Gustavo Helguera

The transferrin receptor and the targeted delivery of therapeutic agents against cancer

BACKGROUND Traditional cancer therapy can be successful in destroying tumors, but can also cause dangerous side effects. Therefore, many targeted therapies are in development. The transferrin receptor (TfR) functions in cellular iron uptake through its interaction with transferrin. This receptor is an attractive molecule for the targeted therapy of cancer since it is upregulated on the surface of many cancer types and is efficiently internalized. This receptor can be targeted in two ways: 1) for the delivery of therapeutic molecules into malignant cells or 2) to block the natural function of the receptor leading directly to cancer cell death. SCOPE OF REVIEW In the present article we discuss the strategies used to target the TfR for the delivery of therapeutic agents into cancer cells. We provide a summary of the vast types of anti-cancer drugs that have been delivered into cancer cells employing a variety of receptor binding molecules including Tf, anti-TfR antibodies, or TfR-binding peptides alone or in combination with carrier molecules including nanoparticles and viruses. MAJOR CONCLUSIONS Targeting the TfR has been shown to be effective in delivering many different therapeutic agents and causing cytotoxic effects in cancer cells in vitro and in vivo. GENERAL SIGNIFICANCE The extensive use of TfR for targeted therapy attests to the versatility of targeting this receptor for therapeutic purposes against malignant cells. More advances in this area are expected to further improve the therapeutic potential of targeting the TfR for cancer therapy leading to an increase in the number of clinical trials of molecules targeting this receptor. This article is part of a Special Issue entitled Transferrins: molecular mechanisms of iron transport and disorders.

Paclitaxel-loaded PCL–TPGS nanoparticles: In vitro and in vivo performance compared with Abraxane®

The purpose of this work was to develop Cremophor(®) EL-free nanoparticles (NPs) loaded with Paclitaxel (PTX) in order to improve the drug i.v. pharmacokinetic profile and to evaluate its activity against commercially available formulations such as Taxol(®) and Abraxane(®). PTX-loaded poly(ε-caprolactone)-alpha tocopheryl polyethylene glycol 1000 succinate (PCL-TPGS) NPs were prepared using three different techniques: (i) by nanoprecipitation (NPr-method), (ii) by emulsion-solvent evaporation homogenized with an Ultra-Turrax(®) (UT-method) and (iii) by emulsion-solvent evaporation homogenized with an ultrasonicator (US-method). The NPs prepared by US-method showed the smallest size and the highest drug content. The NPs exhibited a slow and continuous release of PTX. The in vitro anti-tumoral activity was assessed using two human breast cancer cell lines (MCF-7 and MDA-MB-231) with the WTS assay. Cytotoxicity studies with both cell lines showed that PTX-loaded PCL-TPGS NPs exhibited better anti-cancer activity compared to PTX solution and the commercial formulation Abraxane(®) at different concentrations. Importantly, in the case of triple negative MDA-MB-231 breast cancer cells, the IC50 value for PTX-loaded PCL-TPGS NPs was 7.8 times lower than Abraxane(®). Finally, in vivo studies demonstrated that PTX-loaded PCL-TPGS NPs exhibited longer systemic circulation time and slower plasma elimination rate than Taxol(®) and Abraxane(®). Therefore, the novel NPs investigated might be an alternative nanotechnological platform for PTX delivery system in cancer chemotherapy.

Propulsion of African trypanosomes is driven by bihelical waves with alternating chirality separated by kinks.

Trypanosoma brucei, a parasitic protist with a single flagellum, is the causative agent of African sleeping sickness. Propulsion of T. brucei was long believed to be by a drill-like, helical motion. Using millisecond differential interference-contrast microscopy and analyzing image sequences of cultured procyclic-form and bloodstream-form parasites, as well as bloodstream-form cells in infected mouse blood, we find that, instead, motility of T. brucei is by the propagation of kinks, separating left-handed and right-handed helical waves. Kink-driven motility, previously encountered in prokaryotes, permits T. brucei a helical propagation mechanism while avoiding the large viscous drag associated with a net rotation of the broad end of its tapering body. Our study demonstrates that millisecond differential interference-contrast microscopy can be a useful tool for uncovering important short-time features of microorganism locomotion.

Antibody-cytokine fusion proteins: applications in cancer therapy

Background: Antibody–cytokine fusion proteins consist of cytokines fused to an antibody to improve antibody-targeted cancer immunotherapy. These molecules have the capacity to enhance the tumoricidal activity of the antibodies and/or activate a secondary antitumor immune response. Objective: To review the strategies used to develop antibody–cytokine fusion proteins and their in vitro and in vivo properties, including preclinical and clinical studies focusing on IL-2, IL-12 and GM-CSF. Methods: Articles were found by searching databases such as PubMed and Clinical Trials of the US National Institutes of Health. Results/conclusion: Multiple antibody–cytokine fusion proteins have demonstrated significant antitumor activity as direct therapeutics or as adjuvants of cancer vaccines in preclinical studies, paving the way for their clinical evaluation.

Alternative splicing of Slo channel gene programmed by estrogen, progesterone and pregnancy.

STREX alternative‐exon adds to Slo channel a phosphorylation sequence that can invert protein kinase A (PKA) regulation from excitatory to inhibitory. Because pregnancy switches Slo responsiveness to PKA from inhibitory to excitatory, we hypothesized that STREX expression diminishes with pregnancy and is regulated by sex hormones. Different from total‐rSlo, which is elevated around mid‐pregnancy and decreases at term, STREX transcripts progressively decreased with pregnancy near 80% at term. STREX downregulation was mimicked by estrogen, and opposed by estrogen‐receptor antagonist ICI 182,780 or progesterone (Pg). The regulation of STREX splicing directed by estrogen and Pg provides a mechanism for Slos PKA‐related phenotypic alteration with pregnancy.

A versatile targeting system with lentiviral vectors bearing the biotin-adaptor peptide.

Targeted gene transduction in vivo is the ultimate preferred method for gene delivery. We previously developed targeting lentiviral vectors that specifically recognize cell surface molecules with conjugated antibodies and mediate targeted gene transduction both in vitro and in vivo. Although effective in some experimental settings, the conjugation of virus with antibodies is mediated by the interaction between protein A and the Fc region of antibodies, which is not as stable as covalent conjugation. We have now developed a more stable conjugation strategy utilizing the interaction between avidin and biotin.

Tissue-specific regulation of Ca2+ channel protein expression by sex hormones

The L-type Ca(2+) channel pore-forming alpha subunit, alpha(1C) can be detected in brain and heart as two proteins with molecular masses of approximately 240 kDa and approximately 190 kDa known as alpha(1C-long) and alpha(1C-short), respectively. In brain, the alpha(1C-short) is thought to be the product of a approximately 50 kDa C-terminus calpain-mediated proteolytic deletion. We now show that uterine smooth muscle also possesses alpha(1C-long) and alpha(1C-short) isoforms, and that the relative expression of these two forms is regulated by sex hormones in a tissue-specific manner. Protein expression of alpha(1C) L-type Ca(2+) channels was examined in uterine smooth muscle, brain and heart, comparing non-pregnant (NP) estrus vs. late-pregnant (21 days) rats. The two forms of alpha(1C) were detected in all studied tissues. In late-pregnant uterus, alpha(1C-long) doubled the expression of alpha(1C-short); in NP uterus the opposite occurred. However, these changes were restricted to the uterine muscle, with no changes in brain and heart. To investigate the mechanism of such regulation, ovariectomized rats were treated with sex hormones, progesterone (P4) and/or 17beta-estradiol (estrogen, E2). P4 treatment, which yielded P4 plasma levels of 5 +/- 1 ng/ml and a high P4/E2 ratio (3 +/- 1.5 x 10(3)) similar to the ratio in late-pregnant uterus (1.5 +/- 0.3 x10(3)), facilitated alpha(1C-long) expression. In contrast, E2 or E2+P4 treatment that increased E2 plasma levels to 60 +/- 8 pg/ml and 75 +/- 24 pg/ml, produced low P4/E2 ratios of 0.03 +/- 0.006 and 0.2 +/- 0.1, respectively. These low P4/E2 ratios also found in NP rats at estrus (0.3 +/- 0.1) favored the expression of alpha(1C-short) form in myometrium. Neither hormone treatment altered alpha(1C) expression in brain or heart. Our results indicate that expression of alpha(1C) isoforms depends on P4/E2 ratios. Plasma P4/E2 ratios <1 x 10(3) favor the expression of the alpha(1C-short); whereas ratios >1 x 10(3) facilitate the expression of the alpha(1C-long) form. This regulation is tissue-specific for myometrium since it did not occur in heart and brain tissues.

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.

Cytokines fused to antibodies and their combinations as therapeutic agents against different peritoneal HER2/neu expressing tumors

We have previously generated antihuman HER2/neu–humanized IgG3 fused to interleukin-2 (IL-2), IL-12, or granulocyte macrophage colony-stimulating factor (GM-CSF) [monofunctional fusion proteins (mono-AbFP)] or fused to IL-2 and IL-12 or IL-12 and GM-CSF [bifunctional fusion proteins (bi-AbFP)]. These AbFPs retained cytokine and antigen-binding activities. We have now further characterized the AbFPs and determined the heparin-binding activity of the fused cytokines, their ability to trigger IFN-γ secretion and natural killer (NK) activation, and their direct antitumor efficacy. Flow cytometry revealed heparin-binding activity in the AbFPs containing IL-12 and IL-2, although this activity seems to be decreased in the bi-AbFPs. However, both bi-AbFPs retained the capacity to stimulate IL-12-dependent IFN-γ secretion in the NK cell line KY-1, and IL-12/IL-2 bi-AbFP induced NK activity in splenocytes. The antitumor effectiveness of bi-AbFPs and mono-AbFP combinations was studied in mice challenged i.p. with three different human HER2/neu murine syngeneic models (D2F2/E2, CT26-HER2/neu, and MC38-HER2/neu). Although a significant variability in the profile of antitumor response was observed in the different tumor models, the combination of IL-12 and GM-CSF mono-AbFPs protected 100% of D2F2/E2-challenged and 75% of CT26-HER2/neu–challenged mice. In contrast, bi-AbFPs protected less than the combination of mono-AbFPs and, in some models, even less than mono-AbFPs alone. However, in all cases, most of long-term survivors showed protection after s.c. rechallenge with the tumors and later with the parental tumors not expressing HER2/neu. These results show that, although the pattern of protection is tumor model dependent, treatments with AbFPs can effectively generate high levels of protection against peritoneal tumors expressing HER2/neu, which may be relevant in patients with primary or metastatic peritoneal carcinomatosis that may be observed in ovarian, colon, stomach, bladder, lung, and breast cancers. [Mol Cancer Ther 2006;5(4):1029–40]

Conjugation of an anti–transferrin receptor IgG3-avidin fusion protein with biotinylated saporin results in significant enhancement of its cytotoxicity against malignant hematopoietic cells

We have previously developed an antibody fusion protein composed of a mouse/human chimeric IgG3 specific for the human transferrin receptor genetically fused to avidin (anti-hTfR IgG3-Av) as a universal delivery system for cancer therapy. This fusion protein efficiently delivers biotinylated FITC into cancer cells via TfR-mediated endocytosis. In addition, anti-hTfR IgG3-Av alone exhibits intrinsic cytotoxic activity and interferes with hTfR recycling, leading to the rapid degradation of the TfR and lethal iron deprivation in certain malignant B-cell lines. We now report on the cytotoxic effects of a conjugate composed of anti-hTfR IgG3-Av and biotinylated saporin 6 (b-SO6), a toxin derived from the plant Saponaria officinalis that inhibits protein synthesis. Conjugation of anti-hTfR IgG3-Av with b-SO6 enhances the cytotoxic effect of the fusion protein in sensitive cells and also overcomes the resistance of malignant cells that show low sensitivity to the fusion protein alone. Our results show for the first time that loading anti-hTfR IgG3-Av with a biotinylated toxin enhances the cytotoxicity of the fusion protein alone. These results suggest that anti-hTfR IgG3-Av has great potential as a therapeutic agent for a wide range of applications due to its intrinsic cytotoxic activity plus its ability to deliver biotinylated molecules into cancer cells. [Mol Cancer Ther 2007;6(11):2995–3008]