Jean-François Mirjolet

Abstract 857: Development of a Bruton's tyrosine kinase (Btk) inhibitor - ONO-WG-307, a potential treatment for B-cell malignancies

Purpose: Signals from B cell receptors (BCR) play a central role in signal transduction pathways regulating survival, activation, proliferation, and differentiation of B-lineage lymphoid cells. BCR signaling is implicated in the survival of malignant B cells and recent studies indicate that targeting Btk, an essential component of the BCR pathway, may be effective in the treatment of B-cell lymphoma. ONO-WG-307 is a highly potent and selective Btk inhibitor with an IC50 in the sub-nmol/L range. We evaluated the inhibitory effect of ONO-WG-307 alone and in combination with the chimeric type I anti-CD20 antibody rituximab, using in vitro tumor growth assays and mouse xenograft models. Methods: Two types of tumor cell lines (follicular lymphoma (FL) and activated B-cell-like (ABC) sub-type of diffuse large B cell lymphoma (DLBCL)) wereplated on to micro-titration plates (at doses up to 200 umol/L for ONO-WG-307 and 100 ug/mL for rituximab) for in vitro cytotoxic assays. The same cells were also implanted subcutaneously (SC) into female SCID mice to explore ONO-WG-307 anti-tumor activity in vivo (orally at doses up to 50 mg/kg, bid for ONO-WG-307 and intraperitoneally at doses up to 10 mg/kg, q7d). The IC50 of the in vitro cytotoxic activity was determined using a MTS assay 72 and 96 hours after incubation. Anti-tumor activity was defined as the ratio of the median tumor volume of treatment groups versus control group. The determination of combination index (CI) was calculated by the median-effect method. The CI was used to express synergism ( 1). Results: In the MTS assay, ABC-DLBCL cells were much more sensitive to ONO-WG-307 given as single agent compared with FL cell lines, however, the activity of rituximab single agent had no impact on ABC-DLBCL cell lines in contrast to its inhibitory activity against FL cell lines. When ONO-WG-307 was combined with rituximab, a moderate antagonism was observed in vitro on FL cell lines. In an ABC-DLBCL xenograft model, treatment with single agent ONO-WG-307 resulted in a dose-dependent inhibition of tumor growth and also showed moderate anti-tumor activity in a FL xenograft model. Conclusion: ONO-WG-307 is a highly potent and selective oral Btk inhibitor with evidence of efficacy in both the ABC-DLBCL and the FL cell lines and in xenograft models. These preliminary results suggest that ONO-WG-307 may be an effective therapy in the treatment of B-cell malignancies. To determine whether combination of ONO-WG-307 and rituximab is more efficacious, additional in vivo combination work is underway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 857. doi:1538-7445.AM2012-857

Two new sesquiterpene derivatives from the Tunisian endemic Ferula tunetana Pom.

A new sesquiterpene ester, tunetanin A (1), a new sesquiterpene coumarin, tunetacoumarin A (2), together with eight known compounds, i.e., coladin (3), coladonin (4), isosmarcandin (5), 13‐hydroxyfeselol (6), umbelliprenin (7) propiophenone (8), β‐sitosterol (9), and stigmasterol (10), were isolated from the roots of Ferula tunetana. Their structures were elucidated on the basis of extensive spectroscopic methods, including 1D‐ and 2D‐NMR experiments and MS analysis, as well as by comparison with published data. The cytotoxicity of compounds 1–7 towards two human colon cancer cell lines, HT‐29 and HCT 116, was evaluated. Compounds 3, 4, and 6 showed weak cytotoxic activities.

Unusual oleanane-type saponins from Arenaria montana.

Three oleanane-type saponins, 3-O-β-d-glucopyranosylechinocystic acid 28-O-β-d-xylopyranosyl-(1→4)-[α-l-rhamnopyranosyl-(1→2)]-α-l-rhamnopyranosyl ester (1), 3-O-β-d-glucopyranosylechinocystic acid 28-O-α-l-arabinopyranosyl-(1→3)-β-d-xylopyranosyl-(1→4)-[α-l-rhamnopyranosyl-(1→2)]-α-l-rhamnopyranosyl ester (2), 3-O-β-d-glucopyranosylcaulophyllogenin 28-O-β-d-apiofuranosyl-(1→3)-β-d-xylopyranosyl-(1→4)-[β-d-apiofuranosyl-(1→3)]-α-l-rhamnopyranosyl-(1→2)-α-l-rhamnopyranosyl ester (3) were isolated from the whole plant of Arenaria montana. Their unusual structures for the Caryophyllaceae family were established mainly by 2D NMR techniques and mass spectrometry.

New Adamantane Phenylalkylamines with σ-Receptor Binding Affinity and Anticancer Activity, Associated with Putative Antagonism of Neuropathic Pain

The synthesis of the adamantane phenylalkylamines 2a-d, 3a-c, and 4a-e is described. These compounds exhibited significant antiproliferative activity, in vitro, against eight cancer cell lines tested. The σ(1), σ(2), and sodium channel binding affinities of compounds 2a, 3a, 4a, and 4c-e were investigated. The most interesting analogue, 4a, exhibited significant in vivo anticancer profile on pancreas, prostate, leukemia, and ovarian cancer cell line xenografts together with apoptosis and caspase-3 activation. Inhibition of the cancer cells cycle at the sub-G1 level was also obtained with 4a. Finally, encouraging results were observed with 4a in vivo on mice, suggesting putative antimetastatic and analgesic activities of this compound.

Triterpenoid saponins from Hydrocotyle bonariensis Lam

Phytochemical investigation of the under-ground parts of Hydrocotyle bonariensis led to the isolation of five oleanane-type triterpenoid saponins, 3-O-{β-D-glucopyranosyl-(1 → 2)-[α-L-arabinopyranosyl-(1 → 3)]-β-D-glucuronopyranosyl}-21-O-(2-methylbutyroyl)-22-O-acetyl-R(1)-barrigenol, 3-O-{β-D-glucopyranosyl-(1 → 2)-[α-L-arabinopyranosyl-(1 → 3)]-β-D-glucuronopyranosyl}-21-O-(2-methylbutyroyl)-28-O-acetyl-R(1)-barrigenol, 3-O-{β-D-glucopyranosyl-(1 → 2)-[α-L-arabinopyranosyl-(1 → 3)]-β-D-glucuronopyranosyl}-21-O-acetyl-R(1)-barrigenol, 3-O-{β-D-glucopyranosyl-(1 → 2)-[α-L-arabinopyranosyl-(1 → 3)]-β-D-glucuronopyranosyl}-R(1)-barrigenol, and 3-O-{β-D-glucopyranosyl-(1 → 2)-[α-L-arabinopyranosyl-(1 → 3)]-β-D-glucuronopyranosyl}-22-O-(2-methylbutyroyl)-A(1)-barrigenol, together with the known saniculoside-R1. Their structures were established by 2D NMR techniques and mass spectrometry. Six compounds were evaluated against two human colon cancer cell lines, HCT 116 and HT-29. Two compounds showed weak cytotoxicity with IC(50) 24.1 and 24.0, 83.0 and 83.6 μM against HT-29 and HCT 116, respectively.

Triterpene Saponins from Cyclamen trocopteranthum

Two new triterpene saponins ( 1- 2) together with three known saponins, deglucocyclamin I ( 3), cyclamin ( 4), and mirabilin ( 5), were isolated from the tubers of Cyclamen trocopteranthum. They were elucidated as 3 beta- O-{4- O-[3-hydroxyl-3-methylglutaryl]- beta-D-xylopyranosyl-(1 --> 2)- beta-D-glucopyranosyl-(1 --> 4)-[ beta-D-glucopyranosyl-(1 --> 2)]- alpha-L-arabinopyranosyl}-16 alpha-hydroxy-13 beta,28-epoxy-oleanan-30-al ( 1) and 3 beta- O-{4- O-[3-hydroxyl-3-methylglutaryl]- beta-D-xylopyranosyl-(1 --> 2)-[ beta-D-glucopyranosyl-(1 --> 6)]- beta-D-glucopyranosyl-(1 --> 4)-[ beta-D-glucopyranosyl-(1 --> 2)]- alpha-L-arabinopyranosyl}-16 alpha-hydroxy-20,30-lactone-olean-12-ene ( 2). Their structures were characterized mainly by a combination of 1D- and 2D-NMR techniques ( (1)H- (1)H COSY, TOCSY, NOESY, HSQC, and HMBC) and mass spectroscopy. Saponins 1, 3, and 4 showed a weak cytotoxic activity when tested against HT-29 and HCT 116 tumor colon cancer cells.

Structure elucidation of new oleanane‐type glycosides from three species of Acanthophyllum

From the roots of three species of Acanthophyllum (Caryophyllaceae), two new gypsogenic acid glycosides, 1 and 2, were isolated, 1 from A. sordidum and A. lilacinum, 2 from A. elatius and A. lilacinum, together with three known saponins, glandulosides B and C, and SAPO50. The structures of 1 and 2 were established mainly by 2D NMR techniques as 23‐O‐β‐D‐galactopyranosylgypsogenic acid‐28‐O‐β‐D‐glucopyranosyl‐(1→3)‐[β‐D‐glucopyranosyl‐(1→6)]‐β‐D‐galactopyranoside (1) and gypsogenic acid‐28‐O‐β‐D‐glucopyranosyl‐(1→3)‐[β‐D‐glucopyranosyl‐(1→6)]‐β‐D‐galactopyranoside (2). The cytotoxicity of several of these saponins was evaluated against two human colon cancer cell lines (HT‐29 and HCT 116). Copyright

Abstract 3226: Efficacy of PD-1 - PD-L1 pathway disruptors in syngeneic models

PD-1 - PD-L1 pathway disruptors (e.g. nivolumab, pembrolizumab) are now being approved for treatment of patients with locally evolved or metastatic melanoma and advanced non-small cell lung cancer (NSCLC) who have progressed after first line platinum-based chemotherapy. These drugs have also antitumor efficacy in other tumor types (renal cell carcinoma, bladder, Hodgkin lymphoma, colorectal carcinoma with mismatch-repair deficiency ⋯). However, there is still needs to identify predictive biomarkers of response in order to select patients who will benefit from treatments. PD-L1 expression, mutation numbers, or mismatch repair (MMR) deficiencies were already demonstrated to influence response to PD-1 targeting therapies. However, they did not completely discriminate responders from non-responders or cut off levels are not yet precisely defined. Syngenic models are one of the only experimental models to evaluate immune system targeting therapies. Eight mouse models (4T1, A20, B16-F10, CT-26, EMT-6, MBT-2, LLC and Renca) were tested for response to PD-1 or PD-L1 targeting antibodies. From them, 3 models were characterized as non-responder (i.e. 4T1, LLC and RenCa) while other 7 models were showing to be sensitive to PD-1 inhibitors. PD-L1 expression (using RT-qPCR, WB and flow cytometry) was analyzed on cell lines at baseline and after 24h incubation with IFNg. At baseline, there is no significant difference between responders and non-responders whatever the level of analysis (mRNA, total PD-L1 protein expression or cell surface PD-L1 expression). However, after incubation with IFNg, there is a trend to segregate between responders and non-responders. Increased level of PD-L1 at the cell surface was evidenced for responders. IFNg signaling pathway used interferon regulatory factors (IRFs). IRF1 have been demonstrated to be responsible for both constitutive PD-L1 expression and early induction of PD-L1 after IFNg stimulation. mRNA expression of IRF1, IRF3, IRF7 and IRF9 were analyzed at baseline and after 24h incubation with IFNg. At baseline or after IFNg stimulation, there is no significant difference for IRF3, IRF7 and IRF9 mRNA expression between responders and non-responders. However, IRF1 mRNA is less expressed at baseline in responders as compared to non-responders. Moreover, increase in IRF1 expression after IFNg stimulation is higher in responders when compared to non-responders, which correlates well with changes in PD-L1 cell surface expression after IFNg stimulation. The total number of genomic variations was also analyzed using whole exome sequencing. Responder cell lines have highest number of variations in comparison to non-responder cell lines. In depth characterization of these syngeneic models will increase the understanding of how translatable are the results acquired from preclinical mouse studies. Citation Format: Anais Lagrange, Romain Boidot, Marc Hillairet de Boisferon, Olivier Duchamp, Jean-Francois Mirjolet, Francois Ghiringhelli. Efficacy of PD-1 - PD-L1 pathway disruptors in syngeneic models. [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 3226.

Abstract 5459: Antitumor activity of EP80061, a small-glyco drug in preclinical studies

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Background: Heparan sulphate (HS) containing proteoglycans regulate the activity of many proteins (growth factors, cytokines, adhesion molecules…) involved in pathologies like cancer, inflammation, cardiovascular, metabolic and neurodegenerative diseases, as well as in viral infection. HS mimicking oligosaccharides can interfere with protein/HS interactions and thus modulate the resulting biological effects. The optimization of fully synthetic HS mimetics exhibiting anti-tumour and anti-metastatic properties is currently in progress. Methods: Compound selection was performed using classical in vitro assays (binding and proliferation) and a cellular angiogenesis assay (Angiokit®). In vivo, Swiss nude mice subcutaneously (SC) xenografted with A-673 human rhabdomyosarcoma tumor cells were used to compare compounds showing significant anti-tumour activity. Compounds were intraperitonealy (IP) injected on a daily basis. The most potent compound was then evaluated on syngeneic mouse models: C57 BL/6 mice intravenously (IV) injected with B16-F10 malignant melanoma cells and Balb/c mice bearing orthotopic (OT) Renca kidney carcinoma. Results: Binding assays to FGF-2, PDGF-BB, VEGF-B and SDF-1α in Biacore experiments and growth factor-dependent proliferation tests led to the identification of several inhibitors. Optimized compounds exhibiting anti-angiogenic activities were selected in Angiokit® assays. EP80061 was selected for further studies on the basis of significant activity in this assay. In A-673 SC mouse model, EP80061 was also the most powerful compound. When IP injected at 30 mg/kg/inj. for 28 consecutive days, it induced a marked tumour growth delay. At D22, the optimal T/C value (46%) was reached with a significantly lower tumour volume (p=0.0006, 1672 ± 760 and 765 ± 402 mm3 for vehicle and EP80061 treated mice, respectively). EP80061 induced also a very significant decrease in lung metastasis development in B16-F10 IV injected mice treated at 30 mg/kg/inj. (p<0.0001, 92 ± 24 and 12 ± 8 metastases for vehicle and EP80061 treated mice, respectively). Promising results were also obtained on OT Renca bearing mice where EP80061 induced a decrease of OT tumour size. Conclusion: Current preclinical results support EP80061 as a lead candidate for further developments of small-glyco drugs as anticancer compounds. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5459.

Abstract 2669: Antitumor activity of the CMP-001 (TLR9 agonist) alone or combined with immune modulators in syngeneic tumor models

Targeted blockade of checkpoint inhibitors such as CTLA-4 or PD-1 with antagonist monoclonal antibodies (mAbs) has shown impressive and durable clinical responses in patients with advanced cancer. An alternative strategy to boost anti-tumor immunity is to promote T cell activation through co-stimulatory receptors such as OX40 and 4-1BB. OX40 is of particular interest as treatment with an activating anti-OX40 mAb augments T cell differentiation and cytolytic function leading to enhanced anti-tumor immunity. However, each of these agents benefits only a subset of patients, highlighting the critical need for more effective combinatorial therapeutic strategies. Toll-like receptor 9 (TLR9) agonist CpG oligodeoxynucleotides (ODN) are candidates to promote an anti-tumor immune response. CMP-001, a CpG-A ODN formulated within a virus-like particle, is designed to activate TLR9 (the receptor for CpG-A) in tumor-associated plasmacytoid dendritic cells (pDC) within the tumor or tumor-draining lymph nodes. Resting or immature pDC promote tumor growth, but when activated by CpG-A, the resulting mature pDC promote a robust anti-tumor immune response. Activation of pDC causes secretion of very large quantities of type I interferons, increased expression of costimulatory molecules, and recruitment and activation of other DC subsets to enhance tumor antigen presentation to T cells, culminating in the generation of effective anti-tumor T cell responses. The preclinical efficacy of intratumorally administered CMP-001 alone or in combination with an intraperitoneally administered PD-1 antagonist and/or an OX40 agonist was examined and assessed in a variety of syngeneic tumor models: CT-26 colon tumor model, MBT-2 bladder tumor model, RenCa kidney tumor model, 4T1 breast tumor model and LLC-1 lung tumor model. Tumors were implanted into left and right flanks while only one tumor was injected with CMP-001. In addition to body weight and overall survival, tumor volume was monitored on both flanks to assess direct and abscopal/systemic anti-tumor activity. Some discrepancies were observed between evaluated syngeneic tumor models with non-responders (LLC-1, 4T1) and responders (CT-26, MBT-2, RenCa). The most efficacious results were registered in the CT-26 model. Each therapeutic yielded weak activity as a single agent, which improved when combined with another treatment modality. The best therapeutic efficacy was obtained with the combination of all three agents resulting in cures of both treated and untreated CT-26 tumors in 40% of the mice. The median survival time was increased for these animals compared to those treated with only vehicle or one or two immune modulators (50 vs 18 vs 21 or 23-28 days, respectively). Similar results were generated in the MBT-2 model (and to a less extent in RenCa model) though no complete response was recorded. These data support the clinical investigation of these combinations in cancer patients Citation Format: Francis Bichat, Sylvie Maubant, Jean-Francois Mirjolet, Philippe Slos, Arthur M. Krieg, Aaron Morris. Antitumor activity of the CMP-001 (TLR9 agonist) alone or combined with immune modulators in syngeneic tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2669. doi:10.1158/1538-7445.AM2017-2669