Bispecific GLP-1/GLP-2 agonism in advanced type 2 diabetes: preclinical characterization and a randomized, double-blind, placebo-controlled phase I trial

Domain: biology
Published: 2026-03-24
Editorial outcome: minor_revision

Abstract
PG-102 is a potency-optimized bispecific Fc fusion protein targeting GLP-1 and GLP-2 receptors.
In db/db mouse models of advanced diabetes characterized by uncontrolled hyperglycemia and catabolic
weight loss, PG-102 achieved superior and sustained glycemic control compared with semaglutide or
tirzepatide while preserving body weight, uncoupling glycemic control from catabolic weight loss.
Mechanistic studies indicated that these effects were not driven by acute insulinotropic activity, but by β-
cell preservation and enhanced glucose uptake. These benefits required dual GLP-1R/GLP-2R
engagement, as PG-102 outperformed monospecific Fc fusion agonists or their combination and promoted
coordinated receptor trafficking with delayed internalization. We conducted a randomized, double-blind,
placebo-controlled multiple ascending dose phase 1 study at a single centre in the Republic of Korea in
adults with overweight (BMI 25–30 kg/m²). Twenty-four participants were randomized within three
weekly dose cohorts (15 mg, 30 mg, and 30/60 mg; n=6 per cohort) in a 6:2 ratio to receive PG-102 (n=18)
or placebo (n=6). All randomized participants (PG-102 n=18; placebo n=6) received at least one dose and
were included in the safety analysis. Safety and tolerability were predefined as the primary endpoint and
assessed by treatment-emergent adverse events (TEAEs). TEAEs occurred in 5/6 (83.3%) participants in
each PG-102 cohort and 4/6 (66.7%) in placebo; treatment-related AEs occurred in 5/6 (83.3%) and 3/6
(50.0%), respectively. No serious adverse events or discontinuations due to adverse events occurred.
Gastrointestinal events were mild to moderate. These findings support bispecific GLP-1/GLP-2 agonism
as a mechanistically distinct incretin strategy in advanced T2D. ClinicalTrials.gov identifier:
NCT06309667

Introduction
Glucagon-like peptide-1 receptor agonists (GLP-1RAs) are standard-of-care agents that provide
potent glucose-lowering without hypoglycemia, together with weight reduction and cardiovascular and
renal benefits. Building on this success, the field has progressed toward dual and triple agonists to further
enhance metabolic outcomes, exemplified by GLP-1/GIP co-agonists that deliver superior glycemic and
weight control.1 Nevertheless, fewer than half of patients treated with GLP-1RAs achieve guideline-
recommended glycemic targets (typically HbA1c <7.0%), and treatment discontinuation remains a major
challenge, driven largely by gastrointestinal side effects and inadequate glycemic control, with ~50% of
patients discontinuing therapy within 12 months of initiation.2-4 Moreover, current GLP-1RAs are not
designed for patients experiencing unintentional weight loss—a frequent manifestation of advanced type
2 diabetes marked by catabolism and muscle wasting.5-7 These gaps highlight the need for agents with
improved tolerability that can sustain glycemic control without excessive weight loss, particularly in
elderly, lean, or sarcopenic individuals for whom preservation of body mass is critical.

GLP-2 receptor agonism has emerged as an underutilized yet promising complement to GLP-
1RA therapy, offering unique advantages for T2D treatment. Traditionally recognized for its role in
intestinal growth and repair, GLP-2 has also demonstrated significant potential in metabolic regulation.
Unlike GLP-1, GLP-2 does not substantially enhance acute insulin secretion, but instead supports β-cell
health by promoting proliferation and protecting against cytokine-induced apoptosis under metabolic
stress.8,9 In addition, GLP-2 enhances insulin sensitivity and peripheral glucose disposal, as evidenced by
improved glucose tolerance in vivo without concomitant rises in circulating insulin, as well as increased
glucose uptake in human adipocytes.9,10 GLP-2 also exerts anti-inflammatory actions in islets, including
suppression of pro-inflammatory cytokines such as IL-1β, thereby fostering a milieu conducive to β-cell
function.11 Collectively, these features highlight GLP-2 as a mechanistically complementary partner to
GLP-1, emphasizing β-cell preservation, insulin sensitization, and anti-inflammatory effects rather than
insulinotropic action alone. Nevertheless, potential GLP-2–mediated increases in nutrient absorption,
glucagon secretion, and intestinotrophic effects remain important considerations for therapeutic design.

In this study, we aim to demonstrate that simultaneous activation of GLP-1 and GLP-2 receptors
can overcome the limitations of current incretin-based therapies in advanced type 2 diabetes. To this end,
we use the db/db mouse model, which captures uncontrolled hyperglycemia, β-cell failure, and catabolic
weight loss, to evaluate the metabolic and mechanistic effects of a potency-optimized GLP-1/GLP-2
bispecific agonist, PG-102. We further investigate how dual receptor engagement influences β-cell
preservation, peripheral glucose handling, and receptor trafficking compared with clinically available
incretin agonists and monospecific Fc fusions. Finally, we translate these findings into a phase 1 multiple
ascending dose study to characterize the pharmacokinetics, pharmacodynamics, and tolerability of PG-
102 in humans. Collectively, this integrated preclinical-to-clinical approach establishes the rationale for
PG-102 as a differentiated therapeutic candidate that decouples glycemic control from weight loss in
advanced type 2 diabetes.

Results
Structural design and bioactivity of PG-102
We previously developed PG-105, a prototype GLP-1/GLP-2 Fc fusion protein based on Neo Tri-
ImmunoGlobulin (NTIG) platform, and demonstrated in a metabolic dysfunction-associated
steatohepatitis (MASH) model that dual receptor activation improved glucose homeostasis, strengthened
intestinal barrier integrity, and reduced hepatic steatosis and fibrosis.12 While these findings established
the therapeutic potential of GLP-1/GLP-2 co-agonism, PG-105 exhibited excessive GLP-2R activity,
leading to marked increases in intestinal mass and gallbladder volume (Supplementary Fig. 1), raising
concerns about potential trophic effects upon chronic exposure. To mitigate these liabilities, a panel of
nine GLP-1/GLP-2 NTIG Fc constructs with graded GLP-2R potencies was generated, from which PG-
102 was selected as the optimal candidate. PG-102 preserved the favorable gut barrier effects—reflected
by reduced serum endotoxin levels—while showing attenuated increases in intestinal parameters
compared with PG-105, and notably, no statistically significant gallbladder enlargement, defining a more
balanced and clinically viable pharmacologic profile.

To describe the specific design, PG-102 is a “bivalent” agonist in which GLP-1 and GLP-2
analogues are fused to opposite arms of the NTIG Fc platform (Fig. 1a). The NTIG construct, based on
the hyFc backbone,13 incorporates T250Q and M428L mutations in the FcRn binding region to enhance
binding affinity and prolong half-life, ensuring sustained in vivo activity. Additionally, targeted
mutations—S354C and T366W in one segment and Y349C, T366S, L368A, and Y407V in another—
facilitate heterodimeric assembly through a knob-and-hole mechanism.14 The GLP-1 analogue was
engineered with an A2G mutation to prevent DPP-4 cleavage and further modified by replacing its last
two amino acids with ten C-terminal residues from exenatide at position 30 to enhance receptor binding
affinity.15,16 A tandem GLP-1 construct linked by a GS linker was employed to facilitate post-purification
identification of the heterodimeric GLP-1/GLP-2-NTIG Fc structure and to enhance GLP-1 activity.17 The
GLP-2 analogue carried an A2G mutation to prevent DPP-4 cleavage, together with N17G and L18Q
substitutions to improve structural stability and reduce aggregation (Fig. 1b).18

Receptor potency of PG-102 was evaluated using cAMP production assays in CHO-K1 cells
overexpressing GLP-1R and HEK293 cells overexpressing GLP-2R. PG-102 exhibited an EC₅₀ of 0.68
nM for GLP-1R activation, representing 78% of native GLP-1 potency (0.68 nM vs. 0.53 nM), ~90% of
semaglutide (0.68 nM vs. 0.61 nM), and nearly twice that of tirzepatide (0.68 nM vs. 1.34 nM). For GLP-
2R activation, PG-102 retained an EC₅₀ of 275.33 nM, corresponding to only 3.4% of native GLP-2
potency (275.33 nM vs. 8.84 nM), reflecting deliberate attenuation of GLP-2R activity (Fig. 1c). Given
the structural homology across class-B GPCERs, we next profiled potential off-target activation at human
GIPR and GCGR using matched cAMP assays. PG-102 showed no detectable agonist activity at either
receptor, whereas native ligands (GIP, glucagon) and benchmark multi-agonists (tirzepatide, retatrutide)
produced robust responses, confirming assay sensitivity. Together, these data indicate that PG-102 is
selective for GLP-1R and GLP-2R (Supplementary Fig. 2).

Importantly, a 26-week repeat-dose toxicity study of PG-102 demonstrated the absence of
hyperplasia not only in thyroid C cells but also throughout the gastrointestinal tract. In contrast, rodent
repeat-dose studies of GLP-1–based drugs such as semaglutide and tirzepatide have reported thyroid C
cell hyperplasia, while monkey studies of the GLP-2 analogue teduglutide have shown gastrointestinal
hyperplasia (Supplementary Table 1). These findings establish PG-102 as a bispecific GLP-1/GLP-2
receptor agonist engineered to balance efficacy with improved safety margins.

Dose-dependent glycemic effects of PG-102 in db/db mice
We conducted a dose-dependency study to determine the optimal therapeutic dose of PG-102
using a 14-week db/db mouse model, which recapitulates features of advanced T2D including
uncontrolled hyperglycemia (baseline HbA1c >10%), catabolic weight loss, and progressive β-cell
dysfunction.19 PG-102 demonstrated a clear dose-dependent reduction in both non-fasting blood glucose
and HbA1c levels over a three-month treatment period (Fig. 2a, 2b). The 15 nmol/kg dose modestly
reduced glucose and HbA1c but failed to induce normoglycemia (defined as blood glucose <200 mg/dL)20,
with 0% of mice achieving this benchmark (Fig. 2c). In contrast, the 30 nmol/kg dose enabled over half
of treated mice (57.1%) to achieve normoglycemia, whereas 60 and 120 nmol/kg induced normoglycemia
in nearly all (88.9%) and all (100%) treated mice, respectively, demonstrating substantial improvement
in glycemic control relative to the subtherapeutic 15 nmol/kg dose. Consequently, the 15 nmol/kg dose
was excluded from further analyses due to its suboptimal performance.

In subsequent analyses, PG-102 significantly reduced fasting blood glucose levels across all
effective doses (Fig. 2d) and increased fasting insulin levels at 30 nmol/kg (Fig. 2e), consistent with
improved glucose metabolism driven by enhanced β-cell function. HOMA-IR values demonstrated a
downward trend, suggesting improved insulin sensitivity, though statistical significance was not observed
(Fig. 2f). HOMA-β scores were increased across PG-102 treatment groups, reaching statistical
significance at 30 and 60 nmol/kg doses, suggesting enhanced β-cell preservation and function (Fig. 2g).
The plateau in HOMA-β across 30–120 nmol/kg suggests that β-cell preservation reached a functional
ceiling once normoglycemia was established, consistent with saturating efficacy at higher doses. Together,
these data establish 30 nmol/kg as the minimal efficacious dose that achieves durable normoglycemia
while preserving β-cell function, and it was therefore selected for subsequent head-to-head comparative
studies.

Comparative efficacy of PG-102, semaglutide, and tirzepatide
We next evaluated the efficacy of PG-102 against semaglutide and tirzepatide. A matching dose
of 30 nmol/kg was selected for semaglutide to align with PG-102, whereas 15 nmol/kg was chosen for
tirzepatide, reflecting prior reports that this lower dose yields metabolic efficacy comparable to 30
nmol/kg semaglutide in rodents.21,22 Vehicle-treated db/db mice exhibited persistent severe
hyperglycemia (non-fasting blood glucose ~600 mg/dL; HbA1c >10%). Semaglutide and tirzepatide
reduced blood glucose by ~200 mg/dL and HbA1c by ~2% at two months, but their effects waned by the
third month. In contrast, PG-102 achieved a ~450 mg/dL glucose reduction and ~5% HbA1c decrease by
two-months, restoring normoglycemia and maintaining efficacy throughout the three-month study (Fig.
3a, 3b).

Body weight analysis highlighted an important distinction. Vehicle-treated mice showed rapid
weight loss, reflecting the catabolic state of uncontrolled diabetes. Semaglutide and tirzepatide partially
mitigated this decline, whereas PG-102 more effectively preserved body weight, countering the metabolic
deterioration (Fig. 3c).

To further characterize these effects, we examined pancreatic islets histologically. Vehicle-
treated mice displayed severe islet disorganization and loss of insulin-positive β-cells. Semaglutide
partially improved islet morphology, whereas tirzepatide offered little benefit. By contrast, PG-102
markedly preserved β-cell area and islet architecture, consistent with its robust glycemic effects (Fig. 3d,
3g, 3h). PG-102 also reduced α-cell area compared with vehicle and showed a trend toward lower levels
than semaglutide or tirzepatide, suggesting decreased glucagon production (Fig. 3e, 3i). Moreover, Ki67-
insulin double staining revealed that PG-102 significantly maintained the proportion of proliferating β-
cells, supporting preservation of regenerative potential (Fig. 3f, 3j). We additionally assessed
inflammatory involvement in peri-pancreatic adipose tissue, a visceral fat depot enriched in leukocytes
and implicated as a source of mediators affecting β-cell dysfunction.23,24 Although CD45⁺ staining was
also performed on pancreatic sections, intact islets were largely absent in vehicle-treated mice, precluding
meaningful quantification. Accordingly, peri-pancreatic fat served as the most reliable site to evaluate
inflammation, where vehicle-treated mice displayed abundant CD45⁺ cells that were substantially reduced
by all incretin-based treatments. Notably, PG-102 showed the lowest CD45⁺ area among groups,
suggesting a possible trend toward reduced local inflammation compared to semaglutide or tirzepatide
(Supplementary Fig. 3).

Finally, because tirzepatide’s GIPR agonism is attenuated in rodents,25 we performed an
additional study in db/db mice with moderate hyperglycemia (non-fasting blood glucose 500~600 mg/dL;
9% < HbA1c < 10%), using equal doses of PG-102 and tirzepatide (30 nmol/kg). In this setting, PG-102
consistently achieved superior glucose lowering, HbA1c reduction, body weight preservation, and β-cell
area maintenance compared with tirzepatide (Supplementary Fig. 4), reinforcing its advantage across
different stages of disease severity. Given the species-dependent pharmacology of tirzepatide, these
findings should be interpreted as comparative references rather than direct predictors of clinical efficacy,
yet they highlight the differentiated profile of PG-102.

In vitro mechanisms of PG-102 versus incretin comparators
We next investigated the β-cell–centric mechanisms underlying PG-102–mediated glycemic
control. In perifusion assays using isolated islets from normoglycemic mice, PG-102 augmented glucose-
stimulated insulin secretion (GSIS; 3 mM → 11 mM), compared with vehicle, and trended higher than
semaglutide and tirzepatide, although without statistical significance (Fig. 4a). While semaglutide and
tirzepatide induced comparable GSIS despite differing GLP-1R potency, this likely reflects the limited
contribution of GIPR agonism in rodents.25 Importantly, PG-102 achieved numerically greater insulin
secretion than semaglutide despite equivalent GLP-1R potency, suggesting possible additive
contributions from GLP-2R co-activation.

We next assessed β-cell protection under diabetogenic stress using the streptozotocin (STZ)-
induced cytotoxicity in INS-1 cells, which co-express GLP-1R and GLP-2R (Supplementary Fig. 5a).
PG-102 significantly preserved β-cell viability relative to vehicle, semaglutide, and tirzepatide (Fig. 4b).
Under high-glucose challenge (16.8 mM), PG-102 also promoted robust insulin release, surpassing
semaglutide and far exceeding vehicle control (Fig. 4c). At the molecular level, PG-102 significantly
upregulated -cell transcription factors Pdx1 and Neurod1 (p < 0.05), whereas MafA showed an upward,
non-significant trend; all three factors are essential for insulin gene expression, β-cell identity, and
survival (Supplementary Fig. 6). These findings indicate that PG-102 protects β-cells from cytotoxic
injury while enhancing their insulinotropic function by reinforcing key transcriptional programs.

Finally, to explore systemic metabolic implications, we examined the effect of PG-102 on
peripheral glucose disposal. Both 3T3-L1 adipocytes and L6-GLUT4myc myotubes expressed GLP-1R
and GLP-2R, as confirmed by flow cytometry (Supplementary Fig. 5b and 5c). In glucose uptake assays,
PG-102 markedly enhanced glucose uptake compared with semaglutide or tirzepatide (Fig. 4d–e),
highlighting its superior potential to improve peripheral insulin sensitivity.

Bivalent receptor engagement underlying PG-102 efficacy
To directly evaluate the contribution of dual receptor engagement, we compared monospecific
GLP-1-NTIG Fc, GLP-2-NTIG Fc, their co-administration, and the bispecific PG-102 in an
intraperitoneal glucose tolerance test (ipGTT) in normoglycemic mice. At GLP-1R–matched dosing, PG-
102 enhanced glucose clearance more effectively than GLP-1-NTIG Fc or the co-administered
combination, whereas GLP-2-NTIG Fc alone exerted minimal effect (Fig. 5a). Given the ceiling effect of
ipGTT in healthy mice, these differences likely underestimate the functional advantage of PG-102, but
nonetheless support in vivo synergy arising from simultaneous GLP-1R and GLP-2R activation.

We next assessed β-cell protection under diabetogenic stress. In STZ-challenged INS-1 cells, PG-
102 consistently outperformed monospecific controls and equaled or surpassed the co-treatment of GLP-
1-NTIG Fc and GLP-2-NTIG Fc across glucose-stimulated insulin secretion and preservation of key β-
cell transcription factors, including MafA, Pdx1, and Neurod1 (Fig. 5b and Supplementary Fig. 7a–c). PG-
102 also significantly attenuated STZ-induced Tnf-α expression, exceeding the effect of GLP-1-NTIG Fc
and GLP-2-NTIG Fc co-treatment, consistent with a protective and anti-inflammatory role in β-cell stress
(Supplementary Fig. 7d).

Finally, to explore effects beyond islets, we investigated peripheral glucose metabolism using
differentiated 3T3-L1 adipocytes and L6-GLUT4myc myotubes. PG-102 elicited the greatest increase in
glucose uptake relative to monospecific Fc fusions or their co-treatment (Fig. 5c–d), indicating superior
enhancement of peripheral glucose disposal. Mechanistic dissection with receptor-selective antagonists
revealed that this effect was strongly GLP-2R–dependent, as it was abrogated by GLP-2 (3–33), whereas
exendin (9–39) produced only modest inhibition (Supplementary Fig. 8). Although PG-102 displayed
only ~3% of the native GLP-2R potency in cAMP assays (Fig. 1c), these blockade studies demonstrated
that its glucose uptake effects were predominantly GLP-2R–driven, underscoring a functional
contribution not apparent from potency data alone.

Although these functional studies highlighted a predominant role of GLP-2R in mediating PG-
102’s metabolic effects, the underlying receptor-level mechanism remained to be clarified. To further
investigate receptor dynamics, we performed confocal imaging of GLP-1R and GLP-2R trafficking in
HEK293 cells co-expressing both receptors. GLP-1–NTIG Fc and the GLP-1/GLP-2 co-treatment
triggered rapid GLP-1R internalization (peak ~30–60 min), whereas GLP-2R signals remained diffusely
distributed across the membrane and cytoplasm (Supplementary Fig. 9a–b). In contrast, PG-102 did not
induce rapid receptor loss from the surface but maintained clustered receptor patterns with gradual
redistribution over 120 min, suggesting cis-co-engagement of GLP-1R and GLP-2R that supports
coordinated trafficking and sustained signaling.

Phase 1 MAD trial of PG-102: safety, PK and PD evaluation
A phase 1 multiple ascending dose (MAD) study (NCT06309667) was conducted to evaluate the
safety, tolerability, pharmacokinetic (PK), and pharmacodynamic (PD) profiles of PG-102 in healthy
adults with a body mass index (BMI) of 25 to 30 kg/m ². Safety and tolerability were predefined as the
primary endpoint, assessed by treatment-emergent adverse events (TEAEs), while secondary endpoints
included PK and PD assessments. Exploratory endpoints comprised inflammation-related biomarkers
such as plasma high-sensitivity C-reactive protein (hsCRP).

This randomized, double-blind, placebo-controlled trial included 24 participants aged 18 to 65
years, who were randomized in a 6:2 ratio to receive PG-102 or placebo within three dose cohorts: Cohort
1 (15 mg), Cohort 2 (30 mg), and Cohort 3 (30/60 mg, with an escalating regimen of 30/30/60/60/60 mg),
administered weekly. A schematic overview of the trial design and allocation is provided in Fig. 6 and
Supplementary Table 2. Baseline characteristics were well-balanced (mean age 30.9 ± 6.7 years, BMI
27.0 ± 1.2 kg/m², body weight of 80.6 ± 7.0 kg), with males comprising ~92% of the study population
(Table 1). The 15 mg starting dose was informed by the preceding single ascending dose (SAD) study and
a 4-week toxicology study in cynomolgus monkeys, which established a NOAEL of 6 mg/kg, providing
a wide safety margin (Supplementary Table 3). Participant recruitment was conducted between March
2024 and July 2024, and follow-up was completed by September 2024.

Safety assessments across all dose cohorts demonstrated that PG-102 was well-tolerated. TEAEs
occurred in 19 participants (79.2%), with treatment-related AEs reported in 18 participants (75.0%; Table
2). No serious adverse events (SAEs) or discontinuations due to treatment-related AEs occurred. GI events,
typical of GLP-1RAs,26 represented the majority of TEAEs: dyspepsia (41.7%), diarrhea (20.8%), nausea
(25.0%), and vomiting (12.5%). Dyspepsia showed a dose-related increase (33.3–83.3% across cohorts).
Vomiting was infrequent (0–33.3%) and absent in placebo and 15 mg groups. Nausea incidence was
consistent at 33.3% across all PG-102 doses, while diarrhea showed no clear dose relationship and was
comparable to placebo (16.7%). All GI-related events were mild to moderate, with no evidence of
worsening over time.

PK analyses following the final dose demonstrated dose-proportional exposure and consistent
elimination kinetics (Fig. 7a and Supplementary Table 4). Tmax ranged from 48 hours (30 mg) to 72 hours
(15 mg), with a median of 60 hours in the 30/60 mg cohort, reflecting delayed absorption. AUC0-inf
increased proportionally with dose (259 µg·h/mL at 15 mg; 660 µg·h/mL at 30/60 mg). The terminal half-
life (t1/2) was 107 to 118 hours across cohorts, and clearance (CLss/F) remained low (0.06 to 0.11 L/h),
confirming slow systemic elimination.

PD effects were evaluated by oral glucose tolerance test (OGTT) on day 30, one day post-final
dose. In placebo, glucose peaked at 60 minutes, whereas PG-102 shifted the peak earlier (30 minutes) and
accelerated the return to baseline (Fig. 7b, left panel). All PG-102 cohorts showed numerical reductions
in glucose AUC₀-₂ compared with placebo, with decreases of 26.5%, 24.9%, and 19.8% in the 30 mg,
30/60 mg, and 15 mg groups, respectively (Fig. 6b, right panel). Exploratory exposure–response analysis
suggested a trend toward greater glucose tolerance improvements with higher systemic exposure
(Supplementary Fig. 10). Exploratory biomarkers, including body weight, composition, and inflammatory
indices, showed no changes in this short healthy-volunteer study (Supplementary Tables 5 and 6).

Methods
Ethical approval. All animal experiments were conducted in accordance with relevant ethical
regulations and were approved by the Institutional Animal Care and Use Committee (IACUC) of GI
Biome (approval number: GIB-23-02-007). All cell-based experiments were performed in compliance
with institutional guidelines. The clinical study was conducted in accordance with the Declaration of
Helsinki and Good Clinical Practice (GCP) guidelines, and was approved by the Ministry of Food and
Drug Safety of the Republic of Korea (clinical trial authorization no. 101629) and by the Institutional
Review Board of Seoul St. Mary’s Hospital, the Catholic University of Korea (IRB no. KC23MDSF0619).
Study progression and dose escalation were subject to review and approval by an internal Safety Review
Board at prespecified stages. Written informed consent was obtained from all participants prior to study
participation.

Production of PG-102. The nucleotide sequence encoding PG-102 was synthesized (ATUM, Newark,
CA, USA) and cloned into the pCGS3 expression vector. Stable cell line development was conducted by
Samsung Biologics (Incheon, South Korea) using the CHOZN® ZFN-modified GS-/- cell line, which was
transfected with the purified DNA construct. The transfected cells were cultured in a controlled bioreactor
system, and the expressed protein was purified using a multi-step chromatographic process to ensure high
purity. The final product was formulated in a designated buffer and quantified using UV
spectrophotometry (Implen, CA, USA). Only batches meeting stringent predefined quality criteria,
including purity, structural integrity, and biological activity, were advanced for downstream in vitro and
in vivo studies.

In vitro bioactivity assay. The bioactivity of PG-102 was evaluated using cell lines expressing GLP-1
and GLP-2 receptors, provided by Eurofins DiscoverX Ltd. (Shanghai, China) and MORE-Bio, Inc.
(Hanam-si, South Korea), respectively. Cells were cultured following the manufacturers’ protocols, and
receptor activity assays were conducted to assess PG-102’s potency. For comparison, GLP-1-NTIG Fc,
GLP-2-NTIG Fc, and PG-105 analogs were prepared and tested concurrently.

In vivo study. Twelve-week-old db/db mice (BKS-Leprdb/db/JOriRj, male) were obtained from
Janvier-Labs (Le Genest-Saint-Isle, France) and housed under controlled environmental conditions (22–
24 °C, 50–60% relative humidity, 12-hour light/dark cycle) with ad libitum access to food and water.
Mice were maintained on a standard laboratory chow diet (Teklad SDiets 2918, Inotiv, USA; 6% kcal from
fat, 44.2% kcal from carbohydrates, and 18.4% kcal from protein) throughout the study unless otherwise
indicated. To model advanced-stage T2D, db/db mice were acclimated for two weeks to induce severe
hyperglycemia, defined as baseline HbA1c >10% and non-fasting blood glucose >500 mg/dL, prior to
treatment initiation. At 14 weeks of age, mice were randomized into treatment groups (n = 8 per group)
receiving subcutaneous injections of PG-102 (15, 30, 60, or 120 nmol/kg), semaglutide (30 nmol/kg),
tirzepatide (15 nmol/kg), or vehicle every three days for 12 weeks. Sample sizes were guided by prior
experience with the db/db mouse model and were designed to provide ≥80% power (α = 0.05) to detect
biologically meaningful differences in metabolic outcomes. Non-fasting blood glucose and HbA1c levels
were monitored at baseline and every three weeks. At the end of the study, fasting blood glucose and
insulin levels were measured following a 6-hour fast, and HOMA-IR and HOMA-β scores were calculated
using standard formulas. Only male mice were used in this study to minimize variability associated with
the estrous cycle and because male db/db mice exhibit more severe and consistent hyperglycemia;
therefore, sex was not included as a biological variable in the study design or analysis.

Histological and immunohistochemical analyses. At the end of the study (week 26), pancreatic tissues
were harvested, fixed in 10% neutral-buffered formalin, embedded in paraffin, and sectioned at 4 µm. For
histological evaluation, sections were stained with hematoxylin and eosin (H&E) to assess islet
architecture. Adjacent sections were subjected to immunohistochemical staining for insulin to visualize
insulin-positive islets. For immunohistochemical analysis, sections were incubated with a primary
antibody against insulin (Abcam, ab181547; 1:10,000; rabbit), followed by appropriate HRP-conjugated
secondary antibodies and chromogenic detection. For immunofluorescence analyses, sections were
subjected to antigen retrieval, blocked, and incubated with primary antibodies against insulin (Abcam,
ab181547; 1:10,000; rabbit), glucagon (Abcam, ab10988; 1:200; mouse), and Ki67 (Abcam, ab15581;
1:300; rabbit), followed by species-appropriate fluoresce secondary antibodies and nuclear
counterstaining with DAPI. Images were acquired using a fluorescence microscope (ECLIPSE Ci-L;
Nikon Instruments Inc., Melville, NY, USA). Quantification of positive staining areas and Ki67-positive
cells was performed using ImageJ software (NIH).

Intraperitoneal glucose tolerance test (ipGTT). Ten-week-old C57BL/6J mice (The Jackson
Laboratory, Bar Harbor, ME, USA) were administered the indicated drugs (15 nmol/kg) and maintained
for 24 hours, prior to testing, including a 6-h fasting period immediately before the ipGTT. Glucose (2
g/kg body weight) was injected intraperitoneally, and blood glucose concentrations were measured at the
indicated time points up to 2 h post-injection using a standard glucometer (Accu-Chek Active; Roche
Diagnostics, Mannheim, Germany).

Islet isolation and glucose-stimulated insulin secretion. Pancreatic islets were isolated through
collagenase digestion of the pancreas, followed by gradient purification to obtain intact islets. Fifty size-
matched islets were perifused with Krebs-Ringer bicarbonate buffer (KRB) containing 0.1% bovine serum
albumin at 37 °C and 200 µL/min. After stabilization, islets were exposed to 3 mM glucose (basal)
followed by 11 mM glucose supplemented with 100 nM test drugs. Perifusate fractions were collected
every minute for 45 minutes, and insulin concentrations were quantified using an ELISA kit (ALPCO,
Salem, NH, USA) to evaluate basal and glucose-stimulated insulin secretion.

Cell viability assay. Cell viability was assessed using the MTS assay (CellTiter 96® AQueous One
Solution Cell Proliferation Assay, Promega) according to the manufacturer's instructions. Briefly, INS-1
cells were seeded in 96-well plates at a density of 5 × 10⁴ cells/well and allowed to adhere overnight at
37°C in a humidified atmosphere containing 5% CO₂. Cells were then treated with the indicated
compounds for 24 hours following a 3-hour exposure to streptozotocin (STZ). Subsequently, 20 µL of
MTS reagent was added directly to each well containing 100 µL of culture medium, and the plates were
incubated at 37°C for 4 hours. Absorbance was measured at 490 nm using a microplate reader
(SpectraMax, Molecular Devices). Cell viability was expressed as a percentage relative to the control
group (vehicle-treated cells). All experiments were performed in triplicate and independently repeated at
least three times.

In vitro glucose-stimulated insulin secretion (GSIS). Insulin secretion was measured using the Krebs
Ringer Bicarbonate (KRB) buffer (119 mM NaCl, 5 mM KCl, 2.5 mM CaCl2, 1.2mM KH2PO4, 1.2 mM
MgCl2, 10 mM HEPES, 25 mM NaHCO3, and 0.2% BSA). Cells were pre-incubated for 2 h in the KRB
buffer containing 0.2 mM glucose for glucose starvation. After pre-incubation, cells were incubated in
KRB buffer containing 2- or 16.8-mM glucose (Sigma-Aldrich) at 37 ̊C for 30 min, respectively. Insulin
released into the supernatants was measured using the Mouse Ultrasensitive Insulin ELISA kit (ALPCO)
according to the manufacturer’s

Cell culture and differentiation. 3T3-L1 preadipocytes and L6-GLUT4myc myoblasts were cultured
in Dulbecco's Modified Eagle Medium (DMEM; Thermo Fisher Scientific, Waltham, MA, USA) and α-
MEM (Thermo Fisher Scientific), respectively, supplemented with 10% fetal bovine serum (FBS) and 1%
penicillin-streptomycin (P/S) at 37°C and 5% CO₂. For 3T3-L1 cells, differentiation was initiated upon
confluence (day 0) with DMEM containing 0.5 mM isobutylmethylxanthine (IBMX), 1 µM
dexamethasone, and 10 µg/mL insulin. On day 2, the medium was replaced with DMEM supplemented
with 10% FBS and 10 µg/mL insulin, refreshed every two days. Differentiation was completed within 6–
8 days, confirmed by the presence of lipid droplets. For L6-GLUT4myc cells, differentiation was initiated
at 80–90% confluence using α-MEM supplemented with 2% horse serum (HS), refreshed every 2–3 days.
Differentiation into multinucleated myotubes was achieved within 5–7 days.

Glucose uptake assay. Glucose uptake in 3T3-L1 adipocytes and L6-GLUT4myc myotubes was
measured using a glucose uptake cell-based assay kit (Cayman, Ann Arbor, MI, USA), according to the
manufacturer’s protocol. After 2 h of serum starvation, cells were treated with the indicated drugs (300
nM) and insulin simultaneously for 10 min. Cells were washed three times with Krebs buffer (20 mM
HEPES, 5 mM KH2PO4, 1 mM MgSO4, 1 mM CaCl2, 136 mM NaCl, 4.7 mM KCl, pH 7.4), then
incubated with 500 µg/ml 2-NBDG, a fluorescence-labelled deoxyglucose analog, for 20 min at room
temperature. Reactions were terminated by washing with ice-cold Krebs buffer. Cells were lysed using
Mammalian Protein Extraction Reagent Thermo Fisher Scientific) and centrifuged at 15000 × g for 10
sec. After removing the supernatant, assay buffer added, and 2-NBDG fluorescence was measured using
a microplate reader (excitation/emission = 485/535 nm). Glucose uptake was normalized to protein
content quantified using a bicinchoninic acid (BCA) assay (Thermo Fisher Scientific).

Nonclinical toxicology studies. GLP-compliant repeat-dose toxicity studies were conducted in
cynomolgus monkeys (Macaca fascicularis; males 2–4 years, females 3–5 years) at an AAALAC-
accredited facility (Ina Research Inc., Japan) in accordance with OECD GLP principles and 21 CFR Part
58. All procedures were approved by the Institutional Animal Care and Use Committee (IACUC;
protocols YG21113 and ZO23242) and performed in compliance with applicable animal welfare
regulations.
Two repeat-dose studies were performed: a 4-week study with a 4-week recovery phase and a 26-week
study. In both studies, animals were randomized by sex to receive vehicle (formulation buffer) or PG-102
and were administered subcutaneously once weekly.
In the 4-week study, PG-102 was administered at 2, 6, or 20 mg/kg (n=5/sex for vehicle and 20 mg/kg;
n=3/sex for 2 and 6 mg/kg). In the 26-week study, PG-102 was administered at 2, 5, or 10 mg/kg once
weekly for 26 consecutive weeks.
Animals were housed individually under controlled environmental conditions (22 ± 2 °C; 40–70%
humidity; 12-hour light/dark cycle) with environmental enrichment and ad libitum access to standard
primate diet and water.
Animals were monitored at least twice daily throughout the studies, including pre- and post-dose
observations on dosing days. Clinical evaluations included general condition, posture, activity, food
consumption, gastrointestinal output, and body weight. Humane endpoints were prospectively defined
prior to study initiation and included sustained anorexia, marked body weight loss, persistent
gastrointestinal disturbances, or other signs of clinical deterioration compromising animal welfare. No
animals met predefined humane endpoint criteria requiring unscheduled euthanasia in either study.
At scheduled termination, animals were anesthetized and euthanized under deep anesthesia in accordance
with accepted veterinary practice. Complete gross necropsy was performed, and organs and tissues were
collected, fixed in neutral-buffered formalin, processed routinely, stained with hematoxylin and eosin,
and examined by light microscopy.
Parallel 26-week GLP repeat-dose studies in Sprague–Dawley rats were conducted under comparable
conditions and are described in the Supplementary Methods.

Phase 1 study design; Pharmacokinetic analysis, safety and pharmacodynamic evaluation. A
randomized, double-blind, multiple ascending dose phase 1 study was conducted at a single clinical site
in the Republic of Korea (Seoul St. Mary’s Hospital, The Catholic University of Korea) to evaluate the
safety, tolerability, and pharmacokinetics of subcutaneous PG-102 in healthy individuals with overweight
(body mass index 25–30 kg/m²), with exploratory pharmacodynamic and biomarker assessments.
The reported study corresponds to Part B of a three-part Phase 1 clinical trial. The first participant was
enrolled on March 4, 2024, and the last participant completed enrolment on July 15, 2024.
Progression between dose levels was prespecified and permitted only after safety review and approval by
an internal Safety Review Board at each stage. The trial was preregistered at ClinicalTrials.gov
(NCT06309667) on February 14, 2024, and conducted in accordance with the Declaration of Helsinki and
the International Council for Harmonisation Good Clinical Practice guidelines. The study is reported in
accordance with CONSORT 2010, and a completed checklist is provided in the Supplementary
Information (Supplementary Note 1).
The full clinical study protocol and prespecified statistical analysis plan are available in the
Supplementary Information (Supplementary Notes 2 and 3).
The primary outcomes were safety and tolerability, assessed by the incidence, severity, and causality of
adverse events and by changes in clinical laboratory parameters and physical examinations. Secondary
outcomes included the pharmacokinetic profile of PG-102, assessed using serum concentration–time data
and non-compartmental analyses, with exploratory pharmacodynamic and biomarker endpoints. Safety
assessments were conducted from baseline through end-of-study follow-up, and pharmacokinetic and
exploratory assessments were performed at prespecified time points, including serial sampling up to 672
hours after the final dose.
As is typical for early-phase clinical trials, no formal statistical hypothesis testing or sample size
calculation was performed. Sample size was determined based on practical considerations, prior
experience with similar phase 1 study designs, and the objective of obtaining sufficient safety and
pharmacokinetic data to support dose escalation decisions. Continuous variables were summarized
descriptively (n, mean, standard deviation, median, minimum, maximum), and categorical variables by
frequency and percentage.
Sex and gender were considered in the study design without restrictions on enrollment. All participants
self-reported their sex at screening. Of 67 individuals screened, 31 met eligibility criteria, and 24 were
randomized. Enrollment was defined as randomization, with participants assigned within each cohort in
a 6:2 ratio to receive PG-102 or placebo. Randomization was performed using a predefined allocation
schedule prepared by the study sponsor. Allocation concealment was maintained through centralized
assignment procedures to ensure blinding. Participants, investigators, and study staff involved in clinical
conduct and outcome assessment were blinded to treatment allocation. PG-102 and placebo were identical
in appearance and administered in the same manner to maintain blinding. Cohorts were as follows: Cohort
1 (15 mg weekly), Cohort 2 (30 mg weekly), and Cohort 3 (escalating 30/30/60/60/60 mg weekly). This
allocation resulted in 18 participants receiving PG-102 and 6 receiving placebo. Among the 24
randomized participants, 22 (91.7%) were male and 2 (8.3%) were female (Table 1). Given the very
limited number of female participants, sex- and gender-based analyses were not performed, as they would
not provide meaningful conclusions.
In the 15 mg cohort, one participant discontinued after the fourth dose due to an adverse event considered
unrelated to the investigational product. This participant subsequently withdrew but completed the
scheduled follow-up visit, including all safety assessments. Accordingly, the participant was included in
the safety analysis set but excluded from the efficacy and PK analysis sets.
Participants received five weekly doses of PG-102 or placebo and were followed for two weeks after the
last dose. Serial blood samples were collected for PK analysis at pre-specified intervals up to 672 hours
after the final dose (pre-dose, 4 h, 6 h, 12 h, 24 h, 36 h, 48 h, 72 h, 120 h, 168 h, 336 h, and end-of-study
at 672 h). Serum concentrations of PG-102 were quantified using a validated immunoassay, and non-
compartmental analysis was performed to determine PK parameters. Safety and tolerability were assessed
throughout the study by monitoring adverse events (AEs), clinical laboratory tests, and physical
examinations. AEs were evaluated for severity and causality, and classified as not related, unlikely,
possible, probable, or definite. “Treatment-related” AEs were defined as those assessed as definite,
probable, or possible.

Statistical analysis. Data are expressed as mean ± standard error of the mean (SEM), unless stated
otherwise. Statistical analyses were performed using GraphPad Prism (version 9.0, GraphPad Software,
San Diego, CA, USA). For comparisons among multiple groups, one-way ANOVA followed by Tukey’s
post hoc test was applied to assess all possible pairwise differences. For planned comparisons against a
single treatment of interest, Dunnett’s test was applied using the relevant reference group (PG-102 for
preclinical experiments, placebo for clinical datasets). Quantitative histological data were normalized to
tissue area or cell count using ImageJ (NIH, Bethesda, MD, USA). In the clinical dataset, no formal
correction across multiple PD endpoints was applied, and results should be interpreted as exploratory.
Statistical significance thresholds were *p < 0.05, **p < 0.01, and ***p < 0.001.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript introduces PG-102, which targets both GLP-1 and GLP-2 receptors to treat type 2 diabetes. The manuscript presents results from in vitro studies evaluating the properties of PG-102, mouse studies comparing the effects of PG-102 with semaglutide and tirzepatide, and a phase 1 multiple ascending dose study in humans assessing the safety and pharmacokinetic and pharmacodynamic profiles of PG-102. Overall, the approach of the described studies seems reasonable. My primary concern is that parts of the manuscript need further clarification, including some inconsistencies in different parts of the manuscript. My specific comments are provided below:\n1. In the description of the results in the text: There were a couple places where extra care should be taken to avoid over-interpreting some of the small numerical effects observed. In particular:\na. On line 198, when describing the comparative efficacy and mechanistic evaluation of PG-102 vs. semaglutide and tirzepatide: The text state that “semaglutide and tirzepatide treatment partially improved islet structure and beta-cell area, but restoration was limited.” Given that the islet area and beta-cell area both look nearly identical between the vehicle group and tirzepatide group based on Figures 3g and 3h, I would be hesitant to comment that tirzepatide improved these two outcomes.\nb. In the description of the safety results from the phase 1 multiple ascending dose study (line 280), it states “vomiting … demonstrated dose-dependent increases…” For vomiting, there were a very small number of events in all treatment groups (ranged from 0 to 2 events), with 0 events in both the placebo group and the 15 mg PG-102 group. Therefore, I would be hesitant to emphasize the dose-dependent relationship for vomiting too strongly, due to concern that these results may possibly be due to chance.\n2. In the description of the phase 1 multiple ascending dose study in the results section, on line 266, the reference to “Extended Data Table 1” should be changed to “Extended Data Table 2” (subject disposition across cohorts).\n3. There were a couple sets of previously unpublished results that were referenced briefly in the manuscript, but not described in detail in the methods. In particular:\na. The Discussion section (lines 334-337) refers to results from administering PG-102 to two naturally occurring diabetic feline patients, but these procedures were not described in the methods, nor were the results described prior to the Discussion section.\nb. In Extended Data Table 4, data are presented from a GLP toxicity study in monkeys, but these procedures were not described in the methods, nor were the results described in the manuscript.\n4. For the in vivo study comparing PG-102 to semaglutide and tirzepatide in mice, the sample size should be provided in the methods section.\n5. Also for the in vivo study comparing PG-102 to semaglutide and tirzepatide in mice, the age of the mice is inconsistent in the description of the methods (12-weeks) vs. in the figure legends (14-weeks). Please confirm the correct age of the mice, and ensure that this is correct throughout the manuscript.\n6. In the description of the phase 1 study design and PK analysis in the methods section, on lines 585-586, it states “Serial blood samples were collected for PK analysis at pre-specified intervals up to 696 hours after the final dose.” The “pre-specified intervals” should be described in the text.\n7. In the description of the statistical analysis in the methods section (line 592), it states “one-way ANOVA with Tukey’s post hoc test was used for multi-group comparisons…” Please specify whether all pairs of groups were tested using Tukey’s post hoc test, or if only a subset of tests of interest were assessed (and if so, which subset of tests were assessed). This will be helpful for interpreting whether lack of an indication of statistical significance in the results section and figures indicates a non-statistically significant test result or simply that the comparison was not tested. In addition, the figure legends throughout the manuscript state that statistical significance was assessed using one-way ANOVA, but some of the figure legends do not state the use of Tukey’s post hoc test; the use of Tukey’s post hoc test should be indicated in the figure legends where applicable.\n8. In the figures (e.g., Figure 2), it would be helpful to state the timeframe for cross-sectional results in the figure legend. For example, the legend for Figure Panels 2c and 2d-g state that the results are from end-of-study, but it would be helpful to re-state the timeframe for end-of-study in the legend for easy reference.\n9. Figure 6a includes two plots stacked on top of each other. Based on the figure legend, the top plot is on the linear-scale, and the bottom plot presents the same data on the semilogarithmic-scale. It would be helpful to include headings on these two plots to make it clear what is the difference between the two plots.\n10. Table 1: It is not clear to me what is meant by “treatment-related” adverse events. Please provide a definition for this category of adverse events in the manuscript text and in the description for Table 1.\n11. Was Extended Data Table 1 (Thyroid C cell and gastrointestinal hyperplasia in long-term repeated dose toxicity studies in rats and cynomolgus monkeys) referred to anywhere in the manuscript text?\n12. I am a bit confused about screening and enrollment for the phase 1 multiple ascending dose study, based on the data presented in Extended Data Table 2. In particular:\na. It would be helpful to specify in the table heading and/or description that the numbers presented here are for the phase 1 multiple ascending dose study.\nb. There is no column for the placebo group provided in this table. The relevant information for the placebo group should be added.\nc. The number of volunteers screened and number of volunteers not enrolled are provided in the table by treatment group. Were participants randomized before enrolling in the study? If so, what was the justification for this?\nd. The numbers of subjects enrolled in this table do not seem to match the sample size provided in the methods section text, or in the other tables related to the phase 1 multiple ascending dose study. This table states that there were 8 people enrolled in each of the 3 PG-102 dose cohorts. However, the methods section text states that there were a total of 24 participants, randomized in a 6:2 ratio for the 3 PG-102 dose cohorts and placebo, implying that there should be 6 participants in each treatment group, including each of the PG-102 dose cohorts. The other tables and figures (e.g., Table 1, Extended Data Table 3, Extended Data Figure 2) also imply a total sample size of 24, with 6 participants in each of the 3 PG-102 dose cohorts and in the placebo group. Please confirm that the sample sizes are correct throughout the manuscript.\ne. Similar to the above point, in Extended Data Figure 2, the numbers who were screened, not eligible, and met inclusion/exclusion criteria are consistent with the total numbers across the 3 PG-102 dose cohorts from Extended Data Table 2 (i.e., excluding the placebo group). Should the numbers in Extended Data Figure 2 be revised to include the corresponding numbers from the placebo group, or were the numbers tabulated incorrectly in Extended Data Table 2? Please revise as needed to ensure that the numbers presented in Extended Data Table 2 and Extended Data Figure 2 are both correct.\nf. Under “Reason for screening failure”, there were a total of 6 participants who failed screening for “other” reasons. Are these the same 6 participants who were excluded from the study due to exceeding the subject limit, according to Extended Data Figure 2? If the reason for screening failure is the same for all 6 participants in the “other” category (i.e., due to exceeding the subject limit), then this reason can be stated in the table, instead of labeling the row as “others”.\n13. For the phase 1 multiple ascending dose study, based on Extended Data Table 2, there was one participant who dropped out of the study. Did this participant drop out of the study completely (i.e., stopped participating in data collection procedures), or did they just discontinue the randomized medication? Was the participant who dropped out included in any of the analyses, and if so which ones? Please specify this information in the description of the phase 1 multiple ascending dose study in the methods section.\n14. Extended Data Table 5: There is an asterisk next to T_max in the table, but the meaning of the asterisk is not defined anywhere in the table or legend.\n15. Extended Data Table 6: On line 917 of the figure legend, what is the meaning for the numbers in parentheses following exendin (9-39) and GLP-2 (3-33)?\n16. Extended Data Figure 2: In the second box on the right side, the word “consent” is included twice.\n17. Extended Data Figure 3:\na. Why was semaglutide excluded from this analysis of the moderate and mild hyperglycemia mouse models (even though it was included in the analysis of the severe hyperglycemia mouse model in Figure 3)? This should be explained in the figure legend and/or in the manuscript text.\nb. Panels d-f (results from the mild hyperglycemia mouse model) appear to be missing.\nc. The dose for tirzepatide appears to be incorrect in the legend indicating the colors and symbols for the different treatment groups (it is listed as 30 nmol/kg, but should be 15 nmol/kg).\n18. Extended Data Figure 4: The procedures for this comparison of PG-105 and PG-102 do not appear to be described in the methods section.", "reviewer_2": "Summary\nThis manuscript presents the preclinical and early clinical development of PG-102, a bispecific GLP-1/GLP-2 receptor agonist, for the treatment of type 2 diabetes (T2D). The authors demonstrate PG-102 in vitro characterization and in vivo efficacy in preclinical models and report favorable tolerability and pharmacokinetics in a Phase 1 multiple ascending dose (MAD) study. The central claim is that PG-102 achieves glycemic control without inducing weight loss, distinguishing it from existing GLP-1-based therapies.\nWhile the manuscript is ambitious and addresses an important clinical need, several key mechanistic claims are insufficiently supported. In particular, the dual pharmacology of PG-102 is not convincingly demonstrated in vivo. Additionally, some experimental designs and interpretations require clarification or further validation.\nMajor Comments\n1. The manuscript should include a direct in vivo comparison between PG-102, the GLP-1-NTIG Fc, the GLP-2-NTIG Fc, and a loose combination of the GLP-1 and GLP-2 NTIG Fc’s to validate the synergistic effects of dual receptor targeting, especially for glucose control and islet protection.\na. PG-102 exhibits only ~3.4% of native GLP-2R potency. It is unclear whether this level of activity is sufficient to elicit meaningful in vivo effects. A comparison with a GLP-1-only antibody is essential to isolate the contribution of GLP-2R agonism.\nb. While the adipocyte data support dual receptor synergy, similar experiments in islets or in vivo are lacking. Without these, the role of GLP-2 on the central pharmacologic action of PG-102 remains speculative.\n2. The manuscript claims enhanced tolerability in humans, but it is unclear what this is relative to. No marketed comparators were included in the Phase 1 study. Please clarify the basis for this claim.\n3. The authors should report whether PG-102 activates other class B GPCRs such as GCGR or GIPR. This is particularly important given the structural similarity and potential for off-target effects.\n4. The manuscript uses tirzepatide as a comparator in rodents, but its GIPR activity is limited in this species. This limitation should be acknowledged and discussed.\n5. The reported 40% spontaneous weight loss in vehicle-treated db/db mice is unusually high. The authors should provide citations or additional data to support this observation. The supplementary data show only ~7% weight loss in a milder glucose intolerance model, which raises concerns about reproducibility.\n6. The authors mention reduced CNS exposure as a potential mechanism for improved tolerability. Have they performed ICV injections or biodistribution studies to support this?\n7. The claim that PG-102 enhances insulin secretion over semaglutide and tirzepatide is not statistically significant (line 215). Similarly, MafA expression is not significantly upregulated. These points should be revised or qualified.\n8. The manuscript claims anti-inflammatory effects of PG-102 (line 359), but no supporting data are shown. These claims should be either substantiated or removed.\n9. Plot the AUC for glucose and drug exposure (Figure 6a vs Figure 6b) to show the relationship between exposure and response.\nMinor Comments\nLine 84: Explicitly state the goal of achieving glycemic control without weight loss.\nLine 109: Clarify that no marketed comparators were included in the Phase 1 study.\nLine 218: Cite El, Douros et al. Nature Metabolism 2023 to support the role of GIPR in the pharmacology of tirzeaptide.\nLine 324: Replace “potent” with “effective” to avoid overstatement.\nLines 336–337: Provide data or citations for feline case studies.\nLines 348–354: Add citations to support mechanistic claims.\nLine 358: Specify which other cell types co-express GLP-1R and GLP-2R.\nLine 367–370: Reframe speculative mechanisms as hypotheses rather than conclusions.\nLine 388: Show the data referenced.\nLine 431: If available, include biodistribution data for PG-102 and dulaglutide.", "reviewer_3": "Strengths\nInnovative dual-agonist design with translational potential\nThe study introduces a well-engineered GLP-1/GLP-2 dual-agonist supported by a mechanistically sound rationale. The combination of GLP-1R-mediated glycaemic control and GLP-2R-driven intestinal and systemic homeostasis offers a potential therapeutic niche in advanced type 2 diabetes, cachexia, or malabsorption.\nComprehensive early-phase study design\nThe manuscript captures a broad spectrum of tolerability, exposure, and PD signals in healthy adults. The inclusion of multiple ascending dose arms and detailed PK/PD endpoints (including DEXA, OGTT, HbA1c, FMI/LMI) supports an informative pharmacological characterisation. Albeit, the vast majority of these variables are not presented in the current manuscript.\nPreclinical comparator data strengthen translational appeal\nHead-to-head comparisons with semaglutide and tirzepatide in db/db mice highlight superior glucose-lowering efficacy independent of body weight, suggest a favourable pharmacodynamic dissociation.\nWeakness\nNo formal statistical hypothesis or power justification provided\nAlthough Phase 1 trials are typically exploratory, the manuscript would benefit from explicit clarification regarding sample size rationale and statistical assumptions for key PD endpoints (e.g., body weight change, HbA1c).\nLimited discussion of GLP-2R-specific effects in humans (efficacy and safety)\nWhile the dual-agonist concept is central, the manuscript provides little evidence or speculation about the contribution of GLP-2R agonism per se. For instance, GI permeability markers, gut hormones, or nutrient absorption were not assessed, weakening the mechanistic interpretation of the GLP-2R arm. Especially, the safety concerns related to long-term GLP-2 RAs in humans linked to intestinal neoplasms needs to be addressed.\nPD effects in humans appear modest\nOnly glycaemic effects of PG-102 treatment are presented in this manuscript which is insufficient to thoroughly assess the effects of the compound. Body weight, body composition and hsCRP data needs to be presented as well. But most importantly, the effect of PG-102 appears fairly modest compared to placebo treatment. This may reflect high baseline insulin sensitivity, insufficient duration, or limited GLP-1R potency at early dose levels. The glucometabolic effect of GLP-2 receptor activation in humans remain controversial.\nOverall level of enthusiasm\nThis is a well-conceived and well-executed early-phase translational study introducing a novel dual GLP-1/GLP-2 receptor agonist delivering exciting glycaemic results independent of change in body weight in rodents. Although the human PD effects are modest and the absence supporting data (insulin, Matsuda-index, change in body weight, body composition and inflammatory cytokines) are disappointing. The preclinical results (particularly in the db/db model) are compelling.\nThe manuscript will be of interest to the field of incretin biology and metabolic pharmacology, but it is very heavy on the preclinical which is a disappointment. I recommend publication pending revision and the addition of more human data.\nAdditional comments\nBelow are some suggestions aiming to improve the manuscript.\n• In general\no Line 319: In T2D, metformin is the first-line treatment for hyperglycemia. Please correct the sentence\no Line 325: Please provide insight into how many participants in the STEP-2 or STEP-6 and SURPASS trials obtain ‘treat-to-target’ success with an HbA1c below 5.7%\no Line: 348-353: Please provide references supporting these claims.\no Line 359: The author has not provided data supporting this claim of anti-inflammatory effects of PG-102\no Line 370: Please update this section with a remark alluding to the anti-inflammatory effects observed in the present study. If, please inform the reader, that the anti-inflammatory effects of PG-102 otherwise are purely speculative\no Line 371-374: Please provide reference\no Line 590: Clarify whether any correction for multiple testing was applied across PD endpoints.\no Line 596: Please add supporting information to the sample size calculation\no Line 809 (Fig. 6): Please provide more details with regards to exact N per timepoint if sample size varies\no Line 924 (Extended Data Fig. 2.): please add reason for dropout."}, "author_response": "Comment #1\nIn the description of the results in the text: There were a couple places where extra care should be taken to avoid over-interpreting some of the small numerical effects observed. In particular:\na. On line 198, when describing the comparative efficacy and mechanistic evaluation of PG-102 vs. semaglutide and tirzepatide: The text state that “semaglutide and tirzepatide treatment partially improved islet structure and beta-cell area, but restoration was limited.” Given that the islet area and beta-cell area both look nearly identical between the vehicle group and tirzepatide group based on Figures 3g and 3h, I would be hesitant to comment that tirzepatide improved these two outcomes.\nAnswer #1a\nAs suggested by the reviewer, we have revised the Results section (Line 177 of the revised manuscript) to clarify that tirzepatide did not produce measurable improvements in islet or β-cell area compared with the vehicle group. In addition, we have refined the surrounding paragraph (Lines 175–183) for greater clarity and consistency with the data shown in Figures 3d-3j, while maintaining a professional and cohesive tone throughout the manuscript.\nb. In the description of the safety results from the phase 1 multiple ascending dose study (line 280), it states “vomiting … demonstrated dose-dependent increases…” For vomiting, there were a very small number of events in all treatment groups (ranged from 0 to 2 events), with 0 events in both the placebo group and the 15 mg PG-102 group. Therefore, I would be hesitant to emphasize the dose-dependent relationship for vomiting too strongly, due to concern that these results may possibly be due to chance.\nAnswer 1b\nAs suggested by the reviewer, we have removed the dose-dependent interpretation for vomiting and revised the Results section (Line 287–288 of the revised manuscript) to state that vomiting was infrequent overall and absent in both the placebo and 15 mg PG-102 groups, without implying a dose relationship. In addition, we have refined the subsequent sentences (Lines 287–291) to improve clarity and ensure consistency in the description of other gastrointestinal events within the same paragraph.\n.\nComment #2\nIn the description of the phase 1 multiple ascending dose study in the results section, on line 266, the reference to “Extended Data Table 1” should be changed to “Extended Data Table 2” (subject disposition across cohorts).\nAnswer #2\nAs suggested by the reviewer, we have corrected the Results section reference from ‘Extended Data Table 1’ to ‘Extended Data Table 2’ (Line 276 of the revised manuscript)\nComment #3\nThere were a couple sets of previously unpublished results that were referenced briefly in the manuscript, but not described in detail in the methods. In particular:\na. The Discussion section (lines 334-337) refers to results from administering PG-102 to two naturally occurring diabetic feline patients, but these procedures were not described in the methods, nor were the results described prior to the Discussion section.\nAnswer #3a\nThe exploratory statement regarding naturally diabetic feline patients (Lines 334–337 of the original manuscript) has been removed from the revised manuscript, because of the following reasons.\nThe observations were derived from pilot cases conducted at a collaborating veterinary hospital in Korea, directly performed by the attending veterinarian with verbal consent obtained from pet owners. At the time of the initial manuscript preparation, our understanding was that specific regulatory frameworks for clinical experimentation in companion animals in Korea were not yet established, and such exploratory cases could be reported descriptively.\nHowever, upon further review during the revision process, we recognized that U.S. and international standards require companion-animal studies to adhere to ethical guidelines largely analogous to those for human clinical research, including formal ethical review and documented owner consent.\nAccordingly, we have excluded all feline exploratory statements from the revised manuscript. For transparency in this reviewer response only, we provide a brief descriptive summary of the observations (Figure 1), noting that these were not designed or powered as controlled experiments and therefore are not presented as validated preclinical data. The present manuscript now focuses strictly on validated preclinical models and the controlled human Phase 1 study, which we believe best upholds ethical integrity while ensuring scientific rigor.\nFigure 1. Exploratory glucose-lowering effects of PG-102 in naturally diabetic cats. Two diabetic cats (Cat A: domestic shorthair, 16.8 years, obese; Cat B: Russian Blue, 1.9 years, underweight) were treated with PG-102 (0.3 mg/kg, subcutaneous, once weekly for 4 weeks). Fasting blood glucose, body weight, and serum cholesterol were monitored during follow-up.\nb. In Extended Data Table 4, data are presented from a GLP toxicity study in monkeys, but these procedures were not described in the methods, nor were the results described in the manuscript.\nAnswer #3b\nAs suggested by the reviewer, a description of the GLP toxicity study procedures has been added to the Supplementary Methods, and the corresponding results have been clarified in Extended Data Table 4 (Lines 278–281 of the revised manuscript).\nComment #4\nFor the in vivo study comparing PG-102 to semaglutide and tirzepatide in mice, the sample size should be provided in the methods section.\nAnswer #4\nAs suggested by the reviewer, we have added the sample size for the in vivo study to the Methods section (Line 485 of the revised manuscript), specifying that each treatment group consisted of n = 8 db/db mice\nComment #5.\nAlso, for the in vivo study comparing PG-102 to semaglutide and tirzepatide in mice, the age of the mice is inconsistent in the description of the methods (12-weeks) vs. in the figure legends (14-weeks). Please confirm the correct age of the mice, and ensure that this is correct throughout the manuscript.\nAnswer #5\nAs suggested by the reviewer, we confirm that all in vivo experiments were conducted in 14-week-old db/db mice at treatment initiation. The Methods section has been revised to clarify that mice were obtained at 12 weeks of age and acclimated for two weeks prior to dosing (Lines 482-487 of the revised manuscript)\nComment #6\nIn the description of the phase 1 study design and PK analysis in the methods section, on lines 585-586, it states “Serial blood samples were collected for PK analysis at pre-specified intervals up to 696 hours after the final dose.” The “pre-specified intervals” should be described in the text.\nAnswer #6\nAs suggested by the reviewer, the Methods section has been revised (Lines 585–587 of the revised manuscript) to specify the exact PK sampling timepoints\nComment #7.\nIn the description of the statistical analysis in the methods section (line 592), it states “one-way ANOVA with Tukey’s post hoc test was used for multi-group comparisons…” Please specify whether all pairs of groups were tested using Tukey’s post hoc test, or if only a subset of tests of interest were assessed (and if so, which subset of tests were assessed). This will be helpful for interpreting whether lack of an indication of statistical significance in the results section and figures indicates a non-statistically significant test result or simply that the comparison was not tested. In addition, the figure legends throughout the manuscript state that statistical significance was assessed using one-way ANOVA, but some of the figure legends do not state the use of Tukey’s post hoc test; the use of Tukey’s post hoc test should be indicated in the figure legends where applicable.\nAnswer #7\nAs suggested by the reviewer, all possible pairwise group comparisons were performed using Tukey’s post hoc test following one-way ANOVA. Additionally, when multiple treatment groups were compared against a single control, Dunnett’s multiple comparisons test was applied. We have revised the Methods section (Lines 593–599 of the revised manuscript) to clarify this and updated all relevant figure legends to specify the use of Tukey (all-pairwise) or Dunnett (control-versus-treatment) tests, as appropriate.\nComment # 8\nIn the figures (e.g., Figure 2), it would be helpful to state the timeframe for cross-sectional results in the figure legend. For example, the legend for Figure Panels 2c and 2d-g state that the results are from end-of-study, but it would be helpful to re-state the timeframe for end-of-study in the legend for easy reference.\nAnswer #8\nAs suggested by the reviewer, we have revised the legends for Figures 2 and 3 to explicitly state the timeframe for all cross-sectional analyses by indicating ‘end-of-study (Week 26)\nComment #9.\nFigure 6a includes two plots stacked on top of each other. Based on the figure legend, the top plot is on the linear-scale, and the bottom plot presents the same data on the semilogarithmic-scale. It would be helpful to include headings on these two plots to make it clear what is the difference between the two plots.\nAnswer #9\nAs suggested by the reviewer, we have added headings to Figure 6a: ‘Linear scale’ (upper panel) and ‘Semilogarithmic scale’ (lower panel)\nComment #10\nTable 1: It is not clear to me what is meant by “treatment-related” adverse events. Please provide a definition for this category of adverse events in the manuscript text and in the description for Table 1.\nAnswer #10\nAs suggested by the reviewer, we have added a definition of ‘treatment-related’ adverse events in the Methods section (Lines 590-592 of the revised manuscript). In addition, this definition is now included in the explanatory note below Table 1 for clarity.\nComment #11\nWas Extended Data Table 1 (Thyroid C cell and gastrointestinal hyperplasia in long-term repeated dose toxicity studies in rats and cynomolgus monkeys) referred to anywhere in the manuscript text?\nAnswer #11\nAs suggested by the reviewer, Extended Data Table 1 is referred in the Results section (Lines 129–133 of the revised manuscript) when describing the absence of thyroid C-cell and gastrointestinal hyperplasia in the 26-week repeat-dose toxicity study\nComment #12.\nI am a bit confused about screening and enrollment for the phase 1 multiple ascending dose study, based on the data presented in Extended Data Table 2. In particular:\na. It would be helpful to specify in the table heading and/or description that the numbers presented here are for the phase 1 multiple ascending dose study.\nAnswer #12a\nAs suggested by the reviewer, we have revised the heading and legend of Extended Data Table 2 to specify that the numbers refer to the Phase 1 multiple ascending dose study\nb. There is no column for the placebo group provided in this table. The relevant information for the placebo group should be added.\nAnswer #12b\nAs suggested by the reviewer, placebo group information is incorporated in Extended Data Table 2. For clarity, we have revised the table heading to indicate that values are presented as ‘PG-102 : Placebo’ (e.g., 6:2 indicates 6 subjects received PG-102 and 2 subjects received placebo) across all categories\nc. The number of volunteers screened and number of volunteers not enrolled are provided in the table by treatment group. Were participants randomized before enrolling in the study? If so, what was the justification for this?\nAnswer #12c\nAs suggested by the reviewer, enrollment was defined as randomization (6:2 per cohort). The ‘screened’ and ‘not enrolled’ numbers reflect the pre-randomization process. This has been clarified in the Methods section (Lines 570-572 of the revised manuscript), Extended Data Table 2, and Extended Data Figure 10 of the revised manuscript).\nd. The numbers of subjects enrolled in this table do not seem to match the sample size provided in the methods section text, or in the other tables related to the phase 1 multiple ascending dose study. This table states that there were 8 people enrolled in each of the 3 PG-102 dose cohorts. However, the methods section text states that there were a total of 24 participants, randomized in a 6:2 ratio for the 3 PG-102 dose cohorts and placebo, implying that there should be 6 participants in each treatment group, including each of the PG-102 dose cohorts. The other tables and figures (e.g., Table 1, Extended Data Table 3, Extended Data Figure 2) also imply a total sample size of 24, with 6 participants in each of the 3 PG-102 dose cohorts and in the placebo group. Please confirm that the sample sizes are correct throughout the manuscript.\nAnswer # 12d\nAs suggested by the reviewer, the sample sizes are correct: 24 participants in total, with 8 per cohort randomized in a 6:2 ratio to PG-102 or placebo (18 PG-102, 6 placebo overall). The Methods section (Lines 570-576 of the revised manuscript) has been revised to clarify that randomization occurred within each cohort\ne. Similar to the above point, in Extended Data Figure 2, the numbers who were screened, not eligible, and met inclusion/exclusion criteria are consistent with the total numbers across the 3 PG-102 dose cohorts from Extended Data Table 2 (i.e., excluding the placebo group). Should the numbers in Extended Data Figure 2 be revised to include the corresponding numbers from the placebo group, or were the numbers tabulated incorrectly in Extended Data Table 2? Please revise as needed to ensure that the numbers presented in Extended Data Table 2 and Extended Data Figure 2 are both correct.\nAnswer 12e\nAs suggested by the reviewer, Extended Data Figure 2 (Extended Data Figure 10 of the revised manuscript) has been corrected to align with Extended Data Table 2 of the revised manuscript, and both now include PG-102 and placebo participants with consistent totals\nf. Under “Reason for screening failure”, there were a total of 6 participants who failed screening for “other” reasons. Are these the same 6 participants who were excluded from the study due to exceeding the subject limit, according to Extended Data Figure 2? If the reason for screening failure is the same for all 6 participants in the “other” category (i.e., due to exceeding the subject limit), then this reason can be stated in the table, instead of labeling the row as “others”.\nAnswer 12f\nAs suggested by the reviewer, the 6 participants listed under ‘other’ were those who exceeded the subject limit. Extended Data Table 2 and Extended Data Figure 2 (Extended Data Figure 10 of the revised manuscript) have been revised accordingly for consistency in the revised manuscript\nComment #13\nFor the phase 1 multiple ascending dose study, based on Extended Data Table 2, there was one participant who dropped out of the study. Did this participant drop out of the study completely (i.e., stopped participating in data collection procedures), or did they just discontinue the randomized medication? Was the participant who dropped out included in any of the analyses, and if so which ones? Please specify this information in the description of the phase 1 multiple ascending dose study in the methods section.\nAnswer #13\nAs suggested by the reviewer, one participant in the 15 mg cohort discontinued due to an adverse event unrelated to the investigational product. This participant completed follow-up safety evaluations and was included in the safety analysis set, but excluded from efficacy and PK analyses. This has been clarified in the Methods section (Lines 579–583 of the revised manuscript), Extended Data Figure 2 (Extended Data Figure 10 in the revised manuscript) and Extended Data Table 2\nComment #14\nExtended Data Table 5: There is an asterisk next to Tmax in the table, but the meaning of the asterisk is not defined anywhere in the table or legend.\nAnswer #14\nAs suggested by the reviewer, the unexplained asterisk next to Tmax in Extended Data Table 5 has been removed, and minor typographical errors have been corrected for consistency in the revised manuscript\nComment #15\nExtended Data Figure 1: On line 917 of the figure legend, what is the meaning for the numbers in parentheses following exendin (9-39) and GLP-2 (3-33)?\nAnswer #15\nThe numbers in parentheses indicate the amino acid positions defining the truncated peptide sequences. Exendin (9–39) corresponds to residues 9–39 of exendin-4, acting as a GLP-1 receptor antagonist, and GLP-2 (3–33) corresponds to residues 3–33 of GLP-2, acting as a GLP-2 receptor antagonist. This clarification has been added to the legend of Extended Data Figure 1 (now Extended Data Figure 8 of the revised manuscript).\nComment #16\nExtended Data Figure 2: In the second box on the right side, the word “consent” is included twice.\nAnswer #16\nAs suggested by the reviewer, the duplicated word ‘consent’ in Extended Data Figure 2 (Extended Data Figure 10 of the revised manuscript) has been corrected in the revised manuscript\nComment #17.\nExtended Data Figure 3:\na. Why was semaglutide excluded from this analysis of the moderate and mild hyperglycemia mouse models (even though it was included in the analysis of the severe hyperglycemia mouse model in Figure 3)? This should be explained in the figure legend and/or in the manuscript text.\nAnswer #17a\nAs suggested by the reviewer, we have revised not only the relevant sentence but also the surrounding paragraph (Lines 192–200 of the revised manuscript) to clarify the rationale for excluding semaglutide from the mild and moderate hyperglycemia models.\nSemaglutide was not re-tested in these models, as its efficacy had already been confirmed in the severe hyperglycemia model. Instead, the additional studies focused on a direct head-to-head comparison with tirzepatide at an equivalent dose (30 nmol/kg), given its clinical relevance and the attenuated GIPR agonism of tirzepatide in rodents, which warranted focused evaluation of its relative efficacy.\nb. Panels d-f (results from the mild hyperglycemia mouse model) appear to be missing.\nAnswer #17b\nAs suggested by the reviewer, the data from the mild hyperglycemia model have been added to Extended Data Figure 4 (new panels d–g), and the corresponding figure legend and Results section have been updated accordingly (Lines 195–197 in the revised manuscript).\nc. The dose for tirzepatide appears to be incorrect in the legend indicating the colors and symbols for the different treatment groups (it is listed as 30 nmol/kg, but should be 15 nmol/kg).\nAnswer #17c\nAs suggested by the reviewer, we have re-checked the figure legend and confirm that tirzepatide is correctly labeled as 15 nmol/kg in all relevant panels, including the symbol/color legend for Figure 3. No changes were required in the revised manuscript\nComment #18.\nExtended Data Figure 4: The procedures for this comparison of PG-105 and PG-102 do not appear to be described in the methods section.\nAnswer #18\nAs suggested by the reviewer, the Supplementary Methods section has been updated to include the procedures for the comparison of PG-105 and PG-102 underlying Extended Data Figure 4 (Extended Data Figure 1 of the revised manuscript).\nOf note, the original panel d of Extended Data Figure 4 (gross photograph of the gallbladder) has been removed to prevent potential visual over-interpretation. Gallbladder size at necropsy is inherently variable—affected by bile filling, handling, and viewing angle—and representative images from a small sample (n=4 per group) cannot reliably represent the group distribution. Moreover, the image could be misread as inconsistent with the quantitative data, which showed a significant increase with PG-105 but no statistically significant change with PG-102. In line with Nature Communications guidelines prioritizing quantitative presentation, we now display only the volumetric measurement with individual data points (panel e of Extended Data Figure 4, now panel d of Extended Data Figure 1), which more accurately and reproducibly reflects the dataset.\nReviewer #2\nThis manuscript presents the preclinical and early clinical development of PG-102, a bispecific GLP-1/GLP-2 receptor agonist, for the treatment of type 2 diabetes (T2D). The authors demonstrate PG-102 in vitro characterization and in vivo efficacy in preclinical models and report favorable tolerability and pharmacokinetics in a Phase 1 multiple ascending dose (MAD) study. The central claim is that PG-102 achieves glycemic control without inducing weight loss, distinguishing it from existing GLP-1-based therapies.\nWhile the manuscript is ambitious and addresses an important clinical need, several key mechanistic claims are insufficiently supported. In particular, the dual pharmacology of PG-102 is not convincingly demonstrated in vivo. Additionally, some experimental designs and interpretations require clarification or further validation.\nMajor Comments\nComment #1\nThe manuscript should include a direct in vivo comparison between PG-102, the GLP-1-NTIG Fc, the GLP-2-NTIG Fc, and a loose combination of the GLP-1 and GLP-2 NTIG Fc’s to validate the synergistic effects of dual receptor targeting, especially for glucose control and islet protection.\nAnswer #1\nAs suggested by the reviewer, we generated three complementary data sets directly comparing PG-102 with GLP-1-NTIG Fc, GLP-2-NTIG Fc, and their loose combination:\n1. Glucose control (ipGTT; normoglycemic mice, Figure 5a of the revised manuscript): At GLP-1R–matched dosing, PG-102 improved glucose clearance relative to GLP-1-NTIG Fc and the loose GLP-1/GLP-2-NTIG Fc combination, while GLP-2-NTIG Fc alone showed minimal effect.\n2. Islet protection under diabetogenic stress (INS-1 + STZ; Figure 5b and Extended Data Figure 7 of the revised manuscript): PG-102 outperformed each monospecific construct and matched or exceeded the loose combination across GSIS and β-cell gene expression (restoration of Pdx1 and Neurod1; MafA showed an upward trend; TNFα reduced).\n3. Receptor trafficking (Extended Data Figure 9 of the revised manuscript): Confocal imaging demonstrated cis-acting co-engagement with delayed internalization and persistent signaling, not reproduced by loose combinations.\nTo better reflect the reviewer’s suggestion, we have substantially revised the manuscript by reorganizing the mechanistic figures, introducing a new Results subsection titled “Bivalent receptor engagement underlying PG-102 efficacy,” and restructuring the related text.\nSpecifically, the Results (Lines 201–263) and Discussion (Lines 325–365) sections have been thoroughly revised to provide a clearer and more cohesive mechanistic narrative.\nComment #1a\nPG-102 exhibits only ~3.4% of native GLP-2R potency. It is unclear whether this level of activity is sufficient to elicit meaningful in vivo effects. A comparison with a GLP-1-only antibody is essential to isolate the contribution of GLP-2R agonism.\nAnswer #1a\nAs suggested by the reviewer, direct comparisons with a GLP-1–only construct were conducted (Figure 5a, Lines 228-235 of the revised manuscript), along with receptor trafficking studies (Extended Data Figure 9, Lines 254-263 of the revised manuscript). Despite exhibiting only ~3.4% of native GLP-2R potency in cAMP assays, PG-102 achieved superior in vivo glucose control and demonstrated cis-acting GLP-1R/GLP-2R co-engagement with delayed internalization and sustained signaling. These results confirm a meaningful GLP-2R contribution beyond GLP-1R agonism alone.\nComment #1b\nWhile the adipocyte data support dual receptor synergy, similar experiments in islets or in vivo are lacking. Without these, the role of GLP-2 on the central pharmacologic action of PG-102 remains speculative.\nAnswer #1b\nAs suggested by the reviewer, we now provide β-cell (INS-1 + STZ) and in vivo ipGTT datasets showing that PG-102 outperforms both the GLP-1-only construct and the loose GLP-1/GLP-2 combination in glucose control and β-cell endpoints (Figure 5a-b, Lines 228-243 of the revised manuscript). In addition, receptor trafficking analyses (Extended Data Figure 9, Lines 254-263 of the revised manuscript) further support a direct GLP-2R contribution, establishing that the role of GLP-2 in PG-102’s pharmacology is non-speculative.\nComment #2\nThe manuscript claims enhanced tolerability in humans, but it is unclear what this is relative to. No marketed comparators were included in the Phase 1 study. Please clarify the basis for this claim.\nAnswer #2\nAs suggested by the reviewer, the manuscript has been revised to remove any implicit comparative language. The Title was shortened, and tolerability is now described descriptively (low incidence of gastrointestinal events, no treatment-related discontinuations) in the Discussion (Line 402–407) of the revised manuscript.\nComment #3\nThe authors should report whether PG-102 activates other class B GPCRs such as GCGR or GIPR. This is particularly important given the structural similarity and potential for off-target effects.\nAnswer #3\nAs suggested by the reviewer, we performed the off-target profiling experiments, as described in the revised Results section (Lines 123–128) and Extended Data Figure 2. PG-102 showed no detectable agonist activity at human GIPR or GCGR, confirming selectivity for GLP-1R and GLP-2R.\nComment #4\nThe manuscript uses tirzepatide as a comparator in rodents, but its GIPR activity is limited in this species. This limitation should be acknowledged and discussed.\nAnswer #4\nAs suggested by the reviewer, the limitation of tirzepatide’s attenuated GIPR agonism in rodents has been acknowledged in the Results (Lines 192–200 and 206–208 of the revised manuscript), noting that these data serve as comparative references rather than predictors of clinical efficacy.\nComment #5\nThe reported 40% spontaneous weight loss in vehicle-treated db/db mice is unusually high. The authors should provide citations or additional data to support this observation. The supplementary data show only ~7% weight loss in a milder glucose intolerance model, which raises concerns about reproducibility.\nAnswer #5\nAs suggested by the reviewer, we have cited supporting evidence in the Discussion (Line 372) showing that the ~40% spontaneous weight loss observed in the severe hyperglycemia model is consistent with prior reports in advanced-stage db/db mice (Choi et al., Lab Anim Res. 2015; 31:1–6). The apparent difference from the milder model (~7% loss) is addressed in the revised Discussion (Lines 372–375), which clarifies that it reflects baseline disease severity and independent study conditions.\nIn addition, to more coherently address the mechanisms underlying PG-102’s distinctive profile—achieving normoglycemia while substantially preserving body weight across both severe and moderate hyperglycemia models—we have refined the entire Discussion paragraph (Lines 366–401) for logical flow and mechanistic clarity.\nComment #6\nThe authors mention reduced CNS exposure as a potential mechanism for improved tolerability. Have they performed ICV injections or biodistribution studies to support this?\nAnswer #6\nAs suggested by the reviewer, we have further clarified the rationale regarding potential CNS exposure in relation to tolerability.\nWhile intracerebroventricular (ICV) injections or formal CNS biodistribution studies have not yet been performed for the current PG-102 molecule, exploratory imaging with a precursor form of PG-102 (PG-105) demonstrated negligible brain signal and preferential localization to the small intestine, whereas dulaglutide exhibited measurable brain accumulation and predominant pancreatic uptake (Figure 2 below).\nBecause these findings were exploratory and based on a precursor molecule, they were not included as main data.\nInstead, the conceptual implication—that limited CNS exposure, likely related to the large-molecule nature of Fc-based GLP-1 agonists, may contribute to PG-102’s favorable tolerability profile—has been concisely reflected in the Discussion section (Lines 415–421 of the revised manuscript) in reference to a previous report supporting reduced CNS penetrance of Fc-fused incretin analogs.\nFigure 2. Exploratory biodistribution of PG-105 (a precursor form of PG-102) in Balb/c nude mice. Male BALB/c nude mice (5 weeks old) were administered a single subcutaneous injection of Fc protein, PG-105, or dulaglutide. Whole-body fluorescence imaging was performed at 3, 10, 24, 72, and 168 hours after dosing, followed by ex vivo organ imaging at 10 hours, when blood concentrations were highest. Relative distribution was quantified in major organs including the brain, pancreas, heart, stomach, liver, kidney, small intestine (duodenum, jejunum, ileum), and large intestine. Data are presented as mean ± SD.\nComment #7\nThe claim that PG-102 enhances insulin secretion over semaglutide and tirzepatide is not statistically significant (line 215). Similarly, MafA expression is not significantly upregulated. These points should be revised or qualified.\nAnswer #7\nAs suggested by the reviewer, we repeated the GSIS and MafA analyses under the STZ-induced diabetogenic condition with additional biological replicates. The Results section has been revised to qualify these findings accordingly (Lines 215–218 of the revised manuscript), indicating that insulin secretion (GSIS) was statistically significant, whereas MafA expression showed a non-significant upward trend. Exact n values and statistical details are now provided in the corresponding figure legends.\nComment #8\nThe manuscript claims anti-inflammatory effects of PG-102 (line 359), but no supporting data are shown. These claims should be either substantiated or removed.\nAnswer #8\nAs suggested by the reviewer, we have substantiated the anti-inflammatory claim with new in vivo and in vitro data. The revised manuscript now includes CD45⁺ immunostaining of peri-pancreatic adipose tissue and TNFα expression analyses under β-cell stress, both supporting anti-inflammatory activity of PG-102 (Extended Data Figures 3 and 7d). To integrate these new findings coherently, we have substantially revised the relevant Results (Lines 183–191) and Discussion (Lines 342–352) sections to strengthen the mechanistic narrative regarding PG-102’s anti-inflammatory actions.\nComment #9\nPlot the AUC for glucose and drug exposure (Figure 6a vs Figure 6b) to show the relationship between exposure and response.\nAnswer #9\nAs suggested by the reviewer, an exploratory exposure–response analysis was added, plotting PK exposure (AUC ) against OGTT glucose AUC₀–₂ . The corresponding results are described in the last h\nResults (Lines 303–304 of the revised manuscript) and presented in Extended Data Figure 11.\nMinor Comments\nComment #1. Line 84: Explicitly state the goal of achieving glycemic control without weight loss.\nAnswer #1\nAs suggested by the reviewer, we have revised the Introduction to explicitly state the study objective of achieving glycemic control without weight loss (Line 56-61 of the revised manuscript)\nComment #2. Line 109: Clarify that no marketed comparators were included in the Phase 1 study.\nAnswer #2\nAs suggested by the reviewer, we have revised the manuscript to clarify that no marketed comparators were included in the Phase 1 study and removed any phrasing that could imply otherwise.\nComment #3. Line 218: Cite El, Douros et al. Nature Metabolism 2023 to support the role of GIPR in the pharmacology of tirzapatide.\nAnswer #3.\nAs suggested by the reviewer, we have added a citation to El Douros et al., Nature Metabolism (2023) at Line 192 of the revised manuscript to support the role of GIPR in the pharmacology of tirzepatide.\nComment #4. Line 324: Replace “potent” with “effective” to avoid overstatement.\nAnswer #4\nAs suggested by the reviewer, we have replaced ‘potent’ with ‘effective’ at Line 313 of the revised manuscript to ensure balanced wording.\nComment #5. Lines 336–337: Provide data or citations for feline case studies.\nAnswer #5\nThe exploratory statement regarding naturally diabetic feline patients (Lines 336–337 of the original submission) has been removed from the revised manuscript because of the following reasons.\nThe observations originated from pilot cases conducted independently at a collaborating veterinary hospital in Korea, in which naturally diabetic cats were treated at the discretion of the attending veterinarian with verbal consent obtained from the pet owners. At the time of the initial manuscript preparation, our understanding was that Korea did not yet have clearly defined regulatory pathways for companion-animal research, and such observational cases could be described anecdotally. However, upon further review during the revision process, we recognized that international standards—particularly those aligned with U.S. and European guidelines—require companion-animal studies to undergo formal ethical review and to obtain documented owner consent analogous to human clinical research.\nIn compliance with these ethical principles, all feline-related content has been removed from the revised manuscript. For transparency, we provide here a concise descriptive summary and illustrative image (Figure 3) only within this reviewer response. These pilot observations were not designed or statistically powered as controlled experiments and are therefore not presented as validated preclinical or clinical data. The revised manuscript now focuses exclusively on validated preclinical models and the controlled human Phase 1 study, ensuring both scientific rigor and adherence to ethical research standards.\nFigure 3. Exploratory glucose-lowering effects of PG-102 in naturally diabetic cats. Two diabetic cats (Cat A: domestic shorthair, 16.8 years, obese; Cat B: Russian Blue, 1.9 years, underweight) were treated with PG-102 (0.3 mg/kg, subcutaneous, once weekly for 4 weeks). Fasting blood glucose, body weight, and serum cholesterol were monitored during follow-up.\nComment #6. Lines 348–354: Add citations to support mechanistic claims.\nAnswer #6\nAs suggested by the reviewer, we have added a supporting reference at Line 340 of the revised manuscript to substantiate the mechanistic claims\nComment #7. Line 358: Specify which other cell types co-express GLP-1R and GLP-2R.\nAnswer #7\nAs suggested by the reviewer, we have specified in the revised manuscript that GLP-1R and GLP-2R are co-expressed in enteroendocrine L cells, enteric neurons, intestinal epithelial subsets, pancreatic islets, adipocytes and subsets of vagal afferent neurons (Lines 359–361 of the revised manuscript)\nComment #8. Line 367–370: Reframe speculative mechanisms as hypotheses rather than conclusions.\nAnswer #8\nAs suggested by the reviewer, we have revised the Discussion section to frame the mechanistic interpretations of PG-102 as hypotheses supported by available evidence, rather than as definitive conclusions. In doing so, we extensively restructured the Discussion (Lines 325–365) to more clearly delineate evidence-based findings from conceptual interpretations.\nComment #9. Line 388: Show the data referenced.\nAnswer #9\nAs suggested by the reviewer, we have added the corresponding data as Extended Data Figure 12 and incorporated a brief description into the Discussion section (Lines 388–392 of the revised manuscript).\nComment #10. Line 431: If available, include biodistribution data for PG-102 and dulaglutide.\nAnswer #10\nExploratory biodistribution results comparing a precursor form of PG-102 (PG-105) and dulaglutide have been included in response to Major Comment #6 (Figure 2) and conceptually acknowledged in the revised Discussion (Line 415–421 of the revised manuscript).\nReviewer #3\nStrengths\nInnovative dual-agonist design with translational potential\nThe study introduces a well-engineered GLP-1/GLP-2 dual-agonist supported by a mechanistically sound rationale. The combination of GLP-1R-mediated glycaemic control and GLP-2R-driven intestinal and systemic homeostasis offers a potential therapeutic niche in advanced type 2 diabetes, cachexia, or malabsorption.\nComprehensive early-phase study design\nThe manuscript captures a broad spectrum of tolerability, exposure, and PD signals in healthy adults. The inclusion of multiple ascending dose arms and detailed PK/PD endpoints (including DEXA, OGTT, HbA1c, FMI/LMI) supports an informative pharmacological characterization. Albeit, the vast majority of these variables are not presented in the current manuscript.\nPreclinical comparator data strengthen translational appeal\nHead-to-head comparisons with semaglutide and tirzepatide in db/db mice highlight superior glucose-lowering efficacy independent of body weight, suggest a favorable pharmacodynamic dissociation.\nWeakness\nComment #1\nNo formal statistical hypothesis or power justification provided\nAlthough Phase 1 trials are typically exploratory, the manuscript would benefit from explicit clarification regarding sample size rationale and statistical assumptions for key PD endpoints (e.g., body weight change, HbA1c).\nAnswer #1\nAs suggested by the reviewer, this Phase 1 MAD study was designed to evaluate safety, tolerability, and PK; no formal hypothesis testing or power calculation was performed. The cohort size (n=8 per cohort; total n=24) follows standard early-phase practice. PD and exploratory biomarkers were analyzed descriptively, as clarified in the Methods section (Line 562-565 of the revised manuscript).\nComment #2\nLimited discussion of GLP-2R-specific effects in humans (efficacy and safety)\nWhile the dual-agonist concept is central, the manuscript provides little evidence or speculation about the contribution of GLP-2R agonism per se. For instance, GI permeability markers, gut hormones, or nutrient absorption were not assessed, weakening the mechanistic interpretation of the GLP-2R arm. Especially, the safety concerns related to long-term GLP-2 RAs in humans linked to intestinal neoplasms needs to be addressed.\nAnswer #2\nAs suggested by the reviewer, we have clarified in the Discussion (Lines 431–437 of the revised manuscript) that GLP-2R–specific endpoints such as intestinal permeability, nutrient absorption, or gut hormone secretion were not directly assessed in this phase 1 study. Regarding long-term safety, we have added a statement citing recent real-world data showing no increase in intestinal neoplasms with long-term teduglutide use (Abu Tair et al., Intestine Failure Reports, 2025) and noting that 26-week repeat-dose toxicity studies of PG-102 in rodents and primates showed not only an absence of intestinal hyperplasia but also no evidence of thyroid C-cell hyperplasia commonly associated with GLP-1RAs (Extended Data Table 1 and Lines 437–444 of the revised manuscript).\nComment #3\nPD effects in humans appear modest\nOnly glycaemic effects of PG-102 treatment are presented in this manuscript which is insufficient to thoroughly assess the effects of the compound. Body weight, body composition and hsCRP data needs to be presented as well. But most importantly, the effect of PG-102 appears fairly modest compared to placebo treatment. This may reflect high baseline insulin sensitivity, insufficient duration, or limited GLP-1R potency at early dose levels. The glucometabolic effect of GLP-2 receptor activation in humans remain controversial.\nAnswer #3\nAs suggested by the reviewer, we have added exploratory data on body weight, body composition, and inflammatory biomarkers (hsCRP), along with glycemic endpoints (HbA1c and FPG), to Extended Data Tables 6 and 7. As expected in healthy volunteers with high baseline insulin sensitivity and a short treatment duration, changes in these parameters were modest (Results, Lines 305–306 of the revised manuscript). Importantly, PG-102 consistently reduced glucose AUC during OGTT across active cohorts, demonstrating clear biological activity even at early dose levels (Results, Lines 298-304 of the revised manuscript). Our findings suggest that GLP-2R activation may contribute meaningfully to glucometabolic regulation, a hypothesis that will be further examined in Phase 2 studies involving diabetic patient populations.\nOverall level of enthusiasm\nThis is a well-conceived and well-executed early-phase translational study introducing a novel dual GLP-1/GLP-2 receptor agonist delivering exciting glycaemic results independent of change in body weight in rodents. Although the human PD effects are modest and the absence supporting data (insulin, Matsuda-index, change in body weight, body composition and inflammatory cytokines) are disappointing. The preclinical results (particularly in the db/db model) are compelling.\nThe manuscript will be of interest to the field of incretin biology and metabolic pharmacology, but it is very heavy on the preclinical which is a disappointment. I recommend publication pending revision and the addition of more human data.\nAdditional comments\nBelow are some suggestions aiming to improve the manuscript.\n• In general\nComment #1. Line 319: In T2D, metformin is the first-line treatment for hyperglycemia. Please correct the sentence\nAnswer #1\nAs suggested by the reviewer, we have revised the text to clarify that metformin is the standard first-line therapy for T2D, whereas insulin is recommended for patients with severe hyperglycemia (HbA1c >10%) in line with ADA/EASD guidelines (Lines 308-310 of the revised manuscript).\nComment #2. Line 325: Please provide insight into how many participants in the STEP-2 or STEP-6 and SURPASS trials obtain ‘treat-to-target’ success with an HbA1c below 5.7%\nAnswer #2\nAs suggested by the reviewer, we have revised the Discussion (Line 313-316 of the revised manuscript) to clarify that data on ‘treat-to-target’ success with HbA1c <5.7% are available from the SURPASS trials, where fewer than 50% of participants achieved normoglycemia even at the highest tirzepatide dose. The STEP trials primarily evaluated obesity populations rather than T2D cohorts; therefore, they do not directly report HbA1c-based remission outcomes.\nComment #3. Line: 348-353: Please provide references supporting these claims.\nAnswer #3\nAs suggested by the reviewer, we have added supporting references at Line 340 of the revised manuscript to substantiate these mechanistic claims.\nComment #4. Line 359: The author has not provided data supporting this claim of anti-inflammatory effects of PG-102\nAnswer #4\nAs suggested by the reviewer, we have added new experimental data to substantiate the anti-inflammatory effects of PG-102. These include reduced CD45⁺ immune cell infiltration in peri-pancreatic adipose tissue and suppression of TNFα induction under β-cell stress (Extended Data Figures 3 and 7d of the revised manuscript). In line with these additions, both the Results (Lines 183–191 and 240-243) and Discussion (Lines 342–352) have been revised to more clearly articulate the mechanistic link between PG-102’s dual-receptor engagement and its anti-inflammatory profile.\nComment #5. Line 370: Please update this section with a remark alluding to the anti-inflammatory effects observed in the present study. If, please inform the reader, that the anti-inflammatory effects of PG-102 otherwise are purely speculative\nAnswer #5\nAs suggested by the reviewer, this point was addressed in our response to Comment #4. The revised Discussion section (Lines 342–352) now incorporates the newly added anti-inflammatory findings, thereby moving beyond speculative interpretation.\nComment #6. Line 371-374: Please provide reference\nAnswer #6\nAs suggested by the reviewer, we have added the appropriate reference to support this statement (Line 368 of the revised manuscript).\nComment #7. Line 590: Clarify whether any correction for multiple testing was applied across PD endpoints.\nAnswer #7\nAs suggested by the reviewer, we have clarified in the Methods section (Lines 600–601 of the revised manuscript) that PD assessments were exploratory and summarized descriptively. For endpoints with statistical testing, appropriate post hoc tests were applied as described, but no formal correction across multiple PD endpoints was performed.\nComment #8. Line 596: Please add supporting information to the sample size calculation\nAnswer #8\nAs suggested by the reviewer, we have clarified the basis of the sample size determination in the Statistical Analysis section of the Methods section (Lines 487–489 of the revised manuscript). Specifically, we now state that sample sizes were guided by prior experience with the db/db mouse model and designed to provide at least 80% power at p < 0.05 for detecting biologically meaningful differences in metabolic outcomes.\nComment #9. Line 809 (Fig. 6): Please provide more details with regards to exact N per timepoint if sample size varies\nAnswer #9\nAs suggested by the reviewer, all participants included in the PK analysis completed every planned sampling timepoint without missing data; thus, n was identical across timepoints (15 mg: n = 5; 30 mg: n = 6; 30/60 mg: n = 6; placebo: n = 6). The legend of Figure 6 has been revised to clarify this.\nComment #10. Line 924 (Extended Data Fig. 2.): please add reason for dropout.\nAnswer #10\nAs suggested by the reviewer, we have clarified that in the 15 mg cohort, one participant discontinued due to an adverse event unrelated to the investigational product. This information has been added to the Methods (Line 579–583 of the revised manuscript), Extended Data Figure 2 (Extended Data Figure 10 of the revised manuscript), and Extended Data Table 2"}, {"round": 2, "reviewer_comments": {"reviewer_1": "This is a revised manuscript that evaluates PG-102, which targets both GLP-1 and GLP-2 receptors to treat type 2 diabetes, based on in vitro studies, mouse studies comparing to semaglutide and tirzepatide, and a phase 1 multiple ascending dose study in humans. The revised manuscript is much clearer and improved, and in my opinion it is nearly ready for publication. There are a few minor remaining issues that should be addressed, which are described below:\n1. In the Results, “Phase 1 MAD trial of PG-102: safety, PK and PD evaluation” section, on line 273: I would suggest clarifying that the 6:2 randomization ratio is for PG-102:placebo. For example, the sentence could be revised as “… randomized in a 6:2 ratio to receive PG-102 or placebo within three dose cohorts…”\n2. In the Results, “Phase 1 MAD trial of PG-102: safety, PK and PD evaluation” section, on lines 283-284: Since the AEs can be classified as treatment-related, not the participants, I suggest re-wording the last part of this sentence as “…with AEs considered to be treatment-related in 18 participants (75.0%; Table 1).”\n3. In the Methods, “Phase 1 study design; PK analysis, safety and PD evaluation” section, lines 560-561: As this sentence is currently written, it is a bit confusing since it refers to the three cohorts from the MAD study without providing information defining the cohorts. Most of the information from this sentence is clearly included later in this section on lines 570 – 572, and so the sentence from lines 560 – 561 could be deleted.\n4. Please define all abbreviations from the figures and tables in the corresponding figure legend or table footnote, including SEM, SD, PK, PD, MAD, AUC.\n5. The last sentence in the legend for Figure 6 states “All randomized participants completed every planned PK and PD sampling time point.” However, based on the Methods (lines 579-580) and the sample sizes listed in the figure legend, it seems that this statement is not quite accurate, since 1 randomized participant in the 15 mg group who discontinued treatment was excluded from the PK/PD analyses. Instead, this sentence could be re-written as “All participants included in the analyses completed every planned PK and PD sampling time point.”\n6. In Table 1, the number of participants with discontinuation due to AEs for the 15 mg PG-102 is listed as 0. Is this correct? Based on the Methods (lines 579-580), it seems that the 15 mg group had 1 discontinuation due to AEs (but there were 0 discontinuations due to treatment-related AEs).\n7. In the Supplementary Methods, “In vivo study” section, lines 35-36: This sentence states that the mice were “allocated by baseline glucose, HbA1c, and body weight into three groups.” Does this mean that treatment group allocation was designed to achieve balance of baseline glucose, HbA1c, and body weight across groups (e.g., by “matching” mice based on these factors)? If so, I suggest that the following re-wording of this sentence would more clearly explain how this allocation was done: “…allocated into three groups (vehicle, tirzepatide, PG-102; n=6 per group), ensuring between-group balance on baseline glucose, HbA1c, and body weight.”\n8. In the Clinical Supplementary Results, “Exploratory exposure-response relationship between PK exposure and OGTT glucose excursion” section: Be careful not to overstate the relationship identified between PK exposure and OGTT glucose excursion. The R^2, Pearson r, and Spearman ρ are all small in magnitude, and so I would not have much confidence about whether the negative relationship identified represents a real biological relationship vs. is due to random error.\n9. In Extended Data Table 5: Note that the notation AUC_{0-inf} is used in the table, but the notation AUC_{0-∞} is used in the table description. Please revise so that the notation is consistent between the table and the description.", "reviewer_2": "The authors have adequately addressed my comments and I feel the manuscript is appropriate for publication in this form.", "reviewer_4": "Reviewer Comments on the Revised Manuscript\nThe revised manuscript comprehensively addresses the original comments from Reviewer 3 and strengthens the scientific rigor of the work. The integration of preclinical and clinical data supporting PG-102 as a bispecific GLP-1/GLP-2 receptor agonist for advanced type 2 diabetes (T2D) is compelling, particularly its ability to decouple glycemic control from weight loss. However, 1 key area require further refinement to enhance mechanistic clarity, generalizability, and translational relevance. Below are specific suggestions for revision:\nMechanistic Link Between Low GLP-2R Potency and In Vivo Efficacy\nPG-102 exhibits only ~3.4% of native GLP-2R potency in vitro (Fig. 1c) but delivers meaningful in vivo effects (e.g., enhanced peripheral glucose uptake, β-cell protection). While the manuscript attributes this discrepancy to “cis-co-engagement” of GLP-1R/GLP-2R, direct evidence linking this receptor trafficking phenotype to downstream GLP-2R-mediated signaling in human cells/tissues is lacking.\nAdd a discussion section explicitly addressing how cis-co-engagement compensates for low GLP-2R potency, citing relevant literature on receptor trafficking and signaling crosstalk.\nIn Conclusion, The manuscript presents a novel and clinically relevant therapeutic candidate for advanced T2D. Addressing the above points will enhance mechanistic rigor, generalizability, and translational clarity, strengthening the manuscript’s impact in the field of incretin biology and metabolic pharmacology. With above revisions, the work will be well-positioned for publication and future clinical development."}, "author_response": "Comment #1\nIn the Results, “Phase 1 MAD trial of PG-102: safety, PK and PD evaluation” section, on line 273: I would suggest clarifying that the 6:2 randomization ratio is for PG-102:placebo. For example, the sentence could be revised as “…randomized in a 6:2 ratio to receive PG-102 or placebo within three dose cohorts…”:\nAnswer #1\nAs suggested by the reviewer, the sentence has been revised to clearly specify that the 6:2 randomization ratio corresponds to PG-102 versus placebo allocation within each cohort (Line 281 of the revised manuscript).\nComment #2\nIn the Results, “Phase 1 MAD trial of PG-102: safety, PK and PD evaluation” section, on lines 283-284: Since the AEs can be classified as treatment-related, not the participants, I suggest re-wording the last part of this sentence as “…with AEs considered to be treatment-related in 18 participants (75.0%; Table 1).\nAnswer #2\nAs suggested by the reviewer, we revised the wording to clarify that treatment-relatedness applies to AEs rather than participants, and updated the sentence accordingly (Lines 293 of the revised manuscript).\nComment #3\nIn the Methods, “Phase 1 study design; PK analysis, safety and PD evaluation” section, lines 560-561: As this sentence is currently written, it is a bit confusing since it refers to the three cohorts from the MAD study without providing information defining the cohorts. Most of the information from this sentence is clearly included later in this section on lines 570 – 572, and so the sentence from lines 560 – 561 could be deleted.\nAnswer #3\nAs suggested by the reviewer, the redundant sentence (Lines 560-561 of the original manuscript) referring to cohort composition was removed to improve clarity, as the cohorts are defined later in the Methods section (Lines 632–634 of the revised manuscript).\nComment #4\nPlease define all abbreviations from the figures and tables in the corresponding figure legend or table footnote, including SEM, SD, PK, PD, MAD, AUC.\nAnswer #4\nAs suggested by the reviewer, all abbreviations (SEM, SD, PK, PD, MAD, AUC) are now fully defined in the relevant figure legends and table footnotes.\nComment #5.\nThe last sentence in the legend for Figure 6 states “All randomized participants completed every planned PK and PD sampling time point.” However, based on the Methods (lines 579-580) and the sample sizes listed in the figure legend, it seems that this statement is not quite accurate, since 1 randomized participant in the 15 mg group who discontinued treatment was excluded from the PK/PD analyses. Instead, this sentence could be re-written as “All participants included in the analyses completed every planned PK and PD sampling time point.”\nAnswer #5\nAs suggested by the reviewer, the Figure 6 legend has been revised to specify that all participants included in the PK and PD analyses completed all planned sampling time points (Lines 957-958 of the revised manuscript)\nComment #6\nIn Table 1, the number of participants with discontinuation due to AEs for the 15 mg PG-102 is listed as 0. Is this correct? Based on the Methods (lines 579-580), it seems that the 15 mg group had 1 discontinuation due to AEs (but there were 0 discontinuations due to treatment-related AEs).\nAnswer #6\nAs suggested by the reviewer, we have revised the table (Table 2 of the revised manuscript) to specify “discontinuation due to treatment-related adverse events” and added a clarifying note in the table footnote.\nComment #7\nIn the Supplementary Methods, “In vivo study” section, lines 35-36: This sentence states that the mice were “allocated by baseline glucose, HbA1c, and body weight into three groups.” Does this mean that treatment group allocation was designed to achieve balance of baseline glucose, HbA1c, and body weight across groups (e.g., by “matching” mice based on these factors)? If so, I suggest that the following re-wording of this sentence would more clearly explain how this allocation was done:\n“…allocated into three groups (vehicle, tirzepatide, PG-102; n=6 per group), ensuring between-group balance on baseline glucose, HbA1c, and body weight.”\nAnswer #7\nAs suggested by the reviewer, we have revised the wording in the Supplementary Methods (Lines 34-37) to clarify that mice were allocated into treatment groups in a manner that ensured between-group balance with respect to baseline glucose, HbA1c, and body weight.\nComment # 8\nIn the Clinical Supplementary Results, “Exploratory exposure-response relationship between PK exposure and OGTT glucose excursion” section: Be careful not to overstate the relationship identified between PK exposure and OGTT glucose excursion. The R^2, Pearson r, and Spearman ρ are all small in magnitude, and so I would not have much confidence about whether the negative relationship identified represents a real biological relationship vs. is due to random error.\nAnswer #8\nAs suggested by the reviewer, we have revised Lines 167-174 of the Clinical Supplementary Results to avoid overinterpretation of the exploratory exposure–response analysis by explicitly noting the lack of statistical significance, and the limited sample size.\nComment #9.\nIn Extended Data Table 5: Note that the notation AUC_{0-inf} is used in the table, but the notation AUC_{0-∞} is used in the table description. Please revise so that the notation is consistent between the table and the description.\nAnswer #9\nAs suggested by the reviewer, we have revised the table legend of Extended Data Table 5 (Extended Data Table 4 of the revised manuscript) to ensure consistent notation of AUC. The legend has been updated to use “AUC₀– ,” matching the notation used in the table.\ninf\nReviewer #4\nThe revised manuscript comprehensively addresses the original comments from Reviewer 3 and strengthens the scientific rigor of the work. The integration of preclinical and clinical data supporting PG-102 as a bispecific GLP-1/GLP-2 receptor agonist for advanced type 2 diabetes (T2D) is compelling, particularly its ability to decouple glycemic control from weight loss. However, 1 key area require further refinement to enhance mechanistic clarity, generalizability, and translational relevance. Below are specific suggestions for revision:\nComment #1\nMechanistic Link Between Low GLP-2R Potency and In Vivo Efficacy\nPG-102 exhibits only ~3.4% of native GLP-2R potency in vitro (Fig. 1c) but delivers meaningful in vivo effects (e.g., enhanced peripheral glucose uptake, β-cell protection). While the manuscript attributes this discrepancy to “cis-co-engagement” of GLP-1R/GLP-2R, direct evidence linking this receptor trafficking phenotype to downstream GLP-2R-mediated signaling in human cells/tissues is lacking.\nAdd a discussion section explicitly addressing how cis-co-engagement compensates for low GLP-2R potency, citing relevant literature on receptor trafficking and signaling crosstalk.\nAnswer #1\nAs suggested by the reviewer, we have added a dedicated Discussion paragraph addressing how bivalent cis co-engagement of GLP-1R and GLP-2R could reconcile PG-102’s attenuated intrinsic GLP-2R potency (~3.4% of native GLP-2 in vitro; Fig. 1c) with its robust in vivo efficacy. We also acknowledge that direct evidence linking the phenotype to GLP-2R–mediated downstream signaling in human tissues is currently lacking and identify this as an important area for future validation. These revisions have been incorporated into the revised manuscript in the Discussion (Lines 363–387)."}]

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