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Microenvironment contributes to follicular lymphoma (FL) pathogenesis and impacts survival with macrophages playing a controversial role. In the present study, using FL primary samples and HK follicular dendritic cells (FDC) to mimic the germinal center, together with mouse models, we have analyzed the three-way crosstalk of FL-FDC-macrophages and derived therapeutic opportunities. Ex vivo primary FL-FDC co-cultures (n = 19) and in vivo mouse co-xenografts demonstrated that FL-FDC crosstalk favors tumor growth and, via the secretion of CCL2 and CSF-1, promotes monocyte recruitment, differentiation, and polarization towards an M2-like protumoral phenotype. Moreover, FL-M2 co-cultures displayed enhanced angiogenesis, dissemination, and immunosuppression. Analysis of the CSF-1/CSF-1R pathway uncovered that CSF-1 was significantly higher in serum from grade 3A FL patients, and that high CSF-1R expression in FL biopsies correlated with grade 3A, reduced overall survival and risk of transformation. Furthermore, CSF-1R inhibition with pexidartinib (PLX3397) preferentially affected M2-macrophage viability and polarization program disrupting FL-M2 positive crosstalk. In vivo CSF1-R inhibition caused M2 reduction and repolarization towards M1 macrophages and antitumor effect cooperating with anti-CD20 rituximab. In summary, these results support the role of macrophages in FL pathogenesis and indicate that CSF-1R may be a relevant prognostic factor and a novel therapeutic target cooperating with anti-CD20 immunotherapy. Grants that contributed to this work included: Gilead Sciences Research Funded Agreement, Spanish Ministry of Economy and Competitiveness & European Regional Development Fund (ERDF) “Una manera de hacer Europa” for SAF2014/57708R and SAF2017/ 88275R to PP-G and MCC, RTI2018-094584-B-I00 to DC, CIBERONC (CB16/12/00334 and CB16/12/00225), and finally Generalitat de Catalunya support for AGAUR 2017SGR1009 to DC

Despite the significant contributions of immunocompetent mouse models to the development and assessment of cancer immunotherapies, they inadequately represent the genetic and biological complexity of corresponding human cancers. Immunocompromised mice reconstituted with a human immune system (HIS) and engrafted with patient-derived tumor xenografts are a promising novel preclinical model for the study of human tumor-immune interactions. Whilst overcoming limitations of immunocompetent models, HIS-tumor models often rely on reconstitution with allogeneic immune cells, making it difficult to distinguish between anti-tumor and alloantigen responses. Models that comprise of autologous human tumor and human immune cells provide a platform that is more representative of the patient immune-tumor interaction. However, limited access to autologous tissues, short experimental windows, and poor retention of tumor microenvironment and tumor infiltrating lymphocyte components are major challenges affecting the establishment and application of autologous models. This review outlines existing preclinical murine models for the study of immuno-oncology, and highlights innovations that can be applied to improve the feasibility and efficacy of autologous models.

Objectives Humanised mice have emerged as valuable models for pre‐clinical testing of the safety and efficacy of immunotherapies. Given the variety of models available, selection of the most appropriate humanised mouse model is critical in study design. Here, we aimed to develop a model for predicting cytokine release syndrome (CRS) while minimising graft‐versus‐host disease (GvHD). Methods To overcome donor‐induced variation, we directly compared the in vitro and in vivo immune phenotype of immunodeficient NSG mice reconstituted with human bone marrow (BM) CD34+ haematopoietic stem cells (HSCs), peripheral blood mononuclear cells (PBMCs) or spleen mononuclear cells (SPMCs) from the same human donors. SPMC engraftment in NSG‐dKO mice, which lack MHC class I and II, was also evaluated as a strategy to limit GvHD. Another group of mice was engrafted with umbilical cord blood (UCB) CD34+ HSCs. Induction of CRS in vivo was investigated upon administration of the anti‐CD3 monoclonal antibody OKT3. Results PBMC‐ and SPMC‐reconstituted NSG mice showed short‐term survival, with engrafted human T cells exhibiting mostly an effector memory phenotype. Survival in SPMC‐reconstituted NSG‐dKO mice was significantly longer. Conversely, both BM and UCB‐HSC models showed longer survival, without demonstrable GvHD and a more naïve T‐cell phenotype. PBMC‐ and SPMC‐reconstituted mice, but not BM‐HSC or UCB‐HSC mice, experienced severe clinical signs of CRS upon administration of OKT3. Conclusion PBMC‐ and SPMC‐reconstituted NSG mice better predict OKT3‐mediated CRS. The SPMC model allows generation of large experimental groups, and the use of NSG‐dKO mice mitigates the limitation of early GvHD., In this study, we perform a head‐to‐head comparison between different humanised mouse models in terms of survival, human immune engraftment and response to immunotherapy. We characterise the differences in the immune compartment of these models and demonstrate that some, but not all, can mimic the human immune response to immunotherapies.

Significant advances in immunotherapies have resulted in the increasing need of predictive preclinical models to improve immunotherapeutic drug development, treatment combination, and to prevent or minimize toxicity in clinical trials. Immunodeficient mice reconstituted with human immune system (HIS), termed humanized mice or HIS mice, permit detailed analysis of human immune biology, development, and function. Although this model constitutes a great translational model, some aspects need to be improved as the incomplete engraftment of immune cells, graft versus host disease and the lack of human cytokines and growth factors. In this review, we discuss current HIS platforms, their pathology, and recent advances in their development to improve the quality of human immune cell reconstitution. We also highlight new technologies that can be used to better understand these models and how improved characterization is needed for their application in immuno-oncology safety, efficacy, and new modalities therapy development.

Constitutive activation of B-cell receptor (BCR) signaling is a hallmark of several B-cell non-Hodgkin lymphoma (B-NHL), including diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), follicular lymphoma (FL) and chronic lymphocytic leukemia (CLL). Despite the promising clinical activity of the first BCR kinase inhibitors in some of these entities, the design of new compounds is warranted to improve the survival of B-NHL patients who are poorly responsive, and/or who eventually develop resistance to current BCR-targeting therapies. In this context, we recently described the synthesis of IQS019, a 4-aminopyrido[2,3-d]pyrimidine with improved inhibition pattern against the three BCR-related kinases Lyn, Syk and Btk, when compared to reference BCR kinase inhibitors. Here, by assessing the activity of IQS019 in a panel of 21 human cell lines and 17 primary samples representative of the main B-NHL subtypes, we show that doses of IQS019 in the micromolar range allowed to a rapid and dose-dependent de-phosphorylation of both constitutive and Ig-activated Syk, Lyn and Btk in CLL, MCL, FL and DLBCL cell lines and CLL primary cultures. IQS019 treatment impairs cell proliferation and CXCL12-dependent cell migration, and leads to the induction of caspase-dependent apoptosis. Of interest, in CLL primary cells, these effects were independently of IGVH mutational status. In xenotransplant mouse models of MCL and FL, daily dosing of 2 mg/kg IQS019 for two weeks demonstrated to be safe for the animals and allowed to a 50-60% reduction in tumor outgrowth and glucose uptake, together with a 52% decrease in cell infiltration into the spleen (p < 0.05). These effects were accompanied by a significant (p < 0.05) downregulation of p-Syk, p-Lyn and p-Btk, and consequent mitotic arrest and induction of apoptosis in human malignant B cells. Altogether, these results define the BCR kinase inhibitor IQS019 as a potential candidate in antitumor therapy against a variety of B-NHL subtypes with aberrant activation of BCR pathway. Citation Format: Patricia Balsas, Jocabed Roldan, Laura Jimenez, Vanina Rodriguez, Raimon Puig de la Bellacasa, Jordi Teixido, Alba Matas-Cespedes, Alexandra Moros, Antonio Martinez, Elias Campo, Jose I Borrell, Patricia Perez-Galan, Dolors Colomer, Gael Roue. Preclinical evaluation of IQS019, a novel BCR kinase inhibitor, in in vitro and in vivo models of non-Hodgkin lymphoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5317. doi:10.1158/1538-7445.AM2015-5317

Background B-cell receptor (BCR) signaling contributes to the pathogenesis of B cell malignancies and has emerged as a new target for therapy with special relevance for tumor cell-microenvironment crosstalk. Recently, blockade of the BCR-related kinase Btk (Bruton tyrosin kinase) with the first-in-class inhibitor Ibrutinib, has shown impressive clinical responses in Mantle cell lymphoma (MCL) and Chronic Lymphocytic Leukemia. However, resistances to treatment have already appeared, opening the door to new BTK inhibitors. In this study, we have evaluated the antitumor profile of a novel and highly selective BTK inhibitor CC-292(Celgene)in MCL. Methods Representative MCL lines (MINO, JEKO-1, REC-1, UPN1, Z138, HBL2, GRANTA, JVM-2) were used for in vitro and in vivo studies. The effects of CC-292 on lymphoma cell growth and apoptosis were analyzed by MTT and Annexin V/PI staining, respectively. BTK phosphorylation at Y223 residue was used as a read-out of BTK activity. Microenvironment-derived BTK activation was mimicked by IgM crosslinking or SDF1α/CXCL12 stimulation. Actin polymerization experiments were performed using Phalloidin-TRITC staining, and adhesion assays were done on EIA/RIA 96-well plates coated with Fibronectin or VCAM-1. In vivo activity was assessed in a subcutaneous mouse xenograft model, where daily oral dosing was started when tumors were palpable and extended for a total of 18 days. Results BTK activation (pBtkY(223)) was detected in all MCL cell lines analyzed. CC-292 (10-1000nM) exerted cytostatic effect in part of the cell lines, where REC-1, MINO, JVM2 and UPN1 were the most sensitive cell lines. No correlation between constitutive Btk activation and response to CC-292 was found. Noteworthy, CC-292 potently inhibited constitutive and IgM/CXCL12-induced Btk activation and interfered with tumor cell-microenvironment interactions, by blocking SDF1α/CXCL12-induced actin polymerization, as well as IgM-induced adhesion to VCAM and FN. In CC-292 sensitive cell lines, the combination with the immunomodulatory drug lenalidomide was synergistic and was accompanied with IRF4 decrease in MINO and REC cell lines. Finally, CC-292 (30mg/kg) showed antitumor activity in vivo in a sc mouse xenograft (UPN-1 cell line), reducing tumor outgrowth by 52% Conclusions In summary, these results warrant further investigation of CC-292 for MCL therapy, both alone and in combination with the immunomodulatory agent lenalidomide. Citation Format: Anna Vidal-Crespo, Vanina Rodríguez, Alba Matas-Céspedes, Elías Campos, Armando López-Guillermo, Gael Roué, Dolors Colomer, Patricia Pérez-Galán. The novel BTK inhibitor CC-292 exerts in vitro and in vivo antitumor activity, interferes with adhesion, cell migration, and synergizes with lenalidomide in MCL models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1757. doi:10.1158/1538-7445.AM2014-1757

Daratumumab (DARA) is a human CD38 antibody with broad-spectrum killing activity. DARA induces killing of tumor cells, mainly via complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) (de Weers M, J Immunol 2011). DARA is currently being evaluated in phase I/II clinical trials in patients with multiple myeloma. In these clinical studies the adverse events have been manageable and marked reductions in paraprotein and bone marrow plasma cells have been observed. We have previously reported (Blood (ASH annual meeting abstracts). Nov 2012, 120 (21): 3935) that DARA induces cytotoxic activity in vitro via ADCC in primary cells and cell lines from Mantle Cell Lymphoma (MCL), Follicular Lymphoma (FL) and Chronic Lymphoctic Leukemia (CLL). CDC induction was low, which is associated to high expression of the complement inhibitors and reduced number of CD38 molecules per cell in these indications. This suggests a threshold for CD38-targeted CDC lysis. In addition, based on known interactions between CD38-CXCR4, we also demonstrated that in the CLL subtype, with high CD38 and more migratory capacity, DARA (10-30 µg/mL) inhibited in vitro CXCL12/SDF1α-mediated migration up to 70%. Here, we present the first preclinical in vivo results of DARA in mouse models of MCL, transformed FL(tFL) and CLL. We generated heterotopic and systemic mouse models of these entities by subcutaneous or intravenous inoculation of tumor cell lines in SCID mice, that retain both NK cells and macrophages as potential effector cells. In MCL (REC cell line) and tFL (RL cell line) subcutaneous mouse models, we tested DARA activity in both prophylactic (treatment initiation simultaneous to lymphoma cell inoculation) and therapeutic settings(treatment initiation one week after lymphoma cell inoculation, when tumors were about 100 mm3 in size). In the prophylactic setting, mice received 3 doses of DARA or control IgG bi-weekly (20/10/10 mg/kg). In the therapeutic setting, treatment was intensified to 4 doses of DARA or control IgG weekly (20/10/10/10 mg/kg). In both schedules, mice were sacrificed one week after the last dose. DARA completely abrogated tumor growth of REC or RL cells in the prophylactic setting. Moreover, in the therapeutic setting, DARA induced total tumor regression of REC tumors in 4 out of 6 mice, and prevented the splenomegaly observed in control IgG treated mice. In the case of tFL and therapeutic setting, DARA reduced 60% of tumor growth compared to control IgG treated mice at day 32, when experiment was finished. In CLL, we analyzed the effect of DARA on cell homing to lymphoid organs together with its therapeutic properties in a systemic CLL mouse model. Using NOD/SCID/gamma null mice (lacking NK cells and effective macrophages), we analyzed the effect of DARA on primary CLL cell migration from Peripheral Blood (PB) to bone marrow (BM) and Spleen. In this system, NSG mice were pretreated (day 0) with DARA, control IgG or anti-CXCR4 as positive control for inhibition of cell homing, followed by fresh CLL cell inoculation (50×106 cells/per mice) on day 1. PB, BM and spleen cells were isolated on day 2 and CLL cells were identified by staining for CD45/CD19/CD5 and counted using a flow cytometer. Cell counting showed that CLL cells mainly migrate to the spleen, and that DARA significantly reduced this migration (55% inhibition on average, p Finally, we tested DARA therapeutic activity in a systemic CLL mouse model (MEC2 cell line), following the schedule described before (4 doses of control IgG/ DARA weekly (20/10/10/10 mg/kg)), and assessed efficacy on mice overall survival. Mice treated with control IgG started to lose weight and showed signs of disease between days 30-40 and were sacrificed for ethical reasons. In the DARA treated group, in 7 out of 8 mice survival was extended up to day 90, when the experiment was stopped. In conclusion, DARA demonstrated strong in vivo activity in immunocompromised mouse models of MCL, tFL and CLL cell lines and interfered with homing of primary CLL cells to the spleen. These results warrant further investigation of DARA in clinical trials for these indications. Disclosures: Lopez-Guillermo: Roche: Membership on an entity’s Board of Directors or advisory committees. Lammerts van Bueren:Genmab: Employment, Stocks Other. Bakker:Genmab: Employment, Stocks Other. Parren:Genmab: Employment, Stocks Other. Perez-Galan:Genmab: Research Funding.

Abstract 3935 Daratumumab (DARA) is a human CD38 antibody with broad-spectrum killing activity. DARA induces killing of tumor cells, mainly via complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) (de Weers M, J Immunol 2011). DARA is currently being evaluated in phase I/II clinical trials in patients with multiple myeloma. In these clinical studies the adverse events have been manageable and marked reductions in paraprotein and bone marrow plasma cells have been observed. In the present study, we have analyzed the potential of targeting CD38 using DARA in two types of B-cell non-Hodgkin lymphoma (B-NHL) (follicular lymphoma (FL) and mantle cell lymphoma (MCL)), and in chronic lymphocytic leukemia (CLL). Flow cytometry analysis demonstrated that MCL and CLL tumor cells show heterogeneous expression of CD38, while FL cells showed invariable high CD38 levels. CD38 has attracted special attention in CLL where high CD38 expression is a marker of bad prognosis (Hamblin et al, Blood 1999 and 2002) and is expressed preferentially in the proliferating fraction of the tumor (Damle RN, Blood 2007). In addition, we have recently shown that high CD38 expression in MCL was associated with resistance to the proteasome inhibitor bortezomib (Pérez-Galán P, Blood 2011). Here, we tested the cytotoxic activity of DARA in tumor cell lines and in fresh tumor cells obtained from patients. DARA did not induce CDC in MCL cell lines (MINO, REC, HBL2, JEKO), irrespective of CD38 expression levels. Also, FL cell lines (WSU-FSCCL, RL) expressing relatively high CD38 levels were insensitive to DARA-induced CDC. This low CDC was associated with high expression of the complement inhibitors CD55 and CD59. In addition, the number of CD38 molecules per cell in these MCL and FL cell lines was lower than that found on the CDC-sensitive Daudi Burkitt lymphoma cell line, suggesting a threshold for CD38-targeted CDC lysis. In the presence of PBMC effector cells obtained from healthy donors, DARA showed significant levels of ADCC in cells from MCL, FL and CLL. In CLL primary cases (n=8) tested, DARA (14–43% lysis) was generally superior or at least equally effective (mean+/−SD=28,78 +/− 9,78) in inducing ADCC as compared to the anti-CD20 antibodies ofatumumab (mean+/−SD=21,35 +/− 15,71) and rituximab (mean+/−SD=29,30 +/− 15,90). The immunomodulatory agent lenalidomide shows considerable single agent activity in MCL, FL and CLL. Interestingly, it has been shown that lenalidomide may be able to increase ADCC, probably via activation of NK cells. We therefore tested whether the combination of lenalidomide and DARA could enhance ADCC. Noteworthy, DARA-induced ADCC in MCL, FL cell lines and primary CLL cells was significantly (p Finally, CD38 is important for cell migration and adhesion, especially for CXCR4-CXCL12-induced migration of tumor cells (Vaisitti et al, Leukemia 2010). Our preliminary results suggest that in the CLL subtype with high CD38 and more migratory capacity DARA (10–30 μg/ml) inhibits CXCL12/SDF1α mediated migration up to 70%. These results are of high importance because inhibition of tumor cell trafficking to tissue sites as bone marrow and lymph nodes, may be clinically relevant, as increasing evidence indicates that tumor–microenvironment interactions may play an important role in drug resistance and contribute to clinical failures. We are currently validating this in vitro results in a CLL mouse model. Taken together, these results suggest that DARA may be a promising therapeutic agent both for MCL, FL and CLL, in which DARA exerts its effects mainly via ADCC and for CLL also via inhibition of migration of tumor cells. Interestingly, the cytotoxic activity of DARA by ADCC could be further augmented by addition of lenalidomide in these three models. Disclosures: Lammerts van Bueren: Genmab: Employment. Bakker:genmab: Employment. Parren:genmab: Employment. Perez-Galan:Genmab: Research Funding.