BR112021007508A2 - method for engineering a biological target, composition, cell activation method and microfluidic devices - Google Patents

Abstract

METHOD FOR ENGINEERING MODIFIED A BIOLOGICAL TARGET, COMPOSITION, CELL ACTIVATION METHOD AND MICROFLUID DEVICES. Methods for producing engineered exosomes and other vesicle-like biological targets in a microfluidic device, including allowing a target vesicle-like structure to react and bind to immunomagnetic particles; capture the immunomagnetic particle/vesicle complex by applying a magnetic field; further engineering the captured vesicles by surface modification with additional active portions or internal loading with active agents; and releasing the engineered vesicle-like structures, such as photolytically cleaving a bond between the particle and the engineered vesicle-like structures, thereby releasing intact vesicle-like structures that can act as delivery vehicles for therapeutic treatments.

Description

"METHOD FOR ENGINEERING MODIFIED A BIOLOGICAL TARGET, COMPOSITION, CELL ACTIVATION METHOD AND MICROFLUID DEVICES" CROSS REFERENCE TO RELATED ORDERS

[001] This application claims priority benefit of US Provisional Patent Application Serial No. 62/748,470, filed October 21, 2018, entitled CAPTURE, LOAD AND PHOTO-

MICROFLUID RELEASE ON VESICULAR DEMAND

EXTRACELLULARS AND EXOSOMES AS A VACCINE DELIVERY PLATFORM, incorporated by reference in its entirety herein.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[002] This invention was made with government support in 2017-67021-26600 awarded by the USDA National Institute of Food and Agriculture and GM103638 awarded by the National Institutes of Health. The government has certain rights in the invention.

SEQUENCE LISTING

[003] The following order contains a string listing in computer-readable format (CRF), submitted as an ASCII-formatted text file titled "Sequence_Listing", created October 21, 2019, as 12 KB. The content of the CRF is incorporated herein by reference.

BACKGROUND OF THE INVENTION FIELD OF INVENTION

[004] The disclosure generally refers to microfluidic devices and methods for collecting intact carriers or delivery vehicles for delivering bioactive therapeutics such as peptides or proteins, nucleotides and other active agents (eg, chemicals and drugs).

DESCRIPTION OF RELATED ART

[005] Among all deliverable cells and nanoparticles,

living cell-derived extracellular vesicles, especially exosomes in the nano-size range of 30 ~ 150 nm, have shown important roles in intercellular communications in recent decades. Exosomes derived from immune system cells have been well documented in regulating immune stimulation or suppression, leading to inflammatory, autoimmune, and infectious pathology. The formation of exosomes begins with the creation of endosomes as intracellular vesicles. Exosomes are different from other membrane-derived microvesicles in that they originate from multivesicular bodies (MVBs) for cell secretion. Therefore, exosomes contain specific proteins and nucleic acids and represent their mother cell status and functions at the time of formation in the parent cells. Among many subtypes of exosomes, immunogenic exosomes with an intrinsic payload of class I and II MHC molecules and other costimulatory molecules are capable of mediating immune responses, which opens up opportunities for the development of new delivery platforms that can be used to cancer vaccines, immunotherapy delivery and other delivery associated with in vivo transport.

[006] Compared to other nano-sized delivery systems such as lipids, polymers, gold and silica material, exosomes are derived from living cells, highly biocompatible nano-carriers with intrinsic payload and exhibit much stronger flexibility in loading of desired antigens for effective delivery. Exosomes also eliminate allergen responses without worrying about carrying virulent factors and prevent degradation or loss during delivery. However, the development of vaccines based on exosomes is hampered by substantial technical difficulties in obtaining pure immunogenic exosomes. The different subtypes of exosomes can confound research into the differentiation of different cellular messages. On the other hand, molecular engineering of exosomes through the membrane surface or internal charge may provide an unexplored source for the development of new antigenic exosomes.

[007] Bioengineered-modified exosomes as emerging delivery vehicles have gained substantial attention in the development of a new generation of cancer vaccines, including the recent phase II trial using IFN-DC-derived exosomes loaded with MHC I-restricted cancer antigens /II to promote immune responses based on T cells and natural killer (NK) cells in patients with non-small cell lung cancer. Unfortunately, current exosome engineering approaches, such as transfection or extrusion of parental cells and membrane permeabilization of secreted exosomes, suffer from low yields, low purity and time-consuming operations. There is a need for exosome production methods to solve this bottleneck problem. Due to the intrinsic characteristics of automation and high-efficiency mass transport, microfluidic systems overcome many disadvantages of benchtop systems and provide superior performance in isolation, detection and molecular profiling of exosomes. However, molecular engineering of exosomes using a microfluidic platform has not been explored. Currently, the most reported work on processing exosomes is of poor quality or bonded to solid surfaces/particles, and they are unable to remain intact for further therapeutic preparations.

BRIEF DESCRIPTION OF THE INVENTION

[008] Microfluidic methods and devices are described in this document to design a variety of biological carriers or delivery vehicles, such as cells, extracellular vesicles and exosomes and membranes or lipid particles and polymer particles. These carriers can be captured using the inventive methods and devices, loaded with active agents (either surface-modified or encapsulated) and then released as intact carriers designed to deliver a variety of therapeutic compounds and bioactive agents. Engineered carriers can be used for diagnostics, prognostics, supplemental assays, pharmaceuticals and therapeutics, immunotherapy and vaccine delivery and tissue delivery, and other uses associated with the in vivo delivery of active agents. The embodiments described herein are exemplified with respect to exosomes. However, it will be appreciated that exosomes represent a particularly challenging biological target, so it is anticipated that the platform can be applied to other similar structures – vesicular or vesicle-like structures characterized by a liquid nucleus and membrane or bilayer – including cells ( including T cells), microsomes and the like. These biological targets, which are then engineered into transporters or delivery vehicles, can also be characterized as nano-carriers.

[009] Provided here are microfluidic analytical devices and the method of on-demand capture, loading and photo-release of engineered intact nano-carriers. Microfluidic devices can allow real-time collection and antigenic modification of extracellular vesicles, particularly exosomes, with subsequent release of intact exosomes downstream on demand. Also disclosed are magnetic nanoparticles functionalized with photocleavable affinity probes (active fractions) to capture and release positive MHC-I exosomes on demand via a light trigger. The affinity probe can include an antigenic peptide, antibody, aptamer, nanobody, and other affinity-based probes. Photo-release of the modified/charged exosomes in microfluidic devices can be well spatially and temporally controlled with 95% or more efficiency.

Such a functional flow-line microfluidic cell culture system allows for antigenic engineering of exosomes, either by mediating the growth of their parental cells using stimuli, or by direct molecular engineering on the surface of the produced exosomes.

Heterogeneity of exosome subtypes was found in the same population of parental cells.

Subtypes released from exosomes contain distinct molecular and biological properties for different cellular regulation.

The disclosed methods can capture, carry and release specific exosomal subtypes with more targeted therapeutic functions.

Carriers that can be used in this disclosed method for capture, loading and release include cells,

extracellular vesicles and exosomes, and membrane particles or lipids and polymer particles, which can encapsulate drugs (small molecule compounds), genes, and bioactive therapeutics.

Proof of concept with various tumor antigenic peptides (eg gp-100, MAGE-A3 and MART-

1) which are commonly used in the development of cancer vaccines,

but difficult to manage due to degradation, have been demonstrated here.

Microfluidic devices show high efficiency in engineering immunogenic exosomes (MHC I+), while photo-releasing intact functional exosomes downstream.

Cellular uptake of engineered exosomes by antigen-presenting cells was also demonstrated, which exhibited greatly improved internalization capability compared to unengineered exosomes.

In particular, engineered exosomes show a significantly higher activation rate (at least

30%) for activating T cells challenging purified CD8 T cells from the spleen of 2 Pmel1 transgenic mice, than unengineered exosomes.

We also evaluated the potency of immunogenic exosomes engineered with antimicrobial peptide to stimulate T cells ex vivo using transgenic mice for the treatment of bovine respiratory syncytial virus (BRSV) infections. Exosomes engineered with the BRSV targeting peptide (Peptide 4: peptide M187-195 NAITNAKII, SEQ ID NO: 4) have the ability to activate BRSV M-specific T cells in the presence of activated dendritic cells.

[0010] Consequently, the engineered exosomes are viable and functional for application in immunotherapy against cancer and vaccination for infectious diseases. The easy and cost-effective microfluidic platform for the production of engineered-modified vesicles not only provides a strategy that allows the high-efficiency production of enriched and purified therapeutic vesicles, but also serves as a research tool for understanding the roles of exosomes modified by peptide engineering variables in anti-tumor immune responses, cancer immunotherapy and vaccination for the treatment of infections.

[0011] In specific aspects, the microfluidic devices disclosed in this document may comprise a cell culture chamber sized to maintain biological material in a three-dimensional configuration; a mixing channel fluidly connected to the cell culture chamber and comprising a plurality of sample inlet channels disposed along the mixing channel, wherein the ratio of a cell culture chamber width to the largest cross-sectional dimension of the mix channel is at least 5:1; an isolation channel defining a path for fluid flow from the mixing channel to an isolation outlet; and a collection chamber fluidly connected to the isolation outlet and comprising a magnet operatively coupled to the collection chamber to produce a magnetic field within the collection chamber.

[0012] In other aspects, microfluidic devices may comprise a cell culture chamber comprising a cell culture inlet and a cell culture outlet; a fluid inlet and a particle inlet, wherein the cell culture outlet, the fluid inlet, and the particle inlet fluidly converge at a mixing intersection; a mixing channel fluidly connected to the mixing intersection and defining a path for fluid flow from the mixing intersection to a mixing outlet, in which the ratio of a cell culture chamber width to the largest cross-sectional dimension of the mix channel is at least 5:1; and a collection chamber fluidly connected to the mixing outlet and comprising a magnet operatively coupled to the collection chamber to produce a magnetic field within the collection chamber. In these aspects, the mixing channel may comprise an isolation channel disposed between the mixing intersection and the mixing outlet.

[0013] The isolation channel in microfluidic devices may have a geometry to induce turbulent flow so as to mix fluids flowing in the device. For example, the isolation channel in microfluidic devices can have a serpentine geometry. The isolation channel may further include one or more channel constriction domains that decrease in width to produce a local vortex flow profile. In certain embodiments, the isolation channel may comprise a plurality of channel constrain domains, preferably at least 5 channel constrain domains.

[0014] As described in this document, microfluidic devices comprise a cell chamber and a mixing channel. The cell chamber and mixing channel can have a height and a width. The height and width of the mixing channel can each be at least 50 microns, preferably between 50 and 500 microns. The ratio of the cell culture chamber width to the largest cross-sectional dimension of the mixing channel can be from 5:1 to 500:1, from 5:1 to 200:1, from 5:1 to 100:1, from 5:1 to 20:1, preferably from 6:1 to 12:1. The cell culture chamber can have a volume of about 200 microliters or more, preferably about 200 microliters to about 1 milliliter.

[0015] The microfluidic devices disclosed in this document may further comprise a pump operatively coupled to the device.

[0016] The order also disclosed the method of using the device to capture a target in an on-demand sample, loading and photo-release solution can be used to collect intact delivery carriers. These transporters can be cells, extracellular vesicles and exosomes, and membrane particles or lipid and polymer particles, which can encapsulate bioactive drugs, genes and therapeutics. Specifically, this integrated, continuous method of capture, loading and photo-release can produce the engineered exosomes in a microfluidic device. The methods comprise introducing a biological sample containing exosomes (or other target) into a mixing channel and mixing the exosomes with immunomagnetic particles and a wash buffer to form a mixture; allowing exosomes to react and bind by affinity to immunomagnetic particles; and collecting exosomes bound to immunomagnetic particles by applying a magnetic field inside a collection chamber. The method for producing the engineered exosomes may include introducing cells into a microfluidic device's cell culture chamber and first culturing the cells under conditions that allow the release of exosomes. This can be done in-line, on the same microfluidic device used for capture and loading.

[0017] The cells from which exosomes are released can be selected from dendritic cells, stem cells, immune system cells, megakaryocyte progenitor cells, macrophages or other living cells.

The immunomagnetic particles attached to exosomes may comprise a magnetic particle attached to an affinity probe to capture exosomes by means of a moiety comprising a photocleavable linker. As described herein, the affinity probe to capture exosomes may include an antigenic peptide, antibody, aptamer or antigenic epitope thereof to capture the exosomes. Suitable examples of antigen peptides include MAGE-A3, gp-100, HER-2, p53, PSA-1 or MART-1. The moiety comprising the photocleavable linker may include biotin bound to the immunomagnetic particle and linked at the other end via a photocleavable linker to the affinity probe. The affinity probe targets surface proteins on the target (eg immunostimulatory molecules or markers). Suitable immunostimulatory molecules include an MHC class I molecule, an MHC class II molecule, an interleukin, TNFα, IFNγ, RANTES, G-CSF, M-CSF, IFNα, CTAPIII, ENA-78, GRO, I-309 , PF-4, IP-10, LD-78, MGSA, MIP-1α, MIP-1β and combinations thereof.

Preferably, the affinity probe is itself an antigenic portion (peptide) that preferentially binds to a surface protein on the target.

[0019] After mixing, the immunomagnetic particles capture/bind exosomes (or other target) in the sample. Immunomagnetic particles are immobilized on the device, for example, using a magnet positioned adjacent to a collection chamber. The immobilized bead/exosome complexes are then washed and incubated with a buffer solution containing active fractions for surface loading onto captured exosomes or internal encapsulation, as described in more detail below. The methods may further comprise photolytic cleavage of the captured modified exosomes from the immunomagnetic particles, releasing intact engineered modified exosomes that comprise the active agent (peptide antigen or antigenic epitope thereof).

[0020] Methods to produce the engineered biological targets such as exosomes can be performed using the microfluidic devices disclosed herein. In some embodiments, the methods are performed in real time. In one or more embodiments, the methods are in-line flows (also known as "continuous"), so that the capture, loading, and release of the biological target occur in the same device/container (i.e., without having transfer or move between vessels or reaction tubes, but in-line along a microfluidic channel and in-line engineering capture/modification chamber). Thus, each of the steps can be carried out consecutively, one after the other, using the various inputs that converge in a single microfluidic channel and, preferably, substantially immediately one after the other. In other words, the method involves loading the immunomagnetic beads, followed immediately or almost simultaneously with loading the sample, followed by loading the active agents to be attached or loaded into the captured target. Each component loaded into the microfluidic device converges from its respective inlets into a single microfluidic channel, followed by “automatic” retention in the capture chamber/engineered modification downstream of the inlet by the magnet positioned adjacent to the chamber. As the fluid mixture flows through the channel and then through the chamber, the respective reactions take place in real time (ie capture and loading). Applying light to the chamber can then release the engineered target. It will be appreciated that the in-line flow process is much faster than traditional bench top methods. Preferably, the sample/ball loading process at the respective inlets to collect the release at the outlet, the engineered modified target can be completed in approximately 2 hours in total, more preferably in approximately 90 minutes, and most preferably in approximately 1 hour .

[0021] Compositions comprising engineered exosomes, particularly complexes of immunogenic exosomes are also disclosed. The immunogenic exosome complex may comprise an antigenic peptide or its antigenic epitope conjugated to a surface of an exosome, wherein the immunogenic exosome complex activates the T cell by at least 30% compared to a native exosome. Antigenic loading of such antigenic peptide or antigenic epitopes can be performed after mixing and capturing exosomes to completely coat the exosome surface with antigenic peptides. Compositions can be prepared using the methods disclosed herein. Therefore, the immunogenic exosome complex prepared by a process comprising introducing cells into a cell culture chamber of a microfluidic device; culturing the cells under conditions that allow the release of engineered exosomes; introducing the engineered exosomes into a mixing channel and mixing the engineered exosomes with immunomagnetic particles and a wash buffer to form a mixture for capturing/binding exosomes; then, the application of a magnetic field inside a collection chamber to collect the isolated exosomes, allowing it to react with the loading buffer containing antigenic peptides to form a complex of immunogenic exosomes; and the application of ultraviolet light to break the photo-cleavable ligand to collect the immunogenic exosome complex at the exit of the microfluidic device.

[0022] Pharmaceutical compositions comprising the immunogenic exosome complex are also disclosed. Loading targets can be drugs, genes and bioactive therapeutics. Methods of treating diseases in a subject comprising administering to the subject a pharmaceutical composition comprising an immunogenic exosome complex are disclosed. In certain embodiments, the disease can be an infection. In some instances, the disease could be cancer.

When the disease to be treated is cancer, methods may also include administering a chemotherapeutic agent.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The patent file or application contains at least one drawing executed in color. Copies of this patent publication or patent application with color drawing(s) will be provided by the Institute upon request and payment of the necessary fee.

[0024] Figure 1 illustrates (A) immunomagnetic beads mixed with a sample solution, (B) capturing targets in a sample solution using immunomagnetic beads; and (C) loading antigenic or active agents onto or into the captured target.

[0025] Figure 2A illustrates (A) application of light or activating radiation to the sphere/target complex and (B) photorelease of an engineered (transported) modified target surface with active agents or antigenic fractions.

[0026] Figure 2B illustrates (A) application of light or activating radiation to the sphere/target complex, and (B) photorelease of an engineered modified target (carrier) loaded with active agents within it.

[0027] Figure 3 is an illustration of a process overview for a 3D-printed molded PDMS microfluidic culture chip for in-line flow engineering of antigenic exosomes employed in activating antitumor responses.

[0028] Figure 4 illustrates an embodiment of a microfluidic device.

[0029] Figure 5 shows images of (a) a microfluidic channel and flow; (b) mixing with microspheres; (d) cell morphology; and (e) SEM image of released exosomes.

[0030] Figure 6 illustrates an embodiment of a microfluidic device.

[0031] Figure 7A shows an illustration of on-demand immunomagnetic capture and photo-release of MHC-I positive immunogenic exosomes.

[0032] Figure 7B shows the characterization of three tumor targeting peptide antigens conjugated to photo-cleavable immunomagnetic beads for binding and photo-release of fluorescently labeled immunogenic exosomes. The MHC-I antibody is used as a positive control to compare the binding strength between MHC-I positive exosomes and tumor targeting peptides.

[0033] Figure 8A shows the characterization of on-demand photo-release performance of exosomes captured from immunomagnetic capture beads. The positive control is a fluorescently labeled antibody captured by photo-release immunomagnetic beads. The negative control is the immunomagnetic beads without a photocleavable linker.

[0034] Figure 8B shows the SEM image of a surface of photo-release immunomagnetic beads captured with exosomes.

Exosome particles were seen to be cup-shaped due to sample preparation in vacuum.

[0035] Figure 8C shows the SEM image of the surface of photo-release immunomagnetic beads after photocleavage.

[0036] Figure 8D shows the characterization of the influence of UV exposure time on photo-cleavage efficiency.

[0037] Figure 8E shows the tracking analysis of exosomes size distribution nanoparticles between engineered exosomes and unengineered exosomes.

[0038] Figure 9A shows DC uptake confocal microscope images of Tumor Targeting Antigen Peptide (TTA), gp-100 surface-engineered exosomes, compared to unengineered exosomes. The image was taken every hour to track the uptake of exosomes labeled with green fluorescence by DCs (cell nuclei were stained with DAPI).

[0039] Figure 9B shows cytokine IFN-γ release from cultured DCs measured by ELISA for monitoring for 48 hours, compared between unengineered exosomes and exosomes engineered with gp-100.

[0040] Figure 10A represents representative flow graphs of wells containing T cells + JAWS cells activated with increasing concentrations of the exosomes engineered with gp100.

[0041] Figure 10B represents the cumulative data from all three culture conditions showing the rate of division of CD8+ T cells under stimulation. Results are representative of 2 independent experiments with duplicate wells for each culture condition.

[0042] Figure 11 depicts flow cytometry graphs of wells containing T cells and activated JAWS cells with increasing concentrations of exosomes engineered with bovine respiratory syncytial virus (BRSV) antimicrobial peptide to represent immunogenic potency.

[0043] Figure 12A illustrates a 3D printing approach for 3D mold production integrated with downstream cell culture and exosome isolation, surface engineering, and on-demand photo-release.

[0044] Figure 12B shows the results of replicating the PDMS microfluidic device.

[0045] Figure 13 shows the results of the investigation of the side effect of exposure to ultraviolet rays on the molecular content of the exosome in terms of proteins, DNAs and RNAs.

[0046] Figure 14 shows the culture of dendritic monocytes under different conditions of stimulation: dendritic monocytes without any stimulation (negative control; first image); PWM protein stimulation (positive control; second image); and stimulation of engineered exosomes with gp-100 (last image).

DETAILED DESCRIPTION OF THE INVENTION

[0047] The following description of disclosure is provided as an enabling teaching of disclosure in its best currently known embodiments. To this end, those skilled in the relevant art will recognize and appreciate that many changes can be made in the various embodiments of the invention described herein, while still obtaining the beneficial results of the present disclosure. It will also be apparent that some of the desired benefits of the present disclosure can be obtained by selecting some of the features of the present disclosure without using other features. Accordingly, those working in the art will recognize that many modifications and adaptations to the present disclosure are possible and may even be desirable in certain circumstances and form part of the present disclosure. Thus, the following description is provided as illustrative of the principles of the present disclosure and not as a limitation thereof.

TERMINOLOGY

[0048] Unless defined otherwise, all technical and scientific terms used in this document have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The following definitions are provided for a full understanding of the terms used in this descriptive report.

[0049] The components to be used to prepare the disclosed compositions are disclosed, as well as the compositions themselves to be used in the methods disclosed herein. These and other materials are disclosed in this document, and it is understood that when combinations, subsets, interactions, groups, etc. these materials are disclosed, although specific reference to each of the various individual and collective combinations and permutations of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a specific fluidic channel is disclosed and discussed and a series of modifications that can be made to the fluidic channels are discussed, each combination and permutation of the fluidic channels and the modifications that are possible is specifically contemplated, unless specifically noted in the contrary. Thus, if a class of fluid channels A, B and C are disclosed, as well as a class of fluid channels D, E and F and an example of a combination of fluid channels, or, for example, a combination of fluid channels comprising AD is disclosed, so even though each is not recited individually, each is individually and collectively contemplated meaning combinations, AE, AF, BD, BE, BF, CD, CE and CF are considered disclosed. Likewise, any subset or combination of these is also disclosed. So, for example, the subgroup of A-E, B-F and C-E would be considered disclosed. This concept applies to all aspects of this application, including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed, it is understood that each such additional step can be performed with any specific embodiment or combination of embodiments of the disclosed methods.

[0050] It is understood that the devices disclosed herein have certain functions. Certain structural requirements for performing the disclosed functions are disclosed, and it is understood that there are a variety of structures that can perform the same function that are related to the disclosed structures and that these structures will ultimately achieve the same result.

[0051] Unless expressly stated otherwise, it is in no way intended that any method set forth in this document be construed as requiring its steps to be performed in a specific order. Thus, when a method claim does not expressly recite an order of steps to be followed or it is not specifically stated in the claims or in the description that the steps should be limited to a specific order, it is not intended that an order be inferred in any respect . This applies to any possible unstated basis for interpretation, including: questions of logic regarding the arrangement of steps or operational flow; simple meaning derived from grammatical organization or punctuation; and the number or type of embodiments described in the descriptive report.

[0052] As used in the specification and claims, the singular form "a", "an" and "the" include plural references, unless the context clearly indicates otherwise. For example, the term "an agent" includes a plurality of agents, including mixtures thereof.

[0053] As used herein, the terms "may", "may", "optionally", "may optionally" and "may optionally" are used interchangeably and are intended to include cases where the condition occurs, as well as cases where the condition does not occur. Thus, for example, the statement that a formulation "may include an excipient" is intended to include cases where the formulation includes an excipient, as well as cases where the formulation does not include an excipient.

[0054] Ranges can be expressed as "about" a particular value and/or "about" another particular value. When such a range is expressed, another embodiment includes from a particular value and/or to another particular value. Likewise, when values are expressed as approximations, using the background "about", it will be understood that the particular value forms another embodiment. It is also understood that the endpoints of each of the ranges are significant in relation to the other endpoint and independently of the other endpoint. It is also understood that there are a number of values disclosed in this document, and that each value is also disclosed in this document as “about that” particular value, in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” will also be disclosed.

[0055] The terms "upstream" and "downstream" refer to positions within a device that are relative to another position and a direction of fluid flow. As used in this document, the term "upstream" refers to a first position that is located in a direction opposite to the direction of fluid flow relative to a second position. On the other hand, as used in this document, the term "downstream" refers to a second position that is located in one direction along the direction of fluid flow relative to a first position.

METHODS

[0056] Referring to Figure 1, the general methods described herein involve a plurality of immunomagnetic particles (beads), which are mixed with a sample solution suspected of containing the target. In some embodiments, a biological sample is collected from a subject and prepared for the method, e.g., by dilution with buffer, concentration,

etc.

In some embodiments, cells are harvested and expanded in culture.

In some embodiments, cells are harvested and cultured under conditions to release exosomes or other extracellular vesicles or vesicle-like structures into the culture.

In either case, the immunomagnetic particles contain a photocleavable linker and an affinity probe (and preferably a plurality of photocleavable linkers,

each with a respective affinity probe) extending from the surface of the particle to capture the target.

The immunomagnetic particles are contacted with a sample solution for a period of time sufficient for the target (if present in the sample) to interact with the affinity probes that extend from the immunomagnetic particles.

In Figure 1, a single sphere is shown with a single linker for ease of illustration; however, in practice, each immunomagnetic particle will be coated with a plurality of binders (preferably substantially the entire surface area of the particle/

sphere is coated with binders). Also, the relative sizes in Figure 1 are not to scale but enlarged for illustration purposes.

In practice, the sphere/particle is preferably at least 5 times larger than the target (for example, in the case of exosomes, which range from 30 to 150 nm in diameter, the sphere is preferably 500 nm or larger). In this way, a plurality of targets

(eg exosomes) will be captured on the surface of a single sphere/

particle.

Figure 1 uses a single sphere and target interaction for ease of reference.

As shown in Figure 1(A), the bead and sample solution are mixed for a sufficient period of time (eg, in a mixing chamber or channel in the microfluidic device). If the target is present in the sample solution, it will be captured by the bead (and specifically by an affinity probe that extends from the bead through its photocleavable ligand), as illustrated in Figure 1(B). Exemplary photocleavable linkers are described herein and may include linear chains including biotin or similar moiety at one end for binding to the particle and an amine moiety at the other end for binding to the affinity probe. A preferred linker has the following structure, where the dashed line indicates the bond cleaved during photoexposure, and the "linker group" represents the portion (eg, biotin) used to attach the linker to the bead (directly or via of a functionalized surface coating, eg avidin):

AFFINITY PROBE LINK GROUP

[0057] Although various embodiments are described herein, the affinity "probe" is typically an oligopeptide sequence that has specificity and recognizes the target, such as a peptide that recognizes and acts as a receptor for a surface protein on the target. . As used herein, the phrase "specificity for" is intended to differentiate the affinity probe from non-specific binding or reactions between molecules and means that the set of specific targets to which the affinity probe can interact is limited and in some cases , even exclusive, so that binding does not occur at an appreciable rate with any other molecule, except to the target (and specifically, its designated surface protein(s)). Short oligopeptide sequences are preferably used for the affinity probe, including sequence segments with high target specificity. More preferably, upon binding, the affinity probe and surface protein create a complex that enhances the immunogenic potential of the target, as described in more detail in this document and demonstrated in the working examples.

[0058] The sphere with the captured target (eg exosome) is immobilized in the microfluidic device. The sphere can be immobilized before or after capturing the target. As described elsewhere in this document, this can be achieved by placing a magnet adjacent to an engineering or collection chamber in the microfluidic device. As the sample solution and bead solution flow through the microfluidic channel, the beads and target interact, thus capturing the target. The magnetic spheres are immobilized in the engineering or collection chamber (thus immobilizing the captured target) as the solutions flow. As illustrated in Figure 1(C), the captured target is then engineered by attaching a plurality of active agents (eg, antigenic peptides) to the surface of the target or loading the target with drugs, chemicals, nucleotides, or other bioactive agents ( for example, CRISPR Cas9). For surface modification, the immobilized bead/target complex is washed and incubated in the microfluidic device with a buffer solution containing a plurality of active agents having at least one portion that has specificity for a surface protein displayed on the surface of the target. Preferably, the active agents or portions for surface loading are of the same "type" of compound (e.g., comprise the same oligopeptide) selected for the affinity probe used on the immunomagnetic particles. The immobilized sphere/target complex is incubated with the active agents for a period of time sufficient for the active agents to interact with the captured target. Preferably, the active agent loading into the microfluidic device is at a concentration such that substantially all of the target surface is coated with active agents (e.g., preferably substantially all of the target surface protein is bound by the active agent). Instead of surface modification, Figure 1(C) also represents an alternative where active agents can be loaded onto the target. This can be accomplished by washing and incubating the bead/target complex immobilized on the microfluidic device with a buffer solution containing active agents to be loaded, along with detergents or chemical transfection reagents to induce pore formation in the target for active agent loading, followed by washing with buffer to remove reagents and close pores. In either embodiment, excess active agent is then washed away, leaving the engineered target immobilized with the immunomagnetic bead.

[0059] Referring to Figure 2A, the engineered-modified target can then be released by exposing the immobilized bead/target complex to activating radiation (eg, light) of the appropriate wavelength to cleave the photocleavable ligand. This process releases the engineered-modified target along with the affinity probe that remains attached to the target, which now acts as an engineered-modified carrier or delivery vehicle for the active agents decorated on the target's surface. Likewise, in Figure 2B, the same photorelease process can be used to release targets loaded internally with active agents. A wash buffer can be introduced into the microfluidic device to transport the released targets downstream for collection at the output of the microfluidic device. Advantageously, the light release step in the embodiments of the invention is carried out with exposure times of 15 minutes or less, preferably about 13 minutes or less, more preferably about 12 minutes or less. As demonstrated in the working examples, approximately 100% of the captured target is preferentially released/cleaved within about 10 minutes of exposure time.

[0060] It will be appreciated that since the magnetic beads are already immobilized in the engineering chamber, a separate step is not necessary to separate the target from the magnetic beads in the solution. Instead, after exposure, the ligand between the captured target and the bead is cleaved, thus releasing the engineered modified targets, which flow downstream away from the immobilized beads to the output of the microfluidic device. Released targets, which have been engineered with the active portions (also known as "engineered modified carrier"), can then be collected for analysis and therapeutic use from the output of the microfluidic device or otherwise directly diverted to another chamber or collection device. It will be appreciated that the immunomagnetic beads can then subsequently be collected for reuse by removing the magnetic field from the microfluidic device so that the immunomagnetic beads are no longer magnetically immobilized. The spheres can be washed downstream and collected at the outlet.

[0061] As noted, this process can advantageously occur in a microfluidic device and as a continuous, integrated, in-line approach to isolate, capture, engineer and release intact targets, such as exosomes from a sample, such as therapeutic carriers or delivery vehicles. delivery.

DEVICES

[0062] Extracellular vesicles (≤ 1 µm), particularly exosomes (30 to 150 nm), are the emergent payload to mediate cellular signal transductions. However, standard bench top methods (eg, ultracentrifugation and filtration) do not have the ability to process immunogenic exosomes specifically between other microvesicle subtypes, due to time-consuming (>10 h) and extremely tedious isolation protocols. The present disclosure meets the needs in the art by providing devices that introduce an in-line microfluidic platform for the collection, antigenic modification and photo-release of immunogenic extracellular vesicles and exosomes directly from cell media cultured on chip. These devices provide automatic and rapid production of cell culture antigenic exosomes that can be used in immunotherapy such as cancer immunotherapy. The devices disclosed in this document allow for real-time collection and antigenic modification of exosomes with subsequent downstream photo-release on demand, as depicted in the overview in Figure 3.

[0063] Turning now to Figure 4, there is disclosed here a microfluidic device (200) comprising a cell culture chamber (210) sized to maintain biological material in a three-dimensional configuration; a mixing channel (220) fluidly connected to the cell culture chamber and comprising a plurality of sample inlet channels (222, 224, 226) disposed along the mixing channel, wherein the ratio of a width of the chamber is cell culture (210) for the largest cross-sectional dimension of the mixing channel (220) is at least 5:1; an isolation channel (230) defining a path for fluid flow from the mixing channel (220) to an isolation outlet (234); and a collection chamber (240) fluidly connected to the isolation outlet (234) and comprises a magnet operatively coupled to the collection chamber to produce a magnetic field within the collection chamber (240).

[0064] Devices of the present disclosure can be described by sizes and size comparisons (e.g., proportions) of components within the device. In some embodiments, the cell culture chamber (210) has a volume of about 200 microliters or more (e.g., 200 microliters or more, 250 microliters or more, 300 microliters or more, 350 microliters or more, 400 microliters or more, 450 microliters or more, 500 microliters or more, 550 microliters or more, 600 microliters or more, 650 microliters or more, 700 microliters or more, 750 microliters or more, 800 microliters or more, 850 microliters or more, 900 microliters or more, 950 microliters or more, or 1 milliliter or more). In some embodiments, the cell culture chamber (210) has a volume of about 1000 microliters or less (e.g., 950 microliters or less, 900 microliters or less, 850 microliters or less, 800 microliters or less, 750 microliters or less, 700 microliters or less, 650 microliters or less, 600 microliters or less, 650 microliters or less, 550 microliters or less, 500 microliters or less, 450 microliters or less, 400 microliters or less, 350 microliters or less, 300 microliters or less, 250 microliters or less, or 200 microliters or less). In some embodiments, the cell culture chamber (210) has a volume of from about 200 microliters to about 1 milliliter (e.g., 200 microliters to 900 microliters, 200 microliters to 750 microliters, 200 microliters to 500 microliters, from 300 microliters to 750 microliters or from 350 microliters to about 500 microliters). In some embodiments, the cell culture chamber is of sufficient volume so that the top can be left open for application of a plug (such as a finger pressure buffer made by PDMS) for fluid exchange and pushing fluid into downstream collection channels.

[0065] In some embodiments, the cell culture chamber has a height and a width. The cell culture chamber can have a height of at least 500 microns (e.g. 500 microns or more, 600 microns or more, 650 microns or more, 700 microns or more, 750 microns or more, 800 microns or more, 850 microns or more, 900 microns or more, 950 microns or more, or 1000 microns or more). In some embodiments, the cell culture chamber has a height of 1000 microns or less (e.g., 950 microns or less, 900 microns or less, 850 microns or less, 800 microns or less, 750 microns or less, 700 microns or less, 650 microns or less, 600 microns or less, or 500 microns or less). In some embodiments, the cell culture chamber is 500 microns to 1000 microns high (e.g., 600 microns to 1000 microns, 750 microns to 1000 microns, or 800 microns to 1000 microns).

[0066] The cell culture chamber may have a width of at least 200 microns (for example, 250 microns or more, 275 microns or more, 300 microns or more, 350 microns or more, 400 microns or more, 450 microns or more, 500 microns or more, 550 microns or more, or 600 microns or more).

In some embodiments, the cell culture chamber is 1000 microns or less wide (e.g., less than 1000 microns, 750 microns or less, less than 750 microns, 600 microns or less, 550 microns or less, or 500 microns or less). In some embodiments, the cell culture chamber is 250 microns to 1000 microns wide (e.g., 250 microns to 750 microns, 250 microns to 500 microns, 300 microns to 750 microns, or 300 microns to 500 microns)

[0067] As described in this document, the mixing channel (220) fluidly connects to the cell culture chamber and comprises a plurality of sample inlet channels (222, 224, 226) disposed along the mixing channel. The plurality of sample inlet channels may include a cell culture inlet channel (also referred to herein as inlet B, 222) that fluidly connects to the cell culture chamber and defines a path for introducing fluid from the cell culture chamber. cell culture in the mixing channel. The plurality of sample inlet channels may further include a particle inlet channel (also referred to herein as inlet A, 224) which defines a path for introducing particles into the mixing channel. The plurality of sample inlet channels may further include a fluid inlet channel (also referred to herein as inlet C, 226) that defines a path for introducing fluid (such as a wash buffer) into the mixing channel. The plurality of sample input channels can be in any arrangement. For example, the cell culture inlet can be upstream of the particle inlet that is upstream of the fluid inlet. In other examples, the particle inlet can be upstream of the cell culture inlet that is upstream of the fluid inlet.

[0068] The cell culture inlet, the particle inlet and the fluid inlet can fluidly converge at a mixing intersection. The cell culture inlet channel forms a fluid flow path from the cell culture chamber to the mixing intersection. The particle inlet channel forms a path for fluid flow from a particle inlet to the mixing intersection. The fluid inlet channel forms a path for fluid flow from a fluid inlet to the mixing intersection. As used herein, a fluid flow path may be pictorially represented in the figures by an arrow to indicate the direction of fluid flow through the fluid flow path.

[0069] The mixing channel comprising the cell culture inlet, the particle inlet and the fluid inlet has a height and a width. In some embodiments, the mixing channel has a height of at least 50 microns (e.g., 75 microns or more, 100 microns or more, 120 microns or more, 150 microns or more, 175 microns or more, 200 microns or more, 250 microns or more, 300 microns or more, 350 microns or more, 400 microns or more, or 500 microns or more). In some embodiments, the mixing channel has a height of 500 microns or less (e.g., less than 500 microns or less, 400 microns or less, less than 400 microns or less, 350 microns or less, 300 microns or less, less than 300 microns or less, 275 microns or less, 250 microns or less, 200 microns or less, 150 microns or less, 100 microns or less, or 50 microns or less). In some embodiments, the mixing channel has a height of 50 microns to 500 microns (e.g., 100 microns to 500 microns, 200 microns to 500 microns, 100 microns to 350 microns, or 200 microns to 500 microns ).

[0070] The mixing channel can have a width of at least 50 microns (for example, 75 microns or more, 100 microns or more, 120 microns or more, 150 microns or more, 175 microns or more, 200 microns or more, 250 microns or more, 300 microns or more, 350 microns or more, 400 microns or more, or 500 microns or more). In some embodiments, the mixing channel has a width of 500 microns or less (e.g. less than 500 microns or less, 400 microns or less, less than 400 microns or less, 350 microns or less, 300 microns or less, less than 300 microns or less, 275 microns or less, 250 microns or less, 200 microns or less, 150 microns or less, 100 microns or less, or 50 microns or less). In some embodiments, the mixing channel is 50 microns to 500 microns wide (e.g., 100 microns to 500 microns, 200 microns to 500 microns, 100 microns to 350 microns, or 200 microns to 500 microns ).

[0071] The ratio of the width of the culture chamber to the largest cross-sectional dimension of the mixing channel is at least 2:1, at least 3:1, at least 4:1, at least 5:1, at least 6:1, at least 7:1, at least 8:1, at least 9:1, at least 10:1, at least 12:1, at least 15:1, at least 18:1, at least 20: 1, at least 25:1, at least 50:1, at least 75:1, at least 100:1, at least 150:1, at least 200:1, at least 250:1, at least 300:1, at least 350:1, at least 400:1, at least 450:1 or at least 500:1. In some embodiments, the ratio of the width of the culture chamber to the largest cross-sectional dimension of the mixing channel is from 5:1 to 500:1, from 5:1 to 200:1, from 5:1 to 100 :1, from 2:1 to 25:1, from 5:1 to 20:1, from 5:1 to 15:1, from 6:1 to 25:1, from 6:1 to 20:1, from 6 :1 to 12:1, from 8:1 to 25:1 or from 10:1 to 25:1. A ratio of the culture chamber width to the largest cross-sectional dimension of the mixing channel that is greater than one (e.g. 2:1) defines a narrowing of the channel width at the entrance to the mixing channel.

[0072] One or more channels in the microfluidic device may, in some embodiments, comprise a fluid mixing mechanism that facilitates mixing of fluids flowing through the device. A fluid mixing mechanism induces turbulent flow in order to mix fluids into flow.

Suitable mixing mechanisms include a serpentine or tortuous channel, a channel protrusion or indentation, a channel curvature, among other known mechanisms.

[0073] In some embodiments, a fluid mixing mechanism may be present in the mixing channel and/or in an isolation channel of the microfluidic device. For example, the microfluidic device may include an isolation channel that fluidly connects the mixing channel to an isolation outlet. The isolation channel can be part of the mixing channel or it can be separate. In some embodiments, the isolation channel defines a path for fluid flow from the mixing channel to an isolation outlet. The isolation channel may comprise a coil geometry that increases mixing as the fluids combine. Referring to Figure 4, an isolation channel (230) having a serpentine geometry may be positioned within the device at an advantageous location for fluid mixing, for example, between a mixing intersection and a collection chamber. In some embodiments, the isolation channel is positioned immediately adjacent to a mixing intersection (e.g., immediately downstream).

[0074] The isolation channel can have a similar or narrow width compared to the width of the mixing channel. In some embodiments, the isolation channel may have a narrow width compared to the width of the mixing channel by at least various means.

For example, the isolation channel may comprise one or more channel constriction domains (see, for example, channel constriction domains (232) which form the narrowed width in the isolation channel in Figure 4) disposed within the channel. isolation between the isolation channel input and the isolation channel output. The channel constriction domains can produce a local vortex flow profile of fluid flowing in the device. In some embodiments, the isolation channel comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten channel constriction domains. Any one or more domains of channel constriction can be a bulge and/or indentation in the side walls of the channel. The protrusion and/or indentation can be of any shape, for example rounded, linear, triangular, irregular, etc. The protrusion and/or indentation may surround the inner walls of the channel (e.g., as a ring), or one or more protrusions and/or indentation may be positioned on one or more inner side walls of the channel. The inclusion of a channel constriction domain can increase fluid flow turbulence and fluid mixing when desirable.

[0075] The devices disclosed herein may include a collection chamber (240) hydraulically connected to the isolation outlet (234). In some embodiments, the device may further comprise a magnet operatively coupled to the collection chamber to produce a magnetic field within the collection chamber. The magnet can be any magnet capable of providing a magnetic field within the collection chamber. In some embodiments, the magnetic field can include an oscillating magnetic field. An oscillating magnetic field is a magnetic field that varies regularly (eg, automated periodic regularity) or irregularly (eg, by user-based controls) over time. An oscillating magnetic field includes dynamic changes in the spatial orientation of the north and south magnetic poles so that the direction of the magnetic field changes over time. These changes can be cyclical or irregular. The inclusion of an oscillating magnet capable of providing an oscillating magnetic field within the collection chamber can induce magnetic probes (eg, magnetic spheres or particles) within the collection chamber to dynamically move within the collection chamber along a direction of the magnetic field. As the direction of the magnetic field changes, the directional movement of the magnetic particles within the collection chamber also changes.

This can be used to promote interaction (and association/bonding) between magnetic particles and targets present in a fluid in the collection chamber.

In some embodiments, the magnetic field can be obtained from a permanent magnet. The permanent magnet can be removed when not in use, for example to turn off the magnetic field.

[0076] In some embodiments, the magnet may have any shape, such as a toroidal shape. In some embodiments, the magnet comprises a Helmholtz coil or a permanent magnet.

[0077] The magnetic field can be present across the entire width of the collection chamber. In some embodiments, the magnetic field may be over a portion of the collection chamber, for example, over at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% of the entire width of the collection chamber. Also, the magnetic field may be over other portions of the microfluidic device. For example, the magnetic field can be over one or more of a portion or all of the mixing channel, mixing intersection, particle inlet channel, or other components.

[0078] In some embodiments, the device may comprise a pump operatively coupled to the microfluidic device.

The pump can be any pump known in the art capable of inducing fluid flow within the device. In some embodiments, the pump can transmit negative pressure within the device, thereby drawing fluid through a channel. Examples of suitable pumps can be found in US 2017/0065978 and US 2017/0001197, each of which are incorporated by reference in their entirety.

[0079] Turning now to Figure 6, a microfluidic device (2000) comprising a cell culture chamber (2100) comprising a cell culture inlet (2102) and a cell culture outlet (2104) is also disclosed. ), a fluid inlet channel (2022) and a particle inlet channel (2204), wherein the cell culture outlet (2104), the fluid inlet channel (2022) and the particle inlet channel (2004) fluidly converge at a mixing intersection (2200); a mixing channel (2300) fluidly connected to the mixing intersection (2200) and defining a path for fluid flow from the mixing intersection to a mixing outlet, wherein the ratio of a width of the cell culture chamber (2100) for the largest the cross-sectional dimension of the mixing channel (2300) is at least 5:1; and a collection chamber (2400) fluidly connected to the mixing outlet and comprises a magnet operatively coupled to the collection chamber (2400) to produce a magnetic field within the collection chamber. The mixing channel (2300) may comprise an isolation channel (2302) disposed between the mixing intersection (2200) and the mixing outlet.

[0080] Another aspect of the microfluidic devices provided in this document refers to multiplexed microfluidic devices that contain two or more sets of chambers and/or channels, including the cell culture chamber, the mixing channel, the isolation channel and the chamber of collection. Setting up two or more such channels and/or chambers in a single microfluidic device can increase sample processing throughput and/or allow parallel processing of at least two samples or sample portions for different fractions or manipulations. Two or more chambers and/or channels can be arranged in series, in parallel or in a combination thereof.

[0081] In some embodiments of a parallel multiplexed microfluidic device, two or more mixing channels may have separate sample inputs arranged in the same microfluidic device.

Such an arrangement can be used for multiple fluid samples.

Alternatively, the plurality of mixing channels can be connected to the same sample inputs for parallel processing of the same fluid sample. Furthermore, the two or more mixing channels can have separate outlets arranged on the same microfluidic device or be connected to the same outlet. In one or more embodiments, multiplexed microfluidic devices are contemplated with up to 96 sample inputs.

[0082] The microfluidic devices of the present disclosure can be used in combination with the various compositions, devices, methods, products and applications disclosed herein. In some embodiments, the microfluidic devices can be a standalone microfluidic device. In some embodiments, one or more microfluidic devices can be integrated as part of an equipment, module, or system. In other embodiments, one or more microfluidic devices can be fluidly coupled to an equipment, module, or system.

[0083] By way of example only, one or more microfluidic devices and/or multiplexed microfluidic devices may be fluidly coupled to a detection module. As used in this document, the term “fluidly coupled” refers to two or more devices and/or modules connected in an appropriate manner such that fluid can pass or flow from one device or module to the other device or module. When two or more devices and/or modules are fluidly coupled, additional devices and/or modules may be present between the two or more devices and/or modules.

[0084] Alternatively, two of the two or more devices and/or modules can be connected so that a fluid can pass or flow directly from a first device or module to a second device or module without any intervening devices or modules. Two or more devices or modules can be fluidly coupled, for example, connecting an output of a first device or module to an input of a second device or module using tubing, a conduit, a channel, plumbing, or any combination thereof.

[0085] The detection module can perform any detection method disclosed in this document or other methods known in the art.

In some embodiments, the detection module can include a sample treatment module before the sample is detected for analysis. For example, exosomes (including or excluding immunomagnetic particles) can be subjected to immunostaining prior to detection by microscopy.

Examples of the detection module may include, without limitation, a microscope (eg a brightfield microscope, a fluorescence microscope or a confocal microscope), a spectrophotometer (eg a UV-Vis spectrophotometer), a cell counter, a biocavity laser (see, for example, Gourley et al., J. Phys. D: Appl. Phys. 36: R228-R239 (2003)), a mass spectrometer, an imaging system, an affinity column, a particle classifier, eg a fluorescence activated cell classifier, capillary electrophoresis, a sample storage device and sample preparation device. In some embodiments, a computer system can be connected to the detection module, for example, to facilitate the sample treatment, detection and/or analysis process.

PRODUCTION METHODS

[0086] The devices described herein can be made of any material that is compatible with a fluid sample. In some embodiments, the material for fabricating the devices described herein can be penetrated by a magnetic field. In some embodiments, the material for fabrication of the devices described herein can be substantially transparent so that the sample included therein can be photocleaved in situ or can be viewed under a microscope, for example, for in situ analysis of labeled exosomes magnetically. Exemplary materials that can be used to fabricate the microfluidic devices described herein can include, but are not limited to, glass, copolymer, polymer, or any combinations thereof. Exemplary polymers include, but are not limited to, polyurethanes, rubber, molded plastic, polymethylmethacrylate (PMMA), polycarbonate, polytetrafluoroethylene (TEFLON™), polyvinyl chloride (PVC), polydimethylsiloxane (PDMS), polysulfone, and aliphatic ether-based polyurethane .

[0087] The methods used in the fabrication of any embodiments of the microfluidic devices described in this document may vary with the materials used and include 3D printing methods, soft lithography methods, microassembly, bulk micromachining methods, surface micromachining methods , standard lithographic methods, wet etching, reactive ion etching, plasma etching, stereolithography and laser chemical three-dimensional writing methods, solid object printing, machining, modular assembly methods, replica molding methods, injection molding methods, hot molding methods, laser ablation methods, combinations of methods and other methods known in the art.

[0088] In specific embodiments, the microfluidic devices described herein can be manufactured using a 3D printer.

For example, manufacturing methods for microfluidic devices might include providing three pieces of PDMS molds, including a base, wall, and top magnet holder, as shown in Figure 7. The mold can be printed on a 3D printer. The molds can be coated with Sportline palladium to a thickness of 20 nm followed by assembly using methods known in the art. The PDMS cell chamber can be sized so that, when full, the cell culture chamber has an open end for the chamber plug. PDMS can be molded at a ratio of 10 :1 with a binding reagent and incubated at 40°C for 6 hours. Once the PDMS is cured, it can be removed easily. Chip inputs and outputs can be punched using a punch. The PDMS chips can then be post-glued onto a hot pad at 40°C for 5 minutes. Chips can be cleaned with DI water and sterilized in an autoclave (at 121°C for 30 minutes).

METHODS OF USE

[0089] As discussed in this document, the microfluidic devices disclosed can be used to isolate, capture, manipulate and release engineered extracellular vesicles and various vesicle-like biological structures. In certain embodiments, the engineered extracellular vesicles are immunogenic exosomes. As used in this document, the term "exosome" generally refers to externally released vesicles arising from the endosomal compartment or any cells, eg tumor cells (eg prostate cancer cells) and immune system cells (eg cells antigen presenting cells such as dendritic cells, macrophages, mast cells, T lymphocytes or B lymphocytes). Exosomes are usually membrane vesicles with a size of about 20 to 150 nm that are released from a variety of different cell types, including tumor cells, red blood cells, platelets, lymphocytes, and dendritic cells. Exosomes can be formed by invagination and budding of the membrane of late endosomes. They can accumulate in cytosolic multivesicular bodies (MVBs) from where they can be released by fusion with the plasma membrane. Without wishing to be bound by theory, the vesicle release process is particularly active in proliferating cells, such as cancer cells, where release can occur continuously. When released from tumor cells, exosomes can promote invasion and migration. Thus, in some embodiments, the immunomagnetic particles described herein can be used to capture exosomes originating from cancer cells. Depending on the cellular origin, exosomes can recruit several cellular proteins that may differ from the plasma membrane, including MHC molecules, tetraspanins, adhesion molecules and metalloproteinases. Among many exosomes subtypes, immunogenic exosomes with an intrinsic payload of class I and II MHC molecules and other costimulatory molecules are able to mediate immune responses, which opens up opportunities for the development of new cancer vaccines and delivery in immunotherapy .

[0090] Therefore, methods of producing immunogenic exosomes in a microfluidic device disclosed herein are also provided herein. Methods of producing immunogenic exosome complexes may comprise introducing cells into the cell culture chamber of the microfluidic device. The cells in the cell culture chamber can include any cells from which extracellular vesicles can be obtained. These cells include dendritic cells, stem cells, immune system cells, megakaryocyte progenitor cells, macrophages or combinations thereof.

[0091] The method for the production of immunogenic exosomes can further include culturing the cells under conditions that allow the release of exosomes. In some embodiments, the methods may include enriching or expanding the number of exosomes present in the cell sample by mediating the growth of their parent cells using stimuli known in the art. Conventional methods for culturing a parent cell to produce exosomes are known in the art and can be used in the methods disclosed herein. In some embodiments, cells can be cultured for a period of time, for example, at least about 30 minutes, at least about 45 minutes, at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 5 hours, at least about 6 hours, at least about 8 hours, at least about 10 hours, at least about 12 hours, at least about 15 hours, at least about 18 hours, at least about 20 hours, at least about 24 hours, at least about 30 hours, at least about 36 hours, at least about 40 hours, or at least about 48 hours.

[0092] The method for producing the immunogenic exosomes complex may further comprise mixing the cell culture comprising exosomes with immunomagnetic particles to capture the exosomes and a washing solution to form a mixture. In some embodiments, the methods include introducing the cell culture exosomes into a mixing channel and mixing the exosomes with immunomagnetic particles and a wash buffer to form a mixture.

Immunomagnetic particles can be introduced into the mixing channel via the particle inlet channel and wash buffer can be introduced into the mixing channel via the fluid inlet channel.

[0093] Immunomagnetic particles can selectively bind to exosomes present in cell culture to form immunomagnetic particles bound to the exosome. Therefore, the method can include allowing the exosomes to react with the immunomagnetic particles. Immunomagnetic particles can include a magnetic particle and be of any shape, including, but not limited to, spherical, rod, elliptical, cylindrical and disk. In some embodiments, magnetic particles having a substantially spherical shape and defined surface chemistry can be used to minimize chemical agglutination and non-specific binding. As used in this document, the term "magnetic particles" can refer to a particle on the nanometer or microscale that is attracted or repelled by a magnetic field gradient or has a non-zero magnetic susceptibility. Magnetic particles can be ferromagnetic, paramagnetic or super-paramagnetic. In some embodiments, the magnetic particles can be super-paramagnetic.

[0094] Magnetic particles can range in size from 1 nm to 5 microns. For example, magnetic particles can be from about 500 nm to about 5 microns in size. In some embodiments, magnetic particles can be from about 1 micron to about 5 microns in size. In some embodiments, magnetic particles can be from about 1 micron to about 3 microns in size. Magnetic particles are a class of particles that can be manipulated using magnetic field and/or magnetic field gradient. These particles generally consist of magnetic elements such as iron, nickel and cobalt and their oxide compounds. Magnetic particles (including nanoparticles or microparticles) are well known and methods for their preparation have been described in the art. Magnetic particles are also widely available on the market. A particularly preferred particle is a magnetic particle having a graphene oxide layer or coating that is comprised of graphene oxide nanosheets, as described in US 2018/0100853, filed October 9, 2017, incorporated by reference in its entirety herein. document.

[0095] Magnetic particles can be coated with a plurality of ligands comprising respective affinity probes (molecules) to capture the target (such as an antigenic peptide or antigenic epitope thereof) without adverse effect on the magnetic property. In this regard, the magnetic particle can be functionalized with an organic moiety or functional group and photocleavable linker that can connect the magnetic particle to the respective affinity probes to capture the exosomes. Such organic moiety or functional groups may typically comprise a direct bond or an atom such as oxygen or sulfur, a moiety such as amino groups, carboxylic acid groups, epoxy groups, tosyl groups, silica-like groups, carbonyl groups, amide groups , SO, SO2, SO2NH, SS, or a chain of atoms.

[0096] In certain embodiments, the magnetic particles can be coated with a member of an affinity binding pair that can facilitate the conjugation of the magnetic particles to the affinity probe to capture the exosomes. The term "affinity binding pair" or "binding pair" refers to the first and second molecules that specifically bind to each other. One member of the binding pair is conjugated to the first part to be linked while the second member is conjugated to the second part to be linked. Exemplary binding pairs include any haptenic or antigenic compound in combination with a corresponding antibody or binding portion or fragment thereof (eg, digoxigenin and antidigoxigenin; mouse immunoglobulin and goat anti-mouse immunoglobulin) and non-immunological binding pairs ( eg biotin-avidin, biotin-streptavidin, biotin-neutravidin, hormone [eg, hormone-binding protein thyroxine and cortisol, receptor-receptor agonist, receptor-receptor antagonist (eg, acetylcholine-acetylcholine receptor or an analogue of the same), IgG-protein A, IgG-protein G, Protein AG synthesized by IgG, lectin-carbohydrate, enzyme-enzyme cofactor, enzyme-enzyme inhibitor and pairs of complementary oligonucleotides capable of forming nucleic acid duplexes) and the like. The binding pair can also include a first molecule that is negatively charged and a second molecule that is positively charged.

[0097] An example of the use of binding pair conjugation is the conjugation biotin-avidin, biotin-streptavidin or biotin-neutralavidin. Consequently, in some embodiments, magnetic particles can be coated with avidin-like molecules (e.g., streptavidin or neutrovidin), which can be coupled to biotinylated bonds for use as capture molecules.

[0098] In some embodiments, the magnetic particles can be further functionalized with a chemical cleavable moiety that can bind the magnetic particles to the affinity probe to capture the exosomes and is susceptible to an externally applied cleavage agent/conditions, for example , UV light, pH, redox potential or the presence of degradative molecules such as enzymes. In specific examples, the cleavable linker can be attached to a member of a binding pair (such as biotin) on one functional end to bind to magnetic particles and the other functional end provides an affinity probe to capture exosomes. Thus, after the exosomes attached to the magnetic particles are separated from a fluid sample, the exosomes can be separated from the magnetic particles, if necessary, by cleaving the chemical cleavable portion between the magnetic particles and the affinity probe.

[0099] Exemplary cleavable linking groups include, but are not limited to, photocleavable and redox cleavable linking groups (for example, -OC(O)NH-, -SS- and -C(R)2-SS-, in that R is H or C1-C6 alkyl; phosphate-based cleavable linking groups (for example, -OP(O)(OR)-O-, -O- P(S)(OR)-O-, and -OP(S)(H)-S- , wherein R is optionally substituted straight or branched C 1 -C 10 alkyl); acid cleavable linking groups (for example, hydrazones, amino acid esters and esters, -C=NN- and -OC(O)-); ester-based cleavable linking groups (for example, -C(O)O-); peptide-based cleavable linking groups, (eg linking groups that are cleaved by enzymes such as peptidases and proteases in cells, for example -NHCHRAC(O)NHCHRBC(O)-, where RA and RB are the R groups of the two adjacent amino acids).

[00100] In some embodiments, the cleavable linking group is a photocleavable group that can be cleaved by UV light. Specific examples of photocleavable groups include ortho nitrobenzyl and benzylsulfonyl derivatives such as 6-nitroveratryloxycarbonyl (NVOC), 2-nitrobenzyloxycarbonyl (NBOC), α,α-dimethyl-dimethoxybenzyloxycarbonyl (DDZ), ortho-nitrobenzyl (ONB), 1-( 2-nitrophenyl)ethyl (NPE), alpha-carboxy-2-nitrobenzyl (CNB), 4,5-dimethoxy-2-nitrobenzyl (DMNB), 1-(4,5-dimethoxy-2-nitrophenyl)ethyl (DMNPE) , 5-carboxymethoxy-2-nitrobenzyl (CMNB) and (5-carboxymethoxy-2-nitrobenzyl)oxy)carbonyl (CMNCBZ). It will be appreciated that the substituents on the aromatic nucleus are selected to adjust the absorption wavelength, with electron donor groups (eg, methoxy) generally leading to longer wavelength absorption. For example, nitrobenzyl (NB) and nitrophenylethyl (NPE) are modified by adding two methoxy residues to 4,5-dimethoxy-2-nitrobenzyl and 1-(4,5-dimethoxy-2-nitrophenyl)ethyl, respectively, increasing thus the absorption wavelength range at 340 to 360 nm. Additional examples of photo-removable protecting groups include multiply substituted nitro-aromatic compounds containing a benzyl hydrogen ortho to the nitro group, where the substituent may include alkoxy, alkyl, halo, aryl, alkenyl, nitro, halo or hydrogen. Other materials that can be used include o-hydroxy-α-methyl cinnamoyl derivatives, photocleavable groups based on the coumarin system, such as BHC (as described in US Patent No. 6,472,541, the disclosure of which is incorporated herein by reference), photocleavable group comprising the pHP group (as described in Givens et al., J. Am. Chem. Soc. 122 2687-2697 (2000), the disclosure of which is incorporated herein by reference), linkers derived from ketoprofen, others ortho-nitro aromatic core scaffolds include those that trap nitrous by-products in a hetero Diels Alder reaction (generally discussed in US Patent Application 2010/0105120, the disclosure of which is incorporated herein by reference), nitrodibenzofuran chromophore (NDBF) or a diazo-azide. Other examples of photocleavable groups can be found in, for example, Patchornik, J. Am. Chem. Soc. (1970) 92: 6333 and Amit et al., J. Org. Chem. (1974) 39:192, the disclosures of which are incorporated herein by reference.

[00101] As discussed above, a photocleavable group is one whose covalent attachment to a molecule (such as to a member of a binding pair, eg biotin at one functional end and the other functional end to an affinity probe) is cleaved by exposure to light at an appropriate wavelength. In one aspect, release from the affinity probe and/or binding pair occurs when the conjugate is subjected to ultraviolet light or near ultraviolet light. For example, affinity probe photorelease can occur at a wavelength ranging from about 200 to 380 nm (the exact wavelength or wavelength range will depend on the specific photocleavable group used and may be, for example, 200,

210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370 or 380 or some interval between them). In another aspect, release of the affinity probe can occur when the conjugate is subjected to visible light.

For example, photorelease from the affinity probe can occur at a wavelength ranging from about 380 to 780 nm (the exact wavelength or wavelength range will depend on the specific photocleavable group used and may be, for example, , 380, 400, 450, 500, 550, 600, 650, 700, 750 or 780, or some interval between them).

[00102] As described in this document, magnetic particles further comprise an affinity probe (also referred to herein as a molecule to capture exosomes or a capture molecule). As used herein, the term "affinity probe" or "capture molecule" refers to any molecule, cell or particulate material. Suitable affinity probes comprising a magnetic particle are described in US 2017/0065978 and US 2017/0001197, each of which are incorporated by reference in their entirety. Affinity probes can comprise a binding member that specifically binds to the target (exosome or other extracellular vesicles) of interest. For example, the linker can be a nucleic acid oligomer, antibody, enzyme, hormone, growth factor, cytokine (eg, inflammatory cytokines), proteins, peptide, prion, lectin, oligonucleotide, carbohydrate, lipid, molecular toxin and chemical or other binding element that has high affinity and high specificity for the target, and specificity for a designated surface protein on the target. One or more linkers (for example, a peptide) can be linked to the magnetic particle via the cleavable linker by methods known in the art. Generally, a linker has an affinity constant (Ka) greater than about 105 M-1 (for example, 106 M-1, 107 M-1, 108 M-1, 109 M-1, 1010 M- 1, 1011 M-1 and 1012 M-1 or more) with the target, particularly exosome or other extracellular vesicles.

[00103] In certain embodiments, the affinity probe includes an antigenic peptide or antigenic epitope thereof. As used herein, the term "antigens" refers to a molecule or portion of a molecule capable of being bound by a selective binding agent, such as an antibody, and additionally capable of being used in an animal to induce the production of antibodies capable of binding to an epitope of that antigen. An antigen can have one or more epitopes. The term "antigen" can also refer to a molecule capable of being bound by an antibody or a T-cell receptor (TCR) if presented by MHC molecules. The term "antigen" as used herein also encompasses T cell epitopes. An antigen is additionally capable of being recognized by the immune system and/or being capable of inducing a humoral immune response and/or cellular immune response leading to activation of B and/or T lymphocytes. An antigen can have one or more epitopes (B and T epitopes). The specific reaction referred to above is intended to indicate that the antigen will preferentially react, typically in a highly selective manner, with its corresponding antibody or TCR and not with the multitude of other antibodies or TCRs that may be evoked by other antigens. Antigens, as used here, can also be mixtures of several individual antigens.

[00104] As described above, the affinity probe (antigen) can be a protein or a peptide. In some embodiments, the protein or peptide can be essentially any protein that can activate immune system cells and/or primary immune responses, bind to a rare cell, e.g., a circulating tumor cell, a stem cell, and/ or a microbe. By way of example only, if the target species is cancer, exemplary proteins or peptides or other molecule that can be used to generate cancer affinity probes may include, but are not limited to, MAGE-A3, gp-100, HER- 2, p53, PSA-1 or MART-1, EGFR, ERCC1, CXCR4, EpCAM, CEA, ErbB-2, E-cadherin, mucin-1, cytokeratin, PSA, PSMA, RRM1, androgen receptor, estrogen receptor, progesterone receptor, IGF1, cMET, EML4 or leukocyte associated receptor (LAR).

In some embodiments, the affinity probe may be an antibody or a portion thereof, or an antibody-like molecule. In some embodiments, the capture molecule may be an antibody or a portion thereof, or an antibody-like molecule that is specific for detecting a rare cell, e.g., a circulating tumor cell, a stem cell, and /or a microbe biomarker. In some embodiments, the affinity probe can be an aptamer. In some embodiments, the affinity probe can be a DNA or RNA aptamer. In some embodiments, the affinity probe can be a cell surface receptor ligand. Exemplary cell surface receptor binding includes, for example, a cell surface receptor binding peptide, a cell surface receptor binding glycopeptide, a cell surface receptor binding protein, a surface receptor binding glycoprotein cell surface receptor binding organic compound and a cell surface receptor binding drug. Additional cell surface receptor ligands include, but are not limited to, cytokines, growth factors, hormones, antibodies, and angiogenic factors. In some embodiments, any art-recognized cell surface receptor ligand that can bind to a rare cell, e.g., a circulating tumor cell, a stem cell and/or a microbe, can be used as an affinity probe in the magnetic particles described herein. In one or more embodiments, an affinity probe is selected to target an immunostimulatory molecule displayed on the surface of the target (e.g.,

exosome) such as a class I MHC molecule, a class II MHC molecule, an interleukin, TNFα, IFNγ, RANTES, G-CSF, M-CSF, IFNα, CTAPIII, ENA-78, GRO, I-309 , PF-4, IP-10, LD-78, MGSA, MIP-1α, MIP-1β and combinations thereof. More preferably, after binding of the affinity probe (and subsequent release of the target), the resulting engineered modified target comprising the bound affinity probe increases the immunogenic potential of the released target. For example, binding of an MHC class I surface protein by the affinity probe creates a complex that will (in therapeutic use) enhance the recognition and uptake of the engineered target by an antigen presenting cell for stimulation and activation of the immune system. The peptides are preferably 15 amino acid residues or less in length, more preferably 13 residues or less in length, even more preferably 12 residues or even 11 residues or less in length.

[00106] Examples of affinity probes include those listed in the Table below:

SEQ SEQ

ID ID NO: NO: Peptide 2: Peptide Sequence Peptide 1: SIINFEKL 1 2 RSV M2 82-90 SYIGSINNI Peptide 3: Peptide Sequence Peptide 4: Peptide Sequence 3 4 Fusion 85-93 KYKNAVTEL M187-195 NAITNAKII MAGE- A1 161-169: EADPTGHSY 5 MAGE-A3 168-176: EVDPIGHLY 6 MAGEA-10 254-262: GLYDGMEHL 7 MAGEA3 112-120: KVAELVHFL 8 MAGEA1 278-286: KVLEYVIKV 9 MAGEA3 271-279: FLWGPRALV 10 MAGEA (version 11 MAGEA2 157-166: YLQLVFGIEV 12 alternative): KVAEELVHFL MAGE-A4 230-239: GVYDGREHTV 13 MAGE-C1 1083-1091: KVVEFLAML 14 MAGE-C2 191-200: LLFGLALIEV 15 MAGE-C2 336-344: ALKDVEERV 16 MAGEA3 97-105: TFPDLESEF 17 MAGEA5 5-12: HNTQYCNL 18 Prostate Specific Antigen 146- Embryonic Carcinogenic Antigen 19 20 154: KLQCVDLHV (CEA) 694-702: GVTYACFVSNL Embryonic Carcinogenic Antigen (G250 22CEA Cells) 21 Renal Cell Carcinoma ) 652-660: TYACFVSNL 217-225: HLSTAFARV HER-2/neu 435-443: ILHNGAYSL 23 HER-2/neu 63-71: TYLPTNASL 24 proto-oncoprotein Neu/Her-2/Erbb2 HER-2 434-443: ILHDG AYSL 25 26 66-74: TYVPANASL

SEQ SEQ

ID ID NO: NO: gp100 (pmel17) 209-217: gp100-intron 4 (170-178): 27 28

IMDQVPFSV VYFFLPDHL gp100 (pmel17) 154-162: gp100 (pmel17) 476-485: 29 30

KTWGQYWQV VLYRYGSFSV gp100 (pmel) 280-288 (288V): gp100 (pmel) 209-217: ITDQVPFSV 31 32

YLEPGPVTV gp100 (pmel17) 25-33: gp100: YLEPGPVTA 33 34

KVPRNQDWL gp100-intron 4 (170-178): gp100 (pmel17) 17-25: ALLAVGATK 35 36

VYFFLPDHL HER-2/neu 369-377: KIFGSLAFL 37 p53 264-272: LLGRNSFEV 38 p53 187-197: GLAPPQHLIRV 39 p53 149-157: SLPPPGTRV 40 p53 139-147: KLCPVQLWV 41 p53 65-73: RMPEAAPPV-3 42 Phosphatase of prostatic acid p53 103-111: YLGSYGFRL 43 44 (PAP-3): FLGYLILGV Prostate stem cell antigen PSM P2 (prostate): ALFDIESKV 45 46 (PSCA) 14-22: ALQPGTALL Prostate-specific antigen-1 MelanA/MART 26-35: ELAGIGILTV 47 48 (PSA-1) 141-150: FLTPKKLQCV Human Mena Protein MUC-1 12-20: LLLLTVLTV 49 (overexpressed in breast cancer 50): GLMEEMSAL HER-2/neu 689-697: RLLQETELV 51 HER-2/neu (85-94): LIAHNQVRQV 52 Prostate Specific Antigen-1 Prostate Specific Antigen-1 53 54 (PSA-1) 154-163: VISNDVCAQV 153-161: CYASGWGSI PSA 65-73: HCIRNKSVI 55 EGF -R-479 350-359: KLFGTSGQKT 56 EGF-R 1138-1147: YLNTVQPTCV 57 VEGFR2 400-408: VLTNPISM 58 Fragment of VEGFR2/KDR 1 614-59 624: FSNSTNDILI

[00107] It will be appreciated that the above peptides, suitable for affinity probes, are also examples of active agents or fractions for surface loading on the engineered modified target.

[00108] Immunomagnetic particles (i.e. magnetic particles attached to the affinity probe) are preferably formed prior to the mixing process. For example, the affinity probe (eg, an antigenic peptide bound to biotin via a cleavable linker) and the streptavidin coated magnetic particles are mixed for an effective time period for the biotinylated affinity probe bindings to substantially coat completely the entire surface of the avidin-coated particles.

[00109] Thus, the affinity probe is preferably added to the streptavidin-coated magnetic particles for a period of time, before adding the exosome-containing cell culture.

In such embodiments, the affinity probe may first be added to the streptavidin-coated magnetic particles for a period of time sufficient for at least a portion of the added amount of affinity probe to bind to the streptavidin-coated magnetic particles (and preferably for complete affinity probe binding so as to coat the entire surface of the particle with probe bonds extending therefrom).

[00110] The exosomes present in the cell culture containing exosomes are then added to the same fluid sample, where the exosomes can bind to the affinity probe, which has already formed a conjugate with the magnetic particles coated with streptavidin.

[00111] In some embodiments, the immunomagnetic particles can be formed separately before being introduced into the mixing channel of the device.

[00112] The amount of immunomagnetic particles needed to be added to the sample may depend on a number of factors, including, but not limited to, sample volume to be processed, valence of magnetic particles available for conjugation with the affinity probe, the expected abundance of the exosomes present and any combinations thereof. Very high amounts of immunomagnetic particles added to the device can induce non-specific binding and/or fouling within the microfluidic device. Very low amounts of immunomagnetic particles can result in poor capture efficiency. A person skilled in the art can determine the concentration of immunomagnetic particles and capture molecules.

[00113] The exosomes can be mixed with the immunomagnetic particles for any period of time, for example, seconds, minutes or hours. In some embodiments, exosomes can be mixed with the immunomagnetic particles for at least about 1 min, at least about 2 minutes, at least about 5 minutes, at least about 10 minutes, at least about 15 minutes , at least about 30 minutes, at least about 1 hour, at least about 2 hours or more. A person skilled in the art can easily determine an optimal time for mixing time based on a number of factors including, but not limited to, affinity of the immunomagnetic particles with exosomes, concentrations, mixing temperature, and/or rapidity mixture. However, in one or more embodiments, exosomes are mixed with the particles for 1 hour or less.

[00114] Exosomes and immunomagnetic particles can be introduced into the sample inlets of the microfluidic device at any flow rate that provides sufficient residence time for the mixture to retain in the mixing channel and in the isolation channel of the microfluidic device described herein. In some embodiments, samples can be introduced at a flow rate of between 0.1 µl/min to 1 µl/min. Sample fluids can be introduced into the inlet of the microfluidic device by any methods known to one skilled in the art. For example, a flow generator can be connected to at least one of the inputs and outputs of the microfluidic device described herein. Non-limiting examples of a flow generator can include a peristaltic pump, a syringe pump, and any art-recognized pump that can generally be used to flow a fluid through the microfluidic device.

[00115] The method of producing immunogenic engineering targets may further include capturing exosomes bound to immunomagnetic particles by applying a magnetic field within a collection or engineering chamber. In some embodiments, the magnet has a strong magnetic field strength sufficient to create a magnetic field gradient to cause the magnetically labeled exosomes to separate from the fluid sample in the collection chamber. Immobilized magnetically labeled exosomes can be removed from the microfluidic device for further processing. Preferably, the captured exosomes are further engineered and loaded with additional active portions on the surface or internally, as discussed in this document.

Subsequently, the method includes photolytic cleavage of exosomes bound to the immunomagnetic particles to release intact exosomes coated with active fractions or internally loaded with active agents.

[00116] The released target (exosomes) can be supplied as a pharmaceutical composition. The pharmaceutical composition can include the immunogenic exosomes and a pharmaceutically acceptable excipient. It will be appreciated that the active portions can be tailored to provide a specific adaptive immune response against a target condition, or can be selected more generally to activate the innate immune system against a variety of infections or conditions.

[00117] The methods described in this document can be used to process samples in real time. For example, the methods allow continuous, real-time harvesting and antigenic modification of exosomes with subsequent photo-release on demand.

[00118] As described in this document, the methods can be used to produce an immunogenic exosome complex or other immunogenic vesicle-like structures. In certain embodiments, the immunogenic exosome complex can comprise an antigen peptide conjugated to a surface of an exosome. The methods described in this document for making the complex from immunogenic exosomes provide complexes with a significantly higher activation rate for T cells than unengineered exosomes. In some examples, the immunogenic exosome complex can activate T cells by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, in at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% compared to a native exosome.

Due to the improved activation rate, the immunogenic complexes described here can be used in cancer immunotherapy.

[00119] Consequently, methods of treating disease in a subject using the immunogenic complex are disclosed. The method can include administering to the subject a composition comprising an immunogenic complex. In some embodiments, the disease can be an infection. In some instances, the disease could be cancer. The method can further comprise administering a chemotherapeutic agent that has been loaded into the target.

[00120] Additional advantages of the various embodiments of the invention will be apparent to those skilled in the art upon review of the disclosure herein and the working examples below. It will be appreciated that the various embodiments described herein are not necessarily mutually exclusive, unless otherwise indicated herein.

For example, a feature described or depicted in one embodiment may also be included in other embodiments, but is not necessarily included. Thus, the present invention encompasses a variety of combinations and/or integrations of the specific embodiments described herein.

[00121] As used in this document, the phrase "and/or", when used in a list of two or more items, means that any one of the items listed may be employed by itself or any combination of two or more of the items listed can be employed. For example, if a composition is described as containing or excluding components A, B and/or C, the composition may contain or exclude A alone; B alone; C alone; A and B in combination; A and C in combination; B and C in combination; or A, B and C in combination.

[00122] The present description also uses numerical ranges to quantify certain parameters related to various embodiments of the invention. It should be understood that when numerical ranges are provided, such ranges are to be interpreted as providing literal support for claim limitations that only recite the lower value of the range, as well as claim limitations that only recite the upper value of the range. For example, a disclosed numerical range of about 10 to about 100 provides literal support for a claim that recites "greater than about 10" (no upper limits) and a claim that recites "less than about 100" (no limit lower).

[00123] The devices, systems and methods of the appended claims are not limited in scope by the specific devices, systems and methods described herein, which are intended to be illustrations of some aspects of the claims. Any devices, systems and methods that are functionally equivalent are intended to fall within the scope of the claims. Various modifications of devices, systems and methods other than those shown and described in this document are intended to fall within the scope of the appended claims. Furthermore, although only certain representative devices, systems and method steps disclosed herein are specifically described, other combinations of the devices, systems and method steps are also intended to fall within the scope of the appended claims, even if not specifically recited.

Thus, a combination of steps, elements, components or constituents may be explicitly mentioned in this document or less, however, other combinations of steps, elements, components and constituents are included, although not explicitly stated.

[00124] The term “comprising” and variations thereof, as used in this document, is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms "comprising" and "including" have been used in this document to describe various embodiments, the terms "consisting essentially of" and "consisting of" may be used in place of "comprising" and "including" to provide forms of more specific embodiments of the invention and are also disclosed. Except where indicated, all numbers expressing geometries, dimensions, and so forth used in the specification and claims shall be understood, at a minimum, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, to be interpreted in light of the number of significant digits and common rounding approaches.

[00125] Unless defined otherwise, all technical and scientific terms used in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the disclosed invention belongs. Publications cited in this document and the materials to which they are cited are specifically incorporated by reference.

[00126] By way of non-limiting illustration, examples of certain embodiments of the present disclosure are given below.

EXAMPLES

[00127] The following examples present methods according to the invention. It is to be understood, however, that these examples are provided by way of illustration and nothing in them should be taken as a limitation on the general scope of the invention.

EXAMPLE 1: MODIFICATION BY MICROFLUID ENGINEERING ON DEMAND OF

EXOSOMES FOR IMMUNOTHERAPY AGAINST CANCER

[00128] Abstract: Extracellular nanovesicles ( 1 µm), particularly exosomes (30 to 150 nm), are an emerging delivery system in the mediation of cell communications, which have been observed for the preparation of immune responses by presenting cell signaling proteins parental or tumor antigens to cells of the immune system.

In this example, an in-line microfluidic cell culture flow platform for collection, antigenic modification, and photo-release of surface-engineered modified exosomes directly into a workflow is provided. The PDMS microfluidic cell culture platform is replicated from a 3D printed template. By engineering modifying the antigenic peptides on the exosome surface (eg, gp-100, MART-1, MEGA-A3), effective antigen presentation and T cell activation can be achieved. This has been demonstrated using on-chip culture of human blood-derived leukocytes for engineering modification of secreted exosomes in real-time with melanoma tumor peptides. gp100-specific CD8 T cells that were purified from the spleen of 2 Pmel1 transgenic mice were tested. Significantly higher (~ 30%) T cell activation level induced by engineered exosomes was observed compared to unmodified exosomes.

This microfluidic platform serves as a highly integrated, automated cell culture device for the rapid, real-time production of therapeutic exosomes that can advance cancer immunotherapy.

METHODS AND MATERIALS

[00129] 3D printing and microfluidic device fabrication: three mold pieces were provided for the fabrication of PDMS chips,

including a base, wall and top magnet bracket. The mold was designed using SolidWorks® 2017 and printed by 3D Systems' Project 1200 3D Printer. The multiple pieces had the thinnest structure at 50 µm and the channel height at 50 µm. The cell culture chamber is designed with 1000 µm in diameter and 500 µm in height. All molds were coated with Sportline palladium at a thickness of 20 nm. All three parts were assembled using the PDMS chip. The PDMS was filled to a height below 500 µm, so the cell culture chamber left an open end for the chamber plug. PDMS was molded at a ratio of 10:1 with a binding reagent and incubated at 40°C for 6 hours. After PDMS cures, it can be peeled off easily. The inlets and outlets of the chips were punched using a 0.75mm punch. The Piranha-treated glass and the PDMS were both high voltage plasma for at least 30 seconds. The PDMS chips were then post-glued to the hot pad at 40°C for 5 minutes. The chips were cleaned in DI water and sterilized in an autoclave (at 121 °C for 30 minutes).

[00130] On-chip cell culture and collection, engineering modification and release of exosomes: The cell cartridges (8 mm coverslip) were first cleaned with distilled water and air dried inside the bio-hood. Then, they were autoclaved at 121 °C for 30 minutes. Cartridges were placed in a 24-well plate and 500 µl of 0.1 mg/ml poly-D-lysine hydrobromide (MP Biomedicals) was added to each well and incubated at room temperature for 5 minutes. 1 ml of MD water was added to each well for 3 minutes and repeated twice to clean the cell cartridges and then allowed to air dry inside the bio-hood and stored for future use.

[00131] 4 µl of β2-microglobulin (Sigma-Aldrich) and 10 µl of each protein (gp100, MAGE-A3 and MART-1) were mixed with 186 µl of 1  PBS for the modification solution in a final volume of 200 µl. Inlet B was kept blocked and the modification solution was pumped from inlet A and wash buffer from inlet C through the chip at a volume flow rate of 1 µl/min for 10 minutes and 0.1 µl/min for 10 minutes, and static was set for another 10 minutes. A wash step was processed from both inlet A and inlet C at a volume flow rate of 1 µl/min for 15 minutes. The magnet on the underside was removed and the nearby UV turned on to treat the main chamber for 10 minutes. Another wash step from inlet A and inlet C was applied at a volume flow rate of 1 µl/min for 20 minutes to collect the collected exosome from the approximately 20 µl outlet.

[00132] Ultracentrifugation and staining of exosomes: The collected 20 µl exosomes were added to the ultracentrifuge tube and diluted to the final volume of 1 ml for centrifugation (Thermo Scientific™ Sorvall™ MTX) under 1500 rcf for 30 minutes. The supernatant was removed and transferred to a new ultracentrifuge tube. The mixture was then processed at the rate of 100,000 rcf for 1 hour. Exosomes were stained by the PKH67 Green Fluorescent Cell Linker Midi Kit for General Cell Membrane Labeling (Sigma-Aldrich). The staining solution was prepared with 2 µl of PKH67 and 1 ml of Diluent C. Any solution remaining in the tube was discarded and 1 ml of Diluent C was added to resuspend with gentle pipetting. The stained solution into the ultracentrifuge tube, mixed by pipetting and reacted at room temperature for 3.5 minutes. 2 ml of FBS (exosome spent) was added to quench free dye. 1.5 ml of 0.971 M sucrose solution was added for density gradient centrifugation. Another 6.5 ml of complete medium was added to increase the volume to 10 ml. The ultracentrifuge was set at 100,000 rcf for 1 hour. The supernatant was discarded and the dye ring washed thoroughly without reaching the center of the ring. Another 2 ml of 1X PBS was added to resuspend the pellet. The ultracentrifuge at the speed of

100,000 rcf ran for another 1 hour. The supernatant was sucked off and another 100 µl of 1X PBS was added to resuspend the pellet. All steps were kept under sterile conditions and 1 µl of Penicillin-Streptomycin (ATCC®, Catalog # 30-2300, Lot # 63525409) was added to the collected exosome to inhibit and kill remaining bacteria in the solution. Collected exosomes were stored at 4 °C for less than 1 week and stored at -20 °C for up to one month.

[00133] Exosome absorption: THP-1 cells (ATCC®, TIB-202™) were cultured using RPMI-1640 medium formulated by ATCC (ATCC®, Catalog # 30-2001, Lot # 64331683) plus 10% depleted FBS in exosome to the complete medium. Monocyte cells were subcultured at the number of 8 * 105/ml, and using the alternative medium change method.

Cells were used for exosomes by performing the experiment at a density of 5 * 105/ml. 200 µl of the monocyte cells were transferred to the 48-well plate for a total of 11 wells. 20 μl of normal exosome (NE) was added to 5 wells, also 20 μl of engineered modified (EE) exosome to a further 5 wells, and one well was left as a negative control. Time intervals were defined as 0 hours, 0.5 hours, 1 hour, 2 hours, 3 hours and 4 hours. At each time section, 100 μl of cell suspension medium was removed from the cytocentrifuge at a speed of 400 RPM for 4 minutes. Glass slides were collected and 100 µl of fixative solution (ThermoFisher®, Catalog # R37814, Lot # 17B285301) was added to the cell stain. The mixture was incubated at room temperature for 18 minutes and then the solution was removed. 100 µl of 1x PBS buffer was added to the cell site and left at room temperature for 3 minutes. The 1x PBS buffer was removed and the cell stain was carefully washed with distilled water. The slide was dried with no drop remaining on the slide and 50 µl of 500 nM DAPI (ThermoFisher®, Catalog # D1306, Lot # 1844202) applied to the cell site, covered with light and incubated at room temperature for 4 minutes. The DAPI solution was then quickly removed and followed up with a sufficient amount of PBS buffer 1 x twice for 2 minutes each time. The cell stain was washed with distilled water and briefly dried with no drop remaining on the slide. One drop of Antifade Mountant ProLong™ Gold (ThermoFisher®, Ref # P10144, Lot1887458) was applied and the slide covered with 25x25 #1.5 coverslip without any trapped bubbles. The slide was stored at room temperature for 24 hours before imaging under a confocal microscope.

RESULTS

[00134] 3D printing molded microfluidic cell culture device for in-line collection exosomes: An easy and cost-effective approach to make a PDMS-based chip cell culture microfluidic device using a 3D printed mold has been developed. The culture chip contains an on-chip cell culture chamber 1 mm in diameter and 0.5 mm in height for growing cells on the chip and collecting exosomes derived from the downstream culture medium. The cell culture chamber is left open at the top for application of a buffer made with PDMS with a finger closure plug for changing medium and pushing the medium into the downstream collection channels. The bottom of the cell culture chamber has an exit channel approximately 200 µm wide and 200 µm high (B-Inlet) for introducing the culture medium for mixing with immunomagnetic isolation beads (inlet A). Inlet C is used to introduce wash buffer actuated by a syringe pump. Figure 4 demonstrates the mixing process through inlet A and inlet B and outlet to the exosome isolation channel (coil channel) under fluorescence microscope observation using a fluorescence dye solution. Figure 5(b) records the mixture of immunomagnetic beads within the serpentine channel.

Human blood-derived leukocytes were cultured in the culture device with the morphology shown in Figure 5(c). Few red blood cells were even observed to have a cup shape. Secreted exosomes were isolated, captured, and photo-released from the chip output, and characterized by SEM image shown in Figure 5(d).

[00135] A photo-cleavable linker was conjugated with biotin bi-function and NHS chemistry at both ends. The biotin group anchors the photo-cleavable linker to the surface of streptavidin immunomagnetic beads and the NHS group conjugates the MHC-I peptide via the primary amine, as shown in Figure 7A. Class I MHC molecules are heterodimers that consist of two polypeptide chains, α and β2-microglobulin. The two chains are non-covalently linked through the interaction of the b2m and α3 domains. The other two domains α1 and α2 are folded to form a groove for binding 8-10 amino acid peptides (MHC-I binding peptide). The MHC-I/peptide binding complex will be displayed to cytotoxic T cells, consequently, to trigger an immediate immune system response. Once the MHC-I positive exosomes are captured by the tumor targeting antigen peptide (TTA) and retained by immunomagnetic beads inside the capture chamber with the magnetic field, the antigen loading buffer with saturated TTA peptides will be introduced via input C to completely bind and occupy the remaining MHC-I peptide binding sites available. This antigenic surface engineering modification process can substantially improve the amount of TTA peptide loading to captured MHC-I positive exosomes and increase the potency to activate T cells.

[00136] The strength of binding between the MHC-I peptide-modified photo-release immunomagnetic beads with fluorescently labeled MCH-I positive exosomes was further characterized as shown in Figure 7B. The MHC-I antibody serves as a positive control to assess the strength of binding between tumor-targeting antigen peptides and MHC-I positive exosomes. Because of the stronger binding strength between the MHC-I/peptide complex, it has a higher potential to activate T cell antitumor responses. gp-100 has been shown to have a stronger ability to form the MHC-I complex / peptide and the binding strength is even stronger than the MHC-I antibody (95% vs 84.8%).

[00137] On-demand photo-release performance has been characterized in Figures 8A-8E. With the comparison between the positive control and the negative control, the fluorescently labeled exosomes were captured and released by measuring the fluorescence intensity of aggregated beads under an inverted fluorescence microscope. The SEM imaging approach was used to confirm the photo-release process. When comparing the SEM image of the sphere surface before and after photocleavage, there were no identifiable exosomal particles presented on the surface of the spheres, indicating good photo-release performance. The exposure time to ultraviolet rays was also characterized by reaching a photocleavage rate of 98% in 8 minutes of exposure to ultraviolet rays. The size distribution of engineered exosomes and unengineered exosomes was evaluated, which showed an appropriate size range of exosomes between 50 nm and 200 nm, confirming that the engineered exosomes are maintaining good integrity.

[00138] The side effect of UV exposure of the molecular contents of the exosome was investigated, which shows undetectable changes in terms of exosomal proteins, DNAs and RNAs under 10-minute UV treatment (Figure 13).

[00139] In order to assess the potency and integrity of the engineered-modified exosomes released from a microfluidic cell culture device via on-demand photo-release, exosomes from a chip output were harvested and labeled with green fluorescence. The gp-100 engineered exosomes and the unengineered exosomes were incubated with dendritic monocytes to monitor cell uptake at an interval of one hour. Cells were then fixed and nuclei were stained with DAPI. The green dots shown in Figure 9A are tagged exosomes, which are abundantly distributed around cell nuclei. Cellular uptake starts within an hour and the uptake speed is much faster than unmodified exosomes. After 4 hours, it was observed that both engineered and unengineered exosomes were eliminated via the lysosome pathway. This observation indicated that exosomes modified with gp-100 are much more active for the absorption of dendritic monocytes. Cytokine IFN-γ expression was monitored from the incubation of the modified exosome with gp-100 with dendritic monocytes using ELISA. Compared to incubation of unengineered exosomes, the expression level of IFN-γ was much higher for 48 hours after continuous monitoring, with an increase of almost 2-fold. The gray dashed line in Figure 9B indicates the positive control using the PWM protein as a stimulator. Dendritic cell morphology after stimulation was shown in Figure 14. Compared to the negative control without stimulation, PWM protein and engineered exosomes with gp-100 gave significant influence on the shift to round floating dendritic cells. The exosomes engineered with gp-100 showed a higher rate of stimulation for the production of cytokine IFN-γ than the stimulation of the control PWM protein.

[00140] The potency of the exosome engineered with gp-100 for the activation of CD8+ T cells in proliferation and cytolysis was further investigated. It has been observed that exosomes engineered with gp-100 have the ability to activate transgenic T cells in the presence of activated dendritic cells. gp100-specific CD8 T cells were purified from the spleen of 2 Pmel1 transgenic mice by magnetic cell selection and stained with Cell Trace Violet proliferation dye. Purified T cells were cultured alone (T cells only) and mixed at a 3:1 ratio with naïve JAWS cells (an immature dendritic cell lineage derived from a C57BL/6 mouse), T cells + JAWS cells or JAWS cells which were activated for 48 hours with 200 ng/ml (T cells + activated JAWS cells). Exosomes engineered with the gp100 peptide were added to T cell cultures in increasing proportions of exosomes: dendritic cells (25, 50 and 100). Figure 10A. Cells and exosomes were co-cultured for 5 days and then CD8 T cells were analyzed by flow cytometry for Cell Trace Violet dilution as a measure of proliferation. With the T cell condition only as the negative control, it was observed that the proliferation rate of CD8+ T cells cultured with activated JAWS from gp-100 exosomes showed an increase of more than 30%, which indicated that exosomes modified with gp-100 have strong potency to activate T cell cytolysis. Figure 10B. Developed on-demand microfluidic antigen surface engineering modification and photo-release of exosomes may be a powerful tool for the development of an effective exosome-based vaccine and delivery system for advancing cancer immunotherapy.

[00141] Immunogenic potency has also been investigated for bovine respiratory syncytial virus (BRSV). Activated T cells and JAWS cells were incubated with increasing concentrations of exosomes engineered with antimicrobial peptide BRSV (exosomes engineered with Peptide 4: M187-195 peptide NAITNAKII, SEQ ID NO: 4). Immunological stimulation of CD8+ T cell proliferation is linearly dose-responsive to modified exosomes, which is more effective than the use of high-dose peptide vaccines.

BRSV-engineered exosomes have the ability to activate BRSV M-specific T cells in the presence of activated dendritic cells.

C57BL/6 mice were immunized twice subcutaneously with 20 nM of BRSV M187-196 adjuvanted in QuilA.

At least 4 weeks after the final immunization, animals were sacrificed and spleens were collected.

CD8+ T cells and CD11c+ splenic dendritic cells were isolated by magnetic cell separation.

CD8+ T cells were labeled with the proliferation dye Cell

Trace Violet.

Purified T cells were cultured alone (only cells

T), or were mixed at a 3:1 ratio with CD11c+ splenic DC (cells

T + DC). DC cells were not stimulated or were treated with 200 ng/ml LPS to induce DC activation.

Engineered exosomes loaded with the BRSV peptide using the microfluidic platform described above were added to T cell cultures at increasing exosomes:dendritic cell ratios (25, 50 and 100). Negative control wells received no exosomes.

Positive control wells were treated with pure M187-196 peptide at 1 nM or 5 nM.

Cells and exosomes were co-

cultured for 5 days and then CD8 T cells were analyzed by flow cytometry for Cell Trace Violet dilution as a measure of proliferation.

The results are shown in Figure 11. All results support that the disclosed method for capture, antigenic loading, and photo-release can produce antimicrobial peptide-engineered modified exosomes that lead to successful activation of high potency T cells.

EXAMPLE 2: IN VIVO ADMINISTRATION OF EXOSOMES MODIFIED BY ENGINEERING

[00142] The above immunogenic potency study used transgenic mice that were injected with engineered exosomes using the disclosed patented method. Exosomes were engineered with gp-100 or BRSV M187-196 peptide on the surface using the in-line/continuous microfluidic process described above and suspended in PBS buffer for in vivo intraperitoneal injection via the tail. We did not observe injection site reactions or adverse responses (injection site swelling, irritation, etc.) in the injected mice. The mice were observed twice daily for the first 72 hours after the injections and no adverse reactions were observed, indicating the general safety in vivo of the engineered exosomes and related compositions.

Claims (50)

CLAIMS: 1. METHOD FOR ENGINEERING A BIOLOGICAL TARGET in a microfluidic device, characterized in that it comprises: mixing in a microfluidic mixing channel of the microfluidic device a biological sample suspected of containing the biological target with a plurality of immunomagnetic particles in a fluid to form a mixture; allowing the biological target, if present, to react and bind with the immunomagnetic particles in the mixture to form a particle/target complex; immobilize the particle/target complex in a chamber of the microfluidic device by applying a magnetic field inside the chamber; engineering modifying the target in the particle/target complex by contacting said target with a plurality of active fractions or active agents, wherein the surface of said target is surface modified with said active fractions or wherein said plurality of active agents are loaded into said target to produce an engineered target; and photolytically releasing said engineered modified target from said particle/target complex, wherein said particle remains immobilized in said chamber and wherein said engineered modified target is washed downstream of an outlet of said microfluidic device. 2. METHOD according to claim 1, characterized in that said biological target is selected from the group consisting of cells, extracellular vesicles and fractions of vesicle-like cells. 3. METHOD, according to claim 2, characterized in that said extracellular vesicles are exosomes or microsomes. 4. METHOD, according to claim 3, characterized in that it further comprises: introducing cells into a cell culture chamber of the microfluidic device; cultivate the cells under conditions that allow the release of said exosomes; and introducing exosomes into said mixing channel for mixing with said plurality of immunomagnetic particles. 5. METHOD according to claim 4, characterized in that the cells are selected from the group consisting of dendritic cells, stem cells, immune system cells, progenitor cells of megakaryocytes and macrophages. 6. METHOD, according to claim 1, characterized in that said active fractions recognize and specifically bind to surface proteins on the biological target. The method according to claim 6, characterized in that substantially all of said surface proteins are linked to active moieties after said engineering modification step, so that said target is substantially coated with said active moieties. 8. METHOD according to claim 1, characterized in that said immunomagnetic particles have a diameter of 500 nm or greater. The method according to claim 1, characterized in that each of said immunomagnetic particles reacts with a plurality of said biological targets in said sample, such that said particle/target complex comprises a plurality of said biological targets linked to a single central particle. 10. METHOD, according to claim 9, characterized in that each particle binds to a single type of biological target at a time, so that the same type of biological target is attached to each particle. 11. METHOD, according to claim 1, characterized in that said photolytic release comprises exposing said particle/target complex to light in said microfluidic chamber. 12. METHOD, according to claim 11, characterized in that said target is released within 15 minutes after said exposure. 13. METHOD according to claim 1, characterized in that the method is performed in a continuous process in said microfluidic device, preferably within 90 minutes or less from said mixture until the collection of said engineered modified target in said outlet . 14. METHOD, according to claim 1, characterized in that the active portions are antigenic peptides. 15. METHOD according to claim 1, characterized in that said active agents are drugs, nucleotides, CRISPR Cas9 systems, compounds of small molecules, chemotherapy and the like. The method of claim 1, characterized in that said flat substrate of the microfluidic device comprises an inlet in fluid communication with said chamber through a microfluidic channel extending between the inlet and the chamber, said chamber being at the downstream of said inlet and being in fluid communication with an outlet, said outlet being downstream of said chamber. The method of claim 16, characterized in that said microfluidic device comprises a plurality of inlets for introducing said sample, said immunomagnetic particles and washing buffer into said device. 18. The METHOD of claim 16, characterized in that said microfluidic channel has a serpentine flow path to facilitate mixing of said immunomagnetic particles and said sample. The METHOD according to claim 16, characterized in that said device further comprises a cell culture chamber in fluid communication with said microfluidic channel. The METHOD according to any one of claims 1 to 19, characterized in that each of the immunomagnetic particles comprises a plurality of photocleavable linkers extending from the surface of the particle, each with a respective affinity probe at the terminal end thereof, to capture the biological target. 21. METHOD, according to claim 20, characterized in that the affinity probe comprises an antigenic peptide or its antigenic epitope. 22. METHOD according to claim 21, characterized in that the affinity probe includes an antigenic peptide selected from MAGE-A3, gp-100, HER-2, p53, PSA-1 and MART-1. 23. METHOD according to claim 20, characterized in that the binding of said affinity probe with said biological target forms an immunostimulating complex, which after the release of the particle increases antigen presentation and the activation of cells of the immune system and initiates the immune responses. 24. METHOD according to claim 20, characterized in that the affinity probe is selected as to specificity for an immunostimulatory molecule selected from the group consisting of class I MHC molecules, class II MHC molecules, interleukins, TNFα , IFNγ, RANTES, G-CSF, M-CSF, IFNα, CTAPIII, ENA-78, GRO, I-309, PF-4, IP-10, LD-78, MGSA, MIP-1α, MIP-1β and combinations the same. 25. METHOD according to claim 20, characterized in that the photocleavable linker is conjugated to the surface of the particle by means of a portion of biotin. 26. METHOD, according to claim 20, characterized in that the affinity probes are of the same class or same type of compound as the active portions. 27. COMPOSITION, characterized in that it comprises an engineered biological target, prepared by the method, as defined in claim 1. The COMPOSITION of claim 27, characterized in that said engineered-modified biological target is an exosome, said exosome comprising a plurality of surface-bound active fractions. 29. COMPOSITION according to claim 28, characterized in that said active fractions are antigenic peptides or antigenic epitopes thereof. 30. COMPOSITION according to claim 29, characterized in that said antigenic peptide is selected from MAGE-A3, gp-100, HER-2, p53, PSA-1 and MART-1. 31. COMPOSITION according to claim 29, characterized in that said antigenic peptide is selected as to specificity for an immunostimulatory molecule selected from the group consisting of class I MHC molecules, class II MHC molecules, interleukins, TNFα , IFNγ, RANTES, G-CSF, M-CSF, IFNα, CTAPIII, ENA-78, GRO, I-309, PF-4, IP-10, LD-78, MGSA, MIP-1α, MIP-1β and combinations the same. 32. A COMPOSITION according to claim 31, characterized in that said antigenic peptide and said immunostimulating molecule bind to form an immunostimulating complex on the surface of said exosomes, which increases antigen presentation and activation of immune system cells and the initial immune responses of these exosomes. 33. THE COMPOSITION of claim 27, characterized in that said engineered-modified biological target is an exosome, said exosome comprising a plurality of active agents loaded therein. 34. COMPOSITION according to claim 33, characterized in that said active agents are selected from the group consisting of nucleotides, drugs, chemotherapeutics, small molecule compounds, CRISPR Cas9 systems and the like. 35. METHOD OF ACTIVATION OF CELLS of the immune system and/or initial immune response, characterized in that said method comprises contacting an immune system cell with a composition, as defined in any one of claims 27 to 34. 36. METHOD according to claim 35, characterized in that said contact comprises the administration of said composition to a subject in need thereof. 37. The method according to claim 36, characterized in that said method is effective to activate said subject's innate or adaptive immune system against a condition. 38. METHOD according to claim 37, characterized in that said condition is an infection. 39. METHOD, according to claim 37, characterized in that the condition is cancer. 40. METHOD according to claim 39, characterized in that it further comprises the administration of a chemotherapeutic agent. 41. MICROFLUID DEVICE for use in engineering modification of a biological target, characterized in that said device comprises: a cell culture chamber sized to maintain biological material in a three-dimensional configuration; a mixing channel fluidly connected to the cell culture chamber and comprising a plurality of sample inlet channels disposed along the mixing channel, wherein the ratio of a cell culture chamber width to the largest cross-sectional dimension of the mix channel is at least 5:1; an isolation channel defining a path for fluid flow from the mixing channel to an isolation outlet; and a collection chamber fluidly connected to the isolation outlet and comprising a magnet operatively coupled to the collection chamber to produce a magnetic field within the collection chamber. 42. MICROFLUID DEVICE for use in engineering modification of a biological target, characterized in that said device comprises: a cell culture chamber comprising a cell culture inlet and a cell culture outlet, a fluid inlet channel and a particle inlet, wherein the cell culture outlet, fluid inlet, and particle inlet fluidly converge at a mixing intersection; a mixing channel fluidly connected to the mixing intersection and defining a path for fluid flow from the mixing intersection to a mixing outlet, in which the ratio of a cell culture chamber width to the largest cross-sectional dimension of the mixing channel is at least 5:1; and a collection chamber fluidly connected to the mixing outlet and comprising a magnet operatively coupled to the collection chamber to produce a magnetic field within the collection chamber. 43. MICROFLUIDIC DEVICE, according to claim 42, characterized in that the mixing channel comprises an isolation channel disposed between the mixing intersection and the mixing outlet. 44. MICROFLUIDIC DEVICE, according to any one of claims 41 to 43, characterized in that the isolation channel has a serpentine geometry. 45. The MICROFLUID DEVICE according to any one of claims 41 to 44, characterized in that the isolation channel comprises a channel constriction domain that decreases in width. 46. MICROFLUID DEVICE according to claim 45, characterized in that the isolation channel comprises a plurality of channel constriction domains for producing local vortex flow profile, preferably at least 5 channel constriction domains. 47. MICROFLUIDIC DEVICE, according to any one of claims 41 to 46, characterized in that the ratio of the width of the cell culture chamber to the largest cross-sectional dimension of the mixing channel is from 5:1 to 500:1, from 5 :1 to 20:1 or from 6:1 to 12:1. A MICROFLUID DEVICE according to any one of claims 41 to 47, characterized in that the cell culture chamber has a volume of about 200 microliters or more, preferably from about 200 microliters to about 1 milliliter. A MICROFLUID DEVICE according to any one of claims 41 to 48, characterized in that the mixing channel has a height and a width, where each height and width is at least 50 microns, preferably between 50 and 500 microns. 50. MICROFLUIDIC DEVICE, according to any one of claims 41 to 49, characterized in that it further comprises a pump operatively coupled to the microfluidic device.