Single-chain variable fragment of Anti-integrin antibodies

Abstract

The present invention relates to a new single-chain variable fragment (scFv) agonist of integrin β-1, that interacts with particular epitopes of the human integrin β1 subunit of SEQ ID NO: 1. The invention discloses particular complementarity-determining regions CDR1 to CDR3 of a variable light and a variable heavy chains. The scFv is proposed as an ingredient of stem cell or organoid culture media, as well as an ingredient in compositions comprising collagen or in extracellular matrix compositions. Methods of culturing stem cells or organoids in the presence of this scFv are also disclosed. The invention also provides several uses of scFv, and kits adapted to carry out these several applications.

Description

[0001] Single-chain variable fragment of anti-integrin antibodies

[0002] FIELD OF THE INVENTION

[0003]

[0001] The present invention relates to agonists of integrins for use in in vitro culture methods for culturing stem cells and organoids. It also relates to particular uses of the agonists and the organoids in therapy.

[0004] BACKROUND OF THE INVENTION

[0005]

[0002] The background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0006]

[0003] Organoids are self-organizing, miniature versions of organs that reproduce in vitro the key functional, structural, and biological complexity of that organ. The establishment and growth of 3D Organoids from Adult Stem Cells (ASCs) typically requires a hydrogel of extracellular matrix proteins as substitute for the in vivo basal lamina. Interaction of ap-integrin receptors with the constituent matrix proteins supports polarity, survival, and cell division. The use of Matrigel® as a hydrogel to support 3D epithelial cell culture has been extensively used in the field of organoids culturing. Matrigel®, aka Basement Membrane Extract (BME), is a commercially available mix of extracellular matrix (ECM) proteins, produced by the mouse Engelbreth-Holm-Swarm sarcoma cell line upon growth in mice. However, the use of Matrigel® implies limitations in its applicability. A main drawback is the animal origin of the matrix, which has so far prohibited the production of clinical-grade ECM. The animal production, in addition, leads to high production costs and to ethical issues inherent to animal experimentation.. Moreover, the exact composition of this and other commercially used matrices is not well- defined, which sometimes makes the results of a cell culturing process not reproducible, or not devoid of variability.

[0007]

[0004] In PCT Publication document WO 2020 / 234250 (KONINKLIJKE NEDERLANDSE AKADEMIE VAN WETENSCHAPPEN), the inventors propose a method for culturing an epithelial stem cell or an organoid comprising epithelial stem cells. However, this method does not show or suggest any efficient growth of organoids on collagen hydrogels or in matrices of clinical grade.

[0005] Another requirement for the establishment and growth of 3D Organoids from ASCs is the growth factor cocktail, which typically has to be optimized for each individual tissue, yet typically contains three key ingredients: potent activators of the Wnt pathway (e.g., Wnt3A or its surrogate, plus R-spondins), a tyrosine kinase receptor activator (e.g., EGF) and inhibitors of TGF-p and / or BMP (e.g., Noggin).

[0008]

[0006] The aim of these specific culture conditions is to obtain in vitro-produced organoids with the correct spatial disposition of cells, effectively reproducing the structural and biological complexity of the in vivo organ. In the particular case of 2D -, or 3D organoids comprising epithelial cells, the ultimate structural component underlying survival, growth, and functionality is the basal apical polarity of the organoid cells. The obtention of this polarity is not trivial in in vitro production of any epithelial tissue.

[0009]

[0007] In in vivo conditions, the primary epithelial tissues maintain a close connection with their basal lamina (BM), the complex mixture of extracellular matrix (ECM) proteins mediated by members of the integrin 3-1 subfamily of adhesion receptors. This subfamily consists of 12 members and constitutes the largest subfamily among integrins. In addition to receptors for laminin, the integrin 3-1 subfamily contains receptors for collagens and RGD motif-containing molecules, such as fibronectin, which are present in the stroma adjoining the basal lamina. Interaction of the integrins with the ECM initiate signaling within the epithelial cells which ensures correct basal-apical polarity and supports survival and growth. Other functions include mediating cell migration and localizing cells within a given tissue. Adherence of cells through integrins promotes intracellular signaling pathways, while loss of these signals for some cell types results in apoptosis. Epithelial cells in adult tissues express several different 31 integrins, including those that bind to laminin, collagen, and proteins containing RGD motifs. These receptors are typically expressed on the basal side of the epithelial cells and mediate adhesion to the individual basal lamina components.

[0010]

[0008] For the obtention of functional epithelial organoids, such as intestinal epithelial organoids, the basal side of the cells configuring the organoid need to be in contact with the hydrogel resembling the ECM used for their culturing. This is what is also known in the field “basal-out orientation”. On the other hand, the apical side of the epithelial cells is facing the inner of the lumen of the organoids (i.e., “apical-in orientation”).

[0009] However, and sometimes without an apparent reason, in vitro culture conditions lead to the reversion of this spatial configuration, which results is a non-functional organoid.

[0011]

[0010] Therefore, there is still a need of tools, reagents and matrices in the art that allow the reproducible culturing of organoids and, preferably in a cell environment easily transferable to the clinic.

[0012] SUMMARY OF THE INVENTION

[0013]

[0011] As a result of extensive research, inventors have characterized the role of 01 integrins in various organoid models, and they have probed the utility and essential role of activating (also named stimulatory) 1 integrin antibodies in supporting organoid cultures. They have initially, and surprisingly, found that antagonists of 0-1 block the interactions with ECM proteins leading to complete reversion of basal-apical polarity, and immediate arrest in the cell cycle. Conversely, activating integrin 0-1 antibodies reinforce the interaction of these integrin receptors with the ECM resulting in potentiated adhesion and associated survival and growth of the organoid cells.

[0014]

[0012] Moreover, it is herewith provided a single-chain variable fragment (scFv) agonist of integrin 0-1 with unexpected and advantageous properties in relation to other agonists, such as antibodies, also specific for the integrin 0-1 subunit.

[0015]

[0013] Thus, a first aspect of the invention relates to a single-chain variable fragment (scFv) agonist of integrin 0-1 , preferably wherein the single-chain variable fragment interacts with at least an epitope defined by residues 207 to 218 of the human integrin 01 subunit of SEQ ID NO: 1.

[0016]

[0014] As will be illustrated in the examples below, this scFv agonist of integrin 0-1 performs better than other agonists antibodies of the prior art. Indeed, it allows an enhanced growth of epithelial organoids derived from all parts of the gastrointestinal tract, including liver- and pancreas. It, moreover, potentiates the growth of primary isolated, and BME-established, organoids in collagen-l. It thereby avoids the limitations linked to the use of the BME (Matrigel®).

[0017]

[0015] From all these associated advantages of the scFv agonist of integrin 0-1 , it derives its use in stem cell or organoid culture medium, to promote the efficient establishment of the organoids or cultures of interest, in particular in or in the presence of an extracellular matrix composition, in particular wherein the extracellular matrix comprises or substantially consists of collagen, such as collagen-l, as the extracellular matrix component. Thus, another aspect (second) of the invention is a stem cell or organoid culture medium comprising a single-chain variable scFv agonist of integrin |3-1 as defined in the first aspect, optionally wherein further an extracellular matrix composition is in contact with the stem cell or organoid culture medium. The scFv agonist of integrin -1 according to the invention, including sc T2 / 16, can advantageously be used in culturing stem cells or organoids, in particular in the presence of, or in, or on, an extracellular matrix. In some preferred embodiments, the extracellular matrix composition is collagen based, preferably collagen-l (type I collagen) based. In some embodiments, the extracellular matrix composition is not an extracellular matrix composition formed by living cells. In some embodiments the extracellular matrix composition is not Matrigel or BME. In some embodiments, the extracellular matrix composition is man-made, or synthetic extracellular matrix composition. In some embodiments, the extracellular matrix composition is a chemically defined extracellular matrix composition, indicating that the individual components of the extracellular matrix composition are known and purposely included in the synthetic extracellular matrix composition, in contrast to, for example Matrigel / BME. In some embodiments, the chemically defined extracellular matrix composition comprises collagen, preferably collagen type-l in a concentration (when ready for culturing the cells) of at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6% or more (by weight).

[0018]

[0016] Not only stem cell or organoid culture media can comprise the single-chain variable scFv agonist of integrin 0-1 of the invention, but also other compositions broadly employed in the culture of cells, such as hydrogels made of or comprising collagen, are advantageous for cell culturing, in particular for the culture of organoids. Thus, yet another (third) aspect of the invention is a composition comprising collagen and a scFv agonist of integrin 0-1 as defined in the first aspect.

[0019]

[0017] A fourth aspect of the invention is an extracellular matrix composition that comprises a scFv agonist of integrin 0-1 as defined in the first aspect.

[0020]

[0018] The invention also provides for methods for culturing of stem cells or organoids, in which one or more of the scFv agonist of integrin 0-1 , or any composition comprising it, are used to obtain functional organoids with high yields and directly transferable to the clinical practice.

[0019] Thus, it is another aspect of the invention a method for culturing a stem cell or an organoid, in particular epithelial stem cells or organoids comprising epithelial stem cells, wherein the method comprises culturing said stem cell or organoid in a culture medium suitable for stem cells, wherein the culture method further comprises contacting the cell or organoid with a single-chain variable fragment (scFv) agonist of integrin p-1 as defined in the first aspect, and / or contacting the cell or organoid with a stem cell or organoid culture medium as defined in the second aspect, and / or contacting the cell or organoid with a composition comprising collagen as defined in the third aspect, and / or contacting the cell or organoid with an extracellular matrix composition as defined in the fourth aspect.

[0021]

[0020] From all this, also result stem cell cultures, and / or organoids that in vitro effectively reproduce, and with a high fidelity, all of the functional, and / or structural and biological complexity of that cell tissue or organ. In case of stem cell cultures, they reproduce those functions according to their differentiation stage.

[0022]

[0021] Therefore, another aspect of the invention is a stem cell or organoid, in particular an epithelial stem cell or an organoid comprising epithelial stem cells, which in particular are obtained or obtainable by a method as defined in the method for culturing a stem cell or an organoid of the previous aspect.

[0023]

[0022] These stem cells cultures or organoids are, according to another aspect of the invention for use in therapy and / or in diagnosis. Indeed, these cell aggregates (i.e., stem cell networks or organoids) that reproduce the actual physiology of tissue or organs are therapeutically active structures useful as tissue implants or tissue grafts.

[0024]

[0023] In another aspect, the invention also relates to a stem cell culture or organoid, in particular an epithelial stem cell culture, or an organoid comprising epithelial stem cells, for use in therapy and / or in diagnosis, and wherein the said stem cell or the said organoid are obtained or obtainable by a method as defined above.

[0025]

[0024] Yet another aspect of the invention is the use of a scFv agonist of integrin |3-1 as defined in the first aspect for in vitro culturing a cell, in particular a mammalian, and more in particular a human cell.

[0026]

[0025] In another aspect, the invention also provides the use of a scFv agonist of integrin p-1 as defined in the first aspect for in vitro and / or ex-vivo pre-treating cells, in particular for in vitro and / or ex-vivo pre-treating mammal, preferably human, cells.

[0026] Herewith provided is also a kit of parts, in particular for in vitro and / or ex-vivo pretreating cells, that comprises: a) a scFv agonist of integrin 0-1 as defined in the first aspect, or a composition comprising it; and b) optionally, instructions for the use of the kit and / or for carrying out any of the methods or uses of a scFv agonist of integrin -1 of the invention.

[0027]

[0027] Another aspect of the invention is a method for modulating the polarity of stem cells or of organoids comprising stem cells with apical and basal sides, which comprises a step of contacting the cells or organoids with an antibody or fragment thereof specific for integrin 0-1.

[0028] This later method is, in particular, useful to study the effect of other compounds on the polarity of cells, or even to test compounds that are known to interact with these cells by one of its sides.

[0028] BRIEF DESCRIPTION OF THE DRAWINGS

[0029]

[0029] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

[0030]

[0030] Fig. 1 A, Schematic of integrin functionality in epithelial cells in vivo and in vitro. In vivo, integrin receptors on the basal membrane of epithelial cells, contact a mix of ligand proteins of the basal lamina (basal lam.). In vitro these are replaced by Matrigel / BME® proteins. B, Organoids were grown in BME in the absence (Control) or presence of a strong inhibitory integrin 01 Ab (AIIB2). Subsequent staining for basal marker integrin 01 , in both conditions, was performed on organoids with permeabilized cells. The apical marker Zonula Occludens-1 (ZO-I) and nuclear marker DAPI were used. Dashed marked boxed indicate example for line scan to quantify intensity profile of the markers as shown in C. Scale bar: 50 pm. C, intensity plot of fluorescent apical-, and basal markers in cross-sections of the membrane. Permeabilized control-, and mAb AIIB2-treated intestinal organoids as shown in B, were stained for basal marker integrin 01 , apical marker ZO-I and nuclear marker DAPI as a reference. Apical and basal positions (black vertical lines) were identified based on F-actin staining with phalloidin (not shown) and the mean profile (sharp colored lines) of multiple organoids (n=8 per condition) was calculated (shadowed areas show standard deviation). D, Quantification of KI67 positive nuclei after 2 days of culture of control and mAb AIIB2-treated intestinal organoids, using immunofluorescent staining. E, Survival of intestinal organoids in the presence or absence of AIIB2. Scalebar is 2 mm. F, For quantification of viable cells ATP- sensitive luminescent assay (CellTiter-GLO) was performed at the indicated days. Mean values for consecutive days represent the averages of triple cultures (standard deviation indicated). Data normalized, based on the mean values of day 0.

[0031]

[0031] Fig. 2 A, Integrin recognition-providing Variable regions from the Heavy- (VH), and Light (VL) chains of the mouse hybridoma TS2 / 16 were cloned into a human lgG1 backbone or combined in a single-chain antibody version. Both scFv orientation (VH-VL and VL-VH ) are displayed with N-terminal and C-terminal. The concept of the procedure is schematically displayed. B, SDS-PAGE analysis of purified TS2 / 16 IgG (± 180 kDa) and scFv (± 26 kDa)(shown is VH-VL version) under reducing-, and non-reducing conditions. Proteins visualized by Coomassie Blue staining. Position of separate H-, and L-chains of IgG under reducing conditions are indicated. The molecular weights of markers in kDa (kiloDaltons), are indicated on the left margin. C, Brightfield images of K562 leucocyte cell-based adhesion reporter assay. K562 cells were allowed to adhere to glass-coated fibronectin, for 1 hr at 37°C, in the absence -, or presence of 5 nM of IgG- or scFv-type TS2 / 16 antibody. Cells, remaining after 3 buffer washes under the conditions indicated, are shown. Scalebar: 100 pm. D, A luminescent cell viability assay (CellTiter-Glo®) was used to quantify adhesion of K562 cells towards the fibronectin coated plate under conditions as shown in panel C. Relative light unit (RLU) values are in millions, mean of triplicate values (n=3) and standard deviations are indicated. E, Antibody-induced adhesion of K562 towards Fibronectin. K562 cells were allowed to adhere to plastic-coated Fibronectin (O / N incubation at 4 °C with 20 pg / ml Fibronectin in PBS) in wells of a 96-well plate (triplicate). Plate was subsequently incubated with 2% BSAfor 1 hr at RT. Hereafter, K562 cells in F12 DMEM-advanced medium were incubated for 1 ,5 hrs at 37°C in the presence or absence of 5 nM ITGB1 -specific antibodies, as indicated. Antibodies 12G10 and 9EG7, were Fab versions. The scFv TS2 / 16 applied is of H-L orientation. Upon 3 washes with PBS, attached cells were quantified with a Luciferase-based ATP assay. Mean Relative Light Units (RLU) and STDEV are displayed.

[0032]

[0032] Fig. 3 A, Quantification of colon organoid-derived single cells adhering to coated basement membrane extract (BME) expressed as RLUx105 from a cellular ATP-driven luminescent assay (CellTiter-Glo®). Test conditions include absence of antibody, and nM series of the activating scTS2 / 16, and the inhibitory mAb AIIB2. The graph represents trend lines based on the averages (n=3) for each condition. STDEV indicated. B, Routine cultivation of identically processed fragmented intestinal organoids in absence or presence of integrin subunit a6 blocking mAb GoH3. Quantification of ATP-driven luminescent assay (RLU x 105) after 7 days of culturing is presented with averages and STDEV’s (n=4). C, Routine cultivation of identically processed fragmented intestinal organoids in the absence or presence of scTS2 / 16. The averages and standard deviation (n=4) of total genomic DNA (left vertical axis) and relative luminescence (x106) in the cell viability assay (right vertical axis) at indicated days are depicted. D, Brightfield culture images of intestinal organoids from the experiment shown in C, as present on day 1 and 9. Scalebar is 2 mm. E, Culture images of a cloning assay, starting with a very dilute single cell suspension. Control, GoH3 and scTS2 / 16-supplemented medium are indicated. The scalebar is 2 mm. F, Quantification of the number of organoids (right y-axis) scored after 7 days from organoids as presented in e, the left y-axis displays cell viability expressed as RLUxlO3from a cellular ATP-driven luminescent assay (CellTiter-GLO®). Mean (n=3) and STDEV indicated. G, Clonal outgrowth of intestinal organoids in the presence of different integrin stimulating Fabs. Equal numbers of TrypLE-isolated single cells were embedded in BME and in triplo-cultured in the presence of the indicated integrin 1 antibodies. Tested concentrations are displayed. Antibodies 12G10 (Fab) and TS2 / 16 (H-L) have overlapping epitopes on the ITGB1 exodomain. Antibody 9EG7 (Fab) binds a different region in the same exodomain. The stimulating Fab SNAKA51 binds the integrin a5 chain, which is not expressed in these organoids. Organoid outgrowth was quantified by counting their number at day 10. Average and STDEV indicated.

[0033]

[0033] Fig. 4 Single-cell initiated cultures of stomach (N=1), duodenum (N=2), and ductal organoids from the liver (N=2) and pancreas (N=2), jejunum (N=2), and ileum (N=2), were grown in the presence of either 5 nM inhibitory mAb AIIB2 or activating scTS2 / 16. Organoid-, and medium type are indicated at the left and the top of the figure, respectively. Scalebar is 2 mm. For quantification of the number of viable cells, an ATP-sensitive luminescent assay (CellTiter-GLO®) was performed at the same day the images were taken. Mean RLU (x105) value and STDEV are indicated. The t-test p-values for scTS2 / 16 vs cntrl were; stomach 5x1 O'3, duodenum 1x1 O'6, liver 1x10-5, pancreas 8x1 O'10, jejunum 5x1 O'3, and ileum 8x1 O'4

[0034] Fig 5. A, BME-grown intestinal organoids stained for Integrin a2 (ITGA2) and nuclear marker DAPI. Scale bar: 50 pm. B, Quantification of intestinal organoid-derived single cells adhering to coated Collagen-I, using a cellular ATP-driven luminescent assay (CellTiterGlo). Test conditions include absence of antibody, and a series of nM concentrations of the activating TS2 / 16 single-chain Fv and the inhibitory Al IB2 Ab. The graph represents trend lines based on the averages of three technical replicates (RLU in hundred thousand) for each condition. Standard deviation indicated. C, ITGA2 KO intestinal organoids grown in Collagen-I or BME hydrogels in presence or absence of scTS2 / 16. Images are taken after 5 days of culturing (n=4). Scalebar is 2 mm. Cell viability measured based on ATP-sensitive assay of ITGA2 KO organoids in different hydrogels and conditions mentioned in D. Average and standard deviation of the relative light units (RLU, in thousands) of four replicates are indicated. E, F, Primary biopsies of colon (E) and cholangiocyte (F) tissue was processed and embedded in collagen-l in presence or absence of scTS2 / 16. For colon, images and quantification was done after 10 days of culture (n=4). Scale bar is 2mm. For cholangiocyte, collagen-derived organoids were cultured for 3 weeks, subsequent scTS2 / 16 sensitivity was measured using CellTiterGlo (RLU values in hundred thousand). G, Growth of PureCol EZgel-suspended colon organoids in the presence of inhibitory AIIB2 antibodies or activating scTS2 / 16 antibodies. Triplicate samples of mechanically fragmented colon organoids were suspended in 20 pl drops in roundbottom 96-wells plate. Culture media were supplemented with 5 nM AIIB2 antibody, control medium, or 5 nM scTS2 / 16 antibody, as indicated. Culture images are from day 7. Cellular growth was quantified by an ATP-driven Luminescence assay. Averages and standard deviations indicated. H, BME-grown cholangiocyte organoids were TrypLE-digested to single cells and incubated for 1 ,5 hrs at 37 °C in PureCol EZgel-coated wells (O / N incubation with 250 pg / ml in PBS). Enhancement-, or inhibition of adhesion result from nM concentrations of scTS2 / 16 or AIIB2 antibodies, respectively. Absence of antibody represents uninduced adhesion. Vertical axes indicate the number of adherent cells, displayed as Relative Light Units of an ATP-driven Luminescence assay. Trendlines are based on averages of triplicate values. Standard deviations indicated. I, Day 10 images of cholangiocyte organoid cultures in PureCol EZ hydrogel. Culture was started with TrypLE-isolated single cells derived from a BME- established organoid line. Putative Al IB2, and TS2 / 16 antibody effects on the proliferation of cholangiocyte organoids were compared to control conditions, as indicated. J, Emerging number of cholangiocyte organoids from primary normal liver samples in PureCol EZ hydrogel in the presence and absence of scTS2 / 16. Primary normal liver samples derived from 2 individuals were Collagenase-treated, extensively washed, and plated in hydrogel. The total number of emerging organoids was scored.

[0034]

[0035] Fig. 6: Small-intestinal adhesion towards the BME components LN-111 and collagen type-IV. Colon organoids were enzymatically processed to single cells and resuspended in DMEM-F12 medium. Cells were plated (n=3) to a final concentration of 50.000 cells / 200 pl / well in the absence or nM range presence of scTS2 / 16, as indicated. Wells were coated with BME (100ng / ml), LN-111 (70 ng / ml), collagen typelV (30 ng / ml), or a mix of LN-111 and collagen typelV (70 + 30 ng / ml, resp.). The coat-specific binding of cells was tested by including a nocoat control. For each coating condition the inhibitory AIIB2 ab (30 nM) was also included, to test the relative involvement of integrin B1 . Upon incubation for 1 .5 hrs (37°C), wells were extensively washed with DMEMF12 medium and remaining cells quantified by an ATP-driven luminescence assay (RLU).

[0035]

[0036] Fig. 7: Clonal outgrowth of intestinal organoids in the presence of selected integrin- binding antibodies. Equal numbers of TrypLE-isolated single cells were, in triplo, V embedded in BME® and cultured in the presence of the indicated integrin antibodies. Test concentrations are indicated. Antibodies 12G10 (Fab) and scTS2 / 16 (VH-VL) have overlapping epitopes on the ectodomain of 1 integrin. Antibody 9EG7 (Fab) binds a different region in the same ectodomain. The stimulating Fab SNAKA51 binds the integrin a5 subunit, for which the gene is not expressed in these organoids. Emergence of clones was quantified by counting their number at day 10. Average number of clones and STDEV indicated.

[0036]

[0037] Fig. 8 Growth of PureCol EZgel-suspended colon organoids in the presence of inhibitory mAb AIIB2 -, or activating scTS2 / 16. Triplicate samples of mechanically fragmented colon organoids were suspended in 20 pl drops in round-bottom 96-wells plate. Culture media were supplemented with 5 nM AIIB2 antibody, control medium, or 5 nM scTS2 / 16 antibody, as indicated. Culture images are from day 7. Cellular growth was quantified by an ATP-driven Luminescence assay. Averages and standard deviations indicated.

[0037]

[0038] Fig. 9: BME-grown cholangiocyte organoids were TrypLE-digested to single cells and incubated for 1.5 hrs at 37 °C in PureCol EZgel-coated wells (o / n incubation with 250 pg / ml in PBS). Enhancement-, or inhibition of adhesion result from nM concentrations of scTS2 / 16 or mAb AIIB2, respectively. Absence of antibody represents uninduced adhesion. Vertical axes indicate the number of adherent cells, displayed as Relative Light Units of an ATP-driven Luminescence assay. Trendlines are based on averages of triplicate values. Standard deviations indicated.

[0038]

[0039] Fig. 10: Day 10 images of cholangiocyte organoid cultures in PureCol EZ hydrogel. Culture was started with TrypLE-isolated single cells derived from a BME-established organoid line. AIIB2 Ab, and scTS2 / 16 effects on the proliferation of cholangiocyte organoids were compared to control conditions, as indicated.

[0039]

[0040] Fig. 11 : Emerging number of cholangiocyte organoids from primary normal liver samples in PureCol EZ hydrogel in the presence and absence of scTS2 / 16. Primary normal liver samples derived from 2 individuals were collagenase-treated, extensively washed, and plated in hydrogel. The total number of emerging organoids was scored.

[0040]

[0041] Fig. 12: scTS2 / 16-enhanced growth of duodenum organoids on BME- and Collagen- typel coated transwell inserts. Single cell populations (5x10A4) in intestinal expansion medium (EM) were incubated in upper compartment. ScTS2 / 16-supplemented EM was added to lower compartment. The means of technical triplicates for cell numbers / insert are indicated in the graph.

[0041]

[0042] Fig. 13: scTS2 / 16-enhanced growth of duodenum organoids on BME- and Collagen- typel coated transwell inserts. Single cell populations (5x10A4) in intestinal expansion medium (EM), were incubated in the upper compartments. ScTS2 / 16 was added to EM in the lower compartments. The mean of technical triplicate values for manually determined cell numbers / insert is indicated for each condition in the graph.

[0042]

[0043] Fig. 14: scTS2 / 16-enhanced growth of duodenum organoids on BME- and Collagen- typel coated transwell inserts. Single cell populations (5x10A4) in intestinal expansion medium (EM), were incubated in the upper compartments. ScTS2 / 16 was added to EM in the lower compartments. The mean of technical triplicate values for manually determined cell numbers / insert is indicated for each condition in the graph.

[0043]

[0044] Fig. 15: Essential support of intestinal organoid growth in PureCol® EZ Gel, in particular by the scFv version of the TS2 / 16 antibody. BME®-grown P26N colon organoids were mechanically fragmented and mixed with PureCol® EZ Gel (consisting of purified Type I collagen at a concentration of 0.5% w / w). Intestinal expansion medium, overlaying the hydrogel was supplemented with lgG1 and scFv-version of activating TS2 / 16 antibody, at indicated nM concentrations. At day 7 of culture, images were taken of individual triplicate wells. For quantification of growth we used the CellTiter-GLO® assay. Luminescence values (Relative Light Units-RLU.106) are listed inside the images. The graph summarizes these luminescence assay results and provides SDEV’s and statistical p-values (for t-test ) for the differences in luminescence between 25- and 0 nM of both the IgG -, and scFv supported cultures.

[0044] DESCRIPTION

[0045] Definitions

[0046]

[0045] A portion of this disclosure contains material that is subject to copyright protection (such as, but not limited to, diagrams, device photographs, or any other aspects of this submission for which copyright protection is or may be available in any jurisdiction.). The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or patent disclosure, as it appears in the Patent Office patent file or records, but otherwise reserves all copyright rights whatsoever.

[0047]

[0046] Various terms relating to the methods, compositions, uses and other aspects of the present invention are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art to which the invention pertains, unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definition provided herein. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein.

[0048]

[0047] For purposes of the present invention, the following terms are defined below.

[0049]

[0048] As used herein, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. The indefinite articles “a” and “an” are synonymous with “at least one” or “one or more".

[0050]

[0049] As used herein, the term “and / or” indicates that one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases.

[0050] As used herein, the term "at least" a particular value means that particular value or more. For example, "at least 2" is understood to be the same as "2 or more" i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, .... etc.

[0051]

[0051] As used herein, the term “one or more” of a list of entities means that one or a combination of the said entities is encompassed, in particular a combination with all the entities.

[0052] As used herein, “comprising” or “to comprise” is construed as being inclusive and open ended, and not exclusive. Specifically, the term and variations thereof mean the specified features, steps or components are included. These terms are not to be interpreted to exclude the presence of other features, steps or components. It also encompasses the more limiting “to consist of’.

[0052]

[0053] An “organoid” is an in vitro obtained miniaturized and simplified version of an actual organ, in terms of the key functional, structural and biological complexity of that organ. Organoids may be obtained by culturing adult stem cells of a specific tissue embedded in a 3D cell culture system. The main components consist of a hydrogel, harboring extracellular matrix components, and a cocktail of dedicated growth factors overlaying this hydrogel. Organoids may also be obtained by culturing the same adult stem cells stem cells in a 2D cell culture system. Also this system includes a hydrogel of extracellular matrix components (ECM) , that is here used as a very thin coating on a solid surface. The coating may consist of BME, but also of well-defined sources of collagen as described herin. Cells are “seeded” on top of this coating. For example, in some embodiments this surface is comprised of permeable inserts for transwell cultures. Transwell inserts are used by first adding dedicated organoid medium to the multiwell plate, followed by adding the Transwell inserts, and lastly adding the medium and cells to the inside of the ECM-coated compartment. These inserts provide independent access to both sides of a monolayer of growing cells.

[0053]

[0054] A “single-chain variable fragment” refers to a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, typically connected with a short linker peptide, such as a short linker peptide of 10 to about 25 amino acids. The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa. This protein retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker.

[0055] “Integrins” are heterodimeric transmembrane adhesion receptors, which promote cell- extracellular matrix interactions as well as cell interactions with other cells. They are configured by a non-covalent association of an a (alpha) subunit and a 0 (beta) subunit. These subunits are transmembrane glycoproteins (a type I), and they have a relatively large extracellular domain that mediates ligand binding and a short cytoplasmic tail. Mammals have 18a and 180 subunits. The structure of integrin is shown in FIG. 1 A and includes a “head” region and two rod-like “legs”. Integrin adopts distinct conformations that have different binding affinities to integrin ligands. As previously indicated, among the integrin ligands collagen, laminin, and fibronectin are included. In the known as bent-closed and extended-closed conformations the lower ‘legs’ and the transmembrane regions remain associated, and the ligand-binding is closed. The adoption of a high-affinity extended open conformation involves a series of shape changes, including unbending of the receptor and various inter-module and intra-module movements. When an integrin shifts from the bent-closed / extended-closed conformation to the extended-open conformation is termed that the ‘integrin is in activation state’. This activation is mediated by the a (alpha) subunit or the 0 (beta) subunit.

[0054]

[0056] An “agonist of an integrin", such as the single-chain variable fragment (scFv) agonist of integrin 0-1 of the invention, is a compound that upon interaction with one of the integrin subunits promotes the activation of the protein heterodimer and this way initiate any signaling within which ensures support and growth of the cells, as well as any correct basal-apical polarity if required for that tissue. This activation also allows mediation of cell migration and localization of cells within a given tissue. Many anti-integrin antibodies that stimulate integrin activation and ligand binding function allosterically. This antibodies, or fragments thereof, are also herewith referred as agonists of integrins or “stimulatory antibodies / agonists”. Examples of known stimulatory antibodies of the 0-1 integrin subfamily include those disclosed in WO 2020 / 234250.

[0055]

[0057] On the other hand, an “antagonist of an integrin”, also known and referred in this description as an “inhibitory agonist / antibody”, is a compound that aims to block the interaction between any cell expressing integrins with the extracellular matrix or with other colliding cells. This blockage stops any signaling pathway required for the growth of the cells, or for performing their functions, as well as for migrating within the tissue. The antagonists of integrins promote apoptosis. An example of known inhibitory antibodies of the 0-1 integrin subfamily includes AIIB2 (Hall et al. 1990. The alpha 1 / beta 1 and alpha 6 / beta 1 integrin heterodimers mediate cell attachment to distinct sites on laminin. The journal of cell biology, vol. no.110, 2175-84). An inhibitory antibody of integrin a6 is GoH3 antibody (Sonnenberg et al. 1987. A complex of platelet glycoproteins Ic and Ila identified by a rat monoclonal antibody. Journal of Biological chemistry vol. 262, 10376-10383). All known by the skilled person in the art.

[0056]

[0058] The expression "substantially free”, when in the present description is referred to the presence and amount of a compound or composition, it is to be understood as the compound or composition not being comprised in a particular composition, such as in a collagen composition that also comprises the single-chain variable fragment (scFv) agonist of integrin P-1 of the invention, but also as including those amounts (minor amounts) that do not disturb the effect aimed with a particular composition or environment defined as substantially free of the compound or composition.

[0057]

[0059] As used herein, “conventional techniques” or “methods known to the skilled person” refer to a situation wherein the methods of carrying out the conventional techniques used in methods as disclosed herein will be evident to the skilled worker. The practice of conventional techniques in molecular biology, biochemistry, cell culture, genomics, sequencing, medical treatment, pharmacology, immunology and related fields are well-known to those of skill in the art and are discussed, in various handbooks and literature references.

[0058]

[0060] As used herein, the term "subject" refers to any vertebrate animal, but will typically pertain to a mammal, for example a human, a domesticated animal (such as dog or cat), a farm animal (such as horse, cow, or sheep) or a laboratory animal (such as rat, mouse, non-human primate or guinea pig, rabbit). In preferred examples, the subject is human and includes males, females, adult, elderly, children or infants, in the need of treatment, in particular in the need of treatment involving transplantation of (part of) tissues, in particular of the digestive system tissues.

[0059]

[0061] As used herein, the term “therapeutically-effective amount" or “effective amount” refers to the amount of an organoid, a graft containing the organoid, the cells making up the organoid or the graft containing the organoid, or any other product as disclosed herein, which is effective for producing an effective and desired (therapeutic) effect in a subject at a reasonable benefit / risk ratio applicable and within the context of the treatment of the invention. As used herein, the term “amount” is used interchangeably with the term “dose”.

[0062] As used herein, the terms “therapy”, “treatment” and “treating” refer to therapeutic treatment. The object of the treatment is to at least slow down the disease condition. Those in need of the treatment include those already with the disease condition.

[0060] Detailed description

[0061]

[0063] The invention is defined herein, and in particular in the accompanying claims. Subject- matter which is not encompassed by the scope of the claims does not form part of the present claimed invention.

[0062]

[0064] It is contemplated that any method, use, product, or composition described herein can be implemented with respect to any other method, use, product or composition described herein. Embodiments discussed in the context of methods, use, products and / or compositions of the invention may be employed with respect to any other method, use, product or composition described herein. Thus, an embodiment pertaining to one method, use, product or composition may be applied to other methods, uses, products and compositions of the invention as well.

[0063]

[0065] Any references in the description to methods of treatment refer to the compounds, pharmaceutical compositions, and medicaments of the present invention for use in a method for treatment of the human (or animal) body by therapy.

[0064]

[0066] As embodied and broadly described herein, the present invention is directed to the surprising finding that a single-chain variable fragment (scFv) agonist of integrin p-1 promotes (a) the formation of epithelial stem cell or organoids comprising epithelial stem cells with high yields, which means that the organoids or cells grow fast and in high amounts; and (b) this growth may be achieved in an hydrogel of a defined composition, for example comprising collagen, thus avoiding any of the drawbacks associated to the use of Base Membrane Extracts of non-well defined composition and thus, non-clinically applicable. But also the growth can either be in BME, collagen or a mixture of at least both compositions. In addition, the use of the single-chain variable fragment (scFv) agonist of integrin p-1 according to the invention allows for long-term cultivation of the cells, and allows for the cells to be “passaged” several times (e.g. at least 4 times, at least 5 times, at least 6 times, at least 8 times, or at least 10 times or more) without the cell losing the ability to proliferate and / or differentiate.

[0067] Thus, as previously indicated, a first aspect of the invention relates to a single-chain variable fragment (scFv) agonist of integrin p-1 , preferably wherein the single-chain variable fragment interacts with at least an epitope defined by residues 207 to 218 of the human integrin 1 subunit of (I).

[0065]

[0068] All the sequences in this description are listed in Table 2 below.

[0066]

[0069] pi subunit, as defined in SEQ ID NO: 1, corresponds to the canonical isoform with Uniprot accession number P05556-1, version 2 of 2008-12-16 of the sequence, and release 2023-06-28 of the database.

[0067]

[0070] In a particular embodiment of the single-chain variable fragment according to the first aspect, it is defined by formula (I):

[0068] VN- (L)n- Vc (I), wherein

[0069] L is a peptide linker and n is an integer selected from 0 to 5;

[0070] - VN is the variable chain at the N-terminal position of the single-chain fragment;

[0071] - Vc is the variable chain at the C-terminal position of the single-chain fragment; the line (-) between the linker L and the variable chains, VN and Vc, represent a peptide bond;

[0072] - and wherein VN and Vc are selected from the variable light chain and the variable heavy chain of an anti-integrin P-1 agonist antibody, with the proviso that in formula (I) a variable light (VL) and variable heavy (VH) chain is present.

[0073]

[0071] In a more particular embodiment of the single-chain variable fragment of formula (I), the peptide linker, L, is a peptide that comprises from 5 to 30 amino acids and comprises SEQ ID NO: 2 (GGGGS). Preferably, L is a peptide linker that comprises or consist of SEQ ID NO: 2 and njs an integer that equals 3.

[0074]

[0072] In another particular embodiment of the single-chain variable fragment according to the first aspect, optionally in combination with any of the embodiments above or below, the scFv agonist of integrin p-1 comprises: (a) a variable light chain that comprises an amino acid sequence that comprises a complementarity-determining region 1 (CDR1) consisting of SEQ ID NO: 3 (SVSSIISSNYL), a complementarity-determining region 2 (CDR2) consisting of SEQ ID NO: 4 (RTSNLAS), and a complementarity-determining region 3 (CDR3) consisting of SEQ ID NO: 5 (QQGSDIPLT); and

[0075] (b) a variable heavy chain that comprises an amino acid sequence that comprises a complementarity-determining region 1 (CDR1) consisting of SEQ ID NO: 6 (GFTFSSYTMS), a complementarity-determining region 2 (CDR2) consisting of SEQ ID NO: 7 (TISSGGSYTYYPDSVKG), and a complementarity-determining region 3 (CDR3) consisting of SEQ ID NO: 8 (IGYDEDYAMDH).

[0076]

[0073] In a more particular embodiment, the scFv agonist of integrin p-1 comprises a variable light chain that comprises or consist of the amino acid sequence SEQ ID NO: 9 (EIWTQSPTTMAASPGDKITITCSVSSIISSNYLHWYSQKPGFSPKLLIYRTSNLASGVPPRFS GSGSGTSYSLTIGTMEAEDVATYYCQQGSDIPLTFGDGTKLDLK).

[0077]

[0074] In also another more particular embodiment of the first aspect, optionally in combination with any of the embodiments above or below, the scFv agonist of integrin p-1 comprises a variable heavy chain that comprises or consists of the amino acid sequence SEQ ID NO: 10 ( MDVKLVESGGGLVKPGGSLKLSCAASGFTFSSYTMSWVRQTPEKRLEWVATISSGGSYTYY PDSVKGRFTISRDKAKNTLYLQMGSLKSEDTAMYYCTRIGYDEDYAMDHWGQGTSVTVSS).

[0078]

[0075] In even a more particular embodiment of the scFv agonist of integrin P-1 according to the first aspect, the scFv agonist of integrin P-1 comprises or consists of an amino acid sequence selected from SEQ ID NO: 11 (VH-VL) or SEQ ID NO: 12 (VL-VH). Also, in another even more particular embodiment of the scFv agonist of integrin p-1 according to the first aspect, the scFv agonist of integrin p-1 comprises or consists of an amino acid sequence selected from SEQ ID NO: 13 (VH-VL) or SEQ ID NO: 14 (VL-VH).

[0079]

[0076] SEQ ID NO: 11 corresponds to a compounds of formula (I) in which VN is the VH chain and Vc is the VL chain It is also herewith identified as VH-VL. SEQ ID NO: 12 corresponds to a compounds of formula (I) in which VN is the VL chain and Vc is the VH chain. It is also herewith identified as VL-VN. In the same way, SEQ ID NO: 13 corresponds to a compounds of formula (I) in which VN is the VH chain and Vc is the VL chain that comprises a 6xHis Tag. SEQ ID NO: 14 corresponds to a compounds of formula (I) in which VN is the VL chain and Vc is the VH chain that comprises a 6xHis Tag. As will be understood by the skilled person, also contemplated are the same scFv agonist of integrin 3-1, devoid of the 6xHis Tag.

[0080]

[0077] Advantageously, and as will be illustrated in the Examples section, both scFv (VL-VH and VH-VL) were effective in inducing the growth of organoids.

[0081]

[0078] Another aspect of the invention is a stem cell or organoid culture medium comprising a single-chain variable fragment (scFv) agonist of integrin 3-1 as defined in the first aspects, or in any of its embodiments alone or in combination. In a preferred embodiment, the single-chain variable fragment (scFv) agonist of integrin 3-1 is scTS2 / 16 (VL-VH or VH-VL) as disclosed herein. In another preferred embodiment, the single-chain variable fragment (scFv) agonist of integrin 3-1 is monovalent or is multivalent. In some embodiments, the single-chain variable fragment (scFv) agonist of integrin 3-1 does not induce a conformational change in the absence of ligand. In some embodiments, the single-chain variable fragment (scFv) agonist of integrin 3-1 is a monospecific scFv, for example, not being functionally connected (e.g. linked via a linker) with another antibody of scFV specifically binding to another target.

[0082]

[0079] In a particular embodiment of the culture medium, it is for epithelial stem cells and for epithelial organoids, or, in other words, for the culture of organoids that comprise epithelial stem cells. More in particular the culture medium is for the culture of an epithelial stem cell or epithelial organoid of the mammal, preferably human, digestive system. More in particular, it is a culture medium for epithelial stem cells or epithelial organoids, preferably derived from stem cells of the colon, duodenum, stomach, liver and pancreas.

[0083]

[0080] In a particular embodiment of the stem cell or organoid culture medium that comprises the scFv agonist of integrin 3-1 , the medium further comprises one or more of a Wnt agonist, a BMP inhibitor, a mitogenic growth factor and a TGF- beta inhibitor. Particular examples of Wnt agonist or activators of the Wnt pathway include, among others, Wnt3A or its surrogate, and R-spondins. Examples of BMP inhibitors include noggin.

[0084]

[0081] In another particular embodiment, the stem cell or organoid culture medium that comprises the scFv agonist of integrin 3-1 , also comprises a tyrosine kinase receptor activator, more in particular the epidermal growth factor (EGF).

[0085]

[0082] Also in another particular embodiment, the stem cell or organoid culture medium as previously disclosed, comprises an extracellular matrix composition. In even a more particular embodiment of the stem cell or organoid culture medium, the extracellular matrix composition comprises one or more of collagen, laminin, and fibronectin, vitronectin, and Fibrinogen. Alternatively, a chemically defined hydrogel could consist of chitosan, PLGA, PEG, and more, decorated with peptides mimicking the natural ECM proteins Laminin, collagen or RGD- containing proteins. In another embodiment the extracellular matrix composition is a chemically defined extracellular matrix compositions and / or wherein the extracellular matrix composition is substantially free of any BME, such as the commercially available Matrigel®; or is a Basement Membrane Extract, such as the commercially available Matrigel® .

[0086]

[0083] Preferably used in combination with the scFv agonist of integrin p-1 according to the first aspect, or any of its embodiments, is a composition that comprises collagen. This combination allows for the obtention of stem cell cultures or organoids in an environment substantially free of any BME.

[0087]

[0084] Thus, herewith provided are compositions comprising collagen and a scFv agonist of integrin p-1 as defined in the first aspect or in any of its embodiments. In a particular embodiment, the composition is substantially free of any BME. In a particular embodiment, the composition is chemically defined. In even another particular embodiment, the composition only comprises collagen as integer of an extracellular matrix. These compositions are, preferably, in the form of hydrogels or provided as kits including their components for the preparation of the hydrogel, the kits including the scFv agonist of integrin p-1 , the collagen and, optionally, a buffered aqueous composition for the in-situ preparation of the hydrogel by mixing all the components.

[0088]

[0085] In this compositions comprising collagen and a scFv agonist of integrin P-1 , the collagen is selected from any of the known collagen types. In a more particular embodiment, the collagen is selected from the group consisting of collagen type I, collagen type II and collagen type III. In a preferred embodiment it is collagen type I.

[0089]

[0086] Also, another object of the invention is an extracellular matrix composition (ECM) that comprises a single-chain variable fragment (scFv) agonist of integrin p-1 as defined in the first aspect or in any of its embodiments. This ECM comprises, in a particular embodiment, one or more of a protein constituent of the basal lamina and selected from laminins, collagens and RGD-motif containing molecules, preferably fibronectin. This ECM composition is provided as a ready to use composition already comprising the scFv agonist of integrin p-1 , prepared by mixing all the components. In the alternative, the skilled person in the art will know of commercially available ECM to which the scFv agonist of integrin p-1 is added as additive.

[0090]

[0087] Another aspect of the invention is, as previously disclosed, a method for culturing a stem cell or an organoid, in particular an epithelial stem cells or an organoids comprising epithelial stem cells, wherein the method comprises culturing said stem cell or organoid in a culture medium suitable for stem cells, and wherein the culture method further comprises contacting the cell or organoid with a scFv agonist of integrin P-1 as defined above, and / or contacting the cell or organoid with a stem cell or organoid culture medium as defined above, and / or contacting the cell or organoid with a composition comprising collagen as defined above, and / or contacting the cell or organoid with an extracellular matrix composition as defined above.

[0091]

[0088] For the performance of the method, the skilled person in the art will recognize that conventional techniques are applicable.

[0092]

[0089] In the same way, culture media suitable for stem cells or organoids, in particular epithelial stem cells or epithelial organoids, are well known in the art. In the particular case of a culture medium suitable for culturing epithelial stem cells, it comprises one or more of a Wnt agonist, a BMP inhibitor, a mitogenic growth factor and a TGF-beta inhibitor.

[0093]

[0090] In a particular embodiment of the method, it comprises contacting the stem cell or organoid with one or more additional integrin agonists. These additional one or more integrin agonists are compounds, such as anti-integrin antibodies, that interact with the alpha or the beta subunits of integrin, for example by interacting with the a2, a4, a5, a11 p, aL, aX, pi , P2, P4, and P7. In some embodiments, the additional integrin agonist interacts with one or more of the beta subunits pi , P2, P4, and P7. Particular examples of integrin agonists are anti-integrin antibodies that are stimulatory antibodies selected from the group consisting of TS2 / 16, JBS2, HP1 / 3, SNAKA51 , PTS25-2, PMI-1 , MEM-83, NKI-L16, 496B, 12G10, 8A2, 15 / 7, HUTS-4, 8E3, N29, 9EG7, mAb 24, MEM-148, KIM127, CBR LFA-1 / 2, MEM-48, KIM185, AP3, AP5, LIBS6, LIBS2, 10F8, 2B8 and 2G3 antibodies (See, for example, WO 2020 / 234250).

[0094]

[0091] In another particular embodiment of the method of the invention as previously disclosed, the stem cell or organoid is an epithelial stem cell or organoid of the digestive system or of the kidneys or of the lungs.

[0092] In another particular embodiment of the method of the invention as previously disclosed, the stem cell or organoid is an epithelial stem cell or organoid of the digestive system, in particular of the mammal, preferably of the human, digestive system. In even a more particular embodiment, the stem cell or organoid is an epithelial stem cell or organoid of the digestive system derived from one or more of the colon, duodenum, stomach, liver and pancreas. Therefore, the stem cell or organoid is an epithelial stem cell or organoid of the digestive system. More in particular is an intestinal organoid, a colon organoid, a stomach organoid, a liver organoid or a pancreas organoid.

[0095]

[0093] The method of the invention provides particular features to the by means of it obtained or obtainable stem cells or organoids. Thus, in another aspect the invention relates to a stem cell or organoid, preferably epithelial stem cell or organoid, obtained or obtainable by a method as defined above, in particular obtained or obtainable by a method that comprises culturing said stem cell or organoid in a culture medium suitable for stem cells and that comprises contacting the cell or organoid with (a) a scFv agonist of integrin 0-1 , and / or (b) contacting the cell or organoid with a stem cell or organoid culture medium that comprises a scFv agonist of integrin 0-1 , and / or (c) contacting the cell or organoid with a composition comprising collagen that comprises a scFv agonist of integrin 0-1 , and / or (d) contacting the cell or organoid with an extracellular matrix composition that comprises a scFv agonist of integrin 0-1 .

[0096]

[0094] The obtained stem cells or organoids, in particular epithelial stem cells or epithelial organoids may be used in therapy and / or in diagnosis. For this particular uses in therapy and diagnosis the stem cells or the organoid are, in particular, the ones obtained or obtainable by a method as defined above.

[0097]

[0095] Therapeutic uses or approaches in which the stem cells or organoids can be applied to are selected, in particular, from regenerative medicine, associated or not to partial or total organ extirpation; and organoid transplantation.

[0098]

[0096] The invention thus proposes a method of treatment of a disease by means of regenerative medicine, associated or not to partial or total organ extirpation; and organoid transplantation, wherein a therapeutically effective amount of the herewith obtained stem cells or organoids, in particular epithelial stem cells or epithelial organoids, is administered in a subject in need thereof. This aspect can also be defined as the use of stem cells or organoid as herewith defined, for the preparation of a medicament for the treatment of a disease in which regenerative medicine, associated or not to partial or total organ extirpation; and organoid transplantation is required.

[0099]

[0097] The invention also proposes, therefore, a pharmaceutical composition that comprises a therapeutically effective amount of one or more of the stem cells or organoids of one of the aspects of the invention, together with one or more pharmaceutically acceptable excipients.

[0100]

[0098] The invention also encompasses using the single-chain variable fragment (scFv) agonist of integrin -1 as defined herein for in vitro culturing a cell, in particular an adult stem cell. This cell is in particular a mammal adult stem cell, and more in particular a human adult stem cell. The cells are, in another particular embodiment, stem cells derived from a subject.

[0101]

[0099] Yet in another aspect, the invention relates to the use of a scFv agonist of integrin 0-1 as defined above or any composition comprising it, for in vitro and / or ex-vivo pre-treating cells, preferably mammal, and more preferably human adult stem-cells. From this pre-treated cells organoids or stem-cell derived tissues are obtained which, in another aspect of the invention, are for use in a therapy that includes organ or tissue transplantation and / or tissue regeneration.

[0102]

[0100] To carry out the several methods and uses of the invention and disclosed in connection with the advantageous scFv agonist of integrin 0-1 of the first aspect, is also herewith provided a kit of parts, preferably for in vitro and / or ex-vivo pre-treating cells, that comprises: a) a scFv agonist of integrin 0-1 as defined above, or a composition comprising it; and b) optionally, instructions for the use of the kit, and / or for carrying out any of the methods and uses as defined above.

[0103]

[0101] Several are the options of the kits of the invention. Thus, in a particular embodiment, the kit comprises, preferably in a container, a composition such as a buffered aqueous composition with only the scFv agonist of integrin 0-1 ; and in one or more separate containers any of an ECM composition, a collagen composition, and a stem cell or organoid culture medium. In another alternative embodiment of the kit of the invention, the kit comprises a ready to use composition that comprises the scFv agonist of integrin 0-1 , and which composition is selected from one or more of ECM composition, a collagen composition, and a stem cell or organoid culture medium.

[0104]

[0102] The kits of the invention are, in particular to be used for in vitro and / or ex-vivo pretreating cells. Thus, herewith disclosed is also the use of a kit for in vitro and / or ex-vivo pre- treating cells (i.e., mammal adult stem-cells, preferably human adult stem-cells), which kit comprises: a) a scFv agonist of integrin 0-1 as defined above, or a composition comprising it; and b) optionally, instructions for the use of the kit, and / or for carrying out any of the methods and uses as defined above.

[0105]

[0103] As previously indicated, the inventors surprisingly found that the polarity of organoid cells, in particular of organoids comprising epithelial cells, is reversed in the presence of an antibody that blocks the 1 subunit of integrins, or in the presence of an antagonist of this subunit. In the alternative, they also observed that an antibody or fragment thereof that stimulates or that is agonist of the 01 subunit of integrins, stabilizes the cultured organoids in such a way that the epithelial cells are spatially configured with the basal side out of the organoid, thus in contact with the hydrogel used for their culturing.

[0106]

[0104] Derived from this findings, is herewith disclosed a method for modulating the polarity of stem cells, preferably of epithelial stem cells, or of organoids comprising epithelial stem cells, which cells comprise an apical and a basal side, wherein the method comprises a step of contacting the cells or organoids with an antibody or fragment thereof specific for integrin 0-1.

[0107]

[0105] In a particular embodiment of the method for modulating the polarity of the cells, the method is carried out with an agonist of the integrin 0-1 subunit, preferably with a scFv agonist of integrin 0-1 subunit as defined in the first aspect. In another particular embodiment, the method is carried out with an antagonist of the integrin 0-1 subunit.

[0108]

[0106] In the sense of the invention, the modulation of the polarity of the cells in an organoid, in particular of the epithelial cells in an organoid, means that the spatial distribution and orientation of the asymmetrical cells in relation to the culture media or hydrogel in which they grow is modified or influenced.

[0109]

[0107] Without being bound to any theory, the inventors hypothesize that the effect of an agonist of the integrin 0-1 subunit would be translated to: 1 ) Prevention of apotosis / anoikis; 2) Improved / extended availability of receptors for Wnt (or Wnt Surrogate) and R-spond in, Notch receptor, EG F receptor; and / or 3) Prolonged presence of Integrin receptors at basal-OUT membrane could directly have an effect on integrin mediated kinase signaling downstream from the receptor.

[0108] The effect that was found according to the examples, on improved cloning and growth in general, are probably in part or completely, related to a mix of the effects described in 1 , 2 and 3.

[0110] EXAMPLES

[0111]

[0109] All the materials and methods that apply to the following examples are disclosed at the end of this section for the purpose of simplification.

[0112]

[0110] Materials and methods for all the experiments shown in the examples below can be seen at the end of this description.

[0113] Example 1. Production of single-chain fragment of an anti-integrin fi-1 agonist antibody (TS2 / 16)

[0114]

[0111] As indicated in the section of Materials & Methods a humanized version of the antibody known as TS2 / 16 and a single-chain variable fragment of it was constructed.

[0115]

[0112] TS2 / 16 (ATCC HB-243, commercially available) is an agonist antibody of the |31 chain or subunit of some of the integrins, the membrane proteins used by epithelial cells to interact with the extracellular matrix. TS2 / 16 induces the open configuration of a5 1 receptor on K562. TS2 / 16 binds the same epitope as the inhibitory AIIB2 antibody but is, in contrast, strongly activating. A likely explanation is that this epitope forms a switching element enabling conformational changes of the receptor. Importantly, TS2 / 16 has been reported to enhance intercellular-, or cell-to-ECM adhesion, most notably in the tunable adhesion of leukocytes.

[0116]

[0113] We determined the sequences of the variable regions of the H-, and L-chain of the mouse hybridoma, and used human codon-optimized synthetic DNA (SEQ ID NO: 15 and 16) to clone these into hulgG1 H-, and i^L-chain vectors. The humanized antibody was subsequently purified using standard chromatography procedures. For the single-chain version, the combined V regions of H-, and L-chain were designed in both orientations (Fig. 2a) and connected via a 3x(GGGGS) linker. E.coli codon-optimized DNAs were cloned into a pET expression vector (SEQ ID NO: 13 and 14 correspond to the expressed amino acid sequences). A routine protocol for protein extraction, solubilization and refolding was implemented to obtain pure single-chain versions. On average, we obtained 1 mg pure scTS2 / 16 per 50 ml of bacterial suspension, sufficient for approximately 7 liters of cell culture medium at a final concentration of 5 nM. PAA-gel analysis for both recombinant proteins - the synthetically produced huigGI and scFv version of TS2 / 16 (VH-VL)- are shown in Fig.2 b.

[0117]

[0114] The scTS2 / 16 (VH-VL) proved able to recognize the exodomain of the 01 chain in solution Functionally, both huigGI and scFv versions (VH-VL), induced adhesion at low nM concentration of K562 leucocyte cells towards fibronectin, a standard assay for integrin- mediated adhesion (Fig. 2c-d). The scTS2 / 16 with VL-VH orientation was found to be functionally equivalent to scFv version (VH-VL). The earlier-mentioned extended-open 12G10 and the extended-closed gEG7 activating integrin 1 antibodies, performed significantly less in this classical K562 adhesion assay (Fig.2e).

[0118]

[0115] Based on results described herein, however it was surprisingly found that in particular the scFv version of TS2 / 16 can advantageously be used in methods for culturing a stem cell or an organoid, in particular in the presence of the extracellular matrix protein collagen. In combination with the low-cost bacterial production, the invention is therefor directed to this scFv agonists of integrin 0-1 , including the scFv (VL-VH) and scFv (VH-VL) version of TS2 / 16. Although the VH-VL version was used in subsequent studies, the reversed VL-VH variant performs equally well.

[0119]

[0116] The sequences of the scTS2 / 16 (VH-VL), and of the scTS2 / 16 (VL-VH) are, respectively, SEQ ID NO: 11 and SEQ ID NO: 12. SEQ ID NO: 13 and SEQ ID NO: 14, correspond respectively to the sequences VH-VL, and VL-VH including the 6xHIS tag. The skilled person will understand that in some embodiments the 6XHIS tag is not required and that scTS2 / 16 (VH-VL) and / or scTS2 / 16 (VL-VH) without the 6XHIS tag are likewise according to the invention and claimed.

[0120]

[0117] Human codon-optimized synthetic DNA for each of the VH and VL chains is represented in SEQ ID NO: 15 and 16, respectively.

[0121]

[0118] The amino acid sequence of the VH and VL of the huigGI of TS2 / 16 corresponded to SEQ ID NO: 17 and SEQ ID NO: 9, respectively.

[0122] Example 2. scFv-TS2 / 16 enhances growth of BME-suspended intestinal organoids.

[0123]

[0119] One of the main components of BME® is the protein laminin-111. Four different integrins, a301 , a601 , a701 , and a604 differentially bind to members of the laminin family of proteins. Integrin a6014O,41 binds to laminin-111 , the embryonic laminin in EHS / BME®. The a301 and o604 integrins, both expressed by these organoids, bind to laminin-332 and laminin- 511 , which are absent in EHS / BME®. The specific role of a601 integrin in laminin-111 binding was confirmed by exogenous expression of integrin a6p1 in K562 cells. The newly-acquired laminin-111 recognition was strongly enhanced by the activating TS2 / 16 antibody. We used a series of concentrations of the TS2 / 16 scFv in the low nM range. The observed strong improvement of adhesion in the presence of scTS2 / 16 reached its maximum at 2 nM (Fig. 3a). Of note, the AIIB2 blocking antibody inhibited adhesion of organoid cells, as a validation of this adhesion assay.

[0124]

[0120] To assess if the integrin a6 subunit was also crucial in mediating growth of colon organoids in BME®, we added the inhibitory integrin a6-specific GOH3 antibody 43 while regularly splitting colon organoid cultures. The results shown in Fig.3b, indeed, indicated a strong G0H3-induced reduction of growth.

[0125]

[0121] We then tested whether activation of the 1 component of Integrin receptors could enhance - in addition to adhesion - the growth of colonic organoids. Addition of scTS2 / 16 to standard 3D colon organoid culture in BME at 5 nM, led to increased colon organoid growth. This was confirmed after 10 days of culture by assessment of total genomic DNA, of ATP- dependent luminescent light units (Fig. 3c), and of phase contrast images of the organoid cultures (Fig. 3d). In line, we observed an increased percentage of Ki67+ nuclei.

[0126]

[0122] Single cell cloning from primary tissue or from organoids is generally highly inefficient. We therefore tested the effect of scTS2 / 16 on plating efficiency of such single cells and noted that the clonal outgrowth in the presence of scFv TS2 / 16 (at 5 nM) improved dramatically (Fig. 3e-f). In contrast, the outgrowth of organoids, even in the presence of scTS2 / 16, was completely blocked by adding G0H3 emphasizing the importance of the Integrin a6 component.

[0127]

[0123] The positive effects of Integrin 01 -stimulatory antibodies in the outgrowth of organoids from single cells was much less strong with the two other 01 -chain conformation-stabilizing antibodies, 9EG7 and 12G10 (Fig. 3g).

[0128]

[0124] Thus, while the TS2 / 16-stabilized high affinity conformation of 01 integrins on leukocytes led to enhanced intercellular-, or cell-to-ECM adhesion, its scFv-version also improves establishment and expansion of epithelial organoids by enhancement of a601- mediated interaction with laminin-111 as presented in BME® Example 3. BME-established organoids from other parts of the gastrointestinal tract also rely on integrin pi

[0129]

[0125] We then tested whether the observed integrin 01 -regulated proliferation of intestinal organoids in BME, also held for other parts of the human gastrointestinal tract, i.e., small intestine, stomach organoids, and ductal-type organoids from pancreas and liver. The pertinent organoids were grown in BME supplement with control medium, or with 5nM of either the inhibitory Al IB2 or the activating scTS2 / 16 antibody. AIIB2-induced inhibition and scTS2 / 16- enhancement of growth (as compared to medium without antibody) were observed in all cases (Fig. 4). We concluded that growth of gastrointestinal tract organoids depends on ax01 integrin receptors. We concluded that growth of gastrointestinal tract organoids depends on 01-class integrins and can be enhanced by scTS2 / 16.

[0130] Example 4. scTS2 / 16 antibody potentiates growth of established- and primary isolated colon organoids in collagen-l hydrogel.

[0131]

[0126] Based on the scFv-generated improvement of gastrointestinal organoids in BME, we shifted our focus to the replacement of Matrigel® / BME. Such an alternative should similarly benefit from the addition of scTS2 / 16. We chose Type-I collagen since it is inexpensive and can be produced at clinical grade (for tissue engineering and cosmetic applications). It has been reported previously that exogenous introduction of the a2 subunit integrin-component into K562 cells (which do not naturally express a201 integrins) confers adherence of the cells to collagen, a feature which was highly sensitive to the presence of TS2 / 16

[0132]

[0127] In a first attempt to enhance growth by scTS2 / 16 in a collagen type-l hydrogel, we focused on colon organoids. We tested several classical acidic solutions of collagen-l, which require neutralization and temperature elevation to initiate hydrogel formation (data not shown). Ultimately, a commercially available pre-formulated isotonic form of collagen-l with neutral pH, that forms a 3D hydrogel by simply warming to 37°C, proved to be the most successful preparation. In our hands, this collagen-l preparation did not support organoid expansion beyond 2-3 passages. We confirmed expression of a201 in BME® -grown colon organoids, both at the RNA level (not shown) and by immunofluorescent staining with an antibody (Fig. 5a). In total, 4 a-integrin subunits (a1, -2, -10, and 11 ) harbor a collagen-binding l-domain insert and share a 01-subunit. Of these, the organoids exclusively expressed the a2 subunit (data not shown). The scTS2 / 16 amplified adhesion of organoid cells towards collagen-l (Fig.5b). In contrast, the inhibitory AIIB2 antibody blocked such adhesion (Fig. 5b).

[0133]

[0128] These potent effects of the two antibodies were also reflected in the growth of established colon organoids in the collagen-l hydrogel (Fig. 5g). We also tested pi -mediated growth of freshly isolated colon biopsies in collagen-l hydrogel. Colon tissue from four donors was enzymatically processed and epithelial cell suspensions were embedded in collagen-l in the presence or absence of scTS2 / 16 (5 nM). Culturing in scTS2 / 16-containing medium yielded significantly more organoids as compared to standard colon medium (Fig. 5f).

[0134]

[0129] We then created null-alleles of the gene for the integrin a2 subunit by CRISPR to confirm the relevance of the collagen-recognizing (a2 1 ) integrin for colon organoid growth in collagen hydrogels (Fig. 5c). As predicted, ITGA2 gene mutation blocked organoid growth in collagen-l, even in the presence of scTS2 / 16. Of note, growth in BME® was reduced but not fully blocked, implying that integrins for other ECM components (e.g., a6pi-laminin) act in parallel with a2pi -collagen in maintaining organoid growth (Fig. 5d-e).

[0135]

[0130] Like colon, when a2-expressing liver bile duct organoids were stimulated by scTS2 / 16, increased adhesion towards collagen-l ensued (Fig. 5h), mirrored by scTS2 / 16-enhanced growth of cholangiocyte organoids (Fig. 5i). Guided by these results, we tested the outgrowth of cholangiocyte organoids in collagen-l hydrogels, directly from primary liver biopsies. This consistently yielded more organoids in scTS2 / 16-medium as compared to control medium (Fig. 5j). We subsequently quantified the scTS2 / 16 effects on the newly derived collagen-based cholangiocyte organoids (Fig. 5f).

[0136]

[0131] Examples 1 to 4 allow to affirm that we have showed the functional importance of integrin pi in organoid cultures of human small intestine, colon, stomach, pancreas and liver. Thus, we observe potent growth-blocking effects of the inhibitory integrin pi subunit-specific antibody AIIB2, while the activating integrin pi subunit antibody TS2 / 16 enhances growth. We also describe a bacterially produced single chain version of TS2 / 16 and show that this agonistic single chain antibody binds the human pi subunit with high affinity. Moreover, scTS2 / 16 proves to be highly potent in supporting establishment and growth of organoids in the ‘standard’ BME® hydrogel, simply by addition to the culture medium as a soluble monomer. We observe a crucial role for the integrin a6 component in BME® -dependent growth. We also find that addition of the single chain TS2 / 16 supports the growth of organoids in hydrogels of purified collagen-l. CRISPR-mediated gene knockout demonstrated a2 1 to be responsible for “collagen only”- supported organoid growth. Addition of scFv supports organoid growth but this effect may still be improved. We currently believe that this results from the parallel interaction of a2 1 with collagen-l / lll, and a6 1 with laminin-111 , both natural ECM components present in BME®. The versatility of scTS2 / 16 arises most likely from the fact that the integrin a chains for the ECM proteins laminin and collagen share the 1 signal transduction chain.

[0137]

[0132] The observation that the activation of pi integrins by single-chain TS2 / 16 in leukocytes extends to epithelial organoid cells opens a range of applications. We believe that the availability of this single chain reagent which allows organoids to grow in “collagen only”- hydrogels may avoid the need for costly BME® (Matrigel®). Moreover, bacterial production allows the generation of clinical grade batches of the single chain reagent, while clinical grade collagen preparations are already widely available for tissue repair and cosmetic applications. Mouse and human colon organoids have been shown to be transplantable in mice. The use of the single chain integrin reagent would make expansion of transplantable organoids faster and more cost-efficient. Moreover, a scTS2 / 16-induced high affinity state of the transplanted organoid cells may instruct these to engraft more efficiently. Of note, the first clinical trial using colonic organoid transplantation therapy for intractable cases of ulcerative colitis is currently ongoing.

[0138] Example 5. Integrin (31 is indispensable for organoid growth.

[0139]

[0133] Adult stem cell-derived intestinal organoids exhibiting a basal-OUT phenotype in the presence of external Matrigel® invert their polarity to basal-IN when cultured in the absence of Matrigel® 16. We intended to test the role of integrin pi, dominantly expressed in various organoid models (data not shown), on polarity and associated physiology. The rat anti-integrin pi antibody AIIB2 has previously been shown to allosterically block the recognition of pi integrins for all three major integrin ligand classes (laminin, collagen and RGD-class proteins). This antibody, therefore, enabled us to test the role of the pi integrin component in basal-apical polarization of organoids. Fig.1 (b-f) summarizes the dramatic effects of adding the AIIB2 antibody to human colon organoids. The “basal-OUT” phenotype of these organoids, cultured in the presence of BME®, inverted within 12 hours to basal-IN upon exposure to AIIB2 (Fig. 1b-c). This condition resulted in an immediate abrogation of growth, measured as a strong decrease in the percentage of cells expressing the nuclear proliferation marker Ki67 (Fig.ld), in visual growth kinetics (Fig.le) and in a quantitative cellular ATP assay (Fig.lf). These observations implied that integrin-mediated signals normally maintain growth and are absent in the presence of AIIB2.

[0140] Material & Methods for the examples

[0141] Immunofluorescent analysis of apical / basal polarity and proliferation marker KI-67.

[0142]

[0134] P26N human colon organoids were grown for 2 days in BME + / - the rat anti-ITGB1 antibody AIIB2 at a concentration of 1 ug / ml (gift A.Sonnenberg, Dutch Cancer Institute, Amsterdam, The Netherlands). Organoids were harvested using Cell recovery solution (ThermoFischer) and fixed in 4% formaldehyde (pH 7.4) at 4°C for 1 hour. Hereafter, organoids were washed with PBS 0.1% Tween (PBST)(2% BSA). 3D imaging of organoids was performed essentially as described previously 43. In short, organoids were incubated O / N at 4°C with primary antibodies binding ZO-I (rabbit polyclonal, Thermofischer), ITGB1 (mouse MAB1778, R&D), and Ki67 (rabbit polyclonal AB9260, Merck) or ITGA2 (Abeam, ab30486). Upon extensive washing with PBST, binding of primary reagents was visualized by O / N incubation in PBST / 2%BSA containing Donkey anti-rabbit Alexa 568 (ThermoFischer) and Goat anti mouse Alexa488. Phalloidin Atto 747N (Sigma-Aldrich) was used for direct visualization of F-actin. DNA was stained with DAPI (Thermofischer). Organoids were optically cleared in a glycerolfructose clearing solution prior to imaging. Organoids were imaged on a (microscope, objective, etc.). Analysis was done with Imaged software.

[0143] TS2 / 16 hlgG1 production

[0144]

[0135] We used a simplified workflow to sequence the variable regions of the mouse TS2 / 16 hybridoma (ATCC HB-243) as described. A human codon-optimized coding sequence of the TS2 / 16 VH and VL domains were synthesized by GeneArt (ThermoFischer) and cloned into pFUSEss-CHIg-hG1 and pFUSE2ss-CLIg-hK, respectively (Invivogen). Expression vectors were co-transfected into Expi293F™ human suspension cells using ExpiFectamine™ (Thermofischer). IgG protein was purified by affinity chromatography using CaptivA® Protein A resin (Repligen), followed by size-exclusion chromatography using a Superdex 200 Increase 10 / 300 GL column (Cytiva). TS2 / 16 scFv production in E.Coli

[0145]

[0136] An E.Coli codon-optimized coding sequences of the TS2 / 16 scFv’s (SEQ ID NO: 15 and 16) were synthesized by GeneArt (ThermoFischer) and cloned into a pET21 vector harboring a C-terminal 6x-HIS tag (Novagen) using standard molecular biology techniques. Protein expression induction, using 1 mM IPTG (Sigma), was done in BL21(DE3) cells starting with a OD600 optical density of 0,6 in standard LB medium. Production was at 28 °C for approx. 18 hrs. Bacterial pellets obtained were resuspended in lysis buffer (50 mM Tris pH 7.5, 200 mM NaCI, 10% glycerol, 0.4% Triton x100) and then sonicated (5 cycles; 10 sec ON, 20 sec OFF). The insoluble fraction, obtained by centrifugation at 11.000 rpm for 15 min., was Guanidine-Hydrochloride-solubilized and refolded as described previously 27. The partially purified, refolded, scFv was further purified using Ni-NTA (Qiagen), followed by size-exclusion chromatography using a Superdex S200 increase 10 / 300 GL size-exclusion column (Cytiva) in HBS (10 mM HEPES, pH 7.2, 150 nM NaCI). Analysis of the quality of protein purification was performed running samples, under reducing -, and non-reducing conditions, on a 4-15% TGX precast SDS-PAGE (Biorad) gel. Proteins were visualized using InstantBlue Coomassie Protein stain (Abeam).

[0146] Integrin 1 ectodomain production and Immunoprecipitation.

[0147]

[0137] We used the procedure reported by Takagi et al. 2002 to recombinantly produce the extracellular domain of Integrin betal in the absence of an alpha integrin. Deletion of the ~30 amino acids specific-determining loop (SDL) in the l-like domain is essential for stable secretion of this exodomain. The DNA coding sequence was synthesized by GeneArt (ThermoFischer) and cloned into vector pcDNA3.1 (Addgene). Expression vector was transfected into Expi293F™ human suspension cells using ExpiFectamine™ (Thermofischer). Purification of the C-terminally Flag epitope tagged was performed by absorption of secreted protein to M2- agarose beads (Sigma), and elution by excess 3xFlag peptides (Sigma). Eluted proteins were subsequently subjected to size-exclusion chromatography using a Superdex 200 Increase 10 / 300 GL column (Cytiva). For immunoprecipitation experiments with the ITGB1 -exodomain we used ProtG (Sigma) or M2-agarose (Sigma). All washes and antibody incubations were in standard PBS. ITGB4 antibody ab110167 (Abeam), ITGB1 Ab 1778 (R&D systems), HUTS4 (mab2079z Sigma), M2 anti-Flag (Sigma), anti-Flag HRP (Sigma), anti-6xHIS HRP (R&Dsystems). We used 4-15% PAA gel (Biorad) under reducing conditions, and PVDF membrane (Immobilon) for blotting. ECL (GE-Healthcare).

[0148] Adhesion assays K562 cells

[0149]

[0138] Erythroleukemia cell line K562 cells (ThermoFischer) were cultured in DMEM (Gibco) with 100 U / ml Penicillin-Streptomycin and 5% FBS (Sigma-Aldrich). Bovine Fibronectin (ThermoFischer) was diluted to 20 ug / ml in PBS and incubated O / N at 4°C in a flat-bottom 96- well plate (Greiner). Subsequently, wells were blocked with 2% BSA in PBS for 30 min. at RT. K562 cells were resuspended in DMEM / F12 medium. Wells were loaded with equal amounts of cells, in the absence of antibody, or presence of 5 nM of TS2 / 16 in scFv or IgG format. Upon incubation of cells for 90 minutes at 37°C, unbound cells were removed by 3 washes with DMEM-F12 advanced medium. Number of adherent cells was analyzed by brightfield microscopic images, and a luciferase-based ATP assay (CellTiterGLO: Promega).

[0150] BME® and PureCol EZgel® -based organoid culturing.

[0151]

[0139] Organoids grown in BME® (Cultrex) were passaged by releasing them first from the matrix by incubation for 20 min on ice in Cell recovery solution (ThermoFischer) supplemented with 10 pM of the Rho-kinase inhibitor Y-27632 (Abmole). Subsequent MEM / F12-washed organoids were either mechanically sheared, or TrypLE-digested to single cells. Upon additional washing with DMEM / F12, organoids were either resuspended in fresh BME® or, alternatively, in PureCol EZgel® (Advanced Biometrix). For complete gelation of the BME® -. or PureCol EZgel® gel, plates were incubated at 37°C for 30 or 60 minutes, respectively. Hydrogels were covered with relevant organoid medium and incubated at 37°C and 5% CO2. Passaging of organoids growing in Collagen, was achieved by incubation at 37 °C for 30 min maximally, with Collagenase from Clostridium histolyticum (Sigma-Aldrich) diluted in DMEM / F12 to a concentration of 1 mg / ml. For complete removal of the proteolytic enzyme, organoids were washed twice with an excessive volume of DMEM / F12 medium before re-entry into PureCol EZgel®. The 1-specific inhibitory AIIB2 antibody was added to organoid cultures at 5 nM. The ITGA6-inhibitory antibody was added to organoid cultures as 1 :1000 diluted ascites). The Fab versions of SNAKA51 , 9EG7, and 12G10 antibodies were kindly provided by Jing, Li, Harvard Medical School, Boston Children’s Hospital, US). Culture images were taken with an EVOS cell imaging microscope. Organoid media

[0152] Table 1

[0153] Organoid lines

[0154]

[0140] P26N colon organoids were previously described (Drost, J. et al. Sequential cancer mutations in cultured human intestinal stem cells. Nature 521 , 43-47 (2015)).

[0155]

[0141] Human ileum organoids (Pleguezuelos-Manzano, C. et al. Establishment and Culture of Human Intestinal Organoids Derived from Adult Stem Cells. Curr Protoc Immunol 130, e106 (2020)).

[0156]

[0142] Established human liver- and pancreas ductal lines were described earlier (Broutier, L. et al. Culture and establishment of self-renewing human and mouse adult liver and pancreas 3D organoids and their genetic manipulation. Nat Protoc 11 , 1724-1743 (2016)). New lines from primary biopsies were processed as described (Pleguezuelos-Manzano, C. et al., supra).

[0157] Adhesion assay organoids

[0158]

[0143] Organoids were removed from BME® and dissociated into single cells using TrypLE at 37 °C for 15 min, with repeated mechanical shearing. Single cells were washed and resuspended in DMEM / F12 medium. Coating of wells of a 96-well plates (Greiner), was performed by O / N incubation at 4°C with PBS dilutions of either, 20pg / ml PureCol EZgel® (Advanced Biometrix), 1 :300 dilution of BME®. Subsequently, coated wells were blocked with 2% BSA in PBS for 30 min. at RT. Equal amounts of cells were incubated for 1,5 h at 37 °C to allow cell attachment. After washing twice with DMEM / F12 medium, the number of adherent cells were quantified using CellTiterGlo (Promega).

[0159] ITGA2 CRISPR-Cas9 base editing

[0160]

[0144] sgRNA plasmid preperation:

[0161]

[0145] The following sequence was ordered containing a universal priming part and the sgRNA (underlined): GTTGTGTAATACTGATTCCCCGGTGTTTCGTCCTTTCCACAAG (SEQ ID NO: 18}. The additional bold cytosine residue is included to obtain sufficient transcription from the U6 promoter. Side-directed mutagenesis in SpCas9-vector #47511 was done using Q5 high- fidelity polymerase with mentioned primer and the following universal 57phos / - GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC primer (SEQ ID NO: 19). Upon PCR cleanup, T4-ligase and Dpn1 were used to relegate and generate the plasmid, this was done according to manufactures protocol (NEB). DNA was transformed into DH5alpha bacterial cells, and plasmids were verified using sanger sequencing (Macrogen) with sequence primer GGGCAGGAAGAGGGCCTAT (SEQ ID NO: 20).

[0162] Organoid electroporation

[0163]

[0146] Organoid electroporation was performed as described previously60with some modifications. Wild-type organoids were maintained in expansion medium. Two days prior to electroporation 1 .25% (vol / vol) DMSO was added to the organoid medium. Electroporation was done with 10A6 cells per electroporation with BTXpress solution containing 2.5 ug ITGA2 sgRNA plasmid, 5 ug Piggybac transposase, 5 ug Piggybac hygromycin cassette, 7.5 ug C>T Base editor_eGFP plasmid (Addgene plasmid #112100). Five days post transfection, hygromycin 100 ug / ul (Invivogen) selection was started. Fourteen days after selection, surviving-hygromycin selection clones were individually picked and sequenced with the following primers: ITGA2_fwd GCACCTGCCACCTTCTCCATCA (SEQ ID NO: 21); ITGA2_rev GAAAAGAGGAAAGAAGCAGATT (SEQ ID NO: 22). DNA was obtained using ZYMO Research DNA isolation kit. During clonal outgrowth and selection scTS2 / 16 was always present in the media.

[0164] Western blot

[0165]

[0147] Proteins were run on a 4-15% PAA gel (Biorad) under reducing / non-reducing conditions. Marker proteins (Thermo Scientific #26616, Pager ruler). SDS-PAA gel was transferred to PVDF membrane (Thermo Scientific ref 88518). Transfer efficiency was controlled with ponceau staining. For blocking 5% non-fat milk protein in PBS was used. For direct HIS-tagged protein staining a mouse anti-HIS tag HRP conjugated antibody (R&Dsystems, MAB050H) was used. Indirect staining for the HIS-tag was performed using anti-HIS (Abeam ref 18184) in combination with anti-mouse-HRP (Life Technologies ref A11029). FLAG-tagged proteins were directly visualized using HRP-conjugated M2 anti-FLAG (A8592 Sigma-Aldrich), or indirectly by combining M2 anti-flag (A2220 Sigma) and anti-mouse- HRP (Life Technologies ref A11029) . Chemiluminescent substrate ECL (GE-Healthcare). Table 2. Sequences used in this description

[0166] Example 6. A single-chain derivative of the integrin (31 -activating TS2 / 16 antibody potentiates organoid growth in Matrigel and Collagen hydrogels.

[0167]

[0148] In Example 6, aspects of the experiments described above are again presented, while adding further details and experimentation in addition to the information shown in Examples 1

[0168] - 5 above.

[0169]

[0149] In 2009, a 3D technique to culture adult stem cell-derived, intestinal epithelial tissues was introduced1(references for Example 6 are listed by the end of the Example). These so- called organoids are self-organizing, miniature versions of the intestinal epithelium that can be expanded over long time periods. Since then, culture protocols were published for the isolation of organoids representing epithelial tissues derived from all three germ layers2.

[0150] One essential element of these culture conditions is the growth factor cocktail, which typically has to be optimized for each individual tissue, yet always contains three key ingredients: potent activators of the Wnt pathway (e.g. Wnt3Aor its surrogate3, plus R- spondins4), a tyrosine kinase receptor activator (e.g. EGF) and inhibitors of TGF- and / or BMP (e.g. Noggin)5.

[0170]

[0151] A second critical component for proper 3D organoid formation concerns the interaction of cells with the basement membrane (BM)6. It includes a complex mixture of extracellular matrix (ECM) proteins7, including laminins (LN’s), collagen type-IV and to a lesser degree RGD-motif containing molecules like fibronectin8. LN’s are heterotrimeric proteins assembled from one of five a chains, one of three p, and one of three y chains. In higher organisms these isoforms display varying degrees of cell / tissue specificity9. Collagen IV is a heterotrimeric molecule composed of three a-type chains, which forms polygonal networks through several types of intermolecular interactions10. Both, collagen type IV and laminins are produced by epithelial cells and form independent networks that are connected by nidogen and the heparan sulphate proteoglycan perlecan7.

[0171]

[0152] The embryonic Engelbreth-Holm-Swamn (EHS) tumor produces large amounts of ECM proteins, from which a BM-like hydrogel can be reconstituted11'12 13. Experiments with embryoid bodies revealed a critical role of ai iy1 Ln (Ln-111 ), the major component of the BM-like hydrogel, in the polarization and differentiation of epithelial cells14. Ln-111 in EHS- tumor derived matrices is also responsible for establishing cell polarity and milk production by primary mouse mammary epithelial cells15.

[0172]

[0153] Interaction of epithelial cells with the ECM is mediated by members of the pi -integrin family of adhesion receptors16. These integrins tightly regulate their affinity for ECM ligands through dynamically changing their conformation. Integrin / ECM interaction-induced signaling by epithelial cells ensures not only correct basal-apical polarity, but also plays a critical role in regulating cell migration and in safeguarding cells against anoikis16-2°.

[0173]

[0154] Matrigel®and BME® are trade names for the reconstituted basement membrane matrix derived from Engelbreth-Holm-Swamn (EHS) tumors grown in mice. While these hydrogels have become the universal ECM source for organoids, they have limitations in their applicability21. With nearly 2,000 protein identities, it is not well-defined22. Furthermore, they have the limitation of strict ethical issues and high production costs. Its animal origin has so far prohibited the production of clinical-grade ECM. Various biomaterial-based approaches have been explored to optimize ECM properties that aim to lead to translationally relevant organoid models23.

[0174]

[0155] Here we investigated the role of pi integrins by applying -specific scFv antibodies to a large variety of gastrointestinal organoids. Treatment with pi inhibitory mAb AIIB224resulted in an immediate loss of adherence to laminin-, and collagen types of ECM ligands. The functional importance of the p1 integrins was further demonstrated by a reversal of apical-basal polarity and accompanied growth arrest. Probing the utility of activating pi integrin single chain antibody (scTS2 / 16) produced in this study - reinforced the adhesion and associated growth of all gastro-intestinal type organoids in BME. Adult epithelial tissues mainly contain LN-511 / 521 in their BM. Their integrins show a preference for binding to these isoforms over the EHS / BME-related Ln-11125’26. The authors contemplate that scTS2 / 16 may compensate for this limitation in BME-grown organoids. In line with this, we found a clear scTS2 / 16-mediated potentiation of adhesion and growth potential in all organoid models in contact with collagen type-l.

[0175] Results

[0176] Integrin p1 is indispensable for organoid growth.

[0177]

[0156] Adult stem cell-derived intestinal organoids, exhibiting a basal-OUT phenotype in the presence of external Matrigel®, invert their polarity to basal-IN when cultured in the absence of Matrigel®28. We intended to test the role of integrin pi , dominantly expressed in various organoid models, on polarity and associated physiology. The rat anti-integrin pi antibody AIIB2 has previously been shown to allosterically block the recognition of pi integrins for all three major integrin ligand classes (laminin, collagen and RGD-class proteins)24. This antibody, therefore, enabled us to comprehensively test the role of the pi integrin component in basal-apical polarization of diverse organoid types. Fig.1 (b-f) discussed above summarizes the dramatic effects of adding the AIIB2 antibody to human colon organoids. The “basal-OUT” phenotype of these organoids, cultured in the presence of BME®, inverted within 12 hours to basal-IN upon exposure to AIIB2 (Fig. 1 b-c). This condition resulted in an immediate abrogation of growth, measured as a strong decrease in the percentage of cells expressing the nuclear proliferation marker Ki67 (Fig.1 d), in visual growth kinetics (Fig.1 e) and in a quantitative cellular ATP assay (Fig.1 f). These observations implied that integrin- mediated signals normally maintain growth and are absent in the presence of AIIB2. Further confirmation for cell cycle exit in response to AIIB2, was seen in a time-series experiment analyzing a human ileal-organoid line stably expressing the FUCCI-reporter. A qPCR strongly indicated that these non-cycling organoid cells differentiate to enterocytes and goblet cells. scFv versions of TS2 / 16 activate cell adhesiveness of integrin p1

[0178]

[0157] We next examined the ability of an activating antibody to functionally activate the integrins at the epithelial-organoid cell surface16. A rapid switch from an inactive to an active adhesive state is essential for leukocytes and platelets. Early studies reported conformational changes for the P229'31and P332'33integrins, representing a shift from a low to a higher adhesive state of such cell types. Leukocytes, in addition, express several pi integrins34, which are involved in leukocyte binding to ECM components as well as to other cells. The a2 i , a5 i , and a6 i integrins are prototype adhesion receptors for the ECM proteins collagen type-l, fibronectin, and laminin, respectively. The a4 i integrin is expressed on leukocytes and binds to the VCAM-1 protein, expressed on the membranes of endothelial cells35. The human integrin pi -specific TS2 / 1636antibody promotes a strong enhancement of adhesion by o2 i, a5pi and o6 i integrins with their cognate ECM ligands, and of a4 i with endothelial VCAM protein35. A similar change of cell-substrate adhesiveness by TS2 / 16 was also demonstrated for non-hematopoietic cells. Neuronal cells in defined stages of developing chicken embryos were induced to bind to laminin in vivo in the presence of the activating integrin pi antibody TASC37.

[0179]

[0158] The non-constitutive nature of integrin interactions has promoted the concept that the binding of integrins to their cognate ligands is coupled to extensive structural changes. This was first demonstrated for the aV 3 and al lb|33 integrins38. Electron microscopy (EM) studies subsequently revealed that the pi integrins undergo the same conformational shapes as reported for the 3 integrins39. Thus, integrins exist in an ensemble of three conformational states38: two low affinity states, a bent-closed and extended-closed conformation, and an extended conformation with the ligand binding domain (headpiece) open, which is associated with a higher affinity state. Various integrin pi subunit-specific antibodies, by inducing- or stabilizing discrete conformational states, have allowed measurement of the affinities of the conformational states. The extended headpiece-open conformation of a5pi integrins displays a 5,000-fold higher affinity compared to extended-closed or bent-closed conformations40. Affinity changes for the a401 integrin appeared smaller (600 - 800-fold)41. The activating antibody 9EG742stabilizes the extended conformations )41: its binding to a5p1 leads to a suboptimal affinity compared to antibody 12G1043. This latter antibody binds to the headpiece and stabilizes the headpiece-open conformation. Antibody TS2 / 16 reaches the same level of affinity as mAb 12G1040but, unlike 12G10, does not induce a conformational change in the absence of ligand44. Thus, TS2 / 16 binds to both closed39headpieces in the absence of ligand39, and stabilizes an open headpiece conformation in the presence of ligand44.

[0180]

[0159] We decided to construct a humanized IgG version and single-chain (sc) derivative45of the mouse TS2 / 16 antibody, as well as a single-chain version for ease of production in bacteria (Fig. 2a). We determined the hybridoma-derived sequences of the variable regions of the H-, and L-chain of the mouse Ab TS2 / 1636and used human codon-optimized synthetic DNAto clone these into huigGI H-, and i^L-chain vectors46. The humanized antibody was subsequently purified using standard chromatography purification (Fig 2b). For the sc- version, the combined V regions of H-, and L-chain were linked in two orientations (Fig. 2a) and connected via a 3x(GGGGS) linker. Both protein versions were cloned into a pET expression vector using E.coli codon-optimized DNA. To obtain pure sc-versions, a routine protocol for protein extraction, solubilization and refolding was implemented44'47. On average, we obtained 1 mg pure scTS2 / 16 per 50 ml of bacterial suspension, sufficient for approximately 7 liters of cell culture medium at a final concentration of 5 nM. SDS-PAGE gel analysis for the synthetically produced huigGI and the scFv version of TS2 / 16 (VH-VL), are shown in Fig.2 b. We also developed a protocol to produce the scTS2 / 16 in the human cell line HEK293, a popular line for production of biotherapeutics.

[0181]

[0160] The scTS2 / 16 (VH-VL) bound the ectodomain of the 1 subunit in solution, analogous to its parental IgG version Functionally, both huigGI and scFv versions (VH-VL) induced adhesion at low nM concentrations of K562 (erythroleukemia) cells towards fibronectin, a standard assay for integrin-mediated adhesion48(Fig. 2c-d). The E.Coli- and HEK293 produced versions of the (VH-VL) scFv performed equally well in this assay (data not shown). The scTS2 / 16 with VL-VH orientation was found to be functionally equivalent to the scTS2 / 16 with VH-VL orientation. Based on this outcome, we decided to focus on the scFv (VH-VL) version of TS2 / 16 (scTS2 / 16) for subsequent studies in this Example. scFv-TS2 / 16 enhances growth of BME® -suspended intestinal organoids.

[0161] One of the main components of BME® is the protein LN-111 . Four different integrins, a3 1 , a6 1 , a7pi , and a604 differentially bind to members of the laminin family of proteins25. Integrin a6pi50’51binds to laminin-111 , the embryonic laminin in EHS / BME®. The organoid- expressed o3pi and a6p4 integrins, bind to laminin-332 and laminin-511 , but are absent in EHS / BME®. The specific role of a6pi integrin in laminin-111 binding was confirmed by exogenous expression of integrin a6pi in K562 cells. The newly-acquired LN-111 recognition was evident, but certainly not maximal.

[0182]

[0162] We tested whether this phenomenon would translate to adhesion of colon organoid cells towards coated BME and could be enhanced. We used a series of concentrations of the TS2 / 16 scFv in the low nM range. The observed strong increase of adhesion reached its maximum at 2 nM (Fig. 3a). Of note, the AIIB2 blocking antibody inhibited adhesion of organoid cells, as a validation of this adhesion assay. Since BME contains both LN-111 and collagen IV, we tested the contributions of these components independently in organoid adhesion. Again, we detected a maximum enhancement effect of scTS2 / 16 at 2 nM for the individual components. The inhibitory AIIB2 ab completely inhibited uninduced adhesion with respect to all three coats (Fig. 6).

[0183]

[0163] To assess if the integrin a6 subunit was also crucial in mediating growth of colon organoids in BME®, we added the inhibitory integrin a6-specific GoH3 antibody53while regularly splitting colon organoid cultures. The results shown in Fig.3b indicated a strong GoH3-induced reduction of growth. We then tested whether activation of the pi integrins could enhance (in addition to adhesion) the growth of colonic organoids. Addition of scTS2 / 16 to standard 3D colon organoid culture in BME® at 5 nM led to increased colon organoid growth. This was observed after 10 days of culture by assessment of total genomic DNA, of ATP-dependent luminescent light units (Fig. 3c), and of phase contrast images of the organoid cultures (Fig. 3d). In agreement, we observed an increased percentage of KI67+nuclei.

[0184]

[0164] Single cell cloning from primary tissue or from organoids is generally highly inefficient1 54. We tested the effect of scTS2 / 16 on plating efficiency of such single cells and noted that the clonal outgrowth in the presence of scFv TS2 / 16 (at 5 nM) improved dramatically (Fig. 3e-f).

[0165] Of note, the positive effects of integrin 31 -stimulatory antibodies in the outgrowth of organoids from single cells were much less strong for the two other 01 -chain conformationstabilizing antibodies, 9EG749and 12G1043(Figure 7).

[0185]

[0166] Thus, while the scTS2 / 16-stabilized high affinity conformation of Pi integrins on leukocytes led to enhanced intercellular-, or cell-to-ECM adhesion27’35 39’52’55, it (as scFv- version) also improves establishment and expansion of epithelial organoids by enhancement of a6p1 -mediated interaction with laminin-111 / collagen-IV as presented in BME®.

[0186] BME® -established organoids from other parts of the gastrointestinal tract also rely on integrin p1

[0187]

[0167] We then tested whether the observed integrin 01 -regulated proliferation of intestinal organoids in BME®, also held for other parts of the human gastrointestinal tract, i.e. small intestinal sections duodenum, jejunum, and ileum, stomach organoids56, and ductal-type organoids from pancreas57and liver58. The pertinent organoids were grown in BME® supplemented with control medium, or with 5nM of either the inhibitory mAb AIIB2 or the stimulating scTS2 / 16 antibody. AIIB2-induced inhibition and scTS2 / 16-enhancement of growth (as compared to medium without antibody) were observed in all cases (Fig. 4; data for jejunum not shown). To determine that scTS2 / 16-stimulated growth did not cause a major shifts in the transcriptome of the cells, bulk-sequence experiments were performed on control and antibody stimulated 3D BME cultures of colon, duodenum, and jejunum organoids. The data revealed no significant changes. We concluded that growth of gastrointestinal tract organoids depends on 01 -class integrins and can be safely strengthened by addition of scTS2 / 16 to their medium. scTS2 / 16 antibody potentiates growth of established- and primary isolated colon organoids in collagen-l hydrogel.

[0188]

[0168] Based on the scFv-generated improvement of gastrointestinal organoids in BME®, we tested the hypothesis that organoid growth in an alternative hydrogel would also benefit from the addition of scTS2 / 16. Collagens make up a supra-family of ECM proteins possessing a distinct triple-helical region formed from three polypeptide chains59 60. We chose for Col-I (a1 + a2) , since it is predominant in most body tissues, is inexpensive, and has therefore been extensively explored for in vivo applications (e.g. for tissue engineering and cosmetic applications)61.

[0169] In a first attempt to enhance growth by scTS2 / 16 in a collagen type-l hydrogel, we focused on colon organoids. We tested several classical acidic solutions of collagen type-l, which require neutralization and temperature elevation to initiate hydrogel formation (data not shown). Ultimately, a commercially available pre-formulated isotonic form of collagen-l with neutral pH, that forms a 3D hydrogel by simply warming to 37°C, proved to be the most successful preparationWe confirmed expression of a2 1 in BME®-grown colon organoids, both at the RNA level and by immunofluorescent staining with an antibody (Fig. 5a). In total, 4 a-integrin subunits (a1, -2, -10, and 11) harbor a collagen-binding l-domain insert and share a 1 -subunit. Of these, the colon organoids exclusively expressed the a2 subunit. The scTS2 / 16 amplified adhesion of organoid cells towards collagen-l (Fig.5b). In contrast, the inhibitory AIIB2 antibody blocked such adhesion (Fig. 5b).

[0189]

[0170] These potent effects of the two antibodies were also reflected in the growth of established colon organoids in the collagen-l hydrogel (Figure 8). We also tested pi- mediated growth of freshly isolated colon biopsies in collagen-l hydrogel. Colon tissue from four donors was enzymatically processed and epithelial cell suspensions were embedded in collagen-l in the presence or absence of scTS2 / 16 (5 nM). Culturing in scTS2 / 16-containing medium yielded significantly more organoids as compared to standard colon medium (Fig. 5f).

[0190]

[0171] We then created null-alleles of the gene for the integrin a2 subunit by CRISPR to confirm the relevance of the collagen-recognizing (a2 i) integrin for colon organoid growth in collagen hydrogels. As predicted, ITGA2 gene mutation blocked organoid growth in collagen- I, even in the presence of scTS2 / 16. Although BME® enabled isolation of the ITGA2- / -, growth was reduced compared to the parental organoids. It indicates that BME® growth of organoids is partly controlled by the endogenous collagen-IV.

[0191]

[0172] Like colon, when a2-expressing liver bile duct organoids were stimulated by scTS2 / 16, increased adhesion towards collagen-l ensued (Fig. 9), mirrored by scTS2 / 16- enhanced growth of cholangiocyte organoids (Fig. 10). Guided by these results, we tested the outgrowth of cholangiocyte organoids in collagen-l hydrogels, directly from primary liver biopsies. This consistently yielded more organoids in scTS2 / 16-medium as compared to control medium (Fig. 11). These proved to be Epcamhi9h, as expected for this cell type62.

[0173] Based on the principles established in the 3D cultures, we tested whether collagen- based culturing organoids in 2D would be enhanced by scTS2 / 16, similarly. Various protocols for such cultures have been described previously. In one protocol intestinal cells are cultured on a monolayer of myofibroblasts63. In a second it concerns mouse intestinal organoids64. Protocol three tests only short term human organoid growth65, or applies a more complex collagen sandwich technique in combination with the use of small molecules (CHIR and Valproic acid) to maintain growth66. Here we tested scTS2 / 16 supported growth of duodenum and jejunum organoids in transwells. These were coated with the same collagen used in 3D. To make a comparison the growth, under otherwise identical conditions, was compared with that in BME-coated inserts. These side-by-side experiments are shown in Figure 12, 13 and 14. Herein, duodenum cultures showed an improvement in response to scTS2 / 16, both on BME (amplification ± 2) and collagen (amplification ± 3). For the jejunum line, these amplification factors amounted to ± 7.5 and ± 4.75, respectively. Similarly to the 3D cultures, we tested stability of the transcriptomes of these 2D cultures in the presence of scTS2 / 16. Extensive bulk RNA sequencing clearly justifies the use of scTS2 / 16 for improvement of in vitro intestinal cultures.

[0192]

[0174] Encouraged by the positive results we observed in the 2D cultures up to that point, we then tested whether these effects would be maintained in long term cultures. We started two types of experiments. In the first we cultured Duodenum organoids in collagen-coated transwell inserts in the presence, and absence, of scTS2 / 16. We passaged both types of culture four times, restarting with a fixed number of single cells. The experiment was quantified by scoring the number of cells in inserts (duplicate values) at the end of each passage. By extrapolating these values over four passages we found that approx. 15-times more tissue was produced in the presence of scTS2 / 16. For the second experiment we, cultured jejunum organoids in BME-, and collagen-coated inserts for 6 passages in the presence of scTS2 / 16. We then tested the competency of these cultures to differentiate to the major specialized intestinal cell-types5. Cells grown on BME showed the expected tendency to differentiate towards the major specialized intestinal cell types. Reassuringly, the collagen- grown cells performed at least as well.

[0193] Discussion

[0175] Here we show the functional importance of integrin [31 in epithelial organoid cultures derived from human small intestine, colon, stomach, pancreas, and liver. Thus, we observe potent growth-blocking effects of the inhibitory integrin |31 subunit-specific antibody AIIB2, while the activating integrin |31 subunit antibody scTS2 / 16 enhances growth. We show that this agonistic single chain antibody binds the human [31 subunit with high affinity. Moreover, scTS2 / 16 proves to be highly potent in supporting establishment and growth of organoids in the ‘standard’ BME® hydrogel, simply by addition to the culture medium as a soluble monomer. We observe a crucial role for the integrin a6 component in BME®-dependent growth. We currently believe that organoid growth in BME results from the parallel interaction of a2[31 with collagen-IV, and a6[31 with laminin-111 , both natural ECM components present in BME®. We also find that addition of the single chain TS2 / 16 supports the growth of organoids in hydrogels of purified collagen-l. CRISPR-mediated gene knockout demonstrated a2[31 to be responsible for "collagen only”-supported organoid growth. Our 2D protocol clearly showed that adding the scTS2 / 16 enables potent, and long-term, cultivation of intestinal tissue on collagen. The versatility of scTS2 / 16 arises most likely from the fact that the integrin a chains for the ECM proteins laminin and collagen share the [31 signal transduction chain. The highly proliferative tissue generated under these conditions remains competent to differentiate to cells participating in the absorption of nutrients (enterocytes), production of mucus (goblet cells), or as producers of hormones (enteroendocrine - EE Cells).

[0194]

[0176] The observation that the activation of [31 integrins by single-chain TS2 / 16 in leukocytes extends to epithelial organoid cells opens a range of applications. We believe that the availability of this single chain reagent which allows organoids to grow in "collagen only”- hydrogels may avoid the need for costly BME®(Matrigel®). Moreover, both production in E.Coli and in the HEK293 line for biotherapeutics, allows the generation of clinical grade batches of the single chain reagent. Clinical grade collagen preparations are already widely available for tissue repair61and cosmetic applications67. Mouse and human colon organoids have been shown to be transplantable in mice68-70. The use of the single chain integrin reagent would make expansion of transplantable organoids faster and more cost-efficient. Moreover, a scTS2 / 16-induced high affinity state of the transplanted organoid cells may instruct these to engraft more efficiently. Of note, the first clinical trial using colonic organoid transplantation therapy for intractable cases of ulcerative colitis is currently ongoing71.

[0195] Material and Methods

[0196]

[0177] Immunofluorescent analysis of apical / basal polarity and proliferation marker KI-67.

[0197]

[0178] P26N human colon organoids were grown for 2 days in BME® + / - the rat anti-ITGB1 antibody AIIB2 (DSHB Cat# aiib2, RRID:AB_528306) at a concentration of 1 ug / ml.

[0198] Organoids were harvested using Cell recovery solution (ThermoFischer) and fixed in 4% formaldehyde (pH 7.4) at 4°C for 1 hour. Hereafter, organoids were washed with PBS 0.1% Tween (PBST)(2% BSA). 3D imaging of organoids was performed essentially as described previously72. In short, organoids were incubated O / N at 4°C with primary antibodies binding ZO-I (Thermo Fisher Scientific Cat# 61-7300, RRID:AB_2533938), ITGB1 (R and D Systems Cat# MAB1778, RRID:AB_357909), and Ki67 (Millipore Cat#AB9260, RRID:AB_2142366) or ITGA2 (Abeam Cat# ab30487, RRID:AB_775702). Upon extensive washing with PBST, binding of primary reagents was visualized by O / N incubation in PBST / 2%BSA containing Donkey anti-rabbit Alexa 568 (Thermo Fisher Scientific Cat# A10042, RRID:AB_2534017) and Goat anti mouse Alexa488 (Thermo Fisher Scientific Cat#A-11070, RRID:AB_2534114). Phalloidin Atto 747N (Sigma-Aldrich) was used for direct visualization of F-actin. DNA was stained with DAPI (Thermofischer). Organoids were optically cleared in a glycerol-fructose clearing solution prior to imaging. Organoids were imaged on a (microscope, objective, etc.). Analysis was done with Imaged software.

[0199]

[0179] TS2 / 16 hlgG 1 production

[0200]

[0180] We used a simplified workflow to sequence the variable regions of the mouse TS2 / 16 hybridoma (ATCC HB-243) as described73. A human codon-optimized coding sequence of the TS2 / 16 VH and VL domains were synthesized by GeneArt (ThermoFischer) and cloned into pFUSEss-CHIg-hG1 and pFUSE2ss-CLIg-hK, respectively (Invivogen). Expression vectors were co-transfected into Expi293F™ human suspension cells using ExpiFectamine™ (Thermofischer). IgG protein was purified by affinity chromatography using CaptivA® Protein A resin (Repligen), followed by size-exclusion chromatography using a Superdex 200 Increase 10 / 300 GL column (Cytiva).

[0201]

[0181] TS2 / 16 scFv production in E. coli

[0182] An E.coli codon-optimized coding sequences of the TS2 / 16 scFvs (see supplementary 3b) were synthesized by GeneArt (ThermoFischer) and cloned into a pET21 vector harboring a C-terminal 6x-HIS tag (Novagen) using standard molecular biology techniques. Protein expression induction, using 1mM IPTG (Sigma), was done in BL21 (DE3) cells starting with a OD600optical density of 0,6 in standard LB medium. Production was at 28 °C for approx. 18 hrs. Bacterial pellets obtained were resuspended in lysis buffer (50 mM Tris pH 7.5, 200 mM NaCI, 10% glycerol, 0.4% Triton x100) and then sonicated (5 cycles; 10 sec ON, 20 sec OFF). The insoluble fraction, obtained by centrifugation at 11.000 rpm for 15 min., was Guanidine-Hydrochloride-solubilized and refolded as described previously47. The partially purified, refolded, scFv was further purified using Ni-NTA (Qiagen), followed by size- exlusion chromatography using a Superdex S200 increase 10 / 300 GL size-exclusion column (Cytiva) in HBS (10 mM HEPES, pH 7.2, 150 nM NaCI). Analysis of the quality of protein purification was performed running samples, under reducing -, and non-reducing conditions, on a 4-15% TGX precast SDS-PAGE (Biorad) gel. Proteins were visualized using InstantBlue Coomassie Protein stain (Abeam).

[0202]

[0183] scFv TS2 / 16 production in Expi293 cells.

[0203]

[0184] The coding sequence of the TS2 / 16 scFv (VH-VL orientation) containing the artificial human kappa L-chain signal sequence, and C-terminal tags (8xHis tag, HRV 3C site (LEVLFQ / GP), Fc-tag (huigGI), and a stop codon was cloned into a pcDNA3.1 mammalian expression vector (Supplementary Figure 7). Expression was performed by transient transfection of Expi293 cells using the ExpiFectamineTM transfection kit (Gibco™) according to the manufacturer's instruction. After 4 days, the TS2 / 16 scFv-Fc was captured from the conditioned media with CaptivA protein A affinity resin (Repligen, CA-PRI-0100) and eluted with IgG elution buffer (Thermo Scientific) containing 300 mM NaCI and neutralized with 100 mM Tris pH 8.0 to reach eventually pH 7.0. The sample buffer was exchanged to 1xHBS (20 mM Hepes pH7.3, 300 mM NaCI) using Amicon centrifugal filters (MWCO 30KDa) and treated with PreScissionTM protease (Cytiva) overnight at 4°C. Next, the Fc-tag was captured with CaptivA protein A affinity resin and the PreSissionTM protease with Glutathione Sepharose 4C (Cytiva). Finally, the TS2 / 16 scFv was purified on a size exclusion chromatography (SEC) with SuperdexTM 75 Increase 10 / 300 GL column (Cytivia) on the AKTA pureTM chromatography system (Cytivia) in 1xHBS running buffer (20 mM HEPES pH 7.3, 300 mM NaCI). The TS2 / 16 scFv was then concentrated using Amicon centrifugal filters (Millipore, MWCO 10KDa) and sterilized by filtration over a 0.22 pm centrifugal filter (Merck). Protein concentration was determined via NanoDrop A280 reading using the respective protein molecular weight and extinction coefficient.

[0204]

[0185] Integrin @1 ectodomain production and Immunoprecipitation.

[0205]

[0186] We used the procedure reported by Takagi et al. 2002 to recombinantly produce the extracellular domain of pi subunit in the absence of an a-subunit. integrin. Deletion of the ~30 amino acids specific-determining loop (SDL) in the l-like domain is essential for stable secretion of this ectodomain. The DNA coding sequence was synthesized by GeneArt (ThermoFischer) and cloned into vector pcDNA3.1 (Addgene). Expression vector was transfected into Expi293F™ human suspension cells using ExpiFectamine™ (Thermofischer). Purification of the C-terminally Flag epitope tagged was performed by absorption of secreted protein to M2-agarose beads (Sigma), and elution by excess 3xFlag peptides (Sigma). Eluted proteins were subsequently subjected to size-exclusion chromatography using a Superdex 200 Increase 10 / 300 GL column (Cytiva). For immunoprecipitation experiments of the pi ectodomain we used ProteinG (Sigma) or M2- agarose (Sigma). All washes and antibody incubations were in standard PBS. The integrin P4 antibody (Abeam Cat# ab110167, RRID:AB_10866385), the pi Ab (R and D Systems Cat# MAB1778, RRID:AB_357909), and HUTS4 (Millipore Cat# MAB2079Z, RRID:AB_11214024), M2 anti-Flag (Sigma-Aldrich Cat# F3165, RRID:AB_259529), anti-Flag HRP (Abeam Cat# ab49763, RRID:AB_869428), anti-6xHIS HRP (R and D Systems Cat# MAB050H, RRID:AB_357354). We used 4-15% SDS-PAGE gel (Biorad) under reducing conditions, and PVDF membrane (Immobilon) for blotting. ECL (GE-Healthcare).

[0206]

[0187] Adhesion assays K562 cells

[0207]

[0188] Erythroleukemia cell line K562 cells (ThermoFischer Scientific) were cultured in DMEM (Gibco™) with 100 U / ml Penicillin-Streptomycin and 5% FBS (Sigma-Aldrich). Bovine Fibronectin (ThermoFischer) was diluted to 20 ug / ml in PBS and incubated o / n at 4°C in a flat-bottom 96-well plate (Greiner). Subsequently, wells were blocked with 2% BSA in PBS for 30 min. at RT. K562 cells were resuspended in DMEM / F12 medium. Wells were loaded with equal amounts of cells, in the absence of antibody, or presence of 5 nM of TS2 / 16 in scFv or IgG format. Upon incubation of cells for 90 minutes at 37°C, unbound cells were removed by 3 washes with DMEM-F12 advanced medium. Number of adherent cells was analyzed by brightfield microscopic images, and a luciferase-based ATP assay (CellTiter-Glo®: Promega).

[0208]

[0189] BM8and PureCol EZgeP -based 3D organoid culturing.

[0209]

[0190] Organoids grown in BME® (Cultrex) were passaged by releasing them first from the matrix by incubation for 20 min on ice in Cell recovery solution (ThermoFischer) supplemented with 10 pM of the Rho-kinase inhibitor Y-27632 (Abmole). Subsequent DMEM / F12-washed organoids were either mechanically sheared, or TrypLE™-digested to single cells. Upon additional washing with DMEM / F12, organoids were either resuspended in fresh BME® or, alternatively, in PureCol EZgel® (Advanced Biometrix ). For complete gelation of the BME®-. or PureCol EZgel® gel, plates were incubated at 37°C for 30 or 60 minutes, respectively. Hydrogels were covered with relevant organoid medium and incubated at 37°C and 5% CO2. Passaging of organoids growing in Collagen, was achieved by incubation at 37 °C for 30 min maximally, with Collagenase from Clostridium histolyticum (Sigma-Aldrich C0130) diluted in DMEM / F12 to a concentration of 1 mg / ml. For complete removal of the proteolytic enzyme, organoids were washed twice with an excessive volume of DMEM / F12 medium before re-entry into PureCol EZgel®. The 1 -specific inhibitory AIIB2 antibody was added to organoid cultures at 30 nM. The ITGA6-inhibitory antibody G0H3, was added to organoid cultures as 1 :1000 diluted ascites. The Fab versions of SNAKA51 , 9EG7, and 12G10 antibodies were kindly provided by Jing, Li, Harvard Medical School, Boston Children’s Hospital, US). Culture images were taken with an EVOS cell imaging microscope.

[0210]

[0191] BME® and PureCol EZgel® -based 2D organoid culturing.

[0211]

[0192] Transwell inserts (Thincerts™ ,12-well, pore size 0,4pm, Greiner bio-one 665641) were coated O / N with 2% dilutions of BME® or PureCol EZgel® in DMEM-F12 medium at 4 °C. Hereafter, inserts were washed twice with DMEM-F12 before adding cells. Inserts were loaded with approximately ± 50,000 cells, prepared as a TrypLE™ -dissociated single cell suspension. Upon digestion cells were 40pm-filtered and washed twice in DMEM-F12 medium in the continuous presence of Rho-kinase inhibitor Y-27632 (Abmole). Cells were resuspended in 250 pl relevant culture medium supplemented with Y-27632 (O / N) and optionally long term with 5 nM scTS2 / 16. One ml of identical medium was added to the lower compartment. Passaging of the cultures was started by removing media from both compartments, briefly wash with DMEM-F12, and then filling both compartments with TrypLE™.

[0212]

[0193] Organoid media

[0194] Organoid differentiation media

[0195] ENR medium = Intestinal medium lacking Nicotinamide, p38 inhibitor, A83-01 inhibitor, PGE2, NGS Wnt-Surrogate, Rspondin-3 (but has Rspondin-1). ER medium = ENR medium minus Noggin5.

[0213]

[0196] Organoid lines

[0214]

[0197] The human duodenum organoid line used (donor 4: NKI3) were obtained as normal adjacent tissue from tumor resections. The patients' informed consents were obtained, and the study was approved by the ethics committee of the NKI IRB with code CFMPB582. This study is compliant with all relevant ethical regulations regarding research involving human participants.

[0215]

[0198] Jejunum organoids (organoids, derived from jejunal biopsy, obtained from Prof M. Estes, Baylor College of Medicine, Houston, USA)

[0216]

[0199] ileum organoids were decribed5.

[0217]

[0200] P26N colon organoids were described74

[0218]

[0201] Established human liver- and pancreas ductal lines were described earlier75. New lines from primary biopsies were processed as described5.

[0219]

[0202] Adhesion assay organoids

[0220]

[0203] Organoids were removed from BME® and dissociated into single cells using TrypLE at 37 °C for 15 min, with repeated mechanical shearing. Single cells were washed and resuspended in DMEM / F12 medium. Coating of wells of a 96-well plates (Greiner), was performed by O / N incubation at 4°C with PBS dilutions to 20 g / ml for PureCol EZgel® (Advanced Biometrix), or a 1 :300 dilution of BME®. In one type of experiment coating with BME was compared to collagen-IV (mouse collagen-IV, Biotechne 3410-010-02) and EHS- purified LN-1 (Biotechne 3446-005-01). Subsequently, coated wells were in all cases blocked with 2% BSA in PBS for 30 min. at RT. Equal amounts of cells were incubated for 1 ,5 h at 37

[0221] °C to allow cell attachment. After washing twice with DMEM / F12 medium, the number of adherent cells were quantified using CellTiter-Glo® (Promega).

[0222]

[0204] ITGA2 CRISPR-Cas9 base editing

[0223]

[0205] sqRNA plasmid preparation:

[0224]

[0206] The following sequence was ordered containing a universal priming part and the sgRNA (underlined): GTTGTGTAATACTGATTCCCCGGTGTTTCGTCCTTTCCACAAG 9SEQ The additional bold cytosine residue is included to obtain sufficient transcription from the U6 promoter. Side-directed mutagenesis in SpCas9-vector #47511 was done using Q5 high-fidelity polymerase with mentioned primer and the following universal 57phos / - GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC (SEQ ID NO: 19)primer. Upon PCR cleanup, T4-ligase and Dpn1 were used to relegate and generate the plasmid, this was done according to manufacturers protocol (NEB). DNA was transformed into DH5a bacterial cells, and plasmids were verified using sanger sequencing (Macrogen Europe BV) with sequence primer GGGCAGGAAGAGGGCCTAT (SEQ ID NO:20).

[0225]

[0207] Bulk RNA-sequencing

[0226]

[0208] RNA was extracted using QIAwave RNA Minikit (Cat. No. 74534) according to the manufacturer’s protocol. RNA integrity was measured using the Agilent RNA 6000 Nano kit with the Agilent 2100 Bioanalyzer, and RNA concentrations were determined using the Qubit RNA HS Assay Kit (Thermo Fisher Scientific). RNA integrity number values of RNA samples were typically 9.5-10.0 and never <9.0. RNA libraries were prepared with the TruSeq Stranded messenger RNA polyA kit and single-end (1 x 50 base pairs) sequenced on an Illumina NextSeq 2000. Reads were mapped to the human GRCh37 genome assembly. Differential gene expression analysis was performed using the DESeq2 package in RStudio (v.2022.02.2). Data visualization was performed using the packages ggplot2 and pheatmap in RStudio, or manually plotted using GraphPad Prism (v.8.2.0). The coefficient of determination (R2) was determined either using the ggplot2 package in RStudio or in GraphPad Prism.

[0227]

[0209] N39 ileum FUCCI reporter organoid line

[0228]

[0210] The FUCCI sequence comprising mCherry-Cdt1-T2A-Geminin-hmAzami-Green was PCR-amplified from a construct provided as a kind gift by Geert Kops (Hubrecht Institute, Utrecht). Using Gibson Assembly (NEBuilder HiFi DNAAssembly) it was cloned into a p2Tol- based vector harboring puromycin resistance (Addgene category Cat#714859). Reporter organoids were generated by co-electroporation of 5 pg of the FUCCI expression construct together with 5 pg mT2TP transposase mediating the tol2-dependent random integration into the cell’s genome. Stable transfectants were isolated based on puromycin-resistance and expression of both FUCCI fluorescent signals.

[0229]

[0211] Organoid electroporation

[0230]

[0212] Organoid electroporation was performed as described previously76with some modifications. Wild-type organoids were maintained in expansion medium. Two days prior to electroporation 1.25% (vol / vol) DMSO was added to the organoid medium. Electroporation was done with 10A6 cells per electroporation with BTXpress solution containing 2.5 ug ITGA2 sgRNA plasmid, 5 ug Piggybac transposase, 5 ug Piggybac hygromycin cassette77, 7.5 ug C>T Base editor_eGFP plasmid (Addgene plasmid #112100). Five days post transfection, hygromycin 100 ug / ul (Invivogen) selection was started. Fourteen days after selection, surviving-hygromycin selection clones were individually picked and sequenced with the following primers: ITGA2_fwd GCACCTGCCACCTTCTCCATCA (SEQ ID NO:21); ITGA2_rev GAAAAGAGGAAAGAAGCAGATT (SEQ ID NO:22). DNA was obtained using ZYMO Research DNA isolation kit. During clonal outgrowth and selection scTS2 / 16 was always present in the media.

[0231]

[0213] Western blot

[0232]

[0214] Proteins were run on a 4-15% SDS-PAGE gel (Biorad) under reducing / non-reducing conditions. Marker proteins (Thermo Scientific #26616, Pager ruler). SDS-PAGE gel was transferred to PVDF membrane (Thermo Scientific ref 88518). Transfer efficiency was controlled with ponceau staining. For blocking 5% non-fat milk protein in PBS was used. For direct HIS-tagged protein staining a mouse anti-HIS tag HRP conjugated antibody (R and D Systems Cat# MAB050H, RRID:AB_357354) was used. Indirect staining for the HIS-tag was performed using anti-HIS (Abeam Cat# S2917, RRID:AB_10641949) in combination with anti- mouse-HRP (R and D Systems Cat# MAB050H, RRID:AB_357354). FLAG-tagged proteins were directly visualized using HRP-conjugated M2 anti-FLAG (Abeam, RRID AB_869428), or indirectly by combining M2 anti-flag (Sigma-Aldrich Cat# F3165, RRID:AB_259529) and anti- mouse-HRP (Abeam Cat# ab6808, RRID:AB_955441 ). Chemiluminescent substrate ECL (GE-Healthcare).

[0233]

[0215] Organoid differentiation in 2D

[0234]

[0216] Immunofluorescent staining; to allow differentiation of organoids, the intestinal expansion medium in the bottom compartment was replaced for 5-7 days by ENR medium supplemented with DAPT at 10 pM, or ER medium supplemented with recombinant BMP2 and BMP4 (50 ng / ml each)(Thermo Fischer Peprotech). The upper compartment was exposed to air during this period. Hereafter, medium was removed, tissue 4% PFA-fixed for 20 minutes, washed twice with excess PBS, permeabilized by incubating in PBS / 0,1% Triton X-100 / 0,2% BSAfor 1 hr. Finally, tissue was washed with PBS 0,2% BSA. For visualization of Goblet cells we used the mouse anti-muc2 antibody 11197 (Abcam)(RRID:AB_297837), for enteroendocrine cells rabbit antibody 23342-1 -AP (Proteintech) detecting chromograninA (RRID:AB_2879259), and for enterocytes the marker ApoA1 by using rabbit antibody PAS- 88109 (Thermo Fisher) (RRID:AB_2804657). Alexa-conjugated secondary antibodies (1 :500) used were donkey anti-mouse Alxa488 (1 :500) Thermo Fisher A21202 (RRID:AB_141607), and donkey anti-rabbit Alexa 568 Thermo Fisher A10042 (RRID: AB_2534017). Actin was stained using Phalloidin-Atto 647 from Sigma. All reagents diluted in PBS / 0,1% Triton X- 100 / 0,2% BSA.

[0235]

[0217] References

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[0308]

[0218] Next we compared the IgG and scFv versions of TS2 / 16 in collagen matrix.

[0309]

[0219] The results, shown in Figure 15, make clear that the IgG version did not work well, or did not work at all, as compared to the scFv version of TS2 / 16.

Claims

CLAIMS1 . A single-chain variable fragment (scFv) agonist of integrin -1 , preferably wherein the single-chain variable fragment interacts with at least an epitope defined by residues 207 to 218 of the human integrin pi subunit of SEQ ID NO: 1 .

2. A single-chain variable fragment (scFv) agonist of integrin 3-1 according to claim 1 wherein the single-chain variable fragment interacts with at least an epitope defined by residues 207 to 218 of the human integrin 31 subunit of SEQ ID NO: 1.

3. The single-chain variable fragment according to any of the previous claims, which is defined by formula (I):VN- (L)n- Vc(I), whereinL is a peptide linker and n is an integer selected from 0 to 5;- VN is the variable chain at the N-terminal position of the single-chain fragment;- Vc is the variable chain at the C-terminal position of the single-chain fragment; the line (-) between the linker L and the variable chains, VN and Vc, represent a peptide bond;- and wherein VN and Vc are selected from a variable light chain and a variable heavy chain of an anti-integrin 3-1 agonist antibody, with the proviso that in formula (I) a variable light and variable heavy chain is present.

4. The single-chain variable fragment according to claim 3, wherein the peptide linker, L, is a peptide that comprises from 5 to 30 amino acids and comprises SEQ ID NO: 2 (GGGGS), preferably L is a peptide linker that comprises or consist of SEQ ID NO: 2 and n is 3.

5. The single-chain variable fragment according to any one of the previous claims, which comprises:(a) a variable light chain that comprises an amino acid sequence that comprises a complementarity-determining region 1 (CDR1) consisting of SEQ ID NO: 3 (SVSSIISSNYL), a complementarity-determining region 2 (CDR2) consisting of SEQ ID NO: 4 (RTSNLAS), and a complementarity-determining region 3 (CDR3) consisting of SEQ ID NO: 5 (QQGSDIPLT); and / or(b) a variable heavy chain that comprises an amino acid sequence that comprises a complementarity-determining region 1 (CDR1) consisting of SEQ ID NO: 6 (GFTFSSYTMS), a complementarity-determining region 2 (CDR2) consisting of SEQ ID NO: 7 (TISSGGSYTYYPDSVKG), and a complementarity-determining region 3 (CDR3) consisting of SEQ ID NO: 8 (IGYDEDYAMDH).

6. The single-chain variable fragment according to any one of the previous claims, which comprises a variable light chain that comprises or consist of the amino acid sequence SEQ ID NO: 9 (EIWTQSPTTMAASPGDKITITCSVSSIISSNYLHWYSQKPGFSPKLLIYRTSNLASGV PPRFSGSGSGTSYSLTIGTMEAEDVATYYCQQGSDIPLTFGDGTKLDLK)7. The single-chain variable fragment according to any one of the previous claims, which comprises a variable heavy chain that comprises or consists of the amino acid sequence SEQ ID NO: 10 (MDVKLVESGGGLVKPGGSLKLSCAASGFTFSSYTMSWVRQTPEKRLEWVATISSG GSYTYYPDSVKGRFTISRDKAKNTLYLQMGSLKSEDTAMYYCTRIGYDEDYAMDHW GQGTSVTVSS)8. The single-chain variable fragment according to any one of the previous claims, which comprises or consists of an amino acid sequence selected from SEQ ID NO: 11 (VH- VL), SEQ ID NO: 13 (VH-VL), SEQ ID NO: 12 (VL-VH), and SEQ ID NO: 14 (VL-VH).

9. A stem cell or organoid culture medium comprising a single-chain variable fragment (scFv) agonist of integrin p-1 as defined in any of claims 1 to 8.

10. The stem cell or organoid culture medium according to claim 9, which further comprises one or more of a Wnt agonist, a BMP inhibitor, a mitogenic growth factor and a TGF- beta inhibitor.

11. The stem cell or organoid culture medium according to any one of the previous claims, which further comprises an extracellular matrix composition.

12. The stem cell or organoid culture medium according to claim 11 , wherein the extracellular matrix composition comprises one or more of collagen, laminin, perlecan, fibronectin, and RGD attachment ligand of fibronectin; or is a Basement Membrane Extract13. A composition comprising collagen and a single-chain variable fragment (scFv) agonist of integrin 3-1 as defined in any of claims 1 to 8.

14. An extracellular matrix composition that comprises a single-chain variable fragment (scFv) agonist of integrin p-1 as defined in any of claims 1 to 8.

15. A method for culturing a stem cell or an organoid, wherein the method comprises culturing said stem cell or organoid in a culture medium suitable for stem cells, wherein the culture method further comprises contacting the cell or organoid with a single-chain variable fragment (scFv) agonist of integrin 3-1 as defined in any of claims 1 to 8, and / or contacting the cell or organoid with a stem cell or organoid culture medium as defined in any one of claims 9-12, and / or contacting the cell or organoid with a composition as defined in claim 12, and / or contacting the cell or organoid with an extracellular matrix composition as defined in claims 14.

16. The method according to claim 15, which further comprises contacting the stem cell or organoid with one or more additional integrin agonists.6117. The method according to any one of claims 15 to 16, wherein the stem cell or organoid is an epithelial stem cell or organoid of the digestive system.

18. A stern cell or organoid obtained or obtainable by a method as defined in any one of claims 15 to 17.

19. A stern cell or organoid as defined in claim 18 for use in therapy and / or in diagnosis.

20. A stem cell or organoid for use in therapy and / or in diagnosis, wherein the said stem cell or the said organoid are obtained or obtainable by a method as defined in any one of claims 14 to 16.

21. Use of a single-chain variable fragment (scFv) agonist of integrin p-1 as defined in any one of claims 1 to 8 for in vitro culturing a cell.

22. Use of a single-chain variable fragment of a single-chain variable fragment (scFv) agonist of integrin p-1 as defined in any one of claims 1 to 8 for in vitro and / or ex-vivo pre-treating cells.

23. A kit of parts, preferably for in vitro and / or ex-vivo pre-treating cells, that comprises: a) a scFv agonist of integrin P-1 as defined in any one of claims 1-8, or a composition comprising it; and b) optionally, instructions for the use of the kit, and / or for carrying out any of the methods as defined in any one of claims 15-17, or for the uses of a scFv agonist of integrin p-1 as defined in any one of claims 21-22.

24. A method for modulating the polarity of stem cells or of organoids comprising stem cells, which cells comprise an apical and a basal side, which method comprises a step of contacting the cells or organoids with an antibody or fragment thereof specific for integrin P-1 .

Images