Andrea van Elsas

Uv-induced N- ras mutations are T-cell targets in human melanoma

Human cutaneous melanoma is heterogeneous with respect to the genetic aberrations involved and the genes altered are potential targets for the immune system. The incidence of cutaneous melanoma is known to be linked to UV peak exposure, and the N-ras oncogene is clearly one of the genes involved in the UV carcinogenesis in melanoma. It is mutated in a significant proportion of melanomas and therefore may serve as a target for T cells. Here, we report that an human leukocyte antigen-A2 binding peptide CLLDILDTAGL, encompassing the frequently found 61 -Leu mutation in N -ras, induces cytotoxic T lymphocytes from healthy donor blood that lyse 61 -Leu N-ras transfected melanoma cells. Furthermore, we have found an association between the presence of N-ras mutations and clinical response to immunotherapy with interleukin-2 plus interferon in a group of stage IV melanoma patients. Although the overall survival of these patients was not affected by the N-ras status of their melanomas, these studies suggest that mutated N-ras may provide a target for cytotoxic T lymphocytes in melanoma patients.

N‐ras oncogene expression changes the growth characteristics of human melanoma in two independent SCID‐hu mouse models

Fifteen percent of all human melanomas carry mutations in ras genes, the majority of which are located in codon 61 of the N‐ras gene. However, the biological significance of these mutations is as yet unknown. In this study, we investigated the influence of N‐ras oncogene products mutated in codon 61 on the growth characteristics of human melanoma in vivo by establishing 2 SCID‐hu mouse xenotransplantation models. Tumors grown in SCID mice injected with human melanoma carrying activated N‐ras genes were significantly larger (p < 0.004) than tumors grown in animals injected with the appropriate control transfectants. Additionally, tumors with N‐ras point mutations clearly showed a more pleomorphic phenotype than the control groups. Our results, obtained in 2 independent SCID‐hu xenotransplantation models, suggest that mutated N‐ras oncogene expression may be an important factor influencing growth characteristics of human melanoma without altering metastatic potential. These novel in vivo model systems provide a tool for further study of the biology of mutated ras in melanoma and should also prove useful for testing new and improved treatment strategies for human melanoma carrying mutated ras genes.

Abstract 3283: APRIL/BCMA activation promotes human multiple myeloma progression and further induces immunosuppressive bone marrow microenvironment

Elevated B cell maturation antigen (BCMA) and its ligand a proliferation-inducing ligand (APRIL) may directly advance human multiple myeloma (MM) malignancy in vivo. Here, we first show that BCMA downregulation potently diminishes viability and colony formation of MM cells while BCMA overexpression augments MM cell growth and survival via induction of AKT, MAPK, and NFκB signaling cascades and molecules essential for proliferation and anti-apoptosis. Importantly, BCMA itself forces accelerated tumor growth of xenografted MM cells harboring p53 mutation in mice. BCMA-overexpressing tumors exhibit significantly enhanced CD31/microvessel density and VEGF than paired control tumors. Concurrently, these tumors express increased factors crucial for osteoclast activation, adhesion, and angiogenesis/metastasis, as well as immune inhibition including PD-L1, TGFβ and IL-10 which further triggers 38 IL-10 signaling molecules. In parallel, these identified target genes are induced by paracrine APRIL binding to BCMA in MM cells and they are potently blocked by an antagonistic anti-APRIL monoclonal antibody hAPRIL01A (01A). 01A is cytotoxic against MM cells protected by abnormal bone marrow (BM) myeloid cells, i.e., osteoclasts, macrophages, and plasmacytoid dendritic cells. It further decreases APRIL-induced adhesion and migration of MM cells via blockade of canonical and non-canonical NFκB pathways. It prevents in vivo MM cell growth within implanted human bone chips in SCID mice. 01A-inhibited MM cell viability induced by osteoclasts is further enhanced by lenalidomide and bortezomib. Taken together, these results further characterize new molecular mechanisms of APRIL/BCMA-induced in vivo MM progression and immunosuppressive BM microenvironment, strongly supporting targeting this prominent pathway in MM. Citation Format: Yu-Tzu Tai, Chirag Acharya, Gang An, Michele Moschetta, Mike Y. Zhong, Xiaoyan Feng, Michele Cea, Antonia Cagnetta, Kenneth Wen, Hans van Eenennaam, Andrea van Elsas, Nikhil Munshi, Kenneth Anderson. APRIL/BCMA activation promotes human multiple myeloma progression and further induces immunosuppressive bone marrow microenvironment. [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 3283.

Translating Pembrolizumab to Clinical Practice: Speak Immunology and Learn Fast!

T-cell checkpoint inhibitors treat the cancer patients immune system potentially inducing significant long-term survival. Pembrolizumab demonstrates clinical activity in patients diagnosed with melanoma and other cancers. Its mode of action suggests a rationale for combination with other treatment modalities, urging oncologists to brush up their knowledge of immunology. Clin Cancer Res; 21(19); 4251–3. ©2015 AACR. See related article by Patnaik et al., p. 4286

Abstract 394: A novel anti-a proliferation-inducing ligand hAPRIL.01A monoclonal antibody targets multiple myeloma cells in the bone marrow microenvironment

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA A proliferation-inducing ligand (APRIL), a close member of B-cell-activating factor (BAFF) belonging to the TNF superfamily, is mainly produced by bone marrow (BM) accessory cells to stimulates growth and survival of multiple myeloma (MM) cells. We here characterize molecular mechanisms regulating APRIL activation in the BM microenvironment and further determine whether a novel anti-APRIL monoclonal antibody hAPRIL.01A inhibits its functional sequelae in MM. First, in vitro culture showed that osteoclasts (OC) and macrophages secret significantly higher levels of APRIL than unstimulated CD14+ monocytes and BM stromal cells. All MM cell lines express cell surface BCMA in significantly higher level than TACI (p < 9.06e-15). H929 MM cells (expressing only BCMA, but not TACI), and other MM cell lines were next stimulated with APRIL, in the presence or absence of hAPRIL.01 Ab followed by immunoblotting and TaqMan® Array assays on harvested protein lysates and mRNA. APRIL stimulation activated NF-κB, PI3K/AKT, and ERK1/2 signaling in MM cells. NF-κB-DNA binding activities of p65 and p50 (p52, to a less extent), were significantly upregulated as early as 15 minute after stimulation. Conversely, hAPRIL.01 Ab completely blocked these signaling cascades, consistent with decreased NF-κB-DNA binding activities in BCMA-knock-downed MM cells by shBCMA lentivirus transfection. APRIL further induced pro-survival proteins (Mcl1, Bcl2, BIRC3, XIAP) and MM cell growth-stimulating regulators (CCDN2, CDK4, CDK6, c-myc), which were completely inhibited by hAPRIL.01 Ab. These results correlated with blockade of hAPRIL.01 Ab in APRIL-promoted proliferation and survival of MM cells, in the presence of OCs or macrophages. APRIL also induces adhesion of MM cells to BMSC, which was blocked by hAPRIL.01 Ab. This concurred with hAPRIL.01 Ab-reduced adhesion molecules (CD44, ICAM-1) induced by APRIL. APRIL-increased VEGF-A and PECAM-1 in MM cells was also significantly reduced by this mAb. APRIL-upregulated chemotactic/osteoclast-activating factors (MIP-1α, MIP1β, SDF-1) were also inhibited by this Ab. Other angiogenesis and adhesion/chemoattractant factors were changed in a similar fashion, indicating specific blocking of hAPRIL.01 Ab to APRIL-induced downstream target genes. This mAb further inhibited APRIL-increased viability of plasmacytoid dendritic cells (pDC) and diminished MM cell viability protected by pDC in 3-d cocultures. Finally, hAPRIL.01 specifically overcame APRIL-, but not IL-6, induced protection in lenalidomide- or dexamethasone-treated MM1S and H929 MM cells. This Ab also blocks OC-induced MM cell growth and survival in ongoing SCID-hu model of MM. These studies confirm a constitutive APRIL activation via BCMA and TACI in promoting malignancies of myeloma cells, supporting a novel therapeutics of hAPRIL.01 Ab to target MM in the BM microenvironment. Note: This abstract was not presented at the meeting. Citation Format: Yu-Tzu Tai, Chirag Acharya, Gang An, Mike Y. Zhong, Xiaoyan Feng, Hua Jiang, Hans van Eenennaam, Andrea van Elsas, Nikhil C. Munshi, Kenneth C. Anderson. A novel anti-a proliferation-inducing ligand hAPRIL.01A monoclonal antibody targets multiple myeloma cells in the bone marrow microenvironment. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 394. doi:10.1158/1538-7445.AM2015-394

Abstract 1246: Development of an agonistic antibody against the human T-cell costimulatory receptor CD27 as a potential immunotherapeutic tool.

Immunotherapy of cancer has reached a mature and exciting stage. Clinical activity of monoclonal antibodies (mAb) against the T-cell inhibitory receptors CTLA-4 and PD-1 indicate that cancer patients often have latent tumour-specific T cells that can be mobilized by blocking these receptors. Activation of the T-cell costimulatory receptor CD27, a member of the TNF receptor family, provides another opportunity to promote cytotoxic T cell (CTL)-based anti-tumor immunity. To generate agonistic mAb against human (h)CD27, we used our unique B-cell selection and immortalization technology. Various novel anti-hCD27 mAb were generated and tested for their agonistic properties. We used in vitro read-out systems detecting CD27 (co-)stimulatory function on the basis of NF-κB activity, T cell division and survival. One mAb hCD27.15 (KD ∼1 nM), induced CD27 receptor activation (EC50 ∼1 nM) without the need of cross-linking. Similar to its natural ligand CD70, our hCD27.15 mAb showed bona fide costimulatory properties. hCD27.15 strongly promoted CD8+ T cell expansion under conditions of suboptimal T-cell receptor (TCR) stimulation. hCD27.15 did not stimulate T cell function in absence of TCR engagement. hCD27.15 mAb demonstrated different binding kinetics and a unique epitope, compared to other antibodies that depend on cross-linking to activate CD27. Importantly, hCD27.15 retained its agonistic properties when expressed as a human IgG1 or IgG4 chimera. hCD27.15 was shown to bind and activate primate CD27 with similar potency, providing a rationale for pre-clinical safety testing. In conclusion, to our knowledge we have developed the first mAb to hCD27 that mimics ligand function without the need for cross-linking. Data from mouse models strongly support the application of CD27 agonism to enhance CTL-based anti-tumor immunity, by promoting the generation and survival of CTL, as well as their memory function. We propose therefore that hCD27.15 mAb is a promising tool for cancer immunotherapy. Citation Format: Hans van Eenennaam, Elise Veraar, Winfried Mulder, Ellen Bastiaanssen, Lilian Driessen, David Lutje Hulsik, Paul Vink, Gerda van der Horst, Yanling Xiao, Jannie Borst, Andrea van Elsas. Development of an agonistic antibody against the human T-cell costimulatory receptor CD27 as a potential immunotherapeutic tool. [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 1246. doi:10.1158/1538-7445.AM2013-1246

Abstract 5480: Characterization of anti-human APRIL monoclonal antibodies capable of inhibiting APRIL-dependent B cell function and hyperproliferation.

A Proliferation Inducing Ligand (APRIL) is a TNF ligand that, via its receptors TACI and BCMA, is involved in both B cell physiology as well as in proliferation and survival of malignant B cells. To target APRIL-dependent stimulation of B cell cancers, we recently produced and characterized two monoclonal antagonistic anti-human APRIL antibodies called humanAPRIL.01A (hA.01A) and humanAPRIL.03A (hA.03A). In a first biochemical assay to validate their blocking activity, hA.01A was shown to fully prevent APRIL from binding to its receptors, whereas a substantial difference was detected for hA.03A, which inhibited APRIL binding to BCMA less efficiently than hA.01A. Epitope mapping subsequently revealed that hA.01A and hA.03A bind distinct sites on APRIL, which provided a structural rationale of their different blocking activities. Importantly, this differential inhibition profile can be used to functionally dissect BCMA and TACI-dependent signals and indicated that B cell survival and IgA production are regulated differently by these receptors. Primary CLL cultures were shown to be sensitive to APRIL neutralization using hA.01A. hA.01A also inhibited hyperplasia in a transgenic mouse model reminiscent of B-CLL. Altogether, these data indicate that hA.01A is a novel tool potentially useful for the targeted treatment of B cell-derived cancers such as B-CLL and Multiple Myeloma. Citation Format: Marco Guadagnoli, Katherine Cameron, Elise Veraar, Fiona Kimberley, Andrea van Elsas, Hans van Eenennaam, Jan Paul Medema. Characterization of anti-human APRIL monoclonal antibodies capable of inhibiting APRIL-dependent B cell function and hyperproliferation. [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 5480. doi:10.1158/1538-7445.AM2013-5480