Micro-imprinting of antibodies and biomolecules for cell phenotypic analysis and activation

Abstract

The present invention provides a device comprising a substrate comprising a first region (1) having adsorbed thereon a protein capable of binding to a first membrane molecule, and comprising a second region (2) having adsorbed thereon an antibody targeting a second membrane molecule, the first and second regions extending together in length beyond a size comparable to the length of the cell.

Description

Technical Field

[0001] The present invention relates generally to the field of quantification of membrane molecule expression and cellular function.

[0002] The present invention more particularly relates to functional tests in clinical immunology. Background Art

[0003] In biological sciences and medicine, it is often necessary to identify cell types and quantify their functions. In particular, the presence of specific molecules in the cell membrane is a traditional and effective means of identification. Taking immunity, which is closely related to most diseases, as an example, rapid and accurate quantification of human immune function is very important for early detection of the development of infectious pathologies or the outcome of organ transplantation, the selection of treatments and the monitoring of their efficacy.

[0004] Therefore, understanding the early response of the immune system to pathogens is crucial for diagnosis and therapeutic application of appropriate treatments.

[0005] Thus, various techniques for quantifying lymphocyte function are known from the prior art, including flow cytometry, lymphocyte proliferation and cytokine production.

[0006] Also known from the prior art are the micrometric printing of proteins on antifouling substrates (LIMAP technique) and the reflection interference microscopy (RIM) technique, which enables the visualization of adhesion zones between micrometrically transparent objects.

[0007] However, rapid visualization of immune system responses is challenging for existing technologies, even though there is a long-standing need in this area.

[0008] For the purposes of this application, "CD45 antibody" refers to an antibody that targets a marker designated "CD45" in the differentiation classification or differentiation cluster nomenclature. In general, "CDx antibody" (where x is a natural number such that CDx belongs to the differentiation cluster nomenclature) refers to an antibody that targets a membrane glycoprotein family or marker or antigen or membrane antigen designated by "CDx".

[0009] For the purposes of this application, a membrane protein of a cell is taken to mean any glycoprotein present on the outer surface of a cell and capable of forming a bond with a specific antibody, in particular:

[0010] -CD3 is a membrane protein as described in the paper "A novel Leukocyte Adhesion Deficiency III variant: Kindlin 3 defect results in integrin and non-integrin related defects in different steps of leukocyte adhesion" (Philippe Robert, J Immunol, May 1, 2011, 186 (9) 5273-5283; DOI: https: / / doi.org / 10.4049 / jimmunol.1003141)

[0011] - CD4 is a membrane protein as described in "A microfuidic device for practical label-free CD4+ T cell counting of HIV-infected subjects" (Xuanhong Cheng-Lab on achip 2007, Vol. 7, No. 2, 170-178 doi: 10.1039 / b612966h).

[0012] If the antibody is deposited on the substrate, the membrane protein will be a substrate-adherent membrane protein, the adhesion in question being measured by the energy of the antigen-antibody bond and having the property of keeping the cell at a distance from the substrate equal to the length of the antigen-antibody bond with a detectable optical effect.

[0013] The energy of antigen-antibody binding is often measured by the term "affinity", or the stability of the binding by the "avidity" of multivalent antigens and antibodies.

[0014] For the purposes of this application, "antibody" means specific antibodies and cross-reactive antibodies, whether monovalent or multivalent; the antibody is any known antibody that is capable of being deposited on a biocompatible substrate and binding to a membrane protein.

[0015] In particular, in the context of chimeric or other proteins, the name "antibody" in the sense of the present application may be understood to refer to a molecule that has not been named "antibody" in the prior art but is capable of assembling with another molecule via an antibody-derived protein domain, such as in the case of CAR T lymphocytes, which molecule is attached to its ligand via a chimeric antibody domain.

[0016] Antigen-antibody bonds considered in the present application are those bonds that keep antigens fixed in the antibody site and are essentially non-covalent according to the prior art. These bonds include hydrogen bonds, electrostatic bonds, van der Waals forces and hydrophobic bonds. The multiple bonds between antigen and antibody ensure the stable interaction between the two molecules, and those bonds considered in the present application are those bonds that enable cells to physically maintain a fixed distance from substrates and therefore use physical means (particularly optically) to detect cells that exist near substrates.

[0017] The terms "optical interference reflectometry" or "reflection contrast interference microscopy" refer to a technique known by the acronym IRM (Interference Reflectometry Microscopy). Summary of the invention

[0018] In this context, the present application relates to a device comprising a substrate comprising a first region having adsorbed thereon a protein capable of binding to a first membrane molecule and a second region having adsorbed thereon an antibody targeting a second membrane molecule, the first region and the second region together extending in length beyond a dimension comparable to a cell length.

[0019] Advantageously, the present application relates to a device characterized in that it comprises a substrate comprising a first region on which is adsorbed a protein capable of specifically binding to a cell by interacting with a first membrane molecule and comprising a second region on which is adsorbed an antibody targeting a second membrane molecule expressed on the surface of the cell,

[0020] The first area refers to a first surface defining a first area A1,

[0021] The second area represents a second surface defining a second area A2,

[0022] Wherein the total area represented by the sum of areas A1 and A2 is less than or equal to the area of ​​the projected surface of the suspended cells ( Figure 1 ).

[0023] In the context of the present invention, the device comprises two zones, which are organized so that cells can interact with the first zone or the second zone or with both zones simultaneously. The aim is to detect the overlap of the first zone or the second zone or both zones and thus phenotype the determined cells with the help of proteins and antibodies adsorbed on the first zone and the second zone, respectively.

[0024] To enable overlapping detection, the first region and the second region have a specific configuration so that the total area corresponding to the sum of the areas of the first region and the second region is less than or equal to the area of ​​the projected surface of the cell.

[0025] Thus, if the cell is able to recognize the protein adsorbed on the first region, the cell will adhere thereto, creating an optical microscopic contrast in the first region by reflection interference contrast, which will appear dark, with the second region remaining light in appearance ( Figure 2 In addition, if the cells that have interacted with the first region are able to interact with the antibodies adsorbed on the second region, the cells that adhered to the first region will spread out on the second region to adhere thereto, thereby forming an optical microscopic contrast due to the reflection contrast in the second region (which is a possible way to display the signal generated by the device), which will also appear dark ( Figure 2 In the absence of cells adhering to the device, the entire pattern remains light-colored. ( Figure 2 A in Figure 3 ). The projection surface of a suspended cell means the plane defined by the largest dimension of the cell when viewed from above. For example, if the cell is a perfect sphere, the projection surface will be a disk of radius r, where r is the radius of the perfect sphere. The projection surface will then have πr 2 area and the total area (A1+A2) will be less than πr 2 . Length is the largest dimension of a two-dimensional or three-dimensional geometric shape (as opposed to width or height).

[0026] A two-dimensional shape has an area less than or equal to its length squared; a three-dimensional shape has a projected area less than or equal to its length squared.

[0027] Since the device can be produced in different geometries, it is defined in the present invention according to the length of the cell with which the device may interact. In other words, the device and the area it contains will be defined according to the cell under consideration.

[0028] The cells in suspension correspond to spontaneously non-adherent cells (blood or hematopoietic cells) or to adherent cells which have been detached from their support (eg by trypsin treatment).

[0029] In the variant ( Figure 4 ):

[0030] - the first membrane molecule is a membrane molecule common to a first cell type, and wherein the second membrane molecule is a membrane molecule common to a subtype of the first cell type.

[0031] - the first membrane molecule is a membrane molecule common to a first cell type, and wherein the second membrane molecule is a membrane molecule common to a specific state of the first cell type.

[0032] - the first membrane molecule is a membrane molecule common to a first cell type, and wherein the second membrane molecule is a membrane molecule common to a second cell type capable of interacting with the first cell type.

[0033] - The protein is an antibody targeting CD4, and the antibody is an antibody targeting CD8.

[0034] -The protein is an antibody targeting CD3.

[0035] -The protein is CD19.

[0036] -Antibodies targeting CD69.

[0037] -Antibodies targeting CD25.

[0038] - Antibodies targeting CD107.

[0039] -The antibodies are anti-collagen antibodies.

[0040] -Antibodies targeting CD86.

[0041] Advantageously, the device is capable of identifying cells via the protein adsorbed on the first zone, and the antibody adsorbed on the second zone is also capable of identifying the cell, for example to confirm its phenotype. An example of a device includes a protein adsorbed on the first zone that is an antibody targeting the CD3 antigen, and the antibody adsorbed on the second zone is an antibody targeting either the CD4 or CD8 antigens or a mixture thereof.

[0042] Advantageously, the device is capable of activating cells, for example inducing new cell functions or cell differentiation, by identifying cells via proteins adsorbed on the first zone and antibodies adsorbed on the second zone. Examples of such devices are:

[0043] - the protein adsorbed on the first region is an antibody targeting CD45RO antigen, and the antibody adsorbed on the second region is an antibody targeting CD3 antigen, or a mixture of antibodies targeting CD3 antigen and CD28 antigen, or

[0044] - the protein adsorbed on the first region is an antibody targeting CD56 or CX3CR1 antigen, and the antibody adsorbed on the second region is an antibody targeting CD20 antigen (rituximab), or

[0045] ii- The protein adsorbed on the first region is an antibody targeting CD14 antigen, and the antibody adsorbed on the second region is an antibody targeting any one of CD16, CD32 or CD64 markers or targeting membrane lipopolysaccharide (LPS).

[0046] Advantageously, the device enables cells to be activated via proteins adsorbed on the first zone and antibodies adsorbed on the second zone to detect a cellular response in the form of expressed membrane molecules, such as activation or senescence, i.e. read out the response. Examples of such devices are:

[0047] - the protein adsorbed on the first region is CD19 or a chimeric protein comprising CD19, and the antibody adsorbed on the second region is an antibody targeting any one of the antigens CD69, CD107, CD25, CD57, TIM-3 and LAG-3, or

[0048] - The protein adsorbed on the first region is an antibody targeting the CD20 antigen (rituximab), and the antibody adsorbed on the second region is an antibody targeting the CD107 antigen.

[0049] Advantageously, the device is capable of identifying cells via proteins adsorbed on the first zone and antibodies adsorbed on the second zone to detect cellular responses in the form of expressed membrane molecules, such as activation or senescence, i.e., read the response. An example of such a device is that the protein adsorbed on the first zone is CD19, and the antibody adsorbed on the second zone is an antibody targeting any one of the antigens CD69, CD107, CD25, CD57, TIM-3, and LAG-3.

[0050] Also described is a device as defined above, wherein the first membrane molecule is a membrane molecule expressed at the surface of a first cell type, and wherein the second membrane molecule is a membrane molecule expressed at the surface of a subtype of the first cell type, the subtype of the first cell type expressing the first membrane molecule and the second membrane molecule at its surface.

[0051] The present application further relates to a method comprising the following steps:

[0052] - adsorbing a protein capable of binding to a first membrane molecule on the substrate in a first subcellular size region of the substrate,

[0053] - adsorbing an antibody targeting a second membrane molecule onto the substrate in a second subcellular size region of the substrate.

[0054] The present application also relates to a variant of a method for quantifying and monitoring cell activation kinetics, the variant comprising the following steps:

[0055] - adsorbing a protein capable of binding to a first membrane molecule common to a first cell type onto a substrate in a first subcellular size region,

[0056] - adsorbing to the substrate in the second subcellular size region an antibody targeting a second membrane molecule common to a specific state of the first cell type,

[0057] - contacting a first type of cell population comprising cells of the first type allowing activation kinetics towards a specific state with a substrate,

[0058] - detecting the presence of cells of the first type in the first zone using an optical microscope,

[0059] - quantifying the kinetics of activation of the first type of cells by detecting adhesion of the first type of cells in the second zone using optical microscopy.

[0060] The present application also relates to a variant of the method for selecting a cell type and for triggering its activation, comprising the following steps:

[0061] - adsorbing a protein capable of binding to a first membrane molecule common to a first cell type onto a substrate in a first subcellular size region,

[0062] - in the second subcellular size region, adsorbing to the substrate an antibody targeting a second membrane molecule known to trigger activation of the first cell type,

[0063] - contacting a cell population comprising cells of a first type that are allowed to be activated towards a specific state with a substrate,

[0064] - detecting the presence of cells of the first cell type expressing the first membrane molecule in the first zone using an optical microscope,

[0065] - detecting the triggering of activation of the first type of cells by detecting adhesion of the first type of cells in the second zone using optical microscopy.

[0066] The present application also relates to a variant of a method for detecting cell membrane molecules, the variant comprising the following steps:

[0067] - adsorbing a protein capable of binding to a first membrane molecule common to a first cell type onto a substrate in a first subcellular size region,

[0068] - adsorbing to the substrate in the second subcellular size region an antibody targeting a second membrane molecule common to a subtype of the first cell type,

[0069] - contacting a first type of cell population comprising cells of the subtype with a substrate,

[0070] - detecting the presence of cells of the first cell type expressing the first membrane molecule in the first zone using an optical microscope,

[0071] - Quantification of the cellular expression of the second membrane molecule by detecting cell adhesion in the second zone using light microscopy.

[0072] The present application also relates to a variant of a method for detecting the interaction of two cells, the variant comprising the following steps:

[0073] - adsorbing a protein capable of binding to a first membrane molecule common to a first cell type onto a substrate in a first subcellular size region,

[0074] - adsorbing to the substrate in the second subcellular size region an antibody targeting a second membrane molecule common to a second cell type capable of interacting with the first cell type,

[0075] - contacting a cell population comprising cells of a first type and cells of a second type with a substrate,

[0076] - detecting the presence of cells of the first type in the first zone using an optical microscope,

[0077] - Using light microscopy to detect the presence of cells of the second type or to quantify the expression of a second membrane molecule by second cells in the second region.

[0078] Also described are uses of the substrates, in particular for characterizing the activation or differentiation state of a given cell in a cell population in vitro,

[0079] The substrate comprises a first region on which a protein capable of specifically binding to the determined cell by interacting with a first membrane molecule specific to the determined cell is adsorbed, and a second region on which an antibody targeting a second membrane molecule expressed on the surface of the determined cell is adsorbed,

[0080] The first area refers to a first surface defining a first area A1,

[0081] The second area represents a second surface defining a second area A2,

[0082] wherein the total area represented by the sum of areas A1 and A2 is less than or equal to the area of ​​the projected surface of the suspended cells,

[0083] The second membrane molecule is expressed on the surface of the specific cells when the cells are activated or differentiated.

[0084] In general, the present invention is based on methods that enable phenotypic and single-cell analysis of the activation properties of immune cells, such as Figures 1 to 5 shown.

[0085] This method can be used to generate multiple microdomains (micropatterns) of different proteins and subcellular sizes using optical microscopy ( Figure 3 ), and these microengineered substrates enable a variety of functional assays to be performed on immune cell suspensions or whole blood, such as (i) selection / identification of cell types of interest, (ii) triggering activation signals, and (iii) reading the kinetics of immune activation. The present invention is not limited to such optical microscopy, and any instrument that projects UV light (e.g., confocal microscopy) or any (micro) printing technology may also be used. A person skilled in the art will be able to determine the most appropriate instrument.

[0086] These functions are assessed by the properties of the micropatterned proteins, which are usually antibodies with specific affinity and efficacy. When cell membranes in suspension express the antibody target on the substrate, the cells spread on the corresponding pattern ( Figure 2 ), and cell adhesion smears can be detected by interference microscopy ( Figure 2 ). Detection is not limited to reflection interference contrast imaging, and detection of cell outlines by transmission imaging and image collection can also be used. Here, those skilled in the art will be able to determine which technology is most appropriate.

[0087] By taking a single image and analyzing the spreading of cells in each pattern, the different functions performed by the micropatterns can be assessed. Notably, the readout does not require the tedious manipulations typically involved in immunolabeling-based techniques (cell preparation, incubation, washing). In a typical interference microscopy image, dark areas represent adhesion and bright areas represent non-adhesion ( Figure 2 and Figure 3 ). In practice, the operator's task is limited to placing the smart substrate on a dedicated optical microscope and depositing the cell sample on the substrate. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] Figure 1 : In A, an example of a printed pattern is shown in plan view at the top and in cross section at the bottom. The pattern consists of two concentric zones (1 and 2), each carrying an antibody of a particular specificity, in the example, CD3 in the center (1) and CD69 in the periphery (2). In B, an example of a circulating cell (T lymphocyte) is shown at the same scale, with a membrane marker (here CD3) on its surface, which may interact with the central zone of the pattern. In this example, the largest dimension of the pattern (10 μm) is smaller than the largest dimension of the cell (12 μm).

[0089] Figure 2 :and Figure 1Examples of different cells interacting with the same pattern shown, with the top of the signal obtained in the reflection interference contrast being a side view and the bottom being a plan view (this is one possible way to reveal the signal generated by the device). In A, an example of the interaction between cells that do not carry a membrane marker corresponding to the specificity of the antibodies present. B lymphocytes (showing their CD20 marker) do not express membrane ligands to interact with the antibodies of the pattern and do not adhere to the surface; the resulting appearance in the reflection interference contrast is the absence of signal. In B, an example of the interaction between cells that carry a single type of membrane marker corresponding to one of the specificities of the antibodies present on the pattern. Unactivated T lymphocytes express CD3 but not CD69. The CD3 molecule of this cell is captured by the antibody in the central area of ​​the pattern, so its membrane adheres to this area. This produces interference contrast in the central area of ​​the pattern. In C, an example of the interaction between cells that carry two types of membrane markers corresponding to the specificity of the two types of antibodies present on the pattern. The CD3 molecule of this cell is captured by the antibody in the central area of ​​the pattern, and the CD69 molecule of this cell is captured by the antibody in the peripheral area of ​​the pattern, so its membrane adheres to both areas. This produces interference contrast in the central and peripheral regions of the pattern.

[0090] Figure 3 : In use Figure 1 and Figure 2 Examples of real images obtained during experiments with the type of patterns described in, which were repeated to form a matrix of patterns on a slide. In A, a transmission optical microscopy image shows T lymphocytes individually adhering to a matrix pattern location. Some locations did not capture any cells. In B, a reflection interference contrast image at the same magnification shows that each cell adhering to the pattern in A produces a contrast on the central region of the pattern in B, thereby identifying each cell as expressing CD3 on its surface and therefore a T cell. Some cells produce varying degrees of contrast on the peripheral regions of the pattern to which they adhere, thereby identifying these cells as expressing CD69 on their surface and therefore activated T cells. In C, a fluorescence microscopy image of the same sample at the same magnification, after the cells have been fixed with formaldehyde and incubated with a fluorescent anti-CD69 antibody, shows the membrane expression of the molecule and allows us to observe a matching arrangement with the contrast produced on the peripheral regions of the pattern for each cell: the bright cells in C generally occupy the peripheral regions of the pattern.

[0091] Figure 4 : Example of a possible pattern with two regions, each carrying an antibody type with a particular specificity (with Figure 1 and Figure 2The same pattern as described in ). An example of a pattern with three zones, each carrying an antibody type with a specific characteristic. The total pattern size is kept below 10 μm, which is smaller than the suspension size of the target cell type (here leukocytes).

[0092] Figure 5 : Example of a pattern with three zones, each carrying an antibody type with a specific signature. The total pattern size is kept below 10 μm, which is smaller than the suspension size of the target cell type (here leukocytes). DETAILED DESCRIPTION

[0093] In a first embodiment, reference is made to Figure 1 For element numbers in bold or in parentheses, the present invention can be implemented on a substrate by LIMAP technology, which enables proteins to be arranged and adsorbed on a substrate coated with a PEG (polyethylene glycol) brush isolated according to a UV light pattern, thereby achieving specific adhesion of proteins in the isolated area.

[0094] However, in every case where the adhesion of several proteins is desired, and in particular for two antibodies, it must be possible to specifically deposit a first antibody and then a second antibody, each antibody being specifically deposited in the desired pattern.

[0095] Once specific methods are developed for patterning that works for single cells, the generation of multiple patterns can be replicated in spatial parallel to rapidly obtain a matrix of patterns that enables testing of as many captured cells as there are patterns present on the LIMAP-printed substrate.

[0096] Experimentally, the first zone (1) was locally printed by the combined action of photoinitiators (PLPP, Alveole), placed in solution on a PEG-SVA brush and projected with UV patterns at 375 nm (step 1). The first antibody was then incubated at 4°C for 12 hours (step 2). The second zone (2) was then printed by LIMAP (step 3). The second antibody was added and incubated at 4°C for 12 hours (step 4).

[0097] Here, the second antibody is non-specifically adsorbed to the second zone (which is not limited to the most recently irradiated pattern) because it is also adsorbed in the first zone. The antifouling substrate (PEG brush) is rendered adhesive in the first zone by the first irradiation and in the second zone by the second irradiation, which means that the second antibody is adsorbed not only in the second zone but also in the first zone. These properties can be verified by epifluorescence, for example, using separate fluorescent markers for the antibodies and observing them with interference filters that separate the wavelength of each fluorescent marker.

[0098] In order to avoid non-specific adsorption of the second antibody in the first zone, a person skilled in the art may use a passivation technique known in the art between the two irradiation stages, such as using a passivation solution commonly used in biophysics, in particular:

[0099] -4% BSA in PBS solution was kept at room temperature for 1 hour.

[0100] -4% BSA in PBS for 30 minutes, followed by 1 mg / mL rabbit serum IgG at room temperature for 30 minutes.

[0101] -1% Pluronic F127 in PBS at room temperature for 15 minutes

[0102] The best passivation solution found was 0.23 mg / mL PEG-SVA in Milli-Q water with 10 mM sodium bicarbonate at room temperature for 15 minutes.

[0103] Once a passivation method based on minimal nonspecific adsorption is chosen, the LIMAP technique can be used to generate a periodic pattern consisting of two potential cell adhesion zones: one specific for T lymphocytes as a whole and one specific for activated T lymphocytes. As many adhesion zones as desired can be generated by irradiating each zone, exposing it to the antibody selected for that zone, then passivating and irradiating the next zone, and so on.

[0104] Conveniently, for each zone, the zone as a pattern of spatially periodic structures is replicated in a spatially periodic manner, the pattern being moved across the substrate to enable the same antibody to be deposited and passivated for all illuminated patterns in a single step.

[0105] The replication is conveniently carried out in a known manner using the LIMAP technique, wherein a periodic pattern is produced using an array of micromirrors and imaged all at once onto a substrate in UV light.

[0106] Thus, each antibody is adsorbed only in its zone, i.e. specifically in this zone, the antibodies are adsorbed in continuous zones forming a pattern, the length of the pattern (meaning the maximum geometric dimension) is comparable, it is less than or equal to the length of the cells to be attached (meaning the maximum geometric dimension again), and the patterns are spaced apart so that only one cell can adhere to each pattern. A person skilled in the art can adjust the shape and length of each zone and the distance between the patterns by simple manipulations to ensure that for a specific cell type, only one cell per pattern adheres in a given adhesion order or a given adhesion spatial configuration.

[0107] For example, for phenotyping memory T lymphocytes, a first adhesion zone of the aCD45RO type ("antibody targeting CD45RO") is defined within a circle and a second adhesion zone of the aCD69 type ("antibody targeting CD69") is defined on a ring concentric with the first zone.

[0108] For example, to monitor the activation of memory T lymphocytes, a first adhesion zone of the aCD45RO type ("antibody targeting CD45RO") mixed with aCD3 and aCD28 is selected to allow all memory T lymphocytes to adhere (all expressing CD45RO), and then the activation of the already adhered cells is triggered (via CD3+CD28), the first zone is further defined within a circle, and the same second adhesion zone of the aCD69 type ("antibody targeting CD69") is defined on a ring concentric with the first zone to allow only the activated memory T lymphocytes that specifically express CD69 to adhere.

[0109] Therefore, the device of the present invention can be used in various ways to statically phenotype activated memory T lymphocytes, or dynamically monitor the activation of naive memory T lymphocytes by adding memory T lymphocyte antibodies to adhesion antibodies common to all memory T lymphocytes.

[0110] This device can be easily adapted to any type of cell that can be activated.

[0111] In this embodiment, a substrate is thus obtained on which T lymphocytes can advance and move until they attach to each first zone of the periodically repeating pattern and only when activated do they adhere to each second zone of the periodically repeating pattern.

[0112] By testing zones of variable irradiation diameter on the substrate, the maximum size of the first zone and / or the second zone can be varied to be of the order of a few micrometers, for example 4 μm to 12.5 μm, in order to induce adhesion of a single immune cell.

[0113] As already mentioned, CD45 antibody or CD45 / CD3 / CD28 antibody mixture can be deposited in each first zone as long as it induces non-specific adhesion of any activated or non-activated T lymphocytes, in particular memory T lymphocytes.

[0114] For example, in each second zone, CD69 antibody or a mixture of antibodies containing it, such as CD69+CD25, or a third continuous zone containing CD25 can be deposited, as long as it induces specific adhesion of any activated T lymphocytes, especially memory T lymphocytes. It is also possible to deposit only CD25 to observe secondary T lymphocyte activation with CD69 instead of primary activation (i.e., prior to secondary in time), or both activations with CD69+CD25.

[0115] The activation kinetics can be followed in a known manner using MRI microscopy, where light areas show non-adhesion, meaning that non-activated T lymphocytes will have a first dark area and a second area, the brighter this second area, the weaker their adhesion, the greater their distance from the substrate, or the weaker their activation.

[0116] The shape of the area printed, deposited or coated with the antibody may be contained within a circle or ring.

[0117] For example, a suitable pattern for the first region is a circular disk, and a suitable pattern for the second region is a ring concentric with the circular disk, both having a maximum dimension of about ten microns.

[0118] Then, the adhesion of T lymphocytes to the substrate is observed in the first and second zones, making it possible to count early activated lymphocytes and dynamically monitor their activation intensity as their adhesion strength changes. Thus, camera recording enables precise and quantitative monitoring of kinetics at the early stages of T lymphocyte activation, thus facilitating the delivery of rapid results.

[0119] The interpretation of the invention is particularly straightforward, since adhesion to the rings of the pattern is synonymous with activation of T lymphocytes captured on the pattern, and the absence of adhesion to the rings is characteristic of unactivated T lymphocytes. Using MRI as a method of obtaining contrast, for example, the presence of the rings of the pattern is synonymous with activation of T lymphocytes captured on the pattern, and the absence of the rings is characteristic of unactivated T lymphocytes.

[0120] Using visible optical markers, it will be possible to determine whether a T lymphocyte is present and whether it is activated at each site comprising the first pattern and the second pattern, without relying on fluorescence and therefore requiring no manipulation.

[0121] According to the general inventive principle of the present invention, many variants are possible, which deposit a first antibody that allows any activated or non-activated T lymphocyte to adhere in a first micrometric area (the first antibody is an antibody that is not specific for activated T lymphocytes), and then deposit a second antibody that allows any activated T lymphocyte to adhere in a second micrometric area (the second antibody is an antibody that is specific for activated T lymphocytes).

[0122] Therefore, the present invention allows real-time monitoring of the activation of T lymphocyte immune function from the beginning of infection or transplantation.

[0123] For B lymphocytes, CD20 antibody can be used instead of CD45RO antibody.

[0124] Thus, for a given family of cells that do not specifically express CDx but specifically express CDy (where y is different from x) in a particular state, cells located on cell domains, aCDx, and aCDy (in the sense of extending over the length of the cell) can be deposited successively by additional deposition of aCDz to detect cells in a particular state or cells progressing toward a particular state, where CDz is expressed upon activation of the particular state. Since each cell can occupy at most one domain, or each domain can accommodate at most one cell, quantitative and qualitative techniques can be performed on a cell population according to the teachings of the present application.

[0125] The geometry of the zones described is non-limiting, and in particular, non-concentric zones may be taught in the present application for certain cell types.

[0126] Thus, this application describes a widely available and easily adaptable tool for quantitatively and qualitatively studying various cells and their states, which can be tested using only a microscope equipped with MRI to observe the results and using conventional means to prepare cells with or without a specific state.

[0127] This tool is particularly useful when discovering new clusters of differentiation and their associated antibodies.

[0128] The invention is industrially applicable in the field of testing in clinical immunology and oncology, in the field of cell-based testing for drug research, and in the field of cell-based testing for basic research.

[0129] In addition to the tried and tested methods described above, the present invention can be used in many other applications.

[0130] In some embodiments, the present invention is designed for use in cell phenotyping assays.

[0131] In one embodiment of phenotyping circulating lymphocytes, antibodies targeting CD3 can be deposited in the first zone and antibodies targeting CD4 can be deposited in the second zone, antibodies targeting CD8 can be deposited in the third zone, antibodies targeting CD19 can be deposited in the fourth zone, and antibodies targeting CD16 mixed with antibodies targeting CD56 can be deposited in the fifth zone.

[0132] In embodiments, the invention is designed to perform cell activation assays in response to molecular ligands.

[0133] In an embodiment for quantifying T lymphocyte activation, antibodies targeting CD3 may be deposited in a mixture containing antibodies targeting CD28 in the first zone, antibodies targeting CD4 may be deposited in the second zone, antibodies targeting CD8 may be deposited in the third zone, and antibodies targeting CD69 may be deposited in the fourth zone. In a variation of this embodiment, antibodies targeting CD69 may be replaced by antibodies targeting CD25, or antibodies targeting CD25 may be added to the fifth zone.

[0134] In embodiments for quantifying B cell activation, antibodies targeting the IgM Fc fragment and antibodies targeting CD69 may be deposited in the fourth zone.

[0135] In one embodiment for quantifying NK lymphocyte activation, anti-CD16 antibodies may be deposited in the first zone, antibodies targeting CD56 may be deposited in the second zone, and antibodies targeting CD107 may be deposited in the third zone.

[0136] In one embodiment for quantifying monocyte activation, anti-CD16 antibodies can be deposited in the first zone, antibodies targeting CD14 can be deposited in the second zone, and antibodies targeting CD86 can be deposited in the third zone. In a variant of this embodiment, antibodies targeting CD16 can be replaced by antibodies targeting CD32.

[0137] In one embodiment for the activation of quantitative chimeric antigen receptor T lymphocytes (CAR T-cells), CD19 protein can be deposited in the first zone, and antibodies targeting CD69 can be deposited in the second zone. In a variant of this embodiment, antibodies targeting CD69 can be replaced by antibodies targeting CD25.

[0138] In one embodiment, fibronectin can be deposited in the first zone and antibodies targeting collagen can be deposited in the second zone to detect the fibrotic phenotype of fibroblasts. In one variation, TGF-β (TGF-beta) can be added to the fibronectin in the first zone.

[0139] In some embodiments, the present invention is designed to perform cell activation assays during cell-cell interactions.

[0140] In the embodiment for detecting antigen-specific T lymphocyte activation, anti-CD14 antibodies can be deposited in the first zone, and antibodies targeting CD69 can be deposited in the second zone. In a variant of this embodiment, antibodies targeting CD69 can be replaced by antibodies targeting CD25.

[0141] In one embodiment for detecting NK lymphocyte cytotoxicity, CD16, which is known to bind to K562 target cells, can be deposited in the first zone, and an antibody targeting CD107 can be deposited in the second zone.

[0142] In one embodiment, a checkerboard of sequential protein and antibody zones of varying sizes and subcellular cycles can be printed, and single cell measurements can be made across at least one protein zone and one antibody zone by identifying cell outlines and analyzing adhesion in each zone present in the zone defined by the outlines.

[0143] Throughout this application, the term "subcellular size" will refer to:

[0144] - a region whose length is less than the size of a cell of one type and cells of the same type activated to a particular state, or

[0145] - a zone having a length shorter than the length of the zone spanning the first type of cell and the second type of cell, and not overlapping the two cells when they are in contact. Typically, the size of cells in suspension will be about 8 μm due to the present invention, and may extend to over 50 microns for cells adhered to the substrate.

[0146] In all embodiments, the present invention can be accomplished by adding one or more regions, each region containing a protein targeting a membrane molecule or an antibody targeting a membrane molecule of a cell type selected by the regions of the above embodiments.

[0147] Example:

[0148] Protocols for implementing the teachings of the present application are presented below by way of non-limiting example.

[0149] Application Example 1 : Lymphocyte-selective substrates, activation without substrate and activation with substrate readout.

[0150] Description of the program:

[0151] a-Surface treatment:

[0152] Under the dust cover:

[0153] - Plasma cleaned 22x22 slides: Nexterion Glass D Schott Minifab Cleanroom Clean (Ref 1472309). Plasma cleaner pressure 300mTor

[0154] - Place the plasma cleaner on high for 30 minutes.

[0155] - Remove the slides from the plasma and place each slide in a Petri dish.

[0156] - Prepare APTS solution (200 μL / slide): in milli-Q water + 1% (3-aminopropyl)triethoxysilane (APTS) (reference number A3648 Sigma-Aldrich) + 0.03% acetic acid 196 μL milli-Q water + 2 μL 3% acetic acid + 2 μL APTS

[0157] - Place the solution on a slide in a fume hood and incubate at 4°C for 2 hours

[0158] - Rinse the petri dish 3 times with milli-Q water

[0159] - Dry the slides in a fume hood under a stream of nitrogen gas.

[0160] - Dry on a hot plate at 95°C for 15 minutes

[0161] - Use nitrogen to remove any dust

[0162] - Pasted on PDMS membrane (250μm)

[0163] - Under a fume hood, glue the slide (UV glue) to the bottom of a perforated petri dish (from the outside)

[0164] -Place the petri dish in a UV cube (full surface, 1 minute, 100% laser)

[0165] - Prepare PEG-SVA solution (10 μL / well): milli-Q water, 10 mM carbonate buffer pH 8.5 and 23% PEG-SVA (mPEG-succinimidyl valerate MW 5000Da, reference number: I09681 INTERCHIM). For 40 μL final solution: 36 μL milli-Q water, 4 μL 100 mM carbonate buffer and 0.0092 g PEG SVA

[0166] - Add 10 μL to each well

[0167] - Incubate overnight at 4°C

[0168] - Rinse 8mL / well H2O milli-Q

[0169] b-Printing solution

[0170] Marker (pattern 1):

[0171] - BSA 4% at room temperature for 15 minutes (20 μL / well)

[0172] - Flush 8 mL / well at 4 mL / min

[0173] -Deposit 10 μL / well PLPP

[0174] -Printed under oxygen flow at 3000mj / mm2

[0175] - Flush 8 mL / well at 4 mL / min

[0176] - Deposit BSA-fluorescein and incubate at room temperature for 5 minutes

[0177] - Rinse with PBS 8 mL / well at 4 mL / min

[0178] Passivation:

[0179] -PEG-SVA 5% 10 mM carbonate buffer 20 μL / well at room temperature for 5 minutes

[0180] - Rinse 8mL / well H2O milli-Q

[0181] -BSA 4% 20 μL / well at room temperature for 15 minutes

[0182] - Rinse with PBS 8 mL / well at 4 mL / min

[0183] Printing a 10 μm diameter center circle (pattern 2):

[0184] -Deposit 10 μL / well PLPP

[0185] -Printed under oxygen flow at 3000mj / mm2

[0186] - Flush 8 mL / well at 4 mL / min

[0187] - Anti-CD3-CD28 antibodies were deposited at 100 μg / mL-100 μg / mL-50 μg / mL respectively (final volume 20 μL / well) For one well: 2 μL CD3 and CD28, 1 μL CD45, 15 μL PBS

[0188] - Incubate overnight at 4°C

[0189] - Flush 8 mL / well at 4 mL / min

[0190] Passivation:

[0191] -PEG-SVA 5% 10 mM carbonate buffer 20 μL / well at room temperature for 5 minutes

[0192] - Rinse 8mL / well H2O milli-Q

[0193] -BSA 4% 20 μL / well at room temperature for 15 minutes

[0194] - Rinse with PBS 8 mL / well at 4 mL / min

[0195] Peripheral pattern printing (pattern 3)

[0196] -Deposit 10 μL / well PLPP

[0197] -Printed under oxygen flow at 3000mj / mm2

[0198] - Flush 8 mL / well at 4 mL / min

[0199] - Anti-CD69 antibody was deposited at 100 μg / mL (final volume 20 μL / well) For one well: 4 μL CD69 and 16 μL PBS

[0200] - Incubate overnight at 4°C

[0201] - Flush 8 mL / well at 4 mL / min

[0202] Reference Antibodies:

[0203] - Anti-hCD45 AB: Reference No. NBP2-34804, clone SPM570, NovusBio

[0204] - Anti-hCD3 AB: Ultra leaf purified, reference number 300438, clone UCHT1, Biolegend

[0205] - Anti-hCD28 AB: Ultra leaf purified, reference number 302933, clone CD28.2, Biolegend

[0206] - Anti-hCD69 AB: Ref. MAB2359, clone 298633, R&D system.

[0207] Application Example 2 : Substrates for the selection and identification of memory T lymphocytes with substrate activation and substrate activation readout.

[0208] In this example, the same protocol as Example 1 will be followed, substituting CD45RO for CD3-CD28.

[0209] Application Example 3: T lymphocyte activation measurement

[0210] Figure 3Examples of measurements performed in devices with micropatterns designed to house single T lymphocytes and detect their activation by the TCR complex are shown. These patterns consist of two different zones, each with a specific function: the central zone contains anti-CD3 and anti-CD28 antibodies to select and activate T lymphocytes, and the outer zone contains anti-CD69 antibodies to read CD69 expression of activated cells. Figure 3 The interferometry readout is shown on the right side of B in FIG. It can be seen that some anti-CD69 areas are dark, which means that these captured T lymphocytes were activated in this experiment (confirmed by immunofluorescence). This example shows the concept of a "smart" substrate with multiple antibodies, which is highly flexible and adaptable to different applications and cell types by selecting the printed antibodies accordingly.

[0211] Application Example 4: Other Possible Applications

[0212] The technology described in this invention enables functional assays for personalized use of new immunotherapies by monitoring immune cell activation. Assessing the efficacy of immunotherapies and stratifying patients as potential responders can optimize the use of these expensive treatments. This is an important medical problem. Functional exploration is often too slow and complex to translate into clinical settings, where the cost of manual processing and expertise is highly prohibitive and remains the exclusive domain of academic research. The technology developed by the inventors with "smart" substrates with multiple functional micropatterns enables the measurement of leukocyte activation within hours and can effectively address the unmet need for developing rapid, clinically acceptable functional assays.

[0213] One of the main advantages of this technology is that it can be applied to measure various functions of different leukocyte types. In order to adapt this technology to new specific functions and new types of leukocytes, simply change the properties of the antibodies in the micropattern. The inventors have identified (non-exhaustive) a series of antibodies and molecules to be imprinted, so that various immune cells are selected / identified, activated, triggered, and the functions of cell expression readout (Table 1). For most cell types and functions, the model is based on antibody printing. In some cases, such as in the case of CAR-T lymphocytes, it is important to imprint CD19 as an activation target, and CD19 is not an antibody. In all cases, adapting this technology to new applications relies on the same type of surface chemistry and physicochemistry, and this is just a matter of changing the antibody type in the proven preparation scheme to produce a new functional device.

[0214] Table 1: Used to perform specific functions on different cell types (identification, selection, activation, stimulation, response readout) The micropatterns are of relevant antibodies and proteins.

[0215]

[0216] For example, we may be interested in assessing the activation properties of CAR T cells when combined with a chimeric CD19 target, or NK cells against a rituximab target (see Figure 4 and Figure 5 ).

[0217] In this example, a glass slide (SCHOTT Nexterion) was plasma treated for 15 minutes and covered with a PDMS mold (polydimethylsiloxane, Sylgard 184) to create a channel. A solution of APTES ((3-aminopropyl) triethoxysilane, Sigma) diluted to 1% in milli-Q water containing 0.03% acetic acid was incubated on the glass at 4°C for 2 hours. The surface was then rinsed with water and incubated at 95°C for 15 minutes. A solution of PEG-SVA (MW: 5000Da, INTERCHIM) in 10mM carbonate buffer (NaHCO3) was then incubated on the glass at 4°C for 12 hours. The channel was then rinsed with water, and a PLPP solution (15.5mg.mL-1, Alveole) was introduced into the channel and pumped at 4μL×s with a syringe pump (NEMESYS, Cetoni) throughout the exposure process. -1 The surface was exposed to 3,000 mJ×mm using a PRIMO platform (Alveole). -2 The UV dose was used to produce the binding pattern, and then incubated with 50 μg.mL-1 of 6-x anti-His tag (Life Technologies) at room temperature for 1 hour. The solution was then rinsed and the surface was blocked with 4% BSA (bovine serum albumin, Sigma) for 15 minutes. Then 20 μg × mL-1 of CD19-His-tag (Life Technologies) solution was incubated at 4 ° C for 12 hours. The surface was then blocked again with 4% BSA for 20 minutes, and then isolated with UV light to produce a peripheral pattern for activation reading. Finally, the surface was incubated with human anti-CD69 (Bio Techne, R&D Systems) at room temperature for 1 hour. The cells were then introduced into the channel for 10 minutes and then rinsed so that only the cells remained on the pattern. Readings were taken after incubating the cells for 3 hours in the device.

Claims

1. A device, characterized in that The device comprises a substrate comprising a first region (1) on which a protein capable of binding to a first membrane molecule is adsorbed, and a second region (2) on which an antibody targeting a second membrane molecule is adsorbed, wherein the first region and the second region together extend in length exceeding a dimension comparable to a cell length.

2. The device of claim 1, wherein the first membrane molecule is a membrane molecule common to a first cell type, and wherein the second membrane molecule is a membrane molecule common to a subtype of the first cell type.

3. The device of claim 1, wherein the first membrane molecule is a membrane molecule common to a first cell type, and wherein the second membrane molecule is a membrane molecule common to a specific type of the first cell type.

4. The device of claim 1, wherein the first membrane molecule is a membrane molecule common to a first cell type, and wherein the second membrane molecule is a membrane molecule common to a second cell type capable of interacting with the first cell type.

5. The device of claim 1, wherein the protein is an antibody targeting CD4 and the antibody is an antibody targeting CD8.

6. The device of claim 1, wherein the protein is an antibody targeting CD3.

7. The device of claim 1, wherein the protein is CD19.

8. The device according to claim 1, wherein the antibody is an antibody targeting CD69.

9. The device according to claim 1, wherein the antibody is an antibody targeting CD107.

10. The method is characterized in that the method comprises the following steps: - adsorbing an antibody capable of binding to a first membrane molecule in a first subcellular size region of the substrate, - adsorbing an antibody targeting a second membrane molecule onto the substrate in a second subcellular size region of the substrate.

Images