- Email: [email protected]
671. Potent Anti-Tumor Effects of ApDCs (Aptamer Drug Conjugates) for Targeted Therapeutics in Pancreatic Cancer
Cancer-Targeted Gene and Cell Therapy II treated prostates were significantly diminished when compared to those in the control group. Currently, the experiments are under the way to elucidate the molecular mechanism of overexpressed miR34a to slow down PCa progression. Taken together, our results demonstrated, for the first time, the potentials of rAAV-mediated efficient modulation of miRNA expression in the prostate for inhibiting PCa progression and studying molecular mechanisms in PCa development.
670. A Novel Approach to Expand Antigen Recognition in Chimeric Antigen Receptors
Robert Moot , Sunil Raikar , Lauren Fleischer , Melissa Querrey , Christopher Doering2, Trent Spencer2 1 Emory University, Atlanta, GA, 2Pediatrics, Aflac Cancer and Blood Disorders Center, Atlanta, GA 1
1
1
1
Chimeric antigen receptor (CAR) technology, although very promising, is limited in its application by the availability of target antigens. We propose to expand the pool of potentially targetable cell-surface antigens using the variable lymphocyte receptor (VLR) of the sea lamprey as the antigen recognition region of the CAR. VLRs represent the functional region of the lamprey and hagfish adaptive immune system. They are single chained and variable length, crescent shaped proteins that are produced by assembly of leucine-rich repeat cassettes to form a gene encoding region capable of exceeding 1015 unique variations. Due to their difference in structure compared to Ig based antibodies, VLRs bind antigen in a geometrically dissimilar manner. This unique property of VLRs allows them the ability to bind antigen epitopes that may not typically be bound by scFvs, the result being a potentially expanded repertoire of tumor cell target antigens that may be used in CAR design and application. In our studies, VLRs have been successfully developed from immunized lampreys and target cell specific VLRs have been cloned for several different antigens including VLRs specific for cancer cell lines and purified proteins. This is accomplished using yeast surface display combined with flow sorting and results in monoclonal VLRs specific for the cell line with which the lampreys were immunized. The functionality of the VLR was initially demonstrated in Jurkat cells transduced with a previously generated VLR specific for the B-cell receptor of the mouse tumor line, BCL. CAR protein expression from whole cell lysates of transduced Jurkat cells showed expression of CAR protein. CAR cell surface expression was also confirmed in Jurkat cells using a construct co-expressing GFP preceding a P2A sequence with >90% cells GFP positive. Transduced CAR-Jurkat cells showed upwards of 85% activation in co-culture assays with target cells. In transduced but not co-cultured cells, activation was <5% and activation in naïve cells co-cultured with BCL cells was <2%. These results establish the ability of these cells to effectively produce and express VLR-CAR protein. The BCL VLR-CAR construct was also shown to be functional in several different types of cytotoxic effector cells including gamma delta T-cells and NK-92 cells, where target cell killing was increased significantly over non-transduced cells, indicating that target cell specific toxicity is mediated through the VLR-CAR. From these results, showing that VLRs function effectively as the antigen recognition region of the CAR construct we have concluded that VLRs can serve as a unique alternative for directing CAR activity toward specific effector cells.
Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
671. Potent Anti-Tumor Effects of ApDCs (Aptamer Drug Conjugates) for Targeted Therapeutics in Pancreatic Cancer
Sorah Yoon1, Kai-Wen Huang2, Nagy Habib3, John Rossi1 1 Molecular and Cellular Biology, Beckman Research Institute of City of Hope, Duarte, CA, 2Department of Surgery and Hepatitis Research Center, National Taiwan University College of Medicine, Taipei, Taiwan, 3Department of Surgery and Cancer, Imperial College London, London, United Kingdom Aptamer Drug Conjugates (ApDCs) are a potential class of targeting therapeutics to improve therapeutic index over traditional chemotherapy. As most existing chemotherapies are absorbed into cells non-specifically through lipophilic interaction, to improve the therapeutic index, the drugs were attached to pancreatic cancer specific RNA aptamer, P19, as targeting moieties that preferentially delivered the payload to tumor cells. Active metabolites of gemcitabine (dFdCMP) and 5FdU (5FdUMP) were incorporated P19 intrinsically to make conjugates. The conjugates of P19-dFdCMP or P19-5FdUMP kept their structural functionality getting internalized in their target cells, PANC-1. P19-dFdCMP and P19-5FdUMP was incorporated into replicating DNA, resulting in chain termination and stalling of replication forks in nuclear. They induced the phosphorylation of histone H2AX on Ser139 (γ-H2AX), markers of double-strand DNA breaks, and increased the forms of nuclear foci at the sites of DNA damage. In consequence, the cell proliferation was significantly inhibited 51-53% in PANC-1 and 54-34% in gemcitabine resistance AsPC-1. Targeted anti-mitotic therapies have emerged as a new concept of cancer drugs. Chemotoxine, monomethyl auristatin E (MMAE) is a very potent antimitotic agent that inhibits cell division by blocking the polymerization of tubulin. However, it can’t be used by drug itself because of high toxicity. Herein, toxic molecule, MMAE, was conjugated to P19 for the targeted antimitotic therapies. P19-MMAE caused mitotic G2/M phase arrest, consequently inhibited 56% of cell proliferation on dose-dependent manner comparing control. In vivo assay, P19-MMAE induced the tumor regression via tail vein injection. In this study, the drug attached aptamer significantly decreased non-specific uptake of the drug and increase specific uptake of the conjugate in tumor cells. Our approaches suggest guiding cytotoxic drugs into malignant cancer cells specifically without causing significant harm to the normal cells.
672. Hybrid AAV/Phage Vector Enhances Chemotherapy Efficacy Against Cancer
Keittisak Suwan, Effrosyni Tsafa, Justyna Przystal, Paladd Asavarut, Amin Hajitou Medicine, Imperial College London, London, United Kingdom Chemotherapy has been widely used for cancer treatment, both in early and late stages. However, chemotherapy does not selectively target tumor cells, as normal cells are also harmed by the drugs resulting in several side effects. Another major obstacle to the success of chemotherapy in cancer treatment is the development of tumor drug resistance by cancer cells. In order to avoid these problems, the combination of chemotherapy with other therapeutic strategies has been used in order to lower the chemotherapeutic drug dose. Gene therapy is one of the therapeutic strategies that can be combined with chemotherapy. Mammalian viruses are well recognized vehicles for gene therapy, but major drawbacks of these viral vectors are broad tissue tropism following systemic administration (low specificity for the target cells/tissues), their fragility to harsh environments and difficulty for large scale production. Therefore, our group has developed a vector from bacteriophage, a bacteria virus also named phage. This novel engineered phage, called AAV/Phage, displays the RGD4C peptide to target a specific receptor (αvβ3 integrin) on cancer cell surface, while the phage genome is merged with recombinant S265
670. A Novel Approach to Expand Antigen Recognition in Chimeric Antigen Receptors
Robert Moot , Sunil Raikar , Lauren Fleischer , Melissa Querrey , Christopher Doering2, Trent Spencer2 1 Emory University, Atlanta, GA, 2Pediatrics, Aflac Cancer and Blood Disorders Center, Atlanta, GA 1
1
1
1
Chimeric antigen receptor (CAR) technology, although very promising, is limited in its application by the availability of target antigens. We propose to expand the pool of potentially targetable cell-surface antigens using the variable lymphocyte receptor (VLR) of the sea lamprey as the antigen recognition region of the CAR. VLRs represent the functional region of the lamprey and hagfish adaptive immune system. They are single chained and variable length, crescent shaped proteins that are produced by assembly of leucine-rich repeat cassettes to form a gene encoding region capable of exceeding 1015 unique variations. Due to their difference in structure compared to Ig based antibodies, VLRs bind antigen in a geometrically dissimilar manner. This unique property of VLRs allows them the ability to bind antigen epitopes that may not typically be bound by scFvs, the result being a potentially expanded repertoire of tumor cell target antigens that may be used in CAR design and application. In our studies, VLRs have been successfully developed from immunized lampreys and target cell specific VLRs have been cloned for several different antigens including VLRs specific for cancer cell lines and purified proteins. This is accomplished using yeast surface display combined with flow sorting and results in monoclonal VLRs specific for the cell line with which the lampreys were immunized. The functionality of the VLR was initially demonstrated in Jurkat cells transduced with a previously generated VLR specific for the B-cell receptor of the mouse tumor line, BCL. CAR protein expression from whole cell lysates of transduced Jurkat cells showed expression of CAR protein. CAR cell surface expression was also confirmed in Jurkat cells using a construct co-expressing GFP preceding a P2A sequence with >90% cells GFP positive. Transduced CAR-Jurkat cells showed upwards of 85% activation in co-culture assays with target cells. In transduced but not co-cultured cells, activation was <5% and activation in naïve cells co-cultured with BCL cells was <2%. These results establish the ability of these cells to effectively produce and express VLR-CAR protein. The BCL VLR-CAR construct was also shown to be functional in several different types of cytotoxic effector cells including gamma delta T-cells and NK-92 cells, where target cell killing was increased significantly over non-transduced cells, indicating that target cell specific toxicity is mediated through the VLR-CAR. From these results, showing that VLRs function effectively as the antigen recognition region of the CAR construct we have concluded that VLRs can serve as a unique alternative for directing CAR activity toward specific effector cells.
Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
671. Potent Anti-Tumor Effects of ApDCs (Aptamer Drug Conjugates) for Targeted Therapeutics in Pancreatic Cancer
Sorah Yoon1, Kai-Wen Huang2, Nagy Habib3, John Rossi1 1 Molecular and Cellular Biology, Beckman Research Institute of City of Hope, Duarte, CA, 2Department of Surgery and Hepatitis Research Center, National Taiwan University College of Medicine, Taipei, Taiwan, 3Department of Surgery and Cancer, Imperial College London, London, United Kingdom Aptamer Drug Conjugates (ApDCs) are a potential class of targeting therapeutics to improve therapeutic index over traditional chemotherapy. As most existing chemotherapies are absorbed into cells non-specifically through lipophilic interaction, to improve the therapeutic index, the drugs were attached to pancreatic cancer specific RNA aptamer, P19, as targeting moieties that preferentially delivered the payload to tumor cells. Active metabolites of gemcitabine (dFdCMP) and 5FdU (5FdUMP) were incorporated P19 intrinsically to make conjugates. The conjugates of P19-dFdCMP or P19-5FdUMP kept their structural functionality getting internalized in their target cells, PANC-1. P19-dFdCMP and P19-5FdUMP was incorporated into replicating DNA, resulting in chain termination and stalling of replication forks in nuclear. They induced the phosphorylation of histone H2AX on Ser139 (γ-H2AX), markers of double-strand DNA breaks, and increased the forms of nuclear foci at the sites of DNA damage. In consequence, the cell proliferation was significantly inhibited 51-53% in PANC-1 and 54-34% in gemcitabine resistance AsPC-1. Targeted anti-mitotic therapies have emerged as a new concept of cancer drugs. Chemotoxine, monomethyl auristatin E (MMAE) is a very potent antimitotic agent that inhibits cell division by blocking the polymerization of tubulin. However, it can’t be used by drug itself because of high toxicity. Herein, toxic molecule, MMAE, was conjugated to P19 for the targeted antimitotic therapies. P19-MMAE caused mitotic G2/M phase arrest, consequently inhibited 56% of cell proliferation on dose-dependent manner comparing control. In vivo assay, P19-MMAE induced the tumor regression via tail vein injection. In this study, the drug attached aptamer significantly decreased non-specific uptake of the drug and increase specific uptake of the conjugate in tumor cells. Our approaches suggest guiding cytotoxic drugs into malignant cancer cells specifically without causing significant harm to the normal cells.
672. Hybrid AAV/Phage Vector Enhances Chemotherapy Efficacy Against Cancer
Keittisak Suwan, Effrosyni Tsafa, Justyna Przystal, Paladd Asavarut, Amin Hajitou Medicine, Imperial College London, London, United Kingdom Chemotherapy has been widely used for cancer treatment, both in early and late stages. However, chemotherapy does not selectively target tumor cells, as normal cells are also harmed by the drugs resulting in several side effects. Another major obstacle to the success of chemotherapy in cancer treatment is the development of tumor drug resistance by cancer cells. In order to avoid these problems, the combination of chemotherapy with other therapeutic strategies has been used in order to lower the chemotherapeutic drug dose. Gene therapy is one of the therapeutic strategies that can be combined with chemotherapy. Mammalian viruses are well recognized vehicles for gene therapy, but major drawbacks of these viral vectors are broad tissue tropism following systemic administration (low specificity for the target cells/tissues), their fragility to harsh environments and difficulty for large scale production. Therefore, our group has developed a vector from bacteriophage, a bacteria virus also named phage. This novel engineered phage, called AAV/Phage, displays the RGD4C peptide to target a specific receptor (αvβ3 integrin) on cancer cell surface, while the phage genome is merged with recombinant S265