E. Immunology [E. Immunology] E-1 Hydrophobic Cell Penetrating Peptide-Combined Nuclear Localization Signal Suppresses Pro-Inflammatory Cytokines In Inflammatory Bowel Disease Seokwon Lee¹, Jaehyeon Kim¹, Hyunji Lee¹, Hyeontae Kang¹, Mingu Kang¹, Eunhee Yang¹, Eunna Chung¹, Daewoong Jo¹* ¹Immune Disease Team, Cellivery Therapeutics, Inc., Seoul 03929, Korea Inflammatory bowel disease (IBD) includes two types of inflammatory diseases, ulcerative colitis (UC) and Crohn’s disease (CD). To target the IBD, hydrophobic cell penetrating peptide-combined nuclear localization signal (NLS) was used, as
named improved Cell-Permeable nuclear import inhibitor (iCP-NI). The NLS of iCP-NI was adopted from NF-κB, and then cyclized for enhancement of in vivo stability. iCP-NI competitively binds to the importins, the nuclear transporter, and suppresses the nuclear import of transcription factors upregulating the cytokines. The efficacy of iCP-NI against IBD was evaluated in two different animal models by intrarectal administration of iCP-NI. In dextran sulfate sodium (DSS)-induced UC mouse model, iCP-NI recovered the shortened small intestine by 31% (compare to the vehicle group), and the
inflammatory lesions were also diminished, resulting the restoration of destructured epithelium of small intestine. For the 2, 4, 6-trinitrobenzenesulfonic acid solution (TNBS)-induced CD mouse model, the pathological score (i.e. edema, ulcer, and vascular congestion) was also decreased by 71%, and the internal layer of intestine was restructured. Additionally, the pro-inflammatory cytokines, such as TNF-α, IFN-γ, and IL-6, were reduced in the blood stream by 64%, 97%, and 72%, respectively. Based on the above-described anti-inflammatory effects of iCP-NI, iCP-NI can be a powerful
next-generation. Hydrophobic Cell Penetrating Peptide-Combined Nuclear Localization Signal Suppresses Pro-Inflammatory Cytokines In Inflammatory Bowel Disease Seokwon Lee, Jaehyeon Kim, Hyunji Lee, Hyeontae Kang, Mingu Kang, Eunhee Yang, Eunna Chung and Daewoong Jo Cellivery R&D Institute, Cellivery Therapeutics, Inc., Seoul 03929, Korea. BACKGROUND Inflammatory bowel disease (IBD) includes two types of inflammatory diseases, ulcerative colitis (UC) and Crohn’s disease (CD). A hydrophobic cell-penetrating peptide-combined nuclear localization signal (NLS), named improved Cell-Permeable nuclear
import inhibitor (iCP-NI), was used to target the IBD. The NLS of iCP-NI was adopted from NF-κB and cyclized for in vivo stability enhancement. iCP-NI competitively binds to the importins, the nuclear transporter, and suppresses the nuclear import of transcription factors upregulating the cytokines. AIM METHODS This study aims to develop an iCP-NI as a The efficacy of iCP-NI against IBD was evaluated in the DSS-induced UC mouse model and therapeutic molecule for IBD by determining TNBS-induced CD mouse model. After the mouse was diseased, iCP-NI was intravenously its mode of action and
efficacy in regulating (IV) or intrarectally (IR) administered. The scoring indexes were used to estimate the the expression of pro-inflammatory cytokines. microscopic or histological analysis. The cytokine expressions were also evaluated. RESULTS Figure 1. The Structure And Mode Of Action (MoA) Of Figure 3. Therapeutic Effect Of iCP-NI Improved Cell-Permeable Nuclear Import Inhibitor (iCP-NI) On DSS-Induced Ulcerative Colitis Mouse Model Cytoplasm iCP-NI Treatment (50 mg/kg, IV Injection) Inflammation Activated Once A Day, 7 Days Transcription Factors (IATFs) Initiating Factors NF-κB NFAT
C57BL/6 Day 0 … 6 7 … 9 … 13 Microbial (Female, 8 Weeks) Genetic Factors Environmental STAT AP-1 Factors Dysbiosis 3% DSS In Drinking Water, 7 Days Sampling Sampling Endoscopy & Sampling Antibiotics iCP-NI Immune Cell Recruiting Food Intake Importin α Smoking Competitive H&E Staining : Intestinal Structure Activation Of Inflammatory Cascades Inhibition Upregulating Pro-Inflammatory Cytokines Nucleus Colon Length 3% DSS (i.e. IL-6, IL-12, IL-18, IL-23, IL-1β, And TNF-α) Impaired Epithelial Barrier Functions IATFs No Treatment Diluent iCP-NI Importin α Importin α Development Of IBD No Treatment
Figure 2. Restored Expression Of E-Cadherin In Colonic Epithelial Cell Line 3% DSS Diluent Treated With Dextran Sulfate Sodium (DSS) iCP-NI I.V Day 7 Scale Bar: 200 μm Immuno-Fluorescence Labeling : α-E-Cadherin 10 Day 7 Endoscopy Western Blot : α-E-Cadherin 2% DSS 8 * P<0.05 3% DSS Control Diluent iCP-NI 31% No Treatment Diluent iCP-NI 2% DSS 6 iCP-NI (μM) - - 1 10 50 Colon Length (Cm) 100 4 E-Cadherin 75 E-Cadherin / DAPI 2 50 β-actin 38 0 No Diluent iCP-NI (IV) Treatment 3% DSS Day 13 Colonic Epithelial Cell Line (Caco-2) Scale bar = 20 μm Figure 4. Therapeutic Effect Of iCP-NI On 2, 4,
6-Trinitrobenzenesulfonic Figure 5. Downregulated Cytokine Level In The Plasma Of Acid Solution (TNBS)-Induced Crohn’s Disease (CD) Mouse Model TNBS-Induced CD Mouse Model By iCP-NI Treatment iCP-NI Treatment (50 mg/kg) IV or IR Injection, Twice A Day CBA Assay : Expression Of Pro-Inflammatory Cytokines BALB/c Day 1 2 3 4 5 6 ** P=0.0001 30 ** P=0.007 (Male, 8 Weeks) * ** P=0.78 P=0.007 ** P=0.0017 ** P=0.0003 TNBS (25 mg/kg in 40% EtOH) Sampling 4 5 4 25 20 Pathological Score 3 3 15 9 TNF-α Concentration (pg/ml) 2 IFN-γ Concentration (pg/ml) IL-6 Concentration (pg/ml) Colon Length * * P=0.001
64% 2 10 * * P=0.001 1 1 78% 5 82% 72% Pathological Score (0~9) No Treatment 6 0 No - IV IR 0 No - IV 97% 0 No - IV IR IR Diluent Treatment iCP-NI Treatment iCP-NI Treatment iCP-NI IV 3 65% 71% TNBS TNBS TNBS iCP-NI IR CONCLUSION 0 No - IV IR Treatment iCP-NI TNBS The expression of E-cadherin, the junction protein, was restored with iCP-NI H&E Staining : Intestinal Structure treatment in Caco-2 cells after being damaged by 2% DSS. It implied that the TNBS No Treatment Diluent iCP-NI (IR) iCP-NI (IV) iCP-NI could recover the damaged internal layer of the intestines. This hypothesis was
validated via in vivo study of IBD mouse models, resulting in reduced pathological scores in iCP-NI treatment groups. The expressions of various pro-inflammatory cytokines were also significantly downregulated. Scale bar = 200 μm Wallace Score : Microscopic Score Ameho Score : Histological Score Overall, these results suggest that iCP-NI is a potential treatment for IBD. P<0.001 *** * P<0.05 P<0.01 P<0.01 REFERENCES Contact Information 8 ** 8 ** 22% ** P<0.01 23% * P<0.05 6 6 22% 25% Saez et al. (2021) Int. J. Mol. Sciences Dongho Kim Wallace Score 4 Ameho Score 4 Chung et al. (2020) Science
Advances Cellivery Therapeutics, Inc. 2 2 Kumar et al. (2019) J. Trans. Medicine Kimdh@cellivery.com Liu et al. (2013) Am. Heart Association 0 0 No - IV IR IR No - IV IR IR +82-2-3151-8900 Treatment iCP-NI 5-ASA Treatment iCP-NI 5-ASA Liu et al. (2000) J. Biological Chemistry TNBS TNBS [E. Immunology] E-2 Intracellular Delivery Of Nuclear Localization Signal Alleviates Atopic Dermatitis By Regulating The Inflammatory Skin Environment Jaehyeon Kim¹, Seokwon Lee¹, Hyunji Lee¹, Hyeontae Kang¹, Mingu Kang¹, Eunhee Yang¹, Eunna Chung¹, Daewoong Jo¹* ¹Immune Disease Team, Cellivery Therapeutics,
Inc., Seoul 03929, Korea Atopic dermatitis (AD), which occurs on the skin, is a serious inflammatory autoimmune disease that many people suffer from. Production of pro-inflammatory cytokines/chemokines is regulated by a certain type of inflammation associated transcription factors (IAFTs). Improved Cell-Permeable nuclear import inhibitor (iCP-NI) has been developed by fusing a cyclically-linked NLS of IATFs with advanced macromolecule transduction domain (aMTD), an sequence optimized hydrophobic cell-penetrating peptide (CPP). iCP-NI suppresses the nuclear transport of IRTFs by competitatively
binding to importin, reducing pro-inflammatory cytokines. To verify the anti-AD efficacy of iCP- NI cream (w/v, 5%), DNCB (dinitrochlorobenzene)-induced mouse model was used. iCP-NI was delivered into the epidermal/dermal layers as well as intradermal keratinocyte, following topical administration. Also, iCP-NI restored 1) AD-like skin lesion (50%), as assessed by SCORAD, and 2) the levels of serum IgE (71%), and 3) the expression of filaggrin protein. Moreover, iCP-NI reduced the upregulated cytokines level such as IL-6 (66%), TNF-α (87%). In conclusion, these results suggest that iCP-NI has
a significant therapeutic effect on AD, an inflammatory skin disease, through immune system control. Intracellular Delivery Of Nuclear Localization Signal Alleviates Atopic Dermatitis By Regulating The Inflammatory Skin Environment Jaehyeon Kim, Seokwon Lee, Hyunji Lee, Hyeontae Kang, Mingu Kang, Eunhee Yang, Eunna Chung and Daewoong Jo Cellivery R&D Institute, Cellivery Therapeutics, Inc., Seoul 03929, Korea. BACKGROUND Atopic dermatitis (AD), which occurs on the skin, is a serious inflammatory autoimmune disease that many people suffer from. Production of pro-inflammatory
cytokines/chemokines is regulated by a certain type of inflammation-associated transcription factors (IAFTs) such as NF-κB, STAT1/3, NFAT, and AP-1. Improved Cell-Permeable nuclear import inhibitor (iCP-NI) has been developed by fusing a cyclically-linked NLS of IATFs with advanced macromolecule transduction domain (aMTD), a sequence optimized hydrophobic cell-penetrating peptide (CPP). iCP-NI suppresses the nuclear transport of IRTFs by competitively binding to importin, reducing pro-inflammatory cytokines. AIM METHODS The aim of this study is to develop an iCP-NI as a Nuclear fraction assay
revealed suppressed nuclear translocation of IATFs after therapeutic molecule for autoimmune atopic iCP-NI treatment. The therapeutic effect of iCP-NI cream (w/v, 5%) has been dermatitis by determining its mode of action and verified with a dinitrochlorobenzene (DNCB)-induced AD mouse model. The efficacy on regulating expression of pro-inflammatory cream-formulated iCP-NI was topically applied, twice a day, then expressions of cytokines. inflammatory cytokines and histology of skin barrier were analyzed. RESULTS Figure 1. Improved Cell-Permeable Figure 2. Cell And Mouse Skin Figure 5. iCP-NI
Recovery Skin Lesion Of Atopic Dermatitis Nuclear Import Inhibitor (iCP-NI) Dermal Layer Delivery Of iCP-NI Structure Of iCP-NI Cell Permeability (Keratinocyte) Scoring Atopic Dermatitis (SCORAD) *p<0.05, **p<0.01 HaCaT Cell DNCB 15 Error Bar: SD * R K Q R (20 μM, 1 H) No Treatment - Cream Only iCP-NI Cream V Q Non-CP-NI iCP-NI C K 10 C L 50% P M Scoring Atopic Dermatitis (SCORAD) aMTD cNLS Cy5-Conjugated 5 • 3 rd Generation Hydrophobic • Nuclear Localization Sequence Cell Penetrating Peptide (CPP) (NLS) Of NF-κB 10 μm 0 No - Cream iCP-NI Treatment Only Cream Dermal Transduction DNCB Atopic
Dermatitis Skin Lesion Skin Thickness 160 *p<0.05, **p<0.01 (25 mg/kg, 2 H, Cream, Topical) Error Bar: SD DNCB 120 No Treatment - Cream Only iCP-NI Cream 39 % * Non-CP-NI Epidermal Thickness (μm) 80 68% Cy5-Conjugated Scale bar = 200 μm 40 No iCP-NI 5 μm 0 Treatment - Cream iCP-NI Cream Only CD4+ T Helper Cell DNCB 20 μm *p<0.05, **p<0.01 300 Error Bar: SD * DNCB No Treatment - Cream Only iCP-NI Cream Figure 3. iCP-NI Inhibits Nuclear Import Of IATFs In Human Keratinocyte (HaCaT Cell) 200 150 Atopic Dermatitis Related TARC Secretion In Conditioned Media Helper T Cells Number 250 95% Signaling
Pathways 100 Nucleus Cytoplasm *p<0.05, **p<0.01 800 Error Bar: SD Scale bar = 50 μm 50 TNF-α /IFN-γ - + + + - + + + ** 0 iCP-NI (μM) - - 1 10 - - 1 10 No - Cream iCP-NI p-p65 p-p65 Mast Cell Treatment Only Cream 600 p-STAT6 p65 37% DNCB 150 *p<0.05, **p<0.01 DNCB GATA3 GAPDH Error Bar: SD * Lamin B1 Human TARC (pg/ml) 400 No Treatment - Cream Only iCP-NI Cream 120 TNF-α + IFN-γ 90 66% No Treatment - iCP-NI Mast Cells Number 200 60 NF-κB (p-p65) 0 Scale bar = 100 μm 30 No iCP-NI Treatment - iCP-NI (10 uM) 0 No - Cream iCP-NI (1 uM) Treatment Only Cream Scale Bar = 10 μm TNF-α / IFN-γ (10
ng/ml) DNCB Figure 4. iCP-NI Reduces IgE Levels In Plasma And Pro-Infl
