211 Targeting BTN2A1 modulates anti-tumor activity of Vg9Vd2 T cells

BackgroundVg9Vd2 T constitute the predominant subset among gd T cells in peripheral blood. Their infiltration into malignant tissues is associated with a favorable prognosis. Their anti-tumor activity is triggered by intracellular accumulation of organic phosphoantigens (pAgs) due to tumorigenesis. Recently, BTN2A1 was shown to bind to the Vg9TCR chain allowing immune synapse between cancer and Vg9Vd2T cells, thus initiating the anti-tumoral response. In this study, we generated monoclonal antibodies against BTN2A1 and evaluated their ability to modulate γδT cell cytotoxicity.MethodsAnti-BTN2A1 mAbs were generated by mouse immunization. Their effect on Vg9Vd2 T cell degranulation, secretion of IFNγ/TNFα, and target cell killing as depicted by caspase 3/7 cleavage, were tested in co-cultures with Daudi, HL-60 cell lines and primary acute myelocytic leukemia (AML) blasts with or without zoledronate or the anti-BTN3A mAb 20.1. These readouts were measured by flow cytometry. Endometrial cancer spheroids were used to assess the ability of the anti-BTN2A1 antagonistic mAb to inhibit Vg9Vd2 T cell killing of cancers cells.ResultsWe generated 7 anti-BTN2A1 mAbs and tested their effect on Vg9Vd2 T cell degranulation against Daudi cells with or without zoledronate. Six out of 7 anti-BTN2A1 mAbs significantly inhibited basal Vg9Vd2 T cell degranulation against Daudi up to 17-fold, and 5 of them were able to inhibit Vg9Vd2 T cell degranulation against Daudi in presence of zoledronate. Consistently, anti-BTN2A1 mAbs abrogated zoledronate and anti-BTN3A 20.1-induced apoptosis with different efficiencies. The level of apoptosis inhibition after zoledronate and 20.1 treatment were correlated. Anti-BTN2A1 7.48 mAb was the clone with the highest inhibitory potential. Increasing concentrations of 7.48 abrogated not only Vg9Vd2 T cell degranulation (IC50= 0.033±0.0003 µg/mL) but also TNFα (IC50= 0.03±0.006 µg/mL) and IFNγ (IC50= 0.015±0.004 µg/mL) secretion against Daudi cells in presence of pAgs. The ability of anti-BTN2A1 antibodies to inhibit Vg9Vd2 induced tumor cell apoptosis was also shown in 3D endometrial cancer spheroids. In co-cultures of Vg9Vd2 T cells with primary AML blasts, the anti-BTN2A1 7.48 inhibited Vg9Vd2 T cell degranulation as well as TNFα, IFNγ production and killing of AML blasts.ConclusionsAntagonist antibodies to BTN2A1 highlighted its critical role in Vg9Vd2 anti-tumor responses. BTN2A1 is involved in Vg9Vd2 T cell anti-tumoral activity and can constitute an interesting therapeutic target for gdT cell response immunomodulation in cancer or immune diseases treatment.Ethics ApprovalThe research was approved by the relevant institutional review boards (ethic committee and ANSM, HEMATO-BIO IPC 2013-015, Ref ANSM 131368B-11, Sponsor Institut Paoli Calmettes N° ID RCB 2013-A01437-38).ConsentInformed consent was obtained from all donors in accordance with the 121 Declaration of Helsinki.

Natural Killer (NK) Cell Expression of CD2 as a Predictor of Serial Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)

NK cell ADCC supports monoclonal antibody anti-tumor therapies. We investigated serial ADCC and whether it could be predicted by NK phenotypes, including expression of CD16A, CD2 and perforin. CD16A, the NK receptor for antibodies, has AA158 valine or phenylalanine variants with different affinities for IgG. CD2, a costimulatory protein, associates with CD16A and can augment CD16A-signaling. Pore-forming perforin is essential for rapid NK-mediated killing. NK cells were monitored for their ADCC serial killing frequency (KF). KF is the average number of target cells killed per cell by a cytotoxic cell population. KF comparisons were made at 1:4 CD16pos NK effector:target ratios. ADCC was toward Daudi cells labeled with 51Cr and obinutuzumab anti-CD20 antibody. CD16A genotypes were determined by DNA sequencing. CD2, CD16A, and perforin expression was monitored by flow cytometry. Serial killing KFs varied two-fold among 24 donors and were independent of CD16A genotypes and perforin levels. However, high percentages of CD2pos of the CD16Apos NK cells and high levels of CD16A were associated with high KFs. ROC analysis indicated that the %CD2pos of CD16Apos NK cells can predict KFs. In conclusion, the extent of serial ADCC varies significantly among donors and appears predictable by the CD2posCD16Apos NK phenotype.

Benzoxazole Derivative K313 Induces Cell Cycle Arrest, Apoptosis and Autophagy Blockage and Suppresses mTOR/p70S6K Pathway in Nalm-6 and Daudi Cells

Benzoxazole derivative K313 has previously been reported to possess anti-inflammatory effects in lipopolysaccharide-induced RAW264.7 macrophages. To date, there have been no related reports on the anticancer effects of K313. In this study, we found that K313 reduced the viability of human B-cell leukemia (Nalm-6) and lymphoma (Daudi) cells in a dose-dependent manner without affecting healthy peripheral blood mononuclear cells (PBMCs) and induced moderate cell cycle arrest at the G0/G1 phase. Meanwhile, K313 mediated cell apoptosis, which was accompanied by the activation of caspase-9, caspase-3, and poly ADP-ribose polymerase (PARP). Furthermore, cells treated with K313 showed a significant decrease in mitochondrial membrane potential (MMP), which may have been caused by the caspase-8-mediated cleavage of Bid, as detected by Western blot analysis. We also found that K313 led to the downregulation of p-p70S6K protein, which plays an important role in cell survival and cell cycle progression. In addition, treatment of these cells with K313 blocked autophagic flux, as reflected in the accumulation of LC3-II and p62 protein levels in a dose- and time-dependent manner. In conclusion, K313 decreases cell viability without affecting normal healthy PBMCs, induces cell cycle arrest and apoptosis, reduces p-p70S6K protein levels, and mediates strong autophagy inhibition. Therefore, K313 and its derivatives could be developed as potential anticancer drugs or autophagy blockers in the future.

A Novel Class of Bifunctional Immunotherapeutic That Exploits a Universal Antibody Binding Terminus (uABT) to Recruit Endogenous Antibodies to Cell Expressing CD38 Demonstrate In Vivo efficacy in Three Distinct Animal Models

Background: Antibody recruiting molecules (ARM) represent a new modality in immunotherapy of cancer. These are bifunctional molecules composed of two active termini connected by a linker. One of the termini binds to a target molecule on a cancer cell. The other terminus can recruit endogenous IgG antibodies independent of their antigen binding specificity representing a breakthrough improvement to previous approaches (Murelli et al.J Am Chem Soc. 2009). We named this active moiety a universal antibody binding terminus (uABT). As a result of antibody recruitment to the cell surface, the target cell is "opsonized" by antibodies which then bring in the immune effector cells to eliminate the target through various antibody-dependent destruction mechanisms. Multiple myeloma is a neoplasm that arises from terminally differentiated immunoglobulin producing long-lived plasma cells with 32,000 new cases diagnosed each year. Kleo Pharmaceuticals has developed a series of compounds, CD38-ARM which target human CD38 highly expressed by multiple myeloma cells. CD38 -ARM compounds are therapeutically active in three distinct in vivo models without depleting CD38 expressing immune effector cells like existing therapeutic antibodies such as Daratumumab. Methods: CD38-ARM compounds were tested in three independent in vivo models. In the first model, intraperitoneal Daudi xenograft in SCID mice, 20x106 fluorescently labelled Burkitt lymphoma CD38-expressing Daudi cells were injected into SCID mice and treated with 3 mg/kg of compounds. Peritoneal exudates were examined 24 hours later for percentages and absolute numbers of Daudi cells recovered. Activity of compounds was further tested in a MOLP-8 multiple myeloma cell xenograft model in nude mice. Animals received 106 MOLP8 cells subcutaneously, and were treated with a daily dose of 10 mg/kg after tumor volumes of 150 mm3 were reached. Finally, CD38-ARM efficacy was examined in hu IL-15 transgenic NOG mice that have been preconditioned with busulfan and reconstituted i.p. with 2x106human NK cells. Three weeks after reconstitution, 5x106RAJI cells expressing CD38 were implanted s.c. and treatment commenced a week later with 10 mg/kg QDx14. Mice were monitored for NK cell levels and activation status in the blood during the whole study duration by flow cytometry. Results: CD38-uAbt compounds are able to induce clearance of Burkitt's lymphoma Daudi cells expressing high levels of CD38 in a SCID mouse intraperitoneal model. In addition, we show efficacy of one of these compounds in a multiple myeloma xenograft model in nude mice. Using the MOLP8 subcutaneously implanted tumor model we show that administration of our lead molecule is able to induce 50% tumor growth inhibition (TGI) after a daily dosing schedule against this tumor. This activity is comparable to a pharmacologically relevant dose of Daratumumab. Finally, we demonstrate that CD38-ARM treatment shows significant efficacy in humanized mouse model, where IL-15 Tg NOG mice have been reconstituted with human NK cells prior to tumor implantation. In this model daily dosing with the CD38-ARM resulted in up to 70% TGI when compared to untreated control groups. Using this dose/schedule, the molecule did not elicit NK cell depletion as noted in the Daratumumab group. Conclusions: We demonstrated that CD38-ARM compounds are therapeutically active in three distinct in vivo models. Depletion of Daudi cells in a peritoneal SCID model provides strong evidence for the CD38-ARM's capacity to engage macrophage effector functions. Profound inhibition of tumor growth in the NK cell centered humanized hIL-15 Tg NOG mouse model indicates that killing of target cells is executed by human NK cells and provides a foundation to advancing our compounds towards the clinic. Overall, our results demonstrate value of CD38-ARM molecules both as stand-alone therapeutic as well as a platform to develop compounds tailored to a specific indication, by varying target binding moiety of the molecule. Our results also indicate that CD38-ARM compounds engage a variety of effector mechanisms involved in tumor clearance and tumor growth delay, indicating therapeutic potential across a wide range of clinical settings. Disclosures Bunin: Kleo pharmaceuticals: Employment, Equity Ownership. McGrath:Kleo pharmaceuticals: Employment, Equity Ownership. Rossi:Kleo pharmaceuticals: Employment, Equity Ownership. Welsch:Kleo pharmaceuticals: Employment, Equity Ownership. Vidal:Kleo pharmaceuticals: Employment, Equity Ownership. Trinh:Kleo pharmaceuticals: Employment, Equity Ownership. Spiegel:Kleo pharmaceuticals: Equity Ownership. Rastelli:Kleo pharmaceuticals: Employment, Equity Ownership. Alvarez:Kleo pharmaceuticals: Employment, Equity Ownership.

Targeted Alpha Therapy with 212Pb-NNV003 for the Treatment of CD37 Positive B-Cell Chronic Lymphocytic Leukemia (CLL) and Non-Hodgkin Lymphoma (NHL)

Background Each year more than 90,000 cases of chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma (NHL) are expected in the US. The standard of care is immuno-chemotherapy with alkylating agents in combination with an anti-CD20 monoclonal antibody (mAb). While immuno-chemotherapy regimens are initially effective in inducing responses, most patients inevitably relapse and the same therapies show decreasing efficacy with repeated administration. In recent years, CD37 has been considered as a target for B-cell malignancies. CD37 is strongly and selectively expressed on the surface of mature B lymphocytes and B-cell malignancies. Therapies targeting CD37 expressing cells may become a useful alternative to CD20 targeting agents. Alpha-emitting radionuclides have demonstrated good potential for cancer targeted therapies because of efficient energy deposition along the short alpha track (50-100 µm). The absorbed energy cause irreparable DNA double-strand breaks and localized cytotoxicity while sparing surrounding healthy tissues. We have developed a targeted alpha therapy (TAT) where the CD37-specific antibody NNV003 is coupled to the alpha-particle-emitting radioisotope 212Pb. Materials and Methods Dose-dependent efficacy and tolerability of a single-dose 212Pb-NNV003 treatment was evaluated using escalating activity levels in human disseminated models of Burkitt's lymphoma (Daudi) and CLL (MEC-2). 10 million Daudi cells or 2.5 million MEC-2 cells were intravenously injected in CB17-SCID or R2G2 mice and 212Pb-NNV003 was given two days later. Unspecific, 212Pb-labeled antibody was used as control. A study of the tolerability of 212Pb-NNV003 was performed in R2G2 mice without tumor prior to the efficacy studies. Results 212Pb-NNV003 displays a favorable toxicity profile in tumor-free mice single intravenous dose injections up to 15 µCi showing 100 % survival 4 weeks post-injection. No acute hematological toxicity was observed and animals who received 5, 10 or 15 μCi doses of 212Pb-NNV003 presented only a slight initial reduction in their platelets (PLT) counts which was fully recovered by 20 days after injection. A single intravenous dose of 10, 15 or 20 µCi of 212Pb-NNV003 lead to 70 %, 90 % and 100 % of mice injected with MEC-2 cells being tumor free 20 weeks post cell injection (Figure 1). Control animals that received saline, cold antibody or 212Pb-cetuximab presented a median survival of 4.9, 5.4 and 9.3 weeks, respectively. A single intravenous dose of 2.5, 5 and 7.5 µCi 212Pb-NNV003 lead to over 80% tumor-free mice injected with Daudi cells 15 weeks post cell injection (Figure 2). Control animals that received saline, cold antibody or 212Pb-cetuximab presented a median survival of 7, 7.8 and 7.7 weeks, respectively. Conclusion The results of preclinical studies suggest that TAT using 212Pb-NNV003 is a safe and effective method for the treatment of CD37 positive CLL and NHL. Disclosures Saidi: Orano Med: Employment. Maaland:Nordic Nanovector: Employment. Torgue:Orano Med: Employment, Membership on an entity's Board of Directors or advisory committees. Heyerdahl:Nordic Nanovector: Employment, Equity Ownership. Dahle:Nordic Nanovector ASA: Employment, Equity Ownership, Patents & Royalties.

Mechanisms of Suppressive Effects of MYD88 Inhibitors on the Growth of Lymphoma and Leukemia Cells

Introduction : Myeloid differentiation primary response gene 88 (MYD88) is an adaptor protein that binds to the Toll-like/interleukin-1 receptor to form a homodimer and activates NF-kB pathway. Activating MYD88 mutations are found in 90% of lymphoplasmacytic lymphoma (Waldenström's macroglobulinemia) cases and 30% of activated B-cell type diffuse large B-cell lymphoma (DLBCL) cases. To investigate the role of MYD88 in the growth of lymphoma/leukemia cells, with or without MYD88mutations, and to examine whether MYD88 inhibitors can be used as novel molecular-targeted drugs, we studied their effects on the growth of lymphoma and leukemia cells in vitro. Methods : Seven lymphoma/leukemia cell lines (TMD8 derived from DLBCL with MYD88 mutations, Daudi from Burkitt lymphoma, NALM-6 from B-ALL, Jurkat and KOPT-K1 from T-ALL, THP-1 and TMD7 from AML without MYD88 mutations) and normal lymphocytes from healthy volunteers were obtained, following an informed consent. The effect of the MYD88 inhibitor, ST2825, on the in vitro growth of these cell lines was then examined using the WST-8 colorimetric assay. ST2825 is a synthetic peptide-mimetic compound, which inhibits MYD88 dimerization. A Bruton tyrosine kinase (BTK) inhibitor, ibrutinib, was used as the reference inhibitor. The effect of ST2825 on protein expression was examined by immunoblot analysis. Cell cycle analysis and Annexin V apoptosis assay were performed using flow cytometry. To comprehensively screen the changes in mRNA expression after ST2825 treatment, microarray analysis was performed for TMD8 and Jurkat cells. MYD88knockdown experiments using small interfering RNA were also performed. Results : The MYD88 protein was expressed in all cell lines. ST2825 (30 μM) suppressed the growth of all lymphoma/leukemia cell lines, but did not affect the viability of normal lymphocytes. The IC50 for TMD8 cells was similar to that for other cells, such as Daudi cells. Apoptosis assay revealed that ST2825 induces apoptosis in all the cell lines studied. In Daudi cells, ST2825 induced G2/M cell cycle arrest. Immunoblot analysis revealed that ST2825 suppresses the phosphorylation of certain NF-kB signaling components, such as RELA and IkB, in all cell lines. In TMD8, Daudi, and NALM-6 cells, ST2825 suppressed the phosphorylation of BTK, and MYD88 knockdown suppressed the phosphorylation of RELA and BTK. Microarray analysis revealed that ST2825 treatment downregulates the expression of various genes including MYD88, and upregulates the expression of other genes such as NGFR, FOSB, and HSP. Treatment with ibrutinib (1 nM) suppressed the growth of only TMD8 cells. Treatment with ST2825 plus ibrutinib additively suppressed the growth of TMD8 cells. Conclusions : The MYD88 inhibitor ST2825 suppresses the growth of various lymphoma and leukemia cells, suggesting that MYD88 is involved in regulating the growth of these cells; however, the off-target effects of ST2825 should be considered. Further investigation is required to assess the potential of MYD88 inhibitors as novel molecular-targeted drugs against lymphoma and leukemia. Disclosures No relevant conflicts of interest to declare.

Antiproliferative Efficacy of Kaempferol on Cultured Daudi Cells: An In Silico and In Vitro Study

There is always a constant need to develop alternative or synergistic anticancer drugs with minimal side effects. One important strategy to develop effective anticancer agents is to investigate potent derived compounds from natural sources. The present study was designed to determine antiproliferative activity of Kaempferol using in silico as well as in vitro study. Docking was performed using human GCN5 (hGCN5) protein involved with cell cycle, apoptosis, and glucose metabolism. Cell viability and cytotoxicity on Daudi cells were evaluated by trypan blue and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays in a dose and time dependent manner, respectively. The compound inhibited the proliferation and growth of the Daudi cells, through induced cell death. The pure compound proved lead inhibitors of cell proliferation, thus manifesting significant antiproliferative activity. The docking results revealed that Kaempferol exhibited binding interaction to hGCN5 protein. Further, molecular dynamics using the dock pose of hGCN5-Kaempferol complex were performed to understand the basic structural unit which lead to inefficiency in binding and, therefore, pronounced instability and its possible consequences of reduced binding affinity. The data obtained in this study indicates that Kaempferol is a promising compound leading to inhibition of Daudi cell growth and proliferation.