Method for Assessing a Compound Interacting with a Target on Epithelial Cells

Abstract

Disclosed herein is a method for assessing a compound interacting with a target on polarized epithelial cells. The method comprising the steps of providing an organ chip comprising a main channel and polarized epithelial cells, wherein the main channel is divided into an apical channel and a basal channel separated by the polarized epithelial cells, wherein the apical side of the polarized epithelial cells is directed towards the apical channel and the basolateral side of the polarized epithelial cells is directed towards the basal channel. Determining the localization and optionally the expression level of the target on the polarized epithelial cells. Administering the compound and optionally immune cells, preferably peripheral blood mononuclear cells (PBMC) to the basal channel, when the target is localized on the basolateral side of the epithelial cells or administering the compound and optionally immune cells, preferably peripheral blood mononuclear cells (PBMC) to the apical channel, when the target is localized on the apical side of the epithelial cells. Measuring a parameter of the administration of the compound and the peripheral blood mononuclear cells.

Claims

1. A method for assessing a compound interacting with a target on polarized epithelial cells, the method comprising: providing an organ chip comprising a main channel and polarized epithelial cells, wherein the main channel is divided into an apical channel and a basal channel separated by the polarized epithelial cells, wherein the apical side of the polarized epithelial cells is directed towards the apical channel and the basolateral side of the polarized epithelial cells is directed towards the basal channel; determining the localization and optionally the expression level of the target on the polarized epithelial cells; administering the compound and optionally immune cells, such as peripheral blood mononuclear cells (PBMC) to the basal channel, when the target is localized on the basolateral side of the epithelial cells, or to the apical channel, when the target is localized on the apical side of the epithelial cells; and measuring a parameter of the administration of the compound and optionally the immune cells, such as the peripheral blood mononuclear cells.

2. The method of claim 1, wherein the organ chip is an intestine chip, wherein the polarized epithelial cells are intestine epithelial cells.

3. The method of claim 2, wherein the polarized epithelial cells comprise Caco-2 or primary cells, such as primary human intestinal epithelial cells.

4. The method of claim 1, wherein the organ chip is a lung chip, wherein the polarized epithelial cells are alveolar epithelial cells or primary cells.

5. The method of claim 4, wherein the basal channel comprises pulmonary microvascular endothelial cells.

6. The method of claim 1, wherein determining the localization and optionally the expression level of the target on the polarized epithelial cells is performed by immunofluorescence analysis.

7. The method of claim 1, wherein determining the localization and optionally the expression level of the target on the polarized epithelial cells comprises comparing the localized target on the polarized epithelial cells with the target in a native organ, such as in native intestine or in native lung.

8. The method of claim 1, wherein the compound is a compound to be administered via systemic circulation.

9. The method of claim 1, wherein the polarized epithelial cells and/or the basal channel and/or the apical channel is covered with an extracellular matrix.

10. The method of claim 1, wherein the target on the polarized epithelial cells is an antigen, such as a tumor-overexpressing protein.

11. The method of claim 1, comprising administering the compound to the basal channel and then measuring a parameter of the administration of the compound and the peripheral blood mononuclear cells, and further comprising: administering the compound and peripheral blood mononuclear cells to the apical channel; and measuring a parameter of the administration of the compound and optionally the peripheral blood mononuclear cells.

12. The method of claim 1, wherein the parameter is: a safety parameter associated with a state of the polarized epithelial cells and/or with a state of a tight barrier formed by the polarized epithelial cells; and/or pharmacological parameter.

13. The method of claim 12, wherein the parameter is a safety parameter associated with the integrity or permeability of the barrier formed by the polarized epithelial cells and/or a pharmacological parameter associated with side effects or potency of the compound, peripheral blood mononuclear cell activation or cytokine release.

14. The method of claim 1, wherein the flow rate of the administered compound and optionally the immune cells, through the apical channel or the basal channel is 10 to 60 l/h, 15 to 50 l/h, 20 to 40 l/h, or essentially 30 l/h.

15. The method of claim 1, wherein the compound is an antibody, a bispecific antibody, such as a T-cell bispecific antibody, or a small molecule.

16. The method of claim 15, wherein the compound is a bispecific antibody, such as a T-cell bispecific antibody, that binds an epithelial target and an immune cell.

17. The method of claim 1, wherein the organ chip comprises a first and a second vacuum channel, wherein the main channel is arranged between the first and second vacuum channel, wherein peristaltic movement is modelled by the first and second channels during the administering of the compound and optionally immune cells, such as peripheral blood mononuclear cells (PBMC) and/or during the measuring of the parameter of the administration of the compound and optionally the immune cells, such as the peripheral blood mononuclear cells.

18. The method of claim 1, first performed with a first compound of interest and thereafter again performed with a second compound of interest, and further comprising comparing the parameter for the first compound of interest and the parameter for the second compound of interest are compared.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0081] FIG. 1 shows a flow chart of a method according to an embodiment of the invention.

[0082] FIG. 2 shows a schematic cross-sectional view of an intestine chip as used in the embodiment of FIG. 1.

[0083] FIG. 3a shows pharmacologic data obtained by using a conventional 2D monolayer cell culture in combination with a TCB targeting STEAP1.

[0084] FIG. 3b shows pharmacologic data obtained by using a conventional 2D monolayer cell culture in combination with a TCB targeting CEA.

[0085] FIG. 4a shows pharmacologic data obtained by using the method according to an embodiment of the invention in combination with TCB targeting STEAP1.

[0086] FIG. 4b shows the trans-epithelial permeability of the epithelial cells used for obtaining the pharmacology obtained in FIG. 4a.

[0087] FIG. 5a shows pharmacologic data obtained by using the method according to an embodiment of the invention in combination with TCB targeting CEA.

[0088] FIG. 5b shows the trans-epithelial permeability of the epithelial cells used for obtaining the pharmacology obtained in FIG. 5a.

[0089] FIG. 6a shows the localization of CEA within a conventional 2D monolayer cell culture.

[0090] FIG. 6b shows the localization of CEA within an intestine chip as used in the present invention.

[0091] FIG. 6c shows the localization of STEAP1 within an intestine chip as used in the present invention.

[0092] FIG. 7a shows pharmacologic data obtained by using the method according to an embodiment of the invention using primary epithelial organoids in combination with TCB targeting CEA.

[0093] FIG. 7b shows the trans-epithelial permeability of the epithelial cells used for obtaining the pharmacology obtained in FIG. 7a.

[0094] FIG. 8 shows a comparison of pharmacological data obtained for two different CEA-targeting TCBs.

[0095] FIG. 9 shows a schematic cross-sectional view of a lung chip as according to another embodiment of the invention.

[0096] FIG. 10 shows FOLR1 expression in chip within a lung chip as used in the present invention.

[0097] FIG. 11 shows TCB-mediated cytotoxicity and cytokine release in one embodiment of a lung chip, treated with 20 g/ml of FOLR1 high affinity antibody (FOLR1(Hi) TCB). Activated CD8+CD69+ T cells; apoptotic cells, and cytokine release are represented as granzyme B; IFN; TNF; and IL-6. Nonbinding control antibody=NT. Upper two panels: One-way ANOVA, Tukey's Multiple Comparison Test, *p<0.05, n=4. Lower panels: 2-way ANOVA, Tukey's Multiple Comparison Test, ****p<0.0001, n=4.

[0098] FIG. 12 shows graphs representing T-Cell activation (top left graph); T-Cell (PBMC) attachment to the Alveolus Epithelium (top right graph, after 48 h of treatment); apoptotic cell quantification, lower left and right graphs, respectively, for an assay performed using a lung chip according to one embodiment. Middle right and lower panels demonstrate a selective increase in cytokine secretion between FOLR1(Hi) TCB over FOLR1(Lo) TCB at around 2 g/ml up to 20 g/ml.

EXEMPLARY EMBODIMENTS

[0099] FIG. 1 shows an exemplary embodiment of a method according to the invention. In a first step S1, the location of target antigen 13, respectively 13 is determined in the native intestine 1 and 1. It is noted that only one exemplary antigen is schematically shown. The skilled person is well aware that more than one antigens may naturally be present. Optionally, the expression level of the antigen in the native intestine is determined. In scenario A, shown on the left of FIG. 1, antigen 13 is located on the apical side 11 and not on basolateral side 12 of the epithelial cells. In contrast, in scenario A, which is illustrated on the right side of FIG. 1, antigen 13 is located on the basolateral side 12 of the epithelial cells. Step 1 is optional and only necessary if the localization of the target antigen within the native intestine is unknown or needs to be confirmed. In step S2, an intestine chip 2, respectively 2, is provided. FIG. 1 shows the main channel of the intestine chip as a cross-sectional view. Furthermore, the localization of the target antigen 23, respectively 23, on the epithelial cells within the intestine chip is determined and compared to the localization of the antigen within the native intestine 1, which has either been determined in step S1 or which is already known in the art. Concomitantly, the expression level of the antigen is determined and it is in general verified whether the expression level obtained reflects the expression level of the antigen in the native intestine. In scenario B, the target antigen 23 is located on the apical side 21 and not on the basolateral side 22 of the epithelial cells. In contrast, in scenario B, target antigen 23 is located on the basolateral side 22.

[0100] Following the verification of physiological target expression, TCBs 25 and T-cells 24 are introduced into the basal channel or the apical channel in step S3. Furthermore, TCB-mediated pharmacological parameters and safety parameters are measured. In scenario C, in which antigen 23 is located on the basolateral side, TCBs 25 and T-cells 24 are introduced into the basal channel. As a result, the TCBs can bind to both the T-cells 24 and antigen 23 thus mediating T-cell activation and epithelial cell killing. Therefore, scenario C mimics a patient with a healthy gut receiving TCBs via systemic administration. A healthy gut represents a gut with a tight epithelial barrier, which is thus essentially impenetrable for TCBs and T-cells. In scenario C-1 and C-2 the antigen 23 is located on the apical side of the epithelial cells. In scenario C-1, T-cells 24 and TCBs 25 are administered through the basal channel of intestine chip 2. As a result of the intact tight epithelial barrier, neither the T-cells nor the TCBs can bind to antigen 23. Thus, no TCB mediated T-cell activation and epithelial cell killing is observed. In contrast, in scenario C-2, T-cells 24 and TCBs 25 are administered to the apical channel. As a result, the TCBs bind to both, the T-cells and antigen 23, mediating T-cell activation and epithelial cell killing. Scenario C-2 therefore mimics a pathologically leaky gut, for example that of a clinically plausible cancer patient condition of luminal TCB and T-cell penetration. Typically, immune cell recruitment to the epithelial cell can be visualized by known spectroscopic methods, such as confocal microscopy or Raman spectroscopy. Activation of the immune cells is typically determined by detecting specific cytokine release. A further parameter, which is measured in step S3 is the permeability of the intestine barrier after exposure to TCBs and T-cells. For example, the permeability is measured after a certain time interval, such as 12 h, 24 h or 48 h. A suitable method known in the art for measuring the integrity of the intestine membrane is transepithelilal/transendothelial electrical resistance (TEER), and the use of fluorescent marker (such as FITC-Dextran).

[0101] FIG. 2 shows an exemplary embodiment of an intestine chip 2 as used in the method according to the invention. Intestine chip 2 comprises main channel 30, which is divided into basal channel 31 and apical channel 32 by epithelial cells 20 and membrane 26. Furthermore, main channel 30 is flanked by two vacuum channels 41 and 42, which mimic the peristaltic movement of the gut.

[0102] The workflow described in FIG. 1 was applied in a model study, using TCBs recognizing the tumor-overexpressing proteins STEAP1 (six-transmembrane epithelial antigen of the prostate) and CEA (carcinoembryonic antigen). The TCBs comprised a CD3 arm binding to the CD3 receptor of on the T-cells to be activated and a target arm binding to the target antigen on the surface of the intestinal epithelial cells. Control antibodies comprise similar arms but lacking the ability to either bind to CD3 (herein referred to as non-CD3 antibody) or to the target (herein referred to as non-targeting CD3 antibody). STEAP1 and CEA are also expressed in healthy intestinal epithelial cells on the basolateral and apical surfaces respectively. Caco-2 colonocytes express STEAP1 and CEA and can form a polarized monolayer with apical and basolateral sides when cultivated in the intestine chip, thereby providing a relevant in vitro model for evaluating STEAP1 and CEA TCB intestinal liabilities.

[0103] Prior to profiling the TCBs in the intestine chip, their potency and specificity in conventional Caco-2 monolayer culture in the presence of PBMCs was verified. It has been found that incubation of STEAP1 TCB with Caco-2 cells and PBMCs led to a substantial release of IL-12, INF--, MIP-1-, MIP-1- and TNF-, compared with TCB incubated with Caco-2 cells or PBMCs alone (FIG. 3a). Therefore, and as expected, TCB-mediated activation occurred in a target-dependent manner, requiring the presence of both target (epithelial) and effector (immune) cells. The control antibodies appeared inert (non-CD3 STEAP1) or much less potent (non-targeted CD3) than STEAP1 TCB, further underlining the target specificity of TCBs. As a positive control of T-cell activation, PBMCs co-treated with anti-CD3 and anti-CD28 antibodies triggered activation of PBMCs in the absence of target.

[0104] Surprisingly however, CEA TCB administered to the CEA-expressing Caco-2 cells (FIG. 3b) did not induce a cytokine release. Potential factors that could lead to the observed lack of activation include low potency of the TCB molecule and low CEA expression at the cell surface. To distinguish between these possibilities, the experiment was repeated using the CEA-high SNU-1544 cancer cell line as a positive cell control. CEA TCB treatment in the presence of SNU-1544 cells resulted in substantial cytokine release, confirming the potency of the TCB, and suggesting that the lack of activation in the presence of Caco-2 monolayers may be attributed to low or delocalized CEA expression by the latter. Noticeably, the non-targeted CD3 control antibody led to a substantial cytokine release, including in the PBMC-only conditions and at a higher extent than observed in FIG. 3a. These data suggest a CD3-mediated, target-independent T cell activation that can vary from PBMC donor to donor.

[0105] Intestine chips have been reported to promote the polarization and maturation of Caco-2 barriers, and the establishment of in vivo like morphology, by incorporating key environmental parameters, such as luminal flow and peristalsis, which are absent in conventional monolayer culture. It is hypothesized that intestinal barriers formed within the intestine chip may mimic the native intestinal epithelium with higher fidelity. In particular, it is postulated that CEA expression within the intestine chip may reflect the in vivo pattern more closely. Thus, the localization and expression of CEA in both the 2D monolayer cell culture and the intestine chip has been determined. Indeed, immunofluorescence analysis and confocal microscopy revealed poorly polarized expression of CEA within conventional Caco-2 monolayers, with a peak of CEA expression located more basally than ZO-1 (Zonula occludens-1) (FIG. 6a). In contrast, strong apical enrichment of CEA was detected within the intestine chip (FIG. 6b), verifying the above mentioned hypothesis. The chip likewise recapitulated the physiological expression of STEAP1 which was enriched in the basolateral regions of the epithelium (FIG. 6c).

[0106] After confirming the expression and polarization of CEA and STEAP1 on chip, TCB delivery to an intestine chip rendered immune-competent by the continuous circulation of PBMCs was performed next. Bearing in mind the mode of action of TCBs, which relies upon direct contact between antibody, effector and target cells, it was sought to determine whether PBMCs introduced through the basal channel were able to infiltrate the ECM layer and transmigrate to the epithelial barrier. Monitoring the presence of CFSE-labelled (carboxyfluorescein succinimidyl ester) PBMCs, extensive attachment of immune cells to the bottom of the membrane was observed, along with a small number of cells embedded in the epithelium. Thus, the aptitude of PBMCs to infiltrate the barrier, which is a key step for accessing the target site and mediate the TCB response on epithelial tissue was confirmed.

[0107] To assess whether a systemically administered TCB can engage at a basolaterally-expressed target protein and induce a T cell activation (scenario C in FIG. 1), STEAP1 TCB and PBMCs were introduced in the basal channel of the intestine chip and cytokine release in the supernatant was measured using multicomponent ELISA (FIG. 4a). As expected, basally delivered STEAP1 TCB resulted in substantial cytokine release (FIG. 4a). As reported for similar experiments performed in 2D (FIG. 3), the non-targeted CD3 control antibody elicited a cytokine release response, suggesting a CD3-driven, target-independent T-cell activation. Nevertheless, the potency of STEAP1 TCB was significantly superior at similar concentration (100 g/ml). To assess TCB-mediated tissue damage, epithelial barrier permeability was measured 48 h after the introduction of STEAP1 TCBs and PBMCs. Substantial alterations in barrier function in response to STEAP TCB treatment were detected (FIG. 4b). Epithelial effects appeared to be target-dependent: significant changes in permeability were induced by TCBs in a concentration-dependent manner, and the control antibodies preserved the barrier integrity (FIG. 4b).

[0108] Next, the effect of CEA TCB, which recognizes an apically-expressed target, was evaluated in healthy versus leaky conditions by introducing CEA TCB and PBMCs in the basal channel (scenario C-1 in FIG. 1) or apical channel (scenario C-2 in FIG. 1) of the intestine chip respectively. As expected, CEA TCB administered with PBMCs on the luminal side induced a cytokine release response, best exemplified by TNF concentration in the supernatant (FIG. 5a). However, in this experiment the overall cytokine profile appeared highly variable and unspecific: The non-targeted CD3 control antibody and the CEA TCB in the basal conditions triggered a strong cytokine release response, hypothetically resulting from a donor-specific pre-conditioned state of PBMCs that may confound the target-dependent potency of CEA TCB. Such poised state might be exacerbated by the high T cell density and elevated anti-CD3 drug concentrations used. Nevertheless, and in accordance with STEAP1 TCB data, the effect of CEA TCB on epithelial tissue damage appeared to be target dependent: Significant changes in permeability were induced by CEA TCB in an accessibility-dependent manner (scenario C-1 vs C-2), and the control antibodies preserved the barrier integrity (FIG. 5b). Thus, TCB-mediated cytokine release and tissue damage appeared to be uncoupled, at least partially, under in vitro conditions, and measurement of barrier integrity appears to be a more accurate indicator of target-dependent TCB-mediated effects.

[0109] Overall, the data presented herein have important implications for the predictability and clinical impact of organ-on-a-chip models for drug profiling: Despite CEA expression in Caco-2 cells culture in 2D, CEA TCB failed to elicit immune cell activation (FIG. 3b), whereas CEA TCB treatment of Caco-2 barriers within the intestine chip resulted in activation and barrier leakiness (FIG. 5b). The differential outcomes can likely be attributed to the higher extent of epithelial maturation and physiological pattern of CEA expression observed within the chips, compared with conventional Caco-2 monolayers. This finding highlights a crucial difference between the intestine chip and conventional approaches for the target tissue-specific profiling of TCBs, with profound clinical implications. An assessment of TCB activity based on standard approaches would deem the CEA TCB safe for off-tumor, on-target intestinal effectsa predication with potentially dire consequences in the clinic. The intestine chip, in contrast, would predict low intestinal liability only in patients with intact intestinal barriers, whereas potential on-target TCB effects are foreseen in the clinically plausible and frequent case of patients with leaky guts.

[0110] The application of the immune-competent intestine chip has thus been demonstrated as a platform for TCB profiling in polarized epithelia such as the gut. In particular, the method was able to capture TCB-mediated target tissue damage specifically related to a target antigen. This demonstration was directly enabled by the modularity of the system, which enabled independent access to the luminal and basal epithelial surfaces, thereby allowing to distinguish target-dependent from non-specific, off-target effects, which would otherwise be unapparent in conventional monolayer culture. Moreover, the physiologically-relevant micro-environmental parameters, i.e. presence of ECM and flow, afforded by the intestinal chip promoted the maturation and polarization of the intestinal barrier. The physiologically faithful antigen presentation by intestinal epithelium formed within the intestine chip proved to be essential for revealing TCB effects that were unforeseen using poorly polarized intestinal cells in 2D culture. Whereas TCB safety profiling in the gut was chosen to showcase the utility of this platform, the paradigms introduced here are readily adaptable and extendable to conducting both safety and pharmacology studies of standard antibody or small molecule effects.

[0111] In addition to the intestine chip containing Caco-2 cells, a further embodiment of the invention uses an intestine chip comprising primary epithelial organoids. These were derived from the healthy region of a colonic biopsy and were expanded using standard Matrigel-based culture and subsequently plated on the intestine chip, where they form a tight, polarized and differentiated intestinal epithelial monolayer. The basal channel of the corresponding chip comprises an endothelial barrier, formed from human intestinal microvascular endothelial cells (HIMECs). In order to determine whether the findings relating to CEA TCB with Caco-2 cells can be recapitulated with the intestine chip comprising primary epithelial organoids, expression and the apical localization of CEA was confirmed by immunofluorescence analysis. Additionally, the luminal channel of the device was used to co-deliver PBMCs and CEA TCB, or a control, non-targeting CD3-only engaging TCB (DP47). CEA TCB treatment led to a significantly increased PBMC attachment to the primary epithelium, which is consistent with the TCB mode of action (FIG. 7a). With respect to barrier permeability, a CEA TCB induced dose-dependent increase in barrier permeability is observed, as illustrated in FIG. 7b. This suggests that TCB treatment entail epithelial cell killing and barrier damage. In conclusion, the CEA TCB-related findings were replicated with the intestine chip with primary epithelial organoids, making it a highly physiologically relevant option for the safety profiling if TCBs.

[0112] FIG. 8 further demonstrates that the method disclosed in any of the herein described embodiments is also suitable for comparing and differentiating the safety effects of a related compounds, and thus allows for assessing the safety of a new therapy at a very early stage. In the example shown, two CEA-targeting TCBs were assessed, namely the above mentioned CEA TCB and CEACAM5 TCB. CEACAM5 TCB is a higher affinity molecule, thus resulting in more pronounced intestinal toxicity in patients, which primarily manifests as severe diarrhea. As already discussed in the context of the assessment of CEA TCB (see above), the apical delivery was performed, in order to ensure target engagement. TCB mediated effects were assessed by considering cytokine release and T cell activation (CD69 expression) at 72 h, and through live monitoring of epithelial cell apoptosis (caspase activation) at 24, 48 and 72 h after TCB/PBMC treatment. As can be seen from FIG. 8A, CEACAM5 TCB induced robust, dose dependent cytokine release at relevant doses. FIG. 8B further demonstrates a significantly enhanced T cell activation by CEACAM5 TCB. Additionally, an increase in epithelial cell apoptosis was observed (FIG. 8C). By comparison, CEA TCB resulted in substantially lower cytokine release and T cell activation, and no detectable changes in apoptosis, consistent with its lower binding affinity and more favorable clinical safety profile. These data suggest that the intestine chip is capable of capturing in vitro the different potencies and clinical safety effects of TCB molecules, which further qualifies it as a suitable platform for the safety assessment of new therapies.

[0113] FIG. 9 shows an exemplary embodiment of a lung chip 2 as used in the method according to the invention. Lung chip 2 comprises main channel 30, which is divided into basal channel 31 and apical channel 32 by epithelial cells 20, pulmonary microvascular endothelial cells 27 and membrane 26. Furthermore, main channel 30 is flanked by two vacuum channels 41 and 42, which mimic the breathing movement of the lung. Prior to testing FOLR1-TCBs, expression of FOLR1 in chips was verified by immunofluorescence analysis and surface expression quantified by flow cytometry (FIG. 10). Following the verification of physiological target expression, TCBs and T-cells are introduced into the basal channel or the apical channel. Furthermore, a parameter of the administration is measured, in particular TCB-mediated pharmacological parameters and safety parameters are measured. If the target and T cells are present in the same channel, the TCBs bind to both, the T-cells and antigen, mediating T-cell activation, attachment and epithelial cell killing. That scenario is equivalent to the Scenario C-2 of the gut chip (FIG. 1) and therefore mimics a pathologically leaky alveolus, for example that of a clinically plausible cancer patient condition of luminal TCB and T-cell penetration. Typically, immune cell recruitment to the epithelial cell can be visualized by confocal microscopy. Activation of the immune cells is determined by detecting specific cytokine release and measuring surface expression of activation markers by flow cytometry.

[0114] FIG. 11 shows TCB-mediated cytotoxicity and cytokine release in one embodiment of a lung chip, treated with 20 g/ml of FOLR1 high affinity antibody (FOLR1(Hi) TCB). For performing the assay, the luminal channel was used to co-deliver PBMCs and FOLR1-TCB, or a control, non-targeting CD3-only engaging TCB (NT). Apoptosis detection by caspase 3/7 live imaging revealed a T-cell-dependent killing of alveolar epithelial cells upon FOLR1 TCB treatment. FOLR1-TCB also led to a significantly increased PBMC attachment to the primary alveolar epithelium, in accordance with the expected increased adhesion molecules due to T cell activation. The latter was quantified by flow cytometry detection of activation markers, which demonstrated the specific induction of T cell engagement in presence of FOLR1-TCB. Culture supernatants sampled at different time points were used for multiplex cytokines analysis and showed cytokines release induced by FOLR1-TCB treatment.

[0115] In another assay shown in FIG. 12, the lung chip was used to obtain pharmacological data and to compare to related TCBs with varying affinity. This embodiment of the lung chip captures antibody format differences demonstrating FOLR1(Hi) TCB (high affinity) has a greater lung epithelial toxicity than FOLR1(Lo) TCB (Low affinity). Graphs represent T-Cell activation (top left graph); T-Cell (PBMC) attachment to the Alveolus Epithelium (top right graph, after 48 h of treatment); apoptotic cell quantification, lower left and right graphs, respectively. Middle right and lower panels demonstrate a selective increase in cytokine secretion between FOLR1(Hi) TCB over FOLR1(Lo) TCB at around 2 g/ml up to 20 g/ml. Thus, the measured pharmacological parameters allow a comparison of two related TCBs, thereby demonstrating a safer profile for the low affinity FOLR1-TCP than for the high affinity FOLR1-TCB for apoptosis, PBMC attachment, T cell activation and cytokines release, which corroborats the findings of preclinical cynomolgus toxicity studies. This indeed proves that the lung chip in coculture with PBMCs constitutes a human relevant in-vitro alternative for safety profiling of TCBs.

[0116] Methods

[0117] Intestine Chip

[0118] Chip Activation and Deposition of Collagen I Gel on the Intestine Chip

[0119] The PDMS surface of the chip was activated as follows: the ER-1 reagent was resuspended in the ER-2 solution (Emulate, Inc) at a final concentration of 0.5 mg/ml and administered to fill the top and bottom channels of the chip. The ER-1 & 2 solution was activated for 20 minutes by UV treatment followed by sequential washes with ER-2 and PBS (ThermoFisher #14190144). An extracellular matrix (ECM) solution of 1 mg/ml rat tail collagen I (Corning #354249) to coat the top channel was prepared on ice according to the method of Doyle et al. 2017. Briefly, 10 uL of reconstitution buffer and 10 uL 10DMEM (MilliporeSigma #D7777-101L) and collagen I were diluted in PBS supplemented with calcium and magnesium (PBS++) (ThermoFisher #14040133) and the pH adjusted with 1N Sodium hydroxide (MilliporeSigma #221465) to 7-7.5 for a final volume of 1 ml. A second collagen I ECM solution of 100 g/ml to coat the bottom channel was prepared by diluting the 1 mg/ml solution 10-fold in PBS++. The remaining PBS in the top and bottom channels of the chip was then aspirated and the 1 mg/ml solution was added to the top while the 0.1 mg/ml solution was added to the bottom channel. The ECM solution in the top channel was allowed to polymerize at room temperature for 45 minutes followed by gentle perfusion of 200 l PBS++ with a P200 Gilson Pipet until the semi-polymerized gel began ejecting from the chip. Perfusion was then stopped and the pipet tip was ejected to allow the remaining volume to perfuse by gravity flow. Collagen I gel was allowed to continue setting in a humidified 37 C. incubator for 1.5 hours. The unpolymerized collagen I was then removed from the top and bottom channels by perfusion with PBS++ and stored at 4 C. in a humidified petri dish for up to a day.

[0120] Seeding of the Caco-2 Intestine Chip

[0121] Caco-2 BBE epithelial cells for seeding the intestine chip were routinely subcultured as per vendor recommendations (Millipore Sigma 86010202). For seeding, Caco-2 cells were dissociated with 0.05% trypsin-EDTA (ThermoFisher 25300054) for 5 minutes in a 37 C. humidified incubator and resuspended with excess DMEM (ThermoFisher #10569044) supplemented with 10% fetal bovine serum (FBS) (ThermoFisher #16000) and 100 U penicillin streptomycin (pen/strep) (ThermoFisher 15140122). Cell density and viability was assessed by trypan blue (MilliporeSigma T8154-100ML) exclusion assay followed by centrifugation and resuspendion in DMEM media supplemented with 10% FBS and pen/strep to a final cell density of 1.5 million cells per ml. S1 Organ-Chips (Emulate, Inc.) were then seeded with Caco-2 and incubated for at least 1.5 hours at 37 C. to allow for cell attachment. Both top and bottom channels of the chip were then washed with 200 uL of media to remove unattached or dead cells.

[0122] Media Equilibration

[0123] Before connecting chips to Pods, bottom media, DMEM supplemented with 10% FBS and 100 U pen/strep and top media, bottom media supplemented with 20 g/ml of 3 kDA dextran-cascade blue (ThermoFisher D7132), were equilibrated as follows to remove excess gas from medium and minimize potential bubble formation. 50 mls of top media was warmed in a 37 C. bath for 1 hour prior to being filtered through a 0.45 m Steriflip (FisherSci #SE1M003M00) for at least 15 minutes with a vacuum source of 80 kPa. The equilibrated top media was then used immediately or incubated at 37 C. before use.

[0124] Priming Pods

[0125] Prior to connecting Chips with Caco-2 cells to Pods, the Pods were primed for liquid flow. 1-3 mls of top and bottom media were added to their respective channels reservoirs of the Pod, respectively. 300 l of basal media was then added to both outlet reservoirs to cover the outlet Vias. The Pod ID was set to 054 and the Prime sequence of the Zoe Culture Module (Zoes) was then selected and run.

[0126] Connecting Chips to Pods and Zoes

[0127] Once primed, the Chips with Caco-2 epithelium were connected to the Pods, placed in a Zoe tray, and the tray inserted into the Zoe. The flow rate was set to 30 l/hr and the Regulate cycle was run to initiate flow. Chips were checked for lack of bubbles and flow consistency monitored daily for the next 24-72 hours. If bubbles or inconsistent flow were detected, chips were first manually reprimed by pushing 1 ml of media through the input via and watching for bubble ejection and secondly, the Regulate cycle was re-run.

[0128] Caco-2 Culture on-Chip

[0129] The maturation of the Caco-2 epithelium was monitored daily by brightfield microscopy for the first 72 hours to assess the formation of epithelial villi-like structures following this initial monitoring period. Barrier function was routinely assessed by collecting inlet and outlet samples and measuring the apparent permeability of the dextran-cascade blue 3 kDa. The Caco-2 Intestine-Chip was considered mature between 7-14 days upon formation of a villi-like structures and an apparent permeability between 110-7 and 110-6 cm/s.

[0130] PBMC Isolation

[0131] PBMCs were isolated from citrated, fresh whole blood (Research Blood Components) using Lymphoprep (Stemcell Technologies Catalog #07851) reagent and following the manufactures recommended protocol and cryopreserved in 50% FBS, 10% DMSO (Millipore Sigma D2650-100ML), 40% RPMI-1640 (ThermoFisher #61870-010). A total of million PBMCs per cryotube were frozen in a Mr. Frosty Container (VWR #55710-200).

[0132] 2D TCB Plate Validation Experiment

[0133] 100K Caco-2 Cells were plated in each well of a 24-well plate. Cells were cultured for 2-4 days until a fully confluent monolayer of cells was observed. The day of the experiment, cryopreserved PBMCs were thawed and resuspended in DMEM supplemented with 10% FBS and 100 U of pen/strep. Cells were then labelled with 5 m CSFE (ThermoFisher #C34554) for 20 minutes at room temperature. Excess CSFE was then removed by pelleting cells and washing twice with media. Labelled PBMCs were then resuspended to a final density of 1 million cells per ml. The media in each treatment well was then aspirated and replaced with 0.5 ml of the PBMC solution. Treatment antibodies were then added and the plates incubated overnight at 37 C. The following day, the media from each well was triturated and collected in V-bottom 96-well plates (VWR #29442-068). The PBMCs were pelleted by centrifugation at 300 g5 min and the supernatant transferred to a second 96-well plate for measurement of cytokine levels by multi-analyte ELISA (Meso Scale Discovery #K15067L-1) or viability by LDH (Promega #G1780). Leftover supernatant was decanted from the pelleted PBMCs and fixed with the BD cytofix solution (ThermoFisher #BDB554714). After fixation, PBMCs were spun down at 300 g for 5 minutes and the fixation solution decanted. PBMCs were either stained immediately for analysis by flow cytometry (FACs) or resuspended in 95% FBS, 5% DMSO and stored at 80 C. The remaining Caco-2 wells were then either fixed with 4% paraformaldehyde (PFA) (FisherSci #50-259-98) for 15 minutes at room temperature or lysed for measurement of intracellular LDH according to the manufacturer's recommendations. PFA was decanted from cells, replaced with PBS and stored at 4 C. until staining for immunofluorescence. Fixed Caco-2 wells were prepared for IF staining by first blocking with cell staining solution (Biolegend #420201) for 30 minutes at room temperature. Anti-human CD69 (ab201570-100UG) was diluted 100-fold in cell staining solution and incubated overnight at 4 C. Wells were then washed 3-times with 0.5 ml PBS with 5 min rest periods in between each wash. CD69 was then labelled with donkey anti-mouse conjugated to Alexa 647 secondary antibody (ThermoFisher A-31571) and diluted 250-fold in cell staining solution. Excess stain was washed three times again with PBS and a 5 min rest in between each wash. Cells were then stained with a 100-fold dilution of anti-human ZO-1 conjugated to Alexa 555 (ThermoFisher #MA339100A555) in cell staining solution overnight at 4 C. Excess stain was washed away 3-times with PBS with a 5 min rest in between steps. Nuclei were then stained with 4 drops per ml of NucBlue (ThermoFisher #R37605) in PBS or a 2,000-fold dilution of Hoescht (ThermoFisher H3570). Stained cells were imaged immediately or protected from light and stored at 4 C.

[0134] Administration of PBMCs to Caco-2 Intestine-Chip

[0135] Previously cryostored PBMCs were thawed in a 37 C. bead bath and resuspended in DMEM supplemented with 10% FBS and 100 U of pen/strep to a final concentration of 2 million cells per ml and labelled with a final concentration of 5 M CSFE as described above. After removing excess CSFE, PBMCs resuspended to a density of 16.7 million cells per ml in basal media, aliquoted to 6-well plates, and antibody treatments were added. The final density was 8.3 million PBMCs per ml and the incubation lasted for 4 hours. Aliquots of PBMCs in the treatment conditions were collected, fixed for later FACS analysis, and the supernatants frozen at 20 C. for measurement of cytokine levels. During the PBMC incubation on plates, samples from chips were collected to assess barrier function, and brightfield images were collected to assess epithelial morphology. After 4 hours of incubation, PBMCs to be basally administered were diluted to 50% Percol (17-0891-02) (v/v) for a final density of 4.17 million PBMCs per ml. For PBMCs to be apically administered, apical media supplemented with 40 g/ml dextran 3 kDa-cascade blue was added for a final density of 4.17 million PBMCs per ml. The respective inlet channels were aspirated from each treatment condition and 1.5 ml of PBMC suspension was added to the inlet reservoirs. 200 l of the PBMC treatment conditions were then added to a 96-well plate in triplicate for a side-by-side plate comparison. The chips were equilibrated rapidly with PBMCs by flowing at 300 l/hr for 30 minutes. Both outlet channels were then aspirated and flow was restarted. The flow rate was set to 30 l/hr for the top and bottom channels except for chips with basally administered PBMCs with 50% Percol whose bottom channel flow rate was set to 60 l/hr to account for the increased viscosity. This was considered timepoint 0 for the experiment. Flow was interrupted after 6 and 24 hours and inlet and outlet samples collected for readout analysis on V-bottom plates. For apically administered PBMCs, the inlet PBMCs reservoirs were triturated to resuspend the PBMCs and flow restarted. For cytokine and FACs analysis, 25 l of collected samples were transferred to V-bottom plates and diluted at least 5-fold with PBS. PBMCs were pelleted by centrifugation at 300 g for 5 minutes and the supernatant transferred for cytokine analysis. The PBMC pellets were fixed and stored as described previously for FACs analysis. All cytokine and barrier function samples were sealed in parafilm and stored at 20 C. until ready for analysis.

[0136] After 24 hours of PBMC administration, samples for readouts were collected as described previously. Chips were then disconnected from Pods and fixed with 4% PFA for 15 minutes at room temperature, washed with 200 l PBS for each channel, and cut in half, submerged in PBS supplemented with 0.05% sodium azide (VWR #101446-792), and at 40 C for storage.

[0137] Immunocytochemistry (ICC)

[0138] Chips were blocked with Cell Staining Solution for 0.5 hours at room temperature. Primary antibodies for anti-human antigens STEAP1 (Abcam ab207914), CEA (Abcam ab133633), CD45 (ThermoFisher MA517687), CD69 (Abcam ab201570), ZO-1 conjugated to Alexa-555 (ThermoFisher #MA339100A555) were diluted 100-fold in Cell Staining Solution and incubated on chips overnight at 4 C. Chips were then washed 3-times with PBS at room temperature including a 5-minute rest period between washes. Donkey anti-mouse Alexa-647 (ThermoFisher A-31571), anti-rat Alexa-555 (ThermoFisher SA510027), and anti-rabbit Alexa-647 (ThermoFisher A-31573) secondary antibodies were diluted 500-fold in Cell Staining Solution and incubated at room temperature for 1.5 hours and protected from light. Chips were then washed 3-times with PBS at room temperature with 5-minute rest periods between washes. Nuclei were counter stained with either NucBlue (ThermoFisher R37605) diluted 4 drops per ml of PBS or Hoechst (ThermoFisher H3570) diluted 2000-fold in PBS for 20 minutes at room temperature.

[0139] Confocal Imaging

[0140] Stained Chips were imaged on a LSM880 Confocal microscopy with a 20 objective with a NA=0.6 with Airyscan mode enabled. Z-stack image planes were collected with an optimal step size to match the Nyquist frequency of 1.5 m per plane. For each antigen, 2 chip replicates and 3 fields of view (FOV) were imaged. Airyscan processing was performed with the Zen Blue software package.

[0141] Epifluorescent Imaging

[0142] The stained, half chips were imaged on an Olympus epifluorescent microscope (IX83 Research System) at 10 and 20 magnifications to produce tiled, Z-stacked images of the chip epithelium. Quantification of immune cell migration into the epithelial compartment was assessed by analyzing each plane of a 7-plane Z-stack, 15-tile tile collected at 20 magnification for CD45 signal. Immune cells were detected using the MATLAB imfindcircles function that locates circles using a Hough transform. Analysis was performed on 2-3 Chip replicates per experimental condition.

[0143] Assessment of Epithelial Antigen Polarization

[0144] Confocal Z-stacked images of Chip antigen staining were visualized by the Fiji software suite (Schindelin, J.; Arganda-Carreras, I. & Frise, E. et al. (2012)). The expression of target localization was determined by averaging the intensity values of each XY plane at each Z-position. The Z-profile of each color channel was normalized by the maximum and minimum average intensities according the following equation:

[00001]$\left(\frac{I_i-I_{m.Math..Math.i.Math..Math.n}}{I_{m.Math..Math.a.Math..Math.x}-I_{m.Math..Math.i.Math..Math.n}}\right)$

[0145] Antigen polarization was assessed by comparing the Z-plane of the average maximum intensity (Z.sub.max) to that of the apically polarized tight junction protein ZO-1:

Z.sub.max=(Z.sub.max.sup.antigenZ.sub.max.sup.ZO-1)

[0146] Differences in Z.sub.max0 indicated apical polarization while Z.sub.max<0 indicated basolateral polarization.

[0147] Measurement of Cytokine Levels

[0148] Supernatants collected from chips and plates were analyzed by Meso Scale Discovery multi-analyte kits (K15067L-1) according to manufacturer's instructions.

[0149] Flow-Cytometry (FACs)

[0150] PBMC suspensions stored at 80 C. were thawed and centrifuged at 300 g for 5 minutes at room temperature. Supernatants were decanted and V-bottom plates gently vortexed to loosen the cell pellets. Cell suspensions were then washed twice with 200 l PBS. Antibody staining solutions were prepared by diluting anti-human CD69 (BioLegend 310910) conjugated to APC 250-fold in Cell Staining Solution and incubated 1.5 hours at room temperature or overnight at 4 C. Following antibody staining, cells were washed 3 times with PBS with a 5-minute rest period in between washes.

[0151] Labelled PBMCs were run on a BD FACs Canto II instrument at the Harvard Digestive Disease Center at Boston Children's Hospital and analyzed with the FlowJo V10 software suite.

[0152] Barrier Function

[0153] Barrier function was assessed by measuring the apparent permeability of dextran 3 kDa-cascade blue from the top to the bottom channel. Briefly, samples were collected from the top and bottom channel inlet and outlet reservoirs and stored at 20 C. until the day of measurement. An 8-point standard curve was prepared by 1-to-1 serially diluting the apical inlet media with basal inlet media. All samples and the standard curve were diluted 3-fold in PBS for a final well volume of 150 l and the fluorescence intensity of the labelled dextran was measured on a BioTek Synergy Neo instrument. Autogain was performed on the standard curve to set the appropriate fluorescence dynamic range.

[0154] The apparent permeability of the labelled dextran was calculated using the equation:

[00002]$P_{\mathrm{app}}=\left(\frac{\mathrm{dQ}/\mathrm{dt}}{C_{0}A}\right)$

[0155] Where C.sub.0 is the input concentration of dextran measured in from the apical inlet channel tracer, A is the surface area of the Chip membrane, and dQ/dt is the flux of the dextran transiting from the top to the bottom channel. The flux was calculated by measuring the number of dextran molecules that had moved from one channel to the other.

[0156] Lung Chip

[0157] Chip Activation and Deposition of Collagen I Gel on the Intestine Chip The same procedure than the intestine chip was followed except that ECM of the top channel was constituted of 200 g/mL Collagen IV, 30 g/mL Fibronectin and Laminin 5 g/mL, whereas the ECM of the bottom channel was constituted of 200 g/mL Collagen IV and 30 g/mL Fibronectin.

[0158] Seeding of the Caco-2 Intestine Chip Each channel was washed with 200 L complete SAGM culture medium before seeding human alveolar epithelial cells at 1106 cells/mL density, with 35 to 50 L of the cell suspension into the top channel inlet port while aspirating the outflow fluid from the chip surface. Chips were then placed at 37 C. for 2 hours or until cells have attached. A gentle medium wash was performed to remove excess media and chips placed back in the incubator. On day 1 and 2, complete maintenance medium was replenished in top and bottom channel of each chips. Droplets of complete medium was also added to fully cover all inlet and outlet ports to prevent evaporation. On day 3, 200 L of complete EGM-2MV culture medium was introduced to the bottom channel of each chip before seeding human microvascular endothelial cells at 5106 cells/mL density, with 15 to 20 L of the cell suspension into the bottom channel. A cradle was used to place the chips upside down while endothelial cells needed to attach for one hour at 37 C. A wash with 200 L was then performed for all top and bottom channels with complete maintenance medium or EGM-2MV respectively. Chips were placed backed in the incubator until connection to Zoes.

[0159] Alveolar Epithelial Cells and Endothelial Cells Culture On-Chip

[0160] On day 4 of the culture, chips were connected to Zoes and on day 5 air-liquid-interface was introduced in the upper channel by using complete aspiration technique followed by a one-minute step of 1000 L/hour upper channel flow rate and opt/hour bottom flow rate. Inlets and outlets reservoirs were then emptied and a microscope check performed before repetition of the previous flow step. Inlet and outlet reservoirs were emptied again, but this time leaving a small liquid layer over the bottom inlet reservoir to prevent introduction of unwanted bubbles during the flow. 2-4 mL complete Air Liquid Interface (ALI)-medium was then introduced to bottom channel inlet reservoirs and 1 mL of medium in the air channel Pod inlet reservoirs immediately followed by 1 mL addition in the outlet reservoirs. This equal media distribution in the Pod reservoirs is required to maintain static pressure in the air channel. All trays were then placed back to the Zoes and top channel set to Air while bottom channel flow rate set to 30 L/hour. Medium was refreshed in the bottom channel inlet reservoirs every other day or as needed.

[0161] Administration of PBMCs and TCBs to Lung Chip

[0162] One day prior to PBMC addition, medium from inlet and outlet reservoirs was aspirated and replaced by hydrocortisone-free ALI medium. A bubble check was performed and ALI maintained until the day after. Cryopreserved PBMCs were thawed and kept at 2)(106 cells/mL in complete medium overnight. On dosing day, PBMCs were harvested, counted, washed and stained for 20 minutes at 37 C. with 5 M CMFDA Cell Tracker Green (or equivalent dye) according to manufacturer's instructions. PBMCs were then washed and resuspended in PBMC dosing media (M199+2% FBS) at a concentration of 8106 cells/mL. 2TCB-media solution was added to the cells and incubated for 1 h at 37 C. 500 L of ALI-top channel media (M199+2% FBS) were added to the top inlet reservoirs of all chips before running a flush cycle to re-introduce liquid-liquid interface (LLI). Top channel flow rate was set to woo 4/hour and bottom to 0 and allowed to run for 3 minutes. Zo were paused and all media aspirated from top channel outlet reservoir. 500 L of the dosing solution were then added to the top channel inlets in appropriate chips, a flush cycle run at explained previously before checking PBMCs distribution under the microscope. Chips were connected back to Zoe and the top channel left static for 2-3 hours to allow the PBMCs to settle down. After that time, top channel inlets and outlets were emptied and refreshed with ALI-top channel media. The bottom channel inlets and outlets were also emptied and filled with fresh ALI media (without hydrocortisone). Chips were then connected to flow rate of 30 L/hour for both channels.

[0163] QIFIKIT

[0164] Epithelial cells were detached from the chips with TrypLE and washed before determining cell surface antigens with QIFIKIT with anti-FOLR1 primary mouse monoclonal antibody (R&D systems, Catalog #MAB5646) following manufacturer's instructions (K0078-QIFIKIT, Dako).

[0165] Measurement of Cytokine Levels

[0166] For Luminex assays, cell-free tissue culture supernatants were collected 24 and 48 h post-treatment and analyzed for cytokine/chemokine levels using Custom ProcartaPlex Human 13-plex Cytokine & Chemokine Panel from Thermo Fischer Scientific according to manufacturer's instructions.