Izokibep: Preclinical development and first-in-human study of a novel IL-17A neutralizing Affibody molecule in patients with plaque psoriasis

ABSTRACT

Psoriasis, an immune-mediated inflammatory disease, affects nearly 125 million people globally. The interleukin (IL)-17A homodimer is a key driver of psoriasis and other autoimmune diseases, including psoriatic arthritis, axial spondyloarthritis, hidradenitis suppurativa, and uveitis. Treatment with monoclonal antibodies (mAbs) against IL−17A provides an improvement in the Psoriasis Area and Severity Index compared to conventional systemic agents. In this study, the Affibody^Ⓡ technology was used to identify and optimize a novel, small, biological molecule comprising three triple helical affinity domains, izokibep (previously ABY-035), for the inhibition of IL-17A signaling. Preclinical studies show that izokibep, a small 18.6 kDa IL-17 ligand trap comprising two IL-17A-specific Affibody domains and one albumin-binding domain, selectively inhibits human IL-17A in vitro and in vivo with superior potency and efficacy relative to anti-IL-17A mAbs. A Phase 1 first-in-human study was conducted to establish the safety, pharmacokinetics, and preliminary efficacy of izokibep, when administered intravenously and subcutaneously as single doses to healthy subjects, and as single intravenous and multiple subcutaneous doses to patients with psoriasis (NCT02690142; EudraCT No: 2015–004531–13). Izokibep was well tolerated with no meaningful safety concerns identified in healthy volunteers and patients with psoriasis. Rapid efficacy was seen in all psoriasis patients after one dose which further improved in patients receiving multiple doses. A therapeutic decrease in joint pain was also observed in a single patient with concurrent psoriatic arthritis. The study suggests that izokibep has the potential to safely treat IL17A-associated diseases such as psoriasis, psoriatic arthritis, axial spondyloarthritis, hidradenitis suppurativa, and uveitis.

KEYWORDS:

• ABY-035
• first-in-human
• IL-17A inhibition
• izokibep
• psoriasis

Introduction

Psoriasis is an immune-mediated systemic inflammatory disease affecting nearly 125 million people globally with a prevalence ranging from 0.9% (United States) to 8.5% (Norway).¹ The disease is often associated with other immunological disorders, such as psoriatic arthritis, and with burdensome comorbidities, including metabolic syndrome, diabetes, and cardiovascular disease.² The symptoms of psoriasis can be improved by selective therapeutic blocking of several inflammatory mediators, including tumor necrosis factor (TNF) and interleukins (IL)-12 and 23.³ Emerging data from mouse and human studies highlight the role of a subset of T-helper (Th) cells in the pathogenesis of psoriasis.^3–5 These cells are characterized by the production of IL-17 and are accordingly named Th17 cells.⁵ There is growing appreciation for the role of the Th17/IL-23 axis in psoriatic disease (psoriasis, psoriatic arthritis, and axial spondyloarthritis).^3,4 In addition to biologics targeting TNF, treatment with monoclonal antibodies (mAbs) against IL-17 and IL-23 may provide an improvement in the Psoriasis Area and Severity Index (PASI) compared to conventional systemic agents.^3,4 IL-17 has emerged as an important driver in non-psoriatic diseases as well, with upregulated expression of peripheral Th17 cells and serum IL-17 in patients with autoimmune uveitis.^6,7 Similarly, IL-17 is significantly increased in patients with hidradenitis suppurativa, both in serum and lesional and perilesional skin.^8–12

The IL-17A homodimer (IL-17AA, hereafter referred to as IL-17A) has been identified as a key driver in autoimmune diseases, such as psoriasis.^13,14 In addition to IL-17A, five homologous IL-17 family cytokines elicit similar effects in the target cells but may simultaneously trigger opposing functions in a tissue-specific manner, namely IL-17B, IL-17C, IL-17D, IL-17E (also known as IL-25), and IL-17F.¹⁵ Studies have shown that IL-17A, IL-17C, and IL-17F levels increase by as much as eight-fold in psoriatic lesions, leading to stimulation of keratinocyte proliferation.^3,15,16 The central role of IL-17A in psoriatic disease pathophysiology has been established preclinically, genetically, and via pharmacological inhibition.¹⁵ While the IL-17A and IL-17F homodimers and the IL-17AF heterodimer signal via the IL-17 receptor (IL-17 R), the role of IL-17AF and IL-17F in the pathophysiology of psoriasis remains unclear.^4,17–19 IL-17A inhibition may be more relevant for psoriatic pathogenesis than IL-17C or IL-17F.^15,20 Targeting IL-17 R with mAbs, such as brodalumab, which putatively ablates all IL-17 family cytokine signaling, does not appear to be superior to selective IL-17A inhibitors.²¹ Emerging data from clinical studies indicate that inhibiting both IL-17A and IL-17F is efficacious for treating psoriasis, but can lead to an increase in candidiasis.^22,23 A recent pooled analysis of Phase 2 and Phase 3 study data for bimekizumab (selectively binding to IL-17A and IL-17F) in patients with moderate to severe plaque psoriasis which evaluated the mAb’s 2-year safety profile showed that it was well tolerated, aside from an increased incidence of oral candidiasis.²³ Evidence also suggests that dual IL-17A and IL-17F inhibition may not be more efficacious than blocking IL-17A alone and that more potent inhibition of IL-17A may provide added benefit to patients with IL-17-mediated disease.^{19,20,23–27}

Despite major therapeutic advances in the treatment of these disorders during the last two decades, there remains an unmet need for more efficacious therapies.^20,28 Given the established benefit-risk profile of IL-17A inhibitors, we hypothesize that better treatment efficacy may be achieved by enhancing the potency of anti-IL-17A agents and improving their biodistribution to sites of active inflammation with no dose-related toxicities.¹⁵

Collectively, data to date suggest that increased efficacy in treating IL-17-driven pathology may be achieved with a molecule that: 1) completely and selectively inhibits homodimeric IL-17A signaling, 2) has a relatively small size to allow increased tissue exposure, and 3) has favorable biodistribution properties with a long in vivo half-life, allowing preferential accumulation at sites of inflammation such as skin lesions. Affibody molecules represent a class of small, triple helical high-affinity protein domains that are approximately 6.5 kDa in size and well suited for therapeutic development.^29,30 Affibody molecules are often engineered as a fusion protein with a small albumin-binding domain to extend plasma half-life and achieve better tissue penetration, including high drug exposure at sites of inflammation.^30–35

The Affibody technology was used to identify a novel molecule specific for the inhibition of IL-17A signaling. The molecule was optimized to increase the affinity for IL-17A by combining two IL-17A-specific Affibody domains with one albumin-binding domain to create izokibep, an 18.6 kDa IL-17 ligand trap. Izokibep was shown to specifically bind to and inhibit human IL-17A with high affinity in vitro; its efficacy was demonstrated in in vitro cell-based assays and in an in vivo mouse model. A Phase 1 study of izokibep was conducted to characterize the safety, pharmacokinetics (PK), and preliminary efficacy, when administered intravenously (IV) and subcutaneously (SC) as single doses to healthy subjects, and as a single IV and multiple SC doses to patients with psoriasis (NCT02690142; EudraCT No: 2015–004531–13).³⁵

Results

Preclinical Studies

Design of selective and highly potent IL-17A inhibitor izokibep

Phage display selections and combinatorial mutagenesis were used to isolate IL-17A-specific Affibody molecules.^34,36–39 The final leading candidate was a small 18.6 kDa IL-17 ligand trap comprising one IL-17A-specific Affibody domain at each side of an albumin-binding domain (izokibep) (Figure 1a).³⁹ The ligand trap format of izokibep increased the inhibitory capacity when compared to the monomeric precursor. Additionally, izokibep was more potent than an IL17A-targeting mAb (secukinumab) in a normal human dermal fibroblast (NHDF) cell-based assay (Figure 1b).

Figure 1. Izokibep structure and binding curves. The ligand trap format of izokibep increased the inhibitory capacity of the molecule compared to the monomeric format and with greater inhibition than secukinumab and ixekizumab, where izokibep blocked IL-17A binding to IL-17 R. (A) the proposed binding mode of izokibep involves simultaneous binding of two IL-17A Affibody affinity domains (blue) on each side of the homodimeric IL-17A target protein (gray), thus improving the potency. The albumin binding domain (yellow) enables high-affinity binding to albumin, extending the plasma half-life. (B) Formatting of the IL-17A-binding ligand trap of izokibep, illustrated by an izokibep precursor (dark blue), resulted in a superior inhibitory profile over the unformatted monomer (light blue) as well as secukinumab (red) in an NHDF cell assay. (C) Binding of IL-17A to IL-17 R in the presence of izokibep (dark blue), secukinumab (red), and ixekizumab (gray), as determined by SPR. Human recombinant IL-17 R was immobilized on a chip and human recombinant IL-17A was injected at a concentration of 50 nM in the presence of izokibep (K_D 0.3 pM), secukinumab (K_D 60 pM),⁴⁰ or ixekizumab (K_D 2 pM) at a 1:1 ratio,(a concentration much higher than the reported K_D values).

Abs, absorbance; IL, interleukin; NHDF, normal human dermal fibroblasts; SPR, surface plasmon resonance.

Read the detailed description of this figure

Figure describing the ligand trap format of izokibep that increases the inhibitory capacity of the molecule compared to the monomeric format and to secukinumab and ixekizumab, and blocked IL-17A binding to IL-17R. (a) Figure showing izokibep binding simultaneously to two IL-17A Affibody affinity domains (blue) on each side of the homodimeric IL-17A target protein (gray), thus improving the potency. The albumin-binding domain (yellow) enables high-affinity binding to albumin, extending the plasma half-life. (b) Figure showing izokibep binding to IL-17A using normal human dermal fibroblast cell assay. The izokibep precursor (dark blue), resulted in a superior inhibitory profile over the unformatted monomer (light blue) and secukinumab (red). Figure showing binding of IL-17A to IL-17R using surface plasmon resonance where human recombinant IL-17R was immobilized on a chip and 50 nM human recombinant IL-17A was injected at a 1:1 ratio in the presence of izokibep (dark blue), secukinumab (red), and ixekizumab (grey).

Binding of IL-17A to the IL-17 receptor in presence of izokibep, secukinumab, and ixekizumab

The binding of IL-17A to the IL-17 receptor A (IL-17 R) in the presence of izokibep, secukinumab, or ixekizumab was evaluated by surface plasmon resonance (SPR) analysis. The data showed that simultaneous binding of izokibep to both subunits of IL-17A resulted in almost complete inhibition of IL17A/IL-17 R binding, whereas ixekizumab and secukinumab provided incomplete inhibition of IL-17A/IL-17 R binding (Figure 1c).⁴⁰

Izokibep binding affinity to IL-17A and human serum albumin

Binding affinity of izokibep to IL-17A and human serum albumin (HSA) was measured using a kinetic exclusion assay (KinExA), which allows dissociation constant (K_D) measurements for very high-affinity binding. The KinExA method measures the equilibrium binding affinity and kinetics between unmodified molecules in solution. The analysis demonstrated that izokibep in complex with HSA binds to human IL-17A with K_D values in the femtomolar range (Table 1). In a second experiment, the affinity of izokibep or izokibep mixed with IL-17A to HSA was measured using the KinExA assay. Izokibep and izokibep in complex with IL-17A were both found to bind to HSA with comparable affinity.

Table 1. Binding affinity results by interaction.

Interaction	KD (95% CI)
Izokibep in complex with HSA to IL-17A	0.32 pM (0.15–0.55 pM)
Izokibep in complex with IL-17A to HSA	29 pM (17–46 pM)
Izokibep to HSA	49 pM (32–69 pM)

CI, confidence interval; HSA, human serum albumin; IL, interleukin; K_D: dissociation constant.

Specificity and selectivity

The target-binding specificity of izokibep to IL-17A was tested against a panel of 24 proteins, comprising closely related members of the human IL-17 family (IL17A, IL-17AF, IL-17B, IL-17C, IL-17D, IL-17E, IL-17F), cytokines, receptors, and abundant plasma proteins, using SPR (Biacore) (Figure S1). Besides IL-17A and IL-17AF, no interactions between izokibep and the panel of test proteins were observed. Concentrations up to 250 nM were tested. The K_D of IL-17AF was determined to be 6.9 nM. Thus, izokibep has four orders of magnitude higher affinity for IL-17A than IL-17AF.

In vitro and in vivo efficacy

Multiple studies were performed to evaluate the in vitro and in vivo efficacy of izokibep. A NHDF cell-based assay was used to assess the in vitro potency of izokibep relative to anti-IL-17A antibodies secukinumab and ixekizumab. In the presence of increasing concentrations of izokibep, secukinumab, and ixekizumab, the IL-17A-induced IL-6 production was inhibited in a concentration-dependent manner (Figure 2a). At a fixed concentration of 64 pM IL-17A, izokibep was more potent than secukinumab or ixekizumab, with IC₅₀ values of 0.4 ng/mL (izokibep), 41 ng/mL (secukinumab), and 3.5 ng/mL (ixekizumab).

Figure 2. Izokibep – superior potency in in vitro cell-based assays and in vivo murine model relative to secukinumab and ixekizumab. (a) Potency of izokibep, secukinumab, and ixekizumab in an NHDF cell assay, stimulated with 64 pM IL-17A and 10 fM IL-1beta. All compounds inhibit the IL17Ainduced IL-6 production in a dose-dependent manner. IC50 values generated were 0.4, 3.5, and 41 ng/mL for izokibep, ixekizumab, and secukinumab, respectively. (B) Izokibep prevents the KC secretion in response to stimulation by IL-17A in vivo. KC concentrations were determined by ELISA using a KC-quantification kit. Doses indicated on the x-axis are given in mg/kg and the response on the y-axis in % inhibition ± SD. n = 6 animals per compound and dose level.

Figure describing the efficacy of izokibep compared with secukinumab and ixekizumab in in vitro assay in normal human dermal fibroblasts and in vivo in a C57 mouse model. (a) Figure showing the potency of izokibep, secukinumab, and ixekizumab in a normal human dermal fibroblast cell assay, stimulated with 64 pM IL-17A and 10 fM IL-1b. All compounds inhibited the IL-17A-induced IL-6 production in a dose-dependent manner. (b) Figure showing prevention of keratinocyte chemoattractant secretion by izokibep in response to stimulation by IL-17A in vivo.

IC50, half-maximal inhibitory concentration for a drug; IL, interleukin; KC, keratinocyte chemoattractant; NHDF, normal human dermal fibroblasts; SD, standard deviation.

A comparative in vivo proof-of-mechanism study was performed using the murine keratinocyte chemoattractant (KC) model, wherein SC administration of human IL-17A to mice induces the secretion of the KC (CXCL1).⁴¹ As shown in Figure 2b, the IL17A-induced KC secretion was inhibited in a dose-dependent manner by izokibep, ixekizumab, or secukinumab. Complete inhibition of KC secretion was obtained at doses of 0.1 mg/kg for izokibep, 3 mg/kg for ixekizumab, and 5 mg/kg for secukinumab. On an mg/kg basis, izokibep provided 30- and 50-fold superior efficacy in reducing KC secretion relative to ixekizumab and secukinumab, respectively.

Stability studies

Izokibep samples stored at 40°C for 0, 2, and 4 weeks were evaluated for degradation and dimerization via sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by silver staining and by size-exclusion chromatography (SEC) (Figure S2). Stability data collected under good manufacturing practice (GMP) conditions show that the izokibep drug product was stable during storage at −20°C ±5°C, under accelerated storage conditions at+5°C ±3°C for at least 5 years, and at 25°C ±5°C/relative humidity 55% to 65% for at least 3 months. In addition, izokibep retained its ability to bind to IL-17A after incubation in human serum at 37°C for at least 8 weeks. The superimposed sample titration curves suggest that that izokibep was stable in serum (Figure S3A). The relative-binding activities were calculated, and the resulting graph shows small differences (Figure S3B).

Toxicology

The most relevant toxicology species was determined to be cynomolgus monkeys since izokibep cross-reacts with cynomolgus monkey IL-17A, but not with mouse or rat IL-17A, as determined by SPR (Biacore) studies. In addition, the percent amino acid sequence identity between rhesus/human IL-17A (97%) and cynomolgus/human (95%) is high, but mouse/human (62%), and rat/human (59%) are low, suggesting a low probability of IL-17A binding. A series of toxicity studies were conducted in cynomolgus monkeys, including a 28-day repeated IV dose toxicity study (no observed adverse effect level [NOAEL] 40 mg/kg), a 13-week repeated SC dose toxicity study (NOAEL 20 mg/kg), and a 26-week repeated SC dose toxicity study (NOAEL 20 mg/kg), none of which identified any specific target organ for safety concerns. In the 26-week toxicity study in cynomolgus monkeys, with weekly SC injections of 10, 20, or 40 mg/kg/week, izokibep was generally well tolerated. However, due to the formation of local abscess and systemic sequelae that were considered as adverse event (AE) in one female administered 40 mg/kg/week, the NOAEL was determined to be 20 mg/kg/week, corresponding to a human equivalent dose of 6.5 mg/kg (520 mg in an 80 kg individual). Safety pharmacology parameters were measured as part of the nonclinical toxicology studies in cynomolgus monkeys with no signs of adverse effects of izokibep on central nervous system, cardiovascular, or respiratory functions.

Clinical study

First-in-human study design and demographics

Overall, 46 healthy subjects and 26 patients with psoriasis received izokibep in a Phase 1 clinical trial. The trial was divided into four parts (Figure 3). Part A was a randomized, double-blind, placebo-controlled, single ascending IV dose study in healthy subjects. Part B was an open-label single SC dose study in healthy subjects. Part C was an open-label single IV dose, and Part D an open-label multiple SC dose study, both in patients with psoriasis. In Part C, all patients had moderate-to-severe psoriasis at baseline (defined as PASI ≥12) while in Part D, 10 of the 15 patients had a baseline PASI <12 (i.e., mild to moderate psoriasis). Part A comprised five consecutive dose groups (2, 5, 10, 20, and 40 mg izokibep) with eight healthy subjects each, six dosed with izokibep and two with placebo. Part B comprised six healthy subjects who received 40 mg izokibep. Part C comprised 11 patients with psoriasis who received up to 40 mg izokibep. Part D comprised 15 patients with psoriasis. In D1, two patients received three biweekly (Q2W) doses of 40 mg izokibep. A total of 13 patients were eligible for Part D2, of whom 12 received seven doses of 40 mg izokibep Q2W. One patient received one single dose of 40 mg izokibep before discontinuing the study due to failed drug test. The subject demographics and baseline characteristics are summarized in Table 2.

Figure 3. Design of the first-in-human study with izokibep and exposure. ^amoderate-to-severe psoriasis (PASI≥12). ^ball severities, moderate-to-severe and mild-to-moderate psoriasis (PASI≥12 and PASI<12).

Figure describing the study design of the first-in-human trial with izokibep at different dose levels and route of administration.

IV, intravenous; Q2W, once every second week; PASI, Psoriasis Area and Severity Index; SC, subcutaneous.

Table 2. Demographics and baseline characteristics.

Variable		Placebo	izokibep
			Part A					Part B	Part C1	Part C2	Part D
		0 mg	2 mg	5 mg	10 mg	20 mg	40 mg	40 mg	40 mg	2 mg	40 mg Q2W
		IV	IV	IV	IV	IV	IV	SC	IV	IV	SC
		N = 10	N = 6	N = 6	N = 6	N = 6	N = 6	N = 6	N = 6	N = 5	N = 15
Age – year (Mean ± SD)		41 ± 10.9	43 ± 13.9	42 ± 13.8	35 ± 11.9	43 ± 8.6	45 ± 12.4	53 ± 14.3	42 ± 10.8	35 ± 8.5	39 ± 8.7
Sex – n (%)	Male	3 (30.0)	3 (50.0)	5 (83.3)	6 (100)	1 (16.7)	4 (66.7)	4 (66.7)	4 (66.7)	3 (60.0)	10 (66.7)
	Female	7 (70.0)	3 (50.0)	1 (16.7)	—	5 (83.3)	2 (33.3)	2 (33.3)	2 (33.3)	2 (40.0)	5 (33.3)
Ethnicity - n (%)	Caucasian	10 (100.0)	5 (83.3)	5 (83.3)	4 (66.7)	6 (100.0)	6 (100.0)	5 (83.3)	6 (100.0)	5 (100.0)	12 (80.0)
	Asian	-	—	—	2 (33.3)	—	—	-	-	-	3 (20.0)
	Other	-	1 (16.7)	1 (16.7)	—	—	—	1 (16.7)	-	-	-
Body weight – kg (Mean ± SD)		78.6 ± 15.9	68.9 ± 9.0	85.7 ± 18.8	86.8 ± 12.2	68.6 ± 13.6	79.2 ± 15.7	80.1 ± 4.5	84.9 ± 12.1	83.5 ± 10.3	85.9 ± 18.3
BMI – kg/m^2 (Mean ± SD)		26.2 ± 3.7	23.5 ± 3.0	27.4 ± 3.7	27.2 ± 3.9	24.6 ± 3.1	25.2 ± 3.5	27.7 ± 2.3	28.3 ± 2.4	28.5 ± 4.8	28.3 ± 4.5
PASI score (Mean ± SD)		NA	NA	NA	NA	NA	NA	NA	19.6 ± 5.6	16.6 ± 5.5	12.7 ± 11.3
Duration of psoriasis – months (Mean ± SD)		NA	NA	NA	NA	NA	NA	NA	307.5 ± 190.7	139.8 ± 126.1	206.8 ± 128.2

BMI, body mass index; IV, intravenous; PASI, psoriasis area and severity index; Q2W, once every second week; SC, subcutaneous; SD, standard deviation; NA, not applicable.

Pharmacokinetics

Following a single IV administration of izokibep in healthy volunteers (Part A), the time to maximum plasma concentration (C_max), t_max, was similar between the dose groups, with a median ranging from 0.083 to 2.0 h (Figure 4a). Systemic exposure was largely dose proportional for C_max and for area under the concentration–time curve (AUC) from time 0 to last quantifiable plasma concentration (AUC_0-tlast) and AUC extrapolated to infinity (AUC_0-∞) over the 2 mg to 40 mg IV dose ranges. No clear trends across the dose range were observed for plasma clearance (C_L), with mean values ranging from 0.014 to 0.020 L/h and for volume of distribution at steady state (V_ss) ranging from 4.3 to 5.7 L. Following single SC administrations, t_max was observed between approximately 48 and 72 h (Part B; Figure 4b). The absolute bioavailability of izokibep following a single 40 mg SC dose was approximately 77%. No route of administration dependency was observed in the elimination half-life of izokibep with 278 h for 40 mg SC and 288 h for 40 mg IV. The PK profile upon IV administration of izokibep was similar in both patients and healthy subjects (Part A versus Part C). Steady state was attained within five every other week SC doses (by Day 71; Part D2; Figure 4c), with approximately 2-fold accumulation observed in the AUC over the dosing interval. The mean elimination half-life of izokibep after the last dose was 279 h (12 days), indicating that the PK was linear over time.

Figure 4. Pharmacokinetic profile of izokibep. Mean plasma concentration-time profiles of izokibep following (A) single IV doses in healthy subjects, (B) single SC or IV 40 mg dose in healthy subjects, and (C) repeated 40-mg SC administration in patients, with dosing once every other week on seven occasions (arrows). LLOQ: <0.020 µg/mL. IV, intravenous; LLOQ, lower limit of quantification; SC, subcutaneous.

Figure describing the mean plasma concentration–time profiles of izokibep following, (a) single intravenous doses of izokibep in healthy subjects (b) a single 40 mg subcutaneous or intravenous dose of izokibep in healthy subjects (c) repeated 40 mg subcutaneous dose of izokibep in patients with psoriasis, with dosing once every other week on seven occasions (arrows).

Safety

Izokibep was generally well tolerated. Overall, 59 (81.9%) subjects had treatment-emergent AEs (TEAEs). For both placebo and active groups, all TEAEs were mild or moderate in severity and no serious AEs were reported. There were no discontinuations or deaths due to TEAEs (Table 3).

Table 3. Summary of treatment-emergent adverse events.

	Number of subjects (%) with adverse events
Type of AE	Part A					Part B	Part C1	Part C2	Part D	Total izokibep
	IV	IV	IV	IV	IV	IV	SC	IV	IV	SC	IV/SC
	0 mg	2 mg	5 mg	10 mg	20 mg	40 mg	40 mg	40 mg	2 mg	40 mg Q2W
	N = 10	N = 6	N = 6	N = 6	N = 6	N = 6	N = 6	N = 6	N = 5	N = 15	N = 62
Any TEAE	7 (70.0)	5 (83.3)	3 (50.0)	5 (83.3)	6 (100)	3 (50.0)	5 (83.3)	5 (83.3)	5 (100)	15 (100)	52 (83.9)
Serious TEAEs	-	-	-	-	-	-	-	-	-	1 (6.7)	1 (1.6)
Discontinued due to TEAEs	-	-	-	-	-	-	-	-	-	-	-
Severe TEAEs	-	-	-	-	-	-	-	-	-	1 (6.7)	1 (1.6)
Deaths	-	-	-	-	-	-	-	-	-	-	-
Related TEAEs	5 (50.0)	2 (33.3)	-	1 (16.7)	2 (33.3)	3 (50.0)	3 (50.0)	4 (66.7)	5 (100)	14 (93.3)	34 (54.8)
Severe Related TEAEs	-	–	-	-	-	-	-	-	-	-	-

AE, adverse event; IV, intravenous; Q2W, once every second week; SC, subcutaneous; TEAE, treatment emergent adverse event.

Oropharyngeal pain and nasopharyngitis were the most commonly reported TEAEs across izokibep and placebo treatment groups of Part A (Table S1). Injection site reactions (ISRs) occurred in the SC groups (i.e., Parts B and D): ISRs were transient and mild in all but one subject who reported a moderate AE. One severe TEAE of anaphylactic reaction due to nut allergy was reported in Part D and was determined by the investigator to be unrelated to izokibep treatment.

No adverse trends in clinical laboratory evaluations, vital signs, 12-lead electrocardiograms (ECGs), or physical examinations were observed following single or multiple doses of izokibep to healthy subjects and patients with psoriasis.

Mainly transient or low levels of treatment-emergent anti-drug antibodies (ADAs) were detected in 9/40 subjects in Part A, 4/6 in Part B, and in 3/14 in Part D. No treatment-emergent ADAs were detected in Part C. ADAs detected pre- or post-exposure had no impact on PK parameters, safety (Parts A to D) or efficacy (Parts C and D).

Efficacy

In Part C, the study population comprised 11 patients with moderate-to-severe psoriasis (PASI score ≥12). In Part C2, a 50% reduction in PASI score (PASI50) was achieved by 3 out of 5 patients; two in Week 2 and another in Week 6 following a single 2 mg IV dose, and one patient reached PASI75 in Week 4. In Part C1, a majority of patients reached PASI50 either in Week 2 (4 out of 6 patients) or Week 3 (5 out of 6 patients) following a single 40-mg IV dose (Part C1). The onset of response was very rapid following the 40-mg dose, and clinical response was maintained until Week 6 where 2 out of 6 patients reached PASI75 (Figure 5a). The clinical response of a psoriatic plaque lesion after a single 40-mg IV dose is shown in a selected patient in Figure 5b.

Figure 5. Clinical response to treatment with izokibep. (a)mean (SD) PASI score by week for patients with moderate-to-severe psoriasis (PASI≥12 at baseline). Patients received a single dose of 2 or 40 mg izokibep IV in part C2 and C1, respectively. In part D, patients received multiple doses izokibep SC Q2W 3 × 40 mg (last dose in week 4) or 7 × 40 mg (last dose in week 12). The on- and off-treatment effects are presented until week 12. (b) Psoriatic plaque lesion of selected individual patient prior to (left) and 14 days after (right) a single 40 mg IV dose of izokibep.

IV, intravenous; SC, subcutaneous.

In Part D, five patients with baseline PASI ≥12 (i.e., moderate-to-severe psoriasis, two patients in Part D1 and 3 patients in Part D2) and 10 patients with baseline PASI <12 (i.e., mild-to-moderate psoriasis) were treated with 40 mg SC Q2W. The onset of response in all patients was rapid (Figure 5a). PASI scores of all patients treated in Part D2 by study week is presented in Figure 6. After 12 weeks of treatment, all patients achieved a PASI score <1.5. Six patients maintained a PASI score of 1.5 and all maintained a PASI score <2.5, indicating controlled disease, until the end of study at Week 20.

Figure 6. Individual absolute PASI scores in patients receiving multiple 40 mg doses of izokibep SC Q2W (indicated by arrows). Blue line indicates PASI 1.5.

Figure describes psoriasis area severity index scores (PASI) of all patients treated in Part D2 by study week. After 12 weeks of treatment, all patients achieved a PASI of <1.5. Six patients maintained a PASI score of 1.5 and all maintained a PASI score <2.5, indicating controlled disease, until the end of study at Week 20.

Q2W, once every second week; PASI, psoriasis area and severity index

Static Physician’s Global Assessment

The static Physician’s Global Assessment (sPGA) is a 7-point scale measuring the severity of psoriasis, ranging from 0 (clear) to 6 (very severe). For the two subjects in Part D1 with sPGA of 5 at baseline who received 3 doses of 40 mg izokibep, sPGA decreased to 2 and 3 at Week 6. For the 12 subjects in Part D2 who received 7 doses of 40 mg izokibep, the mean baseline sPGA score of 4 (range: 2 to 5) decreased to 1 (range: 0 to 2) after 12 weeks of izokibep treatment, consistent with decline in the PASI score over this period.

Visual analog scale for pruritus

Patient-reported pruritus generally declined during treatment in both Parts C and D. In Part D, the mean baseline pruritus visual analog scale (VAS, range 0–100 mm with lower scores indicating less severe pruritus) of 64/100 mm declined rapidly to mean values of 8 mm at Week 4 and 2 mm at Week 14.

Visual analog scale joint pain

A single patient in Group D2 had a medical history of psoriatic arthritis. Joint pain for this patient was monitored by VAS throughout the study. The baseline VAS of joint pain declined from 87/100 mm on Day 1 to 53 mm on Day 29, with sustained improvement through Day 85 (58 mm).

Discussion

Izokibep is being developed as a treatment for IL-17-associated inflammatory diseases and was studied in healthy volunteers and patients with psoriasis. The rationale for the molecular design and clinical studies of izokibep was based on the hypothesis that a smaller and more potent inhibitor of IL-17A may provide additional efficacy and benefit patients who are not adequately responding to established treatments. Three properties were hypothesized to provide improved efficacy: 1) complete and selective inhibition of homodimeric IL-17A signaling, 2) a relatively small size to allow increased exposure, and 3) favorable biodistribution properties with a long in vivo half-life, allowing preferential accumulation at sites of inflammation such as skin lesions. All three postulates were met with the design of the izokibep molecule.

IL-17A is a homodimeric protein and the key driver of IL-17-mediated disorders.^4,25 To completely block IL-17A signaling, a molecule was designed that would simultaneously bind to both IL-17A domains. By combining two IL-17A-affinity domains (Affibody molecules) with an albumin-binding domain, a functional dimer was engineered, resulting in an increase in functional affinity (~4 orders of magnitude) for the homodimeric IL-17A protein, with sub-picomolar binding affinity, as compared to izokibep binding with low nanomolar affinity using only one of its IL-17 binding domains to the IL-17AF heterodimer. The principle of using avidity to enhance engineered binding strength has been described previously.⁴² Here, we extended the use of avidity to increase selectivity for a homodimeric (IL-17AA) versus a heterodimeric (IL-17AF) protein variant. Previously, Fyn SH3 domains have been engineered to develop a formatted IL-17 blocking molecule.⁴³ The Fyn domains were fused to the C-terminal part of the light chain of an IgG to enhance the plasma half-life of the product and generate a functional SH3 dimerization for improved blocking of IL17. However, this modification substantially increased the size of the drug. In contrast, we inserted a small albumin-binding domain between the two IL-17A-binding Affibody domains for half-life extension. In addition to a long circulatory half-life, serum albumin is widely distributed in the body, has favorable tissue biodistribution and high uptake at sites of inflammation, all of which are known to increase the efficacy of an anti-inflammatory agents.^30,33,44

The ligand trap structure of izokibep resulted in improved potency with complete and qualitatively superior inhibition of IL-17A signaling compared to the marketed anti-IL-17 mAbs secukinumab and ixekizumab. Izokibep demonstrated potent cell-based assay inhibition and 30- to 50-fold better efficacy in an IL-17A-induced mouse model when compared to secukinumab and ixekizumab. The highest dose in the animal study was selected to match the induction dose of secukinumab based on mg/kg.

Toxicology studies in cynomolgus monkeys with izokibep doses up to 40 mg/kg IV and SC were well tolerated.^45–48 In the 6-month repeat dose study (10, 20, or 40 mg/kg/week SC), an adverse presence of local abscessation in the SC injection site and systemic sequalae was encountered in one female and epithelial/mucosal degeneration with acute inflammation within the glandular stomach was encountered in one male in the 40 mg/kg/week dose group. Both responses were consistent with systemic immune-modulation/suppression. The NOAEL for the 6-month study was 20 mg/kg/week.

The izokibep molecule has been demonstrated to be very stable, both in plasma and in clinically relevant storage conditions. The product has a high purity with no significant aggregation or dimerization even after 4 weeks of incubation at 40°C, 3 months at 25°C, and with at least 5 years of demonstrated storage stability at −20°C and 5°C as per GMP protocols. Experimental data from 70 weeks of storage at room temperature further support the stability profile.

Pre-clinical studies establish that izokibep has the capacity to selectively and potently inhibit IL-17A. Nonclinical toxicology studies provide the rationale for an acceptable toxicologic profile and support clinical development for the treatment of diseases with interleukin 17 (IL-17)-associated pathogenesis.

Initial clinical testing of izokibep investigated the safety, tolerability, and PK profile of single (IV and SC) doses in healthy subjects and single (IV) and multiple (SC) doses in patients with psoriasis. The initial IV dose was chosen to investigate the systemic exposure of izokibep, with SC administration as the intended route, as with established IL-17 inhibitors. The molecular weight of izokibep (18.6 kDa) is approximately eight times smaller than a full-length antibody (~150 kDa). Based on the enhanced potency and smaller size of izokibep (relative to secukinumab), it was hypothesized that a 40 mg dose might achieve a similar clinical response to that observed with 300 mg secukinumab. A starting dose of 2 mg was supported by the NOAEL from the 28-day non-human primate good laboratory practice toxicity study (387-fold safety margin). Additionally, based on PK-pharmacodynamic modeling of exposure and activity of secukinumab, a 2 mg SC izokibep dose was anticipated to provide an incomplete and transient clinical effect. A two-fold dose escalation scheme was used from 5 to 40 mg. The PK profile of izokibep was dose linear and similar in healthy subjects compared to patients with psoriasis. Upon IV administration, V_ss ranged from 4.3 to 5.7 L, SC bioavailability (F) was 77%, and a functional half life of 12 days was observed following seven 40 mg Q2W SC administrations, demonstrating that the albumin-binding domain in izokibep conferred a long plasma half-life and albumin-like distribution properties, while utilizing a minimal drug size.

Treatment-emergent ADAs after Q2W administration in patients with moderate-to-severe psoriasis were detected in 12% of patients, similar to earlier reports of incidence of treatment-emergent ADAs in 14% of patients with moderate-to-severe psoriasis for another IL-17A blocking antibody.²⁵ There were no correlations between ADA and clinical efficacy, altered exposure, or immune-related AEs.

The study investigated the efficacy of izokibep, and dose response was demonstrated with up to seven Q2W administrations of 40 mg. Additional human clinical studies have established that izokibep doses above 40 mg may provide additional benefit to patients, without any additional safety concerns.⁴⁹ Significant reductions in PASI score were observed at doses as low as a single dose of 2 mg. A rapid onset of response was observed after the first dose, and repeated Q2W dosing resulted in a response that was maintained for at least 8 weeks after the last dose. A rapid and sustained effect was also seen on pruritus, an often debilitating symptom, known to be an important factor in the association between improvements in disease severity and quality of life in patients with moderate-to-severe psoriasis.⁵⁰

The SC and IV administrations of izokibep were safe and well tolerated in subjects treated Q2W up to 12 weeks. There were no notable dose-related trends in the number of subjects reporting TEAEs, or in the number of TEAEs, with no relevant difference in the incidence of TEAEs with izokibep compared with placebo.

In one subgroup (D2), patients with psoriasis of any severity were included. All patients showed treatment benefits with izokibep and had absolute PASI scores <1.5 at Week 12 regardless of disease severity at baseline. One patient with concurrent psoriatic arthritis was enrolled, and the VAS score for joint pain decreased in this patient, suggesting a potential therapeutic role of izokibep in patients with psoriatic arthritis. A Phase 2 clinical study (NCT04713072) in psoriatic arthritis patients was completed and a Phase 2/3 study (NCT05623345) in this patient population is currently ongoing.

The IL-17A homodimer is considered a main driver for psoriatic disease, and data from preclinical models indicate that IL-17A is a more important inflammatory signal compared to IL-17AF and IL-17F.⁴ Moreover, serum IL-17F did not correlate with disease activity in psoriasis, in contrast to IL-17A.⁵¹ Importantly, brodalumab, the IL-17 R blocking agent, which should efficiently block all IL-17-AA/AF/FF variants, does not seem to confer better efficacy than mAbs targeting IL-17A, further supporting the critical role of IL-17A.⁵² Similarly, Kaul and coworkers recently showed that more potent IL-17AF inhibition with CJM112 in moderate-to-severe plaque psoriasis did not provide greater clinical efficacy than secukinumab, which blocks IL-17A.²⁵ There are additional approaches to targeting IL-17-induced inflammation, including bispecific IL-17A and IL-17F blocking proteins, such as bimekizumab and sonelokimab.^19,23,24,53 High clinical efficacy has been reported with bimekizumab.^19,23,24 However, it is unclear how much of that effect is attributable to blocking IL-17F, which is 30 times less biologically potent than IL-17A, versus higher drug doses compared to other IL-17A inhibitors.⁵² Results from pooled safety analyses of data from eight randomized clinical trials showed that bimekizumab was well tolerated aside from an increased incidence of mild-to-moderate oral candidiasis.^23,24 Further, a recent study demonstrated that 21.2% of patients treated with bimekizumab developed candidiasis, versus 4.6% of patients treated with secukinumab, suggesting a potential protective role of IL-17F against candida infections, and additional value of targeting IL-17A alone.^22–24 Additionally, an orally administered small molecule (DC-806) IL-17 antagonist is currently in clinical development. Current data are restricted to a short treatment period in mild-to-moderate psoriasis patients.⁵⁴

The efficacy and safety profiles of izokibep were evaluated in a Phase 2 psoriasis trial (NCT03591887) with up to 3 years of patient data of 160 mg SC administration Q2W to every month with no signs of adverse tolerability (data not shown). This suggests that selective and potent IL-17A homodimer blocking can be an efficacious and safe treatment strategy in patients with psoriasis, psoriatic arthritis, and other IL17-mediated diseases. Clinical investigations are underway in psoriatic arthritis (NCT04713072), uveitis (NCT05384249), and hidradenitis suppurativa (NCT05355805).

In summary, izokibep is a small, selective, and potent IL-17A-inhibiting biologic molecule, with demonstrated efficacy in preclinical in vitro and in vivo psoriasis models. In the first-in-human clinical trial, izokibep was well tolerated with no safety concerns identified in healthy volunteers and patients with psoriasis. Efficacy was seen in all patients with psoriatic skin and joint disease. High affinity, combined with small size, extended half-life, and unique selectivity for IL-17A versus IL-17AF make izokibep a novel molecule with potential to safely treat IL17A-associated diseases, including psoriasis, psoriatic arthritis, uveitis, hidradenitis suppurativa, and axial spondyloarthritis. Additional studies are in progress to further characterize the effective dose range in these and other disorders.

Materials and methods

Development of the izokibep molecule

Phage display selections against IL-17A were performed using proprietary Affibody molecule combinatorial libraries mainly as described by Grönwall and colleagues.³⁴ Based on the screening, characterization results, and sequences of promising binders, a maturation library was designed to further assess the sequence space to gain binders with increased affinity and improved properties. A large subset of matured IL-17A-binding Affibody molecules was identified with superior characteristics. Extensive characterization resulted in a best performing IL-17A-binding Affibody molecule that was further formatted. The final molecule, izokibep, is a fusion protein with a dimeric version of the Affibody molecule, where one Affibody molecule is situated on each side of a half-life extending albumin-binding domain.

Characterization assays

Normal human dermal fibroblasts cell assay

An NHDF cell-based assay was used to assess the potency of izokibep, the corresponding monomeric Affibody molecule, and the anti-IL-17A antibodies secukinumab and ixekizumab. A list of all proteins used in different preclinical experiments and studies including supplier and catalog numbers is presented in Table S2. Dilutions of test molecules were prepared in triplicates in an assay buffer composed of fibroblast basal medium with growth promoting factors, either 0.9 nM human IL-17A (Peprotech) and 9 μM HSA (Albucult^Ⓡ, Novozymes) or 0.06 nM human IL-17A (Peprotech), 10 fM IL-1beta (RnD Systems), and 9 μM HSA. Cells were stimulated for 16 h with different concentrations of compounds and IL-17A after which the IL-6 content in supernatants was quantified using an IL-6-specific ELISA (RnD Systems) according to the manufacturer’s instructions.

Kinetic exclusion assay

The kinetic parameters and affinity between IL-17A and izokibep in complex with HSA were determined using KinExA, Sapidyne. Izokibep in complex with HSA (Albucult^Ⓡ, Novozymes) was used as a constant binding partner (CBP) at concentrations of 1 nM, 10 pM, and 1 pM. Human IL-17A (PeproTech) was used as titrant, diluted in 2-fold steps with 5 nM, 100 pM, and 25 pM as starting concentrations. In addition, kinetic parameters and affinity between izokibep and HSA were determined in the presence and absence of IL-17A. Izokibep or izokibep in complex with IL-17A was used as CBP at concentrations 1 nM and 100 pM and HSA was used as a titrant, diluted in 2-fold steps with 1 nM and 10 pM as starting concentrations. The CBPs and the titrants were equilibrated and the remaining amount of free CBP in each equilibrated sample was measured. Data analysis was performed using the KinExA^Ⓡ Pro software and applying a least squares analysis to fit the optimal solutions for the K_D to a curve representative of a 1:1 reversible bi-molecular interaction.

For the determination of the association rate constant (k_a), a direct binding curve analysis was applied. The setup was essentially as described above. However, the amount of free CBP was measured pre-equilibrium, yielding data points that monitored the decrease as the sample moved toward equilibrium. The k_a was determined by a non-linear fit of the theoretical curve to the kinetic data. k_d was calculated based on the measured K_D and k_a.

Surface plasmon resonance analysis to evaluate specificity and selectivity

The specificity of izokibep was determined for IL-17 family members, other cytokines, receptors, and abundant plasma proteins by SPR analysis, using a Biacore 2000 (GE Healthcare). HSA (Albucult^Ⓡ, Novozymes) was immobilized through amine coupling onto the surfaces of a CM5 chip (GE Healthcare) according to the manufacturer’s recommendations. One surface was activated and then deactivated and used as a reference during injections.

Izokibep was injected at a concentration of 200 nM over the sensor chip surfaces followed by injection of 2, 10, and 50 nM IL-17A (PeproTech) or 50 and 250 nM of IL-17AF, IL-17B, IL-17C, IL-17D, IL-17E, IL-17F (all R&D Systems), α-1-antitrypsin (ProSpec), α-2-HS-glycoprotein (ProSpec), α-2-macroglobulin (Sino Biological, Inc.), α-1-microglobulin (Sino Biological, Inc.), β2-microglobulin (Sino Biological, Inc.), GM-CSF (R&D Systems), haptoglobin (ProSpec), hemopexin (Sino Biological, Inc.), holo-transferrin (Sigma), IgA (Bethyl), IgG (Roche), IL-1beta (PeproTech), IL-1R1 (R&D Systems), IL-6 (R&D Systems), IL-17 R (R&D Systems), IL-23 (R&D Systems), and transthyretin (Sino Biological, Inc.), respectively. HBS-EP_0.5 (0.01 M HEPES pH 7.4, 0.5 M NaCl, 3 mM EDTA, 0.005% v/v Surfactant P20) was used for dilutions and as running buffer for IL-17B, IL-17D, IL-17F, and IL-17AF. HBS-EP (•0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v/v Surfactant P20) was used for dilutions and as running buffer for all other proteins.

The chip surfaces were regenerated between each cycle. The reference surface was subtracted from all curves and the sensorgrams were evaluated to determine whether any interaction occurred. Sensorgrams corresponding to the IL-17AF injection were fitted to a kinetic 1:1 Langmuir model in the BiaEvaluation software (GE Healthcare).

Surface plasmon resonance study with izokibep, ixekizumab, and secukinumab: Binding of IL-17A to IL-17 R in the presence of izokibep, secukinumab, or ixekizumab was evaluated by SPR analysis, using a Biacore 2000. IL-17 R (R&D Systems) was immobilized through amine coupling onto the carboxylated dextran layer of the surfaces of a CM5 chip according to the manufacturer’s recommendations. One surface was activated and then deactivated and used as a reference during injections.

IL-17A was injected over the sensor chip surfaces at a concentration of 50 nM in the presence of 50 nM izokibep, ixekizumab, or secukinumab. As a control, IL-17A was injected at a concentration of 50 nM. The chip surfaces were regenerated between each cycle. The reference surface was subtracted from all curves and the sensorgrams were evaluated.

Stability studies

Samples were premixed with five parts 4*LDS (NP0007, Life Technologies) and 11 parts phosphate-buffered saline (PBS) and heated at 70°C for 10 min. A 4% to 12% Bis-Tris gel (NuPAGE, NP0323BOX, Invitrogen) was used and silver stained to visualize bands based on the method by Johansson and Skoog.⁵⁵ Novex Sharp pre-stained protein standard (LC5800, Life Technologies) was used as a molecular weight marker.

SEC was run to detect possible soluble multimeric forms of izokibep in the drug substance. The samples were run on a Superdex 75 10/300 GL size-exclusion column. GMP stability studies were performed according to International Council for Harmonization (ICH) guidelines.

Murine keratinocyte chemoattractant model

In vivo nonclinical efficacy of izokibep was assessed using a murine KC model, where SC administration of human recombinant IL-17A induces secretion of the KC CXCL1. All applicable permissions and ethical approvals for in vivo experiments were obtained. Izokibep (0.01, 0.03, 0.1, and 0.2 mg/kg), ixekizumab (0.04, 0.2, 1, and 3 mg/kg), and secukinumab (0.04, 0.2, 1, and 5 mg/kg) were administered IV to C57BL/6J mice (n = 6 per compound and dose level, Charles River, Germany) 1 h before SC administration of hIL-17A (150 µg/kg). Two hours after administration of hIL-17A, blood samples were taken, and the concentrations of the chemokine KC/CXCL1 were determined by ELISA (KC-quantification kit R&D Systems Cat. No. SMKC00B).

First-in-human clinical study

Clinical study design

ABY-035-001 (NCT02690142) was a Phase 1, first-in-human, multicenter, partially randomized, partially double-blinded study conducted in the United Kingdom to evaluate the safety, tolerability, and PK of izokibep in healthy subjects and patients with psoriasis. The study was performed in 4 parts (Parts A to D). Part A comprised single escalating IV doses of 2, 5, 10, 20, or 40 mg izokibep, or placebo administered to healthy subjects for review of safety, tolerability, and PK data. Part B comprised a single SC dose of 40 mg izokibep administered to healthy subjects. Parts C and D were in patients with psoriasis to allow for a preliminary assessment of efficacy. In Part C, a single dose of 2 or 40 mg izokibep was administered IV. In Part D, either three doses Q2W or seven doses Q2W of 40 mg izokibep were administered SC.

Clinical study population

Adult (18–65 years of age) male and female subjects of any ethnic origin, with a body mass index (BMI) between 18.0 and 32.0 kg/m² and maximum body weight of 120 kg were included for Parts A and B of the study. Adult (18–65 years of age) male and female subjects of any ethnic origin, with BMI between 18.0 and 39.9 kg/m² and minimum body weight of 45 kg were included for Parts C and D of the study. In Part C, patients had a diagnosis of moderate-to-severe plaque-type psoriasis (confirmed by a dermatologist or suitably trained and experienced personnel) at least 6 months prior to administration of izokibep without a documented flare within 30 days prior to screening. Patients also had plaque-type psoriasis covering at least 10% of total body surface area at screening and at baseline (Day −1). In Part D, patients had a diagnosis of plaque-type psoriasis (mild, moderate, or severe) at least 6 months prior to administration of izokibep without a documented significant flare within 30 days prior to screening. Patients also had at least one psoriatic lesion suitable for efficacy assessment at screening and at baseline (Day 1), as judged by the investigator.

Prior to the start of the study, the study protocol and informed consent form were reviewed and approved by an independent ethics committee and the Medicines and Healthcare Products Regulatory Agency. The study was conducted in the United Kingdom, in accordance with the relevant articles of the “Declaration of Helsinki,” the ICH Good Clinical Practice consolidated guidelines, and applicable regional and local legislation. All subjects provided written informed consent prior to their enrollment into the study.

Clinical study assessments

Safety

Safety was assessed via severity of AEs, local tolerability assessments, VAS assessment of injection site pain (Part D only), vital signs, 12-lead ECG, clinical laboratory evaluations, physical examination, ADAs, pro-inflammatory cytokines, and C-reactive protein.

Pharmacokinetics

Izokibep was measured in human plasma samples using a validated liquid chromatography-tandem mass spectrometry assay of peptides obtained after tryptic digestion of izokibep. The quantifier peptide covers the amino acids 101 to 119 of izokibep. The validated range of the assay is 20 to 10,000 ng/mL.

Anti-drug antibody assay

Immunogenicity was assessed using a validated three-tiered, meso-scale discovery-based ADA assay using an izokibep-specific, polyclonal antibody as positive control. The relative sensitivity of the assay was ~100 ng/mL and drug tolerance was ~ 500 ng/mL at 2 µg/mL of drug. Izokibep-specific ADAs were analyzed in predose serum samples and in serum samples taken 4 (Parts A to C) or 7 (Part D) times during the treatment period.

Efficacy

In Parts C and D, the basic characteristics of psoriatic lesions (i.e., redness, thickness, scaliness, and affected surface area) were measured to assess the severity of psoriasis, using the PASI with each characteristic per body area graded on a 0 to 4 scale (total [absolute] PASI score can range from 0 [no psoriasis on the body] to 72 [the most severe case of psoriasis]). Pruritus was evaluated using a VAS assessment of pruritus. Exploratory measures of efficacy in Part D (multiple, SC dosing) were change from baseline in the severity of psoriasis using sPGA; changes from baseline in the severity of joint pain in patients with psoriatic arthritis using VAS (patients with psoriatic arthritis only); and changes from baseline in absolute PASI scores in patients with mild-to-moderate psoriasis.

Clinical study statistical analysis

Summary statistics were generated for safety, tolerability, PK, and efficacy data.

The following PK parameters were determined by non-compartmental analysis using WinNonlin Version 6.4: C_max, t_max, time of last quantifiable plasma concentration (t_last), AUC_0-tlast, AUC_0-∞, plasma terminal elimination half-life (t_½), C_L, V_ss, F, for repeated dose regimen AUC over the dosing interval (AUC_0-τ), and accumulation ratio based on AUC_0-τ. Dose and body weight normalized values were determined by dividing the value of the PK parameter by total dose per kg body weight.

Abbreviations

ADA	=	Anti-drug antibody
AE	=	Adverse event
AUC	=	Area under the curve
BMI	=	Body mass index
CBP	=	Constant binding partner
CI	=	Confidence interval
EDTA	=	Ethylenediamine tetraacetic acid
GMP	=	Good manufacturing practice
GM-CSF	=	Granulocyte macrophage colony-stimulating factor
HBS-EP	=	HEPES buffered saline EDTA Surfactant P20
HEPES	=	N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid
HSA	=	Human serum albumin
ICH	=	International Council for Harmonization
IL	=	Interleukin
ISR	=	Injection site reaction
IV	=	Intravenous
KC	=	Keratinocyte chemoattractant
LLOQ	=	Lower limit of quantification
mAbs	=	Monoclonal antibodies
NA	=	Not applicable
NHDF	=	Normal human dermal fibroblast
NOAEL	=	No observed adverse effect level
PASI	=	Psoriasis area severity index
PK	=	Pharmacokinetics
Q2W	=	Every two weeks
SC	=	Subcutaneous
SD	=	Standard deviation
SEC	=	Size-exclusion chromatography
sPGA	=	Static physician’s global assessment
SPR	=	Surface plasmon resonance
TEAE	=	Treatment emergent adverse event
Th	=	T-helper
TNF	=	Tumor necrosis factor
VAS	=	Visual analog scale

Acknowledgments

The authors would like to thank all patients, healthy volunteers, medical doctors, and clinical staff who made this study possible and acknowledge all Affibody coworkers for scientific and editorial work that support this publicationin particular, Dr Juliette Jansson for pharmacokinetic modeling and Dr Dieter Wetzel for medical expertise.

Disclosure statement

SK, JF, KDB, EG, IHG, DB and FYF are currently, and LG and AW were previously employees of Affibody AB and its affiliates. ACN, APK, and PMP are employees of ACELYRIN.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/19420862.2023.2209920.

Additional information

Funding

Affibody AB, Sweden, and ACELYRIN Inc., USA, funded the work featured in this article.