Efficient lysis of epithelial ovarian cancer cells by MAGE-A3-induced cytotoxic T lymphocytes using rAAV-6 capsid mutant vector.

MAGE-A3 is highly expressed in epithelial ovarian cancer (EOC), making it a promising candidate for immunotherapy. We investigated whether dendritic cells (DCs) transduced with a rAAV-6 capsid mutant vector Y445F could elicit effective MAGE-A3-specific anti-tumor cytotoxic T lymphocyte (CTL) responses in vitro. MAGE-A3 was cloned and rAAV-6-MAGE-A3 purified, followed by proviral genome detection using real-time PCR. Immunofluorescence detection of rAAV-6-Y445F-MAGE-A3-transduced DCs demonstrated 60% transduction efficiency. Fluorescent in situ hybridization analysis confirmed chromosomal integration of rAAV vectors. Flow cytometric analysis of transduced DCs showed unaltered expression of critical monocyte-derived surface molecules with retention of allo-stimulatory activity. Co-culture of autologous T lymphocytes with MAGE-A3-expressing DCs produced CTLs that secreted IFN-γ, and efficiently killed MAGE-A3+ EOC cells. This form of rAAV-based DC immunotherapy, either alone or more likely in combination with other immune-enhancing protocols, may prove useful in the clinical setting for management of EOC.

Large-scale ex vivo expansion and characterization of natural killer cells for clinical applications.

BACKGROUND AIMS: Interest in natural killer (NK) cell-based immunotherapy has resurged since new protocols for the purification and expansion of large numbers of clinical-grade cells have become available. METHODS: We have successfully adapted a previously described NK expansion method that uses K562 cells expressing interleukin (IL)-15 and 4-1 BB Ligand (BBL) (K562-mb15-41BBL) to grow NK cells in novel gas-permeable static cell culture flasks (G-Rex). RESULTS: Using this system we produced up to 19 × 10(9) functional NK cells from unseparated apheresis products, starting with 15 × 10(7) CD3(-) CD56 (+) NK cells, within 8-10 days of culture. The G-Rex yielded a higher fold expansion of NK cells than conventional gas-permeable bags and required no cell manipulation or feeding during the culture period. We also showed that K562-mb15-41BBL cells up-regulated surface HLA class I antigen expression upon stimulation with the supernatants from NK cultures and stimulated alloreactive CD8 (+) T cells within the NK cultures. However, these CD3 (+) T cells could be removed successfully using the CliniMACS system. We describe our optimized NK cell cryopreservation method and show that the NK cells are viable and functional even after 12 months of cryopreservation. CONCLUSIONS: We have successfully developed a static culture protocol for large-scale expansion of NK cells in the gas permeable G-Rex system under good manufacturing practice (GMP) conditions. This strategy is currently being used to produce NK cells for cancer immunotherapy.

Highly activated and expanded natural killer cells for multiple myeloma immunotherapy.

BACKGROUND: Patients with gene expression profiling-defined high-risk myeloma in relapse have poor outcomes with current therapies. We tested whether natural killer cells expanded by co-culture with K562 cells transfected with 41BBL and membrane-bound interleukin-15 could kill myeloma cells with a high-risk gene expression profile in vitro and in a unique model which recapitulates human myeloma. DESIGN AND METHODS: OPM2 and high-risk primary myeloma tumors were grown in human fetal bone implanted into non-obese diabetic severe combined immunodeficiency mice with a deficient interleukin-2 receptor gamma chain. These mice are devoid of endogenous natural killer and T-cell activity and were used to determine whether adoptively transferred expanded natural killer cells could inhibit myeloma growth and myeloma-associated bone destruction. RESULTS: Natural killer cells from healthy donors and myeloma patients expanded a median of 804- and 351-fold, respectively, without significant T-cell expansion. Expanded natural killer cells killed both allogeneic and autologous primary myeloma cells avidly via a perforin-mediated mechanism in which the activating receptor NKG2D, natural cytotoxicity receptors, and DNAX-accessory molecule-1 played a central role. Adoptive transfer of expanded natural killer cells inhibited the growth of established OPM2 and high-risk primary myeloma tumors grown in the murine model. The transferred, expanded natural killer cells proliferated in vivo in an interleukin-2 dose-dependent fashion, persisted up to 4 weeks, were readily detectable in the human bone, inhibited myeloma growth and protected bone from myeloma-induced osteolysis. CONCLUSIONS: These studies provide the rationale for testing expanded natural killer cells in humans.

Epigenetic modulation of MAGE-A3 antigen expression in multiple myeloma following treatment with the demethylation agent 5-azacitidine and the histone deacetlyase inhibitor MGCD0103.

BACKGROUND AIMS: Immunotherapy targeting MAGE-A3 in multiple myeloma (MM) could eradicate highly aggressive and proliferative clonal cell populations responsible for relapse. However, expression of many cancer-testis antigens, including MAGE-A3, can be heterogeneous, leading to the potential for tumor escape despite MAGE-A3-induced immunity. We hypothesized that a combination of the hypomethylating agent 5-azacitidine (5AC) and the histone deacetylase inhibitor (HDACi) MGCD0103 (MGC) could induce MAGE-A3 expression in MAGE-A3-negative MM, resulting in recognition and killing of MM cells by MAGE-A3-specific cytotoxic T lymphocytes (CTL). METHODS: Gene expression analyses of MAGE-A3 expression in primary MM patient samples at diagnosis and relapse were completed to identify populations that would benefit from MAGE-A3 immunotherapy. MM cell lines were treated with 5AC and MGC. Real-time polymerase chain reaction (PCR) and Western blotting were performed to assess MAGE-A3 RNA and protein levels, respectively. Chromium-release assays and interferon (IFN) secretion assays were employed to ascertain MAGE-A3 CTL specificity against treated targets. RESULTS: Gene expression analysis revealed that MAGE-A3 is expressed in MM patients at diagnosis (25%) and at relapse (49%). We observed de novo expression of MAGE-A3 RNA and protein in MAGE-A3-negative cell lines treated with 5AC. MGC treatment alone did not induce expression but sequential 5AC/MGC treatment led to enhanced expression and augmented recognition by MAGE-A3-specific CTL, as assessed by (51)Cr-release assays (P = 0.047) and enzyme-linked immunosorbent assay (ELISA) for IFN-γ secretion (P = 0.004). CONCLUSIONS: MAGE-A3 is an attractive target for immunotherapy of MM and epigenetic modulation by 5AC, and MGC can induce MAGE-A3 expression and facilitate killing by MAGE-A3-specific CTL.

Combinatorial efficacy of anti-CS1 monoclonal antibody elotuzumab (HuLuc63) and bortezomib against multiple myeloma.

Monoclonal antibody (mAb) therapy for multiple myeloma, a malignancy of plasma cells, has not been clinically efficacious in part due to a lack of appropriate targets. We recently reported that the cell surface glycoprotein CS1 (CD2 subset 1, CRACC, SLAMF7, CD319) was highly and universally expressed on myeloma cells while having restricted expression in normal tissues. Elotuzumab (formerly known as HuLuc63), a humanized mAb targeting CS1, is currently in a phase I clinical trial in relapsed/refractory myeloma. In this report we investigated whether the activity of elotuzumab could be enhanced by bortezomib, a reversible proteasome inhibitor with significant activity in myeloma. We first showed that elotuzumab could induce patient-derived myeloma cell killing within the bone marrow microenvironment using a SCID-hu mouse model. We next showed that CS1 gene and cell surface protein expression persisted on myeloma patient-derived plasma cells collected after bortezomib administration. In vitro bortezomib pretreatment of myeloma targets significantly enhanced elotuzumab-mediated antibody-dependent cell-mediated cytotoxicity, both for OPM2 myeloma cells using natural killer or peripheral blood mononuclear cells from healthy donors and for primary myeloma cells using autologous natural killer effector cells. In an OPM2 myeloma xenograft model, elotuzumab in combination with bortezomib exhibited significantly enhanced in vivo antitumor activity. These findings provide the rationale for a clinical trial combining elotuzumab and bortezomib, which will test the hypothesis that combining both drugs would result in enhanced immune lysis of myeloma by elotuzumab and direct targeting of myeloma by bortezomib.