CN113195103A

CN113195103A - Method for generating a therapy delivery platform

Info

Publication number: CN113195103A
Application number: CN201980084740.9A
Authority: CN
Inventors: 何美
Original assignee: University of Kansas; Kansas State University
Current assignee: University of Kansas; Kansas State University
Priority date: 2018-10-21
Filing date: 2019-10-21
Publication date: 2021-07-30
Also published as: KR20210088594A; CA3117030A1; WO2020086471A1; BR112021007508A2; EP3866981A4; IL282538A; JP2022512762A; AU2019368213A1; EP3866981A1

Abstract

A method for producing engineered exosomes and other vesicle-like biological targets in a microfluidic device, comprising reacting and binding target vesicle-like structures with immunomagnetic particles; capturing the immunomagnetic particle/vesicle complexes by applying a magnetic field; further engineering the trapped vesicles by surface modification with additional active moieties or internal loading with active agents; and releasing the engineered vesicle-like structure, such as by photolytically breaking the bond between the particle and the engineered vesicle-like structure, thereby releasing the intact vesicle-like structure, which can serve as a delivery vehicle for therapeutic treatment.

Description

Method for generating a therapy delivery platform

Cross Reference to Related Applications

The priority OF U.S. provisional patent application serial No. 62/748,470 entitled "MICROFLUIDIC ON-DEMAND CAPTURE, LOADING, AND light-induced RELEASE OF EXTRACELLULAR vesicles AND EXOSOMES as vaccine delivery PLATFORM (microfludic ON-DEMAND capsule, load, AND PHOTO-RELEASE OF EXTRACELLULAR membrane VESICLES AND exosmomes AS VACCINE DELIVERY PLATFORM", filed ON day 21/10/2018, the entire contents OF which are incorporated herein by reference.

Federally sponsored research or development

The present invention was made with government support from 2017-. The government has certain rights in the invention.

Sequence listing

The following applications contain a sequence listing in Computer Readable Format (CRF) submitted as an ASCII formatted text file entitled "sequence listing" created on 21 days 10 months in 2019 at 12 KB. The contents of CRFs are incorporated herein by reference.

Background

Technical Field

The present disclosure relates generally to microfluidic devices and methods for harvesting intact carriers or delivery vehicles (delivery vehicles) for delivery of bioactive therapeutic agents, such as peptides or proteins, nucleotides, and other active agents (e.g., chemicals and drugs).

Description of the Related Art

Of all deliverable cells and nanoparticles, extracellular vesicles of viable cell origin, in particular exosomes (exosomes) in the nanometer size range of 30-150nm, have shown an important role in intercellular communication in recent decades. Exosomes of immune cell origin have been well documented in regulating immune stimulation or suppression, driving inflammation, autoimmunity, and infectious disease pathology. Formation of exosomes begins with the creation of endosomes (endosomes) as intracellular vesicles. Exosomes differ from other membrane-derived microvesicles by being derived from a multivesicular body (MVB) for cellular secretion. Thus, exosomes comprise specific proteins and nucleic acids and represent their parental cellular state and function as they are formed in the parental cell. In many subtypes of exosomes, immunogenic exosomes with intrinsic payloads of MHC class I and class II molecules and other costimulatory molecules are able to mediate immune responses, opening opportunities for the development of novel delivery platforms that can be used for cancer vaccines, immunotherapeutic delivery, and other delivery associated with in vivo trafficking.

Exosomes are highly biocompatible nanocarriers of living cell origin with an intrinsic payload, and exhibit greater flexibility in loading the desired antigen for efficient delivery than other nano-sized delivery systems such as lipids, polymers, gold, and silica materials. Exosomes also eliminate allergic reactions, eliminate concerns about carrying virulence factors, and avoid degradation or loss during delivery. However, the development of exosome-based vaccines has been hampered by significant technical difficulties in obtaining pure immunogenic exosomes. Different subtypes of exosomes may confound the study of information to distinguish different cells. On the other hand, molecular engineering of exosomes loaded on the membrane surface or internally can provide an unused resource for the development of novel antigen exosomes.

Bioengineered exosomes as emerging delivery vehicles have received extensive attention in the development of a new generation of cancer vaccines, including recent phase II trials using IFN-DC derived exosomes loaded with MHC I/II restricted cancer antigens to promote T-cell and Natural Killer (NK) cell based immune responses in non-small cell lung cancer patients. Unfortunately, current methods of exosome engineering, such as transfection or extrusion of parental cells, and membrane permeabilization of secreted exosomes, suffer from low yield, low purity, and time-consuming manipulations. Methods of producing exosomes are needed to address this bottleneck problem. Due to the inherent features in automation and efficient mass transport, microfluidic systems overcome many of the disadvantages of desktop systems and show excellent performance in the separation, detection and molecular profiling of exosomes. However, molecular engineering of exosomes using microfluidic platforms has not been explored. Currently, most of the reported work to treat exosomes is either of small mass or bound to solid surfaces/particles, and they do not remain intact for downstream therapeutic agents.

Disclosure of Invention

Methods and microfluidic devices are described herein to engineer various biological carriers or delivery vehicles, such as cells, extracellular vesicles and exosomes, and membrane or lipid particles, as well as polymer particles. These carriers can be captured using the methods and devices of the invention, loaded (surface modified or encapsulated) with active agents, and then released as intact, engineered carriers for delivery of various therapeutic compounds and bioactive agents. Engineered vectors may be used for diagnostic, prognostic, companion assay (companion assay), pharmaceutical and therapeutic agents, immunotherapy and vaccine delivery, and tissue delivery, as well as other uses related to the in vivo transport of active agents. The embodiments described herein are examples with respect to exosomes. However, it will be appreciated that exosomes represent particularly challenging biological targets, such that the platform is expected to be applicable to other similar structures-vesicles or vesicle-like structures characterized by a liquid core and a membrane or bilayer-including cells (including T cells), microsomes, and the like. These biological targets, which are then engineered as carriers or delivery vehicles, can also be characterized as nanocarriers.

Provided herein are microfluidic analytical devices and methods for on-demand capture, loading, and photoreleasing of intact engineered nanocarriers. The microfluidic device may enable real-time harvesting and antigen modification of extracellular vesicles, particularly exosomes, followed by release of intact exosomes downstream as required. Magnetic nanoparticles functionalized with photocleavable affinity probes (active moieties) for capture and on-demand release of MHC-I positive exosomes via light triggering are also disclosed. Affinity probes may include antigenic peptides, antibodies, aptamers, nanobodies, and other affinity-based probes. The light-induced release of the modified/loaded exosomes in the microfluidic device can be well controlled spatially and temporally with an efficiency of 95% or higher. Such a functional streamlined microfluidic cell culture system allows for antigen engineering of exosomes by using stimuli to mediate growth of their parental cells or direct molecular engineering on the surface of the resulting exosomes. Heterogeneity of extracellular somatic subtypes has been found from the same population of parental cells. The released exosome subtypes contain different molecular and biological properties for different cellular regulation. The disclosed methods can capture, load and release specific subsets of exosomes with more targeted therapeutic functions. Carriers for capture, loading and release that can be used in the methods of the present disclosure include cells, extracellular vesicles and exosomes, and membrane or lipid particles and polymer particles, which can encapsulate drugs (small molecule compounds), genes and bioactive therapeutic agents. Proof of concept for several tumor antigen peptides (e.g., gp-100, MAGE-A3, and MART-1) that are commonly used in the development of cancer vaccines but are difficult to deliver due to degradation has been demonstrated herein. Microfluidic devices show high efficiency in engineered immunogenic exosomes (MHC I +) with downstream photoinduced release of intact functional exosomes. Cellular uptake by engineered exosomes of antigen presenting cells has also been demonstrated, exhibiting much improved internalization capacity compared to non-engineered exosomes. In particular, by attacking CD 8T cells purified from the spleen of 2Pmel1 transgenic mice, engineered exosomes showed significantly higher activation rates of activated T cells (at least 30%) compared to non-engineered exosomes. We also evaluated the degree of efficacy of antimicrobial peptide-engineered immunogenic exosomes for in vitro stimulation of T cells using transgenic mice for the treatment of Bovine Respiratory Syncytial Virus (BRSV) infection. Exosomes engineered with BRSV targeting peptides (peptide 4: M187-195 peptide NAITNAKII, SEQ ID No:4) have the ability to activate BRSV M-specific T cells in the presence of activated dendritic cells.

Thus, engineered exosomes are viable and functional for use in cancer immunotherapy and vaccination of infectious diseases. The convenient and low cost microfluidic platform for producing engineered vesicles not only provides a viable strategy for efficient production of purified, enriched therapeutic vesicles, but also serves as a research tool for understanding the role of variable peptide engineered exosomes in anti-tumor immune responses, cancer immunotherapy and vaccination for the treatment of infections.

In particular aspects, the microfluidic devices disclosed herein can include a cell culture chamber sized to hold a 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 a ratio of a width of the cell culture chamber to a maximum cross-sectional dimension of the mixing 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 isolated outlet and comprising a magnet operably coupled to the collection chamber to generate a magnetic field within the collection chamber.

In other aspects, a microfluidic device can include a cell culture chamber including a cell culture inlet and a cell culture outlet; a fluid inlet channel and a particle inlet channel, wherein the cell culture outlet, the fluid inlet channel and the particle inlet channel fluidically converge at a mixing intersection; a mixing channel fluidly connected to the mixing junction and defining a path for fluid flow from the mixing junction to a mixing outlet, wherein a ratio of a width of the cell culture chamber to a maximum 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 operably coupled to the collection chamber to generate a magnetic field within the collection chamber. In these aspects, the mixing channel may comprise an isolation channel disposed between the mixing junction and the mixing outlet.

The isolation channels in the microfluidic device may have a geometry that induces turbulence to mix the fluid flowing in the device. For example, an isolation channel in a microfluidic device can have a serpentine geometry. The isolation channel may further comprise one or more channel constriction regions of reduced width to create a localized swirling flow distribution. In certain embodiments, the isolation channel may comprise a plurality of channel constriction domains, preferably at least 5 channel constriction domains.

As described herein, a microfluidic device includes a cell chamber and a mixing channel. The cell chambers and the mixing channel may have a height and a width. The height and width of the mixing channel may each be at least 50 microns, preferably between 50 and 500 microns. The ratio of the cell culture chamber width to the maximum cross-sectional dimension of the mixing channel may 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 may have a volume of about 200 microliters or greater, preferably about 200 microliters to about 1 milliliter.

The microfluidic devices disclosed herein may further comprise a pump operably coupled to the device.

Also disclosed are methods for capturing a target, loading, and on-demand light release in a sample solution using the device, useful for harvesting intact delivery vehicles. Such carriers can be cells, extracellular vesicles and exosomes, and membrane or lipid particles as well as polymer particles, which can encapsulate drugs, genes and bioactive therapeutic agents. In particular, this integrated and continuous method of trapping, loading, and photo-release can produce engineered exosomes in microfluidic devices. The method comprises introducing a biological sample comprising exosomes (or other targets) into a mixing channel and mixing the exosomes with immunomagnetic particles and a wash buffer to form a mixture; allowing the exosomes to react and associate with the immunomagnetic particles; and collecting exosomes bound to the immunomagnetic particles by applying a magnetic field within a collection chamber. Methods for producing engineered exosomes may include introducing cells into a cell culture chamber of a microfluidic device and first culturing the cells under conditions that allow for release of exosomes. This can be performed in-line in the same microfluidic device used for capture and loading.

The cells from which the exosomes are released may be selected from dendritic cells, stem cells, immune cells, megakaryocytic progenitor cells, macrophages or other living cells.

Immunomagnetic particles bound to exosomes may comprise magnetic particles bound to affinity probes for capturing exosomes via moieties comprising photocleavable linkers. As described herein, affinity probes for capturing exosomes may comprise antigenic peptides, antibodies, aptamers, or epitopes thereof for capturing exosomes. Suitable examples of antigenic peptides include: MAGE-A3, gp-100, HER-2, p53, PSA-1, or MART-1. The moiety comprising the photocleavable linker may comprise biotin bound to the immunomagnetic particle and attached at the other end to the affinity probe via the photocleavable linker. The affinity probes target surface proteins (e.g., immunostimulatory molecules or markers) on the target. Suitable immunostimulatory molecules include MHC class I molecules, MHC class II molecules, interleukins, TNF α, IFN γ, RANTES, G-CSF, M-CSF, IFN α, CTAPIII, ENA-78, GRO, I-309, PF-4, IP-10, LD-78, MGSA, MIP-1 α, MIP-1 β, and combinations thereof. Preferably, the affinity probe itself is the antigenic portion (peptide) of the surface protein that binds preferentially to the target.

After mixing, the immunomagnetic particles capture/bind exosomes (or other targets) in the sample. For example, the immunomagnetic particles are immobilized in the device using a magnet located adjacent to the collection chamber. The immobilized bead/exosome complexes are then washed and incubated with a buffer solution containing active moieties for surface loading onto or encapsulation inside the captured exosomes, as described in more detail below. The method may further comprise photolytic cleavage of the captured, modified exosomes from the immunomagnetic particles, releasing intact, engineered exosomes comprising active agents (antigenic peptides or antigenic epitopes thereof).

Methods for producing engineered biological targets, such as exosomes, may be performed using the microfluidic devices disclosed herein. In some embodiments, the method is performed in real-time. In one or more embodiments, the method is streamlined (also known as "continuous") such that capture, loading, and release of the biological target occur in the same device/vessel (i.e., without having to be transferred or moved between vessels or reaction tubes, but rather in-line along the microfluidic channel and in-line capture/engineering chambers). Thus, each of the steps can be performed sequentially, one after the other, and preferably substantially immediately after the other, using various inlets that converge at a single microfluidic channel. In other words, the method involves immunomagnetic bead loading followed immediately or nearly simultaneously by sample loading followed by loading of the active agent to be attached or loaded into the captured target. Each component loaded into the microfluidic device converges from a respective inlet into a single microfluidic channel, followed by "auto" retention in the capture/engineered chamber downstream of the inlet by a magnet located in an adjacent chamber. As the fluid mixture flows through the channel and then through the chamber, the corresponding reactions occur in real time (i.e., capture and loading). Then, applying light to the chamber may release the engineered target. It should be appreciated that the streamlined process is much faster than the traditional desktop approach. Preferably, the process from loading the sample/beads at the respective inlets to collecting the released engineered target at the outlet can be completed in a total of about 2 hours, more preferably about 90 minutes, and more preferably about 1 hour.

Also disclosed are compositions comprising engineered exosomes, in particular immunogenic exosome complexes. The immunogenic exosome complex may comprise an antigenic peptide or epitope thereof conjugated to the surface of exosomes, wherein the immunogenic exosome complex activates T cells by at least 30% compared to native exosomes. Such antigen loading of the antigenic peptide or antigenic epitope may be performed after mixing and capturing of exosomes, to completely coat the exosome surface with the antigenic peptide. The compositions can be prepared using the methods disclosed herein. Thus, an immunogenic exosome complex is prepared by a method comprising: introducing cells into a cell culture chamber of a microfluidic device; culturing the cells under conditions that allow release of the engineered exosomes; introducing engineered exosomes into a mixing channel and mixing the engineered exosomes with immunomagnetic particles and a wash buffer to form a mixture for exosome capture/binding; then applying a magnetic field within the collection chamber to collect the separated exosomes, allowing reaction with a loading buffer comprising the antigenic peptide to form immunogenic exosome complexes; and applying UV light to break the photocleavable linker for collection of the immunogenic exosome complexes at the outlet of the microfluidic device.

Pharmaceutical compositions comprising the immunogenic exosome complexes are also disclosed. The loading targets can be drugs, genes, and bioactive therapeutic agents. Disclosed are methods of treating a disease in a subject, the method comprising administering to the subject a pharmaceutical composition comprising an immunogenic exosome complex. In certain embodiments, the disease may be an infection. In some examples, the disease may be cancer. When the disease to be treated is cancer, the method may further comprise administering a chemotherapeutic agent.

Brief description of the drawings

This patent or application document contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the office upon request and payment of the necessary fee.

Fig. 1 illustrates (a) mixing of immunomagnetic beads with a sample solution, (B) capturing of targets in the sample solution using immunomagnetic beads; and (C) loading the antigen or active agent onto or into the captured target.

Fig. 2A illustrates (a) the application of light or activating radiation to the bead/target complex, and (B) the light-induced release of the engineered target (of the carrier) whose surface is modified with an active agent or antigenic moiety.

Fig. 2B illustrates (a) the application of light or activating radiation to the bead/target complex, and (B) the light-induced release of the engineered target (carrier) with the active agent loaded inside.

Figure 3 is an illustration of a process overview of 3D printing molded PDMS microfluidic culture chips for streamlined engineering of antigen exosomes for use in activating anti-tumor responses.

Fig. 4 illustrates an embodiment of a microfluidic device.

Fig. 5 shows the following images: (a) microfluidic channels and flow; (b) mixing with the micro-bead; (d) cell morphology; and (e) SEM images of released exosomes.

Fig. 6 illustrates an embodiment of a microfluidic device.

Figure 7A shows an illustration of immunomagnetic capture and on-demand photoreleasing of MHC-I positive immunogenic exosomes.

Figure 7B shows the characterization of three tumor targeting peptide antigens conjugated to photocleavable immunomagnetic beads for binding and photorelease of fluorescently labeled immunogenic exosomes. MHC-I antibodies were used as positive controls to compare the strength of binding between MHC-1 positive exosomes and tumor targeting peptides.

Figure 8A shows a characterization of the performance of on-demand photoreleasing captured exosomes from immunomagnetic capture beads. The positive control was a fluorescently labeled antibody captured by a photo-released immunomagnetic bead. The negative control was immunomagnetic beads without photocleavable linkers.

Fig. 8B shows an SEM image of the surface of the photo-released immunomagnetic beads with exosomes captured. Due to the vacuum sample preparation, the exosome particles appeared to be cupped.

Fig. 8C shows an SEM image of the surface of the photo-released immunomagnetic beads after photo-fragmentation.

Fig. 8D shows a characterization of the effect of UV exposure time on photodisruption efficiency.

Fig. 8E shows nanoparticle tracking analysis of the exosome size distribution between engineered exosomes and non-engineered exosomes.

Fig. 9A shows confocal microscopy images of DC uptake of Tumor Targeting Antigen (TTA) peptide, gp-100 surface engineered exosomes, compared to non-engineered exosomes. One image per hour was taken to track uptake of green fluorescence labeled exosomes by DCs (stained nuclei with DAPI).

Figure 9B shows a comparison between non-engineered exosomes and gp-100 engineered exosomes monitored for 48 hours of cytokine IFN- γ release from DC cultures by ELISA measurements.

Figure 10A depicts representative flow diagrams in wells containing T cell + activated JAWS cells from gp100 engineered exosomes with increasing concentrations.

Fig. 10B depicts cumulative data from all three culture conditions showing the rate of CD8+ T cell division under stimulation. Results are representative of 2 independent experiments using duplicate wells per culture condition.

Figure 11 depicts a flow cytometry plot of wells containing T cells and activated JAWS cells from Bovine Respiratory Syncytial Virus (BRSV) antimicrobial peptide engineered exosomes with increasing concentrations for depicting immunogenic efficacy.

Fig. 12A illustrates a 3D printing method for producing a 3D mold integrated with cell culture and downstream exosome separation, surface engineering and on-demand photo-release.

Figure 12B shows the results of replicating a PDMS microfluidic device.

Figure 13 shows the results of a study of the side effects of UV exposure on the content of exosome molecules in protein, DNA and RNA.

Figure 14 shows dendritic monocyte cultures under different stimulation conditions: dendritic monocytes without any stimulation (negative control; first image); PWM protein stimulation (positive control; second image); and gp-100 engineered extracellular body stimulation (last image).

Detailed Description

The following description of the present disclosure is provided as an enabling teaching of the best mode or modes of carrying out the present disclosure as currently known. To this end, those skilled in the relevant art will recognize and appreciate that many changes can be made to 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 utilizing other features. Accordingly, those who work in the art will recognize that many modifications and adaptations to the present disclosure are possible and can even be desirable in certain circumstances and are a part of the present disclosure. Accordingly, the following description is provided as illustrative of the principles of the present disclosure and not in limitation thereof.

Term(s) for

Unless defined otherwise, all technical and scientific terms used herein 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 complete understanding of the terms used in this specification.

Disclosed are the components used to prepare the disclosed compositions, as well as the compositions themselves, for use in the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if particular fluid channels are disclosed and discussed, and some modifications that can be made to the fluid channels are discussed, then the individual and each combination and permutation of the fluid channels, and the modifications that are possible, are specifically contemplated unless specifically indicated to the contrary. Thus, if an example of one type of fluid channel A, B and C and one type of fluid channel D, E and F and a combination fluid channel are disclosed, or, for example, a combination fluid channel including A-D is disclosed, then even if each is not individually recited, each is individually and collectively contemplated, meaning that combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E and C-F are both considered disclosed. Also, any subset or combination of these is also disclosed. Thus, for example, the subgroups of A-E, B-F and C-E will 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 of these additional steps can be performed with any specific embodiment or combination of embodiments of the methods of the present disclosure.

It should be understood that the devices disclosed herein have certain functionality. Certain structural requirements are disclosed for performing the functions of the present disclosure, and it is to be understood that there are various structures associated with the disclosed structures that can perform the same function, and that these structures will ultimately achieve the same result.

Unless expressly stated otherwise, it is in no way intended that any method described herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not explicitly recite an order to be followed, or where steps are not otherwise specifically stated in the claims or descriptions to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This applies to any possible non-explicit basis for interpretation, including: logical issues 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 specification.

As used in the specification and claims, the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "agent(s)" includes a plurality of agents, including mixtures thereof.

As used herein, the terms "may," "may optionally," and "may optionally" are used interchangeably and are meant to include instances where a condition occurs and instances where a condition does not occur. Thus, for example, a statement that a formulation "may include an excipient" is meant to include both the case where the formulation includes an excipient and the case where the formulation does not include an excipient.

Ranges can be expressed as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. It will be further understood that the respective endpoints of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It will also be understood that there are some values disclosed herein, and that in addition to the value itself, there is also disclosed herein a value for each of which is "about" that particular value. For example, if the value "10" is disclosed, then "about 10" is also disclosed.

The terms "upstream" and "downstream" refer to a location within a device relative to another location and the direction of fluid flow. As used herein, the term "upstream" refers to a first location that is located in a direction opposite to the direction of fluid flow relative to a second location. Conversely, as used herein, the term "downstream" refers to a second location that is located in a direction along the direction of fluid flow relative to the first location.

Method

Referring to fig. 1, the general method described herein involves a plurality of immunomagnetic particles (beads) mixed with a sample solution suspected of containing a target. In some embodiments, a biological sample is collected from a subject and prepared for the method, e.g., by dilution with a buffer, concentration, or the like. In some embodiments, the cells are collected and expanded in culture. In some embodiments, the cells are collected and cultured under conditions that release exosomes or other extracellular vesicles or vesicle-like structures into the culture. In any case, the immunomagnetic particle comprises a photocleavable linker and an affinity probe (and preferably a plurality of photocleavable linkers, each with a corresponding affinity probe) extending from the surface of the particle to capture the target. The immunomagnetic particles are contacted with the sample solution for a time sufficient to allow the target (if present in the sample) to interact with the affinity probes extending from the immunomagnetic particles. In fig. 1, for ease of illustration, a single bead with a single linker is depicted; however, in practice, each immunomagnetic particle will be coated with a plurality of linkers (preferably substantially the entire surface area of the particle/bead is coated with linkers). Further, the relative dimensions in fig. 1 are not to scale, but are exaggerated for illustrative purposes. In practice, the beads/particles are preferably at least 5 times larger than the target (e.g., in the case of exosomes, they are in the 30-150nm diameter range, the beads are preferably 500nm or more). In this way, multiple targets (e.g., exosomes) will be captured on the surface of a single bead/particle. For ease of reference, fig. 1 uses a single bead and target interaction. As shown in fig. 1(a), the beads and sample solution are mixed for a sufficient period of time (e.g., in a mixing chamber or channel in a microfluidic device). If the target is present in the sample solution, it will be captured by the beads (and in particular the affinity probes extending from the beads via their photocleavable linkers), as illustrated in fig. 1 (B). Exemplary photocleavable linkers are described herein and may include a linear chain that includes a biotin or similar moiety at one end for attachment to a particle and an amine moiety at the other end for attachment to an affinity probe. Preferred linkers have the following structure, wherein the dashed line represents a bond that is broken during light exposure, and the "binding group" represents a moiety (e.g., biotin) for attaching the linker (either directly or via a functionalized surface coating, e.g., avidin) to the bead:

although various embodiments are described herein, an affinity "probe" is typically an oligopeptide sequence that is specific for and recognizes a target, such as a peptide that recognizes and serves as a receptor for a surface protein on the target. As used herein, the phrase "specific for … …" is intended to distinguish between non-specific binding or reaction between an affinity probe and a molecule, and means that the set of specific targets that an affinity probe can interact with is limited, and in some cases even exclusive, such that no binding occurs at an appreciable rate with any other molecule other than the target (and in particular, the surface protein(s) it specifies). Short oligopeptide sequences are preferred for affinity probes, which include sequence fragments with high specificity for the target. More preferably, as described in more detail herein and demonstrated in the working examples, upon binding, the affinity probe and the surface protein create a complex that enhances the immunogenic potential of the target.

Beads with captured target (e.g., exosomes) are immobilized in a microfluidic device. The beads may be immobilized before or after target capture. As described elsewhere herein, this can be accomplished by placing a magnet adjacent to a collection or engineered chamber in the microfluidic device. As the sample solution and bead solution flow through the microfluidic channel, the beads and target interact, thereby capturing the target. As the solution flows through, the magnetic beads are immobilized in the collection or engineering chamber (and thus also the captured target). As illustrated in fig. 1(C), the captured target is then engineered by attaching multiple active agents (e.g., antigenic peptides) to the target surface or loading the target with drugs, chemicals, nucleotides or other biologically active agents (e.g., CRISPR Cas 9). For surface modification, in a microfluidic device, the immobilized bead/target complex is washed and incubated with a buffer solution comprising a plurality of active agents having at least one moiety specific for a surface protein present on the target surface. Preferably, the active agent or moiety used for surface loading is of the same "type" (e.g. comprises the same oligopeptide) as the compound selected as an affinity probe for use in the immunomagnetic particle. The immobilized bead/target complex is incubated with the active agent for a period of time sufficient for the active agent to interact with the captured target. Preferably, the concentration of active agent loaded into the microfluidic device is such that substantially the entire surface of the target is coated with the active agent (e.g., preferably, substantially all of the target surface protein is bound by the active agent). Instead of surface modification, fig. 1(C) also depicts an alternative where an active agent can be loaded into the target. This can be implemented by: in a microfluidic device, the immobilized bead/target complex is washed and incubated with a buffer solution containing the active agent to be loaded along with a detergent or chemical transfection reagent to induce pore formation in the target for active agent loading, followed by washing with a buffer to remove the reagent and close the pores. In either embodiment, the excess active agent is then washed away, leaving the engineered target immobilized with immunomagnetic beads.

Referring to fig. 2A, the engineered target may then be released by exposing the immobilized bead/target complex to activating radiation (e.g., light) of an appropriate wavelength to cleave the photocleavable linker. This process releases the engineered target along with the affinity probe still bound to the target, which now acts as an engineered carrier or delivery vehicle for the active agent modified on the surface of the target. Likewise, in fig. 2B, the same photo-release process can be used to release the active agent-loaded target internally. A wash buffer may be introduced into the microfluidic device to transport the released target downstream for collection at the outlet of the microfluidic device. Advantageously, the photoreleasing step in embodiments of the present invention is carried out with an exposure time of 15 minutes or less, preferably about 13 minutes or less, more preferably about 12 minutes or less. As demonstrated in the working examples, about 100% of the captured target is preferably released/fragmented over an exposure time of about 10 minutes.

It will be appreciated that since the magnetic beads have been immobilised in the engineered chamber, no separate step is required to separate the target from the magnetic beads in solution. Rather, after exposure, the linker between the captured target and the bead is broken, releasing the engineered target, which flows downstream from the immobilized bead to the outlet of the microfluidic device. The released targets (also known as "engineered carriers") that have been engineered with active moieties can then be collected from the outlet of the microfluidic device for analytical and therapeutic use or transferred directly to a further chamber or collection device. It will be appreciated that the immunomagnetic beads can then be collected for reuse by removing the magnetic field in the microfluidic device such that the immunomagnetic beads are no longer magnetically immobilized. The beads may be washed downstream and collected from the outlet.

As noted, this process can be advantageously performed in a microfluidic device and as a continuous, integrated, on-line method for isolating, capturing, engineering, and releasing intact targets, such as exosomes, from a sample as therapeutic vectors or delivery vehicles.

Device for measuring the position of a moving object

Extracellular vesicles (< 1 μm), particularly exosomes (30-150nm), are emerging cargo for mediating cell signaling. However, due to time consuming (>10h) and extremely cumbersome isolation protocols, standard benchtop methods (e.g. ultracentrifugation and filtration) lack the ability to specifically process immunogenic exosomes in other microvesicle subtypes. The present disclosure addresses a need in the art by providing a device for introducing a streamlined microfluidic platform for direct harvesting, antigen modification, and light-induced release of immunogenic extracellular vesicles and exosomes from cell culture media cultured on a chip. These devices provide automated and rapid cell culture production of antigen exosomes, useful for immunotherapy, such as cancer immunotherapy. As depicted in the overview of fig. 3, the devices disclosed herein enable real-time harvesting and antigen modification of exosomes with subsequent on-demand photoinduced release downstream.

Turning now to fig. 4, disclosed herein is a microfluidic device (200) comprising: a cell culture chamber (210), the cell culture chamber (210) sized to hold a biological material in a three-dimensional configuration; a mixing channel (220), the mixing channel (220) being fluidly connected to the cell culture chamber and comprising a plurality of sample inlet channels (222, 224, 226) disposed along the mixing channel, wherein a ratio of a width of the cell culture chamber (210) to a maximum cross-sectional dimension of the mixing channel (220) is at least 5: 1; an isolation channel (230), the isolation channel (230) defining a path for fluid flow from the mixing channel (220) to an isolation outlet (234); and a collection chamber (240), the collection chamber (240) fluidly connected to the isolated outlet (234) and comprising a magnet operably coupled to the collection chamber to generate a magnetic field within the collection chamber (240).

The devices of the present disclosure may be described by the size of the components within the device and a comparison (e.g., ratio) of the sizes. 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 microliter 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 about 200 microliters to about 1 milliliter (e.g., 200 microliters to 900 microliters, 200 microliters to 750 microliters, 200 microliters to 500 microliters, 300 microliters to 750 microliters, or 350 microliters to about 500 microliters). In some embodiments, the cell culture chamber has sufficient volume so that the top can remain open for application of a plug (such as a PDMS-made finger plug) for fluid exchange and pushing of fluid to the downstream collection channel.

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 micrometers (e.g., 500 micrometers or more, 600 micrometers or more, 650 micrometers or more, 700 micrometers or more, 750 micrometers or more, 800 micrometers or more, 850 micrometers or more, 900 micrometers or more, 950 micrometers or more, or 1000 micrometers 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 has a height of 500 to 1000 microns (e.g., 600 to 1000 microns, 750 to 1000 microns, or 800 to 1000 microns).

The cell culture chamber can have a width of at least 200 micrometers (e.g., 250 micrometers or more, 275 micrometers or more, 300 micrometers or more, 350 micrometers or more, 400 micrometers or more, 450 micrometers or more, 500 micrometers or more, 550 micrometers or more, or 600 micrometers or more). In some embodiments, the cell culture chamber has a width of 1000 microns or less (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 has a width of 250 to 1000 microns (e.g., 250 to 750 microns, 250 to 500 microns, 300 to 750 microns, or 300 to 500 microns).

As described herein, a mixing channel (220) is fluidly connected to the cell culture chamber and includes 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 B-inlet, 222) fluidly connected to the cell culture chamber and defining a path for introducing fluid from the cell culture chamber to the mixing channel. The plurality of sample inlet channels may further comprise a particle inlet channel (also referred to herein as a-inlet, 224) defining a path for introducing particles to the mixing channel. The plurality of sample inlet channels may further comprise a fluid inlet channel (also referred to herein as C-inlet, 226) defining a path for introducing a fluid, such as a wash buffer, to the mixing channel. The plurality of sample inlet channels may be in any arrangement. For example, the cell culture inlet channel may be upstream of the particle inlet channel, which is upstream of the fluid inlet channel. In other examples, the particle inlet channel may be upstream of the cell culture inlet channel, which is upstream of the fluid inlet channel.

The cell culture inlet channel, the particle inlet channel, and the fluid inlet channel may converge fluidly at a mixing intersection. The cell culture inlet channel forms a path for fluid to flow from the cell culture chamber to the mixing junction. The particle inlet channel forms a path for fluid flow from the particle inlet to the mixing junction. The fluid inlet channel forms a path for fluid to flow from the fluid inlet to the mixing junction. As used herein, the path of fluid flow may be represented graphically in the figures by arrows to indicate the direction of fluid flow through the path of fluid flow.

The mixing channel, which includes the cell culture inlet channel, the particle inlet channel, and the fluid inlet channel, 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, 450 microns or less, 400 microns or less, less than 400 microns, 350 microns or less, 300 microns or less, less than 300 microns, 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 to 500 microns (e.g., 100 to 500 microns, 200 to 500 microns, 100 to 350 microns, or 200 to 500 microns).

The mixing channel can have a width of at least 50 micrometers (e.g., 75 micrometers or more, 100 micrometers or more, 120 micrometers or more, 150 micrometers or more, 175 micrometers or more, 200 micrometers or more, 250 micrometers or more, 300 micrometers or more, 350 micrometers or more, 400 micrometers or more, or 500 micrometers or more). In some embodiments, the mixing channel has a width of 500 microns or less (e.g., less than 500 microns, 450 microns or less, 400 microns or less, less than 400 microns, 350 microns or less, 300 microns or less, less than 300 microns, 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 width of 50 to 500 micrometers (e.g., 100 to 500 micrometers, 200 to 500 micrometers, 100 to 350 micrometers, or 200 to 500 micrometers).

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 maximum 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. The ratio of the culture chamber width to the maximum cross-sectional dimension of the mixing channel is greater than one (e.g., 2:1), which defines a narrowing of the channel width at the entrance of the mixing channel.

In some embodiments, one or more channels in a microfluidic device may include a fluid mixing mechanism that facilitates mixing of fluids flowing through the device. The fluid mixing mechanism induces turbulence to mix the flowing fluid. Suitable mixing mechanisms include serpentine or tortuous channels, channel projections or recesses, channel bends, and other known mechanisms.

In some embodiments, a fluid mixing mechanism may be present in a mixing channel and/or isolation channel of a microfluidic device. For example, the microfluidic device may include an isolation channel fluidly connecting the mixing channel to the isolation outlet. The separation channel may form part of the mixing channel or may be separate. In some embodiments, the isolation channel defines a path for fluid to flow from the mixing channel to the isolation outlet. The isolation channel may include a serpentine geometry that enhances mixing as the fluids merge. Referring to fig. 4, an isolation channel (230) having a serpentine geometry may be placed at a location within the device that facilitates fluid mixing, e.g., between the mixing junction and the collection chamber. In some embodiments, the isolation channel is placed in close proximity to the mixing junction (e.g., in close downstream proximity).

The isolation channel may have a similar or narrowed width compared to the width of the mixing channel. In some embodiments, the isolation channel can have a narrowed width compared to the width of the mixing channel in at least several ways. For example, the isolation channel can include one or more channel constrictions disposed within the isolation channel between the isolation channel inlet and the isolation channel outlet (see, e.g., channel constrictions (232) forming a narrowing width in the isolation channel in fig. 4). The channel constriction region may create a local swirling flow distribution of the 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 of the channel constrictions may be a protrusion and/or a recess in the channel sidewall. The protrusions and/or recesses may have any shape, such as circular, linear, triangular, irregular, etc. The protrusion and/or recess may surround the inner wall of the channel (e.g., as a ring), or one or more protrusions and/or recesses may be located on one or more inner sidewalls of the channel. The inclusion of channel constriction regions can increase fluid flow turbulence and fluid mixing, if desired.

The devices disclosed herein may include a collection chamber (240) fluidly connected to the isolated outlet (234). In some embodiments, the device may further comprise a magnet operably coupled to the collection chamber to generate a magnetic field within the collection chamber. The magnet may be any magnet capable of providing a magnetic field within the collection chamber. In some embodiments, the magnetic field may comprise an oscillating magnetic field. An oscillating magnetic field is a magnetic field that varies regularly over time (e.g., automatically periodically) or irregularly (e.g., by user-based control). The oscillating magnetic field includes dynamic changes in the spatial direction of the north and south magnetic poles such that the direction of the magnetic field changes over time. Such variations may be periodic or irregular. Including an oscillating magnet capable of providing an oscillating magnetic field within the collection chamber can induce within the collection chamber dynamic movement of the magnetic probes (e.g., magnetic beads or particles) within the collection chamber in the direction of the magnetic field. The directional movement of the magnetic particles in the collection chamber will also change due to the change in direction of the magnetic field. This may be used to facilitate the interaction (and association/binding) between the magnetic particles and the target present in the fluid in the collection chamber. In some embodiments, the magnetic field may be obtained from a permanent magnet. The permanent magnet may be removed when not in use, for example, to turn off the magnetic field.

In some embodiments, the magnet may have any shape, such as a ring shape. In some embodiments, the magnet comprises a helmholtz coil or a permanent magnet.

The magnetic field may be present over 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. Further, the magnetic field may be on other portions of the microfluidic device. For example, the magnetic field may be over one or more of a portion or all of the mixing channel, mixing intersection, particle entry channel, or other component.

In some embodiments, the device may include a pump operably coupled to the microfluidic device. The pump may be any pump known in the art capable of inducing fluid flow within the device. In some embodiments, the pump may apply negative pressure within the device, thereby drawing fluid through the channel. Examples of suitable pumps may be found in US20170065978 and US20170001197, each of which is incorporated by reference in its entirety.

Turning now to fig. 6, a microfluidic device (2000) is also disclosed comprising: a cell culture chamber (2100), the cell culture chamber (2100) comprising a cell culture inlet (2102) and a cell culture outlet (2104); a fluid inlet channel (2202) and a particle inlet channel (2204), wherein the cell culture outlet (2104), the fluid inlet channel (2202), and the particle inlet channel (2204) are fluidically merged at a mixing junction (2200); a mixing channel (2300) fluidly connected to the mixing junction (2200) and defining a path for fluid flow from the mixing junction to a mixing outlet, wherein the ratio of the width of the cell culture chamber (2100) to the maximum cross-sectional dimension of the mixing channel (2300) is at least 5: 1; and a collection chamber (2400), the collection chamber (2400) being fluidly connected to the mixing outlet and comprising a magnet operably coupled to the collection chamber (2400) to generate a magnetic field within the collection chamber. The mixing channel (2300) may include an isolation channel (2302) disposed between the mixing junction (2200) and the mixing outlet.

Another aspect of the microfluidic devices provided herein relates to multiplexed microfluidic devices comprising two or more sets of chambers and/or channels comprising: a cell culture chamber, a mixing channel, an isolation channel, and a collection chamber. Configuring two or more such channels and/or chambers on a single microfluidic device can increase sample processing throughput and/or allow parallel processing of at least two samples or portions of samples of different compositions or manipulations. Two or more chambers and/or channels may be arranged in series, parallel, or a combination thereof.

In some embodiments of a parallel multiplexing microfluidic device, two or more mixing channels may have separate sample inlets disposed on the same microfluidic device. Such an arrangement may be applied to a plurality of fluid samples. Alternatively, multiple mixing channels may be connected to the same sample inlet to process the same fluid sample in parallel. In addition, two or more mixing channels may have separate outlets disposed on or connected to the same microfluidic device. In one or more embodiments, a multiplexed microfluidic device is contemplated having up to 96 sample inlets.

The microfluidic devices of the present disclosure may be used in conjunction with the various compositions, devices, methods, products, and applications disclosed herein. In some embodiments, the microfluidic device may be a independently operating microfluidic device. In some embodiments, one or more microfluidic devices may be integrated as part of a device, module, or system. In other embodiments, one or more microfluidic devices can be fluidically coupled to an apparatus, module, or system.

For example only, one or more microfluidic devices and/or multiplexed microfluidic devices may be fluidically coupled to the detection module. As used herein, the term "fluidically coupled" refers to two or more devices and/or modules connected in a suitable manner such that a fluid may be transferred or flowed from one device or module to another. When two or more devices and/or modules are fluidly coupled together, additional devices and/or modules may be present between the two or more devices and/or modules.

Alternatively, two of the two or more devices and/or modules may be connected such that fluid may be transferred or flowed 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 may be fluidly coupled, for example, by connecting an outlet of a first device or module to an inlet of a second device or module using a tube, conduit, channel, pipe, or any combination thereof.

The detection module can perform any of the detection methods disclosed herein or other methods known in the art. In some embodiments, the detection module may comprise a sample processing module prior to detecting the sample for analysis. For example, exosomes (including or not immunomagnetic particles) may be immunostained prior to detection by microscopy. Examples of detection modules may include, but are not limited to, microscopes (e.g., bright field microscopes, fluorescence microscopes, or confocal microscopes), spectrophotometers (e.g., ultraviolet-visible spectrophotometers), cell counters, biological cavity lasers (see, e.g., Gourley et al, J.Phys.D: appl.Phys.36: R228-R239(2003)), mass spectrometers, imaging systems, affinity columns, particle sorters such as fluorescence activated cell sorters, capillary electrophoresis, sample storage devices, and sample preparation devices. In some embodiments, the computer system may be connected to a detection module, for example, to facilitate the process of sample processing, detection, and/or analysis.

Manufacturing method

The devices described herein may be made of any material that is compatible with the fluid sample. In some embodiments, the materials described herein for making the devices can be penetrated by a magnetic field. In some embodiments, the materials used to fabricate the devices described herein can be substantially transparent such that the sample therein can be photodisrupted in situ, or it can be viewed under a microscope, for example, for in situ analysis of magnetically labeled exosomes. Exemplary materials that may be used to fabricate the microfluidic devices described herein may include, but are not limited to, glass, copolymers, polymers, or any combination thereof. Exemplary polymers include, but are not limited to, polyurethane, rubber, molded plastic, Polymethylmethacrylate (PMMA), polycarbonate, polytetrafluoroethylene (TEFLON)^TM) Polyvinyl chloride (PVC), Polydimethylsiloxane (PDMS), polysulfoneAnd ether-based aliphatic polyurethanes.

The methods used in making any of the embodiments of the microfluidic devices described herein may vary with the materials used and include 3D printing methods, soft lithography methods, microassembly, bulk micromachining methods, surface micromachining methods, standard lithography methods, wet etching, reactive ion etching, plasma etching, stereolithography and laser chemical three-dimensional writing methods, solid body printing, machining, modular assembly methods, replication molding methods, injection molding methods, thermoforming methods, laser ablation methods, combinations of methods, and other methods known in the art.

In particular embodiments, the microfluidic devices described herein can be manufactured using a 3D printer. For example, a method of manufacturing a microfluidic device may include providing a three-piece PDMS mold comprising a base, walls, and a top magnet holder, as shown in fig. 7. The mold can be printed out by a 3D printer. The mold may be coated with a 20nm thick Sportline palladium and subsequently assembled using methods known in the art. The dimensions of the PDMS cell chambers can be adjusted so that when filled, the cell culture chambers have open ends for the chamber plugs. PDMS can be cast from linker reagents in a ratio of 10:1 and incubated at a temperature of 40 ℃ for 6 hours. After the PDMS is cured, it can be easily peeled off. The entrance and exit of the chip may be perforated by using a hole puncher. The PDMS chip may then be post-bonded on a hot pad for 5min at a temperature of 40 ℃. The chip can be cleaned using DI water and sterilized by autoclave (30 min at 121 ℃).

Application method

As discussed herein, the disclosed microfluidic devices can be used to separate, capture, engineer, and release engineered extracellular vesicles and various vesicle biostructures. In certain embodiments, the engineered extracellular vesicles are immunogenic exosomes. As used herein, the term "exosome" generally refers to an externally released vesicle derived from: endosomal compartments or any cell, such as tumor cells (e.g., prostate cancer cells) and immune cells (e.g., antigen presenting cells, such as dendritic cells, macrophages, mast cells, T lymphocytes, or B lymphocytes). Exosomes are typically membrane vesicles having a size of about 20-150nm, which are released from a variety of different cell types including: tumor cells, red blood cells, platelets, lymphocytes, and dendritic cells. Exosomes may be formed by membrane invagination and budding from late endosomes. They can accumulate in cytoplasmic vesicles (MVBs), from which they can be released by fusion with the plasma membrane. Without wishing to be bound by theory, the process of vesicle shedding is particularly active in proliferating cells, such as cancer cells, where release may occur continuously. Exosomes, when released from tumor cells, can promote invasion and migration. Thus, in some embodiments, the immunomagnetic particles described herein can be used to capture exosomes derived from cancer cells. Depending on the cell source, exosomes can recruit a variety of cellular proteins that may differ from the plasma membrane, including MHC molecules, tetraspanins, adhesion molecules, and metalloproteinases. In many subtypes of exosomes, immunogenic exosomes with intrinsic payloads of MHC class I and class II molecules and other costimulatory molecules are able to mediate immune responses, opening opportunities for development of novel cancer vaccines and delivery in immunotherapy.

Accordingly, also provided herein are methods of producing immunogenic exosomes in the microfluidic devices disclosed herein. A method of producing an immunogenic exosome complex may comprise introducing cells into a cell culture chamber of a microfluidic device. The cells in the cell culture chamber may include any cell from which extracellular vesicles may be obtained. Such cells include dendritic cells, stem cells, immune cells, megakaryocytic progenitor cells, macrophages, or combinations thereof.

The method for producing immunogenic exosomes may further comprise culturing the cells under conditions that allow for release of the exosomes. In some embodiments, the method may comprise enriching or amplifying the number of exosomes present in the cell sample by mediating their parental cell growth 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, the cells may be cultured for a period of time, such as at least about 30min, at least about 45min, 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.

The method for producing an immunogenic exosome complex may further comprise mixing a cell culture comprising exosomes with immunomagnetic particles for capturing exosomes and a wash solution to form a mixture. In some embodiments, the method comprises introducing exosomes from a cell culture into a mixing channel, and mixing the exosomes with immunomagnetic particles and a wash buffer to form a mixture. The immunomagnetic particles can be introduced into the mixing channel via the particle inlet channel and the wash buffer can be introduced into the mixing channel via the fluid inlet channel.

The immunomagnetic particles can selectively bind to exosomes present in cell culture to form exosome-bound immunomagnetic particles. Thus, the method may comprise allowing exosomes to react with immunomagnetic particles. The immunomagnetic particles can include magnetic particles and can be any shape including, but not limited to, spherical, rod-shaped, elliptical, cylindrical, and disk-shaped. In some embodiments, magnetic particles having a substantially spherical shape and a defined surface chemistry may be used to minimize chemical agglutination and non-specific binding. As used herein, the term "magnetic particle" may refer to a nano-or micro-scale particle that is attracted or repelled by a magnetic field gradient or has a non-zero magnetic susceptibility. The magnetic particles may be ferromagnetic, paramagnetic or superparamagnetic. In some embodiments, the magnetic particles may be superparamagnetic.

The magnetic particles may be in the size range of 1nm to 5 microns. For example, the magnetic particles may be about 500nm to about 5 microns in size. In some embodiments, the magnetic particles may be about 1 micron to about 5 microns in size. In some embodiments, the magnetic particles may be about 1 micron to about 3 microns in size. Magnetic particles are a class of particles that can be manipulated using magnetic fields and/or magnetic field gradients. Such particles are generally composed 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 and commercially available. Particularly preferred particles are magnetic particles having graphene oxide layers or coatings comprising graphene oxide nanoplatelets, as described in US 2018/0100853 filed on 2017, 10, 9, the entire content of which is incorporated herein by reference.

The magnetic particles may be coated with a plurality of linkers including respective affinity probes (molecules) for capturing targets (such as antigenic peptides or antigenic epitopes thereof) that do not adversely affect the magnetic properties. In this regard, the magnetic particles may be functionalized with organic moieties or functional groups and photocleavable linkers that can attach the magnetic particles to corresponding affinity probes to capture exosomes. Such organic moieties or functional groups may generally include a direct bond or atom such as oxygen or sulfur, a unit such as an amino group, a carboxylic acid group, an epoxy group, a tosyl group, a silica-like group, a carbonyl group, an amide group, SO₂、SO₂NH, SS, or a chain of atoms.

In certain embodiments, the magnetic particles may be coated with one member of an affinity binding pair that may facilitate conjugation of the magnetic particles to affinity probes to capture exosomes. The term "affinity binding pair" or "binding pair" refers to a first and a second molecule that specifically bind to each other. One member of the binding pair is conjugated to the first moiety to be linked and the second member is conjugated to the second moiety to be linked. Exemplary binding pairs include any hapten or antigenic compound in combination with a corresponding antibody or binding portion or fragment thereof (e.g., digoxin and anti-digoxin; mouse immunoglobulin and goat anti-mouse immunoglobulin), as well as non-immunological binding pairs (e.g., biotin-avidin, biotin-streptavidin, biotin-neutravidin, hormones [ e.g., thyroxine and cortisol-hormone binding protein, receptor-receptor agonists, receptor-receptor antagonists (e.g., acetylcholine receptor-acetylcholine or analogs thereof), protein AG synthesized from IgG-protein A, IgG-protein G, IgG, lectin-carbohydrates, enzyme-enzyme cofactors, enzyme-enzyme inhibitors, and complementary oligonucleotide pairs capable of forming nucleic acid duplexes), and the like. The binding pair may also include a negatively charged first molecule and a positively charged second molecule.

One example of conjugation using a binding pair is biotin-avidin, biotin-streptavidin, or biotin-neutravidin conjugation. Thus, in some embodiments, the magnetic particles may be coated with an avidin-like molecule (e.g., streptavidin or neutravidin) that can be conjugated with a biotinylated linkage to serve as a capture molecule.

In some embodiments, the magnetic particles may be further functionalized with a cleavable chemical moiety that can attach the magnetic particles to an affinity probe to capture exosomes and is susceptible to externally applied cleavage agents/conditions, e.g., UV light, pH, redox potential, or the presence of a degradative molecule such as an enzyme. In a particular example, the cleavable linker may be conjugated to a member of a binding pair (such as biotin) at one functional end for attachment to a magnetic particle, and the other functional end provides an affinity probe for capturing exosomes. Thus, after separation of the exosome-bound magnetic particles from the fluid sample, exosomes may be separated from the magnetic particles by fragmenting the fragmentable chemical moieties between the magnetic particles and the affinity probes, if desired.

Exemplary cleavable linking groups include, but are not limited to, photocleavable and redox cleavable linking groups (e.g., -OC (O) NH-, -S-and-C (R))₂-S-S-, wherein R is H or C₁-C₆Alkyl groups); phosphate-based cleavable linking groups (e.g., -O-P (O) (OR) -O-, -O-P (S) (OR) -O-and-O-P (S) (H) -S-, wherein R is optionally substituted straight or branched chain C₁-C₁₀Alkyl groups); acid(s)Cleavable linking groups (e.g., hydrazones, esters and esters of amino acids, -C ═ NN-and-oc (o)); an ester group cleavable linking group (e.g., -C (O) O-); peptidyl cleavable linkers (e.g., linkers cleaved by enzymes such as peptidases and proteases in cells, e.g., -NHCHR_AC(O)NHCHR_BC (O) -, wherein R_AAnd R_BIs the R group of two adjacent amino acids).

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 derivatives and benzylsulfonyl groups such as 6-Nitroveratryloxycarbonyl (NVOC), 2-Nitrobenzyloxycarbonyl (NBOC), α -dimethyl-dimethoxybenzyloxycarbonyl (DDZ), ortho-nitrobenzyl (ONB), 1- (2-nitrophenyl) ethyl (NPE), α -carboxy-2-nitrobenzyl (CNB), 4, 5-dimethoxy-2-nitrobenzyl (DMNB), l- (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 tune the wavelength of absorption, and electron donating groups (e.g., methoxy groups) generally result in longer wavelength absorption. For example, the Nitrobenzyl (NB) and the Nitrophenylethyl (NPE) group are modified by adding two methoxy residues to the 4, 5-dimethoxy-2-nitrobenzyl group and the 1- (4, 5-dimethoxy-2-nitrophenyl) ethyl group, respectively, to increase the absorption wavelength range to 340-360 nm. Additional examples of light-removable protecting groups include polysubstituted nitroaromatics comprising a benzylic hydrogen ortho to the nitro group, wherein the substituents may include alkoxy, alkyl, halo, aryl, alkenyl, nitro, halo, or hydrogen. Other materials that may be used include o-hydroxy-a-methylcinnamoyl derivatives, photocleavable groups based on coumarin systems, such as BHC (such as described in U.S. patent No.6,472,541, the disclosure of which is incorporated herein by reference), photocleavable groups including pHP groups (such as described in Givens et al, j.am.chem.soc.1222687-2697 (2000), the disclosure of which is incorporated herein by reference), ketoprofen-derived linkers, other o-nitroaromatic core backbones including those that trap nitroso byproducts in a heterodiels Alder reaction (generally discussed in U.S. patent application No.2010/0105120, the disclosure of which is incorporated herein by reference), Nitrodibenzofuran (NDBF) chromophores or diazo-azides. Further examples of photocleavable groups can be found, for example, in 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.

As described above, a photocleavable group is one whose covalent attachment to a molecule (such as to a member of a binding pair, e.g., biotin, at one functional end and an affinity probe) is cleaved by exposure to light of an appropriate wavelength. In one aspect, release of the affinity probe and/or binding pair occurs when the conjugate is subjected to ultraviolet or near ultraviolet light. For example, the photo-release of the affinity probes may occur at a wavelength in the range of about 200 to 380nm (the exact wavelength or wavelength range will depend on the particular photo-cleavable 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 range therebetween). In another aspect, the release of the affinity probe may occur when the conjugate is subjected to visible light. For example, photo-release of the affinity probes may occur at a wavelength in the range of about 380 to 780nm (the exact wavelength or wavelength range will depend on the particular photo-cleavable group used, and may be, for example, 380, 400, 450, 500, 550, 600, 650, 700, 750, or 780, or some range in between).

As described herein, the magnetic particle further comprises an affinity probe (also referred to herein as a molecule for capturing 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 magnetic particles are described in US20170065978 and US20170001197, each of which is incorporated by reference in its entirety. The affinity probe may include a binding element that specifically binds to a target of interest (exosomes or other extracellular vesicles). Example (b)For example, the binding elements can be nucleic acid oligomers, antibodies, enzymes, hormones, growth factors, cytokines (e.g., inflammatory cytokines), proteins, peptides, prions, lectins, oligonucleotides, carbohydrates, lipids, molecules, and chemical toxins or other binding elements with high affinity and high specificity for a target, as well as binding elements specific for a given surface protein on the target. One or more binding elements (e.g., peptides) can be attached to the magnetic particle via a cleavable linker by methods known in the art. Typically, the binding member has an affinity constant (Ka) for a target, particularly an exosome or other extracellular vesicle, of greater than about 10⁵M^-1(e.g., 10)⁶M^-1、10⁷M^-1、10⁸M^-1、10⁹M^-1、10¹⁰M^-1、10¹¹M^-1And 10¹²M^-1Or higher).

In certain embodiments, the affinity probe comprises an antigenic peptide or an antigenic epitope thereof. As used herein, the term "antigen" refers to a molecule or portion of a molecule that is capable of being bound by a selective binding agent, such as an antibody, and that is otherwise capable of being used in an animal to elicit the production of antibodies that are capable of binding to an epitope of the antigen. An antigen may have one or more epitopes. The term "antigen" may also refer to a molecule capable of being bound by an antibody or a T Cell Receptor (TCR) (if presented by an MHC molecule). As used herein, the term "antigen" also encompasses T cell epitopes. The antigen is additionally capable of being recognized by the immune system and/or capable of inducing a humoral immune response and/or a cellular immune response, leading to the activation of B-and/or T-lymphocytes. The antigen may have one or more epitopes (B-and T-epitopes). The specific responses referred to above are intended to indicate that an antigen will preferably react with its corresponding antibody or TCR, typically in a highly selective manner, and not with a variety of other antibodies or TCRs that may be induced by other antigens. The antigen used herein may also be a mixture of several individual antigens.

As described above, the affinity probe (antigen) may be a protein or a peptide. In some embodiments, the protein or peptide can be substantially any protein capable of activating an immune cell and/or initiating an immune response, binding to a rare cell, such as a circulating tumor cell, stem cell, and/or microorganism. By way of example only, if the target species is cancer, exemplary proteins or peptides or other molecules that may 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 portion thereof, or an antibody-like molecule. In some embodiments, the capture molecule may be an antibody or portion thereof or an antibody-like molecule specific for detection of rare cells, such as circulating tumor cells, stem cells, and/or microbial biomarkers. In some embodiments, the affinity probe may be an aptamer. In some embodiments, the affinity probe may be a DNA or RNA aptamer. In some embodiments, the affinity probe may be a cell surface receptor ligand. Exemplary cell surface receptor ligands include, for example, cell surface receptor binding peptides, cell surface receptor binding glycopeptides, cell surface receptor binding proteins, cell surface receptor binding glycoproteins, cell surface receptor binding organic compounds, and cell surface receptor binding drugs. 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 rare cells such as circulating tumor cells, stem cells, and/or microorganisms can be used as an affinity probe on the magnetic particles described herein. In one or more embodiments, the affinity probes are selected to target immunostimulatory molecules present on the surface of a target (e.g., exosomes), such as MHC class I molecules, MHC class II molecules, interleukins, TNF α, IFN γ, RANTES, G-CSF, M-CSF, IFN α, CTAPIII, ENA-78, GRO, I-309, PF-4, IP-10, LD-78, MGSA, MIP-1 α, MIP-1 β, and combinations thereof. More preferably, upon binding of the affinity probe (and subsequent release of the target), the resulting engineered target comprising the bound affinity probe enhances the immunogenic potential of the released target. For example, binding of MHC class I surface proteins by affinity probes creates a complex that will (in therapeutic use) enhance the recognition and uptake of engineered targets by antigen presenting cells to stimulate and activate the immune system. The peptide is 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.

Exemplary affinity probes include those listed in the following table:

it will be appreciated that the aforementioned peptides suitable for use in affinity probes are also examples of active agents or moieties for surface loading onto engineered targets.

The immunomagnetic particles (i.e. the magnetic particles bound to the affinity probes) are preferably formed prior to the mixing process. For example, affinity probes (e.g., antigenic peptides bound to biotin via a cleavable linker) and streptavidin-coated magnetic particles are mixed together for an effective period of time for biotinylated affinity probe attachment to substantially completely coat the entire surface of the avidin-coated particles.

Thus, preferably, the affinity probes are added to the streptavidin-coated magnetic particles for a period of time before adding the cell culture comprising exosomes. In such embodiments, the affinity probes may be first added to the streptavidin-coated magnetic particles for a period of time sufficient to allow at least a portion of the added amount of affinity probes to bind to (and preferably fully bind to) the streptavidin-coated magnetic particles, thereby coating the entire surface of the particles with probe linkers extending therefrom.

Exosomes present in the cell culture containing exosomes are then added to the same fluid sample, where the exosomes can bind to affinity probes that have formed conjugates with streptavidin-coated magnetic particles.

In some embodiments, the immunomagnetic particles can be formed separately and then introduced into the mixing channel of the device.

The amount of immunomagnetic particles that need to be added to the sample may depend on a number of factors, including but not limited to the volume of the sample to be treated, the valency of the magnetic particles that can be conjugated to the affinity probes, the expected abundance of exosomes present, and any combination thereof. Too high amounts of immunomagnetic particles added to the device may cause non-specific binding and/or clogging within the microfluidic device. Too low an amount of immunomagnetic particles may result in low capture efficiency. The concentration of immunomagnetic particles and capture molecules can be determined by one skilled in the art.

The exosomes may be allowed to mix with the immunomagnetic particles for any period of time, e.g., seconds, minutes, or hours. In some embodiments, exosomes may be mixed with immunomagnetic particles for at least about 1min, at least about 2min, at least about 5min, at least about 10min, at least about 15min, at least about 30min, at least about 1 hour, at least about 2 hours, or longer. The optimal time for mixing can be readily determined by one of ordinary skill in the art based on a number of factors including, but not limited to, affinity of the immunomagnetic particles to exosomes, concentration, mixing temperature, and/or mixing speed. However, in one or more embodiments, the exosomes are mixed with the particles for 1 hour or less.

The exosomes and immunomagnetic particles can be introduced into the sample inlet of the microfluidic device at any flow rate that provides sufficient residence time for the mixture to remain in the mixing channel and isolation channel of the microfluidic device described herein. In some embodiments, can be 0.1uL/min to 1uL/min flow rate into the sample. The sample fluid may be introduced to the inlet of the microfluidic device by any method known to the skilled person. For example, a flow generator may be connected to at least one of the inlet and outlet of the microfluidic devices described herein. Non-limiting examples of flow generators may include peristaltic pumps, syringe pumps, and any art-recognized pump that may be generally used to flow fluids through microfluidic devices.

The method of producing an immunogenically engineered target may further comprise capturing exosomes bound to immunomagnetic particles by applying a magnetic field within the 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. The immobilized magnetically labeled exosomes may be removed from the microfluidic device for further processing. Preferably, the captured exosomes are further engineered and loaded with additional active moieties on the surface or internally as discussed herein. Subsequently, the method comprises photolytic cleavage of exosomes bound to immunomagnetic particles to release intact exosomes coated with active moieties or internally loaded with active agents.

The target of release (exosome) may be provided as a pharmaceutical composition. The pharmaceutical composition may include an immunogenic exosome and a pharmaceutically acceptable excipient. It will be appreciated that the active moiety may be tailored to provide a specific adaptive immune response against a target disorder, or may be more generally selected to activate the innate immune system against a variety of infections or disorders.

The methods described herein may be used to process samples in real time. For example, the method allows for real-time, continuous harvesting and antigen modification of exosomes, and subsequent on-demand photoinduced release downstream.

As described herein, the method can be used to produce immunogenic exosome complexes or other immunogenic vesicle-like structures. In certain embodiments, the immunogenic exosome complex may comprise an antigenic peptide conjugated to the exosome surface. The methods described herein for making immunogenic exosome complexes provide complexes with significantly higher activation rates of T cells than non-engineered exosomes. In some examples, the immunogenic exosome complex may activate at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, 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% of the T cells as compared to native exosomes. Due to the increased activation rate, the immunogenic complexes described herein may be used for cancer immunotherapy.

Accordingly, methods of treating a disease in a subject using the immunogenic complexes are disclosed. The method may comprise administering to the subject a composition comprising an immunogenic complex. In some embodiments, the disease may be an infection. In some examples, the disease may be cancer. The method may further comprise administering a chemotherapeutic agent already loaded into the target.

Additional advantages of various embodiments of the present invention will be apparent to those skilled in the art upon reading the disclosure herein and working examples below. It should be recognized that the various embodiments described herein are not necessarily mutually exclusive, unless otherwise indicated herein. For example, features described or depicted in one embodiment may also be included in other embodiments, but are not necessarily included. Thus, the present invention encompasses various combinations and/or integrations of the specific embodiments described herein.

As used herein, the phrase "and/or," when used in a list of two or more items, means that any one of the listed items can be applied alone, or any combination of two or more of the listed items can be applied. For example, if a composition is described as including or not including component A, B and/or C, the composition may or may not include a alone; b alone; c alone; a combination of A and B; a combination of A and C; a combination of B and C; or a combination of A, B and C.

The present description also uses numerical ranges to quantify certain parameters relating to various embodiments of the invention. It should be understood that when numerical ranges are provided, such ranges are to be construed 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 claims reciting "greater than about 10" (without an upper bound) and claims reciting "less than about 100" (without a lower bound).

The apparatus, systems, and methods of the appended claims are not to be limited in scope by the specific apparatus, systems, and methods described herein, which are intended as illustrations of some aspects of the claims. Any apparatus, system, and method that are functionally equivalent are intended to fall within the scope of the claims. Various modifications of the devices, systems, and methods, in addition to those shown and described herein, are intended to fall within the scope of the appended claims. Further, while only certain representative devices, systems, and method steps disclosed herein have been specifically described, other combinations of devices, systems, and method steps are intended to fall within the scope of the appended claims even if not specifically stated. Thus, combinations of steps, elements, components or ingredients may be referred to herein explicitly or less, however, other combinations of steps, elements, components or ingredients are included even if not explicitly stated.

As used herein, the term "comprising" and variants thereof are used synonymously with the term "comprising" and variants thereof, and are open, non-limiting terms. Although the terms "comprising" and "including" have been used herein to describe various embodiments, the terms "consisting essentially of and" consisting of may be used in place of "comprising" and "including" to provide more particular embodiments of the invention, and are also disclosed. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, all numbers expressing geometric shapes, dimensions, and so forth used in the specification and claims are to be construed in light of the number of significant digits and ordinary rounding approaches unless otherwise indicated.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed invention belongs. The publications cited herein and the materials to which they are cited are specifically incorporated by reference.

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

Examples

The following examples illustrate the process according to the invention. It is to be understood, however, that these examples are provided by way of illustration and nothing therein should be taken as a limitation on the overall scope of the invention.

Example 1: microfluidic on-demand engineering of exosomes for cancer immunotherapy

And (3) abstract: extracellular nanovesicles (< 1 μm), particularly exosomes (30-150nm), are emerging delivery systems that mediate cellular communication, which have been observed to initiate immune responses by presenting parental cell signaling proteins or tumor antigens to immune cells. In this example, a streamlined microfluidic cell culture platform is provided for harvesting, antigen modification and light-induced release of surface engineered exosomes directly in one working stream. The PDMS microfluidic cell culture platform was replicated from a 3D printed mold. By engineering antigenic peptides (e.g., gp-100, MART-1, MEGA-A3) on the surface of exosomes, efficient antigen presentation and T-cell activation can be achieved. This has been demonstrated by engineering secreted exosomes in real time with melanoma tumor peptides using on-chip cultures of human blood-derived leukocytes. Gp 100-specific CD 8T cells purified from the spleen of 2Pmel1 transgenic mice were tested. Significantly higher levels of T cell activation (-30%) induced by engineered exosomes were observed compared to non-engineered exosomes. This microfluidic platform serves as an automated and highly integrated cell culture device for the rapid and real-time production of therapeutic exosomes that can advance cancer immunotherapy.

Method and material

3D printing and microfluidic device fabrication: provides a three-piece mold for manufacturing PDMS chipsA tool includes a base, a wall, and a top magnet holder. The mold is formed by using

2017, and is printed out by a 3D printer of Project 1200 of the 3D System. The pieces had a finest structure of 50 μm and a channel height of 50 μm. The cell culture chamber was designed with a 1000 μm diameter, 500 μm height chamber. All molds were coated with Sportline palladium at a thickness of 20 nm. All three pieces were assembled using PDMS chips. PDMS was filled at a height of less than 500 μm, thus the cell culture chambers left open ends for the chamber plugs. PDMS was cast with linker reagent at a ratio of 10:1 and incubated at a temperature of 40 ℃ for 6 hours. After the PDMS is cured, it can be easily peeled off. The chip inlet and outlet were punched using a 0.75mm punch. Both the Piranha solution (Piranha) treated glass and PDMS were high pressure plasma for at least 30 seconds. The PDMS chips were then post-bonded on a hot pad for 5min at a temperature of 40 ℃. The chip was cleaned by DI water and sterilized by autoclave (30 min at 121 ℃).

On-chip cell culture and exosome collection, engineering and release: the cell cassettes (8mm coverslips) were first cleaned with distilled water and air dried in a biological hood. Then, they were autoclave-treated at 121 ℃ for 30 min. The cassette was placed in a 24-well plate and 500 μ L of 0.1mg/mL hydrobromic acid poly-D-lysine (MP Biomedicals) was added to each well and incubated at room temperature for 5 min. Add 1mL of MD water to each well for 3min and repeat twice to clean the cell box, and then place in a biological hood to air dry and store for future use.

mu.L of beta 2-microglobulin (Sigma-Aldrich) and 10. mu.L of each protein (gp100, MAGE-A3 and MART-1) were mixed with 186. mu.L of 1 XPBS into the modification solution in a final volume of 200. mu.L. The B-inlet was kept closed and the modification solution was pumped through the chip from the A-inlet and the wash buffer from the C-inlet at a volumetric flow rate of 1 μ L/min for 10min, and 0.1 μ L/min for 10min, and set to static for another 10 min. The washing step was carried out for 15min from both the A-inlet and the C-inlet at a volumetric flow rate of 1. mu.L/min. The bottom magnet was removed and the near UV was turned on to treat the main chamber for 10 min. Another wash step from the A-inlet and C-inlet was used, with a volumetric flow rate of 1 μ L/min for 20min, to collect about 20 μ L of disintegrated exosomes from the outlet.

Ultracentrifugation and exosome staining: the collected 20 μ L of exosomes were added to an ultracentrifuge tube and diluted to a final volume of 1mL for centrifugation at 1,500rcf for 30min (Thermo Scientific)^TMSorvall^TMMTX). The supernatant was removed and transferred to a fresh ultracentrifuge tube. The mixture was then treated at 100,000rcf for 1 hour. Exosomes were stained by PKH67 Green Fluorescent Cell-linked Midi Kit (PKH67 Green Fluorescent Cell Linker Midi Kit) (Sigma-Aldrich) for conventional Cell membrane labeling. The staining solution was prepared with 2. mu.L of PKH67 and 1mL of diluent C. Any residual solution in the tube was discarded and 1mL of diluent C was added to resuspend with a soft pipette. The stained solution was transferred to an ultracentrifuge tube, mixed with a pipette, and reacted at room temperature for 3.5 min. 2mL of FBS (extracellular body depletion) was added to quench the free dye. 1.5mL of 0.971M sucrose solution was added for density gradient centrifugation. An additional 6.5mL of complete medium was added to increase the volume to 10 mL. The ultracentrifuge was set at 100,000rcf for 1 hour. The supernatant was discarded and the stained loop was carefully washed without reaching the center of the loop. An additional 2mL of 1X PBS was added to resuspend the pellet. The ultracentrifuge was run at 100,000rcf for an additional 1 hour. The supernatant was aspirated off and an additional 100 μ Ι _ of 1X PBS was added to resuspend the pellet. All steps were maintained under sterile conditions and 1. mu.L of penicillin-streptomycin (A)

Catalog #30-2300, Lot #63525409) was added to the collected exosomes to inhibit and kill the bacteria remaining in solution. The collected exosomes were stored at 4 ℃ for less than 1 week, and at-20 ℃ for up to one month.

Extracellular body uptake: RPMI-1640 medium prepared by using ATCC (

Catalog #30-2001, Lot #64331683) plus 10% exosome-depleted FBS for whole media to culture THP-1 cells ((R)

TIB-202^TM). Monocytes were subcultured in number 8 × 105/mL and by using alternative medium change methods. Cells were treated with 5x 10⁵The density per mL was used for the extracellular body uptake experiments. Transfer 200 μ L of monocytes to 48-well plates for a total of 11 wells. 20 μ L of Normal Exosomes (NE) were added to 5 wells, and 20 μ L of Engineered Exosomes (EE) were also added to another 5 wells, and one well was kept as a negative control. The time intervals were set to 0 hour, 0.5 hour, 1 hour, 2 hours, 3 hours, and 4 hours. At each time period, 100 μ Ι _ of cell suspension medium was removed from the cytospin at 400RPM for 4 min. The glass slides were collected and 100. mu.L of fixative (C)

Catalog # R37814, Lot #17B285301) were added to the spots of cells. The mixture was incubated at room temperature for 18min, and then the solution was removed. 100 μ L of 1 XPBS buffer was added to the cell spots and left at room temperature for 3 min. The 1 × PBS buffer was removed, and the spots of cells were gently washed by distilled water. The slides were dried to leave no droplets on the slides, and 50. mu.L of 500nM DAPI: (R) ((R))

Catalog # D1306, Lot #1844202) were applied to the cell spots, protected from light, and incubated at room temperature for 4 min. The DAPI solution was then removed rapidly and then sufficient 1x PBS buffer was used twice for 2min each. The spots of cells were washed with distilled water and dried briefly to leave no droplets on the slides. Applying one drop of ProLong^TMGold Antifade Mountant(

Ref # P10144, Lot1887458), and covering the slide with 25x25#1.5 coverslip withoutAny trapped air bubbles. Slides were stored at room temperature for 24 hours prior to imaging under confocal microscopy.

Results

3D printing formed microfluidic cell culture device for on-line harvesting of exosomes: simple and low cost methods for fabricating PDMS based on-chip cell culture microfluidic devices using 3D printed molds have been developed. The culture chip comprises an on-chip cell culture chamber having a diameter of 1mm and a height of 0.5mm for growing cells on the chip and collecting exosomes derived from the culture medium downstream. The cell culture chamber was left open at the top to apply a PDMS made finger plug for media exchange and pushing media to the downstream collection channel. The bottom of the cell culture chamber has an outlet channel (B-inlet) approximately 200 μm wide and 200 μm high for introducing culture medium to mix with the immunomagnetic separation beads (A-inlet). The C-inlet was used to introduce a wash buffer driven by a syringe pump. FIG. 4 demonstrates the mixing process through the A-inlet and B-inlet and exit to the exosome isolation channel (serpentine channel) under observation with a fluorescence microscope using a fluorescent dye solution. Figure 5(b) records immunomagnetic beads mixed within serpentine channels. Human blood-derived leukocytes were cultured in a culture apparatus and had the morphology shown in fig. 5 (c). Very few red blood cells were observed to be goblet-shaped. Secreted exosomes were isolated, captured and photo-released from the exit of the chip and characterized by SEM imaging as shown in fig. 5 (d).

Photocleavable linkers are bifunctional conjugation at both ends with biotin and NHS chemistry. The biotin group anchors the photocleavable linker to the surface of streptavidin immunomagnetic beads, and the NHS group is conjugated to the MHC-I peptide via a primary amine, as shown in fig. 7A. MHC class I molecules are heterodimers consisting of two polypeptide chains, alpha and beta 2-microglobulin. The two chains are non-covalently linked via the interaction of b2m and the α 3 domain. The other two domains α 1 and α 2 are folded to form a groove for binding to an 8-10 amino acid peptide (MHC-I binding peptide). The MHC-I/peptide binding complex will be presented to cytotoxic T cells and subsequently used to trigger an immediate response of the immune system. Once MHC-I positive exosomes are captured by Tumor Targeting Antigen (TTA) peptides and retained by immunomagnetic beads in a capture chamber with a magnetic field, an antigen loading buffer with saturated TTA peptides will be introduced via the C-inlet to fully bind and occupy the remaining available MHC-I peptide binding sites. This antigen surface engineering process can greatly increase the loading of TTA peptide on captured MHC-I positive exosomes and enhance the efficacy of activating T cells.

Further characterized is the binding strength between MHC-I peptide modified photoreleased immunomagnetic beads and MCH-I positive exosomes labeled with fluorescence as shown in figure 7B. The MHC-I antibody served as a positive control for assessing the strength of binding between the tumor-targeted antigen peptide and the MHC-I positive exosomes. Due to the stronger binding strength between the MHC-I/peptide complexes, it has a higher potential to activate T cell anti-tumor responses. gp-100 has been shown to have a stronger ability to form MHC-I/peptide complexes and to have even stronger binding strength than MHC-I antibodies (95% versus 84.8%).

The performance of the on-demand photo-release is characterized in fig. 8A-8E. In comparison between the positive and negative controls, fluorescently labeled exosomes were captured and released by measuring the fluorescence intensity of bead aggregates under an inverted fluorescence microscope. SEM imaging methods were used to confirm the photo-release process. By comparing SEM images of the bead surface before and after photodisruption, no identifiable exosome particles were present on the bead surface, indicating good photorelease performance. UV exposure time was also characterized, reaching 98% photodisruption rate within 8 minutes UV exposure. The size distribution of engineered and non-engineered exosomes was evaluated, showing that suitable size ranges for exosomes are between 50nm-200nm, confirming that the engineered exosomes maintained good integrity.

The side effect of UV exposure on the level of exosome molecules was studied, showing that no changes could be detected in exosome proteins, DNA and RNA under 10min UV treatment (fig. 13).

To assess the efficacy and integrity of engineered exosomes released from microfluidic cell culture devices via on-demand light release, exosomes from the chip outlets were harvested and labeled with green fluorescence. Gp-100 engineered exosomes and non-engineered exosomes were incubated with dendritic monocytes to monitor cellular uptake every one hour. Cells were then fixed and stained for nuclei with DAPI. The green dots shown in fig. 9A are labeled exosomes, which are distributed in large numbers around the nucleus. Cellular uptake begins within an hour and uptake rates are much faster than non-engineered exosomes. After 4 hours, clearance of both engineered and non-engineered exosomes by the lysosomal pathway was observed. This observation indicates that gp-100 engineered exosomes are more active for dendritic monocyte uptake. The expression of the cytokine IFN-. gamma.was monitored by incubating gp-100 engineered exosomes with dendritic monocytes using ELISA. The expression level of IFN- γ was much higher, increasing almost 2-fold, 48 hours after continuous monitoring, compared to incubation with non-engineered exosomes. The gray dashed line in fig. 9B represents a positive control using PWM protein as a stimulus. The dendritic cell morphology after stimulation is shown in figure 14. Both PWM protein and gp-100 engineered exosomes had a significant effect on conversion to round floating dendritic cells compared to the non-stimulated negative control. Gp-100 engineered exosomes showed higher stimulation rates for the production of the cytokine IFN- γ compared to control PWM protein stimulation.

The efficacy of gp-100 engineered exosomes to activate CD8+ T cells undergoing proliferation and cytolysis was further investigated. It was observed that gp-100 engineered exosomes have the ability to activate transgenic T cells in the presence of activated dendritic cells. gp 100-specific CD 8T cells were purified by magnetic Cell sorting from spleens of 2Pmel1 transgenic mice and labeled with Cell tracer Violet (Cell Trace Violet) proliferation dye. Purified T cells were cultured alone (T cells only) and compared to naive

JAWS cells (immature dendritic cell line derived from C57BL/6 mouse) (T cells + JAWS cells) or JAWS cells activated with 200ng/mL for 48 hours (T cells + activated JAWS cells) were mixed at a ratio of 3: 1. Engineered exosomes with gp100 peptide were used to increase the ratio of exosomes: dendritic cells (25, 5)0 and 100) to a T cell culture. Fig. 10A. Cells and exosomes were co-cultured for 5 days and then CD 8T cells were analyzed by flow cytometry with cell-tracing purple dilution as a measure of proliferation. With T cell only conditions as a negative control, the observed proliferation rate of CD8+ T cells cultured with JAWS activated with gp-100 exosomes showed an increase of more than 30%, indicating that gp-100 engineered exosomes have a strong potency to activate T cell lysis. Fig. 10B. Microfluidic antigen-on-demand surface engineering and light-induced release for exosome development may be powerful tools for developing effective exosome-based vaccines and delivery systems to advance cancer immunotherapy.

The immunogenic efficacy of Bovine Respiratory Syncytial Virus (BRSV) was also studied. T cells and activated JAWS cells were incubated with increasing concentrations of BRSV antimicrobial peptide-engineered exosomes (exosomes engineered with peptide 4: M187-195 peptide NAITNAKII, SEQ ID NO: 4). Immune stimulation of CD8+ T cell proliferation responds linearly to the dose of engineered exosomes, which is more effective than using 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 20nM BRSV M187-196 in QuilA as adjuvant. At least 4 weeks after the final immunization, animals were euthanized and spleens were collected. CD8+ T cells and CD11c + splenic dendritic cells were isolated by magnetic cell isolation. CD8+ T cells were labeled with cell-tracing purple proliferation dye. Purified T cells were cultured alone (T cells only), or mixed with CD11c + splenic DCs (T cells + DCs) at a 3:1 ratio. DC cells were either left unstimulated or treated with 200ng/mL LPS to induce DC activation. Engineered exosomes loaded with BRSV peptides using the microfluidic platform described above were used to increase the rate of exosomes: dendritic cells (25, 50 and 100) were added to the T cell cultures. Negative control wells received no exosomes. Positive control wells were treated with 1nM or 5nM pure M187-196 peptide. Cells and exosomes were co-cultured for 5 days and then CD 8T cells were analyzed by flow cytometry with cell-tracing purple dilution as a measure of proliferation. The results are shown in FIG. 11. All the results support that the disclosed methods for capture, antigen loading and light-induced release can efficiently produce antimicrobial peptide engineered exosomes, leading to successful activation of T cells with high potency.

Example 2: in vivo administration of engineered exosomes

The above immunogenicity efficacy studies used transgenic mice to which engineered exosomes were injected using a proprietary published method. Exosomes were engineered with gp-100 or BRSV M187-196 peptides on the surface using the streamlined/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 reactions (injection site swelling, irritation, etc.) from injected mice. Mice were observed twice daily for the first 72 hours post-injection, and no adverse effects were observed, indicating the general in vivo safety of engineered exosomes and related compositions.

Sequence listing

<110> Kansas UNIVERSITY (THE UNIVERSITY OF KANSAS)

Kansasis UNIVERSITY RESEARCH FOUNDATION (KANSAS STATE UNIVERSITY RESEARCH FOUNDATION)

<120> method for generating a therapy delivery platform

<130> PPI21170858US

<150> US 62/748,470

<151> 2018-10-21

<160> 59

<170> PatentIn version 3.5

<210> 1

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 1

Ser Ile Ile Asn Phe Glu Lys Leu

1 5

<210> 2

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 2

Ser Tyr Ile Gly Ser Ile Asn Asn Ile

1 5

<210> 3

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 3

Lys Tyr Lys Asn Ala Val Thr Glu Leu

1 5

<210> 4

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 4

Asn Ala Ile Thr Asn Ala Lys Ile Ile

1 5

<210> 5

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 5

Glu Ala Asp Pro Thr Gly His Ser Tyr

1 5

<210> 6

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 6

Glu Val Asp Pro Ile Gly His Leu Tyr

1 5

<210> 7

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 7

Gly Leu Tyr Asp Gly Met Glu His Leu

1 5

<210> 8

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 8

Lys Val Ala Glu Leu Val His Phe Leu

1 5

<210> 9

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 9

Lys Val Leu Glu Tyr Val Ile Lys Val

1 5

<210> 10

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 10

Phe Leu Trp Gly Pro Arg Ala Leu Val

1 5

<210> 11

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 11

Lys Val Ala Glu Glu Leu Val His Phe Leu

1 5 10

<210> 12

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 12

Tyr Leu Gln Leu Val Phe Gly Ile Glu Val

1 5 10

<210> 13

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 13

Gly Val Tyr Asp Gly Arg Glu His Thr Val

1 5 10

<210> 14

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 14

Lys Val Val Glu Phe Leu Ala Met Leu

1 5

<210> 15

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 15

Leu Leu Phe Gly Leu Ala Leu Ile Glu Val

1 5 10

<210> 16

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 16

Ala Leu Lys Asp Val Glu Glu Arg Val

1 5

<210> 17

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 17

Thr Phe Pro Asp Leu Glu Ser Glu Phe

1 5

<210> 18

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 18

His Asn Thr Gln Tyr Cys Asn Leu

1 5

<210> 19

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 19

Lys Leu Gln Cys Val Asp Leu His Val

1 5

<210> 20

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 20

Gly Val Thr Tyr Ala Cys Phe Val Ser Asn Leu

1 5 10

<210> 21

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 21

Thr Tyr Ala Cys Phe Val Ser Asn Leu

1 5

<210> 22

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 22

His Leu Ser Thr Ala Phe Ala Arg Val

1 5

<210> 23

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 23

Ile Leu His Asn Gly Ala Tyr Ser Leu

1 5

<210> 24

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 24

Thr Tyr Leu Pro Thr Asn Ala Ser Leu

1 5

<210> 25

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 25

Ile Leu His Asp Gly Ala Tyr Ser Leu

1 5

<210> 26

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 26

Thr Tyr Val Pro Ala Asn Ala Ser Leu

1 5

<210> 27

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 27

Ile Met Asp Gln Val Pro Phe Ser Val

1 5

<210> 28

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 28

Val Tyr Phe Phe Leu Pro Asp His Leu

1 5

<210> 29

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 29

Lys Thr Trp Gly Gln Tyr Trp Gln Val

1 5

<210> 30

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 30

Val Leu Tyr Arg Tyr Gly Ser Phe Ser Val

1 5 10

<210> 31

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 31

Ile Thr Asp Gln Val Pro Phe Ser Val

1 5

<210> 32

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 32

Tyr Leu Glu Pro Gly Pro Val Thr Val

1 5

<210> 33

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 33

Tyr Leu Glu Pro Gly Pro Val Thr Ala

1 5

<210> 34

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 34

Lys Val Pro Arg Asn Gln Asp Trp Leu

1 5

<210> 35

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 35

Ala Leu Leu Ala Val Gly Ala Thr Lys

1 5

<210> 36

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 36

Val Tyr Phe Phe Leu Pro Asp His Leu

1 5

<210> 37

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 37

Lys Ile Phe Gly Ser Leu Ala Phe Leu

1 5

<210> 38

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 38

Leu Leu Gly Arg Asn Ser Phe Glu Val

1 5

<210> 39

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 39

Gly Leu Ala Pro Pro Gln His Leu Ile Arg Val

1 5 10

<210> 40

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 40

Ser Leu Pro Pro Pro Gly Thr Arg Val

1 5

<210> 41

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 41

Lys Leu Cys Pro Val Gln Leu Trp Val

1 5

<210> 42

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 42

Arg Met Pro Glu Ala Ala Pro Pro Val

1 5

<210> 43

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 43

Tyr Leu Gly Ser Tyr Gly Phe Arg Leu

1 5

<210> 44

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 44

Phe Leu Gly Tyr Leu Ile Leu Gly Val

1 5

<210> 45

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 45

Ala Leu Phe Asp Ile Glu Ser Lys Val

1 5

<210> 46

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 46

Ala Leu Gln Pro Gly Thr Ala Leu Leu

1 5

<210> 47

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 47

Glu Leu Ala Gly Ile Gly Ile Leu Thr Val

1 5 10

<210> 48

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 48

Phe Leu Thr Pro Lys Lys Leu Gln Cys Val

1 5 10

<210> 49

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 49

Leu Leu Leu Leu Thr Val Leu Thr Val

1 5

<210> 50

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 50

Gly Leu Met Glu Glu Met Ser Ala Leu

1 5

<210> 51

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 51

Arg Leu Leu Gln Glu Thr Glu Leu Val

1 5

<210> 52

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 52

Leu Ile Ala His Asn Gln Val Arg Gln Val

1 5 10

<210> 53

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 53

Val Ile Ser Asn Asp Val Cys Ala Gln Val

1 5 10

<210> 54

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 54

Cys Tyr Ala Ser Gly Trp Gly Ser Ile

1 5

<210> 55

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 55

His Cys Ile Arg Asn Lys Ser Val Ile

1 5

<210> 56

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 56

Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr

1 5 10

<210> 57

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 57

Tyr Leu Asn Thr Val Gln Pro Thr Cys Val

1 5 10

<210> 58

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 58

Val Ile Leu Thr Asn Pro Ile Ser Met

1 5

<210> 59

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic peptide

<400> 59

Phe Ser Asn Ser Thr Asn Asp Ile Leu Ile

1 5 10

Claims

1. A method for engineering a biological target in a microfluidic device, the method comprising:

mixing a biological sample suspected of containing the biological target with a plurality of immunomagnetic particles in a fluid to form a mixture in a microfluidic mixing channel of the microfluidic device;

reacting and binding the biological target, if present, to the immunomagnetic particles in the mixture to form particle/target complexes;

immobilizing the particle/target complex in a chamber of the microfluidic device by applying a magnetic field within the chamber;

engineering the target in the particle/target complex by contacting the target with a plurality of active moieties or agents, wherein the surface of the target is a surface modified with the active moieties, or wherein the plurality of agents are loaded within the target to produce an engineered target; and

photolytically releasing the engineered target from the particle/target complex, wherein the particle remains immobilized in the chamber, and wherein the engineered target is washed downstream to an outlet of the microfluidic device.

2. The method of claim 1, wherein the biological target is selected from the group consisting of: cells, extracellular vesicles, and vesicle-like cellular components.

3. The method of claim 2, wherein the extracellular vesicles are exosomes or microsomes.

4. The method of claim 3, the method further comprising:

introducing cells into a cell culture chamber of the microfluidic device;

culturing said cells under conditions that allow for release of said exosomes; and

introducing the exosomes into the mixing channel for mixing with the plurality of immunomagnetic particles.

5. The method of claim 4, wherein the cell is selected from the group consisting of: dendritic cells, stem cells, immune cells, megakaryocytic progenitor cells, and macrophages.

6. The method of claim 1, wherein the active moiety recognizes and specifically binds to a surface protein on the biological target.

7. The method of claim 6, wherein after the engineering step, substantially all of the surface protein is bound to an active moiety such that the target is substantially coated by the active moiety.

8. The method of claim 1, wherein the immunomagnetic particles have a diameter of 500nm or greater.

9. The method of claim 1, wherein each of the immunomagnetic particles reacts with a plurality of the biological targets in the sample such that the particle/target complex comprises a plurality of the biological targets bound to a single central particle.

10. The method of claim 9, wherein each particle binds a single type of biological target at a time, such that the same type of biological target binds to each particle.

11. The method of claim 1, wherein said photolytic release comprises exposing said particle/target complex to light in said microfluidic chamber.

12. The method of claim 11, wherein the target is released within about 15 minutes after the exposing.

13. The method of claim 1, wherein the method is performed in the microfluidic device in a continuous process, preferably within 90 minutes or less from the mixing to the collecting of the engineered target at the outlet.

14. The method of claim 1, wherein the active moiety is an antigenic peptide.

15. The method of claim 1, wherein the active agent is a drug, a nucleotide, a CRISPRCas9 system, a small molecule compound, a chemotherapeutic agent, or the like.

16. The method of claim 1, wherein the microfluidic device planar substrate comprises an inlet in fluid communication with the chamber via a microfluidic channel extending between the inlet and the chamber, the chamber being downstream of the inlet and in fluid communication with an outlet, the outlet being downstream of the chamber.

17. The method of claim 16, wherein the microfluidic device comprises a plurality of inlets for introducing the sample, the immunomagnetic particles, and a wash buffer into the device.

18. The method of claim 16, wherein the microfluidic channel has a serpentine flow path to facilitate mixing of the immunomagnetic particles and the sample.

19. The method of claim 16, the device further comprising a cell culture chamber in fluid communication with the microfluidic channel.

20. The method of any one of claims 1-19, wherein said immunomagnetic particles each comprise a plurality of photocleavable linkers extending from the surface of said particles, each having a respective affinity probe at its terminus for capturing said biological target.

21. The method of claim 20, wherein the affinity probe comprises an antigenic peptide or an antigenic epitope thereof.

22. The method of claim 21, wherein the affinity probe comprises an antigenic peptide selected from the group consisting of: MAGE-A3, gp-100, HER-2, p53, PSA-1, and MART-1.

23. The method of claim 20, wherein binding of the affinity probe to the biological target forms an immunostimulatory complex that enhances antigen presentation and immune cell activation and initiates an immune response upon release from the particle.

24. The method of claim 20, wherein the affinity probes are selected to be specific for an immunostimulatory molecule selected from the group consisting of: MHC class I molecules, MHC class II molecules, interleukins, TNF alpha, IFN gamma, RANTES, G-CSF, M-CSF, IFN alpha, CTAPIII, ENA-78, GRO, I-309, PF-4, IP-10, LD-78, MGSA, MIP-1 alpha, MIP-1 beta and combinations thereof.

25. The method of claim 20, wherein the photocleavable linker is conjugated to the particle surface via a biotin moiety.

26. The method of claim 20, wherein the affinity probe is from the same species or class of compound as the active moiety.

27. A composition comprising an engineered biological target prepared by the method of claim 1.

28. The composition of claim 27, wherein the engineered biological target is an exosome comprising a plurality of active moieties attached to a surface.

29. The composition of claim 28, wherein the active moiety is an antigenic peptide or an antigenic epitope thereof.

30. The composition of claim 29, wherein the antigenic peptide is selected from MAGE-a3, gp-100, HER-2, p53, PSA-1 and MART-1.

31. The composition of claim 29, wherein the antigenic peptide is selected to be specific for an immunostimulatory molecule selected from the group consisting of: MHC class I molecules, MHC class II molecules, interleukins, TNF alpha, IFN gamma, RANTES, G-CSF, M-CSF, IFN alpha, CTAPIII, ENA-78, GRO, I-309, PF-4, IP-10, LD-78, MGSA, MIP-1 alpha, MIP-1 beta and combinations thereof.

32. The composition of claim 31, wherein the antigenic peptide and the immunostimulatory molecule combine to form an immunostimulatory complex on the surface of the exosome, the immunostimulatory complex enhancing antigen presentation and immune cell activation of the exosome and initiating an immune response.

33. The composition of claim 27, wherein the engineered biological target is an exosome comprising a plurality of active agents loaded therein.

34. The composition of claim 33, wherein the active agent is selected from the group consisting of: nucleotides, drugs, chemotherapeutic agents, small molecule compounds, CRISPR Cas9 systems, and the like.

35. A method of activating an immune cell and/or initiating an immune response, the method comprising contacting an immune cell with the composition of any one of claims 27-34.

36. The method of claim 35, wherein said contacting comprises administering said composition to a subject in need thereof.

37. The method of claim 36, which is effective to activate the subject's innate or adaptive immune system for a disorder.

38. The method of claim 37, wherein the condition is an infection.

39. The method of claim 37, wherein the condition is cancer.

40. The method of claim 39, further comprising administering a chemotherapeutic agent.

41. A microfluidic device for engineering a biological target, the device comprising:

a cell culture chamber sized to maintain a 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 a ratio of a width of the cell culture chamber to a maximum cross-sectional dimension of the mixing 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 isolated outlet and comprising a magnet operably coupled to the collection chamber to generate a magnetic field within the collection chamber.

42. A microfluidic device for engineering a biological target, the device comprising:

a cell culture chamber comprising a cell culture inlet and a cell culture outlet,

a fluid inlet channel and a particle inlet channel, wherein the cell culture outlet, the fluid inlet channel and the particle inlet channel fluidically converge at a mixing intersection;

a mixing channel fluidly connected to the mixing junction and defining a path for fluid flow from the mixing junction to a mixing outlet, wherein a ratio of a width of the cell culture chamber to a maximum 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 operably coupled to the collection chamber to generate a magnetic field within the collection chamber.

43. The microfluidic device of claim 42, wherein the mixing channel comprises an isolation channel disposed between the mixing intersection and the mixing outlet.

44. The microfluidic device of any one of claims 41-43, wherein the isolation channel has a serpentine geometry.

45. The microfluidic device of any one of claims 41-44, wherein the isolation channel comprises a channel constriction domain of reduced width.

46. The microfluidic device according to claim 45, wherein the isolation channel comprises a plurality of channel constriction domains, preferably at least 5 channel constriction domains, for generating a local vortex flow distribution.

47. The microfluidic device of any one of claims 41-46, wherein the ratio of the cell culture chamber width to the maximum cross-sectional dimension of the mixing channel is 5:1 to 500:1, 5:1 to 20:1, or 6:1 to 12: 1.

48. The microfluidic device of any one of claims 41-47, wherein the cell culture chamber has a volume of about 200 microliters or greater, preferably about 200 microliters to about 1 milliliter.

49. The microfluidic device of any one of claims 41-48, wherein the mixing channel has a height and a width, wherein each of the height and width is at least 50 microns, preferably between 50 and 500 microns.

50. The microfluidic device of any one of claims 41-49, further comprising a pump operably coupled to the microfluidic device.