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Genetic modification of clinical-grade T cells is undertaken to augment function, including redirecting specificity for desired antigen. We and others have introduced a chimeric antigen receptor (CAR) to enable T cells to recognize lineage-specific tumor antigen, such as CD19, and early-phase human trials are currently assessing safety and feasibility. However, a significant barrier to next-generation clinical studies is developing a suitable CAR expression vector capable of genetically modifying a broad population of T cells. Transduction of T cells is relatively efficient but it requires specialized manufacture of expensive clinical grade recombinant virus. Electrotransfer of naked DNA plasmid offers a cost-effective alternative approach, but the inefficiency of transgene integration mandates ex vivo selection under cytocidal concentrations of drug to enforce expression of selection genes to achieve clinically meaningful numbers of CAR+ T cells. We report a new approach to efficiently generating T cells with redirected specificity, introducing DNA plasmids from the Sleeping Beauty transposon/transposase system to directly express a CD19-specific CAR in memory and effector T cells without drug selection. When coupled with numerical expansion on CD19+ artificial antigen-presenting cells, this gene transfer method results in rapid outgrowth of CD4+ and CD8+ T cells expressing CAR to redirect specificity for CD19+ tumor cells. [Cancer Res 2008;68(8):2961–71]

Glioblastoma multiforme (GBM) is an aggressive tumor with 14.6 months median survival rate. We have previously shown that laminin-411, a vascular basement membrane (BM) protein is a marker of tumor blood vessels that correlates with aggressiveness of GBMs. The laminin-411 pathway involving its β1 chain-containing integrin receptors, and ligand Dll4 for cancer stem cell (CSC) Notch1 was studied in mouse xenograft models to better understand glial tumor growth and new vasculature system development. To confirm the importance of laminin-411 expression for GBM progression and outcome prediction, sections from formalin-fixed paraffin embedded human brain tumors were studied. Immunohistochemical analysis of 107 GBM samples has revealed 87% cases with overexpression of laminin-411, whereas it was only 34% for high-grade (III) and 10.6% for low-grade (I/II) gliomas. The median survival for patients with GBM overexpressing laminin-411 was 10 months, compared to 20.2 months for those expressing “normal” laminin-421. The median recurrence rate was 5.6 and 9.3 months respectively. Morphometric analysis of CSC Notch1, nestin, CD133, and c-myc was correlated with laminin-411 overexpression in patients with high-grade gliomas. Nanobioconjugate PolycefinTM was synthesized to block BM laminin-411 in mice bearing intracranial human U87MG-derived GBM. Two antisense oligonucleotides against laminin-411 α4 and β1 chains were covalently attached on polymalic acid nanoplatform. The nanodrug, PolycefinTM, was able to cross blood brain tumor barrier and delivered drugs into cancer cells (Ding et al. 2010, 2013) using pH-dependent endosome releasing unit Leu-Leu-Leu. Evidence of cross talk was observed between BM, CSCs and tumor proliferation when mice were treated with PolycefinTM. It is shown that blocking synthesis of laminin-411 leads to significally lower tumor expression of integrin β1 chain, Notch ligand Dll4, and Notch1. The CSC markers nestin, CD133, and c-myc that are known indicators of glial tumor progression also showed quantitative reduction by morphometric analysis in tumors of mice treated with anti-laminin-411 Polycefin compared to PBS-treated animals. The data point to the importance of laminin-411-integrin β1-Dll4-Notch1 pathway in GBM development and to the ability of Polycefin to negatively impact CSC as a possible mechamism of GBM inhibition. The results provide new insights in glioma microenvironment and tumor endothelial and parenchymal cell signaling suggesting a novel approach for future therapeutics to target CSCs in vivo through inhibition of laminin-411 to treat highly infiltrating GBMs. Citation Format: Pallavi R. Gangalum, Alexander V. Ljubimov, Alexandra Chesnokova, Bindu Konda, Hui Ding, Jose Portilla-Arias, Adam Mamelak, Serguei Bannykh, Surasak Phuphanich, Jeremy Rudnick, Jethro Hu, Keith L. Black, Julia Y. Ljubimova. Nanoconjugates for inhibition of laminin-411-integrin β1-Dll4-Notch1 pathway to treat glioblastoma multiforme. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2692. doi:10.1158/1538-7445.AM2014-2692

A significant clinical problem with brain metastatic (BM) tumors is drug delivery and diagnostic imaging to verify MRI enhancement(s) for planning treatment. MRI enhancement in cancer patient's brain may result from infection after chemotherapy that impairs immune system; metastasis from primary lung/breast cancer; or a new primary brain tumor. Unlike lung/breast, brain biopsies are often technically impossible. Most drugs or monoclonal antibodies (mAb) are effective for primary tumors but cannot penetrate blood brain barrier (BBB) failing to treat brain metastasis. We used a natural nanobiopolymer, polymalic acid (PMLA), as a platform for the tumor-targeted PolycefinTM drugs for differential brain tumor imaging and treatment. Three xenogeneic orthotropic human brain metastatic tumors, MDA-MB-474, HER2+ breast cancer; A549 lung cancer, and MDA-MB-468, triple negative breast cancer (TNBC), both EGFR+, were injected into the brain of mice. For imaging, PolycefinTM had a covalently attached MRI tracer Gadolinium (Gd-DOTA). Antisense oligonucleotides (AON) were conjugated to PolycefinTM to inhibit gene/protein expression to block tumor growth. The combination of cell surface targeting mAbs, including anti-transferrin receptor (TfR) mAb for drug BBB transcytosis, and AONs to multiple tumor markers on the same delivery polymer was used for tumor treatment. MRI 1H imaging was performed on a 9.4-Tesla MRI system. Treatment groups of animals included (1) HER2+ MDA-MB-474 breast cancer metastases targeted with PMLA-Gd-DOTA/Trastuzumab/TfR mAb; (2) EGFR+ MDA-MB-468 TNBC metastases targeted with PMLA-Gd-DOTA/Cetuximab/TfR mAb; and (3) Controls inoculated with PMLA-IgG mAb and clinical Gd. Imaging: Specific tumor imaging was shown for brain-implanted lung and breast tumors: the inverse of T1-1 relaxation time proportional to Gd concentration was measured in healthy brain and in the tumor. T1-1 time dependence for Gd-DOTA-Polycefin (T1-1 ratio tumor/normal brain) was compared with clinical Gd, MultiHance®. After reaching a maximum, high T1-1 relative values prevailed for several hours for Gd-DOTA-mAb-Polycefin, but declined rapidly for Gd. High contrast for Gd was seen in 20 min, whereas that for Gd-DOTA-Polycefin peaked in 45-60 min, and remained for up to 3 hrs. By differential MRI with anti-HER2 (Trastuzumab) or anti-EFGR (Cetuximab) mAb attached to the nanoplatform, we were able to differentiate HER2+ from EGFR+ metastatic brain tumors. Treatment: Animal survival after Polycefin treatment was significantly higher than in untreated or mAb (Herceptin or Cetuximab) treated animals. Survival increases were as follows: 66% for lung cancer, 47% for HER2+ breast cancer, and 81% for TNBC. We developed a system for differential imaging and successful systemic treatment of various metastatic brain tumors based on specific metastasis targeting, and inhibition of expression of tumor-specific genes/proteins. Citation Format: Julia Y. Ljubimova, Rameshwar Patil, Pallavi Gangalum, Shawn Wagner, Satoshi Inoue, Hui Ding, Jose Portilla-Arias, Arthur Rekechenetskiy, Bindu Konda, Janet Markman, Alexandra Chesnokova, Keith L. Black, Eggehard Holler. Imaging and treatment of brain metastatic tumors using nanopolymers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3911. doi:10.1158/1538-7445.AM2013-3911

Introduction: A significant clinical problem with brain metastatic (BM) tumors is drug delivery and diagnostic imaging to verify MRI enhancement(s) for planning treatment. MRI enhancement in cancer patient's brain might result from infection after chemotherapy that impairs immune system; metastasis from primary lung/breast cancer; or a new primary brain tumor. Unlike lung/breast, brain biopsies are often technically impossible. Therefore, there is urgent need for the development of effective theranostic (dual therapy and diagnostic) systems against brain metastatic cancer. Most chemotherapeutic drugs or therapeutic monoclonal antibodies (mAb), Trastuzumab, Cetuximab, and Rituximab, are effective for primary tumor treatment but cannot penetrate blood brain barrier (BBB) failing to treat brain metastasis. We used a natural nanobiopolymer, polymalic acid (PMLA), as a nanoplatform for the family of tumor-targeted PolycefinTM drugs to provide differential brain tumor imaging and treatment. Methods: Three xenogeneic orthotropic human brain metastatic tumors, MDA-MB-474, HER2+ breast cancer; A549 lung cancer, and MDA-MB-468, triple negative breast cancer (TNBC), both EGFR+, were inoculated stereotactically into the brain of mice. For diagnostic imaging, PolycefinTM was used with covalently attached MRI tracer Gadolinium (Gd-DOTA). Morpholino antisense oligonucleotides (AON) were conjugated to PolycefinTM to specifically inhibit gene/protein expression to block tumor growth. The combination of cell surface targeting mAbs, including anti-transferrin receptor (TfR) mAb for drug BBB transcytosis, and AONs to multiple tumor markers on the same delivery polymer was used for anti-tumor treatment. MRI 1H imaging was performed on a 9.4-Tesla small animal MRI system. Treatment groups of animals included (1) HER2+ MDA-MB-474 breast cancer metastases targeted with PMLA-Gd-DOTA/HER2 mAb/TfR mAb; (2) EGFR+ MDA-MB-468 TNBC metastases targeted with PMLA-Gd-DOTA/EGFR mAb/TfR mAb; and (3) Controls for all treatments inoculated with PMLA-IgG mAb and clinical Gd. Unpublished Results. Imaging: Dynamic T1 analysis. Similar data for specific tumor imaging were obtained for brain-implanted lung and breast tumors: the inverse of T1-1 relaxation time (proportional to Gd concentration) was measured in healthy brain part and in the tumor. T1-1 time dependence for Gd-DOTA-Polycefin (T1-1 ratio tumor/normal brain) was compared with clinically used Gd MRI agent, MultiHance®. After reaching a maximum, high T1-1 relative values prevailed for several hours for Gd-DOTA-mAb-Polycefin, but declined rapidly for Gd. High contrast for Gd was seen in 20 min, whereas that for Gd-DOTA-Polycefin peaked in 45-60 min, and remained for up to 3 hrs. By differential MRI with anti-HER2 (Trastuzumab) or anti-EFGR (Cetuximab) mAb attached covalently to the nanoplatform, we were able to differentiate HER2+ from EGFR+ metastatic brain tumors with corresponding imaging controls. Treatment: Animal survival after Polycefin treatment of various brain metastases was significantly higher than in untreated (PBS) or therapeutic mAb (Herceptin or Cetuximab) treated animals. These survival increases were as follows: 66% for lung cancer metastasis, 47% for HER2+ breast cancer metastasis, and 81% for TNBC metastasis. Conclusions. We have developed a system for differential imaging and treatment of various metastatic brain tumors based on specific metastasis targeting, and inhibition of expression of tumor-specific genes/proteins. Systemic treatment with this system resulted in significantly increased survival of brain metastatic tumor-bearing animals. Citation Format: Julia Y. Ljubimova, R. Patil, P. Gangalum, S. Wagner, S. Inoue, H. Ding, J. Portilla, K. Rekechenetskiy, K. Bindu, J. Markman, A. Chesnokova, K. L. Black, E. Holler. Nanobiocojugates of differential imaging and treatment of brain metastatic tumors. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A50.