PRAME PROTEIN IS LOCATED IN EXTRACELLULAR SIDE OF MEMBRANE AND MIGHT BE A GOOD TARGET FOR ANTIBODY THERAPY OF HEMATOLOGICAL MALIGNANCIES
(Abstract release date: 05/19/16)
EHA Library. Lyzhko N. 06/09/16; 134684; PB1784
Ms. Natalya Lyzhko
Contributions
Contributions
Abstract
Abstract: PB1784
Type: Publication Only
Background
PRAME protein is not expressed in normal cells, but is active in large number of tumor cells. Thus PRAME protein might be a promising target for cancer immunotherapy. It is known that PRAME located in nucleus and cytoplasm of tumor cells. Tajeddine et al (2005) and Wadelin et al (2010) reported that this protein was located in nucleus and cytoplasm of tumor cells. On the other hand, Proto-Siqueira et al (2006) and Quintarelli et al (2008) found this protein on cell membrane.
Aims
To prove that PRAME protein is located on tumor cell surface and can bind with a specific monoclonal antibody.
Methods
We used 11 samples of B-CLL patients blood and cell lines К562, THP-1 and NOMO-1. 5 samples of healthy blood donors were used as negative controls. PRAME expression level was determined by RQ-PCR in blood of 8 B-CLL patients and in cell lines. PRAME epitops in surface of cell from patients and cell lines was determined by flow cytometry and immunocytochemistry experiments. Cell lines were incubated with anty-PRAME monoclonal antibodies (mAbs) 5D3F2 and 6H8F12, together and separately, at concentrations 111 and 112 ug/ml, 11 and 12 ug/ml, respectively. Cell count was carried out after incubation with mAbs. The number of dead cells was evaluated during MTT assay.
Results
PRAME expression level in K562 THP-1 and NOMO-1 cells lines was 104%, 1,6% and 0,5%, respectively. 6 patients were PRAME-negative and 2/8 had PRAME expression median lewel 4,43%. According to flow cytometric data K562, THP-1 and NOMO-1 cell lines had PRAME-positive cells 14%, 3% and 0,9%, respectively. One patient had 0% PRAME-positive cells (flow cytometric data) and none mRNA of PRAME gene expression (RQ-PCR data), one another patient had 100% PRAME-positive cells whith mRNA expression level 8,84%). Immunocytochemistry data showed, that protein PRAME was located in cell’s surface of 11,5% cells in blood of 2/4 patients with B-CLL (we hadn't RQ-PCR data in this cases). К562 and THP-1 cells decreased growth rate by 15-20% and about 15-20% of died cells was observed after incubation with mAbs 5D3F2 and 6H8F12, together and separately, at concentrations 11 and 12 ug/ml, respectively. К562 and THP-1 cells decreased growth rate by 50% and about 4% of cells died after incubation with mAbs 5D3F2 and 6H8F12, together and separately, at concentrations 111 and 112 ug/ml, respectively. NOMO-1 cells decreased growth rate by 30% and around 10% of cells died after incubation with mAbs 5D3F2 and 6H8F12 together at concentrations 11 and 12 ug/ml. NOMO-1 cells decreased growth rate by 30% during incubation with mAb 6H8F12 at concentration 112 ug/ml, but this cells didn’t die. NOMO-1 cell did not demonstrate slowing of cell growth or dying after incubation with mAb 5D3F2 at concentration 111 ug/ml.
Conclusion
We confirmed evidence that PRAME protein is located on surface of cancer cells. The protein is expressed only in cases when mRNA of PRAME is expressed. Cytostatic and cytotoxic effect of mAbs 5D3F2 and 6H8F12 were detected. This mAbs are effective against cell lines К562, THP-1 and NOMO-1. Interestingly, at a lower concentration mAbs have a more pronounced effect. In cases with higher expression level of PRAME gene in cells, we obserwed a greater number of cells with PRAME epitopes on cell surface and more powerful MAbs-depended cytostatic and cytotoxic effect. Our data suggest that PRAME might be a promising target for immunotherapy of PRAME-positive patients with B-CLL.
Session topic: E-poster
Keyword(s): Antibody targeting, B-CLL
Type: Publication Only
Background
PRAME protein is not expressed in normal cells, but is active in large number of tumor cells. Thus PRAME protein might be a promising target for cancer immunotherapy. It is known that PRAME located in nucleus and cytoplasm of tumor cells. Tajeddine et al (2005) and Wadelin et al (2010) reported that this protein was located in nucleus and cytoplasm of tumor cells. On the other hand, Proto-Siqueira et al (2006) and Quintarelli et al (2008) found this protein on cell membrane.
Aims
To prove that PRAME protein is located on tumor cell surface and can bind with a specific monoclonal antibody.
Methods
We used 11 samples of B-CLL patients blood and cell lines К562, THP-1 and NOMO-1. 5 samples of healthy blood donors were used as negative controls. PRAME expression level was determined by RQ-PCR in blood of 8 B-CLL patients and in cell lines. PRAME epitops in surface of cell from patients and cell lines was determined by flow cytometry and immunocytochemistry experiments. Cell lines were incubated with anty-PRAME monoclonal antibodies (mAbs) 5D3F2 and 6H8F12, together and separately, at concentrations 111 and 112 ug/ml, 11 and 12 ug/ml, respectively. Cell count was carried out after incubation with mAbs. The number of dead cells was evaluated during MTT assay.
Results
PRAME expression level in K562 THP-1 and NOMO-1 cells lines was 104%, 1,6% and 0,5%, respectively. 6 patients were PRAME-negative and 2/8 had PRAME expression median lewel 4,43%. According to flow cytometric data K562, THP-1 and NOMO-1 cell lines had PRAME-positive cells 14%, 3% and 0,9%, respectively. One patient had 0% PRAME-positive cells (flow cytometric data) and none mRNA of PRAME gene expression (RQ-PCR data), one another patient had 100% PRAME-positive cells whith mRNA expression level 8,84%). Immunocytochemistry data showed, that protein PRAME was located in cell’s surface of 11,5% cells in blood of 2/4 patients with B-CLL (we hadn't RQ-PCR data in this cases). К562 and THP-1 cells decreased growth rate by 15-20% and about 15-20% of died cells was observed after incubation with mAbs 5D3F2 and 6H8F12, together and separately, at concentrations 11 and 12 ug/ml, respectively. К562 and THP-1 cells decreased growth rate by 50% and about 4% of cells died after incubation with mAbs 5D3F2 and 6H8F12, together and separately, at concentrations 111 and 112 ug/ml, respectively. NOMO-1 cells decreased growth rate by 30% and around 10% of cells died after incubation with mAbs 5D3F2 and 6H8F12 together at concentrations 11 and 12 ug/ml. NOMO-1 cells decreased growth rate by 30% during incubation with mAb 6H8F12 at concentration 112 ug/ml, but this cells didn’t die. NOMO-1 cell did not demonstrate slowing of cell growth or dying after incubation with mAb 5D3F2 at concentration 111 ug/ml.
Conclusion
We confirmed evidence that PRAME protein is located on surface of cancer cells. The protein is expressed only in cases when mRNA of PRAME is expressed. Cytostatic and cytotoxic effect of mAbs 5D3F2 and 6H8F12 were detected. This mAbs are effective against cell lines К562, THP-1 and NOMO-1. Interestingly, at a lower concentration mAbs have a more pronounced effect. In cases with higher expression level of PRAME gene in cells, we obserwed a greater number of cells with PRAME epitopes on cell surface and more powerful MAbs-depended cytostatic and cytotoxic effect. Our data suggest that PRAME might be a promising target for immunotherapy of PRAME-positive patients with B-CLL.
Session topic: E-poster
Keyword(s): Antibody targeting, B-CLL
Abstract: PB1784
Type: Publication Only
Background
PRAME protein is not expressed in normal cells, but is active in large number of tumor cells. Thus PRAME protein might be a promising target for cancer immunotherapy. It is known that PRAME located in nucleus and cytoplasm of tumor cells. Tajeddine et al (2005) and Wadelin et al (2010) reported that this protein was located in nucleus and cytoplasm of tumor cells. On the other hand, Proto-Siqueira et al (2006) and Quintarelli et al (2008) found this protein on cell membrane.
Aims
To prove that PRAME protein is located on tumor cell surface and can bind with a specific monoclonal antibody.
Methods
We used 11 samples of B-CLL patients blood and cell lines К562, THP-1 and NOMO-1. 5 samples of healthy blood donors were used as negative controls. PRAME expression level was determined by RQ-PCR in blood of 8 B-CLL patients and in cell lines. PRAME epitops in surface of cell from patients and cell lines was determined by flow cytometry and immunocytochemistry experiments. Cell lines were incubated with anty-PRAME monoclonal antibodies (mAbs) 5D3F2 and 6H8F12, together and separately, at concentrations 111 and 112 ug/ml, 11 and 12 ug/ml, respectively. Cell count was carried out after incubation with mAbs. The number of dead cells was evaluated during MTT assay.
Results
PRAME expression level in K562 THP-1 and NOMO-1 cells lines was 104%, 1,6% and 0,5%, respectively. 6 patients were PRAME-negative and 2/8 had PRAME expression median lewel 4,43%. According to flow cytometric data K562, THP-1 and NOMO-1 cell lines had PRAME-positive cells 14%, 3% and 0,9%, respectively. One patient had 0% PRAME-positive cells (flow cytometric data) and none mRNA of PRAME gene expression (RQ-PCR data), one another patient had 100% PRAME-positive cells whith mRNA expression level 8,84%). Immunocytochemistry data showed, that protein PRAME was located in cell’s surface of 11,5% cells in blood of 2/4 patients with B-CLL (we hadn't RQ-PCR data in this cases). К562 and THP-1 cells decreased growth rate by 15-20% and about 15-20% of died cells was observed after incubation with mAbs 5D3F2 and 6H8F12, together and separately, at concentrations 11 and 12 ug/ml, respectively. К562 and THP-1 cells decreased growth rate by 50% and about 4% of cells died after incubation with mAbs 5D3F2 and 6H8F12, together and separately, at concentrations 111 and 112 ug/ml, respectively. NOMO-1 cells decreased growth rate by 30% and around 10% of cells died after incubation with mAbs 5D3F2 and 6H8F12 together at concentrations 11 and 12 ug/ml. NOMO-1 cells decreased growth rate by 30% during incubation with mAb 6H8F12 at concentration 112 ug/ml, but this cells didn’t die. NOMO-1 cell did not demonstrate slowing of cell growth or dying after incubation with mAb 5D3F2 at concentration 111 ug/ml.
Conclusion
We confirmed evidence that PRAME protein is located on surface of cancer cells. The protein is expressed only in cases when mRNA of PRAME is expressed. Cytostatic and cytotoxic effect of mAbs 5D3F2 and 6H8F12 were detected. This mAbs are effective against cell lines К562, THP-1 and NOMO-1. Interestingly, at a lower concentration mAbs have a more pronounced effect. In cases with higher expression level of PRAME gene in cells, we obserwed a greater number of cells with PRAME epitopes on cell surface and more powerful MAbs-depended cytostatic and cytotoxic effect. Our data suggest that PRAME might be a promising target for immunotherapy of PRAME-positive patients with B-CLL.
Session topic: E-poster
Keyword(s): Antibody targeting, B-CLL
Type: Publication Only
Background
PRAME protein is not expressed in normal cells, but is active in large number of tumor cells. Thus PRAME protein might be a promising target for cancer immunotherapy. It is known that PRAME located in nucleus and cytoplasm of tumor cells. Tajeddine et al (2005) and Wadelin et al (2010) reported that this protein was located in nucleus and cytoplasm of tumor cells. On the other hand, Proto-Siqueira et al (2006) and Quintarelli et al (2008) found this protein on cell membrane.
Aims
To prove that PRAME protein is located on tumor cell surface and can bind with a specific monoclonal antibody.
Methods
We used 11 samples of B-CLL patients blood and cell lines К562, THP-1 and NOMO-1. 5 samples of healthy blood donors were used as negative controls. PRAME expression level was determined by RQ-PCR in blood of 8 B-CLL patients and in cell lines. PRAME epitops in surface of cell from patients and cell lines was determined by flow cytometry and immunocytochemistry experiments. Cell lines were incubated with anty-PRAME monoclonal antibodies (mAbs) 5D3F2 and 6H8F12, together and separately, at concentrations 111 and 112 ug/ml, 11 and 12 ug/ml, respectively. Cell count was carried out after incubation with mAbs. The number of dead cells was evaluated during MTT assay.
Results
PRAME expression level in K562 THP-1 and NOMO-1 cells lines was 104%, 1,6% and 0,5%, respectively. 6 patients were PRAME-negative and 2/8 had PRAME expression median lewel 4,43%. According to flow cytometric data K562, THP-1 and NOMO-1 cell lines had PRAME-positive cells 14%, 3% and 0,9%, respectively. One patient had 0% PRAME-positive cells (flow cytometric data) and none mRNA of PRAME gene expression (RQ-PCR data), one another patient had 100% PRAME-positive cells whith mRNA expression level 8,84%). Immunocytochemistry data showed, that protein PRAME was located in cell’s surface of 11,5% cells in blood of 2/4 patients with B-CLL (we hadn't RQ-PCR data in this cases). К562 and THP-1 cells decreased growth rate by 15-20% and about 15-20% of died cells was observed after incubation with mAbs 5D3F2 and 6H8F12, together and separately, at concentrations 11 and 12 ug/ml, respectively. К562 and THP-1 cells decreased growth rate by 50% and about 4% of cells died after incubation with mAbs 5D3F2 and 6H8F12, together and separately, at concentrations 111 and 112 ug/ml, respectively. NOMO-1 cells decreased growth rate by 30% and around 10% of cells died after incubation with mAbs 5D3F2 and 6H8F12 together at concentrations 11 and 12 ug/ml. NOMO-1 cells decreased growth rate by 30% during incubation with mAb 6H8F12 at concentration 112 ug/ml, but this cells didn’t die. NOMO-1 cell did not demonstrate slowing of cell growth or dying after incubation with mAb 5D3F2 at concentration 111 ug/ml.
Conclusion
We confirmed evidence that PRAME protein is located on surface of cancer cells. The protein is expressed only in cases when mRNA of PRAME is expressed. Cytostatic and cytotoxic effect of mAbs 5D3F2 and 6H8F12 were detected. This mAbs are effective against cell lines К562, THP-1 and NOMO-1. Interestingly, at a lower concentration mAbs have a more pronounced effect. In cases with higher expression level of PRAME gene in cells, we obserwed a greater number of cells with PRAME epitopes on cell surface and more powerful MAbs-depended cytostatic and cytotoxic effect. Our data suggest that PRAME might be a promising target for immunotherapy of PRAME-positive patients with B-CLL.
Session topic: E-poster
Keyword(s): Antibody targeting, B-CLL
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