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Introduction: Role of angiogenesis in growth, spread and survival of malignant lymphomas remains largely unexplored. Bevacizumab, a monoclonal antibody against vascular endothelial growth factor (VEGF), has been approved for treatment of several types of solid tumors including colorectal carcinoma or breast carcinoma, in all cases in combination with chemotherapy. Mantle cell lymphoma (MCL) is a type of B-cell non-Hodgkin lymphoma characterized by frequent extranodal involvement. Two agents approved for the therapy of MCL including lenalidomide and temsirolimus exert their anti-lymphoma activity at least partially by inhibition of angiogenesis. Our aim was to investigate role of VEGF-dependent angiogenesis on growth and spread of MCL in vivo using different murine models of MCL including patient-derived xenografts (PDXs). We also analyzed a potential anti-lymphoma synergy of a multi-level inhibition of angiogenesis achieved by bevacizumab and temsirolimus. Methods: Experimental therapies were implemented using NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice purchased from Jackson Laboratory (Bar Harbor, Maine, USA). Therapies were initiated three to five days after subcutaneous (SC) or intravenous (IV) injection of MCL cells (= day 1, D1). Therapies were terminated when SC tumor reached 2 cm, or when mice developed signs of terminal disease. Mice were xenografted either with MCL cell lines (JEKO-1, HBL2) or with PDX cells (VFN-M1, VFN-M2) derived in our laboratory from patients with relapsed MCL. Both PDXs were confirmed by NGS to keep majority of somatic mutations with the primary MCL cells. Growth curves of calculated tumor volumes and overall survivals were compared in case of SC and IV-xenografted animals, respectiverly, in the treated mice compared to untreated controls. VEGF levels were analyzed in different tissues (blood, tumor lysate, spleen lysate) by commercially available ELISA kit. TaqMan Angiogenesis Array was used to compare gene expression changes associated with bevacizumab failure. Results: Bevacizumab significantly inhibited growth of SC MCL tumors all tested models (Figure 1) (JEKO-1: 0.71g ±0.24g vs 3.22g ±0.65g, p=0.0061; HBL-2: 0.95g ±0.11g vs 4.2g ±0.28g, p Conclusions: Our data clearly indicate that all anti-angiogenic treatment approaches in malignant lymphomas have to be validated in vivo not only using SC xenografts, but also using systemic (IV) models. Our data also indicate that anti-angiogenic therapies might have auxiliary role (in combination with chemotherapy or targeted agents) in the treatment of malignant lymphomas. The gene expression data after failure of bevacizumab suggest activation of alternate pro-angiogenic pathways. Disclosures No relevant conflicts of interest to declare.

Tumor immunotherapy based on the use of Chimeric Receptor Modified T cells (CAR T cells) is a promising approach for the treatment of a refractory hematological cancer. However, a robust response mediated by CAR T cells is observed only in a minority of patients and the expansion and persistence of CAR T cells in vivo is mostly unpredictable. In order to enhance the effectiveness of CAR-based immunotherapy we tested the immunoadjuvant properities of lenalidomide in combination with CAR19 T cells in a mouse model of B cell lymphoma. CAR19 construct which was used is composed of anti-CD19scFv joined with signaling domain of 4-1BB and TCR zeta and was delivered into T cell via lentiviral transduction. Lenalidomide is an immunomodulatory drug used for the treatment of multiple myeloma and selected B-cell malignancies, e.g. mantle cell lymphoma (MCL) or activated B-cell subtype of diffuse large B-cell lymphoma (ABC-DLBCL). However, the precise mechanism of action is not very well understood and it is believed that is mediated by a modulation of activity of E3 ubiquitin ligase cereblon which leads to increased ubiquitinylation of Ikaros and Aiolos transcription factors resulting in changes of expression of various receptors on the surface of tumor cells. To test our hypothesis, immunodeficient NSG mice (NOD-SCID-gamma chain null mice) were s.c. transplanted with various human B cells lymphoma cells (MCL or ABC-DLBCL) followed by i.v treatment with CAR19 T cells with or without daily i.p. lenalidomide. First, when we measured the growth of tumors following treatment with CAR19 T cells plus lenalidomide we found that this combination more effectively suppressed growth of s.c. B-NHL tumors than treatment with only CAR19 T cells or only lenalidomide (Figure 1, 1x10e7 Nemo tumor cell s.c., followed with 2 doses of 1x10(7) CAR19 T cells + Lenalidomide daily, tumor weight was measured 14 days after treatment). Additionally, in this experiment lenalidomide significantly enhanced infiltration of residual tumors by CD8+CAR19 T cells (not shown). Next, we tested the response of CAR19 T cells in vitro to B-NHL cells in the presence or, absence of lenalidomide to determine the costimulatory effect of lenalidomide on signaling via CAR, our data show that lenalidomide significantly enhanced functional response of CAR19 T cells following recognition of B cells in vitro which is demonstrated by enhanced production of IFN-gamma and by increased expression of CD69 by CAR19 T cells, interestingly, this effect was seen only if CAR19 T cells but not B-NHL cells were pre-treated with lenalidomide or, when we activated CAR19 T cell with antibody to CAR but not with antibody to CD3. Thus, our data indicate that lenalidomide might work through direct effects on T cells and specifically enhance signaling via CAR. The biochemical events underlying this costimulatory effect of lenalidomide on signaling by CAR are currently being investigated. In summary, our data support the use of lenalidomide for augmentation CAR-based immunotherapy in clinical settings. Figure 1 Figure 1. Disclosures Klener: Cellgene: Research Funding.