Eilam Yeini

In vivo comparative study of distinct polymeric architectures bearing a combination of paclitaxel and doxorubicin at a synergistic ratio

ABSTRACT Nowadays, combination therapy became a standard in oncology. In this study, we compare the activity of two polymeric carriers bearing a combination of the anticancer drugs paclitaxel (PTX) and doxorubicin (DOX), which differ mainly in their architecture and supramolecular assembly. Drugs were covalently bound to a linear polymer, polyglutamic acid (PGA) or to a dendritic scaffold, polyglycerol (PG) decorated with poly(ethylene glycol) (PEG), forming PGA‐PTX‐DOX and PG‐PTX‐bz‐DOX‐PEG, respectively. We explored the relationship between the polymeric architectures and their performance with the aim to augment the pharmacological benefits of releasing both drugs simultaneously at the tumor site at a synergistic ratio. We recently designed and characterized a PGA‐PTX‐DOX conjugate. Here, we describe the synthesis and characterization of PG dendritic scaffold bearing the combination of PTX and DOX. The performance of both conjugates was evaluated in a murine model of mammary adenocarcinoma in immunocompetent mice, to investigate whether the activity of the treatments is affected by the immune system. Drug conjugation to a nano‐sized polymer enabled preferred tumor accumulation by extravasation‐dependent targeting, making use of the enhanced permeability and retention (EPR) effect. Both PGA‐PTX‐DOX and PG‐PTX‐bz‐DOX‐PEG nano‐sized conjugates exhibited superior anti‐tumor efficacy and safety compared to the combination of the free drugs, at equivalent concentrations. However, while PGA‐PTX‐DOX was more efficient than a mixture of each drug conjugated to a separate PGA chain, as was previously shown, PG‐PTX‐bz‐DOX‐PEG had similar activity to the mixture of the PG‐PTX‐bz‐PEG and PG‐DOX‐PEG conjugates. Our results show that both conjugates are potential candidates as precision combination nanomedicines for the treatment of breast cancer.

Co-targeting the tumor endothelium and P-selectin-expressing glioblastoma cells leads to a remarkable therapeutic outcome

Glioblastoma is a highly aggressive brain tumor. Current standard-of-care results in a marginal therapeutic outcome, partly due to acquirement of resistance and insufficient blood-brain barrier (BBB) penetration of chemotherapeutics. To circumvent these limitations, we conjugated the chemotherapy paclitaxel (PTX) to a dendritic polyglycerol sulfate (dPGS) nanocarrier. dPGS is able to cross the BBB, bind to P/L-selectins and accumulate selectively in intracranial tumors. We show that dPGS has dual targeting properties, as we found that P-selectin is not only expressed on tumor endothelium but also on glioblastoma cells. We delivered dPGS-PTX in combination with a peptidomimetic of the anti-angiogenic protein thrombospondin-1 (TSP-1 PM). This combination resulted in a remarkable synergistic anticancer effect on intracranial human and murine glioblastoma via induction of Fas and Fas-L, with no side effects compared to free PTX or temozolomide. This study shows that our unique therapeutic approach offers a viable alternative for the treatment of glioblastoma.

Targeting NCAM-expressing neuroblastoma with polymeric precision nanomedicine

&NA; Neural cell adhesion molecule (NCAM) expression is known to be associated with an aggressive biological behavior, increased metastatic capacity and expression of stem‐cell markers in several tumor types. NCAM was also found to be expressed on tumor endothelial cells while forming new capillary‐like tubes, but not on normal endothelial cells. An NCAM‐targeted polymer‐drug conjugate can be used both to target tumors expressing high levels of NCAM as well as the angiogenic vessels and cancer stem cells populations characterized by NCAM expression within tumors. Here, we describe the design, synthesis, physico‐chemical characterization and the biological evaluation of an NCAM‐targeted conjugate of polyglutamic acid with paclitaxel that was developed and evaluated on neuroblastoma, a high NCAM‐expressing tumor. This conjugate inhibited tumor growth to a higher extent compared to the control conjugates and was less toxic than free paclitaxel. The dose of the conjugate could be increased at least twice than the maximum tolerated dose of paclitaxel to achieve better activity without aggravating toxicity. This work presents evidence that NCAM targeting can highly increase the efficacy of nanomedicines in the appropriate tumor models. Graphical abstract Figure. No caption available.

Wilms Tumor NCAM-Expressing Cancer Stem Cells as Potential Therapeutic Target for Polymeric Nanomedicine

Cancer stem cells (CSC) form a specific population within the tumor that has been shown to have self-renewal and differentiation properties, increased ability to migrate and form metastases, and increased resistance to chemotherapy. Consequently, even a small number of cells remaining after therapy can repopulate the tumor and cause recurrence of the disease. CSCs in Wilms tumor, a pediatric renal cancer, were previously shown to be characterized by neural cell adhesion molecule (NCAM) expression. Therefore, NCAM provides a specific biomarker through which the CSC population in this tumor can be targeted. We have recently developed an NCAM-targeted nanosized conjugate of paclitaxel bound to a biodegradable polyglutamic acid polymer. In this work, we examined the ability of the conjugate to inhibit Wilms tumor by targeting the NCAM-expressing CSCs. Results show that the conjugate selectively depleted the CSC population of the tumors and effectively inhibited tumor growth without causing toxicity. We propose that the NCAM-targeted conjugate could be an effective therapeutic for Wilms tumor. Mol Cancer Ther; 16(11); 2462–72. ©2017 AACR.

PO-200 Prevention of melanoma brain colonisation by inhibiting cytokines secretion from activated astrocytes

Introduction The brain microenvironment consists of various cell types such as astrocytes, microglia, neurons and brain endothelial cells. It sustains the normal brain functions and the structures of the cerebral capillaries that form the blood brain barrier (BBB). During tumour progression, malignant cells release vascular endothelial growth factor and other cytokines that increase the permeability of blood vessels allowing them to intravasate into the circulation. In addition, the cells can take advantage of the patho-physiology of the tumour angiogenenic leaky blood vessels at distant organs and extravasate from them at inflamed sites. Such activated site can be the brain parenchyma, which may become a pro-inflammatory metastatic niche. Although little is known about the precise mechanisms and the factors that contribute to the brain metastatic process, there is increasing evidence that astrocytes are involved by mediating a neuroinflammation. They respond by releasing pro-inflammatory cytokines that compromise the integrity of the BBB, increasing the permeability of the capillary endothelium and preparing a metastatic niche for melanoma colonisation. Material and methods We established three spontaneous melanoma brain metastasis mouse models that present a pro-inflammatory microenvironment and a pro-metastatic niche preceding the colonisation of melanoma to the brain. siRNA targeting specific chemokines, neutralizating antibody, or a CRISPR/Cas9 system were used to downregulate the expression and secretion of chemokines for further in vitro studies. Results and discussions We identified MCP-1, a pro-inflammatory chemokine, implicated in the paracrine interaction between melanoma cells and the brain. The positive staining for GFAP and MCP-1 in the tumour area suggests a cooperation between melanoma and the tumour microenvironment, in which MCP-1 plays a pro-tumorigenic role during cell migration. Astrocytes-secreted MCP-1 expression levels increase when co-cultured with melanoma cells or their conditioned media. Downregulation of MCP-1 leads to decreased migration of melanoma cells in vitro. Moreover, neutralising antibody-targeting MCP-1 reduces melanoma invasion in matrigel towards sprouting aortas. Conclusion Activated astrocytes promote the migration of circulating melanoma cells to the brain by increased secretion of pro-inflammatory cytokines, such as MCP-1. Consequently, investigating the changes in melanoma and astrocytes gene-expression could be the key for interfering and preventing melanoma colonisation to the brain.

Molecular Weight-Dependent Activity of Aminated Poly(α)glutamates as siRNA Nanocarriers

RNA interference (RNAi) can contribute immensely to the area of personalized medicine by its ability to target any gene of interest. Nevertheless, its clinical use is limited by lack of efficient delivery systems. Polymer therapeutics can address many of the challenges encountered by the systemic delivery of RNAi, but suffer from inherent drawbacks such as polydispersity and batch to batch heterogeneity. These characteristics may have far-reaching consequences when dealing with therapeutic applications, as both the activity and the toxicity may be dependent on the length of the polymer chain. To investigate the consequences of polymers’ heterogeneity, we have synthesized two batches of aminated poly(α)glutamate polymers (PGAamine), differing in their degree of polymerization, but not in the monomer units or their conjugation. Isothermal titration calorimetry study was conducted to define the binding affinity of these polymers with siRNA. Molecular dynamics simulation revealed that Short PGAamine:siRNA polyplexes exposed a higher amount of amine moieties to the surroundings compared to Long PGAamine. This resulted in a higher zeta potential, leading to faster degradation and diminished gene silencing. Altogether, our study highlights the importance of an adequate physico-chemical characterization to elucidate the structure–function-activity relationship, for further development of tailor-designed RNAi delivery vehicles.

Nanoparticulate vaccine inhibits tumor growth via improved T cell recruitment into melanoma and huHER2 breast cancer

Nanoparticulate vaccines are promising tools to overcome cancer immune evasion. However, a deeper understanding on nanoparticle-immune cell interactions and treatments regime is required for optimal efficacy. We provide a comprehensive study of treatment schedules and mode of antigen-association to nanovaccines on the modulation of T cell immunity in vivo, under steady-state and tumor-bearing mice. The coordinated delivery of antigen and two adjuvants (Monophosphoryl lipid A, oligodeoxynucleotide cytosine-phosphate-guanine motifs (CpG)) by nanoparticles was crucial for dendritic cell activation. A single vaccination dictated a 3-fold increase on cytotoxic memory-T cells and raised antigen-specific immune responses against B16.M05 melanoma. It generated at least a 5-fold increase on IFN-γ cytokine production, and presented over 50% higher lymphocyte count in the tumor microenvironment, compared to the control. The number of lymphocytes at the tumor site doubled with triple immunization. This lymphocyte infiltration pattern was confirmed in mammary huHER2 carcinoma, with significant tumor reduction.

Successful intracranial delivery of trastuzumab by gene-therapy for treatment of HER2-positive breast cancer brain metastases

Background: Trastuzumab is a monoclonal antibody which demonstrates efficacy for HER2 positive breast cancer patients. Recently, an increased incidence of brain metastasis in trastuzumab‐treated patients has been reported. The reason for this may be the effectiveness of systemic trastuzumab allowing patients to survive longer thus providing time for brain metastases to develop, along with the lack of penetration of systemic therapies through the blood brain barrier. In recent years, several administration routes to the brain have been evaluated. Albeit advances in the field, there is still a need for improved delivery of therapeutic antibodies to the brain. To address this challenge, we have developed two gene therapy‐based methods enabling continuous secretion of active trastuzumab in the brain. Methods: We have developed two gene therapy approaches for the delivery of the therapeutic anti‐HER2 monoclonal antibody, trastuzumab, to the brain. We utilized the helper dependent adenovirus vector, containing trastuzumab light and heavy chains coding sequences (HDAd‐trastuzumab). In the first approach, we used the Transduced Autologous Restorative Gene Therapy (TARGT) platform, in which dermal fibroblasts of human and mouse origin, are ex‐vivo transduced with HDAd‐trastuzumab vector, rendering continuous secretion of active trastuzumab from the cells locally. These genetically engineered cells were subsequently implanted intracranially to mice, contralateral to HER2 positive breast carcinoma cells inoculation site, enabling continuous secretion of trastuzumab in the brain. In the second approach, we used the same HDAd‐trastuzumab viral vector, directly injected intracranially, contralateral to the HER2 positive breast carcinoma cells inoculation site. Both methods enabled therapeutic concentrations of local in‐vivo production of active trastuzumab in a mouse model of brain metastatic breast cancer. Results: Trastuzumab secreted from the TARGT platform demonstrated in‐vitro affinity and immune recruitment activity (ADCC) similar to recombinant trastuzumab (Herceptin, Genentech). When implanted in the brain of HER2 positive tumor‐bearing mice, both the TARGT platform of dermal fibroblasts engineered to secrete trastuzumab and direct injection of HDAd‐trastuzumab demonstrated remarkable intracranial tumor growth inhibitory effect. Conclusions: This work presents two gene therapy approaches for the administration of therapeutic antibodies to the brain. The TARGT platform of dermal fibroblasts engineered to secrete active trastuzumab, and the direct injection of HDAd‐trastuzumab viral vector, both rendered continuous in‐vivo secretion of active trastuzumab in the brain and demonstrated high efficacy. These two approaches present a proof of concept for promising gene therapy based administration methods for intracranial tumors as well as other brain diseases.