In vitro methods for TL1A screening

TL1A, commonly called TNFSF15, belongs to the tumor necrosis factor family. TL1A is expressed in a variety of immune cells, including monocytes, macrophages, dendritic cells, T cells, and others. It competitively binds to death receptor 3 (DR3) or decoy receptor 3 (DCR3), providing a stimulatory signal to downstream signaling pathways.

This downstream signaling, in turn, affects cytokine and chemokine production in effector cells through proliferation, activation, and death.¹

Comparatively, DR3, a death domain-containing receptor, is increased after T cell activation. Overexpression causes apoptosis and NF-κ B activation in cell lines.²

The TL1A/DR3 signaling pathway has been identified as a crucial pair for mucosal immunity, contributing to the formation and maintenance of chronic inflammatory responses.

Both TL1A and DR3 are highly localized in inflamed intestinal regions of patients with inflammatory bowel disease (IBD), making them excellent therapeutic targets. This article presents a set of in vitro assay methods for evaluating possible TL1A antagonist candidates, with an emphasis on TL1A/DR3 binding.

Discussion and results

Ensuring TL1A conformation and binding

Natively, TL1A is a stable trimeric molecule produced as a membrane protein that can be broken by matrix metalloproteinases to form a soluble, functional 20kDa protein.³

Subsequent animal models have revealed the distinct functionalities of soluble and membrane-bound TL1A, with the former driving small bowel pathology by increasing levels of regulatory and activated T cells in the spleen and the latter directly acting on T cells to drive inflammatory cytokine production in the lungs.

As a result, finding the appropriate TL1A conformation, whether trimeric or monomeric, can be critical when looking for TL1A antagonist candidates.

Figure 1. Human TL1A, His Tag, Active Trimer (Cat. No. TLA-H5243) has a purity of over 85% in its trimeric form, with a total molecular weight of around 70-90 kDa verified by SEC-MALS. Image Credit: ACROBiosystems

Figure 2. Human TL1A, His Tag, Monomer (Cat. No. TLA-H5246) has a purity of over 90% in its monomeric form, with a total molecular weight of around 25-35 kDa verified by SEC-MALS. Image Credit: ACROBiosystems

After attaining the correct molecular polymeric state, the bioactivity of TL1A recombinant proteins was validated by their binding to DR3. Using SPR, recombinant DR3 and DcR3 were immobilized on a chip and bound to both monomer and trimeric TL1A to demonstrate their binding affinity and native functionality.

Figure 3. (A) Human TL1A, his tag, active trimer (Cat. No. TLA-H5243) binds to immobilized Human DR3 protein, his tag (Cat. No. TN5-H52H3) with an affinity constant of 45.8 nM as determined by an SPR assay. (B) Human TL1A, hig tag, monomer (Cat. No. TLA-H5246) binds to immobilized DcR3, Fc Tag (TNB-H5255) with an affinity constant of 0.497 nM. Image Credit: ACROBiosystems

Affinity validation of TL1A and anti-TL1A monoclonal antibody

After establishing the natural bioactivity of the TL1A recombinant protein by binding to both DR3 and DcR3, TL1A was confirmed to bind to an anti-TL1A neutralizing antibody (Tulisokibart). Again, SPR was used as the preferred approach for evaluating binding.

Figure 4. (A) Human TL1A, his tag, active trimer (Cat. No. TLA-H5243) binds to immobilized Human DR3 protein, his tag (Cat. No. TN5-H52H3) with an affinity constant of 45.8 nM as determined by an SPR assay. (B) Human TL1A, hig tag, monomer (Cat. No. TLA-H5246) binds to immobilized DcR3, Fc Tag (TNB-H5255) with an affinity constant of 0.497 nM. Image Credit: ACROBiosystems

After all components were validated, an ELISA-based inhibitor screening approach was devised. In a solution containing both DR3 and plate-bound TL1A, several doses of anti-TL1A neutralizing antibody were added, with Tulisokibart serving as a positive control.

As the concentration increases, binding activity rapidly decreases, indicating antibody suppression of TL1A:DR3 binding. The sample TL1A antagonist exhibits greater binding inhibition than Tulisokibart.

Figure 5. Serial dilutions of Human Anti-TL1A Neutralizing antibody (Tulisokibart&ACRO) (1:1 serial dilution, from 20 μg/mL to 0.009766μg/mL) was added into Human DR3: Biotinylated Human TL1A binding reactions. Image Credit: ACROBiosystems

Cell-based assay for inhibitor function validation

To further assess the inhibitor activity of anti-TL1A antibodies, a cell-based test was used to assess antibody inhibitor biofunction in a more physiologically relevant setting.

A TL1A reporter cell line was employed and treated with escalating doses of anti-TL1A antibody. Neutralization levels rose with concentration, with an off-target antibody serving as a negative control.

At a dose of 0.1 ug/mL, the reporter cell line is nearly completely neutralized, giving an alternate way to the usual ELISA for screening antibody candidates.

Figure 6. Inhibition of human TL1A protein-induced reporter activity. Image Credit: ACROBiosystems

Conclusion

In conclusion, this article provides valuable validation tests for evaluating TL1A antagonist candidates.

Screening for TL1A antagonist candidates can be a complex activity that begins with SEC-MALS to examine protein structure to verify native conformation, then moves on to SPR to study binding kinetics, ELISA for inhibitory investigations, and lastly a cell-based assay for an in vitro analysis approach.

This method, however, can be used not only for lead screening but also for bioactivity during the development, characterization, lot release, stability, and comparability of anti-TL1A antibodies/inhibitors.

Methods and materials

Surface plasmonic resonance assay

• Running Buffer: 1×HEPES (10 mM HEPES, 150 mM NaCl, 3 mM EDTA) with 0.005 % Tween-20 at pH 7.4.
• Amine Coupling Kit (BR100050, Cytiva): The Amine Coupling Kit (BR100050, Cytiva) included 115 mg of N-Hydroxysuccinimide (NHS), 750 mg of 1-Ethyl-3-(3-dimethylaminopropyl) carbodiamide hydrochloride (EDC), and 10.5 mL of 1M Ethanolamine hydrochloride-NaOH pH8.5. To dissolve the EDC and NHS, 10 mL of filtered and deionized water was added to each vial. The aliquots were kept at -18 ℃ or lower and used within two months. (See Cytiva Amine Coupling Kit booklet of instructions 22-0510-62 AG).

Chip preparation

Human DR3 Protein, His Tag (Cat. No. TN5-H52H3) was diluted to 30 µg/mL in immobilization buffer (10 mM Sodium Acetate, pH 4.5). The activator was made by combining 400 mM EDC and 100 mM NHS (Cytiva) just before injection.

The CM5 sensor chip was active for 420 seconds while the mixture flowed at 10 µL/minute. Injecting 30 µg/mL of Human DR3 Protein, His Tag (Cat. No. TN5-H52H3) in Immobilization buffer 10 mM Sodium Acetate (pH4.5) into the Fc2 sample channel at a flow rate of 10 µL/min resulted in 500 RU immobilization levels.

The chip was deactivated using 1 M Ethanolamine hydrochloride-NaOH (Cytiva) at a flow rate of 10 µL/min for 420 seconds. The reference surface Fc1 channel did not immobilize samples, but the functioning was identical to that of the active surface Fc2 channel.

Desalted

To desalt the buffer of Human TL1A, His Tag (Cat. No. TLA-H5243), use the desalting column (89890, Thermo Fisher) as instructed in the instructions. The concentration can be determined using the UV-Vis technique.

Ran analyte by multi-cycle method

Dilute Human TL1A with the Running Buffer to nine different concentrations (250, 125, 62.5, 31.25, 15.625, 7.813, 3.906, 1.953, and 0 nM). Human TL1A is injected into the channel's Fc1-Fc2 at a flow rate of 30 µL/minute.

The association phase lasts 90 seconds, followed by 210 seconds of dissociation. The Running Buffer handles all the association and dissociation processes.

Repeat eight rounds of analyte concentrations in ascending order. After each cycle of interaction analysis, the sensor chip surface should be thoroughly regenerated with 3 M magnesium chloride injection buffer at a flow rate of 20 µL/minute for 30 seconds to eliminate any bound analyte.

The Analyte injection process must then be repeated during the Analyte Human TL1A's next concentration cycle.

Other details

The entire operation was handled in Running Buffer. The remaining buffers utilized in the SPR Assay method were identical to the injection buffer, which was deposited in the rack tray of the sample compartment.

TL1A : DR3 inhibitor screening protocol

1. Coating: Coat the plate with 1 µg/well (10 µg/ml, 100 µl/well) of Human DR3 Protein, Fc Tag (Cat. No. DR3-H5253) at 4 ℃ overnight (16 hours). The protein is diluted with Coating Buffer (15 mmol/L Na₂CO₃, 35 mmol/L NaHCO₃, 7.7 mmol/L NaN₃, pH 9.6).
2. Washing: Wash each well with 300 µl of Washing Buffer (0.05 % Tween-20 in TBS, pH 7.4) four times. It is important to notice that the washing buffer must be completely removed. After washing, remove the leftover solution by aspirating or decanting. Invert the dish and let it lay on clean paper towels for a time to ensure that it is totally dry.
3. Blocking: To block the wells, add 300 µl of Blocking Buffer (1 % Casein sodium salt from bovine milk in Washing Buffer, pH 7.4) to each well and incubate at 37 °C for 1 hour.
4. Washing: Repeat step 2.
5. Adding Samples: In each well, add 50 µl of 0.009766-20µg/ml Monoclonal Anti-TL1A Antibody, Human IgG1 (Tulisokibart & ACRO), followed by 50 µl of 1ug/ml Biotinylated Human TL1A Protein, His,Avitag (Cat. No. TLA-H52Q1) and incubate at 37 ℃ for 1 hour. The sample is diluted using Sample Dilution Buffer (0.25 % Casein sodium salt from bovine milk in Washing Buffer, pH 7.4).
6. Washing: Repeat step 2.
7. Adding Detection Antibody: Add 100 µl Streptavidin Protein-HRP (Cat. No. STN-NH913) to each well and incubate at 37 ℃ for 1 hour. The antibody is diluted to 0.1 ug in Antibody Dilution Buffer (0.25 % Casein sodium salt from bovine milk in Washing Buffer, pH 7.4).
8. Washing: Repeat step 2.
9. Adding Substrate: Add 200 µl Substrate Solution to each well and incubate at 37 ℃ for 20 minutes. Avoid light. Substrate.
10. Termination: To terminate, add 50 µl of 1 mol/L sulfuric acid to each well.
11. Read OD: When reading OD at 450 nm, the final value is OD450-Blank.

Inhibitor function validation-reporter cell assay protocol

Reagents used

• The Human DR3 (TL1A receptor) (Luc) Jurkat Reporter Cell.
• RPMI Medium 1640 (Gibco, Cat. No. 11875-093).
• Fetal bovine serum (CellMax, Cat. No. SA211.02).
• Hygromycin B (Invitrogen, Cat. No. 10687010).
• Penicillin-Streptomycin (Gibco, Cat. No. 15140-122).
• Human TL1A Protein (AcroBiosystems, Cat. No. TLA-H5243).
• ONE-Glo Luciferase Assay System (Promega, Cat. No. E6120).

Protocol

1. Harvest viable cells and plate them at a density of 5x104 cells/well on 96-well culture plates (Round Bottom) with 30 µL of assay media (1640 containing 10 % FBS).
2. Make serial dilutions of anti-human TL1A neutralizing antibody in assay media.
3. Add serial dilutions of anti-human TL1A neutralizing antibody in 30 µL of assay media to wells activated by the antibody. Meanwhile, add 30 µL of assay media to the control wells that have not been activated.
4. Make serial dilutions of human TL1A protein in assay medium.
5. Add 30 µL of assay media containing serial dilutions of human TL1A protein to wells activated with the protein. Meanwhile, add 30 µL of assay media to the control wells that have not been activated.
6. Incubate at 37 °C with 5 % CO₂ for approximately 6 hours.
7. In each well, add 90 µL of ONE-Glo^™ Luciferase reagent and rock at room temperature for about 2 minutes to complete cell lysis.
8. Transfer 110 µL to a 96-well Bioluminescence detection plate after mixing with a pipette (about 20-25 times). Use a luminescence plate reader to read the plate's RLU.
9. Data analysis. Neutralization (%) = RLU (human TL1A protein stimulated wells without anti-human TL1A neutralizing antibody - human TL1A protein stimulated wells with anti-human TL1A neutralizing antibody) divided by RLU (human TL1A protein stimulated wells without anti-human TL1A neutralizing antibody - unstimulated control wells).
10. GraphPad Prism was used to fit nonlinear regression dose-response curves with log (agonist) versus response variable slope (four parameters).

References

1. Georgios Kokkotis and Giorgos Bamias (2022). TL1A as a therapeutic target in inflammatory bowel disease. Expert Review of Clinical Immunology, 18(6), pp.551–555. https://doi.org/10.1080/1744666x.2022.2074401.
2. Migone, T.S., et al. (2002). TL1A is a TNF-like ligand for DR3 and TR6/DcR3 and functions as a T cell costimulator. Immunity, (online) 16(3), pp.479–492. doi:https://doi.org/10.1016/s1074-7613(02)00283-2.
3. Vassilis Valatas, Kolios, G. and Giorgos Bamias (2019). TL1A (TNFSF15) and DR3 (TNFRSF25): A Co-stimulatory System of Cytokines With Diverse Functions in Gut Mucosal Immunity. Frontiers in Immunology, 10. https://doi.org/10.3389/fimmu.2019.00583.
4. Wang, J., et al. (2014). TL1-A can engage death receptor-3 and activate NF-kappa B in endothelial cells. BMC Nephrology, 15(1). https://doi.org/10.1186/1471-2369-15-178.
5. WATKINS, J. D., DICKERSON, C. T., ROJAS, R., REISSMAN, M. & MCNEELEY, P. 2021. HUMANIZED ANTIBODIES TO TNF-LIKE LIGAND 1A (TL1A) AND USES THEREOF US patent application 17/110004
6. Xu, W.-D., Li, R. and Huang, A.-F. (2022). Role of TL1A in Inflammatory Autoimmune Diseases: A Comprehensive Review. Frontiers in Immunology, 13. https://doi.org/10.3389/fimmu.2022.891328.
7. Zwolak, A., et al. (2022). A stable, engineered TL1A ligand co-stimulates T cells via specific binding to DR3. Scientific reports, (online) 12(1), p.20538. https://doi.org/10.1038/s41598-022-24984-y.

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