- Email: [email protected]
Sa1928 A Humanization and Affinity Maturation of an Anti-CD24 Monoclonal Antibody Targeting Gastrointestinal Cancers
by the cells (p<0.001). However, GABA significantly suppressed both PGE2-induced cell proliferation and cytokine production (<0.01). Conclusions: These data demonstrate that exogenous GABA exerts a direct anti-tumorigenic effect on key parameters of tumorigenesis including proliferation, migration and invasive capacity. Furthermore GABA seems to exert a direct effect through suppression of PGE2 signalling, indicating cross-talk between these signaling pathways.
Sa1928
Background: CD24 is a cell-surface heavily glycosylated GPI-anchored mucin-like protein. We have shown that CD24 is a valid target in gastrointestinal (GI) malignancies. Anti-CD24 mAb treatment induces a significant growth inhibition of CRC and PC cells, in a time- and dose-dependent manner in vitro and in vivo (Gastro 2006, Clin Can Res 2007, Can Res 2008). Affinity maturation is an important process in optimizing therapeutic antibodies. Affinitymatured antibodies can exhibit increased biological efficacy, and allow reduced dosage with less toxic side effects. Aim: To develop mature humanized anti-CD24 monoclonal antibodies (mAbs) and to evaluate their efficacy alone and/or in combination with standard chemotherapy for CRC and PC. Methods: Edman-degradation, cDNA synthesis, sequence and computational analysis (Ig-blast) were performed to reveal the entire DNA sequence of a murine anti-CD24 mAb (SWA11). Replacement of the Fc with the human IgG1 resulted in a mousehuman chimera. A human donor (Ig-blast) was chosen as a scaffold human Ab for grafting critical sequences of the murine antibody into it leading to the generation of humanized Ab derivatives. Sequence analysis of the CDR loops was the base for library designing. Affinity maturation was performed in two steps; CDR walking (two-step selection) and by using phage display technique. Results: In vivo antibody targeting and accumulation within a CD24 positive tumor and its excess clearance was clearly demonstrated using direct imaging (Fig. 1). Combinatorial phage-displayed antibody libraries with varying degrees of diversity at randomized positions from which high-affinity antibodies can be selected were created. A chosen matured clone was isolated and showed higher binding strength (1.8x10-8), compared to the parental murine and humanized Abs (3.3x10-8 and 4.2x10-8). The matured antibody showed selective recognition and binding to the CD24 antigen, as demonstrated by antigen-based (Fig. 2A) and whole-cell ELISA (Fig. 2B) and FACS analysis (Fig. 2C). It stability was enhanced following the maturation process, as demonstrated by in vitro stability test (Fig. 3). The antibody lost only 16% of its activity after 23 days in 37°C. Combined treatment with standard chemotherapeutic agents and natural products, such as monoterpenes (terpinen-4-ol) (Fig. 4), showed significant reduction in cell viability (90% cell death). Conclusion: Targeting CD24 may be a promising treatment for GI malignancies in combination with chemotherapy and natural agents. The resulted matured fully humanized antiCD24 mAb is more effective than the murine parental Ab.
Sa1926 SOX2 and p53 Protein Expression Predicts Response to Preoperative Chemoradiotherapy in Patients With Esophageal Adenocarcinoma Sophie H. van Olphen, Katharina Biermann, bas P. wijnhoven, Manon C. Spaander, Ate van der Gaast, Jan J. lanschot, Leendert Looijenga, Marco J. Bruno Objective Preoperative chemoradiotherapy has recently become common practice in treatment of esophageal cancer with a gain in 5-year survival of 10-15%. However, a significant proportion of patients do not respond well and experiencing unnecessary severe side-effects. Accurate risk-stratification of patients using informative biomarkers before therapy may help to avoid unnecessary morbidity due to ineffective treatment. The aim of this study was to investigate the correlation between the expression of SOX2 and P53 in pre-treatment tumor biopsies and grade of pathological tumor response in resected specimen of patients with esophageal adenocarcinoma (EAC) treated with neoadjuvant chemoradiotherapy (nCRT). Methods All EAC patients who received nCRT according to the CROSS regimen followed by esophagectomy, between January 2003 and July 2011 at the Erasmus University Medical Center, were included. SOX2 and P53 protein expression was visualized by immunohistochemistry on all pre-treatment tumor biopsies and scored independently by two investigators who were blinded for clinical outcome. Aberrant expression was defined as negative expression of SOX2 and overexpression or complete loss of P53 expression. The overall Tumor Regression Grade (TRG) was evaluated using the modified Mandard scoring system. Patients with TRG 1 or TRG 2 were classified as major responders (ie, < 10% of tumor cells remaining), whereas patients with TRG 3 or TRG 4 were classified as minor responders (ie, > 10% of tumor cells remaining). Results In total 77 patients were included. Forty (53%) patients had a major pathological response (TRG 1-2) and 37 (47%) a minor response (TRG 3-4). In pre-treatment biopsies aberrant SOX2 and P53 expression was seen in 40% (31/77) and 83% (64/77), respectively. A major response was significantly associated with an aberrant SOX2 expression (OR 3.9, 95% CI: 1.5 - 10.2, p=0.005) and aberrant p53 expression (OR 4.5, 95% CI: 1.15 - 18.2, p=0.031). Aberrant expression of both biomarkers increased the probability of a major response in the individual patient (OR of 5.6; 95% CI: 2.1 - 14.9, p= 0.001), with a sensitivity of 68%, specificity of 73% and a positive predictive value of 73%. Conclusion SOX2 and P53 expression in the pre-treatment biopsies predict response to nCRT in patients with EAC. These biomarkers might help to identify patients who are likely to benefit most from this multimodality treatment. Sa1927 Significant Genetic Variability Associated With the Evolution of Gastric Cancer Precursor Lesions in a Spanish Population Osmel Companioni, Catalina Bonet, Nadia Garcia, José Miguel Sanz-Anquela, María Berdasco, Magdalena Adrados, Jorge Mendoza, Elena Collantes, G. Ruiz, Enrique Rey, Francisco Sánchez-Ceballos, Elvira Poves, Laura Espinosa, Beatriz Madrigal, Jesus Barrio, Miriam Cuatrecasas, Josep Ignasi Elizalde, Luis Bujanda, Angel Cosme, Angel Ferrandez, Guillermo Muñoz, Victoria Andreu, María José Paules, Sergio Lario, María José Ramírez Lázaro, Javier P. Gisbert, Carlos A. Gonzalez, Núria Sala Background: Gastric carcinogenesis proceeds through several precursor lesions leading to gastric cancer (GC). H. pylori is the main risk factor for this process; however, only ≈1% of infected people develops GC, which indicates that other factors condition the progression from gastric precursor lesions (GPLs) to GC. Previous results of our group indicated that host genetic variability in MUC2 is associated with evolution of GPLs. A few other genes also showed nominal associations. Objective: To replicate previous results in an independent series of patients, and to explore whether genetic variability in other candidate genes plays a role in the evolution of GPL over time. Methods: 559 patients diagnosed with GPL between 1995 and 2004 in nine Spanish hospitals were submitted to a new endoscopy during 20112013, covering a mean follow-up of 12 years. Haploview analysis of HapMap data from Europeans allowed for selection of 141 SNPs of 11 human genes of H. pylori pathways triggered after infection, 7 genes coding for proteins with relevant gastric functions and 12 genes previously associated with gastric carcinogenesis. Sequenom technology was used to genotype SNPs in genomic DNA from saliva. After Hardy-Weinberg equilibrium testing, association between SNPs and haplotypes and the evolution of the lesions was estimated by logistic regression analysis of lesions that progressed versus those that remained stable or regressed, as well as those that regressed versus progression plus stable. The SNPassoc library of R software was used. Results: According to diagnosis at recruitment and at the end of follow-up, 46.3% of the lesions remained stable, 36.5% regressed and 17.2% progressed to a more severe lesion. The strongest association (OR=0.63, 95%CI=0.48-0.81, p=0.0003, additive model) was observed with MUC2 rs10902073 which, as in the previous study, appeared inversely associated with the regression of the GPLs. Another SNP in MUC2, and SNPs in the mucosa TFF1 and TFF2 genes and in H. pylori pathway gene CD14, were also significantly associated with either progression or regression of the lesions (p-val<0.05). Regarding the genes analyzed for the first time, MAPK3 was significantly associated with an increased risk of progression of GPLs while the PTGS2 was inversely associated. SNPs in CDH1, MAP3K14, MUC1, SRC, PTGES, IL1RN, NOD1 and CDX2, were associated with lesion regression. Haplotypes in CD14 and TFF1 were significantly associated with increased risk for progression, while haplotypes in MUC2, PTGES, MAP3K14 and TFF1 were associated with a decreased probability of regression. Conclusion: These results confirm the association of evolution of GPL with genetic variation in MUC2, TFF1 and CD14, and suggest that variation in other genes such as PTGES, MAP3K14, TFF2, MAPK3, PTGS2, CDH1, MUC1, SRC, IL1RN, NOD1 and CDX2, may also influence this process.
Sa1929 Aquaporin-9 Overexpression Activates Akt Signal Pathway and Enhances Chemosensitivity in Colorectal Cancer Zi-Huan Yang, Xing-Zhi Feng, Dan-Dan Huang, Yan-Hong Deng, Hao Chen, Jianping Wang BACKGROUND & AIMS: 5-fluorouracil (5-FU) based chemotherapy is routinely employed to treat colorectal cancer (CRC) patients at high risk for recurrence or those with advanced disease. Aquaporin 9 (AQP9) was known to be associated with arsenic sensitivity in leukemia. But the involvement of AQP9 in chemosensitivity in CRC is still unclear. The present study investigated the role of AQP9 in regulating tumor sensitivity to chemo drugs in CRC. METHODS: We evaluated real-time cell proliferation and cell apoptosis upon application of 5-FU on HCT116 cells transfected with wild-type or mutant AQP9. We colonized the transfected CRC cells into nude mice, and then injected the mice with 5-FU intraperitoneally after tumor formation. We performed immunostaining against AQP9 on tissue microarray that consists of 235 CRC patients with postoperative 5-FU-based chemotherapy. RESULTS:
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AGA Abstracts
AGA Abstracts
A Humanization and Affinity Maturation of an Anti-CD24 Monoclonal Antibody Targeting Gastrointestinal Cancers Shiran Shapira, Dina Kazanov, Sarah Kraus, Itai Benhar, Nadir Arber
Sa1928
Background: CD24 is a cell-surface heavily glycosylated GPI-anchored mucin-like protein. We have shown that CD24 is a valid target in gastrointestinal (GI) malignancies. Anti-CD24 mAb treatment induces a significant growth inhibition of CRC and PC cells, in a time- and dose-dependent manner in vitro and in vivo (Gastro 2006, Clin Can Res 2007, Can Res 2008). Affinity maturation is an important process in optimizing therapeutic antibodies. Affinitymatured antibodies can exhibit increased biological efficacy, and allow reduced dosage with less toxic side effects. Aim: To develop mature humanized anti-CD24 monoclonal antibodies (mAbs) and to evaluate their efficacy alone and/or in combination with standard chemotherapy for CRC and PC. Methods: Edman-degradation, cDNA synthesis, sequence and computational analysis (Ig-blast) were performed to reveal the entire DNA sequence of a murine anti-CD24 mAb (SWA11). Replacement of the Fc with the human IgG1 resulted in a mousehuman chimera. A human donor (Ig-blast) was chosen as a scaffold human Ab for grafting critical sequences of the murine antibody into it leading to the generation of humanized Ab derivatives. Sequence analysis of the CDR loops was the base for library designing. Affinity maturation was performed in two steps; CDR walking (two-step selection) and by using phage display technique. Results: In vivo antibody targeting and accumulation within a CD24 positive tumor and its excess clearance was clearly demonstrated using direct imaging (Fig. 1). Combinatorial phage-displayed antibody libraries with varying degrees of diversity at randomized positions from which high-affinity antibodies can be selected were created. A chosen matured clone was isolated and showed higher binding strength (1.8x10-8), compared to the parental murine and humanized Abs (3.3x10-8 and 4.2x10-8). The matured antibody showed selective recognition and binding to the CD24 antigen, as demonstrated by antigen-based (Fig. 2A) and whole-cell ELISA (Fig. 2B) and FACS analysis (Fig. 2C). It stability was enhanced following the maturation process, as demonstrated by in vitro stability test (Fig. 3). The antibody lost only 16% of its activity after 23 days in 37°C. Combined treatment with standard chemotherapeutic agents and natural products, such as monoterpenes (terpinen-4-ol) (Fig. 4), showed significant reduction in cell viability (90% cell death). Conclusion: Targeting CD24 may be a promising treatment for GI malignancies in combination with chemotherapy and natural agents. The resulted matured fully humanized antiCD24 mAb is more effective than the murine parental Ab.
Sa1926 SOX2 and p53 Protein Expression Predicts Response to Preoperative Chemoradiotherapy in Patients With Esophageal Adenocarcinoma Sophie H. van Olphen, Katharina Biermann, bas P. wijnhoven, Manon C. Spaander, Ate van der Gaast, Jan J. lanschot, Leendert Looijenga, Marco J. Bruno Objective Preoperative chemoradiotherapy has recently become common practice in treatment of esophageal cancer with a gain in 5-year survival of 10-15%. However, a significant proportion of patients do not respond well and experiencing unnecessary severe side-effects. Accurate risk-stratification of patients using informative biomarkers before therapy may help to avoid unnecessary morbidity due to ineffective treatment. The aim of this study was to investigate the correlation between the expression of SOX2 and P53 in pre-treatment tumor biopsies and grade of pathological tumor response in resected specimen of patients with esophageal adenocarcinoma (EAC) treated with neoadjuvant chemoradiotherapy (nCRT). Methods All EAC patients who received nCRT according to the CROSS regimen followed by esophagectomy, between January 2003 and July 2011 at the Erasmus University Medical Center, were included. SOX2 and P53 protein expression was visualized by immunohistochemistry on all pre-treatment tumor biopsies and scored independently by two investigators who were blinded for clinical outcome. Aberrant expression was defined as negative expression of SOX2 and overexpression or complete loss of P53 expression. The overall Tumor Regression Grade (TRG) was evaluated using the modified Mandard scoring system. Patients with TRG 1 or TRG 2 were classified as major responders (ie, < 10% of tumor cells remaining), whereas patients with TRG 3 or TRG 4 were classified as minor responders (ie, > 10% of tumor cells remaining). Results In total 77 patients were included. Forty (53%) patients had a major pathological response (TRG 1-2) and 37 (47%) a minor response (TRG 3-4). In pre-treatment biopsies aberrant SOX2 and P53 expression was seen in 40% (31/77) and 83% (64/77), respectively. A major response was significantly associated with an aberrant SOX2 expression (OR 3.9, 95% CI: 1.5 - 10.2, p=0.005) and aberrant p53 expression (OR 4.5, 95% CI: 1.15 - 18.2, p=0.031). Aberrant expression of both biomarkers increased the probability of a major response in the individual patient (OR of 5.6; 95% CI: 2.1 - 14.9, p= 0.001), with a sensitivity of 68%, specificity of 73% and a positive predictive value of 73%. Conclusion SOX2 and P53 expression in the pre-treatment biopsies predict response to nCRT in patients with EAC. These biomarkers might help to identify patients who are likely to benefit most from this multimodality treatment. Sa1927 Significant Genetic Variability Associated With the Evolution of Gastric Cancer Precursor Lesions in a Spanish Population Osmel Companioni, Catalina Bonet, Nadia Garcia, José Miguel Sanz-Anquela, María Berdasco, Magdalena Adrados, Jorge Mendoza, Elena Collantes, G. Ruiz, Enrique Rey, Francisco Sánchez-Ceballos, Elvira Poves, Laura Espinosa, Beatriz Madrigal, Jesus Barrio, Miriam Cuatrecasas, Josep Ignasi Elizalde, Luis Bujanda, Angel Cosme, Angel Ferrandez, Guillermo Muñoz, Victoria Andreu, María José Paules, Sergio Lario, María José Ramírez Lázaro, Javier P. Gisbert, Carlos A. Gonzalez, Núria Sala Background: Gastric carcinogenesis proceeds through several precursor lesions leading to gastric cancer (GC). H. pylori is the main risk factor for this process; however, only ≈1% of infected people develops GC, which indicates that other factors condition the progression from gastric precursor lesions (GPLs) to GC. Previous results of our group indicated that host genetic variability in MUC2 is associated with evolution of GPLs. A few other genes also showed nominal associations. Objective: To replicate previous results in an independent series of patients, and to explore whether genetic variability in other candidate genes plays a role in the evolution of GPL over time. Methods: 559 patients diagnosed with GPL between 1995 and 2004 in nine Spanish hospitals were submitted to a new endoscopy during 20112013, covering a mean follow-up of 12 years. Haploview analysis of HapMap data from Europeans allowed for selection of 141 SNPs of 11 human genes of H. pylori pathways triggered after infection, 7 genes coding for proteins with relevant gastric functions and 12 genes previously associated with gastric carcinogenesis. Sequenom technology was used to genotype SNPs in genomic DNA from saliva. After Hardy-Weinberg equilibrium testing, association between SNPs and haplotypes and the evolution of the lesions was estimated by logistic regression analysis of lesions that progressed versus those that remained stable or regressed, as well as those that regressed versus progression plus stable. The SNPassoc library of R software was used. Results: According to diagnosis at recruitment and at the end of follow-up, 46.3% of the lesions remained stable, 36.5% regressed and 17.2% progressed to a more severe lesion. The strongest association (OR=0.63, 95%CI=0.48-0.81, p=0.0003, additive model) was observed with MUC2 rs10902073 which, as in the previous study, appeared inversely associated with the regression of the GPLs. Another SNP in MUC2, and SNPs in the mucosa TFF1 and TFF2 genes and in H. pylori pathway gene CD14, were also significantly associated with either progression or regression of the lesions (p-val<0.05). Regarding the genes analyzed for the first time, MAPK3 was significantly associated with an increased risk of progression of GPLs while the PTGS2 was inversely associated. SNPs in CDH1, MAP3K14, MUC1, SRC, PTGES, IL1RN, NOD1 and CDX2, were associated with lesion regression. Haplotypes in CD14 and TFF1 were significantly associated with increased risk for progression, while haplotypes in MUC2, PTGES, MAP3K14 and TFF1 were associated with a decreased probability of regression. Conclusion: These results confirm the association of evolution of GPL with genetic variation in MUC2, TFF1 and CD14, and suggest that variation in other genes such as PTGES, MAP3K14, TFF2, MAPK3, PTGS2, CDH1, MUC1, SRC, IL1RN, NOD1 and CDX2, may also influence this process.
Sa1929 Aquaporin-9 Overexpression Activates Akt Signal Pathway and Enhances Chemosensitivity in Colorectal Cancer Zi-Huan Yang, Xing-Zhi Feng, Dan-Dan Huang, Yan-Hong Deng, Hao Chen, Jianping Wang BACKGROUND & AIMS: 5-fluorouracil (5-FU) based chemotherapy is routinely employed to treat colorectal cancer (CRC) patients at high risk for recurrence or those with advanced disease. Aquaporin 9 (AQP9) was known to be associated with arsenic sensitivity in leukemia. But the involvement of AQP9 in chemosensitivity in CRC is still unclear. The present study investigated the role of AQP9 in regulating tumor sensitivity to chemo drugs in CRC. METHODS: We evaluated real-time cell proliferation and cell apoptosis upon application of 5-FU on HCT116 cells transfected with wild-type or mutant AQP9. We colonized the transfected CRC cells into nude mice, and then injected the mice with 5-FU intraperitoneally after tumor formation. We performed immunostaining against AQP9 on tissue microarray that consists of 235 CRC patients with postoperative 5-FU-based chemotherapy. RESULTS:
S-357
AGA Abstracts
AGA Abstracts
A Humanization and Affinity Maturation of an Anti-CD24 Monoclonal Antibody Targeting Gastrointestinal Cancers Shiran Shapira, Dina Kazanov, Sarah Kraus, Itai Benhar, Nadir Arber