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Blood, Vol. 112, Issue 4, 1329-1337, August 15, 2008
Anti-CS1 humanized monoclonal antibody HuLuc63 inhibits myeloma cell adhesion and induces antibody-dependent cellular cytotoxicity in the bone marrow milieu
Blood Tai et al.
112: 1329
Supplemental Materials for: Tai et al
Files in this Data Supplement:
- Figure S1. CS1 mRNA and protein expression in MM cell lines and patient MM cells (PDF, 29KB) -
(A) Expression profile of CS1 and CD138 in MM lines using Affymetrix U133A array data. Total RNA isolated from 45 MM lines was subject to microarray analysis. Probe set 219159_s for CS1; 201286_s and 201287_s for CD138. (B) Immunostaining with a chimeric anti-CS1 ChLuc90 were performed in 12 MM lines. Results as mean fluorescence intensity (MFI) are shown. (C) Results of CS1 expression by ChLuc90 in CD138-purified cells from 12 MM patients as MFI are shown in the table.
- Figure S2. CS1 expression in CD138-purified plasma cells from MM patient and normal donors (PDF, 35KB) -
CS1 mRNA expression was analyzed with probes 219159_s_at (upper panel) and 222838_at (lower panel), in MM patient cells (MM, n=73) and plasma cell counterparts isolated from normal donors (NPC, n=5).
- Figure S3. Gene expression of CS1 in normal and malignant tissues (PDF, 90KB) -
CS1 mRNA expression in (A) normal body tissues, in (B) primary cancer tissues, and in (C) cancer cell lines was performed using a customized Affymetrix GeneChip® as described previously (Afar et al., 2004). Average intensity units are shown on the Y axis. Samples are shown on the X axis. They are (A): adrenal glands, 1-3; bladder, 4-6; bone marrow, 7-9; breast, 10-12; colonic epithelium, 13-15; cervix, 16-18; brain, 19-21; colon, 22-24; diaphragm, 25-27; epididymis, 28-30; esophagus, 31-33; gallbladder, 34-36; heart, 37-39; kidney, 40-42; liver, 43-45; lung, 46-48; lymph node, 49-51; muscle, 52-54; vagus nerve, 55-57; pancreas, 58-60; prostate, 61-63; skin, 64-66; spleen, 67-69; stomach, 70-72; synovium, 73-75; testis, 76-78; thymus, 79-81; thyroid, 82-84; tonsils, 85-87; trachea, 88-90; ureter, 91-93; uterus, 94-96; (B) bladder cancer, 1-10; breast cancer, 11-20; prostate cancer, 21-30; cervical cancer, 31-40; colon cancer, 41-50; glioblastoma multiforme, 51-60; renal cancer, 61-70; melanoma, 71-80; soft tissue sarcoma, 81-90; lung adenocarcinoma, 91-100; squamous cell carcinoma of the lung, 101-110; esophageal cancer, 111-120; (C) cell lines from bladder cancer - SW780, 1-4; breast cancer - BT474, 5-8; colon cancer - Lovo, 9-12; cervical cancer - Me180, 13-16; glioblastoma - U118, 17-20; head and neck cancer - A253, 21-24; chronic myelogenous leukemia - K562, 25-28; multiple myeloma - L363, 29-32; OPM2, 33-36; B cell lymphoma - Daudi, 37-40; Raji, 41-44; Ramos, 45-48; T cell lymphoma – Karpas 299, 49-52; monocytic leukemia – THP-1, 53-56; U-937, 57-60; lung adenocarcinoma – NCI-H358, 61-64; squamous cell carcinoma of the lung – NCI-H520, 65-68; melanoma – A375, 69-72; ovarian cancer – Ovcar-3, 73-76; pancreatic cancer – BxPC3, 77-80; prostate cancer – LNCaP, 81-84; renal cancer – TR-786-O, 85-88. The data shows that significant CS1 expression is only detected in the myeloma cell lines L363 and OPM2.
- Figure S4. HuLuc63 does not induce lysis of myeloma cells via CDC (PDF, 22KB) -
Complement dependent cell lysis of CS1 positive L363 cells by HuLuc63 was tested using human serum as a source of complement. An anti-HLA-DR antibody was used as a positive control and an isotype control IgG1 was used as a negative control. Maximal lysis was defined by the effect of digitonin (Sigma, D5628) on L363 cell viability, while spontaneous lysis was measured after addition of medium alone. Cell viability was measured using Cell Titer Glo (Promega, G7571) with luminescence as read-out of live cells. Percent cytotoxicity was measured after a 1 hour incubation of cells with antibody in the presence (left panel) or absence (right panel) of 50% human serum and was calculated as (1-(sample - digitonin)/(media-digitonin))*100. In the presence of human serum, the anti-HLA-DR antibody (solid circles) mediated significant cell lysis, while incubation with HuLuc63 (solid squares) or isotype control antibody (open diamonds) exerted no effect. In the absence of human serum, the anti-HLA-DR antibody also lacked cell lysis ability. These results indicate that HuLuc63 does not induce myeloma cell lysis via CDC.
References
Afar DE, Bhaskar V, Ibsen E, Breinberg D, Henshall SM, Kench JG, Drobnjak M, Powers R, Wong M, Evangelista F, O'Hara C, Powers D, DuBridge RB, Caras I, Winter R, Anderson T, Solvason N, Stricker PD, Cordon-Cardo C, Scher HI, Grygiel JJ, Sutherland RL, Murray R, Ramakrishnan V, Law DA. Preclinical validation of anti-TMEFF2-auristatin E-conjugated antibodies in the treatment of prostate cancer. Mol Cancer Ther, 3: 921-932, 2004.
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