mil 21r  (R&D Systems)

Journal: bioRxiv

Article Title: Potent antitumor activity of a designed interleukin-21 mimic

doi: 10.1101/2024.12.06.626481

Figure Lengend Snippet: Computationally designed IL-21 mimic recapitulates receptor interactions with superior stability and human-mouse cross-reactivity. (A) The optimized IL-21 mimic, 21h10, is designed by recapitulating the helical bundle structure of the native hIL-21. Unstructured regions in the hIL-21 are removed, and short helices are extended to accommodate improved intramolecular packing. Some of the interface residues are conserved from the native hIL-21 for the design of initial hits. Other residues from the de novo scaffolds are further optimized, as shown. (B) Association and dissociation of 21h10 to human and murine IL-21R and 𝛾 c show concentration-dependent binding curves of 21h10. 21h10’s K D against hIL-21R is 482 pM, hIL-21R/h𝛾 c is 86.7 nM, mIL-21R is 7.87 nM, and mIL-21R/m𝛾 c is 72.8 nM. For measuring K D against hIL-21R and mIL-21R, IL-21R was immobilized on the biolayer interferometry (BLI) tip. For measuring K D against hIL-21R/h𝛾 c and mIL-21R/m𝛾 c , 𝛾 c was immobilized on the BLI tip and IL-21R was provided in solution with 1.5-fold molar excess to 21h10. Dotted lines show curve fits of raw data using a 1:1 Langmuir binding model. (C) The optimized IL-21 mimic, 21h10, exhibits monodispersed distribution when sized through size-exclusion chromatography using Superdex 75 10/300 GL column. (D) Wavelength scan from 200 nm to 250 nm confirmed 𝛼-helical secondary structure present in 21h10. Thermal melt from 25°C to 95°C with 222 nm scan revealed outstanding thermal stability of 21h10, with a T m around 75°C. (E) Crystal structure of 21h10 in complex with hIL-21R and h𝛾 c (PDB: XXXX). (F) 21h10 conserves key molecular interactions in the site I interface (left) and site IIa interface (right) observed in the structure of the human IL-21 receptor complex. (Human IL-21 receptor complex, PDB ID: 8ENT).

Article Snippet: For IL-21R labeling, either human or murine IL-21R with human Fc-tag (R&D Systems Cat# 991-R2, R&D Systems Cat# 596-MR) were used, and for receptor complex labeling, either human IL-21R with His-tag (R&D Systems Cat# 9249-R2) and human 𝛾 c with Fc-tag (Acro Biosystems Cat# ILG-H5256), or mIL-21R with His-tag (Sino Biological Cat# 51184-M08H) and murine 𝛾 c with Fc-tag (R&D Systems Cat# 784-MR) were used.

Techniques: Concentration Assay, Binding Assay, Size-exclusion Chromatography

Journal: bioRxiv

Article Title: Potent antitumor activity of a designed interleukin-21 mimic

doi: 10.1101/2024.12.06.626481

Figure Lengend Snippet: ( A ) Pre-activated human and murine CD8 + T cells were treated with increasing doses of native IL-21 and 21h10 for 20 minutes and stained with AF488-phospho-STAT1 and AF647-phospho-STAT3 for flow cytometry analysis. ( B ) Genes related to cellular signaling and phenotype are compared for expression levels between murine CD8 + T cells treated with PBS, mIL-21, or 21h10 at 100 pM or 1 nM. * indicates memory-related genes, and ** indicates effector-related genes. Murine CD8 + T cells were pre-activated with TCR signals and treated with the cytokines at different concentrations for 24 hours, and cells were harvested for RNA-sequencing. ( C ) The percentage of IFN-𝛾-secreting cells within isolated CD8 + T cells upon treating PBS, mIL-21, or 21h10 at 1 nM, was quantified using flow cytometry after 5 hours of stimulation with PMA/Ionomycin in the presence of a protein transport inhibitor. ( D ) LCMV was inoculated into healthy mice along with daily injections of PBS, mIL-21 (2.5 µg per mouse), or 21h10 (2.5 µg per mouse). LCMV-specific CD8 + T cells from PBMC and spleen were analyzed for granzyme B expression. ( E ) Computational model of 21AT36, which is redesigned from 21h10 using ProteinMPNN, to bind IL-21R but not bind 𝛾 c . The mutated residues are highlighted in red, which abolish interactions with 𝛾 c . The 𝛾 c is included in the figure to show where the interface is positioned, not to imply that 21AT36 binds to 𝛾 c . ( F ) Western blot for STAT and pSTAT with vehicle (PBS), mIL-21, 21h10, and 21AT36 in TRP1 high CD8 + T cells. ( G ) MC38-bearing mice were treated with PBS, 21AT36, 21h10, or anti-PD-1 for 14 days. ( H ) Mice were inoculated with MC38 and treated with PBS, mIL-21, 10-fold dose mIL-21, and 21h10. ( I ) Western blot for STAT and pSTAT in the spleens of mice treated with vehicle (PBS), mIL-21, and 21h10 ( J ) Normalized pSTAT3/STAT3 in spleens of treated mice over 24 hours from ( I ). ( K ) MC38 rechallenges with MC38-survivor mice from previous 21h10 or anti-PD-1 treatment.

Article Snippet: For IL-21R labeling, either human or murine IL-21R with human Fc-tag (R&D Systems Cat# 991-R2, R&D Systems Cat# 596-MR) were used, and for receptor complex labeling, either human IL-21R with His-tag (R&D Systems Cat# 9249-R2) and human 𝛾 c with Fc-tag (Acro Biosystems Cat# ILG-H5256), or mIL-21R with His-tag (Sino Biological Cat# 51184-M08H) and murine 𝛾 c with Fc-tag (R&D Systems Cat# 784-MR) were used.

Techniques: Staining, Flow Cytometry, Expressing, RNA Sequencing Assay, Isolation, Western Blot

Journal: bioRxiv

Article Title: Potent antitumor activity of a designed interleukin-21 mimic

doi: 10.1101/2024.12.06.626481

Figure Lengend Snippet: Computationally designed IL-21 mimic recapitulates receptor interactions with superior stability and human-mouse cross-reactivity. (A) The optimized IL-21 mimic, 21h10, is designed by recapitulating the helical bundle structure of the native hIL-21. Unstructured regions in the hIL-21 are removed, and short helices are extended to accommodate improved intramolecular packing. Some of the interface residues are conserved from the native hIL-21 for the design of initial hits. Other residues from the de novo scaffolds are further optimized, as shown. (B) Association and dissociation of 21h10 to human and murine IL-21R and 𝛾 c show concentration-dependent binding curves of 21h10. 21h10’s K D against hIL-21R is 482 pM, hIL-21R/h𝛾 c is 86.7 nM, mIL-21R is 7.87 nM, and mIL-21R/m𝛾 c is 72.8 nM. For measuring K D against hIL-21R and mIL-21R, IL-21R was immobilized on the biolayer interferometry (BLI) tip. For measuring K D against hIL-21R/h𝛾 c and mIL-21R/m𝛾 c , 𝛾 c was immobilized on the BLI tip and IL-21R was provided in solution with 1.5-fold molar excess to 21h10. Dotted lines show curve fits of raw data using a 1:1 Langmuir binding model. (C) The optimized IL-21 mimic, 21h10, exhibits monodispersed distribution when sized through size-exclusion chromatography using Superdex 75 10/300 GL column. (D) Wavelength scan from 200 nm to 250 nm confirmed 𝛼-helical secondary structure present in 21h10. Thermal melt from 25°C to 95°C with 222 nm scan revealed outstanding thermal stability of 21h10, with a T m around 75°C. (E) Crystal structure of 21h10 in complex with hIL-21R and h𝛾 c (PDB: XXXX). (F) 21h10 conserves key molecular interactions in the site I interface (left) and site IIa interface (right) observed in the structure of the human IL-21 receptor complex. (Human IL-21 receptor complex, PDB ID: 8ENT).

Article Snippet: Similarly, to measure binding affinity of 21AT36 to hIL-21R or mIL-21R, biotinylated hIL-21R (R&D Systems Cat# AVI9249) or biotinylated mIL-21R (Acro Biosystems Cat# ILR-M82E3), respectively, were immobilized on streptavidin-coated biosensors (SAForteBio) at 5 μg/mL in the octet binding buffer, with 3-fold serial dilutions of the ligand starting at 100 nM, with 300 seconds for association and another 300 seconds for dissociation.

Techniques: Concentration Assay, Binding Assay, Size-exclusion Chromatography

Journal: bioRxiv

Article Title: Potent antitumor activity of a designed interleukin-21 mimic

doi: 10.1101/2024.12.06.626481

Figure Lengend Snippet: ( A ) Pre-activated human and murine CD8 + T cells were treated with increasing doses of native IL-21 and 21h10 for 20 minutes and stained with AF488-phospho-STAT1 and AF647-phospho-STAT3 for flow cytometry analysis. ( B ) Genes related to cellular signaling and phenotype are compared for expression levels between murine CD8 + T cells treated with PBS, mIL-21, or 21h10 at 100 pM or 1 nM. * indicates memory-related genes, and ** indicates effector-related genes. Murine CD8 + T cells were pre-activated with TCR signals and treated with the cytokines at different concentrations for 24 hours, and cells were harvested for RNA-sequencing. ( C ) The percentage of IFN-𝛾-secreting cells within isolated CD8 + T cells upon treating PBS, mIL-21, or 21h10 at 1 nM, was quantified using flow cytometry after 5 hours of stimulation with PMA/Ionomycin in the presence of a protein transport inhibitor. ( D ) LCMV was inoculated into healthy mice along with daily injections of PBS, mIL-21 (2.5 µg per mouse), or 21h10 (2.5 µg per mouse). LCMV-specific CD8 + T cells from PBMC and spleen were analyzed for granzyme B expression. ( E ) Computational model of 21AT36, which is redesigned from 21h10 using ProteinMPNN, to bind IL-21R but not bind 𝛾 c . The mutated residues are highlighted in red, which abolish interactions with 𝛾 c . The 𝛾 c is included in the figure to show where the interface is positioned, not to imply that 21AT36 binds to 𝛾 c . ( F ) Western blot for STAT and pSTAT with vehicle (PBS), mIL-21, 21h10, and 21AT36 in TRP1 high CD8 + T cells. ( G ) MC38-bearing mice were treated with PBS, 21AT36, 21h10, or anti-PD-1 for 14 days. ( H ) Mice were inoculated with MC38 and treated with PBS, mIL-21, 10-fold dose mIL-21, and 21h10. ( I ) Western blot for STAT and pSTAT in the spleens of mice treated with vehicle (PBS), mIL-21, and 21h10 ( J ) Normalized pSTAT3/STAT3 in spleens of treated mice over 24 hours from ( I ). ( K ) MC38 rechallenges with MC38-survivor mice from previous 21h10 or anti-PD-1 treatment.

Article Snippet: Similarly, to measure binding affinity of 21AT36 to hIL-21R or mIL-21R, biotinylated hIL-21R (R&D Systems Cat# AVI9249) or biotinylated mIL-21R (Acro Biosystems Cat# ILR-M82E3), respectively, were immobilized on streptavidin-coated biosensors (SAForteBio) at 5 μg/mL in the octet binding buffer, with 3-fold serial dilutions of the ligand starting at 100 nM, with 300 seconds for association and another 300 seconds for dissociation.

Techniques: Staining, Flow Cytometry, Expressing, RNA Sequencing Assay, Isolation, Western Blot