Extracellular vesicle depleted blood fractions

Abstract

Disclosed herein are human blood fractions depleted of disease-causing extracellular vesicles prepared by plasma exchange, which can be used to treat cancer. In particular, the depleted blood fraction can be used to treat KRAS mutant cancers.

Description

Technical Field

[0001] The present invention provides methods for reducing the extracellular vesicle load in a patient's plasma, as well as methods for treating diseases involving the extracellular vesicles. Background Art

[0002] Extracellular vesicles are lipid bilayer-bounded particles that are released from cells into the extracellular space by budding from the plasma membrane, or alternatively by invagination of endosomal membranes and maturation into multivesicular bodies that fuse with the plasma membrane to release their contents. Extracellular vesicles are thought to provide a means for intercellular communication and intercellular macromolecular delivery.

[0003] Due to their role in the delivery of proteins, lipids, mRNA, miRNA and DNA from one cell to another, extracellular vesicles have been implicated as contributors in the development of a variety of diseases. The packaging of extracellular vesicles appears to be indiscriminate, as such extracellular vesicles can provide for the delivery of both "good" and "bad" cargo to their target cells. Extracellular vesicles have been reported to contain many disease-related cargoes, such as:

[0004] miRNAs in the context of cancer, and

[0005] Neurodegeneration-related peptides such as Aβ, tau, prions, and α-synuclein.

[0006] Although knowledge about EVs is increasing, the means by which disease-associated agents spread between cells remains poorly understood, even if EVs are involved in such processes / transmission. Furthermore, a thorough understanding of the mechanisms by which specific cargoes (proteins, RNAs, etc.) are sorted into specific EVs remains elusive. Thus, conventional drug targeting of EVs continues to be challenging due to the structural heterogeneity of the various different types of EVs, as well as the above-mentioned knowledge gaps regarding the molecular mechanisms of EV packaging and diffusion.

[0007] Therefore, there remains a need for therapies that can consistently and reproducibly reduce the "bad" extracellular vesicle load in patients, thereby providing a reliable means to downregulate the spread of extracellular vesicles and the subsequent pathologies / diseases associated therewith. Summary of the invention

[0008] The words "include / comprise" and the words "have / contain" when used herein with reference to the present invention, are used to specify the presence of stated features, integers, steps or components, but do not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

[0009] It should be understood by those skilled in the art that the specific embodiments disclosed herein should not be understood in isolation, and this specification is intended to understand the disclosed embodiments in combination with each other, rather than individually. Therefore, each embodiment can be used as a basis for modifying or limiting other embodiments disclosed herein.

[0010] Concentration, amount and other numerical data can be represented or presented in range form at this.It should be understood that such range form is used only for convenience and simplicity, and therefore should be flexibly interpreted as not only including the numerical value clearly stated as the limit of range, but also including all single numerical values ​​or sub-ranges contained in the range, as each numerical value and sub-range are clearly stated.As an illustration, the numerical range of "10 to 100" should be interpreted as not only including the value of 10 to 100 clearly stated, but also including the single value and sub-range in the indicated range.Therefore, included in this numerical range is a single value, such as 10,11,12,13 ... 97,98,99,100 and sub-ranges such as 10 to 40,25 to 40 and 50 to 60 etc.This same principle is applicable to the scope of only listing a numerical value, such as "at least 10".In addition, this explanation should be applicable, regardless of the width of the scope or the described characteristics.

[0011] Treatment of the present invention

[0012] In a first aspect, the present invention provides a human blood fraction depleted of extracellular vesicles for use in treating cancer, wherein blood obtained from a patient diagnosed with cancer is subjected to plasma exchange to thereby deplete the extracellular vesicle content prior to re-administration to the patient.

[0013] In a second aspect, the present invention provides the use of plasmapheresis for removing extracellular vesicles from a patient in need thereof, wherein the patient is a patient diagnosed with cancer, and further, wherein no binding agent that specifically binds to extracellular vesicles is used during the plasmapheresis process.

[0014] "Blood fraction" means any derivative of whole blood that has been treated or fractionated to alter the native concentration of red blood cells, white blood cells, platelets, or plasma in whole blood. In some embodiments, "blood fraction" should be interpreted to mean a derivative of whole blood that has been treated to reduce or alter the individual plasma components. Suitable methods for producing blood fractions from whole blood include, but are not limited to, plasma separation.

[0015] "Depletion" in this specification means depletion of disease-causing extracellular vesicles compared to blood samples from patients who have not undergone plasma exchange procedures. In some embodiments, depletion can be equivalent to a reduction of more than 90% in disease-causing extracellular vesicles compared to blood samples from patients who have not undergone plasma exchange procedures. For example, depletion can be equivalent to a reduction of more than 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10% in disease-causing extracellular vesicles compared to blood samples from patients who have not undergone plasma exchange procedures. Disease-causing extracellular vesicles can be identified by targeting a specific set of biomarkers outlined in the specific embodiments of the present invention and assessing the content of extracellular vesicles. These techniques fall within the knowledge of those skilled in the art.

[0016] In some embodiments, prior to treatment with the extracellular vesicle-depleted human blood fraction of the invention, a patient diagnosed with cancer has been evaluated to determine the presence or absence of disease-causing extracellular vesicles in their plasma. For example, in certain embodiments, a patient may have tested positive for disease-causing extracellular vesicles prior to treatment with the invention.

[0017] In other embodiments, prior to treatment with the extracellular vesicle-depleted human blood fraction of the invention, a patient diagnosed with cancer may be unresponsive / refractory to standard of care / first-line pharmacological treatment (for said cancer) using small molecule drugs / biological drugs. The present invention contemplates that the innovative treatments disclosed herein may be given complementary to standard of care treatments, rather than the two being mutually exclusive.

[0018] As used herein, the term "plasmapheresis" means a procedure in which a patient's blood is passed through a device, such as a plasma separation device, and the plasma components filtered through the device are removed and discarded. Red blood cells and other unfiltered blood fractions are reinfused back into the patient, optionally along with a replacement fluid (e.g., fresh frozen plasma or albumin). Generally, the efficacy of plasmapheresis is proportional to the volume of plasma removed relative to the patient's total plasma volume.

[0019] Advantageously, with respect to both aspects of the invention, subjecting a patient's blood to plasma exchange without the incorporation of any specific extracellular vesicle binding agent (e.g., antibodies, antibody fragments, aptamers, etc.) avoids problems associated with non-reproducible binding of structurally heterogeneous extracellular vesicles. In addition, avoiding the need for expensive binding agents greatly reduces the cost of any treatment for patients and healthcare providers. As used herein, "extracellular vesicle binding agent" should be interpreted to mean a material that is capable of selectively binding to extracellular vesicles over other plasma components, and includes antibodies, antibody fragments, other binding proteins, aptamers, etc.

[0020] For the avoidance of any doubt, references to plasmapheresis in this specification do not include the use of any selective extracellular vesicle binding agent as part of the process.

[0021] "Extracellular vesicle" in this specification refers to the particle defined by lipid bilayer, which comprises macromolecules such as proteins, lipids, nucleic acids and combinations thereof that are released from cells into the intracellular space and have a diameter of 20-200nm determined by low temperature transmission electron microscopy (CRYO-TEM) analysis at -179 ° C and an accelerating voltage of 200kV. In one embodiment, the extracellular vesicle can have a diameter of 30-200nm, a diameter of 30-150nm, a diameter of 30-120nm, a diameter of 30-100nm, a diameter of 40-200nm, a diameter of 40-150nm, a diameter of 40-120nm or a diameter of 40-100nm. The extracellular vesicle mentioned in this specification can be rich in transmembrane tetraproteins (tetraspanins) CD9, CD63, CD81 and combinations thereof.

[0022] The extracellular vesicles mentioned in the present specification may have a diameter of 30-200 nm, 30-150 nm, 30-120 nm or 30-100 nm as determined by cryo-TEM analysis [at -179°C and an accelerating voltage of 200 kV], and may be enriched with tetraspanins CD9, CD63, CD81 and combinations thereof. For example, the extracellular vesicles mentioned in the present specification may have a diameter of 40-200 nm, 40-150 nm, 40-120 nm or 40-100 nm as determined by cryo-TEM analysis at -179°C, and may be enriched with tetraspanins CD9, CD63, CD81 and combinations thereof.

[0023] Oncology

[0024] The depleted blood fraction of the present invention may be used to treat cancer. Similarly, with respect to the second aspect of the present invention, where the patient is a patient diagnosed with cancer, the use of plasma exchange for removing extracellular vesicles from the patient may be utilized.

[0025] For example, the cancer can be selected from the group consisting of soft tissue sarcoma, kidney cancer, liver cancer, intestinal cancer, rectal cancer, leukemia, lymphoma, brain cancer, esophageal cancer, uterine cancer, cervical cancer, bone cancer, lung cancer, bladder cancer, breast cancer, laryngeal cancer, colorectal cancer, stomach cancer, ovarian cancer, pancreatic cancer, adrenal cancer, prostate cancer, and combinations thereof.

[0026] In one embodiment, the cancer is selected from the group consisting of colorectal cancer, pancreatic cancer, and lung cancer.

[0027] In some embodiments, the cancer is refractory to chemotherapy.

[0028] In a specific embodiment, the cancer is a KRAS mutant cancer. As used herein, the term "KRAS mutant cancer" should be interpreted to mean any cancer in which the KRAS gene is mutated so that it gets stuck in the "on" position, allowing cells to grow uncontrollably, leading to proliferation of cells, cancer growth, and can ultimately cause metastasis.

[0029] In certain embodiments, the cancer may be a KRAS mutant cancer that is refractory to chemotherapy.

[0030] In one embodiment, the KRAS mutant cancer can be a KRAS mutant colorectal cancer, a KRAS mutant pancreatic cancer, and a KRAS mutant lung cancer. In some embodiments, the KRAS mutant cancer can be a metastatic cancer. For example, the KRAS mutant cancer can be a KRAS mutant metastatic colorectal cancer, a KRAS mutant metastatic pancreatic cancer, and a KRAS mutant metastatic lung cancer.

[0031] In certain embodiments, the depleted blood fractions of the invention are used to treat colorectal cancer. For example, the colorectal cancer may be metastatic colorectal cancer. In other embodiments, the colorectal cancer may be refractory to chemotherapy. In some embodiments, the colorectal cancer may be KRAS mutant metastatic colorectal cancer. For example, the colorectal cancer may be KRAS mutant metastatic colorectal cancer that is refractory to first-line chemotherapy.

[0032] It should be understood by those skilled in the art that the specific embodiments disclosed in paragraphs

[0021] -

[0027] should not be understood separately, and the present specification is intended to disclose these embodiments in combination with other embodiments rather than separately. For example, each of the embodiments disclosed in paragraphs

[0009] -

[0019] should be understood to be explicitly combined with each of the embodiments in paragraphs

[0020] -

[0027] , or any permutation and combination of two or more of the embodiments disclosed therein.

[0033] Plasma exchange volume

[0034] Typically, the patient's total blood volume is calculated according to the Nadler formula ( Nadler SB, Hidalgo JH, BlochT.Prediction of bloodvolumeinnormalhumanadults.Surgery.1962;51(2):224–32 ) calculation, which can be reproduced as follows:

[0035] Total blood volume of the patient (mL):

[0036] Male = (0.006012 x H 3 ) / (14.6x W)+604

[0037] Female = (0.005835 x H 3 ) / (15x W)+183

[0038] H = height (inches), W = weight (pounds).

[0039] Plasmapheresis procedures report the amount of blood / plasma treated based on the patient's total blood volume. Treating 1 volume is equivalent to treating the patient's total blood volume, as determined by the Nadler formula. Similarly, treating 1.5 volumes is equivalent to treating "1.5 x the patient's total blood volume," as determined by the Nadler formula. Naturally, volumes in excess of 1 result in some patients' blood volumes being treated more than once.

[0040] Plasma exchange can also be reported as plasma volume (e.g., 1 plasma volume). Considering that plasma accounts for approximately 55% of the total blood volume, the two nomenclature systems are interrelated and ultimately converge on the total blood volume calculation.

[0041] The present invention contemplates treating about 0.25 to about 2 (fold) blood volumes by plasma exchange. In some embodiments, about 0.25 to about 0.5 blood volumes can be subjected to plasma exchange. For example, about 0.3 to about 0.4 blood volumes can be subjected to plasma exchange. In one embodiment, about 0.33 blood volumes can be subjected to plasma exchange.

[0042] In other embodiments, about 0.75 to about 2 blood volumes can be subjected to plasma exchange. For example, about 1 to about 2 blood volumes can be subjected to plasma exchange. For example, about 1 to about 1.5 blood volumes can be subjected to plasma exchange. In one embodiment, about 1 blood volume can be subjected to plasma exchange.

[0043] It will be appreciated by those skilled in the art that, in addition to the Nadler formula, other less commonly used formulas and formulas centered around the mean blood volume of a specific weight range may be used to determine the total blood volume of a patient. These alternative methods are also within the scope of the present invention. For purposes of the present invention, the relevant variable is blood volume, regardless of the method used to calculate it. Slight changes in the amount of blood volume produced using different formulas will not affect the efficacy of the present invention.

[0044] In one embodiment, a patient diagnosed with cancer can have about 10% to about 95% of their plasma removed from their blood as part of a plasmapheresis procedure. In some embodiments, from about 10% to about 50% of their plasma can be removed from their blood. In other embodiments, about 10% to about 40% of their plasma can be removed from their blood. For example, about 20% to about 40% of their plasma can be removed from their blood. In yet other embodiments, about 15% to about 30% of their plasma can be removed from their blood.

[0045] In other embodiments, about 50% to about 95% of their plasma can be removed from their blood. In other embodiments, about 60% to about 95% of their plasma can be removed from their blood. For example, about 60% to about 90% of their plasma can be removed from their blood. For example, about 60% to about 85% of their plasma can be removed from their blood. In other embodiments, about 60% to about 80% of their plasma can be removed from their blood. In certain embodiments, about 60% to about 75% of their plasma can be removed from their blood.

[0046] In some embodiments, the patient is subjected to multiple repetitions of plasma exchange. For example, each repetition of plasma exchange can occur within 1 to 45 days of the previous repetition. In other embodiments, each repetition of plasma exchange can occur within 1 to 30 days of the previous repetition. For example, each repetition of plasma exchange can occur within 1 to 15 days of the previous repetition. In some embodiments, each repetition of plasma exchange can occur within 1 to 7 days of the previous repetition. In certain embodiments, each repetition of plasma exchange can occur within 1 to 3 days of the previous repetition.

[0047] It should be understood by those skilled in the art that the specific embodiments disclosed in paragraphs

[0029] -

[0037] should not be understood separately, and the present specification is intended to disclose these embodiments in combination with other embodiments rather than separately. For example, each of the embodiments disclosed in paragraphs

[0029] -

[0037] should be understood to be explicitly combined with each of the embodiments in paragraphs

[0009] -

[0028] , or any permutation and combination of two or more of the embodiments disclosed therein.

[0048] Displacement Fluid

[0049] Depending on the blood volume (and therefore the percentage of plasma removed from the patient's blood) processed by the plasma exchange procedure, the patient can receive a replacement fluid after the plasma exchange procedure to avoid hypotension and peripheral edema. Typically, larger blood volumes require replacement fluids to compensate for the plasma volume removed from the patient's blood. Suitable non-limiting examples of replacement fluids include albumin preparations, and fresh frozen plasma diluted with saline. Smaller blood / plasma volumes (e.g., those equal to the plasma donation volume) do not typically require replacement fluids.

[0050] 70% to 80% of the oncotic activity in normal human plasma is attributed to its albumin content, which is in the range of about 35-50 g / L. Therefore, the volume of albumin to be administered as a replacement fluid can be easily calculated based on the volume of blood undergoing plasma exchange / volume of plasma removed from the patient's blood.

[0051] Thus, in certain embodiments, the patient may be administered 10 g to 60 g of albumin per liter of plasma removed by the plasmapheresis procedure. For example, the patient may be administered 30 g to 50 g of albumin per liter of plasma removed by the plasmapheresis procedure.

[0052] Isotonic solutions of human serum albumin are the most common choice for plasma exchange in plasma exchange. Naturally, this replacement fluid strategy can lead to an instantaneous drop in the levels of non-albumin plasma components (including coagulation factors, immunoglobulins, transport proteins and complement components). In fact, redistribution and resynthesis keep most other plasma proteins within a satisfactory range. In some cases, if deficiencies occur, enhanced treatment with other plasma components can be performed.

[0053] It should be understood by those skilled in the art that the specific embodiments disclosed in paragraphs

[0039] -

[0042] should not be understood separately, and the present specification is intended to disclose these embodiments in combination with other embodiments rather than separately. For example, each of the embodiments disclosed in paragraphs

[0039] -

[0042] should be understood to be explicitly combined with each of the embodiments in paragraphs

[0009] -

[0038] , or any permutation and combination of two or more of the embodiments disclosed therein. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Other features and advantages of the present invention will become more apparent from the accompanying drawings, in which:

[0055] Figure 1The results of size exclusion chromatography (SEC) and flow cytometry analyses were plotted and the presence of extracellular vesicles (EVs) in plasma after standard centrifugation, total plasma exchange (TPE), and low-volume plasma exchange (LVPE) was identified;

[0056] Figure 2 is a graph showing the concentration and size of extracellular vesicles in the isolated fractions measured by nanoparticle tracking analysis;

[0057] Figure 3 The relative amounts of EV-associated proteins (including TSG101, raft proteins, and adaptor proteins) detected by Western Blot from plasma obtained by standard centrifugation, LVPE, and TPE are plotted;

[0058] Figure 4 The structure and size distribution of extracellular vesicles in plasma obtained by standard centrifugation, LVPE, and TPE using CRYO-TEM are depicted.

[0059] Figure 5 Venn diagrams of protein EVs in plasma and PDXO are shown. 5a) Venn diagram of EV proteins from metastatic CRC KRAS mutant plasma samples; 5b) Venn diagram of EV proteins identified in PDXO samples; and 5c) Venn diagram of common EV proteins from plasma and PDXO samples. DETAILED DESCRIPTION

[0060] It should be readily apparent to those skilled in the art that the embodiments disclosed below represent only generalized embodiments, and that other arrangements and methods capable of reproducing the present invention are possible and encompassed by the present invention.

[0061] Example 1 - Evaluation of the effect of plasma exchange on the depletion of extracellular vesicles (EVs) from plasma

[0062] A study was conducted to analyze the effect of plasma exchange on extracellular vesicle isolation / removal. For the study, plasma was obtained from five blood donors (n=5) using standard centrifugation, and two different blood separation / plasma separation devices.

[0063] Blood was obtained from five different human donors. From each of the five donors, two whole blood bags (approximately 250 mL each) were filled to ensure uniform sampling. Standard centrifugation [A], Spectra Apheresis System (Terumo BCT, Inc.) [B], and Autopheresis-C TMThe Plasmapheresis System (Fresenius Kabi) [C] obtains a plasma sample from a donor's blood; further details of which are outlined below. Although both blood separation systems separate plasma from blood, they utilize different mechanical principles to do so.

[0064] Plasma obtained by standard centrifugation

[0065] For donor 1, 15 mL of blood from each of blood bag I and blood bag II was pooled into a 50 mL Falcon tube. The resulting 30 mL human blood pool was then centrifuged at 500 x g for 10 min using a centrifuge 5702R (Eppendorf, Hamburg, Germany), followed by centrifugation at 2,500 x g for 15 minutes. This process was repeated for each of the remaining donors 2 to 5. On average, 14 to 16 mL of plasma was separated from the blood samples, aliquoted in cryovials and stored at -80 ° C until evaluation.

[0066] Using Spectra Plasma obtained by Apheresis System (TerumoBCT, Inc.) The SpectraOptia Apheresis System is an automated, continuous blood fractionator that uses centrifugation and optical detection to perform the plasma fractionation procedure. It is typically used for large volume fractionation (>1L), but can also be used for lower volume fractionation (<900mL).

[0067] For donor 1, 200 mL of blood from blood bag 1 was processed using the Spectra Optia Apheresis System. This process was repeated for each of the remaining donors 2 to 5. On average, 70 to 110 mL of plasma was separated from the blood samples, aliquoted in cryovials and stored at -80°C until evaluation.

[0068] Using Autopheresis-C TM Plasma obtained by Plasmapheresis System (Fresenius Kabi)

[0069] The Autopheresis-C system is a blood fractionator that uses a rotating elongated membrane combined with tangential filtration to mechanically separate plasma from blood. It is typically used for lower volume separations up to 880 mL.

[0070] For donor 1, 200 mL of blood from blood bag II was processed using the Autopheresis-C system. This process was repeated for each of the remaining donors 2 to 5. On average, 50 to 80 mL of plasma was separated from the blood samples, aliquoted in cryovials and stored at -80°C until evaluation.

[0071] Isolation and processing of EVs

[0072] To evaluate the effect of plasma exchange on extracellular vesicle isolation, extracellular vesicles from the obtained plasma samples were separated by size exclusion chromatography (SEC).

[0073] 2 ml of sample (plasma) was used to isolate extracellular vesicles. 10 ml of Sepharose TM The CL-2B column was loaded manually and once adapted to room temperature, the sample was carefully loaded onto the column. Different fractions (about 35 fractions, 500 μL each) were collected stepwise using PBS 1X as elution buffer. First, use The protein concentration of all fractions was characterized by UV-Vis spectrophotometer (Abs 280 nm).

[0074] pass The low protein fraction (i.e. lacking albumin) determined by UV-Vis spectrophotometry was analyzed by flow cytometry to identify the extracellular vesicle-enriched fraction. EVs were detected by the presence of three classical extracellular vesicle markers (tetraspanin CD63, CD9, and CD81).

[0075] The four fractions showing the highest MFI fold change were collected and identified as the extracellular vesicle enriched fraction. In addition, the four fractions with the highest protein concentration were collected and identified as the protein-rich fraction. Finally, the four fractions between the protein-rich fraction and the extracellular vesicle-rich fraction were collected and identified as the intermediate fraction. Then, the total protein concentration from the different pooled fractions was determined by bicinchoninic acid assay (MicroBCATM protein assay kit, ThermoFisher Scientific) following the manufacturer's instructions.

[0076] Flow cytometry with extracellular vesicle markers (see above) was used to identify fractions enriched in extracellular vesicles, and these fractions were analyzed by Western blotting for the presence of additional extracellular vesicle markers. Antibodies against TSG101 (ab30871, abcam), raftin-1 (ref: 610820, BD Biosciences) and adaptor protein (ab133267, abcam) were used following standard protocols.

[0077] From each of the three groups (standard centrifugation, Spectra Optia and Autopheresis-C), 20 micrograms of the extracellular vesicle enrichment pool were analyzed for size distribution, and the estimated extracellular vesicle concentration was determined using nanoparticle tracking analysis (NTA) (NanoSightNS300, Malvern Instruments Limited) following the manufacturer's instructions. The sample was diluted 1:40 with sterile and filtered PBS, and each sample was measured three times. The mean value of the three replicates was considered to be the final measurement. Different fractions (EV-rich fractions, protein-rich fractions, and intermediate fractions) of 3-5 μL volume were pooled for CRYO-TEM analysis. Size distribution was quantified by calculating the diameter of all vesicles found in the images obtained by CRYO-TEM. At least 50 vesicles were counted for each sample.

[0078] Results and Discussion

[0079] like Figure 1 As shown, SEC followed by flow cytometry facilitates the identification of extracellular vesicle-enriched fractions in plasma obtained by each of the procedures disclosed in paragraphs

[0058] -

[0062] . The results from each plasma separation procedure were comparable to fractions obtained from standard centrifugation cohorts.

[0080] Figure 1 The presence of the tetraspanins CD81, CD9, and CD63, which are representative extracellular vesicle biomarkers, was demonstrated in a similar sample range for each of the three panels (centrifugation, Spectra Optia, and Autopheresis-C).

[0081] As disclosed in paragraph

[0062] above, nanoparticle tracking analysis is used to determine the extracellular particle concentration and size distribution in the extracellular vesicle-enriched fraction from the donor sample.

[0082] Figure 2 The insets in the figure reveal that the extracellular vesicle-enriched fractions have comparable particle concentrations (particles / mL) and size distributions in plasma obtained by the following different methods: standard centrifugation ("StdC"), SpectraOptia treatment ("TPE"), and Autopheresis-C treatment ("LVPE"), respectively. The concentrations determined are typically within 10 9 -10 12 particles / mL range.

[0083] As disclosed in paragraph

[0061] above, the molecular composition of the extracellular vesicles was determined by Western blotting to further confirm that these isolated materials were in fact extracellular vesicles. The samples were compared with a positive control provided by a lysate of the SH-SY5Y cell line known to contain protein markers.

[0084] from Figure 3 It was clearly demonstrated that additional extracellular vesicle markers (TSG101, raftin-1 and adaptor proteins) were present in the extracellular vesicle enriched fractions from plasma obtained by standard centrifugation, LVPE and TPE as determined by Western blotting. The relative intensity of each biomarker in each of the standard centrifugation, TPE and LVPE groups is plotted as Figure 3 These quantities were consistent across all three of the standard centrifugation, TPE, and LVPE groups.

[0085] Different SEC fractions (EV-rich fractions, protein-rich fractions and intermediate fractions) were subsequently analyzed by CRYO-TEM microscopy. Glass samples were prepared by placing 3 μL of sample on Quantifoil1.2 / 1.3TEM grids, blotted onto a film and inserted into liquid ethane-N2 (l) in a Leica EM CPC cryostation at Centro Nacional de Biotecnologia (Madrid, Spain) and UniversitatAutonoma de Barcelona (UAB, Barcelona, ​​Spain). The grid was transferred to a 626 Gatan cryostat and kept at -179 ° C. The grid was analyzed using a Jeol JEM 2011 transmission electron microscope operating at an accelerating voltage of 200 kV. Images were recorded on a Gatan Ultrascan 2000 cooled charge-coupled device (CCD) camera using a Digital Micrograph software package (Gatan). The diameters of all vesicles determined in the image were calculated and size distribution was quantified. At least 50 vesicles were counted for each sample.

[0086] Figure 4 Results from CRYO-TEM analysis are shown and demonstrate that there is little difference in the structure and size distribution of extracellular vesicles in plasma obtained by standard centrifugation, LVPE or TPE. The majority of extracellular vesicles were determined to be in the size range of 40 nm to 120 nm.

[0087] Example 2 - Identification of proteins in EVs from metastatic KRAS mutant colorectal cancer resistant to chemotherapy.

[0088] Proteomic analysis of EVs from plasma and xenograft samples from metastatic colorectal cancer (CRC) patients with KRAS mutations and resistant to first-line chemotherapy was performed to identify potential tumor-specific EV proteins.

[0089] 2 mL of plasma from three colorectal metastatic cancer patients with KRAS mutations (KRAS mut) and resistant to first-line therapy was obtained from whole blood collected in tubes containing EDTA anticoagulant. Briefly, whole blood was centrifuged at 1500 g for 10 minutes to eliminate cells. The platelet-rich plasma fraction was collected and centrifuged at 2000 g, 4 ° C for 10 minutes to eliminate platelets. The supernatant was collected again and stored at -80 ° C until use. Prior to analysis, the samples were thawed on ice, centrifuged at 12000 g for 30 min at 4 ° C to eliminate debris and particulates and filtered through 0.22 um (Sartorious).

[0090] Patient-derived xenografts (PDX) were obtained from metastatic CRC patients with KRAS mutations and resistant to first-line chemotherapy agents from Vail d'Hebron Hospital (Barcelona, ​​Spain). CMs were obtained from patient-derived xenograft organoids (PDXO) embedded in matrigel (Corning). Briefly, 150,000 cells were mixed in 25uL droplets using a 1:1.5 ratio of matrigel: culture medium. The cell droplets were inoculated and allowed to form a gel at 37°C for 30 minutes. Afterwards, 500uL of culture medium containing the essential factors Noggin, R-spondin and Wnt3a was added to each well, and the plates were incubated at 37°C, 5% CO2. After 48 hours, CMs were collected and new culture medium was added. As the organoids grew, CMs were collected every other day. After reaching confluence, organoids were disassembled from the matrigel using cell recovery solution (Gibco), trypsinized and re-seeded under the same conditions until the collection of the desired CM volume (about 10-15 mL) was completed. CM was processed immediately after collection. In brief, CM from these cultures was collected and centrifuged at 200g for 10 min to eliminate cells. The supernatant was centrifuged at 2000g for 10 min to eliminate debris. Finally, the supernatant was centrifuged at 12000g for 30 minutes to eliminate particles, filtered through 0.22um and stored at -80°C.

[0091] Isolation of EVs

[0092] Size exclusion chromatography was performed using an Izon qeV2 commercial column containing a 35nm pore size. 2mL of sample (plasma or PDXO CM) was loaded into the column. For 10-15mL of PDXO CM, a 10KDa Amicon was used to concentrate the sample. Before use, the column was balanced with 80mL 1X PBS. The sample was loaded into the column and eluted in PBS. The fractions corresponding to the void volume (1 to 13mL) were discarded. The fractions containing EVs were collected, pooled and stored at 4°C. The remaining chromatographic fractions were discarded. The pooled fractions were loaded onto a 20% sucrose cushion and ultracentrifuged at 100 000g for 2 hours at 4°C in a SORVAL SW100+ ultracentrifuge (Thermofisher). The pellet was resuspended in PBS.

[0093] Proteomics Solutions

[0094] The samples were denatured with Laemmli buffer under reducing conditions, heated at 95°C for 10 min and loaded into a 10% polyacrylamide gel. Electrophoresis was performed at 50 V. Once the samples entered the resolving gel, electrophoresis was stopped and the gel was stained o / n with Coomassie blue. The next day, the bands containing the EV protein samples were cut into 1 mm 2 The gel slices were washed with 100% ethanol and transferred to Eppendorf tubes for digestion. The gel slices containing denatured EV-proteins were washed with 50mM ammonium bicarbonate (BA) in 50% ethanol. The first dehydration process included washing the gel with 100% ethanol for 15 minutes. 10mM DTT in 50mM BA was used for residual disulfide bond cleavage. Afterwards, the sample was carbamidomethylated for 30 minutes with 55mM iodoacetamide in 50mM BA in the dark. Then, before protein digestion with 13pg / uL trypsin, 25mM BA, 25mM BA-50% acetonitrile (Acn) were washed and the gel was dehydrated with 100% Acn. The digestion process was performed o / n at 37°C. The next day, the sample was additionally dehydrated with 100% Acn, and peptides were extracted from the gel with 0.2% trifluoroacetic acid at room temperature for 30 minutes. Finally, the sample was evaporated using SpeedVac (Thermofisher).

[0095] The lyophilized tryptic digest is resuspended in a solution comprising 3% Acn and 0.1% fluoroacetic acid, and liquid chromatography-mass spectrometry (LC-MS) is used to analyze using EVOSEP-One chromatograph and Orbitrap Eclipse Tribrid mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Xcalibur software package is used to control mass spectrometer, version 4.2.28.14 (Thermo Fisher Scientific, Bremen, Germany). EVOSEP-One system (EVosep) is used to separate peptide mixtures by online nano-flow liquid chromatography. First, sample is loaded onto capture chromatographic column (Evotip C-18, EV-2001, Evosep) with 250nL / min. Then, reverse phase analytical column (EV1106 post: 150umx150mm, 1.9um, Evosep) is used to analyze peptide mixtures. Peptide elution was performed at a flow rate of 250 nL / min using 0.1% formic acid in water (mobile phase A) and acetonitrile with 0.1% formic acid (mobile phase B) with a linear gradient of mobile phase B from 0 to 35% for 88 min. Ions were generated by applying a voltage of 2 kV to a stainless steel nanopore emitter (Proxeon, Thermo Fisher Scientific) attached to the end of the chromatographic column on a Proxeon nanospray flexible ion source. The Orbitrap Eclipse Tribrid mass spectrometer was operated using data-dependent mode. The scan cycle began with a full scan MS spectrum (from m / z 300 to 1600) obtained in the Orbitrap with a resolution of 30,000 set. When their intensity exceeded 1000 counts, the 20 strongest ions were targeted in a linear ion trap for collision-induced dissociation fragmentation, thereby dissolving single charged ions. Ion collection for both MS and MS / MS scans was achieved in a linear ion trap, and the AGC target was set to 1 x 10 6 ions for survey MS and set to 5000 ions for MS / MS scans. The maximum ion accumulation time was set to 500 and 200 ms in MS and MS / MS modes, respectively. The standardized collision energy was set to 35% and one microscan was obtained for each spectrum. Ions subjected to MS / MS with a relative mass window of 10 ppm were excluded from further sequencing for 20 s. A window of 20 ppm and a separation width of 2 Da were defined for all precursor masses. The lock mass option (m / z 445.120024) for survey scans was enabled in the Orbitrap measurement to improve mass accuracy.

[0096] Proteome data were analyzed using Proteome Discoverer v.2.1 software (Thermo Fisher Scientific). Protein identification was performed using the SwissProt database (2018_11, taxonomy limited to human proteins, 20,413 sequences) using Mascot v.2.5 (Matrix Science, London, UK). MS / MS spectra were searched using a 10 ppm precursor mass tolerance, the fragment tolerance was set to 0.7 Da, trypsin specificity was controlled by setting a maximum of 2 missing cuts, cysteine ​​carbamidomethylation was set as a fixed peptide modification, and methionine oxidation was set as a variable peptide modification.

[0097] The protein identification files (DAT files) produced from Mascot are then loaded into Scaffold software (version 3.00.07; Proteome software, Inc., Portland, OR, USA), for each LC-MS / MS run / sample, a non-redundant list of identified proteins is generated. Whenever a PeptideProphet probability greater than 95% is achieved, peptide identification is verified. Proteins identified with a probability higher than 95% and comprising at least two verified MS / MS spectra are accepted for further analysis. By using described PeptideProphet and ProteinProphet tools, an erroneous protein discovery rate (FDR) lower than 1.0% as estimated by database search is achieved. Using these filters, a protein data set file is generated by "Scaffold software" using "spectral counts" (SpC) as quantitative values.

[0098] Bioinformatics analysis

[0099] Samples were normalized by total counts. Datasets were compared using Venn diagrams (http: / / www.interactivenn.net / , DOI: https: / / doi.org / 10.1186 / s12859-015-0611-3).

[0100] result

[0101] Proteomic data analysis was performed including only proteins identified with at least two spectral counts across datasets containing EVs from both plasma and PDXO.

[0102] Figure 5 A Venn diagram of protein EVs in plasma and PDXO is shown. Figure 5 a) shows a Venn diagram of EV proteins from three metastatic CRC KRASmut plasma samples. Figure 5 b) A Venn diagram showing the EV proteins identified in the three PDXOs.

[0103] For plasma EVs, a non-redundant list of 650 proteins was identified from the 558, 281, and 382 EV proteins identified by each patient. From those 650 EV proteins, 211 proteins were common to all three patients (see Figure 5 a).

[0104] A non-redundant list of 1035 EV proteins was identified in the PDXO-EV samples. A total of 686 proteins were shared by the three PDXO-EVs ( Figure 5 b).

[0105] To identify potential proteins associated with CRC, plasma and PDXO EV proteins were compared to assess overlap. Proteins commonly present in all three samples (plasma and PDXO) were considered for this analysis. From the 211 (plasma) and 686 (PDXO) EV proteins, 134 proteins were present in both groups ( Figure 5 c) Thus, the most abundant plasma proteins that can be found in healthy samples, such as albumin, acute phase proteins, apolipoproteins, immunoglobulins and keratins, were removed from the list. Finally, a total of 97 proteins were found to be present in both samples and therefore potentially associated with EVs from CRC patients.

[0106] The results presented here show the identification of specific proteins in plasma-derived extracellular vesicles from tumor-derived plasma samples, particularly from metastatic colorectal cancer (CRC) KRAS mutant plasma samples. Given this result, when compared to PDXO samples, it can be inferred that these enriched proteins can clearly be derived from extracellular vesicles shed by colorectal tumor cells.

[0107] Thus, the results shown here suggest that by removing circulating EVs in cancer patients, EVs shed by tumors will also be removed. Therefore, removal of EVs in cancer patients by plasmapheresis would represent an effective and reliable means for cancer treatment.

Claims

1. A human blood fraction depleted of extracellular vesicles for use in the treatment of cancer, wherein blood obtained from a patient diagnosed with said cancer is subjected to plasma exchange to thereby deplete the extracellular vesicle content prior to re-administration to said patient.

2. The depleted blood fraction of claim 1, wherein about 0.25 to about 2 blood volumes of the patient diagnosed with the cancer are subjected to plasma exchange.

3. The depleted blood fraction according to any one of the preceding claims, wherein about 0.3 to about 0.4 blood volume of the patient diagnosed with the cancer is subjected to plasma exchange.

4. The depleted blood fraction according to any one of claims 1 to 2, wherein about 1 to about 1.5 blood volumes of the patient diagnosed with the cancer are subjected to plasma exchange.

5. The depleted blood fraction of any one of claims 1 to 2, wherein the patient diagnosed with the cancer has about 10% to about 95% of his plasma removed from his blood as part of a plasmapheresis procedure.

6. The depleted blood fraction of claim 5, wherein the patient diagnosed with the cancer has had about 10% to about 40% of his plasma removed from his blood as part of a plasmapheresis procedure.

7. The depleted blood fraction of claim 5, wherein the patient diagnosed with the cancer has had about 60% to about 95% of his plasma removed from his blood as part of a plasmapheresis procedure.

8. The depleted blood fraction according to any one of the preceding claims, wherein the patient is subjected to multiple repetitions of plasma exchange, each repetition of plasma exchange occurring within 1 to 45 days of the previous repetition of plasma exchange.

9. The depleted blood fraction according to claim 8, wherein each repetition of plasma exchange occurs within 1 to 7 days of the previous repetition of plasma exchange.

10. The depleted blood fraction of any of the preceding claims, wherein the cancer is selected from the group consisting of soft tissue sarcoma, kidney cancer, liver cancer, intestinal cancer, rectal cancer, leukemia, lymphoma, brain cancer, esophageal cancer, uterine cancer, cervical cancer, bone cancer, lung cancer, bladder cancer, breast cancer, laryngeal cancer, colorectal cancer, gastric cancer, ovarian cancer, pancreatic cancer, adrenal cancer, prostate cancer, and combinations thereof.

11. The depleted blood fraction according to any one of the preceding claims, wherein the cancer is a KRAS mutant cancer.

12. The depleted blood fraction of claim 11, wherein the KRAS mutant cancer is a cancer refractory to chemotherapy treatment.

13. The depleted blood fraction of claim 11 or 12, wherein the KRAS mutant cancer is a metastatic cancer.

14. Use of plasmapheresis for removing extracellular vesicles from a patient in need thereof, wherein The patient is a patient diagnosed with cancer, and Further wherein no binding agent that specifically binds to said extracellular vesicles is used during the plasma exchange process.

15. The use according to claim 14, wherein about 0.25 to about 2 blood volumes of the patient diagnosed with cancer are subjected to plasma exchange.

16. Use according to claims 14 to 15, wherein about 0.3 to about 0.4 blood volume of the patient diagnosed with cancer is subjected to plasma exchange.

17. The use according to claims 14 to 15, wherein about 1 to about 1.5 blood volumes of the patient diagnosed with cancer are subjected to plasma exchange.

18. The use according to claims 14 to 15, wherein the patient diagnosed with cancer has about 10% to about 95% of his plasma removed from his blood as part of a plasmapheresis procedure.

19. The use according to claim 18, wherein the patient diagnosed with cancer has about 10% to about 40% of his plasma removed from his blood as part of a plasmapheresis procedure.

20. The use of claim 18, wherein the patient diagnosed with cancer has about 60% to about 95% of his plasma removed from his blood as part of a plasmapheresis procedure.

21. The use according to claims 14 to 20, wherein the patient is subjected to multiple repetitions of plasma exchange, each repetition of plasma exchange occurring within 1 to 45 days of the previous repetition of plasma exchange.

22. The use according to claim 21, wherein each repetition of plasma exchange occurs within 1 to 7 days of the previous repetition of plasma exchange.

23. The use according to claims 14 to 22, wherein the cancer is selected from the group consisting of soft tissue sarcoma, kidney cancer, liver cancer, intestinal cancer, rectal cancer, leukemia, lymphoma, brain cancer, esophageal cancer, uterine cancer, cervical cancer, bone cancer, lung cancer, bladder cancer, breast cancer, laryngeal cancer, colorectal cancer, gastric cancer, ovarian cancer, pancreatic cancer, adrenal cancer, prostate cancer and combinations thereof.

24. The use according to claims 14 to 23, wherein the cancer is a KRAS mutant cancer.

25. The use according to claim 24, wherein the KRAS mutant cancer is a cancer refractory to chemotherapy treatment.

26. The use according to claim 24 or 25, wherein the KRAS mutant cancer is a metastatic cancer.

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