Journal: Frontiers in Immunology

Article Title: Dual blockade of IL-17A and IL-36 pathways via a bispecific antibody exhibits enhanced anti-inflammatory potency

doi: 10.3389/fimmu.2024.1434127

Figure Lengend Snippet: HB0043 retained the affinities and blockade activity to both targets. (A) Schematics of the anti-IL-36R×IL-17A BsAb (HB0043) structure. HB0043 was constructed by linking a scFv targeting IL-36R to the C-terminal domain of the anti-IL-17A IgG backbone. (B) The kinetics profile of HB0043 and HB0017 (the parental anti-IL-17A mAb) binding to human IL-17A, and human IL-36R binding to HB0043 and HB0034 (the parental anti-IL-36R mAb) measured by SPR assay. (C) The ability of HB0043 to neutralize IL-17A signaling was measured and compared to that of HB0017, using an IL-17A blockade cell-based assay. The secretion of IL-6 was monitored, which was induced by human IL-17A (0.3 nM) and TNF-α (0.6 nM) in HT-1080 cell. (D) The inhibition of IL-6 releases from NCI/ADR-RES cells was used as an evaluation of IL-36R signaling inhibition. NCI/ADR-RES cells were stimulated with human IL36α (13.5 nM) in the presence of HB0043 (32 pM to 245 nM) and HB0034 (4 pM to 336 nM).

Article Snippet: Animal use for generation of humanized anti-human IL-36R mAb HB0034 and anti-mouse IL-36R mAb had approvals of IACUC of Shanghai Model Organisms.

Techniques: Activity Assay, Construct, Binding Assay, SPR Assay, Cell Based Assay, Inhibition

Journal: Frontiers in Immunology

Article Title: Dual blockade of IL-17A and IL-36 pathways via a bispecific antibody exhibits enhanced anti-inflammatory potency

doi: 10.3389/fimmu.2024.1434127

Figure Lengend Snippet: Binding affinity of BsAbs and mAbs to the targets from different species.

Article Snippet: Animal use for generation of humanized anti-human IL-36R mAb HB0034 and anti-mouse IL-36R mAb had approvals of IACUC of Shanghai Model Organisms.

Techniques: Binding Assay

Journal: Frontiers in Immunology

Article Title: Dual blockade of IL-17A and IL-36 pathways via a bispecific antibody exhibits enhanced anti-inflammatory potency

doi: 10.3389/fimmu.2024.1434127

Figure Lengend Snippet: HB0043 effectively antagonized the production of IL-6 induced by IL-17A and IL-36 in NHDF cells. (A–C) The ability of HB0043 to block IL-36R signaling and IL-17A signaling was measured and compared to that of HB0034 and HB0017, using IL-17A and IL-36R blockade cell-based assay. The secretion of IL-6 was monitored, which was induced by 0.5 ng/ml human IL-17A and 15 ng/ml IL-36α (A) , 1 ng/ml IL-36β (B) or 2 ng/ml IL-36γ (C) in NHDF cells overnight. Data are expressed as means ± SEM, n=3. *P<0.05, **P<0.01, ***P<0.01, ****P < 0.0001, as determined by one-way ANOVA.

Article Snippet: Animal use for generation of humanized anti-human IL-36R mAb HB0034 and anti-mouse IL-36R mAb had approvals of IACUC of Shanghai Model Organisms.

Techniques: Blocking Assay, Cell Based Assay

Journal: Frontiers in Immunology

Article Title: Dual blockade of IL-17A and IL-36 pathways via a bispecific antibody exhibits enhanced anti-inflammatory potency

doi: 10.3389/fimmu.2024.1434127

Figure Lengend Snippet: Nonclinical safety evaluation in cynomolgus monkeys.

Article Snippet: Animal use for generation of humanized anti-human IL-36R mAb HB0034 and anti-mouse IL-36R mAb had approvals of IACUC of Shanghai Model Organisms.

Techniques: Concentration Assay

Journal: Frontiers in Immunology

Article Title: Dual blockade of IL-17A and IL-36 pathways via a bispecific antibody exhibits enhanced anti-inflammatory potency

doi: 10.3389/fimmu.2024.1434127

Figure Lengend Snippet: Single-dose pharmacokinetic study in cynomolgus monkeys.

Article Snippet: Animal use for generation of humanized anti-human IL-36R mAb HB0034 and anti-mouse IL-36R mAb had approvals of IACUC of Shanghai Model Organisms.

Techniques:

Journal: Frontiers in Immunology

Article Title: Dual blockade of IL-17A and IL-36 pathways via a bispecific antibody exhibits enhanced anti-inflammatory potency

doi: 10.3389/fimmu.2024.1434127

Figure Lengend Snippet: HB0043 retained the affinities and blockade activity to both targets. (A) Schematics of the anti-IL-36R×IL-17A BsAb (HB0043) structure. HB0043 was constructed by linking a scFv targeting IL-36R to the C-terminal domain of the anti-IL-17A IgG backbone. (B) The kinetics profile of HB0043 and HB0017 (the parental anti-IL-17A mAb) binding to human IL-17A, and human IL-36R binding to HB0043 and HB0034 (the parental anti-IL-36R mAb) measured by SPR assay. (C) The ability of HB0043 to neutralize IL-17A signaling was measured and compared to that of HB0017, using an IL-17A blockade cell-based assay. The secretion of IL-6 was monitored, which was induced by human IL-17A (0.3 nM) and TNF-α (0.6 nM) in HT-1080 cell. (D) The inhibition of IL-6 releases from NCI/ADR-RES cells was used as an evaluation of IL-36R signaling inhibition. NCI/ADR-RES cells were stimulated with human IL36α (13.5 nM) in the presence of HB0043 (32 pM to 245 nM) and HB0034 (4 pM to 336 nM).

Article Snippet: Animal use for generation of humanized anti-human IL-36R mAb HB0034 and anti-mouse IL-36R mAb had approvals of IACUC of Shanghai Model Organisms.

Techniques: Activity Assay, Construct, Binding Assay, SPR Assay, Cell Based Assay, Inhibition

Journal: Frontiers in Immunology

Article Title: Dual blockade of IL-17A and IL-36 pathways via a bispecific antibody exhibits enhanced anti-inflammatory potency

doi: 10.3389/fimmu.2024.1434127

Figure Lengend Snippet: Binding affinity of BsAbs and mAbs to the targets from different species.

Article Snippet: Animal use for generation of humanized anti-human IL-36R mAb HB0034 and anti-mouse IL-36R mAb had approvals of IACUC of Shanghai Model Organisms.

Techniques: Binding Assay

Journal: Frontiers in Immunology

Article Title: Dual blockade of IL-17A and IL-36 pathways via a bispecific antibody exhibits enhanced anti-inflammatory potency

doi: 10.3389/fimmu.2024.1434127

Figure Lengend Snippet: HB0043 effectively antagonized the production of IL-6 induced by IL-17A and IL-36 in NHDF cells. (A–C) The ability of HB0043 to block IL-36R signaling and IL-17A signaling was measured and compared to that of HB0034 and HB0017, using IL-17A and IL-36R blockade cell-based assay. The secretion of IL-6 was monitored, which was induced by 0.5 ng/ml human IL-17A and 15 ng/ml IL-36α (A) , 1 ng/ml IL-36β (B) or 2 ng/ml IL-36γ (C) in NHDF cells overnight. Data are expressed as means ± SEM, n=3. *P<0.05, **P<0.01, ***P<0.01, ****P < 0.0001, as determined by one-way ANOVA.

Article Snippet: Animal use for generation of humanized anti-human IL-36R mAb HB0034 and anti-mouse IL-36R mAb had approvals of IACUC of Shanghai Model Organisms.

Techniques: Blocking Assay, Cell Based Assay

Journal: Frontiers in Immunology

Article Title: Dual blockade of IL-17A and IL-36 pathways via a bispecific antibody exhibits enhanced anti-inflammatory potency

doi: 10.3389/fimmu.2024.1434127

Figure Lengend Snippet: The combination of mAbs targeting IL-17A and IL-36R showed stronger inhibition of inflammation relative to mAbs alone in oxazolone-induced mouse atopic dermatitis model. (A) Male C57BL/6J mice were smeared with 5.0% OXA on the shaved back skin and ear on Day0 and followed by a daily topical application of 5.0% OXA on back and 0.5% OXA on ear for thirteen consecutive days from Day7 for acute disease induction. Antibodies (HB0017, HB0034SA, HB0017+HB0034SA) were administered intraperitoneally into mice at 50 mg/kg twice a week. The control group received PBS intraperitoneal injections. (B, C) Combination of anti-IL-36R and anti-IL-17A antibodies significantly ameliorated OXA induced increase of ear thickness (B) and reduced the clinical score of back skin (C) . Clinical score: the comprehensive evaluation of oedema, erythema, dryness and excoriation and each symptom was scored independently on a scale from 0 to 3: 0, none; 1, slight; 2, moderate; 3, severe. Data are expressed as min to max, n=8. *p ≤ 0.05. (D, E) Back skin samples were collected for subsequent histopathological evaluation (D) and H&E staining (E) . Vehicle group: HE staining shows hyperkeratosis, significant thickening of the stratum corneum, acanthosis, and local finger-like protrusions of the epidermis into the dermis. The dermis is edematous, the capillaries are dilated and congested, and accompanied by a small amount of inflammatory cell infiltration. HB0017 and HB0034SA group shows slightly improvement with mild inflammatory cell infiltration, organic lesions in corneum and dermis haven’t shown remission. HB0017+HB0034SA group: Significantly reduced hyperkeratosis and thickening of stratum corneum. Vasodilatation—green arrow; Lymphocytes—blue arrow; Hyperkeratosis/parakeratosis—red arrow. *P<0.05, **P<0.01, ***P<0.01.

Article Snippet: Animal use for generation of humanized anti-human IL-36R mAb HB0034 and anti-mouse IL-36R mAb had approvals of IACUC of Shanghai Model Organisms.

Techniques: Inhibition, Control, Staining

Journal: Frontiers in Immunology

Article Title: Dual blockade of IL-17A and IL-36 pathways via a bispecific antibody exhibits enhanced anti-inflammatory potency

doi: 10.3389/fimmu.2024.1434127

Figure Lengend Snippet: Dual blockade of IL-17A and IL-36R showed stronger inhibition of inflammation relative to mAbs alone in IMQ-induced mouse psoriasis model. (A) Schematic plot showing therapeutic dosing regime of BALB/C mice for experimental data in IMQ-induced psoriasis model. Mice were smeared with 5.0% IMQ (62.5mg) on the shaved back skin from Day1 to Day 7 daily. Antibodies (HB0017, HB0034SA, HB0017+HB0034SA) were administered intraperitoneally into mice at 50 mg/kg twice a week. The control group received PBS intraperitoneal injections. (B) Body weight. (C) PASI score evaluation result, (D, E) Back skin samples were collected for subsequent histopathological evaluation (D) and H&E staining (E) (black arrow: stratum corneum thicken, green arrow: loose structure, red arrow: acanthosis, yellow arrow: trochanterellus lengthen and rodlike, blue arrow: inflammatory cell infiltration, orange arrow: blooding in papillary layer). (F) Relative skin mRNA expression of skin inflammation related cytokines (IL-17A, IL-22, IL-23, IL-36α, IL-36β, IL-36γ). *P<0.05, **P<0.01, ***P<0.01, ****P<0.0001.

Article Snippet: Animal use for generation of humanized anti-human IL-36R mAb HB0034 and anti-mouse IL-36R mAb had approvals of IACUC of Shanghai Model Organisms.

Techniques: Inhibition, Control, Staining, Expressing

Journal: Frontiers in Immunology

Article Title: Dual blockade of IL-17A and IL-36 pathways via a bispecific antibody exhibits enhanced anti-inflammatory potency

doi: 10.3389/fimmu.2024.1434127

Figure Lengend Snippet: Binding affinity of BsAbs and mAbs to the targets from different species.

Article Snippet: For generation of surrogate anti-mouse IL-36R mAb HB0034SA, IL-36R knockout mice on C57BL/6 background (Shanghai Model Organisms Co., Ltd) were immunized with mouse IL-36R protein (R&D Biosystems, Cat# 2354-RP/CF) emulsified in complete Freund’s adjuvant.

Techniques: Binding Assay

Journal: Frontiers in Immunology

Article Title: Dual blockade of IL-17A and IL-36 pathways via a bispecific antibody exhibits enhanced anti-inflammatory potency

doi: 10.3389/fimmu.2024.1434127

Figure Lengend Snippet: The combination of mAbs targeting IL-17A and IL-36R showed stronger inhibition of inflammation relative to mAbs alone in oxazolone-induced mouse atopic dermatitis model. (A) Male C57BL/6J mice were smeared with 5.0% OXA on the shaved back skin and ear on Day0 and followed by a daily topical application of 5.0% OXA on back and 0.5% OXA on ear for thirteen consecutive days from Day7 for acute disease induction. Antibodies (HB0017, HB0034SA, HB0017+HB0034SA) were administered intraperitoneally into mice at 50 mg/kg twice a week. The control group received PBS intraperitoneal injections. (B, C) Combination of anti-IL-36R and anti-IL-17A antibodies significantly ameliorated OXA induced increase of ear thickness (B) and reduced the clinical score of back skin (C) . Clinical score: the comprehensive evaluation of oedema, erythema, dryness and excoriation and each symptom was scored independently on a scale from 0 to 3: 0, none; 1, slight; 2, moderate; 3, severe. Data are expressed as min to max, n=8. *p ≤ 0.05. (D, E) Back skin samples were collected for subsequent histopathological evaluation (D) and H&E staining (E) . Vehicle group: HE staining shows hyperkeratosis, significant thickening of the stratum corneum, acanthosis, and local finger-like protrusions of the epidermis into the dermis. The dermis is edematous, the capillaries are dilated and congested, and accompanied by a small amount of inflammatory cell infiltration. HB0017 and HB0034SA group shows slightly improvement with mild inflammatory cell infiltration, organic lesions in corneum and dermis haven’t shown remission. HB0017+HB0034SA group: Significantly reduced hyperkeratosis and thickening of stratum corneum. Vasodilatation—green arrow; Lymphocytes—blue arrow; Hyperkeratosis/parakeratosis—red arrow. *P<0.05, **P<0.01, ***P<0.01.

Article Snippet: For generation of surrogate anti-mouse IL-36R mAb HB0034SA, IL-36R knockout mice on C57BL/6 background (Shanghai Model Organisms Co., Ltd) were immunized with mouse IL-36R protein (R&D Biosystems, Cat# 2354-RP/CF) emulsified in complete Freund’s adjuvant.

Techniques: Inhibition, Control, Staining

Journal: Frontiers in Immunology

Article Title: Dual blockade of IL-17A and IL-36 pathways via a bispecific antibody exhibits enhanced anti-inflammatory potency

doi: 10.3389/fimmu.2024.1434127

Figure Lengend Snippet: Dual blockade of IL-17A and IL-36R showed stronger inhibition of inflammation relative to mAbs alone in IMQ-induced mouse psoriasis model. (A) Schematic plot showing therapeutic dosing regime of BALB/C mice for experimental data in IMQ-induced psoriasis model. Mice were smeared with 5.0% IMQ (62.5mg) on the shaved back skin from Day1 to Day 7 daily. Antibodies (HB0017, HB0034SA, HB0017+HB0034SA) were administered intraperitoneally into mice at 50 mg/kg twice a week. The control group received PBS intraperitoneal injections. (B) Body weight. (C) PASI score evaluation result, (D, E) Back skin samples were collected for subsequent histopathological evaluation (D) and H&E staining (E) (black arrow: stratum corneum thicken, green arrow: loose structure, red arrow: acanthosis, yellow arrow: trochanterellus lengthen and rodlike, blue arrow: inflammatory cell infiltration, orange arrow: blooding in papillary layer). (F) Relative skin mRNA expression of skin inflammation related cytokines (IL-17A, IL-22, IL-23, IL-36α, IL-36β, IL-36γ). *P<0.05, **P<0.01, ***P<0.01, ****P<0.0001.

Article Snippet: For generation of surrogate anti-mouse IL-36R mAb HB0034SA, IL-36R knockout mice on C57BL/6 background (Shanghai Model Organisms Co., Ltd) were immunized with mouse IL-36R protein (R&D Biosystems, Cat# 2354-RP/CF) emulsified in complete Freund’s adjuvant.

Techniques: Inhibition, Control, Staining, Expressing

Journal: Frontiers in Immunology

Article Title: Dual blockade of IL-17A and IL-36 pathways via a bispecific antibody exhibits enhanced anti-inflammatory potency

doi: 10.3389/fimmu.2024.1434127

Figure Lengend Snippet: Binding affinity of BsAbs and mAbs to the targets from different species.

Article Snippet: For generation of humanized anti-human IL-36R mAb HB0034, BALB/c mice were immunized with recombinant human IL-36R protein (ACRO Biosystems, Cat# IL2-H5254).

Techniques: Binding Assay