Avid Bioservices Inc (CDMO)

[biopharm]

Peregrine Pharmaceuticals : New Patent.. published 8/27/15

Methods and compositions for isolating exosomes

20150241431 08/27/15 new patent


Browse recent Peregrine Pharmaceuticals, Inc. patents
USPTO Applicaton #: #20150241431
Inventors: Alan J. Schroit, Philip E. Thorpe, Shelley P.m. Fussey

Patent Info
Application #
US 20150241431 A1

Publish Date
08/27/2015

Document #
14634607

File Date
02/27/2015

USPTO Class

http://stks.freshpatents.com/Peregrine-Pharmaceuticals-Inc-nm1.php

Website for those that like to seek puzzle pieces and there are many dozens worth of them.

http://stks.freshpatents.com/companies/?sort=3

---------------------------------------------------------

will include the first page below...

--------------------------------------------------------

BACKGROUND OF THE INVENTION

- Top of Page

1. Field of the Invention

The present invention relates to the fields of biotechnology, and particularly to extracellular microvesicles such as exosomes. The invention provides surprising new methods and compositions for isolating disease-related and phosphatidylserine (PS)-positive extracellular microvesicles such as tumor- and viral-derived exosomes, particularly tumor-derived exosomes, which are especially suitable for use with large volumes of biological fluids and produce extracellular microvesicles and exosomes that are antigenically intact.

2. Description of the Related Art

Extracellular microvesicles are a class of membrane bound components released or secreted by cells, and include exosomes, ectosomes, microparticles or microvesicles and apoptotic bodies or blebs (György et al., 2011; Simpson & Mathivanan, 2012). Within this class of extracellular microvesicles, exosomes have gained particular attention in recent years.

Exosomes are typically described as 40-50 to 100 nanometer-sized membrane-derived vesicles and are known to be actively secreted by cells in vivo and in vitro. They are generated from the late endosomes by the inward budding and scission of the endosomal membrane, creating multivesicular bodies (MVBs) that contain intraluminal vesicles. These exosomes are released to the extracellular space upon fusion of the MVB with the plasma membrane. Because they originate from the cell's plasma membrane and are formed by invagination of the endosomal membrane, secreted exosomes possess plasma membrane and endosome proteins that encapsulate a cytosol-derived aqueous space.

Extracellular microvesicles such as exosomes exert a broad array of important physiological functions, e.g., by acting as molecular messengers that traffic information between different cell types. For example, exosomes deliver proteins, lipids and soluble factors including RNA and microRNAs (Thery et al., 2009) which, depending on their source, participate in signaling pathways that can influence apoptosis (Andreola et al., 2002; Huber et al., 2005; Kim et al., 2005), metastasis (Parolini et al., 2009), angiogenesis (Kim et al., 2005; Iero et al., 2008), tumor progression (Keller et al., 2009; Thery et al., 2002), thrombosis (Aharon & Brenner, 2009; Al Nedawi et al., 2005) and immunity by directing T cells towards immune activation (Andre et al., 2004; Chaput et al., 2005) or immune suppression (Szajnik et al., 2010; Valenti et al., 2007; Wieckowski et al., 2009).

Several techniques have been described for the isolation and purification of extracellular microvesicle and exosome populations from different sources, including from malignant effusions and the peripheral blood of cancer patients and from supernatants of in vitro cultivated cell lines and tumor cells. These methods include differential centrifugation, including an ultracentrifugation step (Thery et al., 2006); affinity chromatography (Taylor & Gercel-Taylor, 2008); polymer-mediated precipitation (Taylor et al., 2011), particularly using polyethylene glycol (PEG) of different molecular weights, including the Total Exosome Isolation Reagents from Life Technologies Corporation (U.S. Pat. No. 8,901,284) and ExoQuick™ (US 2013/0337440 A1); and capture on defined pore-size membranes (Grant et al., 2011), such as ExoMir™, which typically uses two filters of different pore-sizes connected in series (US 2013/0052647 A1).

However, the available techniques are limited by drawbacks in two important respects. Firstly, as applied to extracellular microvesicle and exosome preparation in general, they are time-consuming, cumbersome and/or costly, and limited by the amounts of material that can be processed. In particular, the techniques currently available for isolating extracellular microvesicles and exosomes all require a significant reduction in volume to obtain sufficient concentrations for study or use. The typical approach of concentrating the biological medium using ultracentrifugation before proceeding with exosome isolation is very time consuming and requires specialized laboratory equipment.

Secondly, the current techniques are particularly limited as they apply to tumor-derived extracellular microvesicle and exosomes. For example, the extra-corporeal removal of exosomes from the circulation of cancer patients has been proposed, in which patient's blood is pumped through a lectin-affinity column and then returned to the patient (U.S. Pat. No. 8,288,172). It has also been reported that tumor-derived exosomes can be purified using paramagnetic beads coated with antibodies against tumor-specific proteins such as HER2/neu (Koga et al., 2005). Kits using magnetic beads to capture specific exosomes are also available, such as Exo-Flow™ kits. In addition to the general drawbacks described above, such methods and kits are very limiting, requiring both advance knowledge of a particular exosome surface marker to be exploited in the antibody binding, as well as many detailed technical steps in the protocol, such as the preparation and use of biotinylated capture antibodies.

Therefore, there remains in the art a need for new and improved methods of isolating extracellular microvesicles such as exosomes, particularly disease-related and tumor-derived exosomes. The identification of simple and cost-effective new methods of isolating morphologically and antigenically intact extracellular microvesicles and exosomes would be an important advance. What is really needed is a method that is equipped to handle large volumes of biological materials, without specialized laboratory equipment and without the need for an early ultracentrifugation step, and particularly one that can be used to preferentially isolate disease-related and tumor-derived exosomes.
SUMMARY

- Top of Page
OF THE INVENTION

The present invention addresses the foregoing and other needs of the prior art by providing new methods and compositions for isolating extracellular microvesicles such as exosomes, particularly disease-related and phosphatidylserine (PS)-positive exosomes, including tumor-derived exosomes, which methods are rapid, efficient, cost-effective and suitable for use with large volumes of biological fluids. These methods are based on the surprising use of acetate buffers to isolate extracellular microvesicles such as exosomes from solution. The invention provides the ability to isolate large quantities of extracellular microvesicles and exosomes, particularly antigenically intact, disease-related and PS-positive exosomes such as tumor-derived exosomes, which are indistinguishable from those prepared by the current ultracentrifugation methods, the latter of which are time-consuming, cumbersome and volume-limited.

The invention is particularly suitable for purifying or isolating disease-related, viral- and tumor-derived exosomes, which express the negatively-charged phospholipid phosphatidylserine (PS), on their surface, typically in association with non-lipid membrane components, such as membrane proteins. The acetate buffers surprisingly neutralize the surface charge of the extracellular microvesicles and exosomes, thus removing them from solution, without damaging the morphological or functional properties of the resulting extracellular microvesicles and exosomes, and particularly whilst maintaining their original antigenic profile, such that they are “antigenically intact”. Such disease-related exosomes include those from cells infected with a virus or intracellular parasite or pathogen, which cause the host cell to externalize PS, and are preferably tumor-derived exosomes, which typically contain a significant amount of PS on their surface.

A broadly applicable embodiment of the invention is a method of isolating disease-related extracellular microvesicles from a biological fluid, wherein the disease-related extracellular microvesicles have negatively-charged phosphatidylserine on their surface; such a method comprising contacting a sample of the biological fluid with an acetate buffer at a pH and concentration effective to precipitate disease-related extracellular microvesicles from the biological fluid and collecting disease-related extracellular microvesicles from the precipitate, thereby isolating the disease-related extracellular microvesicles.

Such methods include those for isolating extracellular microvesicles from a virally-infected cell, comprising contacting a biological fluid that contains the viral-derived extracellular microvesicles with an acetate buffer at a pH and concentration effective to precipitate the viral-derived extracellular microvesicles from the biological fluid and collecting the viral-derived extracellular microvesicles from the precipitate, thereby isolating the viral-derived extracellular microvesicles. The viral methods preferably isolate the extracellular viral-derived microvesicles substantially free from infectious virus.

The methods of the invention further include those for isolating tumor-derived extracellular microvesicles from a biological fluid, comprising contacting a sample of the biological fluid with an acetate buffer at a pH and concentration effective to precipitate tumor-derived extracellular microvesicles from the biological fluid and collecting tumor-derived extracellular microvesicles from the precipitate, thereby isolating the tumor-derived extracellular microvesicles.

Other examples of disease-related extracellular microvesicles are those derived from a cell infected with an intracellular parasite or pathogen.

In the foregoing methods, the disease-related, viral-derived and tumor-derived extracellular microvesicles are preferably disease-related, viral-derived and tumor-derived exosomes. Preferably, the disease-related, viral-derived and tumor-derived extracellular microvesicles and exosomes are isolated without substantially damaging their morphological or functional properties or cell surface antigens. Human extracellular microvesicles, such as human tumor exosomes can be isolated by the invention.

By applying the invention to all disease-related extracellular microvesicles, such as disease-related exosomes, phosphatidylserine will preferably be present on the surface of the extracellular microvesicles, more preferably in association with non-lipid membrane components, wherein the non-lipid membrane components comprise membrane proteins. In this regard, the acetate buffer is believed to neutralize the surface charge of the phosphatidylserine on the disease-related extracellular microvesicles, thereby precipitating the disease-related extracellular microvesicles from the biological fluid.

Accordingly, the invention particularly provides methods for use with biological fluids that contain a mixed population of extracellular microvesicles comprising disease-related and normal extracellular microvesicles, wherein the methods selectively precipitate the disease-related extracellular microvesicles from the mixed population, as opposed to the normal extracellular microvesicles. Embodiments of this are methods for use with biological fluids that contain a mixed population of exosomes comprising tumor-derived and normal exosomes, wherein the methods selectively precipitate the tumor-derived exosomes from the mixed population, as opposed to the normal exosomes.

Such methods can comprise: (a) obtaining a biological fluid containing a mixed population of exosomes that includes tumor-derived exosomes and non-tumor exosomes; (b) contacting the biological fluid with an acetate buffer at a pH and concentration effective to selectively precipitate tumor-derived exosomes, but not non-tumor exosomes, from the biological fluid; (c) collecting the precipitate from step (b); wherein the precipitate selectively contains the tumor-derived exosomes; and (d) re-suspending the precipitate in a substantially acetate-free buffer at about neutral pH, thereby providing a purified population of tumor-derived exosomes essentially free from non-tumor exosomes.

In more detail, these embodiments may comprise: (a) obtaining a biological fluid containing a mixed population of exosomes that includes tumor-derived exosomes and non-tumor exosomes; (b) performing a first low-speed centrifugation on the biological fluid to provide a clarified fluid essentially free from cells, cell debris and large membrane vesicles; (c) incubating the clarified fluid with an acetate buffer at a pH and concentration effective, and for a time effective, to provide a turbid suspension comprising selectively precipitated tumor-derived exosomes, but substantially no precipitated non-tumor exosomes; (d) subjecting the turbid suspension to a low-speed centrifugation to provide a precipitate and a supernatant; wherein the precipitate selectively comprises the tumor-derived exosomes; (e) collecting the precipitate comprising the tumor-derived exosomes; (f) re-suspending the precipitate in a substantially acetate-free buffer at about neutral pH, thereby providing a purified exosome population that comprises tumor-derived exosomes and is essentially free from non-tumor exosomes; and, optionally (g) subjecting the purified exosome population to a further centrifugation to provide a pellet comprising non-exosome components and removing the pellet, thereby providing an essentially pure composition of tumor-derived exosomes that is substantially free from both non-tumor exosomes and non-exosome components.

As the invention effectively separates disease-related extracellular microvesicles from biological fluids that contain mixed populations of extracellular microvesicles, the invention further provides methods to obtain disease-related, preferably tumor-derived, extracellular microvesicles from the supernatant of diseased or tumor cells cultured in the presence of serum that contains normal extracellular microvesicles, preferably wherein the disease-related or tumor-derived extracellular microvesicles have negatively-charged phosphatidylserine on their surface. These methods comprise contacting the supernatant with an acetate buffer at a pH and concentration effective to selectively precipitate disease-related, preferably tumor-derived, extracellular microvesicles from the supernatant and collecting the precipitate, thereby obtaining disease-related, preferably tumor-derived, extracellular microvesicles from the supernatant without substantial contamination from normal extracellular microvesicles in the serum. A particular example is wherein mouse or human tumor cells are cultured in the presence of bovine or fetal bovine serum.

Another separating embodiment is a method to prepare serum that is substantially free from disease-related, preferably tumor-derived, extracellular microvesicles, wherein the disease-related or tumor-derived extracellular microvesicles have negatively-charged phosphatidylserine on their surface (“depleted serum”); the method comprising: (a) obtaining serum suspected of containing disease-related, preferably tumor-derived, extracellular microvesicles; (b) contacting the serum with an acetate buffer at a pH and concentration effective to precipitate disease-related, preferably tumor-derived, extracellular microvesicles from the serum; and (c) removing from the serum the precipitate formed in step (b), wherein the precipitate contains the disease-related, preferably tumor-derived, extracellular microvesicles, thereby providing a serum that is substantially free from disease-related, preferably tumor-derived, extracellular microvesicles.

These methods are applicable to virally-infected cells and include methods to prepare blood, serum or plasma that is substantially free from infectious virus and extracellular microvesicles derived from virally-infected cells (such as may be performed prior to a blood transfusion), comprising: (a) obtaining blood, serum or plasma suspected of containing infectious virus and extracellular microvesicles derived from virally-infected cells; (b) contacting the blood, serum or plasma with an acetate buffer at a pH and concentration effective to inactivate or precipitate the infectious virus and to precipitate the extracellular microvesicles from the blood, serum or plasma; and (c) removing from the blood, serum or plasma the precipitate formed in step (b), wherein the precipitate contains the inactivated or precipitated virus and the precipitated extracellular microvesicles, thereby providing blood, serum or plasma that is substantially free from infectious virus and extracellular microvesicles derived from virally-infected cells.

In further embodiments, the invention provides methods to detect disease-related, preferably tumor-derived, extracellular microvesicles in a clarified biological fluid, wherein the disease-related or tumor extracellular microvesicles have negatively-charged phosphatidylserine on their surface. These methods comprise contacting the clarified biological fluid with an acetate buffer at a pH and concentration effective to selectively precipitate disease-related, preferably tumor-derived, extracellular microvesicles from the clarified biological fluid and determining the presence of turbidity, as may be detected visually, in the resultant biological fluid; wherein the presence of turbidity in the resultant biological fluid indicates the detection of disease-related, preferably tumor-derived, extracellular microvesicles.

In other embodiments, the invention provides methods to diagnose an animal or patient having at least a first disease, such as cancer, characterized by the presence of disease-related extracellular microvesicles that have negatively-charged phosphatidylserine on their surface. These methods comprise detecting the presence of the disease-related, preferably tumor-derived, extracellular microvesicles in a biological fluid from the patient, thereby diagnosing the patient as having the first disease, such as cancer. Preferably, the disease-related or tumor-derived extracellular microvesicles are detected by a method comprising: (a) contacting the biological fluid with an acetate buffer at a pH and concentration effective to selectively precipitate the disease-related, preferably tumor-derived, extracellular microvesicles from the biological fluid; and (b) determining the presence of the precipitate in the resultant biological fluid; wherein the presence of the precipitate in the resultant biological fluid indicates the detection of the disease-related, preferably tumor-derived, extracellular microvesicles; and (c) optionally, wherein a first such disease is to be identified or differentiated from a second such disease, e.g., wherein cancer is to be identified or differentiated from a viral infection, the presence of the first disease is confirmed by testing for a further and/or independent biomarker or clinical sign of the first disease, such as a further and/or independent biomarker or clinical sign of cancer.

Further aspects of the invention are methods to monitor the disease burden of a patient having a first disease characterized by the amount of disease-related extracellular microvesicles that have negatively-charged phosphatidylserine on their surface, such as to monitor the tumor-burden of a cancer patient. These methods comprise measuring the amount of the disease-related, preferably tumor-derived, extracellular microvesicles in a biological fluid from the patient; wherein the amount of the disease-related, preferably tumor-derived, extracellular microvesicles is measured by a method comprising: (a) contacting the biological fluid with an acetate buffer at a pH and concentration effective to selectively precipitate the disease-related, preferably tumor-derived, extracellular microvesicles from the biological fluid; and (b) measuring the amount of the disease-related, preferably tumor-derived, extracellular microvesicles in the precipitate.

For example, methods to monitor the tumor-burden of a cancer patient comprise:

...
...