ALBERT

Description: Chimeric Antigen Receptors (CARs) are engineered constructs consisting of an antibody-derived antigen recognition domain linked to intracellular T cell signaling domains. Cytotoxic T cells transduced to express tumor-reactive CARs are highly promising tools for immunotherapy of cancer. The CD38 molecule, with its high and homogenous expression on Multiple Myeloma (MM) tumor cells, is considered a suitable target for antibody therapy of MM. Prompted by this, we evaluated the feasibility and efficacy of targeting MM cells with CD38-CAR-transduced T cells (CD38-CAR T cells). To this end, we generated three different retroviral CAR constructs based on human CD38 antibodies as antigen recognition domain, CD3zeta and 41BB (CD137) as signaling domains and transduced them into PBMCs of a healthy donor. After in vitro selection and expansion, all CD38-CAR T cells, either unsorted or CD4/CD8 sorted, effectively lysed MM cell lines in a dose-, and CD38 expression-dependent manner, with a better efficacy for the CD8+ fraction. CD38-CAR T cells also effectively eradicated primary MM cells in the bone marrow mononuclear cells derived from MM patients, indicating their clinical relevancy. Although CD38-CAR T cells also displayed cytotoxic activity against the CD38+ fraction of mature monocytes and NK cells and to a lesser extent CD38+ B and T cells, they did not affect the outgrowth of CD34+ cells into various myeloid lineages. In addition,CD38-CAR T cell activity was effectively controllable by transducing them with a caspase 9-based inducible suicide gene. More interestingly, we discovered that the CD38-CAR T cells were themselves devoid of CD38 surface expression, indicating that CD38 was not essential for T cell expansion and function. Finally, in a novel in vivo xenotransplant model (UM9 cell line), in which myeloma cells were grown in a humanized bone marrow microenvironment, i.v. as well as intra tumor administration of CD38-CAR T cells established significant anti-tumor effects, proving that CD38-CAR endowed cytotoxic T lymphocytes, even with no CD38 expression, can efficiently migrate, infiltrate and eliminate human MM tumors growing in their natural niche. These results demonstrate the feasibility and potency of CAR mediated targeting of CD38+ MM cells. Optimization of CD38-CAR and suicide-gene control of CD38 CAR T cellsmay provide next steps towards safe clinical implementation of CD38-CAR T cell immune therapy. Disclosures Drent: Genmab BV: Guest employee (unpaid) Other. Lammerts van Bueren:Genmab : Employment. Parren:Genmab: Employment, Equity Ownership. van de Donk:Genmab BV: Research Funding; J&J: Research Funding; Celgene: Research Funding. Martens:Genmab BV: Research Funding; J&J: Research Funding; Celgene: Research Funding. Lokhorst:Celgene: Research Funding; J&J: Research Funding; Genmab: Research Funding. Mutis:Celgene: Research Funding; J&J: Research Funding; Genmab BV: Research Funding.

Description: Multiple myeloma (MM) remains an incurable malignancy of clonal plasma cells. Although the new generation of immunomodulatory agents, such as lenalidomide (LEN), and the potent proteasome inhibitor bortezomib (BORT) have significantly improved the overall survival of MM patients, all chemotherapy strategies are eventually hampered by the development of drug-resistance. The outcome of patients who are refractory to thalidomide, lenalidomide (LEN) and bortezomib (BORT) is very poor. Set out with the idea that targeted immunotherapy with human antibodies may offer new perspectives for MM patients, we have recently developed daratumumab (DARA), a CD38 human antibody with broad-spectrum killing activity, mainly via ADCC (antibody dependent cellular cytotoxicity) and CDC (complement dependent cytotoxicity). In our previous preclinical studies and in current clinical phase I/II trials, DARA induces marked anti-MM activity. Based on these encouraging results, we now explored the potential activity of DARA for patients who are refractory to LEN- and/or BORT. In a recently developed human-mouse hybrid model that allows the in vivo engraftment and outgrowth of patient-derived primary myeloma cells in immune deficient Rag2-/-gc-/- mice, single dose DARA treatment appeared to effectively inhibit the malignant expansion of primary MM cells derived from a LEN- and BORT-refractory patient, indicating the potential efficacy of DARA even in LEN/BORT refractory patients. To substantiate the conclusions of these in vivo data, we conducted in vitro assays, in which full BM-MNCs from LEN (n=11) and LEN/BORT (n=8) refractory patients were treated with DARA alone or the combination of DARA with LEN or BORT to induce MM cell lysis. As expected, LEN alone induced no or little lysis of MM cells in the LEN-refractory patients and also BORT was not able to induce any lysis in the BORT-refractory patients. On the contrary, DARA induced substantial levels of MM cell lysis in all LEN and LEN/BORT-refractory patients. This lysis was significantly enhanced by combination with LEN or BORT. The combination of DARA and BORT improved MM lysis by additive mechanisms. However, LEN improved DARA-mediated lysis of MM cells in a synergistic manner through the activation of effector cells involved in DARA-mediated ADCC. In conclusion, our results demonstrate that DARA is also effective against multiple myeloma cells derived from LEN- and BORT-refractory patients. Especially LEN seems to improve responses in a synergistic manner. Our results provide a rationale for clinical evaluation of DARA in combination with LEN to achieve more effective results in LEN- and BORT-refractory patients. Disclosures: Lammerts van Bueren: Genmab: Employment. Bakker:Genmab: Employment. Parren:Genmab: Employment. van de Donk:Celgene: Research Funding. Lokhorst:Genmab A/S: Consultancy, Research Funding; Celgene: Honoraria; Johnson-Cilag: Honoraria; Mudipharma: Honoraria.

Description: Background Durable tumor regression can be achieved in Multiple Myeloma (MM) patients by immunotherapeutic approaches, such as allogeneic stem cell transplantation and donor lymphocyte infusion. However, the low rate of patients, who reach a sustained remission, impedes the success of this therapeutic approach. Currently, much emphasis is placed on the role of the tumor microenvironment in tumor cell immune escape. We previously described that different accessory cells of the microenvironment significantly inhibited the lysis of MM cell lines by T cells in a cell-cell contact dependent manner. Further analysis revealed that this immune escape could be attributed to the induction of a cell adhesion-mediated immune resistance (CAM-IR) of MM against T cell lysis in vitro. Further in vitro studies identified up-regulation of survivin in the MM cells as one of the potential mechanisms of immune resistance. Moreover, co-culture with YM155, a small molecule survivin suppressant, could abrogate accessory cell induced resistance in vitro. Methods and Results We investigated the influence of a human bone marrow microenvironment on the anti tumor activity of T cell therapy in a human-mouse hybrid model (Groen et al. Blood 2012). Immune-deficient RAG2-/- γc-/- -mice were implanted subcutaneously either with uncoated scaffolds or scaffolds coated with human mesenchymal stromal cells (MSC), which generates a human bone marrow environment. Luciferase transduced MM cell line UM9 injected directly in both types of scaffolds grew into MM tumors and were treated with Myeloma reactive minor Histocompatibility antigen specific T cell clones. The T cells induced effective anti-myeloma responses against tumors developed in the uncoated scaffolds. In contrast, and consistent with the in vitro results, no anti-tumor effect was observed in the MSC coated scaffolds. Thus confirming in vivo a microenvironment induced resistance of MM cells to T cell kill. We next investigated if the resistance could be abrogated by combination therapy with T cells and YM155.To this end, mice with tumors in human MSC coated scaffolds were treated with T cells alone, YM155 alone, or the combination of both. YM155- nor T cell-monotherapy induced tumor regression on evaluation of tumor growth based on bioluminescent signal. Moreover, the combination of YM155 with T cells demonstrated a substantial anti-tumor effect. Conclusion YM155 treatment can sensitize myeloma cells to T cell mediated anti-tumor effects and more importantly can overcome microenvironment-mediated resistance of MM cells to T cell treatment. These findings support further development of CAM-IR modulating agents in combination with immune therapy. Disclosures: Minnema: Janssen Cilag: Consultancy, Honoraria. Lokhorst:Genmab A/S: Consultancy, Research Funding; Celgene: Honoraria; Johnson-Cilag: Honoraria; Mudipharma: Honoraria.

Description: T cells retrovirally modified to express therapeutic genes encoding cytokines, exogenous TCRs or suicide molecules represent a novel class of immune therapeutics of great potency. However, recent clinical trials using retrovirally-modified T cells have indicated that T cells exhibit a diminished reactivity upon ex vivo manipulation. In addition, virus-specific memory T cells seem to be lost during gene transfer. In a BNML rat model we have shown that the culture procedure is one of the critical parameters. To preserve T cell reactivity, reliable models are required which permit readout of human T cell activity. We recently developed a huPBMC-RAG2−/−γc−/− mouse model for xenogeneic graft-versus-host disease (xGVHD), in which iv injection of 15 x 106 human T cells into RAG2−/−γc−/− mice consistently leads to high level engraftment and lethal xGVHD within 3 weeks in 80% of mice (van Rijn et al, Blood 2003). We have now used this model to analyze in vivo functionality of human T cells following different ex vivo culture procedures. For this, we cultured human T cells for 7 days with either of the two currently available clinically applicable stimulation conditions: 1) via CD3 and 2) via CD3/CD28. In addition, we included CD3/CD28/4-1BB stimulation to explore the effect of extensive costimulation. Mice were injected with escalating doses T cells. HuCD45+ cells in peripheral blood were measured by FACS. Lethal xGVHD occurred at only 6 times (90.106) the dose of fresh cells for CD3-stimulated T cells and 3 times for CD3/28- or CD3/28/4-1BB-stimulated cells. About 20% of surviving mice developed chronic xGVHD, independent of culture method. While lethal xGVHD was always associated with very high levels of engraftment (up to 95%) engraftment levels in chronic mice ranged from 1–75%. To compare the impact of the different culture conditions on in vivo T cell function, we analyzed engraftment potential. The fraction of huCD45+ cells was plotted against the time and the areas under the curves were compared. Based on a total of 68 mice, statistical analysis showed a 2-fold improvement of engraftment potential for C28-costimulated human T cells compared to CD3-stimulated cells (P

Description: We studied the cell kill induced by granulocyte-macrophage colony-stimulating factor (GM-CSF ) fused to Diphtheria Toxin (DT-GM-CSF ) in acute myeloid leukemia (AML) samples and in populations of normal primitive hemopoietic progenitor cells. AML samples from three patients were incubated in vitro with 100 ng/mL DT-GM-CSF for 48 hours, and AML cell kill was determined in a proliferation assay, a clonogenic assay colony-forming unit-AML (CFU-AML) and a quantitative long-term bone marrow (BM) culture ie, the leukemic-cobblestone area forming cell assay (L-CAFC). To measure an effect on cells with in vivo leukemia initiating potential DT-GM-CSF exposed AML cells were transplanted into immunodeficient mice. In two out of three samples it was shown that all AML subsets, including those with long-term abilities in vivo (severe combined immunodeficient mice) and in vitro (L-CAFC assay) were reduced in number by DT-GM-CSF. Cell kill induced by DT-GM-CSF could be prevented by coincubation with an excess of GM-CSF, demonstrating that sensitivity to DT-GM-CSF is specifically mediated by the GM-CSF receptor. Therefore, binding and internalization of GM-CSF probably occur in immature AML precursors of these two cases of AML. The third AML sample was not responsive to either GM-CSF or DT-GM-CSF. The number of committed progenitors of normal bone marrow (burst-forming unit-erythroid, colony-forming unit granulocyte- macrophage, and cobble stone area forming cell [CAFC] week 2) and also the number of cells with long-term repopulating ability, assayed as week 6 CAFC, were unchanged after exposure to DT-GM-CSF (100 ng/mL, 48 hours). These studies show that DT-GM-CSF may be used to eliminate myeloid leukemic cells with long-term potential in vitro and in immunodeficient mice, whereas normal hemopoietic stem cells are spared.

Description: Expression of the heparan sulfate proteoglycan syndecan-1 is a hallmark of both normal and multiple myeloma (MM) plasma cells. Syndecan-1 could affect plasma cell fate by strengthening integrin-mediated adhesion via its core protein and/or by accommodating and presenting soluble factors via its HS side chains. Here, we show that inducible RNAi-mediated knockdown of syndecan-1 in human MM cells leads to reduced growth rates and a strong increase of apoptosis. Importantly, knockdown of EXT1, a copolymerase critical for HS chain biosynthesis, had similar effects. Using an innovative myeloma xenotransplantation model in Rag-2−/−γc−/− mice, we demonstrate that induction of EXT1 knockdown in vivo dramatically suppresses the growth of bone marrow localized myeloma. Our findings provide direct evidence that the HS chains of syndecan-1 are crucial for the growth and survival of MM cells within the bone marrow environment, and indicate the HS biosynthesis machinery as a potential treatment target in MM.

Description: The NSG [NOD/Lt-scid/IL2Rγnull] xenotransplantation mouse model is currently the model of choice to evaluate human hematopoietic engraftment and to study development of human leukemia. Indeed, we have previously shown that co-expression of BCR-ABL together with the polycomb repression complex 1 (PRC1) member BMI1 in human cord blood (CB) derived CD34+ cells was sufficient to induce a serially transplantable lymphoid leukemia (Rizo et al., Blood 2010). This leukemia was characterized by high levels of CD34+/CD19+/CD20-/IgM-/CD33-/CD15- lymphoid blasts in the bone marrow and a high degree of infiltration of blasts in spleen and liver. Clonal analysis revealed that similar clones gave rise to leukemia in primary and secondary recipients. Although in vivo no myeloid leukemias were observed, in vitro both lymphoid as well as myeloid immortalized long-term cultures could readily be established, in line with phenotypes observed in chronic myeloid leukemia patients whereby a chronic myeloid phase can egress into a myeloid or lymphoid blast crisis. It is very plausible that differences between murine and human hematopoietic stem cell niches underlie these observed differences. Human engraftment in NSG mice is typically lymphoid biased, and since many growth factors and cytokines are species-specific it is clear that the murine niche is not ideal to evaluate human hematopoietic engraftment and leukemic transformation potential. In our current study we have evaluated the in vivo leukemic transformation potential of human CB derived CD34+ cells expressing BCR-ABL and/or BMI1 in NSG mice in which scaffolds coated with culture-expanded human mesenchymal stromal cells (MSCs) were implanted subcutaneously 8 weeks prior to injection of transduced cells, to allow the development of a humanized niche containing mineralized bone-matrix, osteoblasts, stromal cells, as well as appropriate vascularization (Groen et al., Blood 2012). BCR-ABL/BMI1 transduced human CB derived CD34+ cells or primary blast crisis CML patient cells were injected either intravenously or directly into the humanized scaffolds, and leukemia development was evaluated. Our data indicate that in a humanized niche, in contrast to a murine niche, BCR-ABL was sufficient to induce leukemia as a single hit without overexpression of exogenous BMI1. Furthermore, both ALL as well as erythro/myeloid leukemias could be induced. The ALL could be transplanted to secondary recipients and besides the lymphoid marker CD19, the cells also expressed CD33 and CD15, but not CD11b or GPA. These data are in sharp contrast to results obtained in xenograft mouse models without human niches, where BCR-ABL expression alone in human cells was not sufficient to induce leukemia, and secondary hits such as BMI1 were essential. Efficient engraftment of a blast-crisis CML patient sample was also observed in the human niche model, whereby the immature blast-like phenotype was maintained in the human scaffold niche, while more differentiated cells were observed in the mouse bone marrow niche. In vitro, long-term self-renewing cultures could readily be established with cells retrieved from the human scaffold niche of these leukemic mice, while no long-term cultures could be initiated with cells retrieved from the murine bone marrow niche, from the same mouse. These data indicate that a human niche is required to maintain appropriate in vivo self-renewal of human BC CML cells. Interestingly, the endogenous BMI1 levels were significantly higher in cells retrieved from the human scaffold niche as compared to the mouse BM niche. In conclusion, our data indicate that BCR-ABL transformed cells needs secondary event such as over expression of oncogene like BMI1 for its full transformation potential, most likely to overcome or repress oncogene-induced senescence. The mouse environment is not able to provide these secondary events in human cells, whereas the human niche is able to provide signals that together with BCR-ABL are sufficient to fully transform human cells in xenograft models. Disclosures: No relevant conflicts of interest to declare.