Micro - imprinting of antibodies and biomolecules for cell phenotype determination and activation

Abstract

The device includes a substrate comprising a first region (1) to which a protein capable of binding to a first membrane molecule is adsorbed and a second region (2) to which an antibody targeting a second membrane molecule is adsorbed, the first region and the second region extending together over a length corresponding to the length of a cell.

Description

Technical Field

[0001] The present invention generally relates to the field of quantifying the expression of membrane molecules and cell functions.

[0002] More particularly, the present invention relates to functional tests for 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 conventional and effective means of identification. Taking immunity, which is strongly involved in most diseases, as an example, the rapid and accurate quantification of human immune function is very important for the early detection of the onset of infectious diseases or the outcome of organ transplantation, the selection of treatment, and the monitoring of their effectiveness.

[0004] Therefore, knowledge of the initial response of the immune system to pathogens is essential for the therapeutic application of diagnosis and appropriate treatment.

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

[0006] Furthermore, from the prior art, micrometric printing of proteins on antifouling substrates (LIMAP technology) and reflection interference microscopy (RIM) technology are known, which make it possible to visualize the adhesion areas between micrometric transparent objects.

[0007] However, the rapid visualization of immune system responses is a difficult problem for the prior art, despite the long-standing need in this field.

[0008] For the purposes of the present application, a "CD45 antibody" refers to an antibody that targets a marker designated as "CD45" in the nomenclature of differentiation classes or clusters. In general, a "CDx antibody" (where x is a natural number such that CDx belongs to the nomenclature of differentiation clusters) designates an antibody that targets a membrane glycoprotein designated by "CDx", or a family of markers or antigens or membrane antigens.

[0009] For the purposes of the present application, a cell membrane protein means any glycoprotein that is present on the outer surface of a cell and is capable of forming a bond with a specific antibody, and in particular: - CD3 is a membrane protein 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). - CD4 is a membrane protein described in "A microfuidic device for practical label-free CD4+T cell counting of HIV-infected subjects" (Xuanhong Cheng - Lab on a chip 2007, Volume 7 Issue 2 170 - 178 doi:10.1039 / b612966h).

[0010] When an antibody is deposited on a substrate, the membrane protein is a substrate-attached membrane protein, and the attachment in question is of such a nature that it is measured by the energy of the antigen - antibody bond, maintains the cell at a distance from the substrate equal to the length of the antigen - antibody bond, and has a detectable optical effect.

[0011] The energy of antigen-antibody binding is generally measured by the term "affinity" or "binding activity" for multivalent antigens and antibodies, which refers to the stability of the binding.

[0012] For the purposes of this application, "antibody" means both specific antibodies and cross-reactive antibodies, whether monovalent or multivalent. An antibody is any known antibody that can be deposited on a biocompatible substrate and can bind to a membrane protein.

[0013] In particular, when including chimeric proteins or other proteins, the term "antibody" in the context of this application refers to molecules that, although not designated as "antibodies" in the prior art, can associate with another molecule through a protein domain derived from an antibody, such as in the case of CAR T lymphocytes that attach to their ligands through a chimeric antibody domain.

[0014] The antigen-antibody binding considered in this application holds the antigen fixed at the antibody site and is, according to the prior art, essentially non-covalent. These include hydrogen bonds, electrostatic bonds, van der Waals forces, and hydrophobic bonds. Multiple bonds between the antigen and the antibody ensure a stable interaction between these two molecules, and what is considered in this application enables the cell to be physically maintained at a fixed distance from the substrate, thus enabling the use of physical means, particularly optical means, to detect the presence of cells in the vicinity of the substrate.

[0015] The term "optical interference reflection" or "reflection interference contrast microscopy" refers to a technique known by the acronym IRM (interference reflection microscopy). SUMMARY OF THE INVENTION

[0016] In this context, the present application relates to a device comprising a substrate that includes a first region to which a protein capable of binding to a first membrane molecule is adsorbed and a second region to which an antibody targeting a second membrane molecule is adsorbed, wherein the first region and the second region extend together in length over a dimension comparable to the length of a cell.

[0017] Advantageously, the present application relates to a device characterized by comprising a substrate, the substrate including a first region to which a protein capable of specifically binding to a cell by interacting with a first membrane molecule is adsorbed and a second region to which an antibody targeting a second membrane molecule expressed on the surface of the cell is adsorbed. The first region represents a first surface that defines a first region A1. The second region represents a second surface that defines a second region A2. Here, the total area represented by the sum of area A1 and area A2 is less than or equal to the area of the projected surface of the suspended cell (Figure 1).

[0018] In the context of the present invention, the device includes two regions organized such that a cell can interact with the first region or the second region, or both regions simultaneously. The aim is to detect an overlap of the first region or the second region or both regions, and thus to phenotypically determine the cell determined by both the protein and the antibody adsorbed on the first region and the second region, respectively.

[0019] To enable overlap detection, the first region and the second region have a specific configuration such 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.

[0020] Thus, if a cell can recognize the protein adsorbed in the first region, the cell attaches to the first region, forms an optical microscopy contrast by reflection interference contrast in the first region that appears dark, and the second region remains bright (Fig. 2B). Further, if the cell that interacted with the first region can interact with the antibody adsorbed on the second region, the cell attached to the first region spreads over the second region and attaches to the second region, forming an optical microscopy contrast by reflection contrast (one possible way to reveal the signal generated by the device) in the second region, and the second region will also appear dark (Fig. 2C). If there is no cell attachment to the device, the entire pattern remains bright. (Figs. 2A, 3). The projected surface of a suspended cell is interpreted to mean the plane defined by the maximum dimension of the cell when viewed from above. For example, if the cell is a perfect sphere, the projected surface is a disk of radius r, where r is the radius of the perfect sphere. The projected surface has an area of πr 2 and the total area (A1 + A2) is less than πr 2 . The length is the maximum dimension of a two-dimensional or three-dimensional geometric shape (as opposed to the width or height).

[0021] A two-dimensional shape has an area less than the square of its length. A three-dimensional shape has a projected area less than the square of its length.

[0022] Since the device can be manufactured in different geometric shapes, in the present invention, the device is defined with respect to the length of the cells with which it may interact. In other words, the device and the regions it contains are defined according to the cells under consideration.

[0023] Cells in suspension correspond to either spontaneously non-attaching cells (blood cells or hematopoietic cells) or attached cells separated from their support (e.g., by trypsin treatment).

[0024] In a variant (Fig. 4): - The first membrane molecule is a membrane molecule common to the first cell type, and the second membrane molecule is a membrane molecule common to a subtype of the first cell type. - The first membrane molecule is a membrane molecule common to the first cell type, and the second membrane molecule is a membrane molecule common to a specific state of the first cell type. - The first membrane molecule is a membrane molecule common to the first cell type, and the second membrane molecule is a membrane molecule common to a second cell type that can interact with the first cell type. - The protein is an antibody targeting CD4, and the antibody is an antibody targeting CD8. - The protein is an antibody targeting CD3. - The protein is CD19. - The antibody targets CD69. - The antibody targets CD25. - The antibody targets CD107. - The antibody is an anti - collagen antibody. - The antibody targets CD86.

[0025] Advantageously, the device enables cells to be identified via a protein adsorbed on the first region, and the antibody adsorbed on the second region also enables cells to be identified, for example, to confirm their phenotype. An example of such a device includes a protein that is an antibody targeting the CD3 antigen adsorbed on the first region, and the antibody adsorbed on the second region is an antibody targeting either the CD4 or CD8 antigen, or a mixture thereof. adsorbed on the second region is an antibody targeting either the CD4 or CD8 antigen, or a mixture thereof.

[0026] Advantageously, the device enables cells to be identified via a protein adsorbed on the first region, and the antibody adsorbed on the second region activates the cells, for example, enabling the induction of new cell functions or cell differentiation. Examples of such devices are as follows. - The protein adsorbed on the first region is an antibody targeting the CD45RO antigen, and the antibody adsorbed on the second region is an antibody targeting the CD3 antigen, or a mixture of an antibody targeting the CD3 antigen and an antibody targeting the CD28 antigen. - The protein adsorbed on the first region is an antibody targeting the CD56 antigen or an antibody targeting the CX3CR1 antigen, and the antibody adsorbed on the second region is an antibody targeting the CD20 antigen (rituximab). ii) The protein adsorbed on the first region is an antibody targeting the CD14 antigen, and the antibody adsorbed on the second region targets any one of the CD16, CD32, or CD64 markers, or is an antibody targeting membrane lipopolysaccharide (LPS).

[0027] Advantageously, the device enables the activation of cells via the protein adsorbed on the first region and the antibody adsorbed on the second region, and the detection of a cellular response in the form of expressed membrane molecules such as activation or senescence, that is, the reading out of this response. Examples of such devices are as follows. - The protein adsorbed on the first region is CD19 or a chimeric protein containing 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. - 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.

[0028] Advantageously, the protein adsorbed on the first region and the antibody adsorbed on the second region enable the identification of cells and the detection of a cellular response in the form of expressed membrane molecules such as activation or senescence, that is, the reading out of this response. An example of such a device is one in which the protein adsorbed on the first region is 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.

[0029] The device defined above is also described, wherein the first membrane molecule is a membrane molecule expressed on the surface of a first cell type, the second membrane molecule is a membrane molecule expressed on the surface of a subtype of the first cell type, and the subtype of the first cell type expresses the first membrane molecule and the second membrane molecule on its surface.

[0030] The present application further relates to a method comprising the following steps: - adsorbing, in a first intracellular dimension region of the substrate, a protein capable of binding to a first membrane molecule onto the substrate; - adsorbing, in a second intracellular dimension region of the substrate, an antibody targeting a second membrane molecule onto the substrate.

[0031] The present application further relates to a variant of a method for quantifying and monitoring cell activation kinetics, comprising the following steps: - adsorbing, onto the substrate, a protein capable of binding to a first membrane molecule common to a first cell type in a first intracellular dimension region; - adsorbing, onto the substrate in a second intracellular dimension region, an antibody targeting a second membrane molecule common to a specific state of the first cell type; - contacting a population of first type cells comprising first type cells enabling activation kinetics to a specific state with the substrate; - detecting the presence of first type cells in a first region using light microscopy; - quantifying the kinetics of first type cell activation by detecting first type cell attachment in a second region using light microscopy.

[0032] The present application further relates to a variant of a method for selecting a cell type and inducing activation, comprising the following steps: - adsorbing, in a first intracellular dimension region of the substrate, a protein capable of binding to a first membrane molecule common to a first cell type onto the substrate; - Adsorbing an antibody targeting a second membrane molecule known to induce activation of the first cell type on a substrate in a second intracellular dimensional region; - Contacting a cell population containing a first type of cell that enables activation to a specific state with the substrate; - Detecting the presence of cells of the first cell type expressing the first membrane molecule in the first region using light microscopy; - Detecting the induction of activation of the first type of cell by detecting attachment of the first type of cell in the second region using light microscopy.

[0033] This application further relates to a variation of a method for detecting cell membrane molecules, including the following steps: - Adsorbing a protein capable of binding to a first membrane molecule common to the first cell type on a substrate in a first intracellular dimensional region; - Adsorbing an antibody targeting a second membrane molecule common to a subtype of the first cell type on a substrate in a second intracellular dimensional region; - Contacting a population of first type of cells containing subtype cells with the substrate; - Detecting the presence of cells of the first cell type expressing the first membrane molecule in the first region using light microscopy; - Quantifying the cellular expression of the second membrane molecule by detecting cell attachment in the second region using light microscopy.

[0034] This application further relates to a variation of a method for detecting the interaction between two cells, including the following steps. - Adsorbing a protein capable of binding to a first membrane molecule common to the first cell type on a substrate in a first intracellular dimensional region; - Adsorbing an antibody targeting a second membrane molecule common to a second cell type capable of interacting with the first cell type on a substrate in a second intracellular dimensional region; - contacting a population of cells comprising a first type of cell and a second type of cell with a substrate; - detecting the presence of the first type of cell within a first region using light microscopy; - detecting the presence of the second type of cell using light microscopy or quantifying the expression of a second membrane molecule by the second cells within a second region.

[0035] The use of the substrate, particularly in vitro, for characterizing the activation or differentiation state of a given cell within the cell population is also described. The substrate comprises a first region to which a protein capable of specifically binding to a determined cell by interacting with a first membrane molecule specific to the determined cell is adsorbed, and a second region to which an antibody targeting a second membrane molecule expressed on the surface of the determined cell is adsorbed. The first region represents a first surface defining a first region A1. The second region represents a second surface defining a second region A2. The total area represented by the sum of the area A1 and the area A2 is less than or equal to the area of the projected surface of the suspended cells. The second membrane molecule is expressed on the surface of a specific cell when the cell is activated or differentiated.

[0036] Generally speaking, the present invention is based on a method enabling the phenotypic analysis and single-cell analysis of the activation properties of immune cells, as shown in FIGS. 1 to 5.

[0037] This method can generate multiple micro-regions (micro-patterns) of different proteins and intracellular sizes using optical microscopy (Figure 3). These micro-fabricated substrates can perform multiple functional assays on immune cell suspensions or whole blood, such as (i) selecting / identifying the target cell type, (ii) inducing activation signals, and (iii) reading out the dynamics of immune activation. The present invention is not limited to such optical microscopy methods, and any device that projects ultraviolet light (e.g., confocal microscope) or any (micro)printing technology can also be used. Those skilled in the art will be able to determine the most appropriate device.

[0038] These functions are generally evaluated by the nature of the proteins of the micro-patterns, which are antibodies having specific affinity and effective action. When the cell membrane of the suspension expresses the antibody target on the substrate, the cells spread on the corresponding pattern (Figure 2), and the cell adhesion imprint can be detected by interference microscopy (Figure 2). The detection is not limited to reflection interference contrast imaging, and detection by transmission imaging and image collection for detecting cell contours can also be used. Again, those skilled in the art will be able to determine which technique is most appropriate.

[0039] The different functions performed by the micro-patterns can be evaluated by taking a single image and analyzing the spread of cells in each pattern. It is important to note that the readout does not require the long and tedious operations usually involved in immunolabeling-based techniques (cell preparation, incubation, washing). In a typical interference microscopy image, dark regions indicate attachment and bright regions indicate non-attachment (Figures 2 and 3). In practice, the operator's work 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

[0040]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

[0041] In the first embodiment, referring to FIG. 1 for the elements in bold or the element numbers in parentheses, the present invention aligns and adsorbs proteins on a substrate coated with a polyethylene glycol (PEG) brush insulated according to an ultraviolet light pattern. This can be carried out on the substrate using the LIMAP technique, thereby achieving specific attachment of proteins in the insulated regions.

[0042] However, in each case where the attachment of several proteins is required, especially for two antibodies, it is necessary to be able to specifically deposit the first antibody and then the second antibody on the desired pattern respectively.

[0043] When a specific process is developed for a pattern suitable for a single cell, it is possible to spatially replicate the generation of multiple patterns in parallel in order to quickly obtain a pattern matrix that enables testing on the same number of captured cells as the pattern present on the LIMAP-printed substrate.

[0044] Experimentally, the first region (1) is locally printed by the combined action of a photoinitiator (PLPP, Alveole), placed in a solution on the PEG-SVA brush, and subjected to the projection of a 375 nm ultraviolet light pattern (step 1). Then, the first antibody is incubated at 4°C for 12 hours (step 2). Next, the second region (2) is printed by LIMAP (step 3). The second antibody is added and incubated at 4°C for 12 hours (step 4).

[0045] In this context, the second antibody adsorbs also to the first region and thus adsorbs non-specifically onto the second region (not limited to the most recently irradiated pattern). The anti-fouling substrate (PEG brush) is made adherent in the first region by the first irradiation and in the second region by the second irradiation, which means that the second antibody adsorbs not only in the second region but also in the first region. These properties can be verified, for example, by epifluorescence using a separate fluorescent marker for the antibody and observing them using an interference filter that separates the wavelengths of each fluorescent marker.

[0046] To avoid non-specific adsorption of the second antibody in the first region, one skilled in the art may use passivation techniques known in the prior art, for example, the use of passivation solutions common in biophysics, in particular, between the two irradiation steps shown below: - 4% BSA in PBS for 1 hour at room temperature, - 4% BSA in PBS for 30 minutes, followed by 1 mg / mL rabbit serum IgG for 30 minutes at room temperature, - 1% Pluronic F127 in PBS for 15 minutes at room temperature.

[0047] The best passivation solution found was 0.23 mg / mL PEG-SVA in milli-Q water containing 10 mM sodium bicarbonate for 15 minutes at room temperature.

[0048] When a passivation method is selected based on minimal non-specific adsorption, periodic patterns containing two potential cell adhesion regions can be created using the LIMAP technique: one is specific for T lymphocytes as a whole and one is specific for activated T lymphocytes. It is also possible to create as many adhesion regions as necessary, for example, by irradiating each region, exposing it to the antibody selected for the region, and then passivating and irradiating the next region.

[0049] Conveniently, for each region, the region taken as a pattern of a spatially periodic structure is replicated in a spatially periodic manner, shifting the pattern on the substrate to enable the deposition of the same antibody and the immobilization of all irradiated patterns in a single step.

[0050] Replication is conveniently carried out in a known manner using the LIMAP technique in which an array of micromirrors is used to generate a periodic pattern and is imaged onto the substrate at once with ultraviolet light.

[0051] Thus, each antibody is adsorbed only in its region, that is, specifically in this region, the antibody is adsorbed in an adjacent region forming a pattern whose length, meaning the maximum geometric dimension, is less than or equal to the length of the cell to which it attaches (again meaning the maximum geometric dimension), and the patterns are spaced apart such that only one cell can attach to each pattern. A person skilled in the art can adapt the shape and length of each region and the distance between the patterns by simple operations to ensure that for a specific cell type, only one cell attaches to each pattern in a given attachment order or a given attachment spatial configuration.

[0052] For example, to phenotype memory T lymphocytes, the first attachment region of the aCD45RO type ( "antibody targeting CD45 RO") is defined inside a circle, and the second attachment region of the aCD69 type ( "antibody targeting CD69") is defined on a circular ring concentric with the first region.

[0053] For example, to monitor the activation of memory T lymphocytes, the first attachment region of the αCD45RO type (an "antibody targeting CD45RO") mixed with αCD3 and αCD28 is selected to attach all memory T lymphocytes (all of which express CD45RO) and then induce the activation of the attached cells (by CD3+CD28). The first region is further defined within a circle. The same second αCD69 type attachment region (an "antibody targeting CD69") is defined on a circular ring concentric with the first region to attach only the activated memory T lymphocyte cells that specifically express CD69.

[0054] Thus, the device of the present invention can be used generally, either statically to determine the phenotype of activated memory T lymphocytes or dynamically to monitor the activation of naive memory T lymphocytes by adding a memory T lymphocyte antibody to an attachment antibody common to all memory T lymphocytes.

[0055] Such a device can be easily adapted to any type of cell that can be activated.

[0056] Thus, in this embodiment, a substrate is obtained on which T lymphocytes can move and progress until they attach to each first region of a periodically repeated pattern and only when activated, attach to each second region of the periodically repeated pattern.

[0057] By performing a region test with a variable illumination diameter on the substrate, it is possible to change the maximum size of the first region and / or the second region. This size is on the order of a few micrometers, for example, 4 μm to 12.5 μm, to induce the attachment of a single immune cell.

[0058] As already mentioned, the CD45 antibody or the CD45 / CD3 / CD28 antibody mixture can be deposited in each first region as long as it induces non-specific attachment of activated or non-activated T lymphocytes, especially memory T lymphocytes.

[0059] For example, in each of the second regions, a CD69 antibody or a mixture of antibodies containing it (e.g., CD69+CD25) or a third adjacent region containing CD25 can be deposited as long as it induces specific adhesion of activated T lymphocytes, particularly memory T lymphocytes. It is also possible to deposit only CD25 and observe secondary T lymphocyte activation rather than primary activation by CD69 (preceding secondary activation in time), or to observe both by CD69+CD25.

[0060] The activation kinetics can be tracked in a known manner using MRI microscopy, where bright regions reveal non-adhesion, suggesting that non-activated T lymphocytes have a first dark region and a second region that becomes brighter the weaker the adhesion, the greater the distance from the substrate, or the weaker the activation.

[0061] The shape of the region printed, deposited, or coated with the antibody can be included within a circle or a ring.

[0062] For example, a pattern suitable for the first region is a disk, and a pattern suitable for the second region is a ring concentric with the disk, both having a maximum dimension of about 10 microns.

[0063] Subsequently, the observation of T lymphocyte adhesion to the substrate in the first and second regions enables kinetic monitoring of their activation intensity as a function of the count of initially activated lymphocytes and their adhesion strength. Thus, camera recording helps to enable accurate and quantitative monitoring of the kinetics in the initial stages of T lymphocyte activation and provides rapid results.

[0064] ​Attachment of the pattern to the ring is synonymous with activation of T lymphocytes captured on the pattern, and the absence of attachment to the ring is characteristic of inactivated T lymphocytes, so the interpretation of the present invention is particularly simple. When using MRI as a method to obtain contrast, for example, the appearance of the ring of the pattern is synonymous with activation of T lymphocytes captured on the pattern, and the absence of the appearance of the ring is characteristic of inactivated T lymphocytes.

[0065] Using a visible light marker, for each site containing the first and second patterns, it is possible to determine whether T lymphocytes are present and whether they are activated without relying on fluorescence and thus without manipulation.

[0066] Numerous variations are possible with respect to the general inventive principle of the present invention, which is to deposit a first antibody (i.e., an antibody not specific to activated T lymphocytes) that allows attachment of any T lymphocytes, regardless of activation or inactivation, to the first micrometer region, and then deposit a second antibody (i.e., an antibody specific to activated T lymphocytes) that allows attachment of any activated T lymphocytes to the second micrometer region.

[0067] Therefore, the present invention enables real-time monitoring of the activation of T lymphocyte immune function from the very start of infection or transplantation.

[0068] For B lymphocytes, a CD20 antibody can be used instead of a CD45RO antibody.

[0069] Thus, for a given cell family that does not specifically express CDx in a particular state but specifically expresses CDy (where y is different from x), by further depositing aCDz, it is possible to deposit adjacent to a cell domain, aCDx, and aCDy (in the sense of extending across the length of the cell) for detecting cells in a particular state or cells progressing towards a particular state, where CDz is expressed upon activation of a particular state. Since each cell can occupy at most one domain or each domain can accommodate at most one cell, it is possible to perform quantitative and qualitative techniques on a cell population according to the teachings of the present application.

[0070] The geometric shape of the described region is non-limiting, and in particular, non-concentric regions can be taught in the present application for a particular type of cell.

[0071] Thus, the present application describes a widely available and easily adaptable tool for quantitatively and qualitatively studying various cells and their states using an MRI-equipped microscope for observing results and a test that can be performed with only conventional means of preparing cells that either have or do not have a particular state.

[0072] This tool is particularly useful as new differentiation clusters and their related antibodies are discovered.

[0073] The present invention is industrially applicable in the fields of tests for clinical immunology and oncology, cell-based tests for pharmaceutical research, and cell-based tests for basic research.

[0074] In addition to the above-described methods that have already been attempted and tested, the present invention can also be used for many other applications.

[0075] In some embodiments, the present invention is designed for cell phenotyping.

[0076] In one embodiment for phenotyping circulating lymphocytes, a CD3-targeting antibody can be deposited in a first region, a CD4-targeting antibody in a second region, a CD8-targeting antibody in a third region, a CD19-targeting antibody in a fourth region, and an antibody targeting CD16 can be mixed with an antibody targeting CD56 and deposited in a fifth region.

[0077] In an embodiment, the present invention is designed to perform a cell activation assay in response to a molecular ligand.

[0078] In an embodiment for quantifying T lymphocyte activation, the CD3-targeting antibody can be mixed and deposited with a CD28-targeting antibody in a first region, a CD4-targeting antibody in a second region, a CD8-targeting antibody in a third region, and a CD69-targeting antibody in a fourth region. In a variation of this embodiment, the antibody targeting CD69 can be replaced with an antibody targeting CD25, or an antibody targeting CD25 can be added to the fifth region.

[0079] In an embodiment for quantifying B cell activation, an antibody targeting the IgM Fc fragment and an antibody targeting CD69 can be deposited in the fourth region.

[0080] In one embodiment for quantifying NK lymphocyte activation, an anti-CD16 antibody can be deposited in the first region, an antibody targeting CD56 in the second region, and an antibody targeting CD107 in the third region.

[0081] In one embodiment for quantifying monocyte activation, an anti-CD16 antibody can be deposited in the first region, an antibody targeting CD14 in the second region, and an antibody targeting CD86 in the third region. In a variation of this embodiment, the antibody targeting CD16 may be replaced by an antibody targeting CD32.

[0082] In one embodiment for quantifying the activation of chimeric antigen receptor T lymphocytes (CAR T cells), the CD19 protein can be deposited in the first region, and an antibody targeting CD69 can be deposited in the second region. In a variation of this embodiment, the antibody targeting CD69 may be replaced by an antibody targeting CD25.

[0083] In one embodiment, to detect the fibrotic phenotype of fibroblasts, fibronectin can be deposited in the first region, and an antibody targeting collagen can be deposited in the second region. In one variation, TGF-β (transforming growth factor beta) can be added to the fibronectin within the first region.

[0084] In some embodiments, the present invention is designed to perform a cell activation assay during cell-cell interaction.

[0085] In an embodiment for detecting antigen-specific T lymphocyte activation, an anti-CD14 antibody can be deposited in the first region, and an antibody targeting CD69 can be deposited in the second region. In a variation of this embodiment, the antibody targeting CD69 may be replaced by an antibody targeting CD25.

[0086] In one embodiment for detecting NK lymphocyte cytotoxicity, CD16, which is known to bind to K562 target cells, can be deposited in the first region, and an antibody targeting CD107 can be deposited in the second region.

[0087] In one embodiment, a checkerboard pattern of protein regions and antibody regions of different sizes and consecutive cell cycles can be printed, the cell contour can be identified, and single-cell measurements can be performed across at least one protein region and one antibody region by analyzing the attachment in each region present within the region defined by the contour.

[0088] Throughout this application, the term "intracellular dimension" refers to the following: - A region having a length smaller than the dimensions of one type of cell and the dimensions of the same type of cell activated in a specific state, or - A region having a length shorter than the length of a region that spans between the first type of cell and the second type of cell but does not overlap both cells when they are in contact. Typically, the size of cells in suspension is on the order of 8 μm, and thanks to the present invention, cells attached to the substrate can extend beyond 50 microns.

[0089] In all embodiments, the present invention can be completed by adding one or more regions respectively containing a protein that targets the membrane molecules of the cell type selected by the regions of the above embodiments or an antibody that targets the membrane molecules.

Example

[0090] A protocol for implementing the teachings of the present application is presented below as a non-limiting example.

[0091] Example 1: Lymphocyte-selective substrate, no substrate activation and substrate activation readout available.

[0092] Description of the protocol: a - Surface treatment: Under a dust hood: - Plasma clean 22×22 glass slides. Nexterion glass D Schott Minifab cleanroom washed (ref 1472309). The plasma cleaner pressure was 300 mTor. - Operate the plasma cleaner at "high" for 30 minutes. - Remove the slides from the plasma and place each slide in a Petri dish. - Prepare an APTS solution (200 μL / slide): milli-Q water + 1% (3-aminopropyl)triethoxysilane (APTS) (ref A3648 Sigma-Aldrich) + 0.03% acetic acid 196 μL milli-Q water + 2 μL 3% acetic acid + 2 μL APTS - Place the solution on the slides in the draft and incubate at 4°C for 2 hours - Rinse the Petri dish three times with milli-Q water. - Dry the slides under a nitrogen stream in the draft. - Dry at 95 °C for 15 minutes on a hot plate. - Use nitrogen to remove dust. - Affix it onto a PDMS film (250 μm). - Under the draft, adhere a glass slide (UV adhesive) to the bottom of the perforated Petri dish (from the outside). - Place the Petri dish in a UV cube (entire surface, 1 minute, 100% laser). - PEG-SVA solution (10 μL / well): Prepare in milli-Q water, 10 mM carbonate buffer pH 8.5 and 23% PEG-SVA (mPEG-valeric acid succinimidyl, molecular weight 5000 daltons, ref: I09681 INTERCHIM). For 40 μL of For the final solution: Used 36 μL of milli-Q water, 4 μL of 100 mM carbonate buffer and 0.0092 g of PEG SVA. - Add 10 μL to each well. - Incubate overnight at 4 °C. - Rinse with 8 mL / well of milli-Q water. b - Printing protocol Marker (pattern 1): - 4% BSA, 15 minutes at room temperature (20 μL / well) - Rinse 8 mL / well at 4 mL / min. - Deposit 10 μL / well of PLPP. - Print at 3000 mj / mm2 under an oxygen stream. - Rinse 8 mL / well at 4 mL / min. - Deposit BSA-fluorescein and incubate for 5 minutes at room temperature. - Rinse 8 mL / well of PBS at 4 mL / min. Immobilization: - 5% PEG-SVA, 10 mM carbonate buffer, 20 μL / well, 5 minutes at room temperature - Rinse with 8 mL / well of milli-Q water. - 4% BSA, 20 μL / well, 15 minutes at room temperature - Rinse 8 mL / well of PBS at 4 mL / min Print a central circle with a diameter of 10 μm (Pattern 2): - Deposit 10 μL / well of PLPP - Print at 3000 mj / mm2 under a stream of oxygen - Rinse 8 mL / well at 4 mL / min - Deposition of anti-CD3-CD28 antibody at 100 μg / mL~100 μg / mL~50 μg / mL respectively (final volume 20 μL / well). For one well: 2 μL of CD3 and CD28, 1 μL of CD45, 15 μL of PBS - Incubate overnight at 4°C - Rinse 8 mL / well at 4 mL / min Immobilization: - 20 μL / well of 5% PEG-SVA in 10 mM carbonate buffer for 5 minutes at room temperature - Rinse with 8 mL / well of milli-Q water - 20 μL / well of 4% BSA for 15 minutes at room temperature - Rinse 8 mL / well of PBS at 4 mL / min Peripheral pattern printing (Pattern 3) - Deposit 10 μL / well of PLPP - Print at 3000 mj / mm2 under a stream of oxygen - Rinse 8 mL / well at 4 mL / min - Deposition of anti-CD69 antibody at 100 μg / mL (final volume 20 μL / well). For one well: 4 μL of CD69 and 16 μL of PBS - Incubate overnight at 4°C - Rinse 8 mL / well at 4 mL / min Reference antibody: - Anti-hCD45 AB: ref NBP2-34804, clone SPM570, NovusBio - Anti-hCD3 AB: Ultra-LEAF purified, ref 300438, clone UCHT1, Biolegend - Anti-hCD28 AB: Ultra-LEAF purified, ref 302933, clone CD28.2, Biolegend - Anti-hCD69 AB: ref MAB2359, clone 298633, R&D system.

[0093] Example 2: Substrate for selecting and identifying memory T lymphocytes with substrate activation and substrate activation readout.

[0094] In this example, CD3 - CD28 is replaced with CD45RO and the same protocol as in Example 1 is followed.

[0095] Example 3: Measurement of T lymphocyte activation Figure 3 shows an example of a measurement in a device having a micropattern designed to contain single T lymphocytes and detect their activation by the TCR complex. These patterns were each composed of two distinct regions having specific functions: the central region contained anti - CD3 and anti - CD28 antibodies for selecting and activating T lymphocytes, and the lateral region contained anti - CD69 antibody for reading out CD69 expression of activated cells. The interferometer readout is shown on the right side of Figure 3B. Some of the anti - CD69 regions are dark, which can be understood to mean that the captured T lymphocytes are activated in this experiment (confirmed by immunofluorescence). This example demonstrates the concept of a "smart" substrate with multiple antibodies, which is very flexible and can be adapted to different implementations and cell types by appropriately selecting the printed antibodies.

[0096] Example 4: Other possible implementations The technology described in the present invention enables a functional assay for personalized use of new immunotherapies by monitoring immune cell activation. By evaluating the effectiveness of immunotherapy and stratifying patients as potential responders, the use of these costly treatments can be optimized. This is an important medical problem. Functional investigations are often too time-consuming and complex to translate into clinical situations where the cost of human handling and expertise is highly restrictive, remaining in the realm of academic research. The technology developed by the inventors of a "smart" substrate with multiple functional micro-patterns enables the measurement of leukocyte activation within a few hours and can effectively address the unmet need for the development of rapid, clinically acceptable functional assays.

[0097] One of the main advantages of this technology is that it can be adapted to measure various functions of different leukocyte types. To adapt this technology to new specific functions and new types of leukocytes, it is only necessary to simply change the nature of the antibodies in the micro-pattern. The inventors have identified a (non-exhaustive) series of antibodies and molecules imprinted to perform the functions of selection / identification, activation induction, and cell expression readout for various immune cells (Table 1). For most cell types and functions, the model is based on antibody printing. In some cases, for example, in 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 implementations depends on the same type of surface chemistry and physical chemistry and only involves changing the type of antibody in the preparation protocol that has already been proven for creating new functional devices.

[0098] [Table 1]

[0099] For example, the inventors may be interested in evaluating the activation characteristics of CAR T cells when bound to a chimeric CD19 target, or the activation characteristics of NK cells against a rituximab target (see FIGS. 4 and 5).

[0100] In this example, a glass slide (SCHOTT Nexterion) is plasma-treated for 15 minutes and covered with a PDMS mold (polydimethylsiloxane, Sylgard 184) to create channels. A solution of APTES ((3-aminopropyl)triethoxysilane, Sigma) diluted to 1% with milli-Q water containing 0.03% acetic acid is incubated on the glass for 2 hours at 4 °C. The surface is then rinsed with water and incubated at 95 °C for 15 minutes. Next, a solution of PEG-SVA (molecular weight: 5000 daltons, INTERCHIM) in 10 mM carbonate buffer (NaHCO3) is incubated on the glass for 12 hours at 4 °C. The channels are then rinsed with water, and a solution of PLPP (15.5 mg·mL−1, Alveole) is introduced into the channels and stirred at a flow rate of 4 μL·s -1 −1 using a syringe pump (NEMESYS, Cetoni) during the exposure. The surface is exposed to a UV dose of 3000 mJ·mm -2 −2 to create a binding pattern using the PRIMO platform (Alveole), and then incubated with 50 μg·mL−1 of anti-His Tag (Life Technologies) for 1 hour at room temperature. The solution is then rinsed, and the surface is blocked with 4% BSA (bovine serum albumin, Sigma) for 15 minutes. Next, a solution of 20 μg·mL−1 of CD19-His-tag (Life Technologies) is incubated at 4 °C for 12 hours. After the surface is blocked again with 4% BSA for 20 minutes, it is re-insulated with UV light to create a peripheral pattern for activation readout. Finally, the surface is incubated with human anti-CD69 (Bio Techne, R&D Systems) for 1 hour at room temperature. The cells are then introduced into the channels for 10 minutes and then rinsed so that only the cells remain on the pattern. The readout is performed after incubating the cells in the device for 3 hours.

Claims

1. A substrate comprising a first region (1) to which a protein capable of binding to a first membrane molecule is adsorbed and a second region (2) to which an antibody targeting a second membrane molecule is adsorbed, wherein the first region and the second region extend together over a length comparable to the length of a cell. A device characterized by this.

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

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

4. The device according to claim 1, wherein the first membrane molecule is a membrane molecule common to a first cell type, and 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 according to claim 1, wherein the protein is a CD4-targeting antibody and the antibody is a CD8-targeting antibody.

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

7. The device according to 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 following: - Adsorbing an antibody capable of binding to a first membrane molecule to a first intracellular dimension region of the substrate; - Adsorbing an antibody targeting a second membrane molecule on the substrate in a second intracellular dimension region of the substrate; A method characterized by including these steps.

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