WO2024227924A1

WO2024227924A1 - Method for obtaining a cytokine response

Info

Publication number: WO2024227924A1
Application number: PCT/EP2024/062268
Authority: WO
Inventors: Albin JAKOBSSON; Maximilian OTTOSSON
Original assignee: Cellevate AB
Current assignee: Cellevate AB
Priority date: 2023-05-03
Filing date: 2024-05-03
Publication date: 2024-11-07
Anticipated expiration: 2025-11-03

Abstract

The present invention relates to a method for obtaining an immune response comprising one or more cytokines from a sample. In particular, an immune response of high quality is obtained by probing immune cells placed on a three-dimensional nanofibrous network.

Description

Method for obtaining a cytokine response

Technical field of the invention

Background of the invention

The human immune system is designed for protecting the human organism against disease. It comprises a range of highly specialized immune cells programmed to recognize and eliminate foreign substances that are thought to be harmful. This is achieved by several modes of actions, one of which is the protective response known as inflammation. The function of inflammation is to remove the initial cause of cell injury, clear out necrotic cells and damaged tissue, and initiate tissue repair. The reaction includes release of pro- inflammatory signalling molecules from immune cells to drive the inflammatory response. An important type of pro-inflammatory signalling molecules are cytokines.

Inflammation has to be tightly controlled by the human organism. Too little inflammation can lead to progressive damage to the tissue, and too much inflammation may lead too chronic inflammation, such as rheumatoid arthritis, which is caused in part by production of pro-inflammatory cytokines. A special case is when a foreign substance causes the immune system to respond by a massive release of pro-inflammatory cytokines into the circulatory system and the inflammatory response flares out of control. This situation is referred to as cytokine release syndrome (CRS) or cytokine storm. This highly unwanted situation may be caused by pathogens or as an adverse effect of administration of a therapeutic agent. CRS is by example suspected to be one of the leading causes of Acute Respiratory Distress Syndrome (ARDS) suffered by some patients infected with SARS- CoV-2.

The severity of CRS is indisputable, and it is regarded as top of the list of undesired immunotoxological adverse effects in drug development. Accordingly, it is standard practice to evaluate the cytokine release response induced by candidate therapies as part of the toxicology assessment. This need has become particularly eminent because regulators have questioned the reliability of preclinical testing strategies of new therapies, hereunder the projected safety based on animal models. Any therapeutic agent with the potential to interact with the immune system should be evaluated, including small molecules, biologies and substances targeting membrane-bound receptors. Cytokine release assays (CRAs) are in vitro assays in which immune cells collected from healthy subjects are exposed to a candidate therapeutic agent to evaluate any cytokine response caused by the interaction. Typically, whole blood or isolated lymphocyte samples are dosed with a candidate therapeutic agent which is benchmarked against a known control substance, such as a well-characterized immunoregulatory agent or another drug. The benchmark substance is preferably working via a similar mechanism of action. After exposure of the immune cells to the candidate therapeutic agent, the supernatant is recovered and the content of key cytokines, such as IL-2, IL-6, IL-10, IFNy and TNFo, is determined by multiplex technology.

Unfortunately, the presently available CRA platforms do not consistently produce cytokine responses that are reflective of the native response of the immune cells. Either the CRAs do not generate strong enough cytokine responses or they lack the capability to generate a representative diverse cocktail of cytokines. The result is an inability to discriminate between weak and moderate cytokine responses, and a failure to accurately determine relevant thresholds which when exceeded may be associated with unacceptable adverse effects.

Thus, there is an unmet need for provision of CRAs that more accurately resemble the native cytokine response of immune cells.

Hence, it would be advantageous to provide a cytokine release assay (CRA) that consistently facilitates strong and diverse cytokine secretion.

Specifically, it would be advantageous with an efficient method that can rapidly determine the content of every key cytokine secreted from a sample comprising immune cells when dosed with a candidate therapeutic agent.

Summary of the invention

The method for obtaining an immune response presented herein is based on seeding of immune cells on a three-dimensional scaffold comprising nanofibers. The nanofiber matrix offers an environment upon which the immune cells readily proliferate and where the native physiological properties of the immune cells are retained. The activity of the immune cells is therefore preserved, resulting in a strong and native-like cytokine response upon exposure foreign substances. Thus, an object of the present invention relates to the provision of a method in which immune cells upon exposure to a therapeutic agent or a known immunoregulatory agent produce a native-like immune response.

Another object of the present invention relates to provision of an efficient method for obtaining a high quality cytokine response from a sample comprising immune cells, leading to improved sensitivity of a cytokine release assay and the ability to predict with higher certainty the risk of adverse effects associated with a therapeutic agent.

Thus, an aspect of the present invention relates to a method for obtaining an immune response comprising one or more cytokines from a sample, said method comprising the steps of:

(i) provision of a container comprising one or more nanofibers,

(ii) addition of a sample comprising immune cells to said container, and

(iii) addition of a therapeutic agent to said container, thereby obtaining said immune response from said sample.

Another aspect of the present invention relates to use of a container comprising one or more nanofibers for obtaining an immune response comprising one or more cytokines from a sample comprising immune cells, wherein said sample are subjected to a therapeutic agent.

A further aspect of the present invention relates to a kit comprising: a container comprising one or more nanofibers,

- at least one immunoregulatory agent,

- optionally, instructions for use.

Brief description of the figures

Figure 1 shows quantification of selected cytokines following stimulation of PBMCs cultured in either a two-dimensional (2D) tissue culture plate (Market standard) or a three- dimensional (3D) nanofibrous network (3D nanofiber). (A) Quantification of (A) MIP-ip, (B) IL-5, (C) IL-6, and (D) IL-lra response from PBMCs following stimulation with CD3/CD28 beads. The no stimuli control is the response when no stimuli has been added (data pooled from both market standard plates and 3D nanofiber plates).

Figure 2 shows quantification of cytokines following stimulation of PMCs with CD3/CD28 beads when cultured in either a two-dimensional (2D) tissue culture plate (Market standard) or a three-dimensional (3D) nanofibrous network (3D nanofiber). Absolute numbers have been normalised to the concentration of cytokines obtained when using the 3D nanofiber plate. The control comprises media but no stimulant (data pooled from both market standard plates and 3D nanofiber plates).

Figure 3 shows quantification of selected cytokines following stimulation of PBMCs cultured in either a two-dimensional (2D) tissue culture plate (Market standard) or a three- dimensional (3D) nanofibrous network (3D nanofiber). (A) Quantification of (top) IL-ip and G-CSF response from PBMCs following stimulation with LPS, and (bottom) IL-1 [3 and IL-6 response from PBMCs following stimulation with ConA. The no stimuli control is the response when no stimuli has been added (data pooled from both market standard plates and 3D nanofiber plates).

Figure 4 shows quantification of cytokines following stimulation of PMCs with LPS when cultured in either a two-dimensional (2D) tissue culture plate (Market standard) or a three- dimensional (3D) nanofibrous network (3D nanofiber). Absolute numbers have been normalised to the concentration of cytokines obtained when using the 3D nanofiber plate. The control comprises media but no stimulant (data pooled from both market standard plates and 3D nanofiber plates).

Figure 5 shows quantification of cytokines following stimulation of PMCs with ConA when cultured in either a two-dimensional (2D) tissue culture plate (Market standard) or a three- dimensional (3D) nanofibrous network (3D nanofiber). Absolute numbers have been normalised to the concentration of cytokines obtained when using the 3D nanofiber plate. The control comprises media but no stimulant (data pooled from both market standard plates and 3D nanofiber plates).

Detailed description of the invention

Definitions

Prior to outlining the present invention in more details, a set of terms and conventions is first defined:

Cytokines

In the present context, the term "cytokines" refers to a group of small proteins that are involved in cell signalling. They may be produced by immune cells, such as macrophages, B lymphocytes, T lymphocytes and mast cells, in response to an extracellular stimuli. Cytokines may have autocrine, paracrine, and/or endocrine activity, which can influence immune and inflammatory responses. Cytokines include, but are not limited to, chemokines, interferons, interleukins, lymphokines, colony-stimulating factors (CSFs), and tumour necrosis factors (TNFs).

Cytokine response

In the present context, the term "cytokine response" refers to the set of cytokines secreted by a cell or population of cells upon extracellular stimuli. The extracellular stimuli can be a pathogen or a therapeutic agent.

A cytokine response may comprise a single cytokine or a plurality of different cytokines. The particular combination of cytokines in the response is referred to as the cytokine profile.

Nanofiber

In the present context, the term "nanofiber" refers to fibers with diameters in the range of 50-1500 nm. The fibers may be generated from different types of polymers, such as polycaprolactone (PCL).

The nanofibers may be prepared by any method. Such methods include, but are not limited to, electrospinning, meltblowing, drawing, self-assembly, template synthesis, and thermal-induced phase separation.

Container

In the present context, the term "container" refers to any vessel suitable for storing and/or culturing of cells. The container is preferably a conventional culturing vessel, including, but not limited to, a cell culturing plate, multiwell plate, a petri dish, a tube, and a cell culturing device.

Immune cells

In the present context, the term "immune cell" refers to any cell that is part of the immune system and assist in combatting infections and other diseases. Immune cells include, but are not limited to, B lymphocytes, T lymphocytes, neutrophils, eosinophils, basophils, mast cells, monocytes, macrophages, dendritic cells, and natural killer cells.

A subsection of the immune cells are peripheral blood mononuclear cells (PBMCs) which are peripheral blood cells with a round nucleus. PBMCs include lymphocytes, monocytes, and dendritic cells. Immune response

In the present context, the term "immune response" refers to a reaction in an organism to a substance that is recognised as harmful or foreign. Through the activation of immune cells, the organism seeks to eliminate the cause of the immune response, such as bacteria or viruses. The reaction includes release of soluble pro-inflammatory molecules, such as cytokines.

In the present context, the key readout of the immune response is the mix and amount of cytokines. Therefore, the term cytokine response" may be used instead of "immune response" herein.

Therapeutic agent

In the present context, the term "therapeutic agents" refers to any substance that may be used in inhibition, amelioration, or treatment of one or more diseases or disorders. Therapeutic agents include, but are not limited to, biologies, antibodies, monoclonal antibodies, proteins, peptides, anti-inflammatory agents, small molecule drugs, and cell therapies.

In the present context, the term "biologies" refers to biopharmaceuticals that are manufactured in or extracted from a biological source. The biological source may be human, animal cells, microorganisms, or fungi. Examples of biologies include, but are not limited to, monoclonal antibodies, blood factors, thrombolytic agents, hormones, growth factors, and antigens.

The therapeutic agents may be added to a container comprising nanofibers and a sample comprising immune cells with the purpose of assaying the immune response elicited by the immune cells upon contact with the therapeutic agent.

Immunoregulatory agent

In the present context, the term "immunoregulatory agent" refers to a substance which stimulates, modulates or suppresses the immune system. The immunoregulatory agents may be categorised as either specific which affect specific parts of the immune system or non-specific which affect the immune system in a general manner.

For the purpose of evaluating the immune response elicited by a sample in response to a therapeutic agent, an immunoregulatory agent giving rise to a known immune response may be used as a benchmark. In this manner, the immunoregulatory agent may act as either a positive or negative control. Immunoregulatory agents suitable for use as benchmark include, but are not limited to, proinflammatory agents, anti-inflammatory agents, superantigens, antibodies, drugs, and combinations thereof.

Examples of immunoregulatory agents include the drugs resiquimod, imiquimod, and gardiquimod, which stimulate cells through a toll-like receptors (TLR) 7 dependent pathway.

Immunoregulatory agents also include lipopolysaccharide (LPS), Concanavalin A (ConA), anti-CD3 antibodies, anti-CD28 antibodies, anti-CD 52 antibodies, and cholesterol crystals, and combinations thereof.

Mean diameter

In the present context, the term "mean diameter" refers to the average diameter of the nanofibers. The mean diameter may be determined from SEM images of the nanofibrous network. Preferably, the mean diameter is determined from measurement of at least 100 individual nanofibers within the sample, e.g. by use of image analysis software, such as Image!

The mean diameter of the nanofibers can be adjusted in the process of preparing the nanofibers. This may be achieved by varying the parameters of e.g. the electrospinning process.

Preferably, the mean diameter of the nanofibers in the nanofibrous network is from about 200 nm to about 1500 nm, such as about 600 nm to about 800 nm.

Surface area

In the present context, the term "surface area" refers to the Brunauer-Emmett-Teller (BET) surface area. The BET surface area may be determined by measurement of the physiosorption of a gas, usually nitrogen, to give a value of the sample. The BET method can accurately determine the surface area of the nanofibers.

The surface area is given as area per unit mass (e.g. m²/g) and can be measured according to ISO 9277:2022 - Determination of the specific surface area of solids by gas adsorption — BET method.

About Wherever the term "about" is employed herein in the context of amounts, for example absolute amounts, such as numbers, purities, weights, sizes, etc., or relative amounts {e.g. percentages, equivalents or ratios), timeframes, and parameters such as temperatures, pressure, etc., it will be appreciated that such variables are approximate and as such may vary by ±10%, for example ± 5% and preferably ± 2% (e.g. ± 1%) from the actual numbers specified. This is the case even if such numbers are presented as percentages in the first place (for example 'about 10%' may mean ± 10% about the number 10, which is anything between 9% and 11%).

Method for obtaining a cytokine response

Herein are described a method which improves the quality of presently available cytokine release assays (CRAs). The method relies on utilization of nanofibers as a three- dimensional scaffold upon which immune cells can be seeded and retain their physiological activity. Importantly, the method disclosed herein improves the quality of the cytokine response of immune cells upon stimulation and significantly increases the sensitivity with which a diverse set of cytokines can be detected.

(i) provision of a container comprising one or more nanofibers,

(ii) addition of a sample comprising immune cells to said container, and

It is to be understood that the method is performed ex vivo, meaning that the bioassay is conducted in an external environment to the natural habitat of the sample, i.e. outside the living body. The ex vivo setup is suitable for testing the biological responses of the immune cells within the sample in an environment where variables can be controlled and allow for comparative studies.

Thus, an embodiment of the present invention relates to the method as described herein, wherein the method is an ex vivo method.

Another aspect of the of the present invention relates to an ex vivo method for obtaining an immune response comprising one or more cytokines from a sample, said method comprising the steps of: (i) provision of a container comprising one or more nanofibers,

(ii) addition of a sample comprising immune cells to said container, and

The method facilitates native-like testing of the immune response elicited by the sample when exposed to the therapeutic agent. In particular, the content of cytokines released into the extracellular environment (/.e. the supernatant) can be determined by multiplex analysis, and a cytokine profile and strength of the cytokine response can be recorded. Accordingly, the risk of a candidate therapeutic agent causing cytokine release syndrome (CRS) can be assessed prior to any clinical testing. This may be achieved by comparing the values with known reference values, or by running a positive and/or negative control with a known therapeutic agent or immunoregulatory agent in parallel for comparison.

The method presented herein provides a significantly improved platform for evaluating the risk of CRS in that it detects more cytokines and at a higher signal-to-noise ratio than a comparable market standard assay. Surprisingly, release of cytokines that was barely detectable or not detectable at all with a market standard assay, was clearly identifiable with the method herein. This was also the case for cytokines such as IL- 1 b and IL-6, which are notoriously difficult to get sufficient response on. The method performed consistently over a set of samples and with a selection of different stimuli.

Without being bound by theory, it is contemplated that the inclusion of nanofibers in the sample wells create an environment which advantageously activates immune cells, resulting in a more native-like cytokine response than can be achieved with commercially available cytokine release assays.

The content of cytokines in an immune response depends on the substance triggering the immune response. Cytokines can have many and to some extent unrelated functions that can depend on the presence or absence of other cytokines. However, it is possible to largely divide cytokines into five major groups, all of which can influence the course of a cytokine storm. The major groups are interferons, interleukins, chemokines, colonystimulating factors (CSF), and tumor necrosis factor (TNF).

Thus, an embodiment of the present invention relates to the method as described herein, wherein the one or more cytokines are selected from the group consisting of interferons, tumor necrosis factor a (TNFa), colon-stimulating factors (CSF), interleukins, and chemokines, and combinations thereof. Among the many different cytokines, some have been tapped as key cytokines of cytokine storms. Having a method that can accurately reconstruct the content of these key cytokines is especially valuable as they are major determinants for assessing the risk of a specific candidate therapeutic agent producing a cytokine storm.

Therefore, an embodiment of the present invention relates to the method as described herein, wherein the one or more cytokines are selected from the group consisting of IL-6, IL-8, IL-lb, IP-10, IFN-g, MCP-1, IL-4, IL-10, IL-17, MIP-lb, Eotaxin, FGF basic, G-CSF, GM-CSF, IL-lra, IL-2, IL-5, IL-9, IL-12(p70), IL-15, MCP-l(MCAF), MIP-la, PDGF-bb, RANTES, TNF-a and VEGF, and combinations thereof.

A preferred embodiment of the present invention relates to the method as described herein, wherein the one or more cytokines are selected from the group consisting of IL-6, IL-8, IL-2, IL-17 TNF-a, IFN-g, IL-lb, and MIP-lb.

The nanofibers create a three-dimensional scaffold in the container upon which the immune cells can attach and proliferate. The nanofibers may be either natural or synthetic, and can also include combinations of more than a single type of nanofiber.

Therefore, an embodiment of the present invention relates to the method as described herein, wherein the one or more nanofibers are selected from natural polymers and synthetic polymers, and combinations thereof.

Another embodiment of the present invention relates to the method as described herein, wherein the synthetic polymers are selected from the group consisting of polycaprolactone (PCL), poly(lactic acid) (PLA), polystyrene, polyurethane (PU), poly(lactic-co-glycolic acid) (PLGA), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), and poly(ethylene-co- vinylacetate) (PEVA).

A preferred embodiment of the present invention relates to the method as described herein, wherein the one or more nanofibers comprises polycaprolactone (PCL).

A further embodiment of the present invention relates to the method as described herein, wherein the natural polymers are selected from the group consisting of cellulose, collagen, silk fibroin, keratin, gelatin and polysaccharides. Although the present method is not restricted to nanofibers of a particular diameter, it has been found that nanofibers of some mean diameters produce a three-dimensional environment that better recreate the collagen and elastin fiber bundles that make up the natural extracellular matrix. Without being bound by theory, it is contemplated that the spatial arrangement of the immune cells improves their proliferation and influences their mutual interaction and activity via the increased number of interaction points. This will influence the quality of the cytokine response.

Moreover, without being bound by theory, it is expected that the porosity of the nanofibrous network can influence the quality of the cytokine response. If the scaffold is too dense, the cells cannot migrate into the scaffold, and only the surface or the top layers become available. Too small fiber diameters will lead to mechanically "weak" fibers that will collapse under the weight, and the pore volume in the z-axis will decrease. Too large fiber diameter will not mimic the in vivo environment, and the cells will interact with the fiber as it would be a "flat" and not curved surface.

Accordingly, an embodiment of the present invention relates to the method as described herein, wherein the one or more nanofibers have a mean diameter of about 200 nm to about 1500 nm, such as about 300 to about 1200 nm, such as about 400 to about 1000 nm, such as about 500 to about 900 nm, such as about 600 to about 800 nm.

Another embodiment of the present invention relates to the method as described herein, wherein the one or more nanofibers have a mean diameter of about 700 nm.

A further embodiment of the present invention relates to the method as described herein, wherein the one or more nanofibers form a three-dimensional scaffold.

In a preferred variant, the three-dimensional nanofiber scaffold comprises only polymeric components. In this variant, the three-dimensional nanofiber scaffold does not comprise other biological molecules or coating substances, such as proteins or peptides. Coating substances include, but is not limited to extracellular matrix (ECM) proteins and adhesive peptides. ECM protein include, but is not limited to, fibronectin, collagen and laminin.

Thus, an embodiment of the present invention relates to the method as described herein, wherein the three-dimensional nanofiber scaffold does not comprise any proteins or peptides. Another embodiment of the present invention relates to the method as described herein, wherein the three-dimensional nanofiber scaffold does not comprise any non-polymeric proteins or proteins.

A further embodiment of the present invention relates to the method as described herein, wherein the three-dimensional nanofiber scaffold does not comprise extracellular matrix (ECM) proteins, such as fibronectin, collagen and laminin, in particular fibronectin.

In a preferred variant , the three-dimensional nanofiber scaffold comprises only a single type of nanofiber. The single nanofiber is preferably PCL.

Therefore, an embodiment of the present invention relates to the method as describe herein, wherein the three-dimensional nanofiber scaffold does not comprise any further polymers.

A still further embodiment of the present invention relates to the method as described herein, wherein the immune cells are arranged in three dimensions on the one or more nanofibers.

The nanofibers are not limited by the method from which they are produced. Many well- developed methods for fabricating nanofibers already exist, and it is contemplated that they will all be suitable for producing the nanofibers for the present method. For high throughput production of nanofibers some methods may be preferred. Also the production method may affect the physical properties of the nanofibers, and therefore it may be desirable to utilise one production method over another depending on the sample to be seeded on the nanofibers.

Therefore, an embodiment of the present invention relates to the method as described herein, wherein the one or more nanofibers are generated by a process selected from the group consisting of electrospinning, meltblowing, drawing, self-assembly, template synthesis, and thermal-induced phase separation.

Another embodiment of the present invention relates to the method as described herein, wherein the one or more nanofibers are electrospun nanofibers.

A further embodiment of the present invention relates to the method as described herein, wherein the one or more nanofibers are provided in the form of sheets. The nanofibers can be functionalized to tune the interaction between the nanofibers and the immune cells of the sample. Functionalization may be either physical or chemical. Examples of properties that can be added to the nanofibers via functionalization include, but are not limited to, hydrophilicity/hydrophobicity, charge alteration, or engrafting of moieties that have activity towards entities on the immune cell surface. Such functionalization may improve cell attachment, proliferation and/or activation of the immune cells.

Therefore, an embodiment of the present invention relates to the method as described herein, wherein the one or more nanofibers are functionalized with one or more functional groups selected from chemical functional groups, chemical moieties or biological molecules.

Securing sufficient cell attachment to the three-dimensional nanofiber scaffold is important to establishing an native-like environment for assessing the risk of cytokine storms triggered by therapeutic agents. However, many polymers, including nanofibers, are hydrophobic by nature. This is also the case for a polymer such as polycaprolactone (PCL). Cell attachment can be promoted by increasing the hydrophilicity of the nanofibers. Thus, the nanofibers may be treated chemically or physically to improve hydrophilicity leading to enhanced immune cell colonisation. One preferred treatment is plasma treatment, wherein ionised gas, such as oxygen or nitrogen, bombard the polymer surface to induce chemical reactions between native groups of the polymer and the reactive plasma species. The end result is a more hydrophilic surface.

Accordingly, an embodiment of the present invention relates to the method as described herein, wherein the wherein the one or more nanofibers are plasma treated.

Another embodiment of the present invention relates to the method as described herein, wherein the wherein the one or more nanofibers are functionalized by air/oxygen plasma treatment.

The container receiving the sample and the therapeutic drug may comprise more than a single type of nanofibers. For some applications it may be beneficial to combine two or more different types of nanofibers to fine tune the physical properties of the nanofiber scaffold and/or exploit chemical properties of a set of fibers.

Thus, an embodiment of the present invention relates to the method as described herein, wherein the container comprises at least two nanofibers. The nanofibers have the physical advantage that they provide a large surface area for the immune cells to attach onto which leads to increased proliferation over cell cultures grown in two dimensions.

Therefore, an embodiment of the present invention relates to the method as described herein, wherein the surface area per unit mass of said one or more nanofibers is in the range of 1-10 m²/g, preferably 2-8 m²/g, more preferably 3-6 m²/g.

In addition to the nanofibers, the container may comprise one or more "activators" included to assist the immune cells in retaining their in v/ o-like function. The activators may be included as part of the coating of the container or as part of the culture medium. Two advantageous activators are anti-CD3 antibodies and anti-CD28 antibodies that provide primary and co-stimulatory signals, thereby assisting activation and expansion of the immune cells, such as T lymphocytes.

Thus, an embodiment of the present invention relates to the method as described herein, wherein the container and/or sample comprises anti-CD3 antibody and/or anti-CD28 antibody.

Another embodiment of the present invention relates to the method as described herein, wherein the container and/or sample comprises anti-CD3 antibody, anti-CD28 antibody and anti-CD137 antibody.

A further embodiment of the present invention relates to the method as described herein, wherein the container comprises anti-CD3 antibody.

Yet another embodiment of the present invention relates to the method as described herein, wherein the sample comprises anti-CD28 antibody.

A preferred embodiment of the present invention relates to the method as described herein, wherein the container comprises anti-CD3 antibody and the sample comprises anti- CD28 antibody.

The sample may in principle be any type of sample which comprises immune cells from a healthy donor. Typical samples used for CRAs include whole blood, peripheral blood mononuclear cells (PBMCs) and splenocytes. A PBMC sample may comprise lymphocytes, monocytes, and dendritic cells or can be a pure isolation of lymphocytes and monocytes. Accordingly, a PBMC sample does not comprise all the physiological components of whole blood, but may be more sensitive to revealing a particular cytokine signal. Thus, for some applications, it may be preferred to test PBMCs as weak cytokine signals may get lost/overlooked in whole blood samples.

Therefore, an embodiment of the present invention relates to the method as described herein, wherein the sample is selected from the group consisting of whole blood, peripheral blood mononuclear cells (PBMCs) and splenocytes.

A preferred embodiment of the present invention relates to the method as described herein, wherein the sample comprises peripheral blood mononuclear cells (PBMCs).

Cytokine storms may occur in any organism with a developed immune system. Thus, the method described herein is not limited to humans. The amount of cells seeded into the container (e.g. the individual wells of a multiwell plate) can be adjusted to achieve the best signal-to-noise ratio when detecting the cytokine response. The method presented herein is advantageous in that a high amount of cells can be seeded on the nanofibers before the culture become confluent. The increased number of cells results in a stronger immune response in each sample well. The sample comprising the immune cells is preferably provided as a liquid sample comprising also culturing medium, and potentially additional immune cell activators.

Thus, an embodiment of the present invention relates to the method as described herein, wherein the sample is of mammalian origin, preferably human origin.

Thus, an embodiment of the present invention relates to the method as described herein, wherein the sample comprises about 0.05 x 10⁶ to about 2.5 x 10⁶ immune cells, such as about 0.1 x 10⁶ to about 0.5 x 10⁶ immune cells.

Another embodiment of the present invention relates to the method as described herein, wherein the sample comprises about 2 x 10⁶ to about 20 x 10⁶ immune cells per ml, such as 4 x 10⁶ to about 10 x 10⁶ immune cells per ml.

Yet another embodiment of the present invention relates to the method as described herein, wherein the sample is provided as a liquid sample.

A further embodiment of the present invention relates to the method as described herein, wherein the liquid sample comprises a culturing medium. A still further embodiment of the present invention relates to the method as described herein, wherein the sample comprises about 100 to about 10000 cells per mm² of nanofiber, such as about 500 to about 5000 cells per mm² of nanofiber, such as about 1000 to about 2000 cells per mm² of nanofiber.

An even further embodiment of the present invention relates to the method as described herein, wherein the sample comprises about 2 x 10⁹ to about 20 x 10⁹ cells per g of nanofiber, such as about 4 x 10⁹ to about 10 x 10⁹ cells per g of nanofiber, such as about 5 x 10⁹ to about 6 x 10⁹ cells per g of nanofiber.

Prior to addition of the therapeutic agent, the immune cells may be incubated for a period of time to establish a cell culture of a sufficient size to be capable of eliciting a native-like immune response. The incubation period may be performed for at least 12 hours, such as at least 18 hours, such as at least 24 hours. Incubation may be performed under standard culturing conditions.

Thus, an embodiment of the present invention relates to the method as described herein, wherein addition of the sample is followed by an incubation period prior to addition of said therapeutic agent.

Another embodiment of the present invention relates to the method as described herein, wherein said incubation period is at least 12 hours, such as at least 18 hours, such as at least 24 hours, preferably for 24 hours.

The container harbouring the sample may be any vessel that is suitable for storing and/or culturing of cells. The method is therefore suitable for use with standard laboratory equipment. Examples of containers include, but are not limited to, a cell culturing plate, multiwell plate, a petri dish, a tube, and a cell culturing device. To increase throughput of the method, it is preferred to utilise a container that enables multiplex analysis of the samples, such as containers with a plurality of sampling wells that can be used with multiplex analysis workflow. Multiwell plates are the preferred option. Individual sampling wells of the multiwell plate can be coated with nanofibers and optionally any activators prior to loading of the sample.

Accordingly, an embodiment of the present invention relates to the method as described herein, wherein the container is selected from the group consisting of a multiwell plate, a petri dish and a cell culturing device. Thus, a preferred embodiment of the present invention relates to the method as described herein, wherein the container is a multiwell plate comprising 4, 6, 12, 24, 48, 96, 384, 1536 or 3456 sample wells, preferably a 96 well plate.

The method described herein is not limited to detection of a particular set of cytokines, but may in principle be utilised to determine the content of any cytokine. The presence of cytokines is determined after addition of the therapeutic agent via any suitable detection readout. Many options for detecting cytokines in CRAs is already available. Preferably, detection of the cytokines is by multiplex assay.

Therefore, an embodiment of the present invention relates to the method as described herein further comprising a step (iv) of detection of said one or more cytokines.

Another embodiment of the present invention relates to the method as described herein, wherein the detection of the one or more cytokines is by a readout selected from the group consisting of luminescence, fluorescence, chemiluminescence, electrochemical, and radioactive.

A further embodiment of the present invention relates to the method as described herein, wherein the detection of the one or more cytokines is achieved by a techniques selected form the group consisting of cytometry, immunoassays, multiplex assays, and ELISA.

The requirement for an incubation period prior to the detection of the cytokines, and the length hereof, depends on the sensitivity of the detection assay and the strength and quality of the cytokine response generated by the method. For commercially available CRAs the incubation period prior to detection of cytokines is typically around 24 hours. However, it is contemplated that the method herein is capable of producing cytokine responses that can be accurately detected much faster. If needed, the method herein can increase the turnover of sample analysis markedly compared to commercially available alternatives.

Therefore, an embodiment of the present invention relates to the method as described herein, wherein steps (iii) and (iv) are separated by at least about 4 hours, such as at least about 6 hours, such as at least about 8 hours, such as at least about 10 hours, such as at least about 12 hours, such as at least about 18 hours, about 24 hours, such as at least about 36 hours, such as at least about 48 hours, such as at least about 72 hours. Another embodiment of the present invention relates to the method as described herein, wherein steps (iii) and (iv) are separated by no more than about 4 hours, such as no more than about 6 hours, such as no more than about 8 hours, such as no more than about 10 hours, such as no more than about 12 hours, such as no more than about 18 hours, about 24 hours, such as no more than about 36 hours, such as no more than about 48 hours, such as no more than about 72 hours.

A further embodiment of the present invention relates to the method as described herein, wherein steps (iii) and (iv) are separated by no more than about 4 hours, such as no more than about 6 hours, such as no more than about 8 hours.

Cytokine release syndrome (CRS) is an exceptionally severe and undesired adverse effect of any therapy. Rightfully, there is consequently massive focus on de-risking the trigger of CRS caused by candidate therapeutic agents prior to any preclinical use, let alone commercial distribution of the therapeutic agent. Any therapeutic agent that have the slimmest risk of interfering with the immune system should be tested in a CRA. The group of therapeutic agents relevant to the method herein is therefore quite large and diverse. Also, the method is applicable for testing the cytokine response triggered by any type of therapeutic agent.

Thus, an embodiment of the present invention relates to the method as described herein, wherein the therapeutic agent is selected from the group consisting of biologies, antibodies, monoclonal antibodies, proteins, peptides, anti-inflammatory agents, small molecule drugs, and cell therapies, and combinations thereof.

Another embodiment of the present invention relates to the method as described herein, wherein the therapeutic agent is selected from the group consisting of antibodies, monoclonal antibodies, proteins, peptides, anti-inflammatory agents, small molecule drugs, and cell therapies, and combinations thereof.

A further embodiment of the present invention relates to the method as described herein, wherein the therapeutic agent is a biologies selected from the group consisting of monoclonal antibodies, blood factors, thrombolytic agents, hormones, growth factors, and antigens.

A still further embodiment of the present invention relates to the method as described herein, wherein the therapeutic agent is not a cytokine, such as stem cell factor (SCF). Yet another embodiment of the present invention relates to the method as described herein, wherein the therapeutic agent is not IgE.

The method described herein is particularly suitable for testing pharmaceutical compounds (drugs) as part of the process of ensuring that no pharmaceuticals causing severe adverse effects are put on the market. Thus, the therapeutic agent are preferably a compound that is intended to be used in human medicine. Among relevant drugs are those that are non- naturally occurring, semi-synthetic, or synthetic.

Therefore, an embodiment of the present invention relates to the method as described herein, wherein the therapeutic agent is a therapeutic compound intended for treatment or amelioration of a disease or medical condition.

Another embodiment of the present invention relates to the method as described herein, wherein the therapeutic agent is a non-naturally occurring substance.

The absolute levels of secreted cytokines may vary depending on the immune cells comprised in the sample. For this reason it can be beneficial to run relevant positive and negative controls in parallel with the candidate therapeutic agents. In particular, it is preferred to use positive controls that have a similar mechanism of action as the therapeutic agent in question. The common for the controls are that they are considered standard references with a known/expected cytokine response. The negative and/or positive control can be performed in parallel in separate containers, e.g. in separate sampling wells of a multiwell plate. In that way, the method may include repeating the initial steps of the method but with loading of a negative or positive control instead of a candidate therapeutic agent.

Thus, an embodiment of the present invention relates to the method as described herein, further comprising repeating all steps of the method one or more times wherein one or more immunoregulatory agents are added instead of said therapeutic agent.

It is to be understood that the negative and/or positive control is performed in a separate container, such as a separate sampling well on a multiwell plate, from the one in which the therapeutic agent is added.

Thus, an embodiment of the present invention relates to the method as described herein, wherein the method comprises repeating all steps in one or more separate containers, and wherein one or more immunoregulatory agents are added to each of said separate containers instead of said therapeutic agent.

Another embodiment of the present invention relates to the method as described herein, wherein the immunoregulatory agents are selected from the group consisting of proinflammatory agents, anti-inflammatory agents, superantigens, antibodies, drugs, and combinations thereof.

A further embodiment of the present invention relates to the method as described herein, wherein the immunoregulatory agent is selected from the group consisting of lipopolysaccharide (LPS), Concanavalin A (ConA), anti-CD3 antibodies, anti-CD28 antibodies, anti-CD 52 antibodies, resiquimod, imiquimod, gardiquimod, and cholesterol crystals, and combinations thereof.

A still further embodiment of the present invention relates to the method as described herein, wherein the immunoregulatory agent is selected from the group consisting of immunoglobulins, such as IgGl, IgG2a, and IgG4, and combinations thereof.

The cytokine response triggered by the control or reference substance can be used in the assessment of the risk of a candidate therapeutic agent triggering a cytokine storm. Positive controls which are known to trigger strong responses can be used as a benchmark for assigning a risk profile to the candidate therapeutic agents. Therapeutic agent that are generating cytokine responses stronger than those of positive controls known to be in the risk of triggering a cytokine storm should be labelled as high risk substances, for which further investigation and/or repeat of the method is recommended.

Accordingly, an embodiment of the present invention relates to the method as described herein, wherein the immune response induced by addition of said therapeutic agent to the sample is compared to the immune response induced by addition of said one or more immunoregulatory agents to the sample.

Another embodiment of the present invention relates to the method as described herein further comprising a step of comparing the cytokine response caused by the therapeutic agent with the cytokine response caused by one or more immunoregulatory agents.

A further embodiment of the present invention relates to the method as described herein, comprising the following steps:

(i) provision of a container comprising one or more nanofibers, (ii) addition of a sample comprising immune cells to said container,

(iii) addition of a therapeutic agent to said container,

(iv) detection of said one or more cytokines, and

(v) comparing the immune response detected in step (iv) with a reference immune response.

Yet another embodiment of the present invention relates to the method as described herein, wherein the reference immune response is obtained by performing steps (i)-(iv), wherein an immunoregulatory agents are added instead of said therapeutic agent.

A still further embodiment of the present invention relates to the method as described herein further comprising a step of assigning the therapeutic agent with a risk value for triggering CRS.

An even further embodiment of the present invention relates to the method as described herein, wherein the therapeutic agent is assigned a first risk value if the immune response detected in step (iv) are equal to or stronger than the reference immune response, and a second risk value if the cytokine response detected in step (iv) are weaker than the reference cytokine responses.

Immunoregulatory agents with a known immune response can be used as benchmark response against which the response induced by the therapeutic agent can be compared.

Accordingly, an embodiment of the present invention relate to as described herein, wherein said one or more immunoregulatory agents induce a known immune response.

The risk value assigned to the therapeutic agent can be used as part of the assessment of the adverse effect profile. The risk value may be used for predicting the likelihood that the therapeutic agent induce CRS if administered to a subject. The subject is preferably a human subject.

Therefore, an embodiment of the present invention relates to the method as described herein, wherein said risk value is an indicator for the likelihood of the therapeutic agent inducing a cytokine storm if administered to a subject.

Another embodiment of the present invention relates to the method as described herein, wherein the subject is a human. The method may be used for evaluation of the risk that a therapeutic agent trigger CRS when administered to a subject.

Thus, an aspect of the present invention relates to an ex vivo method of evaluating the risk that a therapeutic agent triggers CRS if administered to a subject, said method comprising the following steps:

(i) provision of a container comprising one or more nanofibers,

(ii) addition of a sample comprising immune cells to said container,

(iii) addition of a therapeutic agent to said container to obtain an immune response comprising one or more cytokines,

(iv) detection of said one or more cytokines, and

It is to be understood that this aspect of the invention is also compatible with the other embodiments described herein.

The evaluation of the risk for triggering CRS can be done by comparing the immune response induced by the therapeutic agent to an immune response induced by a known benchmark compound.

Thus, an embodiment of the present invention relates to the method as described herein, wherein the reference immune response is obtained by performing steps (i)-(iv), wherein an immunoregulatory agent is added instead of said therapeutic agent, and wherein the immunoregulatory agent induce a known immune response.

Another embodiment of the present invention relates to the method as described herein, wherein the therapeutic agent is assigned a first risk value if the immune response detected in step (iv) is equal to or higher than the reference immune response, and a second risk value if the cytokine response detected in step (iv) is lower than the reference cytokine responses.

Yet another embodiment of the present invention relates to the method as described herein, wherein if the immune response detected in step (iv) is: higher than the reference immune response then it is indicative of a high risk that the therapeutic agent trigger CRS if administered to a subject, or lower than the reference immune response then it is indicative of a low risk that the therapeutic agent trigger CRS if administered to a subject. In the present context, the magnitude of the immune response is measured by detection and quantification of one or more cytokines as described herein. Thus, a higher immune response corresponds to an immune response wherein the level of cytokine(s) is increased compared to the reference immune response, and a lower immune response corresponds to an immune response wherein the level of cytokine(s) is decreased compared to the reference immune response.

The benchmark compound can be an immunoregulatory agent that is known to induce either a strong immune response or a weak immune response. If the reference immune response is strong, then therapeutic agents inducing an immune response of equal or greater magnitude are at high risk of causing CRS. These therapeutic agents should be further evaluated to make sure they are safe for administration to a subject. If the reference immune response is weak, then therapeutic agents inducing an immune response of equal or decreased magnitude are at low risk of causing CRS. These therapeutic agents can be considered safe and with and low probability of causing CRS.

Therefore, an embodiment of the present invention relates to the method as described herein, wherein the immune response detected in step (iv) is compared with:

- a reference immune response obtained from an immunoregulatory agent that is known to induce a strong immune response, and/or

- a reference immune response obtained from an immunoregulatory agent that is known to induce a weak immune response.

Another embodiment of the present invention relates to the method as described herein, wherein the immune response detected in step (iv) is compared with a reference immune response obtained from an immunoregulatory agent that is known to induce a strong immune response.

Yet another embodiment of the present invention relates to the method as described herein, wherein the therapeutic agent is assigned a high risk value if the immune response detected in step (iv) is equal to or higher than the reference immune response.

A further embodiment of the present invention relates to the method as described herein, wherein the immune response detected in step (iv) is compared with a reference immune response obtained from an immunoregulatory agent that is known to induce a weak immune response. A still further embodiment of the present invention relates to the method as described herein, wherein the therapeutic agent is assigned a low risk value if the immune response detected in step (iv) is equal to or lower than the reference immune response.

An even further embodiment of the present invention relates to the method as described herein, wherein: if the therapeutic agent is assigned a high risk value it is recommended to carry out further evaluation of the therapeutic agent, and

- if the therapeutic agent is assigned a low risk value it is not necessary to carry out further evaluation of the therapeutic agent.

Another aspect of the present invention relates to a container for a cytokine release assay comprising:

- one or more nanofibers, and

- one or more activators selected from anti-CD3 antibody and/or anti-CD28 antibody.

A further aspect of the present invention relates to use of a container comprising one or more nanofibers for obtaining an immune response comprising one or more cytokines from a sample comprising immune cells, wherein said sample are subjected to a therapeutic agent.

The CRA may also be packaged as a kit-of-parts. The kit includes a container comprising nanofibers upon which a sample may be seeded. Moreover, one or more negative or positive controls can be included in the kit, so that the user only need to supply a sample with immune cells and a therapeutic agent to be evaluated.

Thus, a still further aspect of the present invention relates to a kit comprising: a container comprising one or more nanofibers,

- at least one immunoregulatory agent,

- optionally, instructions for use.

It is to be understood that the terms "one or more cytokines", "one or more nanofibers", "sample", "container", and "therapeutic agent" when used in the context of the use or kit described above can be the same as described for the method herein.

The listing or discussion of an apparently prior published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge. Preferences, options and embodiments for a given aspect, feature or parameter of the invention should, unless the context indicates otherwise, be regarded as having been disclosed in combination with any and all preferences, options and embodiments for all other aspects, features and parameters of the invention. This is especially true for the description of the method for obtaining an immune response from a sample and all its features, which may readily be part of a use or kit for obtaining an immune response from a sample. Embodiments and features of the present invention are also outlined in the following items.

Items

XI. A method for obtaining an immune response comprising one or more cytokines from a sample, said method comprising the steps of:

(i) provision of a container comprising one or more nanofibers,

(ii) addition of a sample comprising immune cells to said container, and

X2. The method according to item XI, wherein the one or more cytokines are selected from the group consisting of interferons, tumor necrosis factor a (TNFa), colon-stimulating factors (CSF), interleukins, and chemokines, and combinations thereof.

X3. The method according to any one of items XI or X2, wherein the one or more cytokines are selected from the group consisting of IL-6, IL-8, IL-lb, IP-10, IFN-g, MCP-1, IL-4, IL- 10, IL-17, MIP-lb, Eotaxin, FGF basic, G-CSF, GM-CSF, IL-lra, IL-2, IL-5, IL-9, IL- 12(p70), IL-15, MCP-l(MCAF), MIP-la, PDGF-bb, RANTES, TNF-a and VEGF, and combinations thereof.

X4. The method according to any one of items X1-X3, wherein the one or more cytokines are selected from the group consisting of IL-6, IL-8, IL-2, IL-17 TNF-a, IFN-g, IL-lb, and MIP-lb.

X5. The method according to any one of the preceding items, wherein the one or more nanofibers are selected from natural polymers and synthetic polymers, and combinations thereof.

X6. The method according to item X5, wherein the synthetic polymers are selected from the group consisting of polycaprolactone (PCL), poly(lactic acid) (PLA), polystyrene, polyurethane (PU), poly(lactic-co-glycolic acid) (PLGA), poly(3-hydroxybutyrate-co-3- hydroxyvalerate) (PHBV), and poly(ethylene-co-vinylacetate) (PEVA).

X7. The method according to any one of the preceding items, wherein the one or more nanofibers comprises polycaprolactone (PCL).

X8. The method according to any one of items X5-X7, wherein the natural polymers are selected from the group consisting of cellulose, collagen, silk fibroin, keratin, gelatin and polysaccharides.

X9. The method according to any one of the preceding items, wherein the one or more nanofibers have a mean diameter of about 200 nm to about 1500 nm, such as about 300 to about 1200 nm, such as about 400 to about 1000 nm, such as about 500 to about 900 nm, such as about 600 to about 800 nm.

X10. The method according to any one of the preceding items, wherein the one or more nanofibers have a mean diameter of about 700 nm.

XI 1. The method according to any one of the preceding items, wherein the one or more nanofibers form a three-dimensional scaffold.

X12. The method according to any one of the preceding items, wherein the one or more nanofibers are provided in the form of sheets.

X13. The method according to any one of the preceding items, wherein the one or more nanofibers are generated by a process selected from the group consisting of electrospinning, meltblowing, drawing, self-assembly, template synthesis, and thermal- induced phase separation.

X14. The method according to any one of the preceding items, wherein the one or more nanofibers are electrospun nanofibers.

X15. The method according to any one of the preceding items, wherein the one or more nanofibers are functionalized with one or more functional groups selected from chemical functional groups, chemical moieties or biological molecules.

X16. The method according to any one of the preceding items, wherein the one or more nanofibers are functionalized by air/oxygen plasma treatment. X17. The method according to any one of the preceding items, wherein the container comprises at least two nanofibers.

X18. The method according to any one of the preceding items, wherein the surface area per unit mass of said one or more nanofibers is in the range of 1-10 m²/g, preferably 2-8 m²/g, more preferably 3-6 m²/g.

X19. The method according to any one of the preceding items, wherein the sample is selected from the group consisting of whole blood, peripheral blood mononuclear cells (PBMCs) and splenocytes.

X20. The method according to any one of the preceding items, wherein the sample comprises peripheral blood mononuclear cells (PBMCs).

X21. The method according to any one of the preceding items, wherein the immune cells are arranged in three dimensions on the one or more nanofibers.

X22. The method according to any one of the preceding items, wherein the sample is of mammalian origin, preferably human origin.

X23. The method according to any one of the preceding items, wherein the sample comprises about 0.05 x 10⁶ to about 2.5 x 10⁶ immune cells, such as about 0.1 x 10⁶ to about 0.5 x 10⁶ immune cells.

X24. The method according to any one of the preceding items, wherein the sample comprises about 2 x 10⁶ to about 20 x 10⁶ immune cells per ml, such as 4 x 10⁶ to about 10 x 10⁶ immune cells per ml.

X25. The method according to any one of the preceding items, wherein the sample is provided as a liquid sample.

X26. The method according to item X25, wherein the liquid sample comprises a culturing medium.

X27. The method according to any one of the preceding items, wherein the sample comprises about 100 to about 10000 cells per mm² of nanofiber, such as about 500 to about 5000 cells per mm² of nanofiber, such as about 1000 to about 2000 cells per mm² of nanofiber.

X28. The method according to any one of the preceding items, wherein the sample comprises about 2 x 10⁹ to about 20 x 10⁹ cells per g of nanofiber, such as about 4 x 10⁹ to about 10 x 10⁹ cells per g of nanofiber, such as about 5 x 10⁹ to about 6 x 10⁹ cells per g of nanofiber.

X29. The method according to any one of the preceding items, wherein addition of the sample is followed by an incubation period prior to addition of said therapeutic agent.

X30. The method according to any one of the preceding items, wherein the container is selected from the group consisting of a multiwell plate, a petri dish and a cell culturing device.

X31. The method according to any one of the preceding items, wherein the container is a multiwell plate comprising 4, 6, 12, 24, 48, 96, 384, 1536 or 3456 sample wells, preferably a 96 well plate.

X32. The method according to any one of the preceding items further comprising a step (iv) of detection of said one or more cytokines.

X33. The method according to item X32, wherein the detection of the one or more cytokines is by a readout selected from the group consisting of luminescence, fluorescence, chemiluminescence, electrochemical, and radioactive.

X34. The method according to any one of items X32 or X33, wherein the detection of the one or more cytokines is achieved by a techniques selected from the group consisting of cytometry, immunoassays, multiplex assays, and ELISA.

X35. The method according to any one of items X32-X34, wherein steps (iii) and (iv) are separated by at least about 4 hours, such as at least about 6 hours, such as at least about 8 hours, such as at least about 10 hours, such as at least about 12 hours, such as at least about 18 hours, about 24 hours, such as at least about 36 hours, such as at least about 48 hours, such as at least about 72 hours.

X36. The method according to any one of items X32-X35, wherein steps (iii) and (iv) are separated by no more than about 4 hours, such as no more than about 6 hours, such as no more than about 8 hours, such as no more than about 10 hours, such as no more than about 12 hours, such as no more than about 18 hours, about 24 hours, such as no more than about 36 hours, such as no more than about 48 hours, such as no more than about 72 hours.

X37. The method according to any one of the preceding items, wherein the therapeutic agent is selected from the group consisting of biologies, antibodies, monoclonal antibodies, proteins, peptides, anti-inflammatory agents, small molecule drugs, and cell therapies, and combinations thereof.

X38. The method according to any one of the preceding items, further comprising repeating all steps of the method one or more times wherein one or more immunoregulatory agents are added instead of said therapeutic agent.

X39. The method according to item X38, wherein the immunoregulatory agents are selected from the group consisting of proinflammatory agents, anti-inflammatory agents, superantigens, antibodies, drugs, and combinations thereof.

X40. The method according to any one of items X38 or X39, wherein the immunoregulatory agent is selected from the group consisting of lipopolysaccharide (LPS), Concanavalin A (ConA), anti-CD3 antibodies, anti-CD28 antibodies, anti-CD 52 antibodies, resiquimod, imiquimod, gardiquimod, and cholesterol crystals, and combinations thereof.

X41. The method according to any one of items X38 or X39, wherein the immunoregulatory agent is selected from the group consisting of immunoglobulins, such as IgGl, IgG2a, and IgG4, and combinations thereof.

X42. The method according to any one of items X38-X41, wherein the immune response induced by addition of said therapeutic agent to the sample is compared to the immune response induced by addition of said one or more immunoregulatory agents to the sample.

X43. The method according to any one of the preceding items, wherein the method is an ex vivo method.

X44. The method according to any one of the preceding items, wherein the therapeutic agent is selected from the group consisting of antibodies, monoclonal antibodies, proteins, peptides, anti-inflammatory agents, small molecule drugs, and cell therapies, and combinations thereof. X45. The method according to any one of the preceding items, wherein the therapeutic agent is a biologies selected from the group consisting of monoclonal antibodies, blood factors, thrombolytic agents, hormones, growth factors, and antigens.

X46. The method according to any one of the preceding items, wherein the therapeutic agent is a therapeutic compound intended for treatment or amelioration of a disease or medical condition.

X47. The method according to any one of the preceding items, wherein the therapeutic agent is a non-naturally occurring substance.

X48. The method according to any one of items X38-X47, wherein said one or more immunoregulatory agents induce a known immune response.

X49. The method according to any one of the preceding items, said method comprising the following steps:

(i) provision of a container comprising one or more nanofibers,

(ii) addition of a sample comprising immune cells to said container,

(iii) addition of a therapeutic agent to said container,

(iv) detection of said one or more cytokines, and

X50. The method according to item X49, wherein said the reference immune response is obtained by performing steps (i)-(iv), wherein an immunoregulatory agents are added instead of said therapeutic agent.

X51. The method according to any one of items X49 or X50, said method further comprising a step of assigning the therapeutic agent with a risk value for triggering CRS.

X52. The method according to any one of items X49-X51, wherein the therapeutic agent is assigned a first risk value if the immune response detected in step (iv) are equal to or stronger than the reference immune response, and a second risk value if the cytokine response detected in step (iv) are weaker than the reference cytokine responses. X53. The method according to any one of items X51 or X52, wherein said risk value is an indicator for the likelihood of the therapeutic agent inducing a cytokine storm if administered to a subject.

X54. The method according to item X53, wherein the subject is a human.

X55. The method according to any one of items X11-X54, wherein the three-dimensional nanofiber scaffold does not comprise any proteins or proteins.

X56. The method according to any one of items X11-X55, wherein the three-dimensional nanofiber scaffold does not comprise any non-polymeric proteins or peptides.

X57. The method according to any one of items X11-X56, wherein the three-dimensional nanofiber scaffold does not comprise any further polymers.

Wl. An ex vivo method of evaluating the risk that a therapeutic agent triggers CRS if administered to a subject, said method comprising the following steps:

(i) provision of a container comprising one or more nanofibers,

(ii) addition of a sample comprising immune cells to said container,

(iv) detection of said one or more cytokines, and

W2. The method according to item Wl, wherein the reference immune response is obtained by performing steps (i)-(iv), wherein an immunoregulatory agent is added instead of said therapeutic agent, and wherein the immunoregulatory agent induce a known immune response.

W3. The method according to any one of items Wl or W2, wherein the therapeutic agent is assigned a first risk value if the immune response detected in step (iv) is equal to or higher than the reference immune response, and a second risk value if the cytokine response detected in step (iv) is lower than the reference cytokine responses.

W4. The method according to any one of items W1-W3, wherein if the immune response detected in step (iv) is: higher than the reference immune response then it is indicative of a high risk that the therapeutic agent trigger CRS if administered to a subject, or lower than the reference immune response then it is indicative of a low risk that the therapeutic agent trigger CRS if administered to a subject.

W5. The method according to any one of items W1-W4, wherein the immune response detected in step (iv) is compared with:

W6. The method according to any one of items W1-W5, wherein the immune response detected in step (iv) is compared with a reference immune response obtained from an immunoregulatory agent that is known to induce a strong immune response.

W7. The method according to item W6, wherein the therapeutic agent is assigned a high risk value if the immune response detected in step (iv) is equal to or higher than the reference immune response.

W8. The method according to any one of items W1-W5, wherein the immune response detected in step (iv) is compared with a reference immune response obtained from an immunoregulatory agent that is known to induce a weak immune response.

W9. The method according to item W8, wherein the therapeutic agent is assigned a low risk value if the immune response detected in step (iv) is equal to or lower than the reference immune response.

W10. The method according to any one of items W3-W9, wherein: if the therapeutic agent is assigned a high risk value it is recommended to carry out further evaluation of the therapeutic agent, and

Yl. Use of a container comprising one or more nanofibers for obtaining an immune response comprising one or more cytokines from a sample comprising immune cells, wherein said sample are subjected to a therapeutic agent. Y2. The use according to item Yl, wherein the one or more cytokines are selected from the group consisting of interferons, tumor necrosis factor a (TNFa), colon-stimulating factors (CSF), interleukins, and chemokines, and combinations thereof.

Y3. The use according to any one of items Y1 or Y2, wherein the one or more cytokines are selected from the group consisting of IL-6, IL-8, IL-lb, IP-10, IFN-g, MCP-1, IL-4, IL- 10, IL-17, MIP-lb, Eotaxin, FGF basic, G-CSF, GM-CSF, IL-lra, IL-2, IL-5, IL-9, IL- 12(p70), IL-15, MCP-l(MCAF), MIP-la, PDGF-bb, RANTES, TNF-a and VEGF, and combinations thereof.

Y4. The use according to any one of items Y1-Y3, wherein the one or more cytokines are selected from the group consisting of IL-6, IL-8, IL-2, IL-17 TNF-a, IFN-g, IL-lb, and MIP- lb.

Y5. The use according to any one of items Y1-Y4, wherein the one or more nanofibers are selected from natural polymers and synthetic polymers, and combinations thereof.

Y6. The use according to item Y5, wherein the synthetic polymers are selected from the group consisting of polycaprolactone (PCL), poly(lactic acid) (PLA), polystyrene, polyurethane (PU), poly(lactic-co-glycolic acid) (PLGA), poly(3-hydroxybutyrate-co-3- hydroxyvalerate) (PHBV), and poly(ethylene-co-vinylacetate) (PEVA).

Y7. The use according to any one of items Y1-Y6, wherein the one or more nanofibers comprises polycaprolactone (PCL).

Y8. The use according to any one of items Y5-Y7, wherein the natural polymers are selected from the group consisting of cellulose, collagen, silk fibroin, keratin, gelatin and polysaccharides.

Y9. The use according to any one of items Y1-Y8, wherein the one or more nanofibers have a mean diameter of about 200 nm to about 1500 nm, such as about 300 to about 1200 nm, such as about 400 to about 1000 nm, such as about 500 to about 900 nm, such as about 600 to about 800 nm.

Y10. The use according to any one of items Y1-Y9, wherein the one or more nanofibers have a mean diameter of about 700 nm. Yll. The use according to any one of items Y1-Y10, wherein the one or more nanofibers form a three-dimensional scaffold.

Y12. The use according to any one of items Yl-Yll, wherein the one or more nanofibers are provided in the form of sheets.

Y13. The use according to any one of items Y1-Y12, wherein the one or more nanofibers are generated by a process selected from the group consisting of electrospinning, meltblowing, drawing, self-assembly, template synthesis, and thermal-induced phase separation.

Y14. The use according to any one of items Y1-Y13, wherein the one or more nanofibers are electrospun nanofibers.

Y15. The use according to any one of items Y1-Y14, wherein the one or more nanofibers are functionalized with one or more functional groups selected from chemical functional groups, chemical moieties or biological molecules.

Y16. The use according to any one of items Y1-Y15, wherein the wherein the one or more nanofibers are functionalized by air/oxygen plasma treatment.

Y17. The use according to any one of items Y1-Y16, wherein the container comprises at least two nanofibers.

Y18. The use according to any one of items Y1-Y17, wherein the surface area per unit mass of said one or more nanofibers is in the range of 1-10 m²/g, preferably 2-8 m²/g, more preferably 3-6 m²/g.

Y19. The use according to any one of items Y1-Y18, wherein the sample is selected from the group consisting of whole blood, peripheral blood mononuclear cells (PBMCs) and splenocytes.

Y20. The use according to any one of items Y1-Y19, wherein the sample comprises peripheral blood mononuclear cells (PBMCs).

Y21. The use according to any one of items Y1-Y20, wherein the immune cells are arranged in three dimensions on the one or more nanofibers. Y22. The use according to any one of items Y1-Y21, wherein the sample is of mammalian origin, preferably human origin.

Y23. The use according to any one of items Y1-Y22, wherein the sample comprises about 0.05 x 10⁶ to about 2.5 x 10⁶ immune cells, such as about 0.1 x 10⁶ to about 0.5 x 10⁶ immune cells.

Y24. The use according to any one of items Y1-Y23, wherein the sample comprises about 2 x 10⁶ to about 20 x 10⁶ immune cells per ml, such as 4 x 10⁶ to about 10 x 10⁶ immune cells per ml.

Y25. The use according to any one of items Y1-Y24, wherein the sample is provided as a liquid sample.

Y26. The use according to item Y25, wherein the liquid sample comprises a culturing medium.

Y27. The use according to any one of items Y1-Y26, wherein the sample comprises about 100 to about 10000 cells per mm² of nanofiber, such as about 500 to about 5000 cells per mm² of nanofiber, such as about 1000 to about 2000 cells per mm² of nanofiber.

Y28. The use according to any one of items Y1-Y27, wherein the sample comprises about 2 x 10⁹ to about 20 x 10⁹ cells per g of nanofiber, such as about 4 x 10⁹ to about 10 x 10⁹ cells per g of nanofiber, such as about 5 x 10⁹ to about 6 x 10⁹ cells per g of nanofiber.

Y29. The use according to any one of items Y1-Y28, wherein addition of the sample is followed by an incubation period prior to addition of said therapeutic agent.

Y30. The use according to any one of items Y1-Y29, wherein the container is selected from the group consisting of a multiwell plate, a petri dish and a cell culturing device.

Y31. The use according to any one of items Y1-Y30, wherein the container is a multiwell plate comprising 4, 6, 12, 24, 48, 96, 384, 1536 or 3456 sample wells, preferably a 96 well plate.

Y32. The use according to any one of items Y1-Y31, wherein the therapeutic agent is selected from the group consisting of biologies, antibodies, monoclonal antibodies, proteins, peptides, anti-inflammatory agents, small molecule drugs, and cell therapies, and combinations thereof.

Y33. The use according to any one of items Y1-Y32, wherein the therapeutic agent is selected from the group consisting of antibodies, monoclonal antibodies, proteins, peptides, anti-inflammatory agents, small molecule drugs, and cell therapies, and combinations thereof.

Y34. The use according to any one of items Y1-Y33, wherein the therapeutic agent is a biologies selected from the group consisting of monoclonal antibodies, blood factors, thrombolytic agents, hormones, growth factors, and antigens.

Y35. The use according to any one of items Y1-Y34, wherein the therapeutic agent is a therapeutic compound intended for treatment or amelioration of a disease or medical condition.

Y36. The use according to any one of items Y1-Y35, wherein the therapeutic agent is a non-naturally occurring substance.

Y37. The use according to any one of items Y11-Y36, wherein the three-dimensional nanofiber scaffold does not comprise any proteins or proteins.

Y38. The use according to any one of items Y11-Y37, wherein the three-dimensional nanofiber scaffold does not comprise any non-polymeric proteins or peptides.

Y39. The use according to any one of items Y11-Y38, wherein the three-dimensional nanofiber scaffold does not comprise any further polymers.

Zl. A kit comprising: a container comprising one or more nanofibers,

- at least one immunoregulatory agent,

- optionally, instructions for use.

Z2. The kit according to item Zl, wherein the container is selected from the group consisting of a multiwell plate, a petri dish and a cell culturing device. Z3. The kit according to any one of items Z1 or Z2, wherein the container is a multiwell plate comprising 4, 6, 12, 24, 48, 96, 384, 1536 or 3456 sample wells, preferably a 96 well plate.

Z4. The kit according to any one of items Z1-Z3, wherein the one or more nanofibers are selected from natural polymers and synthetic polymers, and combinations thereof.

Z5. The kit according to item Z4, wherein the synthetic polymers are selected from the group consisting of polycaprolactone (PCL), poly(lactic acid) (PLA), polystyrene, polyurethane (PU), poly(lactic-co-glycolic acid) (PLGA), poly(3-hydroxybutyrate-co-3- hydroxyvalerate) (PHBV), and poly(ethylene-co-vinylacetate) (PEVA).

Z6. The kit according to any one of items Z1-Z5, wherein the one or more nanofibers comprises polycaprolactone (PCL).

Z7. The kit according to any one of items Z4-Z6, wherein the natural polymers are selected from the group consisting of cellulose, collagen, silk fibroin, keratin, gelatin and polysaccharides.

Z8. The kit according to any one of items Z1-Z7, wherein the one or more nanofibers have a mean diameter of about 200 nm to about 1500 nm, such as about 300 to about 1200 nm, such as about 400 to about 1000 nm, such as about 500 to about 900 nm, such as about 600 to about 800 nm.

Z9. The kit according to any one of items Z1-Z8, wherein the one or more nanofibers have a mean diameter of about 700 nm.

Z10. The kit according to any one of items Z1-Z9, wherein the one or more nanofibers form a three-dimensional scaffold.

Zll. The kit according to any one of items Z1-Z10, wherein the one or more nanofibers are provided in the form of sheets.

Z12. The kit according to any one of items Zl-Zll, wherein the one or more nanofibers are generated by a process selected from the group consisting of electrospinning, meltblowing, drawing, self-assembly, template synthesis, and thermal-induced phase separation. Z13. The kit according to any one of items Z1-Z12, wherein the one or more nanofibers are electrospun nanofibers.

Z14. The kit according to any one of items Z1-Z13, wherein the one or more nanofibers are functionalized with one or more functional groups selected from chemical functional groups, chemical moieties or biological molecules.

Z15. The kit according to any one of items Z1-Z14, wherein the wherein the one or more nanofibers are functionalized by air/oxygen plasma treatment.

Z16. The kit according to any one of items Z1-Z15, wherein the container comprises at least two nanofibers.

Z17. The kit according to any one of items Z1-Z16, wherein the surface area per unit mass of said one or more nanofibers is in the range of 1-10 m²/g, preferably 2-8 m²/g, more preferably 3-6 m²/g.

Z18. The kit according to any one of items Z1-Z17, wherein the immunoregulatory agents are selected from the group consisting of proinflammatory agents, anti-inflammatory agents, superantigens, antibodies, drugs, and combinations thereof.

Z19. The kit according to any one of items Z1-Z18, wherein the immunoregulatory agent is selected from the group consisting of lipopolysaccharide (LPS), Concanavalin A (ConA), anti-CD3 antibodies, anti-CD28 antibodies, anti-CD 52 antibodies, resiquimod, imiquimod, gardiquimod, and cholesterol crystals, and combinations thereof.

Z20. The kit according to any one of items Z1-Z19, wherein the immunoregulatory agent is selected from the group consisting of immunoglobulins, such as IgGl, IgG2a, and IgG4, and combinations thereof.

The invention will now be described in further details in the following non-limiting examples.

Examples

Example 1: Evaluation of nanofibrous network for obtaining a cytokine response from human peripheral blood mononuclear cells (PBMCs) following stimulation The purpose of the example was to evaluate the cytokine response produced by PBMCs cultured on plates comprising a three-dimensional nanofibrous network (3D nanofiber) compared to PBMCs cultured on two-dimensional (2D) tissue culture plates (Market standard).

Method

Preparation of assay plates comprising three-dimensional (3D) nanofibrous network (3D nanofiber)

Electrospun polycaprolactone (PCL) nanofibers with a mean diameter of 700 nm were added into a MicroWell 96-Well Optica I -Bottom Plate. The fibers were secured at the bottom of each well. The plates were sterilized with 70% EtOH followed by UV-C. Finally, the plates were oxygen plasma coated for 5 minutes at 30% power with a low pressure plasma system "Atto" from Diener Electronic. The plates were pre-incubated with cell culture media for 30 min at 37 °C (5% CO2) prior to seeding with cell samples.

Preparation of sample

Human PBMCs from three different donors were isolated from leukocyte concentrate. Shortly, at the day of assay, leukocyte concentrate from three different donors was diluted 1:4 with PBS and added to Ficoll Paque Plus to generate a Ficoll gradient by centrifugation. The gradient comprised five layers after centrifugation in the following order from top to bottom of the tube: Plasma (PBS), PBMCs, Ficoll, granulocytes and erythrocytes. Accordingly, the PBMC layer was located at the interphase between the plasma layer and the Ficoll layer.

The PBMC layer were transferred to new tubes and washed in PBS. Cells were counted using a Scepter cell counter. Cells were counted and diluted to double working concentration (4xl0⁶ cells/ml) in cell culture medium supplemented with 10% Fetal Bovine Serum and Penicillin/Streptomycin. PBMCs (125 pl/well) were plated in 96-well plates comprising either the three-dimensional (3D) nanofibrous network (3D nanofiber) or no nanofibers (Market standard). The market standard plate was a TPP tissue culture plate. Stimuli or control media were then added to the wells. Stimuli were selected from LPS (lOOng/ml), ConA (3pg/ml), CD3/CD28 beads (ratio 1 : 1), or control media. After 24 hours, 150 pl medium was removed and evaluated for cytokine release using Luminex.

The pathogen-associated molecular pattern (PAMP) LPS is recognized by immune cells through the activation of the toll-like receptor 4 (TLR4). Binding of LPS to TLR4 activates NF-KB through the recruitment and activation of MyD88, IL-1R kinase, TNFR associated factor 6, as well as NADPH oxidase. LPS is a strong activator of immune cells including B cells, dendritic cell, monocytes, macrophages, and activates the production of cytokines such as TNFo, IL-ip, IL-6, IL8, IL-10, IL- 12, IL-15 and GM-CSF.

The use of CD3/CD28 beads for T cell activation via the o[3-T cell receptor complex results in a specific T cell response, causing the secretion of cytokines and chemokines i.e. IFN- y, IL-2, IL-4, IL-5, IL-10, IL-13, IL-17A and Granzyme B.

ConA is an antigen-independent mitogen and can be used as an alternative way to stimulate T cells. This lectin is frequently used as a surrogate for antigen-presenting cells in T cell stimulation experiments, however not as specific as the use of CD3/CD28 beads. ConA stimulate transcription factors and cytokine production resulting in the secretion of cytokines i.e. IFN-y, IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17A, IL-21, G-CSF, GM-CSF, Granzyme B and TNF-o.

Cytokine quantification (Luminex assay)

After 24 hours of incubation as described above, supernatants were transferred to new plates and kept at -80°C until cytokine analysis. Cytokine analysis and quantification was performed using Luminex 27-plex kits. All kit reagents, kit standard and the cytokine samples were prepared according to manufacturer's instruction.

Briefly, the kit standard and cytokine samples were subjected to the following sequential process. Magnetic microparticles were added to plates and washed 2x. 50pl of kit standard or cytokine sample (diluted 1:4) were added to wells and all samples were incubated for 30 minutes at room temperature on a horizontal orbital shaker (800rpm). Each plate was washed 3x in washing buffer using a magnetic plate. Biotin-antibody was added and incubated dark for 30 minutes at room temperature on a horizontal orbital shaker (800rpm). Each plate was washed 3x in washing buffer using a magnetic plate. Streptavidin-PE was added to each well and incubated dark at room temperature on a horizontal orbital shaker (800rpm) for 10 minutes. Each plate was washed 3x in washing buffer using a magnetic plate. Microparticles were resuspended in 125pl assay buffer and incubated for 30 seconds on a horizontal orbital shaker (800rpm). All samples were analyzed on a Bio-Rad Luminex 200 analyzer.

Individual results (6-7 repeats/donor) are pooled and presented as mean value ± SEM. The no stimuli control for market standard plates and 3D nanofiber plates are pooled and presented as a single value of no stimuli control. The effect of the type of plate on cytokine release is analyzed with a one-way anova test, followed by Tukey’s multiple comparisons test. Results

Stimuli with either CD3/CD28 beads (Figure 1A-D), LPS (Figure 3, top) or ConA (Figure 3, bottom) generated significantly increased cytokine responses when cultured on plates comprising the nanofibrous network (3D nanofiber) as demonstrated for MIP-ip, IL-5, IL- 6, and IL-lra (Figure 1A-D), IL-ip and G-CSF (Figure 3, top) and IL-ip and IL-6 (Figure 3, bottom), respectively. Noticeably, IL-6 was not detectable with the market standard plate when PBMC were stimulated by CD3/CD28 beads (Figure 1C).

The concentration of a broad selection of cytokines were determined following stimulation of PBMCs with either CD3/CD28 beads, LPS, or ConA. Results were normalised to the cytokine concentration obtained from PBMCs cultured on 3D nanofiber plates for easy comparison to PBMCs cultured on market standard plates (Figures 2 and 4-5). The cytokine concentration was increased for PBMCs cultured on 3D nanofiber plates irrespective of the immunostimulatory agent used, and in particular PBMCs stimulated by CD3/CD28 beads yielded markedly higher cytokine concentrations. Importantly, some cytokines that were hardly detectable using the market standard plates, such as IL-6, gave strong signals when cultured on the 3D nanofiber plates.

Conclusion

This example demonstrates that cytokine concentrations induced in response to exposure to an immunostimulatory agent can be significantly increased by culturing PBMCs on plates comprising a three-dimensional (3D) nanofibrous network (3D nanofiber). Accordingly, the sensitivity is improved compared to the two-dimensional (2D) tissue culture plates (Market standard). Moreover, the 3D nanofiber plates allow detection of a diverse set of cytokines, including cytokines that are not detectable by conventional methods.

Claims

1. An ex vivo method for obtaining an immune response comprising one or more cytokines from a sample, said method comprising the steps of:

(i) provision of a container comprising one or more nanofibers,

(ii) addition of a sample comprising immune cells to said container, and

2. The method according to claim 1, wherein the one or more cytokines are selected from the group consisting of interferons, tumor necrosis factor a (TNFa), colon-stimulating factors (CSF), interleukins, and chemokines, and combinations thereof.

3. The method according to any one of claims 1 or 2, wherein the one or more nanofibers are selected from natural polymers and synthetic polymers, and combinations thereof.

4. The method according to claim 3, wherein the synthetic polymers are selected from the group consisting of polycaprolactone (PCL), poly(lactic acid) (PLA), polystyrene, polyurethane (PU), poly(lactic-co-glycolic acid) (PLGA), poly(3-hydroxybutyrate-co-3- hydroxyvalerate) (PHBV), and poly(ethylene-co-vinylacetate) (PEVA).

5. The method according to any one of the preceding claims, wherein the one or more nanofibers comprises polycaprolactone (PCL).

6. The method according to any one of the preceding claims, wherein the one or more nanofibers form a three-dimensional scaffold.

7. The method according to claim 6, wherein the three-dimensional nanofiber scaffold does not comprise any proteins or proteins.

8. The method according to any one of the preceding claims, wherein the surface area per unit mass of said one or more nanofibers is in the range of 1-10 m²/g, preferably 2-8 m²/g, more preferably 3-6 m²/g.

9. The method according to any one of the preceding claims, wherein the sample is selected from the group consisting of whole blood, peripheral blood mononuclear cells (PBMCs) and splenocytes.

10. The method according to any one of the preceding claims, wherein the container is selected from the group consisting of a multiwell plate, a petri dish and a cell culturing device.

11. The method according to any one of the preceding claims further comprising a step (iv) of detection of said one or more cytokines.

12. The method according to claim 9, wherein steps (iii) and (iv) are separated by no more than about 4 hours, such as no more than about 6 hours, such as no more than about 8 hours, such as no more than about 10 hours, such as no more than about 12 hours, such as no more than about 18 hours, about 24 hours, such as no more than about 36 hours, such as no more than about 48 hours, such as no more than about 72 hours.

13. The method according to any one of the preceding claims, wherein the therapeutic agent is selected from the group consisting of biologies, antibodies, monoclonal antibodies, proteins, peptides, anti-inflammatory agents, small molecule drugs, and cell therapies, and combinations thereof.

14. The method according to any one of the preceding claims, further comprising repeating all steps of the method one or more times wherein one or more immunoregulatory agents are added instead of said therapeutic agent.

15. The method according to any one of claims 13 or 14, wherein the immune response induced by addition of said therapeutic agent to the sample is compared to the immune response induced by addition of said one or more immunoregulatory agents to the sample.

16. The method according to any one of the preceding claims, said method comprising the following steps:

(i) provision of a container comprising one or more nanofibers,

(ii) addition of a sample comprising immune cells to said container,

(iii) addition of a therapeutic agent to said container,

(iv) detection of said one or more cytokines, and

17. The method according to claim 16, wherein said the reference immune response is obtained by performing steps (i)-(iv), wherein an immunoregulatory agent are added instead of said therapeutic agent.

18. The method according to claim 17, wherein said immunoregulatory agent induce a known immune response.

19. The method according to any one of the preceding claims, said method further comprising a step of assigning the therapeutic agent with a risk value for triggering Cytokine Release Syndrome (CRS).

20. The method according to any one of claims 16-19, wherein the therapeutic agent is assigned a first risk value if the immune response detected in step (iv) are equal to or stronger than the reference immune response, and a second risk value if the cytokine response detected in step (iv) are weaker than the reference cytokine responses.

21. An ex vivo method of evaluating the risk that a therapeutic agent triggers Cytokine Release Syndrome (CRS) if administered to a subject, said method comprising the following steps:

(i) provision of a container comprising one or more nanofibers,

(ii) addition of a sample comprising immune cells to said container,

(iv) detection of said one or more cytokines, and

22. The method according to claim 21, wherein the immune response detected in step (iv) is compared with:

23. Use of a container comprising one or more nanofibers for obtaining an immune response comprising one or more cytokines from a sample comprising immune cells, wherein said sample are subjected to a therapeutic agent.

24. A kit comprising: a container comprising one or more nanofibers,

- at least one immunoregulatory agent, optionally, instructions for use.