Purpose: We generated a humanized antibody, HuLuc63, which specifically targets CS1 (CCND3 subset 1, CRACC, and SLAMF7), a cell surface glycoprotein not previously associated with multiple myeloma. To explore the therapeutic potential of HuLuc63 in multiple myeloma, we examined in detail the expression profile of CS1, the binding properties of HuLuc63 to normal and malignant cells, and the antimyeloma activity of HuLuc63 in preclinical models. Experimental Design: CS1 was analyzed by gene expression profiling and immunohistochemistry of multiple myeloma samples and numerous normal tissues. HuLuc63-mediated antimyeloma activity was tested in vitro in antibody-dependent cellular cytotoxicity (ADCC) assays and in vivo using the human OPM2 xenograft model in mice.Results: CS1mRNA was expressed in >90% of 532 multiple myeloma cases, regardless of cytogenetic abnormalities. Anti-CS1antibody staining of tissues showed strong staining of myeloma cells in all plasmacytomas and bone marrow biopsies. Flow cytometric analysis of patient samples using HuLuc63 showed specific staining of CD138+ myeloma cells, natural killer (NK), NK-like Tcells, and CD8+ Tcells, with no binding detected on hematopoietic CD34+ stem cells. HuLuc63 exhibited significant in vitro ADCC using primary myeloma cells as targets and both allogeneic and autologous NK cells as effectors. HuLuc63 exerted significant in vivo antitumor activity, which depended on efficient Fc-CD16 interaction as well as the presence of NK cells in the mice. Conclusions: These results suggest that HuLuc63 eliminates myeloma cells, at least in part, via NK-mediated ADCC and shows the therapeutic potential of targeting CS1with HuLuc63 for the treatment of multiple myeloma.

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 × 109 functional NK cells from unseparated apheresis products, starting with 15 × 107 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.

Summary Killer immunoglobulin-like receptor (KIR)-ligand mismatched natural killer (NK) cells play a key role in achieving durable remission after haplo-identical transplantation for acute myeloid leukaemia. We investigated the feasibility of transfusing haplo-identical, T-cell depleted, KIR-ligand mismatched NK cells, after conditioning therapy with melphalan and fludarabine, to patients with advanced multiple myeloma (MM) followed by delayed rescue with autologous stem cells. No graft-versus-host disease or failure of autologous stem cells to engraft was observed. There was significant variation in the number of allo-reactive NK cells transfused. However, all NK products containing allo-reactive NK cells killed the NK cell target K562, the MM cell line U266, and recipient MM cells when available. Post NK cell infusion there was a rise in endogenous interleukin-15 accompanied by increasing donor chimaerism. Donor chimaerism was eventually lost, which correlated with the emergence of potent host anti-donor responses indicating that the immunosuppressive properties of the conditioning regimen require further optimization. Further, blocking of inhibitory KIR-ligands with anti-human leucocyte antigen antibody substantially enhanced killing of MM cells thus highlighting the potential for modulating NK/MM cell interaction. Encouragingly, 50% of patients achieved (near) complete remission. These data set the stage for future studies of KIR-ligand mismatched NK cell therapy in the autologous setting.

The presence of a metaphase cytogenetic abnormality (CA) is the key negative predictor of outcome in patients with multiple myeloma (MM). Gene expression profiling (GEP) of such patients showed increased expression of NY-ESO-1 compared to patients with normal cytogenetics (60% versus 31%; P ‫؍‬ .004). NY-ESO-1 was also highly expressed in relapsing MM especially patients with CA (100% versus 60.7%; P < .001). GEP findings were confirmed at the protein level by immunostaining of marrow biopsies for NY-ESO-1. We detected spontaneous NY-ESO-1-specific antibodies by enzymelinked immunosorbent assay in 33% of patients with NY-ESO-1 ؉ MM, especially in CA patients (9 of 13; 70%), but in none of the NY-ESO-1 ؊ patients with MM (n ‫؍‬ 27) or healthy donors (n ‫؍‬ 21 IntroductionDuring the past 10 years, numerous human tumor-associated antigens (Ags) have been identified, either by screening cDNA libraries with sera derived from cancer patients containing an antibody (Ab) to a tumor-associated Ag (SEREX) or by using T lymphocytes specific for tumor peptides presented in the context of specific HLA alleles. [1][2][3][4][5][6][7][8][9][10][11] The most rapidly expanding group of tumor Ags are the cancer/testis (C/T) Ags, which are either not expressed or are present at very low levels in normal tissues except the testes and perhaps the placenta. 12,13 Because the testes are not patrolled by the immune system, expression of C/T Ags in this environment is not harmful.Of the C/T Ags described thus far, NY-ESO-1 is among the most immunogenic with not only well-documented spontaneous [14][15][16][17][18][19][20] and vaccine-induced immunity, but also clinical responses in a substantial percentage of chemorefractory cancers. 19,21 NY-ESO-1 mRNA is found in approximately 20% to 40% of tumors including melanoma, prostate, transitional cell bladder, breast, lung, medullary thyroid, squamous head and neck, and cervical carcinoma. 12,14,[22][23][24][25][26][27] Because it is expressed in such a wide variety of tumors, NY-ESO-1 offers a unique opportunity to develop a broad-spectrum tumor-specific cancer vaccine.High-dose chemotherapy with autologous peripheral blood stem cell transplantation (auto-PBSCT) has significantly improved the outcome of patients with multiple myeloma (MM). [28][29][30][31][32][33][34][35][36][37] We and others have shown that the presence of cytogenetic abnormalities (CAs) is the most powerful prognosticator for poor outcome. [38][39][40][41][42][43][44][45] Intensification of treatment in our Total Therapy II (TTII) protocol has resulted in additional improvement in event-free (EFS) and overall survival (OS) of patients without CAs (67% of patients). However, no such improvement has yet been observed for patients with CAs (33% of patients). 41,43,46 Fewer than 10% of patients treated with tandem auto-PBSCT protocols remain in long-term remission and are considered "operationally cured." 40 These data highlight the urgent need for new approaches to improve diseasefree survival in such patients.We analyzed ou...

IntroductionMultiple myeloma (MM) is a prototypic B-cell malignancy with overall survival varying from a few months to more than 15 years with melphalan-based autologous stem cell transplantation. 1 It is well established that patients with abnormal, especially hypodiploid karyotypes, and high lactic dehydrogenase have a worse prognosis. 2 Recently, we have reported that approximately 13% of MM patients have a high-risk prognostic score defined by the differential expression of 70 key genes, many of which are located on chromosome 1. 3 These patients have a poor outcome compared with low-risk patients, with inferior actuarial event-free survival (18% vs 60%, hazard ratio (HR) ϭ 4.51) and overall survival (28% vs 78%, HR ϭ 5.16) at 5 years despite intensive treatment on total therapy tandem transplantation regimens and the incorporation of novel agents, such as bortezomib into up-front management. 3 There is therefore a need for novel treatment approaches that target the melphalan-refractory myeloma stem cell pool in high-risk patients and synergize with currently available cytoreductive regimens.Killer-cell immunoglobulin-like receptor (KIR)-ligand (KIR-L) mismatched natural killer (NK) cells have potent antileukemic effects in the setting of heavily T cell-depleted haplo-identical allogeneic transplantation, but the majority of MM patients cannot tolerate the associated toxicity of such regimens. 4 However, we have recently demonstrated in a pilot trial that T cell-depleted haplo-identical KIR-L mismatched NK cells can be safely given to high-risk MM patients in the setting of an autologous transplantation without evidence of graft-versus-host disease. 5 Unfortunately, not all NK cells transfused are alloreactive; indeed, the majority are inhibited by patient human leukocyte antigen (HLA) class I, particularly HLA-C and -Bw4 molecules. 6 NK-cell activity is regulated by a dynamic balance between inhibitory and activating receptors that recognize ligands on target cells, with the inhibitory signals typically being dominant to prevent destruction by NK cells of healthy cells. [7][8][9][10][11] It has been postulated that mature NK cells express at least one inhibitory receptor for autologous HLA class I, thus preserving self-tolerance. In contrast, NK cells avidly lyse tumor cells that do not display such inhibitory KIR-L. A classic example is the cell line K562, which does not express HLA class I.HLA class I molecules consist of a major histocompatibility complex-encoded heavy chain, the light chain ␤ 2 -microglobulin (␤ 2 M), 12 and a third subunit, a peptide of 8 to 10 amino acids. 12,13 In humans, class I heavy chains are encoded by 3 loci, HLA-A, -B, and -C. The proteasome is responsible for the generation of peptides, which are transported to the endoplasmic reticulum. There they combine with HLA class I and ␤ 2 M, and are transported to the cell surface via the Golgi apparatus. 14 Binding of peptides to HLA class I is essential for the stability of HLA class I at the cell surface. HLA class I molecules/␤ 2...

Highly activated/expanded natural killer (NK) cells can be generated via stimulation with the HLA-deficient cell line K562 genetically modified to express 41BB-ligand and membrane-bound interleukin (IL)15. We tested the safety, persistence and activity of expanded NK cells generated from myeloma patients (auto-NK) or haplo-identical family donors (allo-NK) in heavily pretreated patients with high-risk relapsing myeloma. The preparative regimen comprised bortezomib only or bortezomib and immunosuppression with cyclophosphamide, dexamethasone and fludarabine. NK cells were shipped overnight either cryopreserved or fresh. In 8 patients, up to 1×108 NK cells/kg were infused on day 0 and followed by daily administrations of IL2. Significant in vivo expansion was observed only in the 5 patients receiving fresh products, peaking at or near day 7, with the highest NK cell counts in 2 subjects who received cells produced in a high concentration of IL2 (500 units/mL). Seven days after infusion, donor NK cells comprised > 90% of circulating leukocytes in fresh allo-NK cell recipients, and cytolytic activity against allogeneic myeloma targets was retained in vitro. Among the 7 evaluable patients, there were no serious adverse events that could be related to NK cell infusion. One patient had a partial response and in another the tempo of disease progression decreased; neither patient required further therapy for 6 months. In the 5 remaining patients, disease progression was not affected by NK cell infusion. In conclusion, infusion of large numbers of expanded NK cells was feasible and safe; infusing fresh cells was critical to their expansion in vivo.

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. [Mol Cancer Ther 2009;8(9):2616-24]

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