Susann Szmania

CS1, a Potential New Therapeutic Antibody Target for the Treatment of Multiple Myeloma

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: CS1 mRNA was expressed in >90% of 532 multiple myeloma cases, regardless of cytogenetic abnormalities. Anti-CS1 antibody 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 T cells, and CD8+ T cells, 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 CS1 with HuLuc63 for the treatment of multiple myeloma.

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. [Mol Cancer Ther 2009;8(9):2616–24]

Infusion of haplo-identical killer immunoglobulin-like receptor ligand mismatched NK cells for relapsed myeloma in the setting of autologous stem cell transplantation

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.

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.

Protein Transduction of Dendritic Cells for NY-ESO-1-Based Immunotherapy of Myeloma

Myeloma vaccines, based on dendritic cells pulsed with idiotype or tumor lysate, have been met with limited success, probably in part due to insufficient cross-priming of myeloma antigens. A powerful method to introduce myeloma-associated antigens into the cytosol of dendritic cells is protein transduction, a process by which proteins fused with a protein transduction domain (PTD) freely traverse membrane barriers. NY-ESO-1, an immunogenic antigen by itself highly expressed in 60% of high-risk myeloma patients, was purified to near homogeneity both alone and as a recombinant fusion protein with a PTD, derived from HIV-Tat. Efficient entry of PTD-NY-ESO-1 into dendritic cells, confirmed by microscopy, Western blotting, and intracellular flow cytometry, was achieved without affecting dendritic cell phenotype. Experiments with amiloride, which inhibits endocytosis, and N-acetyl-l-leucinyl-l-norleucinal, a proteasome inhibitor, confirmed that PTD-NY-ESO-1 entered dendritic cells by protein transduction and was degraded by the proteasome. Tetramer analysis indicated superior generation of HLA-A2.1, CD8+ T lymphocytes specific for NY-ESO-1(157-165) with PTD-NY-ESO-1 compared with NY-ESO-1 control protein (44% versus 2%, respectively). NY-ESO-1-specific T lymphocytes generated with PTD-NY-ESO-1 secreted IFN-gamma indicative of a Tc1-type cytokine response. Thus, PTD-NY-ESO-1 accesses the cytoplasm by protein transduction, is processed by the proteasome, and NY-ESO-1 peptides presented by HLA class I elicit NY-ESO-1-specific T lymphocytes.

Optimizing dendritic cell-based immunotherapy in multiple myeloma: intranodal injections of idiotype-pulsed CD40 ligand-matured vaccines led to induction of type-1 and cytotoxic T-cell immune responses in patients

Vaccination with idiotype (Id) protein‐pulsed dendritic cells (DCs) has been explored in multiple myeloma and the results have been disappointing. To improve the efficacy of DC vaccination in myeloma, we investigated the use of Id‐ and keyhole limpet haemocyanin (KLH)‐pulsed, CD40 ligand‐matured DCs administered intranodally. Nine patients with smouldering or stable myeloma without treatment were enrolled and DC vaccines were administered at weekly intervals for a total of four doses. Following vaccination, all patients mounted Id‐specific γ‐interferon T‐cell response. Interleukin‐4 response was elicited in two, and skin delayed‐type hypersensitivity reaction occurred in seven patients. More importantly, Id‐specific cytotoxic T‐cell responses were also detected in five patients. Most if not all patients mounted a positive T‐cell response to KLH following vaccination. At 1‐year follow‐up, six of the nine patients had stable disease, while three patients had slowly progressive disease even during the vaccination period. At 5‐year follow‐up, four of the six patients continued with stable disease. No major side effects were noted. In summary, intranodal administration of Id‐pulsed CD40 ligand‐matured DCs was able to induce Id‐specific T and B‐cell responses in patients. Current efforts are geared towards breaking tumour‐mediated immune suppression and improving clinical efficacy of this immunotherapy.

Ex vivo-expanded natural killer cells demonstrate robust proliferation in vivo in high-risk relapsed multiple myeloma patients.

Highly activated/expanded natural killer (NK) cells can be generated by stimulation with the human leukocyte antigen-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 haploidentical 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 U/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.

Immunization With a Recombinant Mage-a3 Protein After High-dose Therapy for Myeloma

MAGE-A3 is frequently expressed in high-risk multiple myeloma (MM). We immunized a healthy donor with MAGE-A3 protein formulated in AS02B to transfer immunity to her identical twin, diagnosed with MAGE-A3-positive MM. After a melphalan 200 mg/m2 syngeneic peripheral blood stem cell transplant, primed donor cells collected after immunizations were transferred and followed by repeated patient immunizations. MAGE-A3 immunizations were well tolerated. Strong MAGE-A3–specific antibody, cytotoxic T-lymphocyte (CTL), and T-helper responses were induced in both twins. A humoral response was transferred to the patient with the donor peripheral blood stem cells and increased by booster immunization. The CTL response targeted a previously undescribed HLA-A*6801 binding MAGE-A3115-123 peptide. MAGE-A3115-123 CTLs were detected in the patient more than 1 year after the last immunization. Multiple T-helper cellular responses were detected with the dominant response to an HLA-DR11 restricted MAGE-A3 epitope. The patient remains in remission 2.5 years after the second transplant. This report shows for the first time that immunization of a healthy donor with a defined cancer-testis protein induces immune responses that can be transferred and expanded posttransplant in the recipient. MAGE-A3 immunization may be a useful adjunct to high dose melphalan-based peripheral blood stem cell transplant, providing a new therapeutic option for high-risk MM.

Interleukin-6 Receptor Polymorphism Is Prevalent in HIV-negative Castleman Disease and Is Associated with Increased Soluble Interleukin-6 Receptor Levels

Multicentric Castleman Disease is largely driven by increased signaling in the pathway for the plasma cell growth factor interleukin-6. We hypothesized that interleukin-6/interleukin-6 receptor/gp130 polymorphisms contribute to increased interleukin-6 and/or other components of the interleukin-6 signaling pathway in HIV-negative Castleman Disease patients. The study group was composed of 58 patients and 50 healthy donors of a similar racial/ethnic profile. Of seven polymorphisms chosen for analysis, we observed an increased frequency between patients and controls of the minor allele of interleukin-6 receptor polymorphism rs4537545, which is in linkage disequilibrium with interleukin-6 receptor polymorphism rs2228145. Further, individuals possessing at least one copy of the minor allele of either polymorphism expressed higher levels of soluble interleukin-6 receptor. These elevated interleukin-6 receptor levels may contribute to increased interleukin-6 activity through the trans-signaling pathway. These data suggest that interleukin-6 receptor polymorphism may be a contributing factor in Castleman Disease, and further research is warranted.