POTENTIAL GOOD TARGET FOR IMMUNOTHERAPY ? PRAME ANTIGEN HAS SELF-ACTIVATING ABILITY
(Abstract release date: 05/19/16)
EHA Library. Misyurin V. 06/09/16; 134505; PB1605
Mr. Vsevolod Misyurin
Contributions
Contributions
Abstract
Abstract: PB1605
Type: Publication Only
Background
According to literature, PRAME (preferentially expressed antigen in melanoma) protein may be exposed on extracellular membrane. Interestingly, that PRAME is structural homolog of TLR2 protein. TLR2 activates NF-kB signaling pathway and NF-κB activates an expression of TLR2. As a result, TLR2-mediated activation of expression TLR2 is possible event. If PRAME is similar to TLR2, it’s possible to suggest PRAME-mediated activation of its own expression. PRAME protein is expressed in a wide range of cancers. In addition, this protein is non-active in cells of somatic tissues and is highly immunogenic. If PRAME is expressed in a tumor cell, it’s possible to increase an expression level of PRAME; this can lead to higher immunogenicity of tumor cells. This method could be a powerful in context of specific anti-PRAME immunotherapy.
Aims
To show that intracellular PRAME protein expression level is increased in case of binding anti-PRAME antibody with cell membrane.
Methods
We created a mouse monoclonal antibody 5D3F2, specifically recognizing PRAME antigen. Tree cell lines were used for incubation with anti-PRAME antibody: acute monocytic leukemia cell line THP-1, acute myeloid leukemia cell line NOMO-1 and disseminated melanoma cell line mel IBR. Expression level of PRAME gene was evaluated by RQ-PCR. Cell lines THP-1 and NOMO-1 were incubated in RPMI 1640 with addition of anti-PRAME antibody. Antibody concentration was 10 ug/ml, and amount of cells counted 10000/ml. Samples of cells was analyzed after 1, 2 and 4 hours and after 1, 2 and 3 days of incubation. Number of PRAME-expressing cells was determined by flow cytometry using a FITC-labeled antibodies 5D3F2. All experiments were repeated 3 times.
Results
PRAME gene expression level was 1,67% in THP-1; 0,46% in NOMO-1 and 963% in mel IBR relative of ABL expression level before experiment. According to flow cytometry data, number of PRAME-expressing cells THP-1 and NOMO-1 before experiment was 11,4% and 3,6%, respectively. Intensity of PRAME-expressing cells in THP-1 fluorescence after incubation with antibody 5D3F2 was 101,21% (after 1 hour), 75,79% (2 hour), 88,45% (4 hour), 421,28% (1 day), 433,33% (2 day) and 820,37% (3 day). Fluorescence intensity of PRAME-expressing cells in NOMO-1 after incubation with antibody 5D3F2 was 93,88% (1 hour), 87,07% (2 hour), 74,67% (4 hour), 175,45% (1 day), 562,67% (2 day) and 146,33% (3 day).
Conclusion
PRAME protein is expressed on THP-1 and NOMO-1 cell surface. Antibody 5D3F2 binds with cell membrane and it could explain decrease of fluorescence intensity in first hours. FITC-labeled 5D3F2 show this reduction because many PRAME epitopes are closed by non-labeled 5D3F2. But after 1 and more days number of PRAME epitopes on cell surfase grows, and flow cytometry reaction displays it. This finding suggests that it is possible to develop an anti-PRAME monoclonal antibody for therapy PRAME-positive disease. Moreover, PRAME expression level may be increased during therapy with these antibodies. It can be useful, because PRAME-expressing cell becomes more immunogenic after binding with a potential therapeutic anti-PRAME antibody. We did not investigate the role of NF-kB, but it will be our next challenge.
Session topic: E-poster
Keyword(s): Acute leukemia, Antibody response, Antibody targeting, Cell line
Type: Publication Only
Background
According to literature, PRAME (preferentially expressed antigen in melanoma) protein may be exposed on extracellular membrane. Interestingly, that PRAME is structural homolog of TLR2 protein. TLR2 activates NF-kB signaling pathway and NF-κB activates an expression of TLR2. As a result, TLR2-mediated activation of expression TLR2 is possible event. If PRAME is similar to TLR2, it’s possible to suggest PRAME-mediated activation of its own expression. PRAME protein is expressed in a wide range of cancers. In addition, this protein is non-active in cells of somatic tissues and is highly immunogenic. If PRAME is expressed in a tumor cell, it’s possible to increase an expression level of PRAME; this can lead to higher immunogenicity of tumor cells. This method could be a powerful in context of specific anti-PRAME immunotherapy.
Aims
To show that intracellular PRAME protein expression level is increased in case of binding anti-PRAME antibody with cell membrane.
Methods
We created a mouse monoclonal antibody 5D3F2, specifically recognizing PRAME antigen. Tree cell lines were used for incubation with anti-PRAME antibody: acute monocytic leukemia cell line THP-1, acute myeloid leukemia cell line NOMO-1 and disseminated melanoma cell line mel IBR. Expression level of PRAME gene was evaluated by RQ-PCR. Cell lines THP-1 and NOMO-1 were incubated in RPMI 1640 with addition of anti-PRAME antibody. Antibody concentration was 10 ug/ml, and amount of cells counted 10000/ml. Samples of cells was analyzed after 1, 2 and 4 hours and after 1, 2 and 3 days of incubation. Number of PRAME-expressing cells was determined by flow cytometry using a FITC-labeled antibodies 5D3F2. All experiments were repeated 3 times.
Results
PRAME gene expression level was 1,67% in THP-1; 0,46% in NOMO-1 and 963% in mel IBR relative of ABL expression level before experiment. According to flow cytometry data, number of PRAME-expressing cells THP-1 and NOMO-1 before experiment was 11,4% and 3,6%, respectively. Intensity of PRAME-expressing cells in THP-1 fluorescence after incubation with antibody 5D3F2 was 101,21% (after 1 hour), 75,79% (2 hour), 88,45% (4 hour), 421,28% (1 day), 433,33% (2 day) and 820,37% (3 day). Fluorescence intensity of PRAME-expressing cells in NOMO-1 after incubation with antibody 5D3F2 was 93,88% (1 hour), 87,07% (2 hour), 74,67% (4 hour), 175,45% (1 day), 562,67% (2 day) and 146,33% (3 day).
Conclusion
PRAME protein is expressed on THP-1 and NOMO-1 cell surface. Antibody 5D3F2 binds with cell membrane and it could explain decrease of fluorescence intensity in first hours. FITC-labeled 5D3F2 show this reduction because many PRAME epitopes are closed by non-labeled 5D3F2. But after 1 and more days number of PRAME epitopes on cell surfase grows, and flow cytometry reaction displays it. This finding suggests that it is possible to develop an anti-PRAME monoclonal antibody for therapy PRAME-positive disease. Moreover, PRAME expression level may be increased during therapy with these antibodies. It can be useful, because PRAME-expressing cell becomes more immunogenic after binding with a potential therapeutic anti-PRAME antibody. We did not investigate the role of NF-kB, but it will be our next challenge.
Session topic: E-poster
Keyword(s): Acute leukemia, Antibody response, Antibody targeting, Cell line
Abstract: PB1605
Type: Publication Only
Background
According to literature, PRAME (preferentially expressed antigen in melanoma) protein may be exposed on extracellular membrane. Interestingly, that PRAME is structural homolog of TLR2 protein. TLR2 activates NF-kB signaling pathway and NF-κB activates an expression of TLR2. As a result, TLR2-mediated activation of expression TLR2 is possible event. If PRAME is similar to TLR2, it’s possible to suggest PRAME-mediated activation of its own expression. PRAME protein is expressed in a wide range of cancers. In addition, this protein is non-active in cells of somatic tissues and is highly immunogenic. If PRAME is expressed in a tumor cell, it’s possible to increase an expression level of PRAME; this can lead to higher immunogenicity of tumor cells. This method could be a powerful in context of specific anti-PRAME immunotherapy.
Aims
To show that intracellular PRAME protein expression level is increased in case of binding anti-PRAME antibody with cell membrane.
Methods
We created a mouse monoclonal antibody 5D3F2, specifically recognizing PRAME antigen. Tree cell lines were used for incubation with anti-PRAME antibody: acute monocytic leukemia cell line THP-1, acute myeloid leukemia cell line NOMO-1 and disseminated melanoma cell line mel IBR. Expression level of PRAME gene was evaluated by RQ-PCR. Cell lines THP-1 and NOMO-1 were incubated in RPMI 1640 with addition of anti-PRAME antibody. Antibody concentration was 10 ug/ml, and amount of cells counted 10000/ml. Samples of cells was analyzed after 1, 2 and 4 hours and after 1, 2 and 3 days of incubation. Number of PRAME-expressing cells was determined by flow cytometry using a FITC-labeled antibodies 5D3F2. All experiments were repeated 3 times.
Results
PRAME gene expression level was 1,67% in THP-1; 0,46% in NOMO-1 and 963% in mel IBR relative of ABL expression level before experiment. According to flow cytometry data, number of PRAME-expressing cells THP-1 and NOMO-1 before experiment was 11,4% and 3,6%, respectively. Intensity of PRAME-expressing cells in THP-1 fluorescence after incubation with antibody 5D3F2 was 101,21% (after 1 hour), 75,79% (2 hour), 88,45% (4 hour), 421,28% (1 day), 433,33% (2 day) and 820,37% (3 day). Fluorescence intensity of PRAME-expressing cells in NOMO-1 after incubation with antibody 5D3F2 was 93,88% (1 hour), 87,07% (2 hour), 74,67% (4 hour), 175,45% (1 day), 562,67% (2 day) and 146,33% (3 day).
Conclusion
PRAME protein is expressed on THP-1 and NOMO-1 cell surface. Antibody 5D3F2 binds with cell membrane and it could explain decrease of fluorescence intensity in first hours. FITC-labeled 5D3F2 show this reduction because many PRAME epitopes are closed by non-labeled 5D3F2. But after 1 and more days number of PRAME epitopes on cell surfase grows, and flow cytometry reaction displays it. This finding suggests that it is possible to develop an anti-PRAME monoclonal antibody for therapy PRAME-positive disease. Moreover, PRAME expression level may be increased during therapy with these antibodies. It can be useful, because PRAME-expressing cell becomes more immunogenic after binding with a potential therapeutic anti-PRAME antibody. We did not investigate the role of NF-kB, but it will be our next challenge.
Session topic: E-poster
Keyword(s): Acute leukemia, Antibody response, Antibody targeting, Cell line
Type: Publication Only
Background
According to literature, PRAME (preferentially expressed antigen in melanoma) protein may be exposed on extracellular membrane. Interestingly, that PRAME is structural homolog of TLR2 protein. TLR2 activates NF-kB signaling pathway and NF-κB activates an expression of TLR2. As a result, TLR2-mediated activation of expression TLR2 is possible event. If PRAME is similar to TLR2, it’s possible to suggest PRAME-mediated activation of its own expression. PRAME protein is expressed in a wide range of cancers. In addition, this protein is non-active in cells of somatic tissues and is highly immunogenic. If PRAME is expressed in a tumor cell, it’s possible to increase an expression level of PRAME; this can lead to higher immunogenicity of tumor cells. This method could be a powerful in context of specific anti-PRAME immunotherapy.
Aims
To show that intracellular PRAME protein expression level is increased in case of binding anti-PRAME antibody with cell membrane.
Methods
We created a mouse monoclonal antibody 5D3F2, specifically recognizing PRAME antigen. Tree cell lines were used for incubation with anti-PRAME antibody: acute monocytic leukemia cell line THP-1, acute myeloid leukemia cell line NOMO-1 and disseminated melanoma cell line mel IBR. Expression level of PRAME gene was evaluated by RQ-PCR. Cell lines THP-1 and NOMO-1 were incubated in RPMI 1640 with addition of anti-PRAME antibody. Antibody concentration was 10 ug/ml, and amount of cells counted 10000/ml. Samples of cells was analyzed after 1, 2 and 4 hours and after 1, 2 and 3 days of incubation. Number of PRAME-expressing cells was determined by flow cytometry using a FITC-labeled antibodies 5D3F2. All experiments were repeated 3 times.
Results
PRAME gene expression level was 1,67% in THP-1; 0,46% in NOMO-1 and 963% in mel IBR relative of ABL expression level before experiment. According to flow cytometry data, number of PRAME-expressing cells THP-1 and NOMO-1 before experiment was 11,4% and 3,6%, respectively. Intensity of PRAME-expressing cells in THP-1 fluorescence after incubation with antibody 5D3F2 was 101,21% (after 1 hour), 75,79% (2 hour), 88,45% (4 hour), 421,28% (1 day), 433,33% (2 day) and 820,37% (3 day). Fluorescence intensity of PRAME-expressing cells in NOMO-1 after incubation with antibody 5D3F2 was 93,88% (1 hour), 87,07% (2 hour), 74,67% (4 hour), 175,45% (1 day), 562,67% (2 day) and 146,33% (3 day).
Conclusion
PRAME protein is expressed on THP-1 and NOMO-1 cell surface. Antibody 5D3F2 binds with cell membrane and it could explain decrease of fluorescence intensity in first hours. FITC-labeled 5D3F2 show this reduction because many PRAME epitopes are closed by non-labeled 5D3F2. But after 1 and more days number of PRAME epitopes on cell surfase grows, and flow cytometry reaction displays it. This finding suggests that it is possible to develop an anti-PRAME monoclonal antibody for therapy PRAME-positive disease. Moreover, PRAME expression level may be increased during therapy with these antibodies. It can be useful, because PRAME-expressing cell becomes more immunogenic after binding with a potential therapeutic anti-PRAME antibody. We did not investigate the role of NF-kB, but it will be our next challenge.
Session topic: E-poster
Keyword(s): Acute leukemia, Antibody response, Antibody targeting, Cell line
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