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Module 4.2.2 — Pharmacokinetic Study Reports (TILA-278)

Field	Value
Document ID	M4-2.2
Version	1.0
Compound	TILA-278 (anti-TL1A antagonist / IL-22R agonist bispecific)
Standard	ICH M4S / S3
Confidentiality	Confidential

Nonclinical study documentation for TILA-278.

Change History

Version	Date	Author	Summary
1.0	2026-07-08	Nonclinical	Initial issue

4.2.2.0 Introduction and Overview

This section summarizes the nonclinical pharmacokinetic (PK), toxicokinetic (TK), and disposition data supporting the clinical development of TILA-278, a humanized IgG1 bispecific monoclonal antibody comprising an anti–TL1A (TNFSF15) antagonist arm and an IL-22 receptor (IL-22RA1/IL-10RB) agonist arm, administered subcutaneously (SC) for the treatment of moderate-to-severe ulcerative colitis (UC). Sponsor: Virtual Biopharma Inc.

The nonclinical PK/ADME program was designed in accordance with ICH S6(R1) (Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals) and ICH S3A (Toxicokinetics: The Assessment of Systemic Exposure in Toxicity Studies). Consistent with the guidance for monoclonal antibodies, the program focuses on characterization of systemic exposure, absorption after SC dosing, target-mediated disposition, tissue distribution/target localization, and immunogenicity, rather than on classical small-molecule ADME (mass balance, metabolite identification, cytochrome P450 [CYP] phenotyping, transporter interactions, and radiolabeled excretion), which are neither required nor scientifically informative for an intact IgG.

Selection of the pharmacologically relevant species. Both binding arms of TILA-278 are directed against human targets. In vitro binding/potency assessment established that TILA-278 cross-reacts with cynomolgus monkey (Macaca fascicularis) TL1A and IL-22R with affinities/potencies comparable to the human targets (anti–TL1A arm K_D ≈ 0.2 nM human / 0.4 nM cynomolgus; IL-22R agonist arm functional EC_50 within ~2-fold across species), whereas no meaningful binding to mouse or rat orthologues was detected. Rodents are therefore not pharmacologically relevant, and standard rodent PK studies were not conducted. The cynomolgus monkey is the sole pharmacologically relevant species and served as the species for single-dose PK, repeat-dose TK, and safety assessment. Where only one relevant species exists, ICH S6(R1) supports conduct of the pivotal repeat-dose toxicity/TK program in that single species.

Nonclinical PK/TK studies supporting the submission

Study/Report No.	Type	Species / System	Route / Regimen	GLP	CTD location
TILA278-BA-001	Bioanalytical validation — total TILA-278 (LBA)	Cynomolgus serum	—	Yes	4.2.2.1
TILA278-BA-002	Bioanalytical validation — anti-drug antibody (ADA)/NAb	Cynomolgus serum	—	Yes	4.2.2.1
TILA278-PK-001	Single-dose PK	Cynomolgus monkey	IV + SC, single dose	Non-GLP	4.2.2.2
TILA278-TOX-002	4-week repeat-dose toxicity with TK	Cynomolgus monkey	SC, once weekly (QW)	Yes	4.2.2.2 / 4.2.3.2
TILA278-TOX-003	13-week repeat-dose toxicity with TK (pivotal)	Cynomolgus monkey	SC, QW + recovery	Yes	4.2.2.2 / 4.2.3.2
TILA278-TCR-001	Tissue cross-reactivity (IHC)	Human + cynomolgus tissue panels	—	Yes	4.2.2.3 / 4.2.3.7

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4.2.2.1 Analytical Methods and Validation Reports

Serum concentrations of TILA-278 were determined using a validated ligand-binding assay (LBA) on an electrochemiluminescence (ECL, Meso Scale Discovery) platform. To account for the bispecific format, the bioanalytical strategy comprised a quantitative assay for total (intact) TILA-278 and two functional/target-capture assays confirming that both binding arms remained active in circulation:

• Total TILA-278 assay: anti-idiotype capture directed at a framework/hinge epitope common to the molecule, with an anti-human IgG (Fc) detection, quantifying total drug irrespective of target engagement.
• Anti–TL1A arm functional assay: capture with recombinant human TL1A, detection with labeled anti-idiotype, confirming free/active TL1A-binding capacity.
• IL-22R arm functional assay: capture with recombinant IL-22R subunit (IL-22RA1), detection with labeled anti-idiotype, confirming free/active IL-22R-binding capacity.

The total-drug assay was validated for cynomolgus serum with respect to accuracy, precision, selectivity, dilutional linearity, parallelism, hook effect, and matrix interference in accordance with FDA (Bioanalytical Method Validation, 2018) and ICH M10 principles.

Table 4.2.2.1-1. Summary of validated bioanalytical and immunogenicity methods

Assay	Analyte	Matrix	Format	Range (LLOQ–ULOQ)	Inter-assay precision / accuracy
TILA278-BA-001	Total TILA-278	Cynomolgus serum	ECL sandwich LBA	0.100–100 µg/mL	≤12.4% CV / within ±11%
TILA278-BA-001 (functional)	Active anti–TL1A / IL-22R arms	Cynomolgus serum	ECL, target-capture	0.200–100 µg/mL	≤14% CV / within ±13%
TILA278-BA-002	Anti-drug antibodies (ADA)	Cynomolgus serum	Bridging ECL, tiered (screen/confirm/titer)	Sensitivity ~50 ng/mL; drug tolerance ~25 µg/mL at 100 ng/mL positive control	Cut points statistically derived
TILA278-BA-002 (NAb)	Neutralizing antibodies	Cynomolgus serum	Competitive ligand-binding NAb	Sensitivity ~250 ng/mL	—

Immunogenicity was assessed using a tiered strategy (screening → confirmatory → titer) with a competitive-ligand-binding neutralizing-antibody (NAb) assay. Assay cut points were established from drug-naïve cynomolgus serum. Individual toxicity study reports (Module 4.2.3.2) contain the study-specific bioanalytical documentation; full method validation reports are provided under this section.

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4.2.2.2 Absorption

4.2.2.2.1 Single-Dose Pharmacokinetics (Study TILA278-PK-001)

Single-dose PK was characterized in cynomolgus monkeys following a single intravenous (IV) reference dose and single SC doses spanning the anticipated pharmacologic and toxicologic range. IV administration provided reference clearance and volume parameters and enabled estimation of absolute SC bioavailability; the SC dose range was selected to interrogate the transition from target-mediated (nonlinear) to predominantly linear elimination.

Design: Male and female cynomolgus monkeys (n = 3/sex/group) received a single dose of TILA-278: 5 mg/kg IV, or 5, 25, or 150 mg/kg SC (mid-scapular). Serial serum samples were collected through Day 42; total drug was measured by the validated LBA, with concurrent ADA sampling.

Table 4.2.2.2-1. Single-dose PK parameters of TILA-278 in cynomolgus monkey (sex-combined mean)

Parameter	5 mg/kg IV	5 mg/kg SC	25 mg/kg SC	150 mg/kg SC
C_max (µg/mL)	118 (C_0, extrapolated)	12	78	560
T_max (day)	—	3	3	4
AUC_0–∞ (µg·day/mL)	800	520	3120	21 600
t_½,z (day)	8.2	7.8	9.5	11.6
CL or CL/F (mL/day/kg)	6.3 (CL)	9.6	8.0	6.9
V_ss (mL/kg)	68	—	—	—
Absolute bioavailability, F (%)	—	~65	~78	~90 (apparent)

Key findings.

• After SC dosing, absorption was slow, with T_max at approximately 3–4 days, consistent with lymphatic/interstitial uptake of a large IgG.
• Systemic exposure (C_max, AUC) increased in a greater-than-dose-proportional manner across 5→25→150 mg/kg SC (a 30-fold dose increase produced an ~42-fold increase in AUC), and apparent clearance (CL/F) decreased with increasing dose. This nonlinearity is the expected signature of saturable target-mediated drug disposition (TMDD; Section 4.2.2.2.3).
• Terminal half-life increased with dose (~7.8 → 11.6 days), reflecting the diminishing fractional contribution of the high-capacity, saturable target-mediated clearance pathway as dose increases and the emergence of the slower, FcRn-protected linear elimination phase.
• Absolute bioavailability estimated against the single 5 mg/kg IV reference is confounded by dose-dependent (nonlinear) clearance. Because target-mediated clearance is unsaturated at the low IV reference dose, the IV reference AUC is disproportionately low relative to the higher SC doses, so the apparent bioavailability rises artefactually with SC dose (to ~90% at 150 mg/kg). This apparent increase reflects progressive saturation of target-mediated clearance rather than a true change in the absorbed fraction. When the nonlinearity is accounted for by population PK/TMDD modelling of the combined IV and SC data, the true absolute SC bioavailability is estimated at approximately 65–70%, typical for an SC-administered IgG1.

4.2.2.2.2 Repeat-Dose Toxicokinetics (Studies TILA278-TOX-002 and TILA278-TOX-003)

TK was integrated into the GLP repeat-dose toxicity studies (Module 4.2.3.2). The pivotal 13-week study (TILA278-TOX-003) administered TILA-278 by SC injection once weekly at 10, 50, and 150 mg/kg/week, with an 8-week treatment-free recovery period; exposure was characterized on Day 1 and at Week 13 (Day 85).

Table 4.2.2.2-2. Toxicokinetic summary — 13-week SC study TILA278-TOX-003 (sex-combined mean)

Dose (mg/kg/week)	Day	C_max (µg/mL)	AUC_0–168h (µg·day/mL)	Accumulation ratio, R_ac (AUC)
10	1	24	118	—
10	85	41	210	1.8
50	1	135	720	—
50	85	250	1450	2.0
150	1	430	2450	—
150	85	820	4900	2.0

Key findings.

• Exposure increased with dose in an approximately dose-proportional to modestly greater-than-proportional manner across 10–150 mg/kg/week, consistent with partial saturation of target-mediated clearance across this high-dose range.
• Modest accumulation was observed with once-weekly dosing (R_ac ≈ 1.8–2.0). Observed accumulation was somewhat lower than predicted from the terminal half-life alone, consistent with time-varying (target-mediated and, in a minority of animals, ADA-associated) clearance.
• No consistent sex difference in exposure was observed.
• Exposure margins at the no-observed-adverse-effect level (NOAEL) relative to the anticipated clinical SC exposure are presented in the integrated nonclinical overview (Module 2.4) and toxicology written summary (Module 2.6.6); the 13-week study supports the clinical dosing interval and margin assessment.

Comparable exposure and accumulation behavior was observed in the 4-week study (TILA278-TOX-002).

4.2.2.2.3 Nonlinear Kinetics and Target-Mediated Drug Disposition

The disposition of TILA-278 is governed by two parallel elimination pathways characteristic of a therapeutic IgG that engages membrane-associated/soluble targets:

1. Nonspecific (linear) clearance via fluid-phase pinocytosis and proteolytic catabolism, opposed by FcRn-mediated recycling of the intact IgG1 backbone, which confers the long terminal half-life.
2. Target-mediated (saturable, nonlinear) clearance arising from high-affinity engagement of TL1A and IL-22R, followed by target-mediated cellular internalization and lysosomal degradation of the antibody–target complex (antigen sink).

At low concentrations, target-mediated clearance predominates, producing rapid elimination, low bioavailability estimates, and short apparent half-life. As dose/concentration increases, the finite target pool saturates, target-mediated clearance becomes a progressively smaller fraction of total clearance, and the kinetics approach linearity dominated by the FcRn-protected pathway. This mechanism accounts for the greater-than-proportional exposure increases, the dose-dependent decline in CL/F, and the dose-dependent increase in terminal half-life described above. The dual-target format means the antigen sink reflects the combined capacity of both the TL1A and IL-22R pools. These properties informed the target-mediated PK/PD framework and the minimum anticipated biological effect level (MABEL)–based first-in-human dose projection (Section 4.2.2.7).

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4.2.2.3 Distribution

4.2.2.3.1 Volume of Distribution

Consistent with the physicochemical properties of a ~150 kDa IgG1, TILA-278 exhibited a limited volume of distribution. The steady-state volume (V_ss ≈ 68 mL/kg) approximated plasma volume plus a modest interstitial contribution, indicating that distribution is confined largely to the vascular and interstitial compartments, with tissue access governed by convective transport and, at sites of target expression, by receptor-mediated uptake. No classical radiolabeled quantitative tissue-distribution or whole-body autoradiography study was conducted; per ICH S6(R1), such studies are not informative for an unmodified therapeutic antibody, whose biodistribution is driven by target expression and FcRn biology rather than by lipophilic partitioning.

4.2.2.3.2 Tissue Cross-Reactivity (Study TILA278-TCR-001)

A GLP tissue cross-reactivity (TCR) study was conducted by immunohistochemistry using biotinylated TILA-278 (with an isotype-matched control antibody) applied to a full panel of frozen human tissues (FDA-recommended panel of ~32–38 tissues from ≥3 donors) and to the corresponding cynomolgus tissue panel, to characterize on-target localization, screen for unexpected (off-target) binding, and confirm human/cynomolgus concordance in support of species relevance.

The observed staining pattern was consistent with the known distribution of the two targets: IL-22RA1 on epithelial cells of the gastrointestinal tract, skin, respiratory tract, liver, pancreas, and kidney; and TL1A on vascular endothelium, mononuclear/lymphoid cells, and germinal centers. Staining was predominantly membranous and/or cytoplasmic in the expected cell types, with no unexpected binding to tissues lacking target expression.

Table 4.2.2.3-1. Representative tissue cross-reactivity results (biotinylated TILA-278)

Tissue / cell type	Associated target arm	Human	Cynomolgus
GI tract epithelium (colon, small intestine)	IL-22R (agonist arm)	+ (membranous/cytoplasmic)	+
Skin (epidermis)	IL-22R	+	+
Liver (hepatocytes)	IL-22R	+	+
Pancreas (acinar/ductal)	IL-22R	+	+
Lung (bronchial epithelium)	IL-22R	+	+
Kidney (tubular epithelium)	IL-22R	+	+
Lymphoid tissue (germinal centers, mononuclear cells)	TL1A (antagonist arm)	+	+
Vascular endothelium (multiple tissues)	TL1A	+	+
Tissues without target expression (e.g., cardiac myocyte, CNS neuropil)	—	No specific staining	No specific staining

Interpretation. The staining pattern was concordant between human and cynomolgus tissues, reinforcing the cynomolgus monkey as the pharmacologically and toxicologically relevant species and predicting on-target localization to the gastrointestinal/epithelial (mucosal-repair, IL-22R) and lymphoid/endothelial (anti-inflammatory, TL1A) compartments relevant to the UC indication. No off-target binding of toxicological concern was identified. Consistent with regulatory points-to-consider, TCR findings are complementary and hypothesis-generating: in vitro tissue binding does not by itself establish in vivo accessibility or toxicity, and the results are interpreted alongside the in vivo repeat-dose toxicology (Module 4.2.3.2).

4.2.2.3.3 Plasma Protein Binding and Placental Transfer

Plasma protein binding studies are not applicable to a therapeutic antibody, which is itself a plasma protein and does not partition via conventional protein-binding equilibria. No dedicated nonclinical placental-transfer study was conducted; IgG1 crosses the placenta via FcRn in a gestational-age-dependent manner (predominantly late gestation), and this class property is addressed in the reproductive/developmental toxicology assessment and in labeling considerations rather than by a stand-alone distribution study.

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4.2.2.4 Metabolism

No metabolism (biotransformation) studies were conducted, and none are warranted for TILA-278 in accordance with ICH S6(R1). As an intact IgG1, TILA-278 is not a substrate for cytochrome P450 enzymes or phase II conjugation. Its expected metabolic fate is proteolytic catabolism to small peptides and constituent amino acids, which are recycled into the endogenous amino-acid pool. Catabolism occurs by two routes: (i) nonspecific proteolysis following fluid-phase pinocytosis into cells of the reticuloendothelial system and vascular endothelium, opposed by FcRn-mediated salvage of intact antibody, and (ii) target-mediated internalization of antibody–target complexes with subsequent lysosomal degradation. No pharmacologically active or toxicologically relevant metabolites are anticipated, and metabolite identification/profiling (e.g., by mass spectrometry) is not scientifically meaningful for this modality. This position is consistent with ICH S6(R1) and with FDA and EMA guidance on therapeutic proteins.

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4.2.2.5 Excretion

No excretion (mass-balance) studies were conducted, and none are warranted, consistent with ICH S6(R1). Intact IgG of ~150 kDa is not eliminated by renal filtration (well above the glomerular molecular-weight cutoff) or by biliary excretion; elimination proceeds via intracellular catabolism to amino acids (Section 4.2.2.4), which re-enter normal anabolic and catabolic pathways. A radiolabeled excretion-balance study would recover label as recycled amino acids and endogenous protein rather than as intact drug or drug-derived metabolites, and would provide no interpretable disposition information. The waiver of quantitative excretion studies is therefore scientifically justified.

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4.2.2.6 Pharmacokinetic Drug Interactions (Nonclinical)

No dedicated nonclinical small-molecule drug–drug interaction (DDI) studies were conducted, consistent with ICH S6(R1) and with FDA (In Vitro Drug Interaction Studies, 2020; Clinical Drug Interaction Studies, 2020) and EMA guidance. As a therapeutic antibody, TILA-278 does not interact directly with CYP450 enzymes, UGTs, or membrane drug transporters, and in vitro CYP inhibition/induction and transporter substrate/inhibitor assays are neither applicable nor predictive for this modality.

The scientifically relevant DDI consideration for TILA-278 is the cytokine-modulation (therapeutic protein–drug interaction) pathway. Chronic inflammation and elevated proinflammatory cytokines (e.g., IL-6, TNF) can suppress hepatic CYP450 expression; agents that normalize the inflammatory milieu may partially reverse this suppression and thereby alter the exposure of concomitant CYP substrates, particularly those with a narrow therapeutic index. For TILA-278, the anti–TL1A arm dampens TH1/TH17-driven inflammation, and the IL-22R agonist arm acts on hepatocytes and epithelial cells (IL-22RA1) to drive STAT3-dependent regenerative and acute-phase responses; both mechanisms have a plausible, though indirect and generally modest, potential to modulate the inflammatory/hepatic environment. Nonclinical (in vitro or animal) models are recognized as poorly predictive of this cytokine-mediated interaction in humans; accordingly, a nonclinical DDI study was not performed. The potential for a cytokine-mediated effect on CYP substrates is instead evaluated within the clinical pharmacology program (Module 5) and, as warranted, addressed through population-PK analysis and product labeling. No direct pharmacokinetic drug-interaction liability is expected for this bispecific antibody.

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4.2.2.7 Other Pharmacokinetic Studies

4.2.2.7.1 Impact of Immunogenicity on Toxicokinetics

Anti-drug antibodies were monitored in all repeat-dose studies using the validated tiered assay (Section 4.2.2.1). A treatment-emergent ADA response was detected in a subset of cynomolgus monkeys, with incidence and measurable impact inversely related to dose owing to high-dose drug tolerance: ADA-positive animals were observed predominantly in the low- and mid-dose groups, whereas exposure was generally maintained at the high dose. In the minority of ADA-positive animals, ADA was associated with increased clearance and reduced trough exposure over the dosing period, contributing to inter-animal variability and to the lower-than-predicted accumulation ratios (Section 4.2.2.2.2). ADA in cynomolgus monkeys reflects the expected immune response of a non-tolerant species to a humanized protein and is not predictive of human immunogenicity; these data are used to interpret nonclinical TK and exposure margins rather than to project clinical immunogenicity, which is assessed in the clinical program.

4.2.2.7.2 Interspecies Considerations and Human Dose Projection

The nonclinical PK dataset, together with the target-mediated PK/PD framework, supported first-in-human dose selection. Because the pharmacology is target-driven and nonlinear—and because one arm is a receptor agonist—the first-in-human starting dose was derived using a MABEL approach that integrated in vitro binding/potency, receptor-occupancy modeling for both arms, and the cynomolgus PK/TMDD characterization, rather than by simple allometric scaling of clearance alone. Human clearance and half-life were projected from the cynomolgus linear-phase parameters using allometric principles appropriate for IgG (with FcRn-conserved recycling), and the anticipated SC bioavailability was informed by the cynomolgus estimate of ~65–70%. Detailed human PK, exposure–response, and immunogenicity analyses are provided in Module 5 (Clinical Pharmacology).

4.2.2.7.3 Cardiovascular and QT Assessment

Consistent with ICH S7B and E14 (and the associated Q&A on their applicability to biotechnology-derived products), no stand-alone in vitro hERG or dedicated cardiovascular safety-pharmacology study was conducted; core cardiovascular, respiratory, and central-nervous-system safety-pharmacology endpoints were instead integrated into the repeat-dose toxicity program (TILA278-TOX-003) in accordance with ICH S6(R1). A large, target-specific IgG that does not distribute intracellularly and has no interaction with cardiac ion channels carries no direct proarrhythmic (hERG) liability; on this basis a dedicated clinical thorough QT/QTc (TQT) study is likewise not warranted, as addressed in the clinical pharmacology program (Module 5). The waiver of a dedicated QT assessment is scientifically justified for this modality.

4.2.2.7.4 Integrated Conclusions

The nonclinical PK/ADME characterization of TILA-278 is complete and appropriate for a humanized IgG1 bispecific antibody under ICH S6(R1):

• SC absorption in cynomolgus monkeys is slow (T_max ~3–4 days) with true absolute bioavailability of ~65–70% after correction for nonlinear clearance.
• Disposition is dominated by parallel FcRn-protected linear clearance and saturable target-mediated (dual-target) clearance, producing the expected nonlinear, dose-dependent exposure, half-life, and clearance behavior.
• Distribution is confined to the vascular/interstitial space; tissue cross-reactivity is on-target, concordant between human and cynomolgus tissues, and free of off-target findings of concern.
• Metabolism (catabolism to amino acids) and excretion follow standard IgG biology, and dedicated metabolite/excretion studies are appropriately waived.
• No direct small-molecule DDI liability exists; the indirect cytokine-modulation pathway is addressed clinically.
• Immunogenicity in the non-tolerant animal species affected exposure in a dose-dependent, drug-tolerance-limited manner and is not predictive of human immunogenicity.

These data, together with the repeat-dose toxicology (Module 4.2.3.2) and the integrated summaries (Modules 2.4 and 2.6.4/2.6.5), support the clinical use of subcutaneous TILA-278 in patients with moderate-to-severe ulcerative colitis.