Xian-Feng Li

Immunomodulatory drug lenalidomide (CC-5013, IMiD3) augments anti-CD40 SGN-40-induced cytotoxicity in human multiple myeloma: Clinical implications

SGN-40, a humanized immoglobulin G1 (IgG1) anti-CD40 monoclonal antibody, mediates cytotoxicity against human multiple myeloma (MM) cells via suppression of interleukin (IL)-6-induced proliferative and antiapoptotic effects as well as antibody-dependent cell-mediated cytotoxicity (ADCC). Here, we studied the clinical significance of an immunomodulatory drug lenalidomide on SGN-40-induced cytotoxicity against CD138(+)CD40(+) MM lines and patient MM cells. Pretreatment with lenalidomide sensitized MM cells to SGN-40-induced cell death. Combined lenalidomide and SGN-40 significantly induced MM apoptosis, evidenced by enhanced cleavage of caspase-3/8/poly(ADP-ribose)polymerase and increased sub-G(0) cells, compared with either single agent at the same doses. Pretreatment of effector cells with lenalidomide augmented SGN-40-induced MM cell lysis, associated with an increased number of CD56(+)CD3(-) natural killer (NK) cells expressing CD16 and LFA-1. Importantly, pretreatment with lenalidomide or lenalidomide and SGN-40 markedly enhanced NK-cell-mediated lysis of autologous patient MM cells triggered by SGN-40. Lenalidomide also up-regulated CD40L on CD56(+)CD3(-) NK cells, facilitating IL-2-mediated activation of NK cells. In addition, lenalidomide induced the CD56(dim) NK subset, which are more potent mediators of ADCC against target MM cells than the CD56(bright) NK subset. Finally, pretreatment of both effector and target MM cells with lenalidomide markedly enhanced SGN-40-mediated ADCC against CD40-expressing MM cells. These studies, therefore, show that the addition of lenalidomide to SGN-40 enhances cytotoxicity against MM cells, providing the framework for combined lenalidomide and SGN-40 in a new treatment paradigm to both target MM cells directly and induce immune effectors against MM.

Human anti-CD40 antagonist antibody triggers significant antitumor activity against human multiple myeloma.

Monoclonal antibodies (mAb) directed against lineage-specific B-cell antigens have provided clinical benefit for patients with hematologic malignancies, but to date no antibody-mediated immunotherapy is available for multiple myeloma. In the present study, we assessed the efficacy of a fully human anti-CD40 mAb CHIR-12.12 against human multiple myeloma cells. CHIR-12.12, generated in XenoMouse mice, binds to CD138-expressing multiple myeloma lines and freshly purified CD138-expressing cells from >80% multiple myeloma patients, as assessed by flow cytometry. Importantly, CHIR-12.12 abrogates CD40L-induced growth and survival of CD40-expressing patient multiple myeloma cells in the presence or absence of bone marrow stromal cells (BMSC), without altering constitutive multiple myeloma cell proliferation. Immunoblotting analysis specifically showed that PI3-K/AKT, nuclear factor-kappaB (NF-kappaB), and extracellular signal-regulated kinase activation induced by CD40L (5 mug/mL) was inhibited by CHIR-12.12 (5 mug/mL). Because CD40 activation induces multiple myeloma cell adhesion to both fibronectin and BMSCs, we next determined whether CHIR-12.12 inhibits this process. CHIR-12.12 decreased CD40L-induced multiple myeloma cell adhesion to fibronectin and BMSCs, whereas control human IgG1 did not. Adhesion of multiple myeloma cells to BMSCs induces interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) secretion, and treatment of multiple myeloma cells with CD40L further enhanced adhesion-induced cytokine secretion; conversely, CHIR-12.12 blocks CD40L-enhanced IL-6 and VEGF secretion in cocultures of multiple myeloma cells with BMSCs. Finally, CHIR-12.12 triggered lysis of multiple myeloma cells via antibody-dependent cellular cytotoxicity (ADCC) but did not induce ADCC against CD40-negative multiple myeloma cells, confirming specificity against CD40-expressing multiple myeloma cells. These results provide the preclinical rationale for clinical trials of CHIR-12.12 to improve patient outcome in multiple myeloma.

Blockade of the MEK/ERK signalling cascade by AS703026, a novel selective MEK1/2 inhibitor, induces pleiotropic anti-myeloma activity in vitro and in vivo

This study investigated the cytotoxicity and mechanism of action of AS703026, a novel, selective, orally bioavailable MEK1/2 inhibitor, in human multiple myeloma (MM). AS703026 inhibited growth and survival of MM cells and cytokine‐induced osteoclast differentiation more potently (9‐ to 10‐fold) than AZD6244. Inhibition of proliferation induced by AS703026 was mediated by G0‐G1 cell cycle arrest and was accompanied by reduction of MAF oncogene expression. AS703026 further induced apoptosis via caspase 3 and Poly ADP ribose polymerase (PARP) cleavage in MM cells, both in the presence or absence of bone marrow stromal cells (BMSCs). Importantly, AS703026 sensitized MM cells to a broad spectrum of conventional (dexamethasone, melphalan), novel or emerging (lenalidomide, perifosine, bortezomib, rapamycin) anti‐MM therapies. Significant tumour growth reduction in AS703026‐ vs. vehicle‐treated mice bearing H929 MM xenograft tumours correlated with downregulated pERK1/2, induced PARP cleavage, and decreased microvessels in vivo. Moreover, AS703026 (<200 nmol/l) was cytotoxic against the majority of tumour cells tested from patients with relapsed and refractory MM (84%), regardless of mutational status of RAS and BRAF genes. Importantly, BMSC‐induced viability of MM patient cells was similarly blocked within the same dose range. Our results therefore support clinical evaluation of AS703026, alone or in combination with other anti‐MM agents, to improve patient outcome.

Abstract 400: Establishment and characterization of a novel IL6-dependent HLA-A2+ human myeloma cell line LNT1

The novel multiple myeloma (MM) cell line LNT1 was established from peripheral blood of a 66-year-old female patient with recurrent plasma cell leukemia of IgG/kappa type. Peripheral blood mononuclear cells from the heparinized peripheral blood separated by Ficoll-Hypaque density gradient centrifugation were cultured in RPMI 1640 medium/20% FBS, 100 U/ml penicillin and 50 μg/ml streptomycin, without additional growth factors. Cells were cultured by replacing ½ of the volume of the culture with fresh medium every 3-4 days for 3 months. In the 4 th month, a slow yet sustained growth of cultured cells was noted, and designated LNT1. These cells grow as single cell suspension; IL-6-induced proliferation in a dose-dependent manner with 2 ng/ml of IL-6 inducing maximal DNA synthesis (by [ 3 H]thymidine uptake) and survival (by MTT). In contrast, IGF-1 had no effect. Subsequently, 2 ng/ml of IL-6 was added to cultures and growth now sustained at 7 months. LNT1 was negative for Epstein-Barr virus, as evidenced by PCR using primers specific for EBNA-1. May-Grunwald-Giemsa staining showed that LNT1 cells have a high nucleocytoplasmic ratio with excentrically located nuclei, prominent nucleoli, and a deeply basophilic and vacuolated cytoplasm. Some cells were bi- or multi-nucleated. Surface antigen analysis showed that LNT1 cells express CD138, CD38, HM1.24, CS1, and HLA-A2, but did not express CD19, CD20, CD3, CD16, and CD14. Chromosome analysis at 7 months revealed the karyotype: 43, X, -X, del(1)(p13p22), psu dic(6;1)(q13;p11), del(12)(p11.2), −13, add(19)(q13.4), −22[2]/43, idem, del(2)(q11.2q2?3), der(6)ins(6;?)(q21;?)[7]. DNA fingerprinting was performed by simultaneously amplifying eight short tandem repeat (STR) loci and the amelogenin gene in a multiplex PCR reaction using specific primers to confirm the authentication of cell line. The established LNT1 cell line and the primary tumor showed identical band patterns of these 9 markers and the same immunoglobulin H rearrangement. An unique and identical VH3/IgG fragment was amplified using the appropriate V H family-specific framework region primer in conjunction with C H isotype specific (IgG) primers in DNA samples of both LNT1 cell line and the original tumor. Moreover, identical DNA sequence of VH3/IgG PCR from cell line and primary tumor further confirms clonality. LNT1 cell line also bears a N-RAS G12D mutation, as in the original tumor. Finally, LNT1 cell line is responsive to conventional (Dex, Malphalan) and novel or emerging (bortezomib, lenalidomide, perifosine, AS703026) anti-MM therapies. Therefore, the novel LNT1 cell line will provide a useful model system to study genetics and biology of MM, as well as to evaluate novel therapeutic strategies. Since LNT1 cells highly express HLA-A2, this line also serves a useful model to validate novel tumor-associated antigens for immunotherapy in MM. 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 400.