Detection of car t cell activation by granzyme

Abstract

The invention is directed to a method for detecting cell activation by incubating cells with a solid surface provided with at least one activation moiety capable of activating the cells, and at least one fluorescence dye bound to a quenching unit, wherein the cells are activated by recognizing the at least one activation moiety and wherein after activation, the cell secretes granzyme molecules which cleaves the quenching molecule from the at least one fluorescence dye, thereby enabling detection of fluorescence emission from the at least one fluorescence dye characterized in that the at least one activation moiety and the at least one fluorescence dye bound to a quenching unit are provided on the solid surface within an area of less than 500 µm2.

Description

DETECTION OF CAR T CELL ACTIVATION BY GRANZYMEBACKGROUND

[0001] The present invention is directed to a method for detecting cell activation by detecting the enzymatic activity of secreted granzyme(s).

[0002] In vivo, following lymphocyte priming and activation, the activated lymphocytes (e.g. T cells) recognises, through a receptor (e.g. T cell receptor (TCR)), antigen(s) expressed on the surface of the target cell. This initial recognition induces the formation of connection between the lymphocyte and target cell and can cumulate in the selective lysis of the target cell. A key mechanism of lymphocyte-induced cytotoxicity consists of the targeted release of lytic proteins (pore forming proteins (i.e. perforins) and proteolytic proteins (i.e. Granzymes)). In the case of genetically modified lymphocytes (e.g. CAR T cells), the process of activation and target cell lysis is believed to be very similar, however, the recognition of the targeted antigen is initiated by the genetically engineered receptor (e.g. chimeric antigen receptor (CAR)) expressed on the surface of the genetically engineered lymphocyte.

[0003] The utilisation of a granzyme cleavable sequence for the detection of active granzyme has been around for some time and different iterations of it are disclosed in the following publications:

[0004] Beverly Z. Packard, and al., The Journal of Immunology, Granzyme B Activity in Target Cells Detects Attack by Cytotoxic Lymphocytes, 2007. In this publication the authors describe a cell permeable sequence which is composed of a fluorophore separated by a granzyme B sequence VGPD’FGR. Following granzyme cleavage the self-quenching fluorophore separates, and fluorescence’s can be detected by microscopy. In this publication the authors use the measured fluorescence as a readout for T cell mediated targeted killing. There is no mention of coupling the granzyme-cleavable sequence and / or the quenched fluorophore to a bead or using it as a readout for -cell- activation or using it for detection of granzyme activity outside of the cells.

[0005] Clarissa Liesche and al., Frontiers in Immunology, Single-Fluorescent Protein Reporters Allow Parallel Quantification of Natural Killer Cell-Mediated Granzyme and Caspase Activities in Single Target Cells, 2018. In this publication, the authors describe a fluorescent protein biosensor which was cellularly expressed and composed of a cytoplasmic localisation sequence, a granzyme cleavable sequence (IEPD and VGPD’FGR are described)and a fluorophore. Following granzyme cleavage, the fluorophore separates from the cytoplasmic localisation signal and the fluorescent signal accumulates in the nucleus which can be quantified by microscopy.

[0006] Ning Zhao and al., ACS Central Science, In Vivo Measurement of Granzyme proteolysis from Activated Immune Cells with PET, 2021. In this publication, the authors describe a radioactive peptide which can be administered in vivo, which is composed of a radioactive probe that is attached to a membrane binding domain, a granzyme cleavable sequence (IEPDVSQV) and a peptide masking domain. Upon granzyme B cleavage, the membrane binding sequence which is attached to the radioactive probe is separated from the peptide masking domain, effectively enabling the membrane binding domain to burry itself into the membrane of the neighboring cells (target and immune) which can then be detected by positron emission tomography (PET).

[0007] Jeehun Park, and al., ACS Sensors, Multifunctional Microparticles with Stimulation and Sensing Capabilities for Facile NK Cell Activity Assay, 2021. In this publication, the authors describe a bead which is specific for NK cells and which is loaded with fluorescent probe that is attached to a granzyme cleavable sequence “VGPD’FGR”. The NK specific activation antibodies and fluorescent probes are all attached to the bead via biotinylation and following the specific activation of NK cells, the loss / reduction of the fluorescence signal can be measured as a readout for biological activity.

[0008] WO2022 / 258997A1 describes a cell permeable peptide which is composed of a quencher, a granzyme cleavable sequence (lEPD’AL) and a fluorophore. Following granzyme-mediated cleavage, the fluorophore is separated from the quencher and the cells with active granzyme can be detected by imaging.

[0009] Still, there is a need for a method for a standardized, fast and easy-to-use readout for testing the effector functions of naturally occurring or genetically modified lymphocytes.SUMMARY

[0010] It was therefore an object of the invention to provide a method for detecting cell activation by incubating cells with a solid surface provided with at least one activation moiety capable of activating the cells, and at least one fluorescence dye bound to a quenching unit, wherein the cells are activated by recognizing the at least one activation moiety and wherein after activation, the cell secretes granzyme molecules which cleave the quenching molecule from the at least one fluorescence dye, thereby enabling detection of fluorescenceemission from the at least one fluorescence dye characterized in that the at least one fluorescence dye is covalently bound via a spacer unit capable of being cleaved by granzyme molecules to a quenching unit and is provided on the solid surface adjacent to the at least one activation moiety within an area of less than 1500 pm2preferable less than 500 pm2

[0011] More precisely, the method of the invention for detecting cell activation comprises incubating cells with a composition according to general formula (I)(A)-S-(F-C-Q) (I)WhereinA: activation moiety capable of activating cellsS: solid surfaceF : fluorescence dyeC: spacer unit capable of being cleaved by granzyme moleculesQ: quenching unitwherein the cells are activated by recognizing the at least one activation moiety and wherein after activation, the cell secretes granzyme molecules which cleave the quenching molecule from the at least one fluorescence dye, thereby enabling detection of fluorescence emission from the at least one fluorescence dye characterized in that the at least one fluorescence dye F is covalently bound via a spacer unit C capable of being cleaved by granzyme molecules to a quenching unit Q and wherein the units (F-C-Q) and (A) are provided on the solid surface S in an area of less than 500 pm2.

[0012] Another object of the invention is a composition for detection of cell activation as disclosed characterized by general formula (I)(A)-S-(F-C-Q) (I)WithA: activation moiety capable of activating cellsS: solid surfaceF : fluorescence dyeC: spacer unit capable of being cleaved by granzyme moleculesQ: quenching unitwherein the units (F-C-Q) and (A) are provided on the solid surface S in an area of less than 500 pm2.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Fig. 1 shows a graphical representation of the granzyme sensor containing i) a bead linker (R-), ii) a dye and iii) a quencher as well as iv) a granzyme labile peptide sequence. Cleavage in the presence of a specific granzyme leads to release of the quencher and appearance of the fluorescent signal.

[0014] Fig. 2 shows a schematic mechanism of action for simultaneous activation and read-out of lymphocytes via granzyme-specific cleavage of a peptide tethered to all-in-one beads.

[0015] Fig. 3 shows identification of granzyme specific sequences candidates.

[0016] Fig. 4 and b show that in the presence of the 3 required components (cells, granzyme sensors and stimulation) a specific signal can be rapidly detect.DETAILED DESCRIPTION

[0017] The term “granzyme sensors” refers to the granzyme cleavable peptide sequence, including fluorophore, quencher and coupling site.cell-activating ligands A / activation moieties capable of activating cells A

[0018] In the following, the method of the invention using such surfaces is described by way of example for the activation of CAR T cells, but it should be clear that method of the invention is capable to activate and detect the activation status of other lymphocytes or cells too.

[0019] The solid surface may be loaded with covalently attached cell-activating ligands A such as a CAR ligand or an anti-CAR specific antibody (e.g., anti-CD19 CAR (REA1297 - FMC63) or anti-CD20 CAR (REA1341) and optionally one or more costimulatory antibodies (e.g., anti-CD2 (REA972 and / or anti-CD28 (REA612)).

[0020] Such cell activating particle specifically engage the CAR and co-stimulation co-receptors to mimic the creation of an immune synapse and induce the specific activation of CAR T cells in a heterogeneous T cell population. The specific selection and combination of the antibodies tethered on the surface of the beads can theoretically enable the specific activation of any CAR T cell or immune cell type of interest.

[0021] The solid surface as utilized in the method of the invention provides both, cell activating ligands and granzyme sensing units, each covalently attached to the surface. In order to activate the cells and downstream detection of the specific activation through thefluorescence emitted from the cleaved granzyme sensor(s), those entities need to be in close proximity.

[0022] Fig 2 shows the method of the general concept of the invention by way of example for CAR cells. The cell activating ligands bind to and specifically activates the CAR T cells by engaging the CAR as well as potential co-stimulatory molecules, creating an immunological synapse and activating the downstream signaling cascade. The activated CAR T cell will then subsequently degranulate and release granzyme into the immunological synapse formed on the solid surface. The granzyme will then subsequently come into contact with the granzyme cleavable sequence between the fluorophore and the quencher. Cleaving the granzyme cleavable sequence enables the quencher to migrate away from the fluorophore and the emission of detectable fluorescent signal.

[0023] The at least one activation moiety is selected from the group consisting of antigens (e.g. CD19, CD20, CD22, BCMA, GPRC5D, CD33, NG2, GD2, MUC1. MSLN, PSMA, FOLR1, EGFR, HER2, etc ), proteins (e.g. CD 19, CD20, CD22, BCMA, GPRC5D, CD33, NG2, GD2, MUC1. MSLN, PSMA, FOLR1, EGFR, HER2, etc ), peptide(s) (e.g. CD19, CD20, CD22, BCMA, GPRC5D, CD33, NG2, GD2, MUC1. MSLN, PSMA, FOLR1, EGFR, HER2, etc ), glycoprotein(s) (e g. CD19, CD20, CD22, BCMA, GPRC5D, CD33, NG2, GD2, MUC1. MSLN, PSMA, FOLR1, EGFR, HER2, etc ), antibodies (e g. anti-CD19 CAR, anti-CD20 CAR. anti-CD22 CAR, anti-BCMA CAR. anti-GPRC5D CAR, anti-CD33 CAR, anti-MUCl CAR, anti-PSMA CAR, anti-MSLN CAR, anti-NG2, anti-GD2 CAR, anti-FOLR1 CAR, anti-EGFR CAR, anti-HER2 CAR, anti-CAR linker (eg. Whitlow, G4S), etc ), antibody fragments (e.g. FABs), bispecific antibodies (BsAbs), bispecific nanobodies, Bispecific T cell engagers (BiTEs), or nanobodies (e.g. anti-CD19 CAR, anti-CD20 CAR. anti-CD22 CAR, anti-BCMA CAR. anti-GPRC5D CAR, anti-CD33 CAR, anti-MUCl CAR, anti-PSMA CAR, anti-MSLN CAR, anti-NG2, anti-GD2 CAR, anti-FOLRl CAR, anti-EGFR CAR, anti-HER2 CAR, anti-CAR linker (eg. Whitlow, G4S),) directed against any part of a naturally occurring or genetically introduced receptor (e.g. chimeric antigen receptor (CAR), Adapter CARs, T cell receptor (TCR), T cell receptor fusion constructs (TRuCs), synthetic TCRs and antigen receptors (STARs) and HLA-independent TCRs (HITs), etc.), anti-CD19, anti-CD20, anti-CD22, anti-BCMA, anti-GPRC5D, anti-CD38, anti-CD79a / b, anti-CD37, anti-CD33, anti-CD64, anti-CDllc, anti-CD3, anti-CD5, anti-CD7, anti-CD28, anti-CD278, anti-CD134, anti-CD137, anti-NKG2D, anti-CD16, anti-NKp30, anti-NKp44, anti-NKp46, anti-CD56, anti-CD94 / NKG2A, anti-CD226, anti-MSLN, anti-CD123, anti-HER2, anti-GD2, anti-EGFR, anti-PSMA, anti-MUCl, anti-CD318, anti-PD-Ll, anti-TSPAN8, anti-CD66c,anti-CEA, anti-CLA, anti-CD276, anti-FolRl, anti-CLEC12A, anti-CLL-1, anti-CTLA-4, anti-TIM-3, anti-LAG-3, anti-VISTA, anti-CCR7, anti-SIRPa, anti-B7-H4, anti-CD371, antigens (e.g. CD 19, CD20, BCMA, NG2, MSLN, PSMA, MUC1, FOLR1, EGRF, HER2, GD2, GPRC5D), recombinant proteins or peptide sequences (e.g., CD 19, CD20, BCMA, NG2, MSLN, PSMA, MUC1, FOLR1, EGRF, HER2) and peptides or proteins presented on artificial MHC molecule (e.g. MACSimers).

[0024] It is further possible that the solid surface is provided with at least one costimulatory moiety capable of enhancing the activation of cells by recognizing the at least one activation moiety.

[0025] The at least one costimulatory moiety may selected from the group consisting of anti-CD3, anti-CD28, anti-CD2, anti-CD6, anti-CD26, anti-ICOS (CD278), anti-CD53, anti-LFA-l(aLp2, CDlla / 18), anti-MAC-1 (aM 2, CDllb / 18), anti-CDl lc / 18, anti-VLA-4 (a4pi, CD49d / 29), anti-VLA-5(a5pi, CD49e / 29), anti-NKp46 (CD335), anti-CD16 (FcgRIIIA), anti-NKG2C (CD94 / 159c), anti-KIR2DSl-2, anti-KIR2DS3-6, anti-KIR3DSl, anti-KIR2DL4 (CD158d), anti-CD84, anti-NKp30 (CD337), anti-NKp44, anti-NKp80, anti-KIR-S, anti-mNKR-PIC, anti-mNKG2D-S, anti-mAct.Ly49, anti-NKG2D (CD314), anti-CRACC (CD319), anti-Ly9, anti-NTB-A, anti-DNAM-1 (CD226), anti-CD7, anti-CD59, anti-BY55 (CD160), anti-CD44, anti-2B4 (CD244), anti-adhesion molecules (e.g. anti-cadherin, anti-selectin, anti-integrin (e.g. anti-LFA-l(aLp2, CDlla / 18)), or any of the antigens, proteins, glycoproteins, peptides, antibodies, bispecific antibodies (BsAbs), bispecific nanobodies, bi-specific T cell engagers (BiTEs), or nanobodies. sSolid surface S

[0026] In order to specifically activate cells, particularly to avoid the pitfalls of cancer cell line-mediated activation, solid surfaces for cell activating were created onto which the activation and senor units are covalently bound in close proximity. Preferable, the at least one activation moiety and the at least one fluorescence dye bound to a quenching unit or the units F-C-Q and A of formula (I) are provided on the solid surface S in an area of less than 1500 pm2. Preferable, the units are provided on the solid surface S within an area of 0.01 to 1499 pm2, more preferable within an area of 2 to 400 pm2most preferred within an area of 5 to 100 2| m

[0027] The solid surface may be provided as planar surface or as particle, but the important factor is that the units are provided in close proximity. The solid surface itself may be larger than 1500 pm2.

[0028] Preferable, the solid surface S is provided as particle with a mean diameter (d50) of less than 20 pm, like 19 to 0.01 pm. More preferable, the solid surface S is provided as particle with a mean diameter (d50) of less than 10 - 3 pm, most preferred less than 5 to 3 pm .

[0029] In the embodiment wherein the solid surface S is provided as particle, the average amount of units (F-C-Q) and (A) of formula (I) depend on the size of the particle and can be estimated as follows:Particle diameter Surface area / particle (pm2) # of units per particle0.02 pm 1.3 E-3 1-300.5 pm 0.785 125-20003 pm 28 4.5E4-7E55 pm 79 1.3E5-2E610 pm 314 5E5-8E6

[0030] The solid surface S may be provided al least with a surface consisting of materials selected from the group consisting of polystyrene, silica, polyolefins, polysaccharides, polyesters, polyacrylates, polylactic, iron oxide, Liposomes, or Lipid-Nanoparticle (LNPs). In the variant of the invention wherein the solid surface S is provided as particle, the whole particle may be provided from these materials.

[0031] More specific, the solid surface used in the method of the invention can be defined by general formula (II)(A - LI) - S - (L2 - L3(F) - C - L4(Q)) (II)

[0032] Wherein F, A, S, C have the same meaning as in formula (I)

[0033] LI is the linker of A to the solid surface, e.g., an amino acid such as cysteine or lysine, or a chemical linker e.g., polyethylene glycol, with chemical functionality for binding beads, e.g., via click chemistry, Michael addition or amide bond formation.

[0034] L2 is the linker of L3(F)-C-L4(Q) to the solid surface, e.g., an amino acid such as cysteine or lysine, or a chemical linker e.g., polyethylene glycol, with chemical functionality for binding beads, e.g., via click chemistry, Michael addition or amide bond formation.

[0035] L3 is a linker to which one or more fluorophores are linked to C - L4(Q), e.g., an amino acid such as cysteine or lysine, or a chemical linker e.g., polyethylene glycol, with chemical functionality for binding dyes, e.g., via click chemistry, Michael addition or amide bond formation. For attachment of multiple fluorophores, branched polymer backbones can be used. Furthermore, the L3 linker can also contain an additional amino acids or series ofanimo acids with a defined chemical composition which are included to contribute / modify or alter some of the chemical properties of the granzymes sensor, including but not limited to enhancing solubility, increasing the distance between fluorophore and quencher, altering the isoelectric properties, etc.

[0036] L4 is a linker to which one or more quenchers are linked to C - L3(F), via e.g., an amino acid such as cysteine or lysine, or a chemical linker e.g., polyethylene glycol, with chemical functionality for binding quenchers, e.g., via click chemistry, Michael addition or amide bond formation. For attachment of multiple quenchers, branched polymer backbones can be used. Furthermore, the L4 linker can also contain an additional amino acids or series of animo acids with a defined chemical composition which are included to contribute / modify or alter some of the chemical properties of the granzymes sensor, including but not limited to enhancing solubility, increasing the distance between fluorophore and quencher, altering the isoelectric properties, etc.

[0037] F may be a fluorophore from the family of xanthenes, like fluorescein, or rhodamines, cyanines, phthalocyanines, oxazines, coumarins, acridines, naphthalimides, oxadiazoles, pyrenes, porphyrins, stilbenes, anthraquinones, squaraines, indoles and oxazoles, pyrromethenes, or pyridyloxazole, with excitation / emission between 350-1100 nm, such as 5-TAMRA or Vio515.

[0038] The solid surface may be provided with different fluorescence dyes covalently bound via different spacer units capable of being cleaved by different granzyme molecules to a quenching unit. With this variant, multiplexing of different types of granzyme molecules is possible.Spacer unit C capable of being cleaved by granzyme molecules

[0039] In the method of the invention, various spacer units capable of being cleaved by granzyme molecules can be used. Preferable, the spacer units C comprise a granzyme-specific protein sequence (linear or cyclic) and may comprise one or more cleavable units (CU), with a general formula C = (CU)n, and n = 1-5.

[0040] The cleavable units (CU) may be a building block composed of 4 - 8 amino acids with the following positions / sequence: P4-P3-P2-P1-P1’-P2’-P3’-P4’ (orientated in the N-terminus to C-terminus). Accordingly, the “ ’ ” denotes the amino acids which are attached to the C-terminus following cleavage. Hence, Pl’, P2’, P3’ and P4’ are amino acids which remain tethered to the quencher following enzymatic cleavage.

[0041] Since cleavage occurs between Pl and Pl’, cleavable units (CU) according to the invention may comprise amino acids only on position / sequence P4-P3-P2-P1-P1’. To increase the applicability of the method, cleavable units (CU) according to the invention may comprise amino acids further on position / sequence P4-P3-P2-P1-PU-P2’ or P4-P3-P2-P1-P 1 ’ -P2’ -P3 ’ or P4-P3 -P2-P 1 -P U -P2’ -P3 ’ -P4’

[0042] Each of the positions P4-P3-P2-P1-P1’-P2’-P3’-P4’ may be occupied by a certain amino acid. The permissible amino acids for the respective positions are shown in the following table:

[0043] The spacer unit C / CU comprises or consists of sequences selected from the group consisting of SEQ ID 1 to SEQ ID 2773 as listed in the accompanying Sequence Listing, wherein X = non-natural amino acid or alternative compound (e.g. Poly(ethylene glycol) 2-aminoethyl ether acetic acid).Quencher unit unit O

[0044] The quenching unit may be selected from the group consisting of the family of black hole quenchers (e.g., BHQ-1, BHQ-2, BHQ-3), QSY quenchers (e.g., QSY7, QSY21, QSY35), dark quenchers (e.g., Dabcyl, DDQ-1, Eclipse), DY quenchers (e.g., DYQ-2M), atto quenchers (e.g., ATTO612Q), BMN quenchers (e.g., BMN-Q460), TQ quenchers (e.g., TQ2, TQ3), BBQ quenchers (e g., BBQ650), IRDye quenchers (e g., IRDyeQCl) IOWA black quenchers (e.g., IBFQ) and / or indocarbocyanine-based quenchers (e.g., Cy-Q dyes).Cells to be detected / use of the method of the invention

[0045] The method of the invention is especially suitable to detect cells selected from the group consisting of T-cells, CAR T-cells, NK cells, tumor infiltrating lymphocytes(TILs), genetically engineered T cells, CARNK cells, dendritic cells, macrophages, MAIT cells, iNKT cells and B cells.EXAMPLESFeasibility validation of proof of principle (POP)Cell preparation

[0046] The CAR T cells are generated in the CliniMACS Prodigy® instrument, which is an automated closed cell processing instrument commercially available from Miltenyi Biotec BV&Co. KG.

[0047] The steps required to obtain the functional CAR T cells are illustrated in Fig. 3. Fig. 3 shows different sequences that were generated and tested by incubating the indicated sequence with activated recombinant granzyme (black triangles) as compared to buffer (open grey circles). This approach was used to screen the sequences listed in the table and the sequence listing, which derived from publicly available primary literature or publicly available consensus sequences.

[0048] Briefly the T cells are magnetically separated by CD4+and CD8+selection from starting material (e.g. whole blood, PBMC or fresh Leukapheresis). The purified T cells are subsequently activated with MACS® GMP T Cell TransAct™. The T cells are subsequently virally transduced with gamma-retroviral vectors (RV) or lentiviral vectors and the transduced T cells are subsequently expanded with T cell cultivation media which is composed of TexMACS™ GMP Medium and MACS GMP Cytokines. Following the expansion, the cells are collected, quantified and formulated at a fixed concentration depending on patient and body weight for downstream applications. The CAR T cells can be used directly from the Prodigy “Fresh” or frozen down in freezing media and stored in liquid nitrogen until use.

[0049] Cultured cells were prepared by thawing previously frozen CAR T cells and placing them in culture for 4 days in T cell culture media (TexMACS + 3% human Serum + (40 lU / mL) recombinant human IL-7 and (40 ZU / mL) recombinant human IL-15). Media was changed on day 1, 2 and 4 after thawing cells. Media changes were performed by centrifuging cells at 300g for 5min and resuspending the cells in fresh T Cell culture media at 1E6 CD3+cells / mL. Frozen cells were prepared by thawing the cells and allowing them to rest at room temperature (RT) for 1 hour prior to use.Granzyme sensorSeveral granzyme specific sequences (Table

[0036] ) originating from publicly available primary literature or publicly available consensus data were generated in small scale in house and screened for functionality. The screening was performed by incubating the free floating granzyme sensor (0.15ug / well) with an activated recombinant granzyme (16nM - purchase from commercially available vendors) specific to the sequences or in buffer (Figure 3).Optimal functional sequence candidates for a specific granzyme were produced by Peptides & Elephants GmbH in larger scale. Two granzymes sensors were tested, which are both composed of 4 main components; 1) a quencher (i.e. DYQ-2-M) bound to a cysteine, 2) a granzyme specific cleavable sequence (Peptide 1 = VGPD’FRAL, Peptide 2 = lEPD’AL), 3) fluorescent dye (i.e. 5-TAMRA) tethered to a lysine and 4) a lead sequence (i.e. cysteine) to tether the sensor to the cell activating beads (see also Figure 1).Bead loading

[0050] Three different types of beads were created for comparison:1. Cell activating beads: beads that have only cell activating ligands covalently attached to the surface (not according to the invention)2. Sensing beads: beads that have only granzyme sensing units covalently attached to the surface, (not according to the invention)3. So called “All-in-one beads”: beads that have both, cell activating ligands as well as granzyme sensing units covalently attached to the surface (according to the invention)

[0051] In short, stimulating and co-stimulating molecules (e.g., anti-CD19 CAR (REA1297), anti-CD2 (REA972), anti-CD28 (REA612) antibodies) and / or Granzyme sensing units (e.g., CK(5-TAMRA)IEPDALC (DYQ-2-M)) were reduced using tris(2-carboxyethyl)phosphine hydrochloride (TCEP, Sigma-Aldrich) for one hour prior to the addition to mal eimide (Succinimidyl-4-(N-maleimidomethyl)cyclohexan-l-carboxylat, SMCC) activated beads. Conjugation was stopped after 2 h by addition of a 50 mM solution of b-mercaptoethanol (Calbiochem) followed by a 40 mM solution of N-ethylmaleimide (NEM, Sigma-Aldrich). Then modified particles were collected via centrifugation (3000 x g, 5 min), washed with PBS / Pluronic (0.03%) and resuspended, (see also Front. Immunol. 15:1254162. doi: 10.3389 / fimmu.2024.1254162)Cells and Bead quantification

[0052] The number of viable CD3+cells and transduction efficiency (%CAR+) was quantified by flow cytometry. These values were used to calculate the concentration of CAR T cells / mL for proper loading of the experimental cell culture plates. Similarly, the count of particles was determined by MACSQuant (Miltenyi Biotec). The beads (particles) hat a mean diameter d50 of 3 pm, i.e. a surface area of 28 pm2.Plate reader

[0053] 1E4 to 1E6 CAR T cells (CD2019.1 or CD19 CAR T cells) suspended in TexMACS media (Miltenyi) were dispensed into a black, glass bottom 96 well plate.Afterwards, 1E6 to 1E7 beads were added to the respective wells. The beads were the all-in-one beads, which is a fusion of the granzyme sensor unit to cell activating beads. Control beads were also included, composed of cell-activating beads (beads with only stimulating molecules and without the granzyme sensor molecule) and / or sensing beads (containing only the granzyme sensor and without the stimulating molecules). The plate was sealed and placed into a Synergy Hl microplate reader and heated to 37°C. The fluorescent read was performed for 4 hours, within which the fluorescence (ex. 540nm and em. 580nm) was measured every 2 to 5 min through the bottom of the glass plate. The replicates of the relative fluorescence emission from the different conditions measured were averaged and plotted (Y axis) over time (X axis).Investigation of the bead functionality

[0054] Fig. 4 shows that the detection of a specific signal is observed when all 3 required components are combined separately (dark grey triangles) or when CAR T cells are combined with the all-in-one beads (squares). However, the signal is much higher when the stimulating and sensing units are attached to the same bead platform. This specific signal separates from the non-specific signal as seen in the condition of CAR T cells cultured with granzyme sensors loaded beads (light grey triangles) or all-in-one beads only (hollow diamonds). Controls include CAR T cells only (hollow triangles), cell activating beads only (hollow spheres) and cell activating beads combined with CAR T cells (hollow squares) which generate no signal (flat lines).

[0055] As demonstrated in Fig. 4, cell activating beads only (Fig. 4 - hollow circles), CAR T cells only (Fig. 4 - hollow triangles) and cell activating beads combined with CAR T cells (Fig. 4 hollow squares) generated no observable signal. When CAR T cells werecultured with beads that only carried the granzyme sensor (Fig. 4 - closed light grey triangles), a background signal which gradually increased over time was observed.. Also, all-in-one beads composed of SEQ ID 30 (4 A) or SEQ ID 31 (4B), by themselves generated a low background signal which varied between the 2 tested granzyme sensor constructs (Fig. 4 - hollow diamonds), an additional observation warranting further investigation. Interestingly, when we included CAR T cells with a two bead stimulation-detection system, a cell activating beads in combination with a granzyme sensor loaded onto a separate beads (Fig. 4 - dark grey triangle), we clearly observed a specific signal which separated from the bead (All-in-one) only (Fig. 4 - hollow diamond) or CAR T cells incubated with sensor loaded beads (Fig.4 - triangle light grey). Interestingly, when we combined the granzyme sensor and cell activating moieties on one bead, generating the all-in-one bead, and combined it with CAR T cells, we rapidly detected a signal which separated from all other conditions (Fig. 4 - closed squares).

Claims

CLAIMS1. Method for detecting cell activation by incubating cells with a solid surface provided with at least one activation moiety capable of activating the cells, and at least one fluorescence dye bound to a quenching unit, wherein the cells are activated by recognizing the at least one activation moiety and wherein after activation, the cell secretes granzyme molecules which cleave the quenching molecule from the at least one fluorescence dye, thereby enabling detection of fluorescence emission from the at least one fluorescence dyecharacterized in that the at least one fluorescence dye is covalently bound via a spacer unit capable of being cleaved by granzyme molecules to a quenching unit and is provided on the solid surface adjacent to the at least one activation moiety within an area of less than 1500 pm2.Method according to claim 1 characterized in that the solid surface is provided as planar surface or as particle with a mean diameter of less than 20pm.3 Method according to claim 1 or 2 characterized in that the solid surface is provided with a surface consisting of materials selected from the group consisting of polystyrene, silica, polyolefins, polysaccharides, polyesters, polyacrylates, polylactic, iron oxide, Liposomes, or Lipid-Nanoparticle (LNPs).Method according to any of claims 1 to 3 characterized in that the quenching unit Q is selected from the group consisting of the family of black hole quenchers, QSY quenchers, dark quenchers, DY quenchers, atto quenchers, BMN quenchers, TQ quenchers, BBQ quenchers, IRDye quenchers, IOWA black quenchers and indocarbocyanine-based quenchers.5 Method according to any of claims 1 to 4 characterized in that the spacer unit is comprised of sequences selected from the group consisting of SEQ ID 1 to SEQ ID 2773.6 Method according to any of claims 1 to 5 characterized in that the at least one activation moiety is selected from the group consisting of antigens, proteins, peptides, antibodies, antibody fragments, bispecific antibodies (BsAbs), bispecific nanobodies, Bi-specific T cell engagers (BiTEs), nanobodies, Adapter CARs, T cell receptor (TCR), T cell receptor fusion constructs (TRuCs), synthetic TCRs and antigen receptors (STARs), HLA-independent TCRs (HITs).

7. Method according to any of claims 1 to 6 characterized in that the solid surface is provided with at least one costimulatory moiety capable of enhancing the activation of cells by recognizing the at least one activation moiety.

8. Method according to claim 7 characterized in that the at least one costimulatory moiety is selected from the group consisting of anti-CD3, anti-CD28, anti-CD2, anti-CD6, anti-CD26, anti-ICOS (CD278), anti-CD53, anti-LFA-l(aLp2, CDlla / 18), anti-MAC-1 (aMp2, CDllb / 18), anti-CDllc / 18, anti-VLA-4 (a4pi, CD49d / 29), anti-VLA-5(a5pi, CD49e / 29), anti-NKp46 (CD335), anti-CD16 (FcgRIIIA), anti-NKG2C (CD94 / 159c), anti-KIR2DSl-2, anti-KIR2DS3-6, anti-KIR3DSl, anti-KIR2DL4 (CD158d), anti-CD84, anti-NKp30 (CD337), anti-NKp44, anti-NKp80, anti-KIR-S, anti-mNKR-PIC, anti-mNKG2D-S, anti-mAct.Ly49, anti-NKG2D (CD314), anti-CRACC (CD319), anti-Ly9, anti-NTB-A, anti-DNAM-1 (CD226), anti-CD7, anti-CD59, anti-BY55 (CD160), anti-CD44, anti-2B4 (CD244), anti-cadherin, antiselectin, anti-integrin.Method according to any of claims 1 to 8 characterized in that the solid surface is provided with different fluorescence dyes covalently bound via different spacer units capable of being cleaved by different granzyme molecules to a quenching unit.10 Method according to any of claims 1 to 9 characterized in that the cells to be detected are selected from the group consisting of T-cells, CAR T-cells, NK cells, tumor infiltrating lymphocytes (TILs), genetically engineered T cells, CAR NK cells, dendritic cells, macrophages, MAIT cells, iNKT cells and B cells.11 Composition for detection of cell activation characterized by general formula (I)(A)-S-(F-C-Q) (I)WhereinA: activation moiety capable of activating cellsS: solid surfaceF : fluorescence dyeC: spacer unit capable of being cleaved by granzyme moleculesQ: quenching unitWherein the units (F-C-Q) and (A) are provided on the solid surface S in an area of less than 500 pm2.

12. Composition according to claim 12 characterized in that the solid surface is provided as particle with mean diameter of less than 12.6 pm.

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