Kate Sasser

Upregulation of CD38 expression on multiple myeloma cells by all- trans retinoic acid improves the efficacy of daratumumab

Daratumumab is an anti-CD38 monoclonal antibody with lytic activity against multiple myeloma (MM) cells, including ADCC (antibody-dependent cellular cytotoxicity) and CDC (complement-dependent cytotoxicity). Owing to a marked heterogeneity of response to daratumumab therapy in MM, we investigated determinants of the sensitivity of MM cells toward daratumumab-mediated ADCC and CDC. In bone marrow samples from 144 MM patients, we observed no difference in daratumumab-mediated lysis between newly diagnosed or relapsed/refractory patients. However, we discovered, next to an expected effect of effector (natural killer cells/monocytes) to target (MM cells) ratio on ADCC, a significant association between CD38 expression and daratumumab-mediated ADCC (127 patients), as well as CDC (56 patients). Similarly, experiments with isogenic MM cell lines expressing different levels of CD38 revealed that the level of CD38 expression is an important determinant of daratumumab-mediated ADCC and CDC. Importantly, all-trans retinoic acid (ATRA) increased CD38 expression levels but also reduced expression of the complement-inhibitory proteins CD55 and CD59 in both cell lines and primary MM samples. This resulted in a significant enhancement of the activity of daratumumab in vitro and in a humanized MM mouse model as well. Our results provide the preclinical rationale for further evaluation of daratumumab combined with ATRA in MM patients.

Antitumor efficacy of the anti-interleukin-6 (IL-6) antibody siltuximab in mouse xenograft models of lung cancer

Introduction: Interleukin-6 (IL-6) can activate downstream signaling pathways in lung cancer cells, such as the STAT3 pathway, and is reported to be produced by tumor cells with activating EGFR mutations. We examined IL-6/STAT3 in lung cancer tumor tissues and the effects of siltuximab, a neutralizing antibody to human IL-6, in mouse models of lung cancer. Methods: IL-6 and STAT3 activation levels were compared with tumor histology and presence of KRAS mutations in snap-frozen, non–small-cell lung cancer tumors. The effects of siltuximab alone or in combination with erlotinib were examined in mouse xenograft models constructed using three cell line xenograft models and one primary explant mouse model. We examined the influence of cancer-associated fibroblasts (CAFs) on tumor growth and siltuximab effects. Results: IL-6 levels were higher in tumors of squamous cell versus adenocarcinoma histology and were not associated with presence of KRAS mutations. Tyrosine phosphorylation status of STAT3 did not correlate with tumor IL-6 levels. Serine phosphorylation of STAT3 was correlated with KRAS mutation status. Both tumor and stromal cells contributed to total IL-6 within tumors. Siltuximab had minimal effect as a single agent in xenografts with tumor cells alone; however, in models coadministered with CAFs, siltuximab had more potent effects on tumor inhibition. We observed no effects of combined erlotinib and siltuximab. Conclusions: IL-6 is elevated in subsets of human NSCLCs, especially with squamous cell histology. Tumors supported by stromal production of IL-6 seem to be the most vulnerable to tumor growth inhibition by siltuximab.

IL6 sensitizes prostate cancer to the antiproliferative effect of IFNα2 through IRF9

Development and progression of prostate cancer (PCa) are associated with chronic inflammation. The cytokine interleukin 6 (IL6) can influence progression, differentiation, survival, and angiogenesis of PCa. To identify novel pathways that are triggered by IL6, we performed a gene expression profiling of two PCa cell lines, LNCaP and MDA PCa 2b, treated with 5 ng/ml IL6. Interferon (IFN) regulatory factor 9 (IRF9) was identified as one of the most prevalent IL6-regulated genes in both cell lines. IRF9 is a mediator of type I IFN signaling and acts together with STAT1 and 2 to activate transcription of IFN-responsive genes. The IL6 regulation of IRF9 was confirmed at mRNA and protein levels by quantitative real-time PCR and western blot respectively in both cell lines and could be blocked by the anti-IL6 antibody Siltuximab. Three PCa cell lines, PC3, Du-145, and LNCaP-IL6+, with an autocrine IL6 loop displayed high expression of IRF9. A tissue microarray with 36 PCa tissues showed that IRF9 protein expression is moderately elevated in malignant areas and positively correlates with the tissue expression of IL6. Downregulation and overexpression of IRF9 provided evidence for an IFN-independent role of IRF9 in cellular proliferation of different PCa cell lines. Furthermore, expression of IRF9 was essential to mediate the antiproliferative effects of IFNα2. We concluded that IL6 is an inducer of IRF9 expression in PCa and a sensitizer for the antiproliferative effects of IFNα2.

Sepantronium Bromide (YM155) improves daratumumab-mediated cellular lysis of multiple myeloma cells by abrogation of bone marrow stromal cell-induced resistance.

Targeted immunotherapy with the human anti-CD38 antibody daratumumab has recently emerged as a promising strategy for the treatment of multidrug resistant patients with multiple myeloma (MM). In experimental in vitro and in vivo settings, and in recently completed clinical trials, daratumumab

Abstract 3530: The role of IL-6 in ERα+ breast cancer and potential use for Siltuximab, an anti-IL-6 antibody, in ERα+ breast cancer treatment.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Interleukin-6 (IL-6) is an important growth factor for estrogen receptor-alpha (ERα) positive breast cancer. High IL-6 serum levels are associated with poor prognosis in ERα+ breast cancer patients, and IL-6 related polymorphisms that lead to elevated IL-6 expression are associated with decreased overall survival (PMID 17261184, 12771987). Preclinical data suggest that in contrast to ERα negative breast cancer cell lines, ERα positive cell lines rarely produce autocrine IL-6, and are therefore dependent on paracrine IL6 produced by the microenvironment. Furthermore, when ERα positive cell lines are exposed to paracrine IL-6, phosphorylation of STAT3-Y705, increased growth rates and more aggressive tumor phenotypes are observed (PMID 17586727, 18974155, 19581928). We utilized 3D co-cultures and heterotypic xenograft models to investigate the ability of siltuximab, an anti-IL-6 antibody in clinical development, to attenuate paracrine IL-6 effects across a panel of 8 ERα positive breast cancer cell lines and heterotypic xenograft models. From a panel of 8 ERα postive breast cancer lines, 75% responded to recombinant human IL-6 (hIL-6) protein by phosphorylation of STAT3 (Ty705), but not AKT, MEK1/2, or ERK1/2. Siltuximab treatment blunted pSTAT3 induction in all IL-6 responsive ERα positive breast cancer lines but failed to reduce pSTAT3 phosphorylation in three ERα negative cell lines that produced autocrine IL-6. The 3D tumor growth assay demonstrated accelerated growth rates for ERα positive breast cancer lines in the presence of IL-6 or human mesenchymal stem cells (hMSC), which returned to baseline with siltuximab treatment. In addition, when hIL-6 was supplemented in vivo, the ERα positive tumor cell line, MCF-7 engrafted without the need for estrogen supplementation and tumor growthblunted with siltuximab treatment. When tumors were allowed to establish before treatment, siltuximab was able to induce tumor regressions in all treated animals (10/10). Tumor regression was associated with decreased mitotic counts in tumors. In addition to these experiments, we investigated STAT3 activation in a panel of primary patient-derived ERα positive breast cancer samples. 43% of samples demonstrated activated STAT3 as determined by Y705 phosphorylation. These same ERα positive breast cancer patients displayed an activated IL-6 network as assessed by an a priori 32-gene IL-6 network gene expression profile signature and was also associated with increased IL-6 serum levels. This activated IL-6 gene signature was also associated with ERα positive tumors and increased metastasis as assessed by positive node status in four independent breast cancer data sets. Taken together, these data suggest a key role for IL-6 in estrogen independent ERα positive breast cancer progression. Citation Format: Amy Axel, Tineke Casneuf, Peter King, JD Alvarez, Brett Hall, Kate Sasser. The role of IL-6 in ERα+ breast cancer and potential use for Siltuximab, an anti-IL-6 antibody, in ERα+ breast cancer treatment. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3530. doi:10.1158/1538-7445.AM2013-3530

CD38+ M-MDSC expansion characterizes a subset of advanced colorectal cancer patients

BACKGROUND Myeloid-derived suppressor cells (MDSCs) are a population of immature immune cells with several protumorigenic functions. CD38 is a transmembrane receptor-ectoenzyme expressed by MDSCs in murine models of esophageal cancer. We hypothesized that CD38 could be expressed on MDSCs in human colorectal cancer (CRC), which might allow for a new perspective on therapeutic targeting of human MDSCs with anti-CD38 monoclonal antibodies in this cancer. METHODS Blood samples were collected from 41 CRC patients and 8 healthy donors, followed by peripheral blood mononuclear cell (PBMC) separation. Polymorphonuclear (PMN-) and monocytic (M-) MDSCs and CD38 expression levels were quantified by flow cytometry. The immunosuppressive capacity of M-MDSCs from 10 CRC patients was validated in a mixed lymphocyte reaction (MLR) assay. RESULTS A significant expansion of CD38+ M-MDSCs and a trend of expansion of CD38+ PMN-MDSCs (accompanied by a trend of increased CD38 expression on both M- and PMN-MDSCs) were observed in PBMCs of CRC patients when compared with healthy donors. The CD38+ M-MDSCs from CRC patients were found to be immunosuppressive when compared with mature monocytes. CD38+ M- and PMN-MDSC frequencies were significantly higher in CRC patients who previously received treatment when compared with treatment-naive patients. CONCLUSIONS This study provides a rationale for an attempt to target M-MDSCs with an anti-CD38 monoclonal antibody in metastatic CRC patients. FUNDING NCI P01-CA14305603, the American Cancer Society, Scott and Suzi Lustgarten Family Colon Cancer Research Fund, Hansen Foundation, and Janssen Research and Development.

Could daratumumab be used to treat severe allergy

In the management of allergic diseases, targeting IgE using biologicals is a current research focus. This proof of concept shows that plasma cell depletion using daratumumab results in decreased total and allergen-specific IgE levels.