# EVO301: an IL18BP-SAFA biologic demonstrates prolonged residence in inflamed skin and efficacy in a preclinical atopic dermatitis model

Jamie L. Harden *, Robert Whitener , Amanda Jacobson , Alexandra Pavel , Jinjoo Park , Susan Chi , Sang-Hoon Cha , Seung Goo Kang , Jeegar Patel , and Lorena Riol-Blanco  
*Evommune, Inc. Palo Alto, CA, USA ; AprilBio Co., Ltd., Rm602, Biomedical Science Building, Kangwon National University, Chuncheon 24341, Republic of Korea*

## Introduction

IL-18 is a pleotropic cytokine that amplifies inflammation and may contribute to the pathogenesis  
of multiple chronic inflammatory diseases. IL-18 is robustly expressed in dermal epithelial tissue,  
and thus is a target of high interest for treating atopic dermatitis, due to several strong scientific  
rationale listed below:

EVO301 is a novel biological therapeutic targeting IL-18 mediated inflammation and is currently  
in a Phase 2 clinical trial in atopic dermatitis (NCT06723405). EVO301 is comprised of the native  
IL18 binding-protein (IL18BP) and a human serum albumin Fab (SAFA) moiety, which increases  
the half-life of this therapeutic and may provide a benefit of homing to sites of inflammation.

## EVO301 10 mpk 0 0

In this study, we evaluated EVO301 PK and preferential homing to oxazolone-inflamed ear skin  
in human serum albumin (HSA), human neonatal Fc receptor (hFcRn) double transgenic mice.  
Additionally, we evaluated the ability of EVO301 to improve disease parameters in the chronic  
oxazolone dermatitis model.

## Materials and Methods

***HSA/hFcRn mice and PK study:*** *HSA/hFcRn mice were purchased from Genoway; these mice have the gene for murine serum*  
*albumin replaced with human serum albumin, and the murine FcRn gene replaced with the human FcRn gene. In brief, 8-12 week old*  
*mice were given a single intravenous (i.v.) injection of EVO301. Serum samples were collected at various time points post i.v. dosing. An*  
*ELISA bioanalysis method for quantification of EVO301 in mouse serum was developed and performed at Charles River Laboratories.*

***Fluorescent labeling of EVO301 and FLI in oxazolone-inflamed ear skin:*** *Fluorescent labeling of EVO301 and the FLI in the*  
*oxazolone-induced dermatitis model was performed at Perceptive. EVO301 was fluorescently labeled with CF-750 per manufactures*  
*instructions. Male HSA/hFcRN mice (n = 5) (Genoway) were sensitized to oxazolone (1.0% Oxazolone solution (vehicle: ethanol and*  
*acetone (1:4)) applied epicutaneously (150 µL) to shaved abdomen 7 days prior to disease induction (D-7). For disease induction (D0),*  
*0.5% Oxazolone solution (vehicle: ethanol and acetone (1:4)) applied topically to the right ear (10 µL), and vehicle solution was applied*  
*topically to the left ear, on Days 0, 1, 2, 3, 4, and 5. On Day 3, mice were administered i.v. 10mpk fluorescently labeled EVO301. FLI*  
*imaging was performed at 1hr, 4hr, 6hr, 24hr, 48hr, and 72hrs after fluorescently labeled EVO301 injection.*

***Cryo-fluorescence tomography (CFT):*** *CFT was performed at Perceptive. Animals were frozen in n-hexane/dry ice. All samples were*  
*stored at -80°C prior to embedding. CFT Image Acquisition: Whole body mice were embedded into one Optimal Cutting Temperature*  
*(OCT) compound block. The sample blocks were serially sectioned at 55 µm in a cryomacrotome along with sequential imaging of the*  
*block face at each sectioning plane. High resolution images with white light and test article compatible fluorescent channels were*  
*acquired using the CFT Xerra system.*

***Chronic oxazolone induced dermatitis model:*** *additional information about this model can be found in the publication by Jang et al.,*  
*Cytokine 172 (2023) 156413. In brief, SKH-1 mice were sensitized with 100μL 3% oxazolone solution on day 0. From days 7 to 27, mice*  
*were challenged with 100μL 0.2 % oxazolone solution thrice weekly. Mice were assigned to eight groups and administered treatment*  
*from days 7 to 27. Anti-IL4Rα Ab and anti-IL18 Ab were intraperitoneally administered twice weekly from day 7. EVO301 was*  
*intraperitoneally administered every other day from day 7. Skin erythema, scaling, and skin thickness were scored from 0 to 4.*

## EVO301 demonstrates longer half-life in HSA/hFcRN mice versus C57Bl/6 mice

|  | C57BL6 | HSA/hFcRN |
| --- | --- | --- |
| AUClast(hr*μg/mL) | 3,896 | 17,500 |
| AUCinf(hr*μg/mL) | 4,102 | 17,700 |
| t1/2(hr) | 30.2 | 47.4 |
| CL(mL/hr/kg) | 2.44 | 0.564 |
| Vss(mL/kg) | 81.4 | 43.2 |

## Figure 1. EVO301

**demonstrates longer half-life in**  
**HSA/hFcRn vs C57Bl6 mice.** A  
single intravenous (i.v.) dose of  
10mpk of EVO301 was  
administered to HSA/hFcRn mice  
or C57Bl/6 mice and the systemic  
PK profile of EVO301 was  
evaluated through at least 120  
hours. Table presents the  
pharmacokinetic assessment of  
EVO301 in C57Bl6 mice versus  
HSA/hFcRn mice

## EVO301 preferentially homes to and resides in oxazolone- inflamed ear tissue

**A**

## A rationale listed below:

**B**

**C**

**D**

**FLI vs systemic PK**

**Figure 2. Fluorescence lifetime imaging (FLI) of EVO301 in**  
**HSA/hFcRn mice.** (A) Schematic of FLI study in oxazolone-induced  
inflammation in HSA/hFcRn mice. (B) Images of mice at 1, 4, 6, 24, 48,  and 72 hrs post 10 mpk i.v. CF-750 labeled EVO301 (C)  
Quantification of relative fluorescence (Efficiency) in the right and left  
ear over the duration of the study (D) Comparison of EVO301 FLI  
results with previous EVO301 systemic PK in HSA/hFcRn mice

## Cryo-fluorescence tomography of EVO301

## A

**B**

**Figure 3. EVO301 demonstrates preferential homing to inflamed**  
**tissue compared to internal organs as determined by CFT** (A)  
Images of mice without (left) and with (center and right) fluorescently  
labeled EVO301 at 10 mpk, 72 hours post dosing (B)  
Quantification of fluorescence intensity in right (oxazolone inflamed) ear compared to internal tissues

## EVO301 reduces disease in the chronic oxazolone-induced dermatitis model in mice

**G1: Control**  
**G2: OXA + Vehicle**  
**G3: OXA + EVO301 (1 mpk)**  
**G4: OXA + EVO301 (3mpk)**  
**G5: OXA + EVO301 (10mpk)**  
**G6: OXA + anti-IL4Ra mAb (3 mpk)**  
**G7: OXA + anti-IL-18 mAb (10 mpk)**

**Figure 4. EVO301 demonstrates efficacy in the chronic oxazolone-induced atopic dermatitis model in**  
**SKH-1 hairless mice.** (A) Experimental design  (B) Representative images of mice in each group at Day 0 and  
Day 27 (C) Cumulative disease score (erythema, scaling, and thickness) of each group (D) AUC of cumulative  
score. (E) Histopathological features of skin lesions and (F) epidermal thickness of H&E stained skin tissues of  
each group of mice. This data was also previously published in Jang et al., Cytokine 172 (2023) 156413.

## Conclusions

• IL-18 is a proinflammatory cytokine that is proposed to contribute to multiple  
inflammatory pathways, and is abundantly expressed in epithelial tissues, such as skin.

• EVO301 is comprised of the native IL18 binding-protein (IL18BP) and a human serum  
albumin Fab (SAFA) moiety. PK studies in C57Bl6 mice versus HSA/hFcRn mice  
confirmed HSA binding *in vivo,* extending the half-life of EVO301

• In HSA/hFcRn mice, EVO301 preferentially homed to inflamed tissue (oxazoloneinduced dermatitis), compared to uninflamed tissue and internal organs

• EVO301 demonstrated efficacy in the chronic oxazolone-induced model of atopic  
dermatitis in mice

• These data support that EVO301 may be an effective therapeutic for the treatment of  
chronic inflammatory diseases, such as atopic dermatitis

## Acknowledgements and Disclosures

We would like to thank Genoway for their support on PK in the HSA/hFcRn mice, Charles River Laboratories   
(Randil Weerasuriya and Aruni Karunanayake) for their support in developing the EVO301 ELISA bioanalytical   
method, and the Perceptive team (Emmy Do and Filipa Mota) for their support with the FLI and CFT project.  
Additionally, we would like to thank our AprilBio colleagues for the support of this project, including the chronic   
oxazolone dermatitis model (Jang et al., Cytokine 172 (2023) 156413).  
Some illustrations were created with   
BioRender.com. JLH, RW, AJ, AP, JP, and LRB  are employees of, and hold stock in, Evommune.