Development and Therapeutic Potential of an Anti-CD79b Antibody-Drug Conjugate, Anti-CD79b-Vc-MMAE, for the Treatment of Non-Hodgkin’s Lymphoma

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2618-2618
Author(s):  
Andrew G. Polson ◽  
Fiona L Bennett ◽  
Yvonne Chen ◽  
Mark Dennis ◽  
Dan Eaton ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Surface ◽  
Cell Lines ◽  
Surface Expression ◽  
Hodgkin's Lymphoma ◽  
Cell Surface Expression ◽  
Sensitive Cell ◽  
Large Panel ◽  
Xenograft Models ◽  
Antibody Drug

Abstract Antibody-drug conjugates (ADCs), antibodies linked to potent cytotoxic drugs via specialized chemical linkers, provide a means to increase the effectiveness of chemotherapy by targeting the drug to neoplastic cells while reducing side effects. We have previously shown that ADCs targeted to CD79b are highly effective in xenograft models of non-Hodgkin’s lymphoma. Here we report the development of an ADC consisting of a humanized anti-CD79b antibody (hu-anti-CD79b) conjugated to monomethylauristatin E (MMAE) through engineered cysteines (THIOMABS) by a maleimidocaproyl-valinecitrulline-p-aminobenzyloxycarbonyl (MC-vcPAB) linker (hu-anti-CD79b-thioMAb-MC-vc-PAB-MMAE) that is designed to be cleaved by cathepsins. To determine the potential of this ADC, hu-anti-CD79b-thioMAb-MC-vc-PAB-MMAE (referred to as anti-CD79b-vcMMAE henceforth), as a therapeutic for NHL we interrogated its potency across a large panel of NHL cell lines. Strikingly, anti-CD79b-vcMMAE has very potent activity across a large panel of NHL cell lines in vitro with 68% (23 out of 34) cell lines having greater than 50% reduction in cell viability. Quantitative FACS across the cell line panel revealed that of the 11 insensitive cell lines, 9 had negligible surface CD79b, suggesting a threshold effect in that below a specific level of antigen on the cell surface resulted in cells being insensitive to anti-CD79b-vcMMAE. Within the sensitive cell lines, there was not a direct correlation per se with anti-CD79b-vcMMAE IC50 values and cell surface expression levels and this prompted us to investigate other potential molecular parameters. Gene expression profiling and gene set enrichment analysis revealed that genes predominantly involved in antigen processing and presentation and genes induced by IFN-gamma were significantly enriched in the less sensitive cell lines to CD79b-vcMMAE. Classifying our NHL cell lines as GCB or ABC subtypes by gene expression revealed that both ABC and GCB were responsive. In addition, the activity of anti-CD79b-vcMMAE was very potent in p53 mutant as well as p53 wild-type cell lines. Since the pre-clinical evidence suggests that anti-CD79b-vcMMAE will be a very promising drug candidate for NHL and we have established that cell surface expression is perhaps the best predictor of response, we wished to determine the prevalence of expression of CD79b on the cell surface of primary human lymphoma and CLL samples to estimate a patient population that may gain benefit. Strikingly, CD79b was detected in all cases of CLL, MZL, HCL, DLBCL, FL, and MCL. Furthermore, CD79b expression was detected in all cases that had relapsed from prior chemotherapy regimens, highlighting the clinical relevance of this target and potential therapeutic utility of anti-CD79b-vcMMAE. To assess the potential of anti-CD79b-vcMMAE in vivo we compared its efficacy to R-CHOP in three xenograft models of NHL. In all three models, a single dose of anti-CD79b-vcMMAE resulted in complete sustained tumor remission and was more effective than R-CHOP. These data suggest that anti-CD79b-vcMMAE could be broadly efficacious as a treatment for NHL.

Cell surface expression of LDL receptor in chronic hepatitis C: correlation with viral load

2007 ◽  
Vol 293 (1) ◽  
pp. E416-E420 ◽  
Author(s):  
Jean-Michel Petit ◽  
Anne Minello ◽  
Laurence Duvillard ◽  
Valérie Jooste ◽  
Serge Monier ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chronic Hepatitis ◽  
Viral Load ◽  
Hepatitis C ◽  
Cell Surface ◽  
Chronic Hepatitis C ◽  
Mononuclear Cells ◽  
Ldl Receptor ◽  
Surface Expression ◽  
Cell Surface Expression

The LDL receptor (LDL-R) has been proposed as the viral receptor for Hepatitis C virus (HCV). This hypothesis has been based exclusively on in vitro studies. In human mononuclear cells, LDL-R gene expression has been demonstrated to be parallel and be coordinately regulated to gene expression in the human liver. The purpose of the current study was to determine the mononuclear cell surface expression of the LDL receptor in patients with HCV chronic infection according to viral load. Sixty-eight consecutive untreated chronic hepatitis C patients were studied to determine the mononuclear cell surface expression of the LDL-R. LDL-Rs were quantified at the surface of mononuclear cells in fresh blood samples taken after fasting using flow cytometry. LDL-R expression was significantly associated with LDL-cholesterol ( r = −0.25; P = 0.03) and HCV-viral load ( r = 0.37, P = 0.002). In multivariate analysis, the LDL-R expression was significantly associated with HCV viral load, whereas genotype, age, body mass index, and fibrosis were not. In conclusion, our data provided by a human study, suggest that the LDL-R may be one of the receptors implicated in HCV replication.

Ku80 autoantigen as a cellular coreceptor for human parvovirus B19 infection

Blood ◽  
2005 ◽  
Vol 106 (10) ◽  
pp. 3449-3456 ◽  
Author(s):  
Yasuhiko Munakata ◽  
Takako Saito-Ito ◽  
Keiko Kumura-Ishii ◽  
Jie Huang ◽  
Takao Kodera ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Surface ◽  
Cell Lines ◽  
Bone Marrow Cells ◽  
Parvovirus B19 ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Human Parvovirus B19 ◽  
Erythroid Cells ◽  
Interfering Rna

AbstractHuman parvovirus B19 (B19) infects human erythroid cells expressing P antigen. However, some cell lines that were positive for P antigen failed to bind B19, whereas some cell lines had an ability to bind B19 despite undetectable expression of P antigen. We here demonstrate that B19 specifically binds with Ku80 autoantigen on the cell surface. Furthermore, transfection of HeLa cells with the gene of Ku80 enabled the binding of B19 and allowed its entry into cells. Moreover, reduction of cell-surface expression of Ku80 in KU812Ep6 cells, which was a high-sensitive cell line for B19 infection, by short interfering RNA for Ku80 resulted in the marked inhibition of B19 binding in KU812Ep6 cells. Although Ku80 originally has been described as a nuclear protein, human bone marrow erythroid cells with glycophorin A or CD36, B cells with CD20, or T cells with CD3 were all positive for cell-surface expression of Ku80. B19 infection of KU812Ep6 cells and bone marrow cells was inhibited in the presence of anti-Ku80 antibody. Our data suggest that Ku80 functions as a novel coreceptor for B19 infection, and this finding may provide an explanation for the pathologic immunity associated with B19 infection.

Identification of a Novel Auto-Antibody Highly Prevalent in Patients with Hepatitis-Associated and Idiopathic Aplastic Anemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3200-3200
Author(s):  
Hiroyuki Takamatsu ◽  
Zhirong Qi ◽  
Tomoyuki Sakurai ◽  
Luis Espinoza ◽  
Naomi Sugimori ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Peptide Mass Fingerprinting ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Healthy Individuals ◽  
Peptide Mass ◽  
Leukemia Cell Lines ◽  
Cd34 Cells

Abstract Abstract 3200 Poster Board III-137 Hepatitis-associated aplastic anemia (HAA) is a subset of acquired AA that is highly responsive to immunosuppressive therapy. The target antigens of the immune system attack in HAA are thought to be a protein shared by both liver and hematopoietic stem cells, since it is usually associated with severe hepatitis of unknown etiology. Screening sera from patients with HAA for the presence of antibodies (Abs) recognizing liver cell-derived proteins may be useful in identifying novel auto-antigens in AA. To test this hypothesis, sera from HAA patients were examined using immunoblotting with a lysate of a hepatocellular carcinoma cell line Huh7 and subsequent peptide mass fingerprinting. Methods and Results The serum of a patient with typical HAA (a 23 year-old male) possessing a small population of paroxysmal nocturnal hemoglobinuria (PNH)-type cells was used for Western blotting (WB) with the lysates of Huh7. A distinct band of 70 kDa protein was revealed. The same band was revealed when the culture supernatant of Huh7 cells was subjected to WB. The peptide mass fingerprinting of the 70 kDa band identified this protein to be heat shock protein (HSP) 72. HSP72 is a stress-inducible protein and extracellular HSP72 enhances the cytotoxicity of CD4+ T cells and NK cells. An examination of the sera from HAA patients, idiopathic acquired AA (IAA) patients and healthy individuals with WB revealed the anti-HSP72 Abs to be detected in 10 of 12 (83%) HAA patients and in 57 of 80 (71%) IAA patients while it was detected only in 8 of 59 (14%) healthy individuals. The prevalence of anti-HSP72 Abs in AA was markedly higher than that of anti-kinectin Abs (39%), anti-PMS1 Abs (10%), anti-DRS-1 Abs (38%) or anti-moesin Abs (37%) reported previously. Anti-HSP72 Abs were frequently detectable both in patients with IAA possessing PNH-type cells (63%) and in patients without PNH-type cells (86%), a finding contrasting to the higher prevalence of anti-DRS-1 Abs and anti-moesin Abs in patients with PNH-type cells than in those without PNH-type cells reported previously. Although anti-HSP72 Abs were detectable in the sera of patients with rheumatoid arthritis and systemic lupus erythematosus, the prevalence was 15% (4 of 27) and 20% (1 of 5), respectively. In contrast to a previous report that detected anti-HSP72 Abs in 24% of patients with chronic hepatitis C, WB failed to detect the Abs in the sera of 4 patients with autoimmune hepatitis and 5 with hepatitis B or C. Ten patients with HAA were treated with immunosuppressive therapy, and 7 of the 8 responders expressed anti-HSP72 Abs. The quantification of the gene expression level of HSP72 by blood cells using real-time PCR demonstrated that the HSP72 mRNA levels were markedly higher in myeloid leukemia cell lines as well as CD34+ cells isolated from 3 healthy individuals in comparison to that in lymphoid or monocytoid leukemia cell lines. HSP72/GAPDH ratios of PBMCs and CD34+ cells from 3 healthy individuals, K562, KH88, OUN-1 were 0.51, 1.31, 1.02, 0.07 and 0.09 respectively. Other leukemia cell lines such as Daudi, Molt-4 and THP-1 did not display detectable levels of HSP72 mRNA. The cell surface expression of HSP72 was examined in various kinds of leukemia cell lines and CD34+ bone marrow (BM) cells derived from 3 healthy individuals using Ab to HSP72 (Clone C92F3A-5) because previous studies demonstrated heat-inducible expression of HSP72 by K562. Flow cytometry detected cell surface HSP72 on immature CML cell lines such as K562 but not on CD34+ BM cells, acute promyelocytic leukemia cell lines such as NB-4 and HL-60, and lymphoid leukemia cell lines such as Molt-4 and Daudi. Exposure to 42°C for 2 h increased the HSP72 expression on K562 cells and Molt-4 cells but not on CD34+ cells. Conclusion Anti-HSP72 Ab is the most prevalent auto-Ab in AA among the auto-Abs previously detected. Given the increased expression of HSP72 by immature myeloid cells as well as stress-inducible cell surface expression of the molecule, immune responses to HSP72 may thus play an essential role in the pathogenesis of HAA and IAA. Disclosures No relevant conflicts of interest to declare.

Perfusion with sickle erythrocytes up-regulates ICAM-1 andVCAM-1 gene expression in cultured human endothelial cells

Blood ◽  
2000 ◽  
Vol 95 (10) ◽  
pp. 3232-3241 ◽  
Author(s):  
Yan-Ting Shiu ◽  
Mark M. Udden ◽  
Larry V. McIntire
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Cell Adhesion ◽  
Cell Surface ◽  
Sickle Cell ◽  
Cell Adhesion Molecules ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Human Endothelial Cells ◽  
Sickle Erythrocytes

Sickle cell anemia is characterized by periodic vasoocclusive crises. Increased adhesion of sickle erythrocytes to vascular endothelium is a possible contributing factor to vasoocclusion. This study determined the effect of sickle erythrocyte perfusion at a venous shear stress level (1 dyne/cm2) on endothelial cell (EC) monolayers. Sickle erythrocytes up-regulated intercellular adhesion molecule-1 (ICAM-1) gene expression in cultured human endothelial cells. This was accompanied by increased cell surface expression of ICAM-1 and also elevated release of soluble ICAM-1 molecules. Expression of vascular cell adhesion molecule-1 (VCAM-1) messenger RNA (mRNA) was also strikingly elevated in cultured ECs after exposure to sickle cell perfusion, although increases in membrane-bound and soluble VCAM-1 levels were small. The presence of cytokine interleukin-1β in the perfusion system enhanced the production of ICAM-1 and VCAM-1 mRNA, cell surface expression, and the concentrations of circulating forms. This is the first demonstration that sickle erythrocytes have direct effects on gene regulation in cultured human ECs under well-defined flow environments. The results suggest that perfusion with sickle erythrocytes increases the expression of cell adhesion molecules on ECs and stimulates the release of soluble cell adhesion molecules, which may serve as indicators of injury and/or activation of endothelial cells. The interactions between sickle red blood flow, inflammatory cytokines, and vascular adhesion events may render sickle cell disease patients vulnerable to vasoocclusive crises.

scholarly journals Cholangiocyte expression of α2β1-integrin confers susceptibility to rotavirus-induced experimental biliary atresia

2008 ◽  
Vol 295 (1) ◽  
pp. G16-G26 ◽  
Author(s):  
Mubeen Jafri ◽  
Bryan Donnelly ◽  
Steven Allen ◽  
Alex Bondoc ◽  
Monica McNeal ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Cell Surface ◽  
Biliary Atresia ◽  
Cell Lines ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Newborn Mice ◽  
A Cell

Inoculation of BALB/c mice with rhesus rotavirus (RRV) in the newborn period results in biliary epithelial cell (cholangiocyte) infection and the murine model of biliary atresia. Rotavirus infection of a cell requires attachment, which is governed in part by cell-surface expression of integrins such as α2β1. We hypothesized that cholangiocytes were susceptible to RRV infection because they express α2β1. RRV attachment and replication was measured in cell lines derived from cholangiocytes and hepatocytes. Flow cytometry was performed on these cell lines to determine whether α2β1 was present. Cholangiocytes were blocked with natural ligands, a monoclonal antibody, or small interfering RNA against the α2-subunit and were infected with RRV. The extrahepatic biliary tract of newborn mice was screened for the expression of the α2β1-integrin. Newborn mice were pretreated with a monoclonal antibody against the α2-subunit and were inoculated with RRV. RRV attached and replicated significantly better in cholangiocytes than in hepatocytes. Cholangiocytes, but not hepatocytes, expressed α2β1 in vitro and in vivo. Blocking assays led to a significant reduction in attachment and yield of virus in RRV-infected cholangiocytes. Pretreatment of newborn pups with an anti-α2 monoclonal antibody reduced the ability of RRV to cause biliary atresia in mice. Cell-surface expression of the α2β1-integrin plays a role in the mechanism that confers cholangiocyte susceptibility to RRV infection.

scholarly journals Addition of truncated oligosaccharides to influenza virus hemagglutinin results in its temperature-conditional cell-surface expression.

1989 ◽  
Vol 108 (2) ◽  
pp. 355-365 ◽  
Author(s):  
J Hearing ◽  
M J Gething ◽  
J Sambrook
Keyword(s):  
Influenza Virus ◽  
Cell Surface ◽  
Cell Lines ◽  
Mutant Cell ◽  
Surface Expression ◽  
Ovary Cell ◽  
Cell Surface Expression ◽  
Permissive Temperature ◽  
Influenza Virus Hemagglutinin ◽  
Conditional Expression

In the preceding paper (Hearing, J., E. Hunter, L. Rodgers, M.-J. Gething, and J. Sambrook. 1989. J. Cell Biol. 108:339-353) we described the isolation and initial characterization of seven Chinese hamster ovary cell lines that are temperature conditional for the cell-surface expression of influenza virus hemagglutinin (HA) and other integral membrane glycoproteins. Two of these cell lines appeared to be defective for the synthesis and/or addition of mannose-rich oligosaccharide chains to nascent glycoproteins. In this paper we show that at both 32 and 39 degrees C in two mutant cell lines accumulate a truncated version, Man5GlcNAc2, of the normal lipid-linked precursor oligosaccharide, Glc3Man9GlcNAc2. This is possibly due to a defect in the synthesis of dolichol phosphate because in vitro assays indicate that the mutant cells are not deficient in mannosylphosphoryldolichol synthase at either temperature. A mixture of truncated and complete oligosaccharide chains was transferred to newly synthesized glycoproteins at both the permissive and restrictive temperatures. Both mutant cell lines exhibited altered sensitivity to cytotoxic plant lectins when grown at 32 degrees C, indicating that cellular glycoproteins bearing abnormal oligosaccharide chains were transported to the cell surface at the permissive temperature. Although glycosylation was defective at both 32 and 39 degrees C, the cell lines were temperature conditional for growth, suggesting that cellular glycoproteins were adversely affected by the glycosylation defect at the elevated temperature. The temperature-conditional expression of HA on the cell surface was shown to be due to impairment at 39 degrees C of the folding, trimerization, and stability of HA molecules containing truncated oligosaccharide chains.

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