Christina Kousparou

Antibodies targeting cancer stem cells: A new paradigm in immunotherapy?

Antibody targeting of cancer is showing clinical and commercial success after much intense research and development over the last 30 years. They still have the potential to delivery long-term cures but a shift in thinking towards a cancer stem cell (CSC) model for tumour development is certain to impact on how antibodies are selected and developed, the targets they bind to and the drugs used in combination with them. CSCs have been identified from many human tumours and share many of the characteristics of normal stem cells. The ability to renew, metabolically or physically protect themselves from xenobiotics and DNA damage and the range of locomotory-related receptors expressed could explain the observations of drug resistance and radiation insensitivity leading to metastasis and patient relapse. Targeting CSCs could be a strategy to improve the outcome of cancer therapy but this is not as simple as it seems. Targets such as CD133 and EpCAM/ESA could mark out CSCs from normal cells enabling specific intervention but indirect strategies such as interfering with the establishment of a supportive niche through anti-angiogenic or anti-stroma therapy could be more effective. This review will outline the recent discoveries for CSCs across the major tumour types highlighting the possible molecules for intervention. Examples of antibody-directed CSC therapies will be given and the outlook for the future development of this emerging area will be given.

Antibody-guided enzyme therapy of cancer producing cyanide results in necrosis of targeted cells.

A number of enzyme/prodrug activation approaches for the treatment of cancer have been reported to date with varying success. We describe progress in the development of a system based on a β‐glucosidase enzyme in combination with a naturally occurring “prodrug,” the sugar linamarin, which releases the cytotoxin cyanide. A recombinant fusion protein, composed of an scFv (MFE‐23) reactive against carcinoembryonic antigen (CEA) and a plant‐derived β‐glucosidase (linamarase), was produced and its cytotoxic potential was investigated. The fusion protein was expressed in a supersecretory mutant strain of Saccharomyces cerevisiae and purified by affinity chromatography. Extensive functional in vitro characterisation of the fusion protein showed that it retained antigen binding activity but that its catalytic activity was impaired, a problem not related to its fusion with the scFv. Nevertheless, we demonstrated complete tumour cell killing at doses of prodrug that are completely nontoxic to nontargeted cells. Preliminary in vivo characterisation showed that extensive glycosylation of the fusion protein caused its rapid clearance through the hepatic route. Aggregational properties also led to poor pharmacokinetics. Furthermore, we present some data analysing the mode of cell death resulting from exposure to this system. Enzymic catalysis of the substrate generates cyanide, a metabolic poison that asphyxiates cells and leads them to a necrotic‐like cell death. This system has been called antibody‐guided enzyme nitrile therapy (AGENT).

Generation of a selectively cytotoxic fusion protein against p53 mutated cancers

BackgroundA significant number of cancers are caused by defects in p21 causing functional defects in p21 or p53 tumour-suppressor proteins. This has led to many therapeutic approaches including restoration by gene therapy with wild-type p53 or p21 using viral or liposomal vectors, which have toxicity or side-effect limitations. We set out to develop a safer, novel fusion protein which has the ability to reconstitute cancer cell lines with active p21 by protein transduction.MethodsThe fusion protein was produced from the cell-translocating peptide Antennapedia (Antp) and wild-type, full-length p21 (Antp-p21). This was expressed and refolded from E. coli and tested on a variety of cell lines and tumours (in a BALB/c nude xenograft model) with differing p21 or p53 status.ResultsAntp-p21 penetrated and killed cancer cells that do not express wild type p53 or p21. This included cells that were matched to cogenic parental cell lines. Antp-p21 killed cancer cells selectively that were malignant as a result of mutations or nuclear exclusion of the p53 and p21 genes and over-expression of MDM2. Non-specific toxicity was excluded by showing that Antp-p21 penetrated but did not kill p53- or p21- wild-type cells. Antp-p21 was not immunogenic in normal New Zealand White rabbits. Recombinant Antp peptide alone was not cytotoxic, showing that killing was due to the transduction of the p21 component of Antp-p21. Antp-p21 was shown to penetrate cancer cells engrafted in vivo and resulted in tumour eradication when administered with conventionally-used chemotherapeutic agents, which alone were unable to produce such an effect.ConclusionsAntp-p21 may represent a new and promising targeted therapy for patients with p53-associated cancers supporting the concept that rational design of therapies directed against specific cancer mutations will play a part in the future of medical oncology.

Production and binding analyses of a humanised scFv against a cryptic epitope on tumour-associated fibronectin.

Tumour-associated splice variants of fibronectin are a major source of tumour-matrix associated targets and are proving very successful in the development of clinical agents to treat cancer. One of the first monoclonal antibodies to be produced to this target, murine BC-1, recognises a cryptic epitope in domain 7 of the B-form splice variant (EDB-FN). Antibody fragments based on this immunoglobulin (IgG) were unstable, but BC-1 humanisation provided an opportunity to produce a more stable single-chain Fv (scFv). The variable domains of the humanized BC-1 IgG were sub-cloned and constructed into a scFv (HuBC-1 scFv) which was successfully expressed in Escherichia coli. The scFv retained its conformationally-sensitive epitope recognition and demonstrated a good affinity to the target of around 50 nM as measured by ELISA, Surface Plasmon Resonance and Flow Cytometry. Furthermore, the scFv was thermostable and stable in serum allowing substantial localisation to human tumours grown in mouse xenograft models. This scFv could form the basis of future tumour-specific biopharmaceuticals.

Abstract 1977: Therapeutic miRNAs targeted selectively to tumors by mesenchymal stem cell derived microparticles

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Introduction: MiRNAs have been implicated in the development of some if not all cancer types and have been identified as attractive targets for therapy. However, systemic delivery of bare miRNAs faces its own set of limitations because of degradation by RNases and filtration and excretion by the kidneys. In this work we propose miRNAs as therapeutic agents. We have developed a novel system for miRNA delivery applicable for both local and systemic administration with the use of mesenchymal stem cell (MSC) microparticles (MPs). Methodology: MSCs were isolated from the Whartons jelly of human umbilical cords. The MSC cultures were subjected to serum deprivation, leading to the formation of MPs (secreted membrane vehicles <1μm) which were harvested and characterized by SEM, PCR, FACS and Fluorescence Microscopy. Breast (MDA-MB-231 & MCF-7), colon (RKO & HT-29) and ovarian adenocarcinoma (SKOV3) cell lines were then exposed to MPs. The response to treatment was evaluated by cell morphology, proliferation, migration, gene expression and apoptosis. Furthermore, the therapeutic potential of MPs was tested in vivo in xenograft tumor mouse models. Innovative imaging modalities such as in vivo flow cytometry and whole body fluorescence-bioluminescence were employed to dynamically investigate the biodistribution and homing kinetics of MPs in mice. Results: In vitro experiments confirmed that MSC-derived MPs can be internalized by the various cancer cell lines and induce a biological effect as evidenced by membrane damage, cell shrinkage and blebbing in the recipient cell. Significantly, there was evidence that MPs induce apoptosis, inhibit cell proliferation and tumor growth attenuation in a dose/time-dependent manner. The pro-apoptotic and anti-migration effects of MPs in cancer cells were almost completely abrogated by RNase treatment before administration to cultures. In vivo studies demonstrated that we were able to monitor and quantify fluorescently labelled MPs in circulation and to detect and image the biodistribution and incorporation in cells and organs in healthy and tumor-bearing mice. Conclusion: MSC-derived MPs containing miRNAs possess tumor inhibitory properties both in vitro and in vivo. Administration of MPs after RNase treatment induces the loss of anti-cancer properties suggesting a horizontal transfer of small RNAs from MPs to cancer cells. MPs formulated to contain specific miRNAs, could affect the action of genes associated with carcinogenesis, neovascularization, metastasis and other cancer characteristics, thus leading to therapeutic benefit. Citation Format: Marianna Prokopi, Agamemnon Epenetos, Andreas Anayiotos, Costas Pitsillides, Konstantinos Kapnisis, Christina Kousparou. Therapeutic miRNAs targeted selectively to tumors by mesenchymal stem cell derived microparticles. [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 1977. doi:10.1158/1538-7445.AM2014-1977

Abstract 2987: Generation of a selectively cytotoxic fusion protein against p53 mutated cancers

The most common genetic alteration in human cancer involves the p53 tumor suppressor gene resulting in defective control of cell cycle arrest and death. The p53 protein induces a cyclin-dependent kinase (cdk) inhibitor, p21 which occupies a central position in cell cycle regulation. Cdk inhibition by p21 results in a lack of progression from the G1 to the S-phase due to the prevention of retinoblastoma (Rb) phosphorylation and subsequent inhibition of transcription factors that regulate the genes involved in DNA replication and cell-cycle progression. It is hypothesized that the re-introduction of p21 into tumor cells will regenerate the pathway to apoptosis (programmed cell death) and inhibit proliferation since it has been suggested that overexpression of p21 results in suppression of tumor growth in vitro and in vivo.Unfortunately, p21 protein alone is unable to be administered directly in vivo and progress to the cell nuclei of cancer cells since there is no active mechanism to transport the protein. However, a number of proteins are known which can translocate across the cytoplasmic membrane of mammalian cells and into the nuclei, carrying additional cargoes with them. One of these translocating proteins is a 60 amino acid peptide corresponding to the homeodomain of the Drosophilia protein Antennapedia [ANTP]. A fusion protein (TR1) has been developed where the full-length p21 protein is attached to the antennapedia protein with a view to delivering the p21 protein into cancer cells to restore cell cycle control. TR1 penetrated and killed cancer cells that do not express wild type p53 or p21. This included cells that were matched to cogenic parental cell lines. Antp-p21 killed cancer cells selectively that were malignant as a result of mutations or nuclear exclusion of the p53 and p21 genes and over-expression of MDM2.Non-specific toxicity was excluded by showing that TR1 penetrated but did not kill p53- or p21- wild-type cells. TR1 was not immunogenic in normal New Zealand White rabbits. Recombinant Antp peptide alone was not cytotoxic, showing that killing was due to the transduction of the p21 component of TR1. TR1was shown to penetrate cancer cells engrafted in vivo and resulted in tumour eradication when administered with chemotherapy. TR1 may represent a new and promising targeted therapy for patients with p53-associated cancers supporting the concept that rational design of therapies directed against specific cancer mutations will play a part in the future of cancer therapeutics. Citation Format: Agamemnon A. Epenetos, Christina Kousparou, Aleksandra Filipovic. Generation of a selectively cytotoxic fusion protein against p53 mutated cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2987.

Abstract 234: Inhibition of the NOTCH transcription factor complex by a novel hybrid protein leads to effective tumor therapy.

The Notch pathway has been implicated in the generation and propagation of cancer stem cells and is thus an attractive target for therapy. We have generated a hybrid protein (Antp-DNMAML) consisting of the truncated version of Mastermind‐like (MAML) that behaves in a dominant negative (DN) fashion inhibiting Notch, and the cell penetrating peptide Antennapedia (Antp). Results indicate that the Antp-DNMAML translocates into the nucleus, suppresses Notch activation, reverts the transformed phenotype, inhibits the anchorage‐dependent growth and induces self contact inhibition and apoptosis in tumorigenic human breast cancer cells. More significantly, there is direct evidence that inhibiting Notch signaling at the transcriptional level with the Antp-DNMAML protein, suppresses the expression of downstream Notch targets and inhibits tumor growth in nude mice, without organ or systemic toxicity. In summary, intracellular delivery of dominant-negative transcription complex proteins using the Antp platform is a new and specific approach for cancer therapy . Citation Format: Agamemnon A. Epenetos, Christina Kousparou, Spyros Stylianou, Mahendra Deonarain, Aleksandra Filipovic. Inhibition of the NOTCH transcription factor complex by a novel hybrid protein leads to effective tumor therapy. [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 234. doi:10.1158/1538-7445.AM2013-234

Abstract 4364: TR4 for the treatment of human neoplasms: Novel antiNotch agent

It has been widely demonstrated that the Notch receptor signaling pathway is required for regulation and differentiation of stem cells in many tissues cell types. It is also well established that aberrant Notch signaling can lead to several human forms of cancer, including leukaemia, breast, colon, skin, cervical, and lung cancer, making Notch an attractive target for new cancer therapies. It has become clear that one of the major therapeutic targets in the Notch pathway is the cleavage of four Notch receptors, in which gamma-secretase inhibitors play a role. Unfortunately inhibition of γ-secretase complex is accompanied by many serious side effects. In order to avoid the side effects associated with γ-secretase inhibitors and achieve an improved therapeutic effect, a more selective Notch inhibition is desirable. We have genetically engineered a fusion protein, consisting of the Drosophila transcription factor Antennapedia (ANTP) and the truncated version of Mastermind-like (MAML) that behaves in a dominant negative (DN) fashion and inhibits the Notch transactivation complex. This fusion protein, (ANTP/DN-MAML) or TR4, has been tested for its ability to target tumor cells in vitro and in vivo. Our results indicate that targeting Notch signaling with the ANTP/DN-MAML (TR4) suppresses Notch activation, translocates into nucleus and inhibits tumor growth in mice, without significant organ or systemic toxicity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4364. doi:10.1158/1538-7445.AM2011-4364

Abstract 4441: Intracellular delivery of therapeutic siRNA via an antennapedia-RNA-binding domain fusion peptide

A key characteristic in tumorigenesis is the aberrant expression of various oncogenes, whose cellular products drive various anti-apoptotic/pro-survival pathways. The RNAi pathway is an endogenous cellular pathway that transiently inhibits the translation of mRNA into protein. It therefore has the potential to selectively arrest the production of mutated proteins. As the Intracellular delivery of bioactive molecules is still a major challenge, we are exploring the possible ability of the Antenapedia homeodomain (Antp) to act as an effective transporter of conjugated cargo across biological membranes. We have previously shown that Antp delivers both p21 into p21-null tumors in order to restore cell-cycle regulation, as well as a truncated form of MAML to inhibit Notch activation and induce apoptosis. To test whether in silico-designed siRNA has the ability to silence mutated forms of genes, we have created a bifunctional fusion protein, TR7, which incorporates Antp and an RNA-binding protein. TR7 attempts to both neutralize the anionic nature of siRNA as well as introduce it to the cytoplasm, thus initiating the RNAi process in a non-cytotoxic manner. In summary, this project, utilizing Antp, aims to solve the issue of systemic RNAi delivery, as well as harness the endogenous RNAi pathway to effectively halt the production of abnormal proteins in cancer and other life threatening illnesses. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4441. doi:10.1158/1538-7445.AM2011-4441