respectively. Lower SES patients were more likely to be ≥ 60 years (16 vs. 8%, p = 0.003), and to present higher risk International Prognostic score (IPS) (44 vs. 31%, p = 0.004) and advanced disease (71 vs. 58%, p = 0.003). After adjustments for potential confounders, lower SES remained independently associated with poorer survival (HR = 3.12 [1.86‐5.22] for OS and HR = 1.66 [1.19‐2.32] for PFS). The fatality ratio during treatment was 7.5 and 1.3% for lower and higher SES (p = 0.0001). Infections and treatment toxicity accounted for 81% of these deaths. SES is an independent factor associated with shorter survival in HL in Brazil. Potential underlying mechanisms associated with the impact of SES are delayed diagnosis and poorer education. Educational and socio‐economic support interventions must be tested in this vulnerable population.

Chronic Graft-Versus-Host Disease: Survival Impact on Patients with Severe Aplastic Anemia (SAA) and Paroxysmal Nocturnal Hemoglobinuria (PNH)

S308 Setting: Stem cell transplantation centers. Patients: From 867 patients who underwent to an ASCT in our center, 21 (2.42%) were cMPN: 8 (38.1%) were primary myelofibrosis (MF), 11 (52.4%) secondary MF and 2 CMN different from MF. Main Outcomes Measures: Our data are similar to other institutions based on literature that have been published. Results: The clinical and biological features and ASCT characteristics are summarized in table 1. All patients achieved neutrophil and platelet engraftment. Concerning the complications related to ASCT: 71.4% of patients suffered from mucositis (86.7% grade I and II), 23.8% developed CMV reactivation, 9.5% haemorrhagic cystitis, 4.8% obstruction sinusoidal syndrome and none developed transplant associated microangiopathy. A total of 16 patients (76.2%) developed acute graft versus host disease (GVHD) (18.8 % grades III-IV) with a median of presentation of 26 days (15-141) and 11 patients (52.4%) developed chronic GVHD (63.6% extensive) with a median of 203 days (120-566). Transplant related mortality (TRM) at day +100 was 4.8% and the global TRM. At day +100 post ASCT, 17 patients (80.9%) achieved complete remission (CR) and 3 (14.3%) patients were in relapse. Six (28.6%) of these 20 patients relapsed after a median of 150 days (53-2295) from ASCT. In four of them, the approach of relapse was modulating immunosuppression +/donor lymphocyte infusion and hypomethylating agents in the cases of MRD, reaching CR and complete chimerism. In the last follow-up, 13 patients (61.9%) were alive (11 in CR and 2 with active disease) and 8 (38.1%) were dead from different causes. With a median follow-up of 35 months (16-121) the overall survival (OS) and event free survival (EFS) at 3 years was 61% and 57%, respectively. Conclusions: The results of our series confirms the potential curative of ASCT. We observed that more than a half of patients are long survivors. Modulating immunosuppression plays an important role in the setting of post ASCT relapse.

Abstract 493: Antitumoral efficacy of therapeutic human anti-KIR antibody (BMS-986015/IPH2102) in a preclinical xenograft tumor model.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Natural Killer cells (NK cells) are lymphocytes able to recognize and kill tumors for which the expression of Major Histocompatibility Complex (MHC) class I molecules is altered. This “missing self” recognition is mediated in humans by the lack of engagement of MHC class I i.e. Human Leucocyte Antigens (HLA) molecules with NK cell inhibitory receptors that include Killer Immunoglobulin-like Receptors (KIR). Some tumors escape NK cell immune surveillance by increasing the expression of HLA molecules on their surface. Consequently, blocking interactions between KIR and HLA molecules constitutes an interesting therapeutic strategy. The anti-KIR2DL1/2/3-specific monoclonal antibody, BMS-986015/IPH2102, is a human IgG4 that is being developed for treating both hematologic malignancies and solid tumors. In rodents, the MHC class I inhibitory system regulating NK cell activation is based on lectin-like family Ly49 but the KIR molecules are not expressed. The objective of this study was to develop a preclinical model to assess the efficacy of the drug candidate used in clinical trials, BMS-986015/IPH2102. Mice expressing the human NK inhibitory KIR2DL3, on the surface of NK cells were generated on a RAG-1deficient background (KIRtgRAG mice). The human B cell lymphoma cell line, 721.221, transduced with HLA-Cw3 molecule, a ligand of KIR2DL3, was intra-venously engrafted in these mice. The expression of HLA-C by tumor cells was sufficient to allow them to escape control of NK cells, leading to mice death in around 30 days. IPH2102 treatment increased mice survival in a dose dependent manner when injected at the same time as the tumor challenge. This protective effect was NK cell mediated and directly correlated with the duration of KIR saturation. Interestingly, BMS-986015/IPH2102 treatment also improved survival in therapeutic conditions i.e. when the antibody was injected 5 days after the tumor, also in a NK cell dependent manner. In conclusion, this study showed efficacy of BMS-986015/IPH2102 as single agent in a HLA-Cw3-expressing tumor model and this xenogenic pre-clinical model will be an excellent tool to investigate the therapeutic benefits of combination treatments. Citation Format: Caroline Sola, Fabien Chanuc, Ariane Thielens, Nicolas Fuseri, Isabelle Palacios, Mathieu Blery, Pascale Andre, Eric Vivier, Sophie Ugolini, Robert Graziano, Francois Romagne, Cecile Bonnafous. Antitumoral efficacy of therapeutic human anti-KIR antibody (BMS-986015/IPH2102) in a preclinical xenograft tumor model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 493. doi:10.1158/1538-7445.AM2013-493