Safety, pharmacokinetics, and pharmacodynamics of MK-6194, an IL-2 mutein designed to selectively activate regulatory T cells: single ascending dose and multiple ascending dose trial data

Abstract

MK-6194, an interleukin-2 mutein designed to selectively activate regulatory T cells (Tregs), was evaluated for safety, pharmacokinetics (PK), immunogenicity, and pharmacodynamics in healthy participants. In a single ascending dose trial (N = 56), participants received subcutaneous MK-6194 or placebo (3:1 ratio) across dose levels ranging from 1 to 10 mg. In a multiple ascending dose trial (N = 54), participants received subcutaneous MK-6194 or placebo (3:1 ratio) at dose levels ranging from 0.5 to 5 mg every 2 wk (total 3 doses) as well as 5 mg every 4 wk (total 2 doses). Baseline characteristics were comparable between trials, with participants mostly male with a mean age of 36 yr. There were no serious adverse events or dose-limiting toxicities. The most common adverse events were injection site erythema and eosinophil count elevations (with no indication of severe eosinophilia or eosinophilia-related organ damage). PK showed dose-proportionality and repeated doses of MK-6194 did not result in accumulation or time-dependent PK. Immunogenicity was low with no impact on PK or safety. Treg expansion as assessed by flow cytometry and Treg-specific demethylation region analysis was observed in a dose-dependent manner during both trials and expanded within about 8 d postdose up to about 5-fold and returned to baseline by 14 to 29 d postdose. Minimal impact was observed on other lymphocytes including total T lymphocyte and natural killer cell counts. These findings support the further development of MK-6194 as a potential treatment for autoimmune disorders.

Introduction

Autoimmune and inflammatory diseases can result from disrupted immune homeostasis resulting in chronic inflammation and tissue damage.¹ Among the various immune cells implicated in the pathogenesis of autoimmune disorders, regulatory T cells (Tregs) play a pivotal role in maintaining immune homeostasis and preventing autoimmunity.² However, in autoimmune disorders, the function and number of Tregs are often compromised, contributing to disease progression.³ Thus, expansion of Tregs holds promise as a means to treat various autoimmune disorders.

Interleukin (IL)-2 is a cytokine involved in immune regulation that has both pro- and anti-inflammatory properties, and that mediates its activity through interaction with IL-2 receptors (IL-2Rs) of different configurations. Three subunits can contribute to IL-2Rs, IL-2Rα (CD25), IL-2Rβ (CD122), and IL-2Rγc (CD132), and the composition of IL-2R subunits determines receptor affinity to IL-2. The intermediate-affinity IL-2R, which is dimeric and consists of the 2 subunits IL-2Rβ and IL-2Rγc, is present on effector T lymphocytes and natural killer (NK) cells. Therefore, this configuration can promote cytotoxic activity. Conversely, the high-affinity IL-2R, which is trimeric and includes IL-2Rα, IL-2Rβ, and IL-2Rγc,⁴ is present on activated effector T cells as well as on Tregs, which are IL-2–dependent T cells with regulatory or suppressive functions that selectively express transcription factor FOXP3.^5,6 Tregs express elevated levels of IL-2Rα/CD25 in which IL-2 signaling promotes immune homeostasis. Notably, mice deficient in IL-2 or IL-2Rα/CD25 exhibit pathologic autoimmunity and inflammation.^4,7,8

High-dose therapeutic IL-2 administration that engages both intermediate and high-affinity receptors has been used in oncology, particularly for advanced-stage melanoma and renal cell carcinoma to enhance NK cells and conventional T cells that detect and kill cancer cells. In contrast, low doses of IL-2 preferentially bind to the high-affinity trimeric IL-2Rs, which have a 10 to 100 times higher affinity for IL-2 compared with the dimeric IL-2Rs.⁹

Preclinical studies in mouse models of autoimmune diseases have demonstrated the beneficial effects of low-dose IL-2 therapy in restoring immune tolerance and impeding disease progression through Treg expansion.^10,11 Based on these studies the use of low-dose IL-2 therapy has been explored in clinical trials where clinical improvements have been observed in autoimmune diseases such as systemic lupus erythematosus^12,13 and graft-versus-host disease.^14,15 However, factors such as low selectivity leading to limited therapeutic window and short half-life requiring frequent subcutaneous dosing have impeded progress of IL-2 therapies in autoimmune disorders and imposed limitations to therapeutic potential.^16–22 Modified variants of IL-2 (i.e., IL-2 muteins [IL-2Ms]) have been developed and offer a novel method for expanding Tregs by enhancing CD25 (IL-2Rα) binding while reducing CD122 (IL-2Rβ) binding. This results in selective activation of trimeric CD25-containing IL-2Rs over dimeric receptors with the intent of activating Tregs over conventional T cells.^23,24

MK-6194 (formerly PT101) is a novel IL-2M fused to an effector-function ablated human IgG domain (i.e., the Fc region of the IgG molecule) for half-life extension.²⁵ MK-6194 selectively induces IL-2R–mediated signal transduction in Tregs through trimeric IL-2R activation in vitro and preferentially expands them in vivo, making it a potential therapeutic approach in the treatment of autoimmune and inflammatory diseases.²⁶ In this report, we describe two phase 1 clinical trials of MK-6194, including a single ascending dose (SAD) trial and a multiple ascending dose (MAD) trial, that were conducted to investigate safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of MK-6194 in healthy participants.

Methods

Protocol 001 (SAD trial) was conducted from January 31, 2020 to November 25, 2020, at a single site in Canada. Protocol 003 (MAD trial) was conducted from November 22, 2021, to December 12, 2022, at a single site in Belgium. The studies were conducted according to principals of Good Clinical Practice and were approved by the clinical trial sites’ Institutional Review Board and Ethics Committee, respectively. All participants provided written informed consent.

Study designs

The SAD trial was a randomized double-blind, placebo-controlled, single-dose escalation trial. Seven cohorts of 8 participants per cohort received a single administration of MK-6194 or placebo subcutaneously in a SAD design (Fig. 1). Each participant was eligible to participate in 1 cohort only. Sentinel dosing was used in each cohort (1 participant who received MK-6194 and 1 participant who received placebo) to ensure adequate safety and tolerability prior to administration of study drug to the remainder of the cohort. Available safety, PK, and PD data from preceding panels were reviewed prior to dose escalation.

Figure 1.

Schematics for the SAD and MAD studies showing design of the studies. D, day.

The MAD trial was a randomized double-blind, placebo-controlled, MAD escalation trial. The trial enrolled 54 participants in 7 sequential panels of up to 8 participants per panel who received multiple subcutaneous doses of MK-6194 or placebo (randomized 3:1) over 29-d treatment periods (Fig. 1). Treatment was administered every 2 wk up to 3 doses or every 4 wk up to 2 doses. Each participant was eligible to participate in 1 panel only. Dosing initiated at 0.5 mg and escalated through 5 mg. Refer to Fig. 1 for dosing assignments and dosing intervals. Available safety, PK, and PD data from preceding panels were reviewed prior to dose escalation.

Participants

Eligible participants were healthy subjects between the ages of 18 and 55 yr, body mass index (BMI) within 18 and 30 kg/m² (SAD) or 18 and 32 kg/m² (MAD) and weighed at least 50 kg. Key exclusion criteria included pregnancy or nursing, certain diseases, surgeries or infections, recent immunization, abnormal laboratory values or physical exam findings, and any other medical or personal condition that may affect safety or compliance. Participants were randomized via a computer-generated randomization schedule.

Assessments

Safety and tolerability

Safety and tolerability were assessed through multiple measures including adverse events (AEs), systemic and injection site reactions, physical examination findings, vital sign measurements, measurement of antidrug antibody (ADA), clinical laboratory results, and electrocardiograms. Severity, seriousness, and causality of AEs were assessed independently by the study investigators.

Pharmacokinetics

In the SAD trial, blood samples for serum concentration of MK-6194 were collected for determination of serum MK-6194 levels at predose and multiple postdose time points for each cohort (1, 4, 8, 24, 48, 72, 168, 336, and 672 h for cohorts 1–3 and 1, 4, 8, 12, 24, 48, 72, 120, 168, 216, 264, 336, 504, and 672 h for cohorts 4–7). Serum MK-6194 PK samples were obtained in the MAD trial on the following days: 1, 2, 3, 5, 8, 15, 16, 17, 19, 22, 29, 30, 31, 33, 36, 43, and 57 (poststudy visit). On days 1 and 29, additional PK samples were collected predose and at 1, 4, and 12 h postdose, and on day 15 samples were collected predose and 12 h postdose. PK analyses were conducted at Charles River Laboratories. MK-6194 concentrations were determined from human serum using a validated enzyme-linked immunosorbent assay with a capture reagent specific to MK-6194 and a lower limit of detection of 0.15 ng/mL.

Immunogenicity

In the MAD trial, serum samples were collected at predose and on days 15, 29, and 57 prior to dosing on treatment days. Samples were screened for antibodies binding to MK-6194 and the titers of confirmed positive samples were recorded. Samples at matched time points were also evaluated for MK-6194 serum concentration. Samples that were positive for antibody binding to MK-6194 were further characterized for neutralizing activity for MK-6194. Cross-reactivity and neutralizing activity to wild-type IL-2 was also evaluated in the MAD trial. Serum samples for the evaluation of antibodies to MK-6194 were analyzed by Charles River Laboratories.

Pharmacodynamics

In the SAD trial immunophenotyping samples for Tregs and other immune cells were analyzed using fresh whole blood samples. Treg and other cell type measurements in the MAD trial were done using flow cytometry using a novel lyse-fix-freeze method.²⁷ This method involved the use of antibodies targeting specific cell surface markers, allowing for the identification and analysis of different cell populations by flow cytometry. The assay involves direct staining of whole blood samples with a cocktail of antibodies against CD45, CD3, CD4, CD8, CD56, CD14, CD19, CD127, CD25, and FOXP3 for the SAD trial and frozen lysed/fixed K2 EDTA anticoagulated whole blood samples from the MAD trial with a cocktail of antibodies against CD45, CD3, CD4, CD8, CD56, CD14, CD20, CD127, CD25, and FOXP3. A fluorescence-minus-two control sample for CD25 FOXP3 was prepared for each sample and fully stained samples were processed in duplicate. Coefficient of Variance (%CV) was calculated among the replicates for all parameters. Frozen lysed/fixed K2 EDTA anticoagulated whole blood samples were received and stored at approximately −80 °C.

DNA methylation status of CpG sites in the FOXP3 Treg-specific demethylation region (TSDR) and CD4 were evaluated with the fit-for-purpose validated Epiontis ID quantitative polymerase chain reaction–based assay to monitor CD4 and Treg cell counts using 75 mL of whole blood collected in PaxGene DNA tubes (stored at 20 °C until use) in both studies. The assay uses oligonucleotides (forward/reverse primers and probe) designed to detect demethylated (TpG) and methylated (CpG) templates by quantitative polymerase chain reaction. Resulting data are reported as relative quantitation based on parallel measurements of GAPDH methylation status (% Tregs in total leukocytes) and absolute quantitation (cells/µL) with the evaluation of plasmid-based calibrators. In the MAD trial, a total of 331 whole blood samples from 54 healthy participants were collected at multiple time points (day 1 predose, day 8, day 15 predose, day 22, day 29 predose, day 36, and day 57), encompassing 6 dose panels for epigenetic immune cell counting. Overall, 99.7% (n = 330 of 331) of sample results passed Epiontis ID assay quality control criteria.

PD analysis was done by Charles River Laboratories for the SAD trial and PPD Inc. for the MAD trial.

Total sCD25 (free sCD25 and MK-6194 bound sCD25 together) in serum was measured by Quantikine ELISA Human CD25/IL-2 Rα (catalog number: DR2A00; Bio-Techne/R&D Systems) in the MAD trial. This assay employs the quantitative sandwich enzyme immunoassay technique. It was confirmed as a total sCD25 assay because MK-6194 was shown not to interfere with the measurement of sCD25 in human serum. Fit-for-purpose validation and sample analysis of total sCD25 was executed at Q2 Solutions Laboratories. In total, 698 serum samples at predose and multiple postdose time points were successfully analyzed using this method passing prespecified precision and accuracy (nonbias) acceptance criteria.

Statistical analysis

The sample sizes were based on clinical and practical considerations to meet objectives for a phase 1 trial while minimizing overall exposure to healthy participants.

The safety analysis set included all participants who were enrolled and received at least 1 dose of the study drug. Data from participants in the safety analysis set were used for demographic characteristics, baseline characteristics, and safety summaries. The PK Set consisted of all participants who received study drug and had at least 1 measurable serum concentration.

Standard noncompartmental analysis methods were used to calculate PK parameters for both the SAD and MAD studies. Area under the concentration-time curve was determined using the linear-up/log-down trapezoidal rule. For each PK parameter in the MAD trial, individual values of MK-6194 serum PK parameters from participants in all panels were pooled, natural log transformed, and analyzed based on a linear model containing a fixed effect for dose level, day, and the interaction of dose and day, and a random effect for participants.

Dose proportionality of MK-6194 PK was assessed for the SAD trial. Analysis was performed using a linear regression fit of the natural log-transformed area under the concentration-time curve, or natural log-transformed Cmax, versus the natural log-transformed dose to estimate the slope and the confidence interval of the slope. To minimize overall exposure to healthy participants, no formal dose-proportionality analysis was performed for the MAD trial. Exposures were observed to be approximately dose proportional in that trial as well.

In the MAD trial, Tregs, conventional T lymphocytes, and NK cells were evaluated using the fold change from baseline by flow cytometry. The absolute change from baseline in FOXP3 TSDR was also evaluated. Placebo participants were pooled across panels. The mean estimates (MK-6194 doses vs. placebo) and their corresponding 95% confidence interval were calculated.

Results

Participants

SAD study

A total of 56 participants were randomized and completed with no discontinuations. Males constituted 62.5% of the participants, and 85.7% were White. The mean age of all participants was 36.1 yr, with a range of 18 to 55 yr. The mean height, weight, and BMI of all participants were 169.1 cm, 73.3 kg, and 25.6 kg/m², respectively (Table 1).

Table 1.

Baseline characteristics for the SAD trial.

	Pooled placebo (n = 14)	MK-6194 1 mg (cohort 1) (n = 6)	MK-6194 3.5 mg (cohort 2) (n = 6)	MK-6194 10 mg (cohort 3) (n = 6)	MK-6194 3.5 mg (cohort 4) (n = 6)	MK-6194 5 mg (cohort 5) (n = 6)	MK-6194 7.5 mg (cohort 6) (n = 6)	MK-6194 5 mg (cohort 7) (n = 6)	MK-6194 pooled (n = 42)	Total (n = 56)
Sex
Male	11 (78.6)	5 (83.3)	5 (83.3)	4 (66.7)	2 (33.3)	3 (50.0)	3 (50.0)	2 (33.3)	24 (57.1)	35 (62.5)
Female	3 (21.4)	1 (16.7)	1 (16.7)	2 (33.3)	4 (66.7)	3 (50.0)	3 (50.0)	4 (66.7)	18 (42.9)	21 (37.5)
Age, y
Mean ± SD	34.9 ± 6.62	36.3 ± 11.98	35.5 ± 12.24	35.2 (± 9.11	32.5 ± 8.22	42.8 ± 13.79	32.5 ± 11.64	40.2 ± 8.77	36.4 ± 10.78	36.1 ± 9.87
Median	35.0	33.5	39.5	33.0	30.5	50.0	28.5	39.0	34.5	35.0
Range	24–50	24–51	18–49	26–49	24–43	18–53	23–55	30–51	18–55	18–55
Race/ethnicity
Asian	0	1 (16.7)	0	1 (16.7)	0	0	0	0	2 (4.8)	2 (3.6)
Black or African American	1 (7.1)	1 (16.7)	1 (16.7)	0	1 (16.7)	1 (16.7)	0	0	4 (9.5)	5 (8.9)
White	13 (92.9)	4 (66.7)	5 (83.3)	5 (83.3)	5 (83.3)	5 (83.3)	6 (100.0)	6 (100.0)	36 (85.7)	49 (87.5)
Height, cm
Mean ± SD	171.3 ± 6.8	172.0 ± 8.8	172.4 ± 5.7	169.5 ± 8.4	165.7 ± 6.9	170.2 ± 11.5	165.8 ± 10.2	162.7 ± 8.7	168.3 ± 8.8	169.1 ± 8.4
Median	170.1	169.7	173.9	169.7	164.1	170.9	162.2	160.7	167.4	169.4
Range	158.7–184.5	162.3–184.7	161.4–177.5	158.6–178.2	157.1–177.9	155.3–187.0	155.1–182.6	150.8–174.5	150.8–187.0	150.8–187.0
Weight, kg
Mean ± SD	76.9 ± 10.8	76.6 ± 7.2	76.4 ± 10.9	71.1 ± 7.5	70.8 ± 13.0	68.2 ± 9.6	69.0 ± 13.1	72.8 ± 9.6	72.1 ± 10.1	73.3 ± 10.4
Median	78.25	75.65	77.90	71.20	65.05	68.45	71.30	74.20	72.60	73.65
Range	55.2–91.2	67.0–85.4	62.0–87.5	62.6–83.5	58.8–94.3	54.0–79.6	51.6–88.4	55.5–84.2	51.6–94.3	51.6–94.3
BMI, kg/m2
Mean ± SD	26.2 ± 3.5	25.9 ± 2.6	25.6 ± 2.8	24.7 ± 1.8	25.6 ± 3.2	23.5 ± 2.1	24.9 ± 2.6	27.4 ± 2.1	25.4 ± 2.551	25.6 ± 2.8
Median	26.90	25.55	25.60	24.55	25.00	22.90	25.60	28.10	25.20	25.55
Range	18.3–30.9	22.7–30.1	21.9–29.1	23.0–27.2	21.8–29.7	21.5–26.9	21.4–27.5	24.4–29.5	21.4–30.1	18.3–30.9

Values are n (%), unless otherwise indicated.

MAD study

A total of 54 participants were randomized to study treatments with 7 discontinued after the first injection (due to COVID-19 infection [n = 4], influenza-like illness [n = 1], and withdrawal of consent [n = 2]). Of note, participants in the 5 mg every 2 wk (Q2W) and 5 mg every 4 wk (Q4W) cohorts only received 2 doses of MK-6194 (Fig. 1). The participants were predominantly male (83.3%) and White (94.4%). The mean age of participants was 36.1 yr, with a range of 18 to 56 yr. The mean height, weight, and BMI of the participants were 176.4 cm, 75.7 kg, and 24.4 kg/m², respectively (Table 2).

Table 2.

Baseline characteristics for the MAD trial.

	Pooled placebo (n = 13)	0.5 mg MK-6194 Q2W (panel A) (n = 6)	1 mg MK-6194 Q2W (panel B) (n = 7)	2 mg MK-6194 Q2W (panel C/D) (n = 10)	3.5 mg MK-6194 Q2W (panel E) (n = 6)	5 mg MK-6194 Q2W (panel F) (n = 6)	5 mg MK-6194 Q4W (panel G) (n = 6)	Total (n = 54)
Sex
Male	11 (84.6)	5 (83.3)	7 (100.0)	8 (80.0)	3 (50.0)	5 (83.3)	6 (100.0)	45 (83.3)
Female	2 (15.4)	1 (16.7)	0	2 (20.0)	3 (50.0)	1 (16.7)	0	9 (16.7)
Age, y
Mean ± SD	32.8 ± 12.1	37.0 ± 7.6	35.0 ± 8.1	33.6 ± 10.7	44.3 ± 13.9	36.7 ± 14.0	39.3 ± 12.1	36.1 ± 11.4
Median	28.0	36.5	37.0	32.0	52.0	39.5	39.5	35.0
Range	18–54	28–48	25–46	21–50	26–55	19–53	24–56	18–56
Race/ethnicity
Asian	1 (7.7)	0	0	0	0	0	0	1 (1.9)
Black or African American	2 (15.4)	0	0	0	0	0	0	2 (3.7)
White	10 (76.9)	6 (100.0)	7 (100.0)	10 (100.0)	6 (100.0)	6 (100.0)	6 (100.0)	51 (94.4)
Height, cm
Mean ± SD	176.8 ± 10.3	177.8 ± 9.4	180.2 ± 7.9	176.8 ± 10.5	170.5 ± 5.9	174.4 ± 7.3	177.4 ± 5.3	176.4 ± 8.8
Median	177.4	180.8	181.8	180.0	173.1	174.0	176.2	176.8
Range	158.0–196.0	160.0–187.5	165.1–188.5	159.5–189.0	161.9–176.0	165.5–184.6	172.0–186.2	158.0–196.0
Weight, kg
Mean ± SD	78.2 ± 8.1	77.3 ± 12.2	70.6 ± 12.6	76.2 ± 9.5	67.2 ± 4.9	77.2 ± 8.0	81.8 ± 13.8	75.7 ± 10.2
Median	79.4	78.5	73.4	72.6	66.2	77.7	84.0	75.6
Range	65.2–95.2	60.8–93.8	51.8–90.7	64.8–91.0	62.4–76.0	67.2–86.6	60.4–95.4	51.8–95.4
BMI, kg/m2
Mean ± SD	24.5 ± 3.7	21.6 ± 2.5	24.4 ± 2.6	23.1 ± 1.5	25.4 ± 3.2	25.93 ± 8	25.3 ± 4.3	24.4 ± 3.4
Median	24.2	21.4	24.6	23.6	24.7	26.4	24.8	24.3
Range	18.9–30.1	18.1–25.6	20.8–27.8	20.8–24.5	22.8–31.6	20.4–31.0	18.3–32.2	18.1–32.2

Values are n (%), unless otherwise indicated.

Safety

SAD study

A total of 17 (40.5%) of 42 participants receiving MK-6194 and 3 (21.4%) of 14 participants receiving placebo reported at least 1 treatment-emergent AE (TEAE). There were no serious AEs, deaths, or AEs leading to discontinuation of treatment. The most common AEs reported were injection site reactions such as erythema and pruritus. The TEAEs were generally mild to moderate in severity. All AEs that were reported were transient in nature and resolved or were resolving by the end of the trial (Table 3).

Table 3.

Summary of safety results.

	Pooled MK-6194 treatment arms	Pooled placebo
SAD trial	n = 42	n = 14
Participants with AEs	17 (40.5%)	3 (21.4%)
With serious AEs	0	0
With AEs leading to withdrawal	0	0
Most common (AEs)
Injection site erythemaa	7 (16.7%)	0
Pruritus	3 (7.1%)	0
Dyspnea	3 (7.1%)	0
MAD trial	n = 41	n = 13
Participants with AEs	41 (100%)	12 (92.3%)
With serious AEs	0	0
With AEs leading to withdrawal	3 (7.3%)	2 (15.4%)
Most common (AEs)
Injection site erythemaa	36 (87.8%)	2 (15.4%)
Headache	14 (34.1%)	6 (46.2)
Eosinophil counts increased	21 (51.2%)	1 (7.7%)

^aThe Common Terminology Criteria for Adverse Events grading scale was used to grade injection site erythema the SAD trial. In the MAD trial, the FDA Guidance used for Healthy Adult and Adolescent Volunteers Enrolled in Preventive Vaccine Clinical Trials was used to grade injection site erythema.

MAD study

Forty-one (100%) of 41 participants receiving MK-6194 and 12 (92.3%) of 13 participants receiving placebo reported at least 1 TEAE. There were no serious AEs or deaths. Three (7.3%) out of 41 participants receiving MK-6194 and 2 (15.4%) out of 13 participants receiving placebo discontinued the drug due to AEs (Table 3). Consistent with the SAD trial, injection site erythema was the most common AE. In the MK-6194 treatment arms, the eosinophil count increase was observed more frequently than in participants receiving placebo (i.e., 51% vs. 8%). The eosinophil count increases were dose responsive, not assessed as severe (absolute eosinophil count >5,000/µL), and not associated with organ damage (Table 2).

Pharmacokinetics

Overall, the PK of MK-6194 was observed to be generally consistent across studies, with PK profiles after the first dose being similar to the final dose (Fig. 2). Repeated doses of MK-6194 did not result in accumulation or time-dependent PK, and exposures were approximately dose proportional.

Figure 2.

Mean MK-6194 serum concentration (log scale) vs. time by dose in the (A) SAD and (B) MAD studies. Error bars have been omitted for clarity of the figure. Relative standard error of the maximum mean concentrations ranged between approximately 8% and 34% in the SAD study, and approximately 13% and 57% in the MAD study.

SAD study

The geometric mean t1/2 of MK-6194 ranged from 20.4 to 28.3 h and the Tmax ranged from 10.5 to 13.5 h. MK-6194 exposure was dose proportional across the range studied (from 1 to 10 mg). The geometric mean AUC_0–inf (%CV) at doses of 1, 3.5, 5, 7.5, and 10 mg were 309 (94.1%), 1750 (48.1%), 2150 (54.3%), 3430 (44.2%), and 4160 (24.6%) h•µg/L, respectively. Geometric mean Cmax (%CV) values were 8.12 (82.3%), 39.6 (49.5%), 43.2 (49.7%), 67.2 (42.3%), and 88.7 (18.3%) µg/L, respectively. Interparticipant variability was moderate, with the 1 mg dosing group being notable for having 1 subject with relatively high exposure as compared with the rest of the group.

MAD study

In the MAD trial, the geometric mean t1/2 of MK-6194 following final dose administration was approximately 40 h for the 0.5 mg dose and ranged between about 20 and 32 h for the higher doses. The median Tmax was approximately 12 h, with the following exceptions: in the 0.5 mg group, the median Tmax was approximately 18 h for the first and second doses, and in the 5 mg Q4W group, the median Tmax was approximately 24 h. Similar to the SAD PK data, MK-6194 exposures increased in an approximately dose proportional manner. The interparticipant variability was moderate, with individual variability in the 0.5, 1, and 2 mg dosing groups being higher as compared with the other dosing groups.

The longitudinal time course for the 0.5, 1, 2, 3.5, and 5 mg MK-6194 dose cohorts exhibited a monoexponential decline for about 1 wk after injection. MK-6194 did not accumulate, and the PK had no apparent dependence on time (i.e., the PK for the first dose was generally representative of subsequent doses) (Fig. 2).

Immunogenicity

SAD study

The incidence of ADA-positive participants among participants who received MK-6194 was 25% (3 of 12 participants), 16.7% (2 of 12 participants), and 50% (3 of 6 participants) in the MK-6194 3.5 mg, 5 mg, and 10 mg dose groups, respectively. In 3 participants (2 participants for the MK-6194 3.5 mg dose and 1 participant for MK-6194 10.0 mg dose), ADAs were detected in the predose sample. No ADAs were detected in participants who received the MK-6194 1 mg and 7.5 mg doses. Only 1 participant had detectable ADAs at the end of the trial, and this participant also had ADAs detected predose. The incidence of ADA-positive participants receiving placebo was 21.4% (3 of 14 participants). There was no clear effect of ADA on PK or ADA-related safety findings.

MAD study

The incidence of treatment-emergent ADA was 5.3% (2 of 38 evaluable participants receiving MK-6194). The occurrence of ADA was intermittent or transient within each ADA-positive participant and did not exhibit any association with dose level or timing of administration. One participant who received MK-6194 0.5 mg Q2W had ADA titers ≤20, the lowest possible measurable ADA, on days 15 and 57. One participant who received MK-6194 1 mg Q2W had ADA titers ≤80 and ≤20 on days 15 and 29, respectively. There was 1 non–treatment-emergent positive ADA in the MK-6194 2 mg group (i.e., the participant was positive prior to treatment with a titer ≤20 at predose and day 15). There was no clear effect of ADA on PK or ADA-related safety findings. All 3 active-arm participants who had positive ADA results showed evidence of neutralization against MK-6194. The 2 treatment-emergent ADAs also showed evidence of neutralization against wild-type IL-2.

Pharmacodynamics

SAD study

PD is primarily presented for the MAD trial. Briefly, in the SAD trial, fold increases from baseline in sCD25 and Treg counts were observed in a dose-dependent manner, peaking at day 8 after single-dose administration (Fig. S1).

MAD study

In the MAD trial, dose-related fold-increases from baseline in total Tregs were observed with peak responses around 8 d after dosing with consecutive return toward baseline (Fig. 3). The mean fold increases from baseline for CD4+ CD25+ FOXP3+ Tregs on day 8 (i.e., 7 d after the first dose) were 1.76, 1.68, 1.59, 3.96, 1.97, and 2.90 for MK-6194 0.5, 1, 2, 3.5, and 5 mg Q2W and 5 mg Q4W, respectively, and 0.81 for placebo. These dose-related Treg fold increases from baseline continued with repeated dosing. Slightly lower than expected fold increase from baseline in Tregs in the 5 mg Q2W panel was caused by a higher mean baseline Treg count in that panel, and expansion of absolute Treg counts from baseline in that panel was similar to what was observed in the 3.5 mg Q2W and 5 mg Q4W panels (not shown). The mean fold increases from baseline for CD4+ CD25+ FOXP3+ Tregs 7 d after the last dose were 2.13, 1.84, 1.57, 5.08, 3.14, and 3.34 for MK-6194 0.5, 1, 2, 3.5, and 5 mg Q2W and 5 mg Q4W, respectively, and 1.27 for placebo. The mean maximal Treg fold change from baseline was consistently above 2 for MK-6194 doses of 3.5 and 5 mg (Fig. 3). For total sCD25, the mean fold increases from baseline on day 8 were 1.12, 1.54, 1.80, 2.14, 2.65, and 2.81 for MK-6194 0.5, 1, 2, 3.5, and 5 mg Q2W and 5 mg Q4W, respectively, and 1.04 for placebo. The mean fold increases from baseline for total sCD25 7 d after the last dose were 1.24, 1.56, 2.25, 2.54, 3.29, and 2.82 for MK-6194 0.5, 1, 2, 3.5, and 5 mg Q2W and 5 mg Q4W, respectively, and 1.13 for placebo. While the mechanism and potential clinical relevance of sCD25 increase has not been fully elucidated, dose-responsive increases in serum CD25 have been observed in the context of treatments targeting IL-2R for Treg expansion.²⁸

Figure 3.

Effect of MK-6194 on Tregs and protective cell types over time in the MAD trial (gating strategy shown in Fig. S2) (A) Mean ± 95% confidence interval (CI) fold change from baseline over time in select cells showing expansion of Tregs by flow cytometry (CD4+ CD25+ FOXP3+ Tregs) and by immunoassay (total soluble CD25) and mean ± 95% CI absolute change from baseline in FOXP3 TSDR methylation status over time. (B) Mean ± 95% CI fold change from baseline over time in NK cells and total T lymphocytes.

No significant dose-responsive expansion was observed for T lymphocytes or NK cells (Fig. 3). Absolute changes in demethylation of the FOXP3 TSDR over time was consistent with expansion of functional Tregs and generally corresponded with flow cytometry–based Treg assessments (Fig. 3).

Discussion

Research with low-dose IL-2 treatments has shown diverse outcomes in managing various autoimmune and inflammatory conditions. An analysis of 5 clinical trials of low-dose IL-2 in patients with chronic graft-versus-host disease showed an overall response rate of 53% after up to 12 wk of treatment. These studies indicated an expansion of Tregs peaking at 2 to 4 wk into treatment. The increased ratio of Tregs over conventional T cells in these studies was associated with overall response. However, NK cells also increased throughout the treatment periods.²⁹ Further research in the use of low-dose IL-2 in systemic lupus erythematosus patients also demonstrated Treg expansion, but the trials missed their primary endpoints. Insufficient selectivity and/or suboptimal PK of low-dose IL-2 may have contributed to these findings as well, suggesting that modification of these factors may improve outcomes.^30,31

Subsequent development in therapeutic IL-2s has focused on IL-2 agonists with prolonged half-life^32,33 and greater specificity for CD25 over CD122.^34,35 Which features of IL-2 agonists translate into improved clinical efficacy is currently being investigated. MK-6194 is an IL-2M with preserved CD25 but decreased CD122 binding. The results of these SAD and MAD studies show increases in Treg expansion with 3.5 and 5 mg doses of approximately 3- to 5-fold expansion with little to no dose-dependent effects on conventional T cells or NK cells, which can be expanded during recombinant IL-2 treatments. Moreover, the PK profile showed a mean estimated half-life of 27 h after a single dose and up to about 32 h based on the final dose in our multidose trial (i.e., SC injections once every 2 wk or once every 4 wk). Overall, PK assessment of MK-6194 showed a prolonged half-life when compared with low dose IL-2 or wild-type IL-2, which has a half-life of only several minutes,³⁶ as a result of the fusion with the Fc IgG domain.

High-dose IL-2 therapy used in cancer treatment has been associated with serious safety issues. These include reports of acute thromboembolic events, particularly venous thromboembolism due to increase in platelet adherence with IL-2 and activation of the intrinsic coagulation pathway, as well as autoimmune reactions.^37,38 Additional notable effects with high-dose IL-2 therapy include capillary leak syndrome as well as severe eosinophilia.^39–42 Low-dose IL-2 treatment, in contrast, has been associated with an improved safety profile.^23,43 MK-6194 was generally well tolerated in the SAD and MAD studies. The most common AEs were injection site reactions, most of which were deemed mild in severity, and mild, asymptomatic elevations in eosinophil counts in the MK-6194–treated groups. There were no trial discontinuations related to injection site reactions or eosinophilia. There were no cases of severe eosinophilia (>5,000 eosinophils/µL) or eosinophilia-mediated organ damage. At the same time, MK-6194 selectivity for CD25 allowed for 3- to 5-fold expansion of Tregs without expansion of other cell types susceptible to IL-2–induced stimulation such as conventional T cells or NK cells.

There are some important limitations to consider regarding this research. These were small, phase 1 trials designed to assess safety, PK, and PD in a population of healthy participants. Preliminary data from low-dose IL-2 studies suggest that clinical efficacy might correlate with the degree of Treg expansion in peripheral blood.⁴⁴ However, whether the degree of Treg expansion through MK-6194 treatment in peripheral blood translates to clinical efficacy in different indications has not yet been established. Later-stage, larger randomized controlled trials are needed to characterize efficacy and the potential correlation between pharmacodynamic activity and efficacy and safety in participants who have specific autoimmune disorders. These data provide an important initial assessment of MK-6194 in healthy participants.

In summary, the PK profile from these studies demonstrated that MK-6194, through fusion to a human Fc domain, had a longer half-life than traditional low-dose IL-2 treatments and has the potential benefit of less frequent dosing. The PD analysis showed that the higher doses of MK-6194 treatment were associated with robust Treg expansion with minimal impact on conventional T cells or NK cells. MK-6194 treatment was generally well tolerated, with no serious AEs observed and a low incidence of discontinuations due to AEs. These results demonstrate the therapeutic potential of MK-6194 and support further development as a potential treatment for autoimmune disorders.

Author contributions

J.F.S., K.C.-B., N.K., S.A., G.N., J.C., S.A.S., M.I., T.L., J.R., T.B., N.H.-S., K.K.-T., K.L.O., J.L.V., J.S.S., R.W., and E.L. contributed to the conception or design of the work, and analysis and interpretation of the data. L.L., T.D., L.M., E.P., L.L.-G., A.V.C., X.Z., L.F.-D., L.P., G.A.B., M.B., K.S., K.V.D., T.L., P.L., and S.S., contributed to the acquisition, analysis, or interpretation of data. E.S. and S.R. were study investigators and contributed to the acquisition of data. All authors drafted the work or reviewed it critically for important intellectual content.

Johannes F. Scheid (Conceptualization [Lead], Data curation [Equal], Methodology [Lead], Project administration [Equal]), Kiki Cunningham-Bussel (Conceptualization [Equal], Methodology [Equal]), Nancy Kim(Methodology [Equal]), Shiuli Agarwal (Conceptualization [Equal], Methodology [Equal]), Garrett Nieddu (Data curation [Equal], Formal analysis [Lead], Methodology [Equal]), Josee Cote (Data curation [Equal], Project administration [Equal]), Lieselotte Lemoine (Data curation [Equal], Methodology [Equal]), Tatjana Decaesteker (Data curation [Equal], Methodology [Equal]), Luis Mendez (Formal analysis [Equal], Methodology [Equal]), Erina Paul (Data curation [Lead], Formal analysis [Lead], Methodology [Equal]), Latisha Love-Gregory (Data curation [Equal], Formal analysis [Equal]), Alejandra Virginia Contreras (Data curation [Equal]), Xuemei Zhao (Data curation [Equal], Formal analysis [Equal]), Lucia Franco-Dilone (Data curation [Equal], Formal analysis [Equal]), Ling Pang (Data curation [Equal], Formal analysis [Equal]), Gretchen A. Baltus (Funding acquisition [Equal]), Maribel Beaumont (Data curation [Equal]), Ketal Shah (Data curation [Equal]), Nathan Higginson-Scott (Conceptualization [Equal], Data curation [Equal], Formal analysis [Equal], Methodology [Equal]), Katalin Kis-Toth (Data curation [Equal], Formal analysis [Equal], Methodology [Equal]), Kevin L. Otipoby (Conceptualization [Equal], Data curation [Equal], Formal analysis [Equal], Methodology [Equal]), Joanne L. Viney (Conceptualization [Equal], Data curation [Equal], Formal analysis [Equal], Methodology [Equal]), Eric Sicard (Investigation [Lead]), Sylvie Rottey (Investigation [Lead]), John Sundy (Conceptualization [Equal], Data curation [Equal], Methodology [Equal]), Kristien Van Dyck (Data curation [Equal], Methodology [Equal]), Tine Laethem (Data curation [Equal]), Patrick Larson (Data curation [Equal], Formal analysis [Equal], Supervision [Equal]), Santosh Sutradhar (Data curation [Equal], Formal analysis [Equal]), Richard Wnek (Data curation [Equal], Formal analysis [Equal]), Tjerk Bueters (Formal analysis [Equal], Supervision [Equal]), Eseng Lai (Conceptualization [Equal], Data curation [Equal], Formal analysis [Equal]), S. Aubrey Stoch (Project administration [Equal], Supervision [Equal]), and Marian Iwamoto (Data curation [Equal], Formal analysis [Equal]), and Jonathan A. Robbins (Conceptualization [Lead], Methodology [Lead], Project administration [Equal], Supervision [Lead])

Supplementary material

Supplementary material is available at ImmunoHorizons online.

Funding

Funding for this research was provided in part by Pandion Therapeutics, Inc., which was was acquired by Merck & Co., Inc., Rahway, NJ, USA, and by Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, USA.

Conflicts of interest

J.F.S., K.C.-B., N.K., S.A., G.N., J.C., L.L., T.D., L.M., E.P., L.L.-G., A.V.C., X.Z., L.F.-D., L.P., G.A.B., M.B., K.S., K.V.D., T.L., P.L., S.S., R.W., T.B., E.L., S.A.S., M.I., and J.A.R. are or were employees of Merck & Co., Inc. (Rahway, NJ, USA). N.H.-S., K.K.-T., K.L.O., J.L.V., and J.S. were employees of Pandion Therapeutics, Inc., which was acquired by Merck & Co., Inc. (Rahway, NJ, USA). E.S. and S.R. report no conflicts of interest.

Data availability

The data sharing policy, including restrictions, of Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, USA (MSD), is available at https://trialstransparency.msdclinicaltrials.com/policies-perspectives.aspx. Requests for access to the clinical study data can be submitted via email to the Data Access mailbox (mailto:dataaccess@msd.com).