Exosome removal as a therapeutic adjuvant in cancer.

Exosome secretion is a notable feature of malignancy owing to the roles of these nanoparticles in cancer growth, immune suppression, tumor angiogenesis and therapeutic resistance. Exosomes are 30-100 nm membrane vesicles released by many cells types during normal physiological processes. Tumors aberrantly secrete large quantities of exosomes that transport oncoproteins and immune suppressive molecules to support tumor growth and metastasis. The role of exosomes in intercellular signaling is exemplified by human epidermal growth factor receptor type 2 (HER2) over-expressing breast cancer, where exosomes with the HER2 oncoprotein stimulate tumor growth and interfere with the activity of the therapeutic antibody Herceptin®. Since numerous observations from experimental model systems point toward an important clinical impact of exosomes in cancer, several pharmacological strategies have been proposed for targeting their malignant activities. We also propose a novel device strategy involving extracorporeal hemofiltration of exosomes from the entire circulatory system using an affinity plasmapheresis platform known as the Aethlon ADAPT™ (adaptive dialysis-like affinity platform technology) system, which would overcome the risks of toxicity and drug interactions posed by pharmacological approaches. This technology allows affinity agents, including exosome-binding lectins and antibodies, to be immobilized in the outer-capillary space of plasma filtration membranes that integrate into existing kidney dialysis systems. Device therapies that evolve from this platform allow rapid extracorporeal capture and selective retention of target particles < 200 nm from the entire circulatory system. This strategy is supported by clinical experience in hepatitis C virus-infected patients using an ADAPT™ device, the Hemopurifier®, to reduce the systemic load of virions having similar sizes and glycosylated surfaces as cancer exosomes. This review discusses the possible therapeutic approaches for targeting immune suppressive exosomes in cancer patients, and the anticipated significance of these strategies for reversing immune dysfunction and improving responses to standard of care treatments.

Nanoparticle analysis of circulating cell-derived vesicles in ovarian cancer patients.

Cell-derived vesicles are recognized as essential components of intercellular communication, and many disease processes are associated with their aberrant composition and release. Circulating tumor-derived vesicles have major potential as biomarkers; however, the diagnostic use of exosomes is limited by the technology available for their objective characterization and measurement. In this study, we compare nanoparticle tracking analysis (NTA) with submicron particle analysis (SPA), dynamic light scattering (DLS), and electron microscopy (EM) to objectively define size distribution, number, and phenotype of circulating cell-derived vesicles from ovarian cancer patients. Using the NanoSight LM10 instrument, cell-derived vesicles were visualized by laser light scattering and analyzing Brownian motion of these vesicles captured by video. The NTA software calculates the size and total concentration of the vesicles in solution. Using vesicles isolated from ovarian cancer patients, we demonstrate that NTA can measure the size distributions of cell-derived vesicles comparable to other analysis instrumentation. Size determinations by NTA, SPA, and DLS were more objective and complete than that obtained with the commonly used EM approach. NTA can also define the total vesicle concentration. Furthermore, the use of fluorescent-labeled antibodies against specific markers with NTA allows the determination of the "phenotype" of the cell-derived vesicles.

Modeling hepatitis C virus therapies combining drugs and lectin affinity plasmapheresis.

Hepatitis C virus (HCV) infection can be cured by standard pegylated interferon (IFN) + ribavirin drug therapy in 30-50% of treatment-naïve genotype 1 HCV patients. Cure rate is defined as a sustained viral response measured 6 months after the end of treatment. Recently, Fujiwara et al. [Hepatol Res 2007;37:701-710], using a double-filtration plasmapheresis (DFPP) technique, showed that simple physical reduction in circulating HCV using a 1-week pretreatment increased the cure rate for treatment-naïve type 1 HCV patients from 50 (controls) to 78% (treated). For previous nonresponders, the cure rate increased from 30 to 71%. This effect occurs even though the DFPP per treatment HCV viral load reduction averaged 26%. In clinical studies discussed here, a lectin affinity plasmapheresis (LAP) device caused an estimated 41% decrease in viral load as previously reported. A more detailed analysis using normalized data to correct for any variations in initial viral load gave an average 29% per treatment viral load reduction in 5 HCV-positive dialysis patients. The latter data indicate that continuous application of LAP could bring HCV viral load to undetectable levels in 4.1 days. Compared to DFPP, the LAP approach has the advantage that no plasma losses are incurred. In addition hemopurification can be carried out for extended periods of time analogous to continuous renal replacement therapy for the treatment of acute kidney failure, making the process much more effective. Calculations based on these data predict that continuous hemopurification would substantially increase the rate of viral load reduction (approx. 14-fold) and therefore increase the cure rate for HCV standard-of-care drug therapies without adding additional drugs and their associated side effects.

Reduction of hepatitis C virus using lectin affinity plasmapheresis in dialysis patients.

BACKGROUND/AIMS: To test the safety and efficacy of the Aethlon Hemopurifier, a lectin affinity cartridge, in clearing hepatitis C virus (HCV) from the blood of HCV-positive end-stage renal disease patients undergoing dialysis. Viral RNA was measured using real-time quantitative reverse transcriptase polymerase chain reaction. RESULTS: HCV clearance from plasma or blood was measured using either direct capture on immobilized Galanthus nivalis agglutinin (GNA) or using miniature plasmapheresis cartridges containing immobilized GNA. HCV in plasma samples was rapidly cleared by direct affinity capture (t(1/2) = approx. 20 min) and HCV in human blood was cleared using the Hemopurifier (t(1/2) = 2-3 h). Institutional-review-board-sanctioned clinical safety studies were conducted at the Apollo and Fortis Hospitals in India. At Apollo, 4 patients were treated 3 times/week for 2 weeks. HCV captured on the Hemopurifier averaged 8.9 x 10(8) viral copies/cartridge (n = 5), representing approximately 30% of the initial viral body burden. At Fortis, 3 patients treated 3 times/week for 1 week completed the viral load studies. Two patients showed measurable viral load reduction, while the third showed both increases and decreases in viral load. After Hemopurifier treatment, average HCV viral load was reduced by 57%. Surprisingly, average viral load was also 82% lower 7 days after treatment. Control samples also showed a marked transient reduction in HCV viral load as previously reported. CONCLUSION: The Hemopurifier rapidly cleared HCV from blood treated in vitro. In patients, the combination of the Hemopurifier plus dialysis decreased HCV viral load by 57% in 1 week. Moreover, viral load reduction continued up to 7 days after treatment.

Exosomes as a tumor immune escape mechanism: possible therapeutic implications.

Advances in cancer therapy have been substantial in terms of molecular understanding of disease mechanisms, however these advances have not translated into increased survival in the majority of cancer types. One unsolved problem in current cancer therapeutics is the substantial immune suppression seen in patients. Conventionally, investigations in this area have focused on antigen-nonspecific immune suppressive molecules such as cytokines and T cell apoptosis inducing molecules such as Fas ligand. More recently, studies have demonstrated nanovesicle particles termed exosomes are involved not only in stimulation but also inhibition of immunity in physiological conditions. Interestingly, exosomes secreted by cancer cells have been demonstrated to express tumor antigens, as well as immune suppressive molecules such as PD-1L and FasL. Concentrations of exosomes from plasma of cancer patients have been associated with spontaneous T cell apoptosis, which is associated in some situations with shortened survival. In this paper we place the "exosome-immune suppression" concept in perspective of other tumor immune evasion mechanisms. We conclude by discussing a novel therapeutic approach to cancer immune suppression by extracorporeal removal of exosomes using hollow fiber filtration technology.

Induction of antitumor immunity through xenoplacental immunization.

Historically cancer vaccines have yielded suboptimal clinical results. We have developed a novel strategy for eliciting antitumor immunity based upon homology between neoplastic tissue and the developing placenta. Placenta formation shares several key processes with neoplasia, namely: angiogenesis, activation of matrix metalloproteases, and active suppression of immune function. Immune responses against xenoantigens are well known to break self-tolerance. Utilizing xenogeneic placental protein extracts as a vaccine, we have successfully induced anti-tumor immunity against B16 melanoma in C57/BL6 mice, whereas control xenogeneic extracts and B16 tumor extracts where ineffective, or actually promoted tumor growth, respectively. Furthermore, dendritic cells were able to prime tumor immunity when pulsed with the placental xenoantigens. While vaccination-induced tumor regression was abolished in mice depleted of CD4 T cells, both CD4 and CD8 cells were needed to adoptively transfer immunity to naïve mice. Supporting the role of CD8 cells in controlling tumor growth are findings that only freshly isolated CD8 cells from immunized mice were capable of inducing tumor cell caspases-3 activation ex vivo. These data suggest feasibility of using xenogeneic placental preparations as a multivalent vaccine potently targeting not just tumor antigens, but processes that are essential for tumor maintenance of malignant potential.

Mathematical model of the effect of affinity hemodialysis on the T-cell depletion leading to AIDS.

HIV-derived envelope proteins appear to be intimately involved in the destruction of uninfected T cells that leads to AIDS in a process known as the 'bystander effect'. Affinity hemodialysis has been proposed as an effective means of reducing these viral toxins. Using deterministic mathematical models based on the well-known Perelson formulations, the effectiveness of affinity hemodialysis in reducing the levels of viral gp120 was analyzed. Incorporating experimental data on the function of the affinity dialysis system and data from published analyses of HIV viral dynamics, two different models of HIV and AIDS were analyzed. Both models predict a rapid and sustained reduction in gp120 levels. In the model incorporating stem cell dynamics, affinity hemodialysis treatment under several different scenarios was associated with a significant increase in T-cell levels independent of any release from lymphatic tissues. The calculations support the contention that affinity hemodialysis is a potentially useful adjunctive therapy, which can be employed to treat HIV-infected patients in conjunction with drug therapy. For those patients resistant to anti-retroviral drugs or those unable to take the drugs due to the side effects of those medications, affinity hemodialysis treatment may become a viable option.

Affinity hemodialysis for antiviral therapy. II. Removal of HIV-1 viral proteins from cell culture supernatants and whole blood.

BACKGROUND: HIV-1 gp120 may play a role in the progression from HIV infection to AIDS. AIMS: We investigated affinity hemodialysis for removing gp120 from cell culture and whole blood. METHODS: Anti-gp120 antibodies covalently coupled to agarose beads were packed into columns or hollow-fiber hemodialysis cartridges. Supernatants from HIV-infected HL2/3 cells or gp120 containing whole blood were pumped over the columns and gp120 measured by ELISA. RESULTS: Anti-gp120 agarose removed approximately 90% of HIV-1 gp120 from HL2/3 cultures in 30-60 min. Capture was antibody-dependent (F105 > IDX 1121 > ABI 13-108). Affinity hemodialysis also efficiently captured gp120 from buffer in a first-order, flow-rate-dependent fashion (t((1/2)) = 13 min at 0.9 ml/min). Clearance was faster than calculated diffusion (t((1/2)) approximately 2.5 h) suggesting significant convective transport. gp120 removal from blood was slower (t((1/2)) = 1.4 h). CONCLUSION: Affinity hemodialysis efficiently clears gp120 from cell culture fluids and blood and may be useful in slowing the progression to AIDS.

Affinity hemodialysis for antiviral therapy. I. Removal of HIV-1 from cell culture supernatants, plasma, and blood.

We tested an affinity hemodialysis technique designed to efficiently remove HIV and toxic viral proteins from blood. Miniature polyethersulfone hollow-fiber dialysis cartridges (200-500 nm pore) were packed with anti-HIV antibodies covalently coupled to agarose beads and sealed inside the cartridge. Cell culture fluids, plasma, or infected blood (7-15 ml) containing HIV-1 were circulated over the cartridge at 0.7-10 ml/min and the rate of removal of HIV measured by PCR and p24 ELISA. The technique removed up to 98% of HIV-1 particles from cell culture supernatants. Affinity hemodialysis also efficiently captured cultured HIV from human blood plasma (90%) and native HIV from infected blood (83% to 100%). Viral capture followed first-order kinetics (t(1/2) = 2.8 h). Variations in antibody type, matrix linkage (protein G versus direct coupling), bead pore size, and temperature of operation (25-37 degrees C) had only small effects. Although some binding was nonspecific, direct binding to the immobilized antibodies appeared to be the predominant mechanism.