Cells, cell lines, and cultures
Peripheral blood was collected from healthy donors, and fresh blood or bone marrow was collected from patients with MM after informed consent in accordance with the Declaration of Helsinki. Approval was obtained from the University of Arkansas for Medical Sciences Institutional Review Board for these studies. NK cells were enriched from whole blood samples either with the RosetteSep human NK cell enrichment cocktail (StemCell Technologies Inc., Vancouver, BC) or using microbeads coated with CD56 mAb (Miltenyi Biotech, Auburn, CA). CD138-positive plasma cells were purified from patient-derived bone marrow aspirates via positive selection with anti-CD138 microbeads (Miltenyi Biotech). After magnetic bead selection, cells were confirmed to be >90% pure by flow cytometry for CD56 or CD138, respectively. The OPM2 and K562 cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA).
Clinical grade bortezomib (VELCADE®
) was purchased from or gifted by Millennium Pharmaceuticals, Inc., (Cambridge, MA). Anti-human CS1 mAbs were generated as previously described, including murine IgG2a MuLuc63 and its humanized IgG1 version elotuzumab (formerly HuLuc63) both specific for the extracellular region of human CS1, and 1G9 mAb which recognizes the intracellular region.15
The human IgG1
isotype control mAb, MSL109, used in all in vitro experiments, is a fully human anti-cytomegalovirus mAb.21
Murine IgG2a isotype control mAb used for all in vivo experiments is a mAb directed against VP7 of bluetongue virus (BTV) and is available as a hybridoma from the ATCC (clone 8A3B.6).
SCID-hu mouse model
C.B-17/IcrHsd-Prkdcscid (Harlan Sprague, Indianapolis, Indiana) were implanted with human fetal bone (Advanced Bioscience Resources, Alameda, CA) subcutaneously as previously described.22,23
Upon bone engraftment, 2-8 × 106
cells from heparinized bone marrow aspirates obtained from myeloma patients containing at least 20% CD138+ plasma cells were injected directly into the human fetal bone in 50 μL phosphate-buffered saline (PBS). For each experiment, cells from the same patient were injected into the fetal bone of 5 to 6 SCID-hu hosts. Changes in levels of circulating human immunoglobulin (hu Ig) of the M-protein isotype, monitored by ELISA as previously described,22
was used as an indicator of myeloma growth. When hu Ig levels reached 10 μg/mL or higher in 2 consecutive measurements, 2 mice with comparable tumor load (tumor derived from the same patient) were randomized to receive murine isotype control mAb (anti-BTV) or MuLuc63 (parental mouse anti-CS1 mAb). Dosing was 10 mg/kg of mAb administered intraperitoneally two times a week for a total of 6 doses. Tumor growth was monitored for a period of about 6 weeks by ELISA for hu Ig. The statistical significance of differences was assessed with the student t
< 0.05 was considered significant. All animal work was carried out according to the National Institutes of Health's Guide for the Care and Use of Laboratory Animals, with protocols approved by the University of Arkansas for Medical Sciences Institutional Animal Care and Use Committee.
Gene expression profiling
CD138-positive plasma cells were purified from samples of bone marrow from patients with MM, as described above, before and 48 hours (h) after a single dose of 1.0mg/m2
Gene expression profiling for CS1
expression (probe set 219159_s_at) was performed using the Affymetrix U133 Plus 2.0 arrays platform (Affymetrix Inc. Santa Clara, CA) as previously described.24
The student's t test was used to determine whether CS1
gene expression was significantly different in plasma cells collected before versus after bortezomib administration.
CD138-purified MM cells from bone marrow collected from patients before and 48h after a dose of 1.0mg/m2 bortezomib were purified as above, cryopreserved, then subsequently thawed and analyzed in the same experiment by flow cytometry for CS1 expression. Dead and apoptotic cells were gated out using propidium iodide and annexin V stains (Invitrogen, Carlsbad, CA) and the resulting viable cell population was analyzed for CS1 expression using elotuzumab conjugated to fluorescein isothiocyanate (FITC). Samples were run on a FACSARIA cytometer (BD Pharmingen, San Diego, CA). Histogram overlays were created using FCS express software (De Novo Software, Los Angeles, CA).
Antibody-dependent cellular cytotoxicity (ADCC) assay
Target cells were suspended at a density of 1.0 × 106 cells/mL in RPMI media with a fixed dose of 10 nM bortezomib or titrated doses of bortezomib (20 nM, 10 nM, 5 nM, 1 nM) or in media alone and incubated in a 5% CO2 incubator at 37° for 18 h. Cells were collected, washed, assessed for viability, re-suspended at a density of 20 × 106 viable cells/mL, and labeled for 1 h with 50 mCi Na2 [51Cr]O4 per 106 cells. 51Cr-labeled cells were washed and added to a 96-well, V-bottomed polystyrene plate at a cell density of 15,000 cells per 75 μL. Elotuzumab or MSL109 (isotype control mAb) was added in a volume of 25 μl to the target cells for a final concentration of 0.001 to 10 μg/mL. NK cells or PBMC from healthy donors were added in a volume of 100 μl to bortezomib-treated or mock-treated target cells at a ratio of 10:1 (NK:targets) or 25:1 (PBMC:targets). Pretreatment of targets for 0.5 h with 50 mcg/ml Fc receptor blocking antibody (Serotec, Oxford, UK) was performed for some assays. After a 4-hour incubation at 37°C, cells were centrifuged at 1200 rpm for 5 minutes, and released 51Cr was measured in the 100 uL supernatants. Maximum release was determined from target cells lysed with 100 mg/ml digitonin or 1% Triton X-100. Antibody independent cellular cytotoxicity (AICC) was determined using target cells, plus media, plus NK cells; spontaneous lysis was determined using 51Cr-labeled cells plus media without NK or PBMC effectors. Percent cytotoxicity was calculated as ([sample - AICC]/[Maximum - AICC]) * 100. The concentration of elotuzumab necessary to produce 50% of the maximal elotuzumab-mediated ADCC response (EC50) was determined from a curve fit using the sigmoidal dose-response non-linear curve regression in GraphPad Prism. EC50 values from the untreated cells were compared to those cells treated with bortezomib in the same experiment. A two tailed paired t-test was used to determine the p value.
Primary myeloma cells were purified from patient bone marrow, as described above, and treated with 5 or 20 nM bortezomib or vehicle for 18 hours to assess the effects of bortezomib on elotuzumab-mediated lysis of patient-derived autologous NK cells. Cr51 release assays were performed as above with NK cell : myeloma cell ratios of 1:1, 10:1, and 30:1. Elotuzumab and isotype control mAb MSL109 were used at a concentration of 10 μg/mL. The NK cell-sensitive cell line K562 was included as a positive control. Percent cytotoxicity was calculated as ([sample - spontaneous lysis]/[Maximum - spontaneous lysis]) * 100. In vitro experiments were performed in triplicate, and the results are reported as mean ± standard error of the mean (SEM).
Mouse xenograft model
Female IcrTac:ICR-Prkdcscid mice (6-8 weeks of age) were obtained from Taconic Farms (Germantown, NY) and were inoculated in the lower right flank with 1 × 107 OPM2 cells (American Type Culture Collection, Manassas, VA) in RPMI-1640 media from HyClone (Logan, UT). Caliper measurements were performed thrice weekly for the calculation of tumor volume, and tumor growth was monitored for a period of 1 to 2 months. The following formula was used to calculate tumor volume: L×W×H/2, where L (length) is the longest side of the tumor in the plane of the animal's back; W (width) is the longest measurement perpendicular to the length and in the same plane; and H (height) is the highest point perpendicular to the back of the animal.
Mice with an average tumor size of 100 mm3 were allocated randomly into treatment groups of 10-15 mice each. Bortezomib was administered intraperitoneally in phosphate-buffered saline (PBS) at a dose of 1 mg/kg or twice weekly for weeks 1 and 2, no treatment for week 3, and 1 mg/kg twice weekly for weeks 4 and 5 (total of 8 doses). Elotuzumab or MSL 109 (at doses of 1 or 10 mg/kg in PBS) were administered intraperitoneally twice weekly for 5 weeks (total of 10 doses) on days offset from bortezomib dosing. One-way analysis of variance with a Tukey post-test was used to determine differences between elotuzumab and control treatment groups. All animal work was carried out according to the National Institutes of Health's Guide for the Care and Use of Laboratory Animals, with protocols approved by the Institutional Animal Care and Use Committee at PDL BioPharma.
Engrafted tumor with human fetal bones in the SCID-Hu model were removed, formalin fixed for 24 h and then decalcified with EDTA, pH 7.0. Decalcified, formalin-fixed, paraffin-embedded tissues were cut into 4μM sections for Hematoxylin and Eosin (H&E) staining. OPM2 xenograft tumors were harvested at the end of study and fixed in formalin for 24 hours. De-paraffined tissue slides were immersed in DAKO Target retrieval solution (pH 6) in a decloaking chamber (Biocare Medical, Concord CA) for antigen epitope retrieval. CS1 expression in OPM2 xenografts and primary myeloma tumor grafts was determined by staining the tissues with 1G9 mouse mAb at 1 μg/ml for 30minutes and EnVision® secondary polymer System (Dako, Carpinteria, CA). Magnification is at 50×. Percent tumor burden was determined by a certified pathologist using histological analysis of the tumor grafts.