Pallavi R. Gangalum

HER2-positive breast cancer targeting and treatment by a peptide-conjugated mini nanodrug.

HER2+ breast cancer is one of the most aggressive forms of breast cancer. The new polymalic acid-based mini nanodrug copolymers are synthesized and specifically characterized to inhibit growth of HER2+ breast cancer. These mini nanodrugs are highly effective and in the clinic may substitute for trastuzumab (the marketed therapeutic antibody) and antibody-targeted nanobioconjugates. Novel mini nanodrugs are designed to have slender shape and small size. HER2+ cells were recognized by the polymer-attached trastuzumab-mimetic 12-mer peptide. Synthesis of the nascent cell-transmembrane HER2/neu receptors by HER2+ cells was inhibited by antisense oligonucleotides that prevented cancer cell proliferation and significantly reduced tumor size by more than 15 times vs. untreated control or PBS-treated group. We emphasize that the shape and size of mini nanodrugs can enhance penetration of multiple bio-barriers to facilitate highly effective treatment. Replacement of trastuzumab by the mimetic peptide favors reduced production costs and technical efforts, and a negligible immune response.

Polymalic acid nanobioconjugate for simultaneous immunostimulation and inhibition of tumor growth in HER2/neu-positive breast cancer.

Breast cancer remains the second leading cause of cancer death among women in the United States. Breast cancer prognosis is particularly poor in case of tumors overexpressing the oncoprotein HER2/neu. A new nanobioconjugate of the Polycefin(TM) family of anti-cancer drugs based on biodegradable and non-toxic polymalic acid (PMLA) was engineered for a multi-pronged attack on HER2/neu-positive breast cancer cells. An antibody-cytokine fusion protein consisting of the immunostimulatory cytokine interleukin-2 (IL-2) genetically fused to an antibody specific for human HER2/neu [anti-HER2/neu IgG3-(IL-2)] was covalently attached to the PMLA backbone to target HER2/neu expressing tumors and ensure the delivery of IL-2 to the tumor microenvironment. Antisense oligonucleotides (AON) were conjugated to the nanodrug to inhibit the expression of vascular tumor protein laminin-411 in order to block tumor angiogenesis. It is shown that the nanobioconjugate was capable of specifically binding human HER2/neu and retained the biological activity of IL-2. We also showed the uptake of the nanobioconjugate into HER2/neu-positive breast cancer cells and enhanced tumor targeting in vivo. The nanobioconjugate exhibited marked anti-tumor activity manifested by significantly longer animal survival and significantly increased anti-HER2/neu immune response in immunocompetent mice bearing D2F2/E2 murine mammary tumors that express human HER2/neu. The combination of laminin-411 AON and antibody-cytokine fusion protein on a single polymeric platform results in a new nanobioconjugate that can act against cancer cells through inhibition of tumor growth and angiogenesis and the orchestration of an immune response against the tumor. The present Polycefin(TM) variant may be a promising agent for treating HER2/neu expressing tumors and demonstrates the versatility of the Polycefin(TM) nanobioconjugate platform.

Toxicity and efficacy evaluation of multiple targeted polymalic acid conjugates for triple-negative breast cancer treatment

Abstract Engineered nanoparticles are widely used for delivery of drugs but frequently lack proof of safety for cancer patient’s treatment. All-in-one covalent nanodrugs of the third generation have been synthesized based on a poly(β-l-malic acid) (PMLA) platform, targeting human triple-negative breast cancer (TNBC). They significantly inhibited tumor growth in nude mice by blocking synthesis of epidermal growth factor receptor, and α4 and β1 chains of laminin-411, the tumor vascular wall protein and angiogenesis marker. PMLA and nanodrug biocompatibility and toxicity at low and high dosages were evaluated in vitro and in vivo. The dual-action nanodrug and single-action precursor nanoconjugates were assessed under in vitro conditions and in vivo with multiple treatment regimens (6 and 12 treatments). The monitoring of TNBC treatment in vivo with different drugs included blood hematologic and immunologic analysis after multiple intravenous administrations. The present study demonstrates that the dual-action nanoconjugate is highly effective in preclinical TNBC treatment without side effects, supported by hematologic and immunologic assays data. PMLA-based nanodrugs of the Polycefin™ family passed multiple toxicity and efficacy tests in vitro and in vivo on preclinical level and may prove to be optimized and efficacious for the treatment of cancer patients in the future.

Curcumin Targeted, Polymalic Acid-Based MRI Contrast Agent for the Detection of Aβ Plaques in Alzheimer's Disease

Currently, there is no gadolinium-based contrast agent available for conventional magnetic resonance imaging (MRI) detection of amyloidal beta (Aβ) plaques in Alzheimers disease (AD). Its timely finding would be vital for patient survival and quality of life. Curcumin (CUR), a common Indian spice effectively binds to Aβ plaques which is a hallmark of AD. To address this binding, we have designed a novel nanoimaging agent (NIA) based on nature-derived poly(β-l-malic acid) (PMLA) containing covalently attached gadolinium-DOTA(Gd-DOTA) and nature-derived CUR. The all-in-one agent recognizes and selectively binds to Aβ plaques and is detected by MRI. It efficiently detected Aβ plaques in human and mouse samples by an ex vivo staining. The method can be useful in clinic for safe and noninvasive diagnosis of AD.

Gene expression changes in rat brain after short and long exposures to particulate matter in Los Angeles basin air: Comparison with human brain tumors.

Air pollution negatively impacts pulmonary, cardiovascular, and central nervous systems. Although its influence on brain cancer is unclear, toxic pollutants can cause blood-brain barrier disruption, enabling them to reach the brain and cause alterations leading to tumor development. By gene microarray analysis validated by quantitative RT-PCR and immunostaining we examined whether rat (n=104) inhalation exposure to air pollution particulate matter (PM) resulted in brain molecular changes similar to those associated with human brain tumors. Global brain gene expression was analyzed after exposure to PM (coarse, 2.5-10μm; fine, <2.5μm; or ultrafine, <0.15μm) and purified air for different times, short (0.5, 1, and 3 months) and chronic (10 months), for 5h per day, four days per week. Expression of select gene products was also studied in human brain (n=7) and in tumors (n=83). Arc/Arg3.1 and Rac1 genes, and their protein products were selected for further examination. Arc was elevated upon two-week to three-month exposure to coarse PM and declined after 10-month exposure. Rac1 was significantly elevated upon 10-month coarse PM exposure. On human brain tumor sections, Arc was expressed in benign meningiomas and low-grade gliomas but was much lower in high-grade tumors. Conversely, Rac1 was elevated in high-grade vs. low-grade gliomas. Arc is thus associated with early brain changes and low-grade tumors, whereas Rac1 is associated with long-term PM exposure and highly aggressive tumors. In summary, exposure to air PM leads to distinct changes in rodent brain gene expression similar to those observed in human brain tumors.

Simultaneous blockade of interacting CK2 and EGFR pathways by tumor-targeting nanobioconjugates increases therapeutic efficacy against glioblastoma multiforme

Glioblastoma multiforme (GBM) remains the deadliest brain tumor in adults. GBM tumors are also notorious for drug and radiation resistance. To inhibit GBMs more effectively, polymalic acid-based blood-brain barrier crossing nanobioconjugates were synthesized that are delivered to the cytoplasm of cancer cells and specifically inhibit the master regulator serine/threonine protein kinase CK2 and the wild-type/mutated epidermal growth factor receptor (EGFR/EGFRvIII), which are overexpressed in gliomas according to The Cancer Genome Atlas (TCGA) GBM database. Two xenogeneic mouse models bearing intracranial human GBMs from cell lines LN229 and U87MG that expressed both CK2 and EGFR at different levels were used. Simultaneous knockdown of CK2α and EGFR/EGFRvIII suppressed their downstream prosurvival signaling. Treatment also markedly reduced the expression of programmed death-ligand 1 (PD-L1), a negative regulator of cytotoxic lymphocytes. Downregulation of CK2 and EGFR also caused deactivation of heat shock protein 90 (Hsp90) co-chaperone Cdc37, which may suppress the activity of key cellular kinases. Inhibition of either target was associated with downregulation of the other target as well, which may underlie the increased efficacy of the dual nanobioconjugate that is directed against both CK2 and EGFR. Importantly, the single nanodrugs, and especially the dual nanodrug, markedly suppressed the expression of the cancer stem cell markers c-Myc, CD133, and nestin, which could contribute to the efficacy of the treatments. In both tumor models, the nanobioconjugates significantly increased (up to 2-fold) animal survival compared with the PBS-treated control group. The versatile nanobioconjugates developed in this study, with the abilities of anti-cancer drug delivery across biobarriers and the inhibition of key tumor regulators, offer a promising nanotherapeutic approach to treat GBMs, and to potentially prevent drug resistance and retard the recurrence of brain tumors.

Polymalic Acid-based Nano Biopolymers for Targeting of Multiple Tumor Markers: An Opportunity for Personalized Medicine?

Tumors with similar grade and morphology often respond differently to the same treatment because of variations in molecular profiling. To account for this diversity, personalized medicine is developed for silencing malignancy associated genes. Nano drugs fit these needs by targeting tumor and delivering antisense oligonucleotides for silencing of genes. As drugs for the treatment are often administered repeatedly, absence of toxicity and negligible immune response are desirable. In the example presented here, a nano medicine is synthesized from the biodegradable, non-toxic and non-immunogenic platform polymalic acid by controlled chemical ligation of antisense oligonucleotides and tumor targeting molecules. The synthesis and treatment is exemplified for human Her2-positive breast cancer using an experimental mouse model. The case can be translated towards synthesis and treatment of other tumors.

Abstract 2692: Nanoconjugates for inhibition of laminin-411-integrin β1-Dll4-Notch1 pathway to treat glioblastoma multiforme

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

Abstract A50: Nanobiocojugates of differential imaging and treatment of brain metastatic tumors

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 patient9s 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.

Abstract 3911: Imaging and treatment of brain metastatic tumors using nanopolymers.

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 patient9s 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