📚 Part of the TILA-278 Regulatory Dossier — Reader's Guide. This article shows the live document; edits to the source appear here automatically.

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Immunogenicity Summary Report (TILA-278)

Field	Value
Document ID	IMMUNO-001
Version	1.0
Compound	TILA-278 (anti-TL1A antagonist / IL-22R agonist bispecific)
Standard	FDA/EMA immunogenicity
Confidentiality	Confidential

Clinical documentation for the TILA-278 program.

Change History

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

1. Introduction

1.1 Purpose and Scope

This report presents the integrated immunogenicity assessment for TILA-278 (Virtual Biopharma Inc.), a humanised IgG1 bispecific monoclonal antibody in which one binding arm is an antagonist of TL1A (TNFSF15) and the second is an agonist of the interleukin-22 receptor (IL-22R). The data derive from the pivotal Phase 2b induction study TILA278-201 in adults with moderate-to-severe ulcerative colitis (UC). The report describes the tiered anti-drug antibody (ADA) bioanalytical strategy, the observed incidence, kinetics, titres and neutralising character of ADA over the 12-week induction period, and the impact of treatment-emergent immunogenicity on pharmacokinetics (PK), efficacy and safety.

Because TILA-278 is administered subcutaneously (SC) on a chronic-intent regimen and carries a novel receptor-agonist domain, immunogenicity is a defined element of the clinical pharmacology and benefit-risk characterisation. The assessment supports the interpretation of exposure-response relationships and the safety profile, in particular injection-site and hypersensitivity findings.

1.2 Regulatory Basis

The immunogenicity programme was designed and executed in accordance with:

• FDA Guidance for Industry, Immunogenicity Testing of Therapeutic Protein Products — Developing and Validating Assays for Anti-Drug Antibody Detection (January 2019).
• FDA Guidance for Industry, Immunogenicity Assessment for Therapeutic Protein Products (2014).
• EMA Guideline on Immunogenicity Assessment of Therapeutic Proteins (EMEA/CHMP/BMWP/14327/2006 Rev. 1) and Guideline on Immunogenicity Assessment of Monoclonal Antibodies Intended for In Vivo Clinical Use (EMEA/CHMP/BMWP/86289/2010).
• The multi-tiered analytical paradigm and statistical cut-point methodology described in the published bioanalytical consensus literature (Shankar and Devanarayan recommendations), and USP General Chapters <1106> and <1106.1>.

Consistent with ICH S6(R1), no genotoxicity or carcinogenicity concern applies to this monoclonal-antibody modality; the immunogenicity risk is addressed empirically through clinical ADA monitoring rather than nonclinical prediction, since rodent-derived ADA data are not predictive of human immunogenicity.

1.3 Product Attributes Relevant to Immunogenicity

TILA-278 is a recombinant humanised IgG1 bispecific antibody expressed in a Chinese hamster ovary (CHO) cell line and purified by Protein A capture, polishing chromatography and dedicated viral-clearance steps. Drug substance is controlled for aggregate content, charge variants and glycosylation, each of which is a recognised product-related determinant of immunogenic potential. Drug product is a sterile SC solution presented in a single-use prefilled syringe/autoinjector in a polysorbate-containing buffer. Aggregate levels at release and on stability are maintained within specification to minimise the aggregation-driven immunogenicity risk.

2. Immunogenicity Risk Assessment

A pre-study risk assessment was performed to establish the sampling schedule, assay sensitivity/drug-tolerance targets, and the scope of neutralising-antibody (NAb) characterisation. The classification integrates product-, treatment- and patient-related factors.

Table 2-1. Immunogenicity risk assessment.

Domain	Factor	Assessment	Direction of risk
Product	Humanised IgG1 framework	Low intrinsic sequence-derived risk	↓
Product	Bispecific format with novel IL-22R agonist arm	Non-native geometry and agonist epitope may present neo-epitopes	↑
Product	Aggregates / charge / glyco-variants	Controlled by CMC specifications	Neutral (controlled)
Treatment	SC route of administration	Dermal antigen-presenting cell exposure raises risk relative to IV	↑
Treatment	12-week induction, repeat dosing	Sustained antigen exposure	↑
Patient	Moderate-to-severe UC; frequent background immunomodulators/corticosteroids	Concomitant immunosuppression tends to lower measured ADA	↓
Patient	Prior biologic exposure (stratification factor)	Possible cross-reactive priming	↑

Overall classification: moderate immunogenicity risk. This classification mandated (i) a validated tiered ADA assay with sensitivity ≤100 ng/mL and clinically relevant drug tolerance, (ii) domain-resolved NAb assessment against both the anti-TL1A and IL-22R agonist arms, and (iii) prospective evaluation of the impact of ADA on PK, efficacy and safety.

3. Bioanalytical Strategy — Tiered ADA Assay

3.1 Overview and Platform

A validated three-tier binding-ADA scheme (screening → confirmation → titre) followed by neutralising-antibody characterisation was applied. Binding ADA were measured by an electrochemiluminescence (ECL) bridging immunoassay on the Meso Scale Discovery (MSD) platform, using biotinylated TILA-278 as capture and SULFO-TAG–labelled TILA-278 as detection. The bridging format detects IgG and IgM isotypes and is agnostic to which arm the antibody binds. Acid dissociation was incorporated in sample pre-treatment to improve drug tolerance in the presence of circulating TILA-278.

Because TILA-278 is bispecific, the strategy additionally resolves the domain specificity of confirmed ADA (anti-idiotype directed at the anti-TL1A paratope, at the IL-22R agonist paratope, at both, or at framework/linker regions) and the neutralising potential against each functional arm independently (Section 3.5).

3.2 Tier 1 — Screening Assay

The screening tier identifies potentially positive samples. The screening cut point (SCP) was established statistically from drug-naïve UC and healthy-donor serum to yield a nominal 5% false-positive rate (95th percentile of the normalised signal distribution), using a floating cut point applied per plate. Samples with signal at or above the SCP were designated screening-positive (potentially positive) and advanced to confirmation; samples below were reported negative.

3.3 Tier 2 — Confirmatory Assay

Screening-positive samples were confirmed by competitive inhibition (immunodepletion): the sample was pre-incubated with excess unlabelled TILA-278, and signal suppression indicated drug-specific antibody. The confirmatory cut point (percentage signal inhibition) was set to a 1% false-positive rate (99th percentile of inhibition in drug-naïve samples). Samples meeting or exceeding the inhibition threshold were reported confirmed ADA-positive.

3.4 Tier 3 — Titre Assay

Confirmed-positive samples were serially diluted and re-assayed; the titre was reported as the reciprocal of the highest dilution yielding a response at or above the titre cut point. Titres are reported as ordinal titre steps and summarised as low / moderate / high categories.

3.5 Neutralising-Antibody (NAb) Assays — Domain-Resolved

All confirmed ADA-positive samples were characterised for neutralising activity using two orthogonal, arm-specific assays, reflecting the bispecific mechanism (TL1A antagonism plus IL-22R agonism):

• Anti-TL1A arm (antagonist): a competitive ligand-binding (CLB) assay measuring inhibition of TILA-278 binding to immobilised recombinant TL1A. Neutralising ADA reduce the drug–TL1A interaction signal below the NAb cut point.
• IL-22R arm (agonist): a cell-based bioassay in an IL-22R–expressing reporter line measuring TILA-278–driven STAT3 pathway activation; neutralising ADA suppress the agonist-induced reporter signal. A CLB confirmatory format (inhibition of TILA-278 binding to IL-22R) was used for samples with matrix interference in the cell-based readout.

Confirmatory drug-specificity and, where sample volume permitted, immunodepletion with single-arm reagents were used to attribute neutralising activity to the anti-TL1A paratope, the IL-22R paratope, or both. To maintain neutralising-antibody detection in the presence of circulating drug, NAb samples underwent the same acid-dissociation pre-treatment together with a target-capture bead-extraction step, achieving drug tolerance in excess of the highest observed Week-12 trough concentration.

3.6 Assay Validation Summary

Key validation parameters are summarised in Table 3-1; all met pre-defined acceptance criteria under the risk-based validation plan.

Table 3-1. ADA and NAb assay validation summary.

Parameter	Screening (ECL bridging)	Confirmatory	Titre	NAb anti-TL1A (CLB)	NAb IL-22R (cell-based)
Cut point (false-positive target)	5.0% (95th pct)	1.0% (99th pct)	Titre cut point (0.1%)	1.0%	1.0%
Sensitivity (affinity-purified positive control)	22.8 ng/mL	—	—	478 ng/mL	642 ng/mL
Drug tolerance (at 100 ng/mL PC)	25 µg/mL	25 µg/mL	—	20 µg/mL	15 µg/mL
Intra-/inter-assay precision (CV)	<12% / <18%	<12% / <18%	≤1 titre step	<15% / <20%	<15% / <20%
Hook effect	None to 500 µg/mL	—	—	None to 250 µg/mL	None to 250 µg/mL
Selectivity/matrix interference	Acceptable (individual and disease-state sera)	Acceptable	Acceptable	Acceptable	Acceptable

Drug tolerance = highest TILA-278 concentration at which the 100 ng/mL positive control remains detectable. Selectivity was demonstrated in individual and disease-state (UC) sera, including evaluation of rheumatoid factor and disease-associated autoantibody interference. NAb sample pre-treatment (acid dissociation plus target-capture bead extraction) provided drug tolerance exceeding the highest observed Week-12 mean trough concentration (11.8 µg/mL).

The screening sensitivity of 22.8 ng/mL and drug tolerance to 25 µg/mL exceed the ≤100 ng/mL sensitivity expectation and cover observed pre-dose (trough) drug concentrations, supporting reliable ADA detection across the sampling schedule; the NAb assays likewise retained detection above the highest observed trough.

3.7 Sampling Schedule and Analysis Definitions

ADA samples were collected pre-dose at Weeks 0, 4, 8 and 12, and at early termination, aligned with the study visit schedule (Weeks 0, 2, 4, 8, 12). PK trough (Ctrough) samples were collected concurrently to permit exposure–ADA correlation. The following prospectively defined categories were applied:

• Pre-existing ADA: confirmed-positive at baseline (Week 0).
• Treatment-induced ADA: baseline-negative and confirmed-positive at ≥1 post-baseline visit.
• Treatment-boosted ADA: baseline-positive with a post-baseline titre increase of ≥2 titre steps (≥4-fold).
• Treatment-emergent ADA (TE-ADA): treatment-induced or treatment-boosted (the primary incidence metric).
• Persistent TE-ADA: confirmed-positive at ≥2 post-baseline sampling times spanning ≥4 weeks, or positive at the last available sampling time.
• Transient TE-ADA: TE-ADA not meeting the persistent definition (typically a single positive result).
• Neutralising ADA (NAb): confirmed TE-ADA positive with neutralising activity against ≥1 functional arm.

The ADA-evaluable population comprised randomised subjects with a valid baseline result and ≥1 evaluable post-baseline ADA result.

4. Immunogenicity Results

4.1 Analysis Populations and Sample Accountability

Of 900 randomised subjects (299 TILA-278 High, 300 TILA-278 Low, 301 Placebo), the ADA-evaluable population comprised 830 subjects (281 High, 279 Low, 270 Placebo). Post-baseline ADA sample retrieval exceeded 96% at each scheduled visit; the small attrition was consistent with the overall study discontinuation pattern (17/17/29 for High/Low/Placebo) and was non-differential with respect to ADA status.

4.2 ADA Incidence

Table 4-1. Anti-drug antibody incidence over the 12-week induction period (ADA-evaluable population).

Category	TILA-278 High (N=281)	TILA-278 Low (N=279)	Placebo (N=270)
Screening-positive, ever	41 (14.6%)	44 (15.8%)	12 (4.4%)
Confirmed ADA-positive, ever	24 (8.5%)	28 (10.0%)	6 (2.2%)
Pre-existing (baseline) ADA	4 (1.4%)	5 (1.8%)	4 (1.5%)
Treatment-emergent ADA (TE-ADA)	22 (7.8%)	25 (9.0%)	2 (0.7%)
— treatment-induced	20 (7.1%)	23 (8.2%)	2 (0.7%)
— treatment-boosted	2 (0.7%)	2 (0.7%)	0

Treatment-boosted subjects are a subset of the pre-existing (baseline-positive) group; confirmed-positive-ever equals treatment-induced plus pre-existing.

Pooled across active arms, TE-ADA incidence was 47/560 (8.4%). Incidence was not dose-ordered: the numerically higher rate in the Low arm is consistent with the lower circulating drug concentrations reducing assay drug interference and with the expected absence of a strong dose–ADA relationship for a therapeutic mAb. Confirmed positivity in the Placebo arm (2.2%, largely pre-existing/baseline reactivity) reflects background anti-idiotype-like reactivity in the UC population and defines the assay-attributable false-positive floor.

4.3 Onset and Persistence

Median time to first TE-ADA detection was Weeks 4–8, consistent with a primary adaptive response over the induction period. The majority of TE-ADA responses were transient.

Table 4-2. Persistence of treatment-emergent ADA (active arms).

Persistence	TILA-278 High (n=22)	TILA-278 Low (n=25)	Active pooled (n=47)
Transient	16 (72.7%)	18 (72.0%)	34 (72.3%)
Persistent	6 (27.3%)	7 (28.0%)	13 (27.7%)

4.4 ADA Titres

Titres were predominantly low. Median reciprocal titre among TE-ADA-positive subjects was 40 (range 10–1280); high titres were confined to a small persistent subset.

Table 4-3. Titre distribution among TE-ADA-positive subjects (active pooled).

Titre category (reciprocal)	Active pooled TE-ADA (n=47)
Low (<100)	34 (72.3%)
Moderate (100–1000)	11 (23.4%)
High (>1000)	2 (4.3%)

4.5 Neutralising Antibodies (Domain-Resolved)

Of 47 active-arm TE-ADA-positive subjects, 11 (23.4%) had neutralising activity against ≥1 arm; NAb positivity therefore represented 11/560 (2.0%) of the ADA-evaluable active population. Neutralising responses were predominantly low-titre and transient.

Table 4-4. Neutralising-antibody characterisation (active pooled).

NAb characterisation	Active pooled (of 47 TE-ADA+)
NAb-positive (any arm)	11 (23.4%)
— anti-TL1A arm neutralising	8
— IL-22R arm neutralising	5
— both arms neutralising	2
NAb-positive and persistent	5

Domain resolution confirmed that neutralising activity could be directed at either paratope independently; no ADA response selectively abolished one arm while enhancing the other, and no evidence of ADA-mediated agonist potentiation of the IL-22R arm was observed.

4.6 Pre-existing ADA

Baseline (pre-existing) confirmed positivity was low and comparable across arms (1.4%/1.8%/1.5%), indicating no material pre-treatment sensitisation. Boosting of pre-existing responses was infrequent (4 subjects across active arms) and low-titre.

5. Impact of Immunogenicity

5.1 Impact on Pharmacokinetics

TILA-278 PK is dominated by target-mediated drug disposition (TMDD). Week-12 pre-dose Ctrough was compared by TE-ADA status. Low-titre, transient ADA had no discernible effect on exposure; a reduction in trough concentration was confined to the small persistent/NAb-positive subset.

Table 5-1. Week-12 trough concentration by ADA status (active arms).

Group	Week-12 Ctrough, geometric mean (µg/mL)
High — TE-ADA-negative (n=259)	11.8
High — TE-ADA-positive, all (n=22)	9.6
High — persistent and/or NAb-positive subset	4.9
Low — TE-ADA-negative (n=254)	5.4
Low — TE-ADA-positive, all (n=25)	4.6

The ~15–20% lower mean trough in the pooled ADA-positive group, and the more marked reduction in the persistent/NAb-positive subset, are consistent with modestly enhanced clearance in a minority of subjects. Given TMDD-dominated disposition and the low NAb frequency (2.0%), the population-level effect of immunogenicity on TILA-278 exposure over induction is small.

5.2 Impact on Efficacy

Clinical remission (modified Mayo ≤2, no subscore >1) at Week 12 was cross-tabulated by TE-ADA status within the ADA-evaluable population. For reference, remission in the primary FAS analysis was 37.3% (106/284) High, 16.2% (46/283) Low and 0.7% (2/273) Placebo, with LS-mean change in modified Mayo of −3.36 / −2.76 / −1.00 and differences versus placebo of −2.36 (95% CI −2.49, −2.23; p<0.0001) High and −1.77 (95% CI −1.90, −1.64; p<0.0001) Low.

Table 5-2. Week-12 clinical remission by ADA status (ADA-evaluable population).

Group	Remission n/N (%)
High — TE-ADA-negative	100/259 (38.6%)
High — TE-ADA-positive	6/22 (27.3%)
Low — TE-ADA-negative	43/254 (16.9%)
Low — TE-ADA-positive	3/25 (12.0%)

The numerically lower remission rate in ADA-positive subjects is based on very small positive-subgroup counts with wide, overlapping confidence intervals and is driven by the persistent/NAb-positive subset with reduced exposure. There was no meaningful loss of the highly significant, dose-ordered efficacy effect attributable to immunogenicity at the population level over the 12-week induction period.

5.3 Impact on Safety — Injection-Site and Hypersensitivity

Injection-site reactions (ISR) were the principal drug-attributable finding in TILA278-201 and were more frequent on active SC drug than placebo, with no dose-dependence. ISR and hypersensitivity events were examined by TE-ADA status.

Table 5-3. Injection-site and hypersensitivity events by ADA status.

Safety parameter	Active — ADA-negative (n=513)	Active — ADA-positive (n=47)	Placebo (n=270)
Injection-site reactions	38 (7.4%)	4 (8.5%)	6 (2.2%)
Hypersensitivity events (any)	8 (1.6%)	1 (2.1%)	3 (1.1%)
Serious hypersensitivity / anaphylaxis	0	0	0

ISR incidence was essentially identical in ADA-positive and ADA-negative subjects, indicating that ISR are attributable to the SC route/formulation rather than to a systemic anti-drug immune response. Hypersensitivity events were uncommon, mild-to-moderate, non-serious and temporally unrelated to ADA detection; no anaphylaxis, serum-sickness/immune-complex disease, or systemic injection-related reactions occurred. These findings are consistent with the overall safety dataset (≥1 TEAE 109/131/130; SAE 3/0/4; deaths 2/0/1, all assessed as unrelated to study drug; no dose-dependent safety signal).

5.4 Immune-Complex and Cross-Reactivity Considerations

No clinical or laboratory findings suggestive of immune-complex-mediated toxicity were identified. Given the endogenous IL-22R agonist arm, potential cross-reactivity of ADA with endogenous IL-22 signalling was specifically considered; the domain-resolved NAb analysis provided no evidence of ADA that neutralised or potentiated endogenous-pathway signalling beyond neutralisation of the drug itself, and no adverse events consistent with disruption of endogenous IL-22 biology (e.g., mucosal or dermatological deterioration attributable to loss of epithelial-repair signalling) were observed in ADA-positive subjects.

6. Discussion and Conclusions

Over the 12-week induction period in TILA278-201, TILA-278 demonstrated a low-to-moderate, largely transient and low-titre immunogenicity profile characteristic of a humanised IgG1 antibody administered subcutaneously:

• Incidence: TE-ADA occurred in 8.4% of active-treated subjects (7.8% High, 9.0% Low), without dose-ordering. Pre-existing ADA were rare and balanced across arms.
• Character: ~72% of TE-ADA responses were transient and ~72% were low-titre; neutralising antibodies were detected in 2.0% of the ADA-evaluable active population and were resolved by functional arm using domain-specific assays.
• Impact on PK: population exposure was minimally affected; a modest reduction in trough concentration was confined to a small persistent/NAb-positive subset, consistent with TMDD-dominated disposition.
• Impact on efficacy: the robust, highly significant, dose-ordered remission effect was preserved; the small numerical decrement in ADA-positive subjects reflects the low-exposure persistent/NAb-positive minority and is not statistically or clinically material at the population level.
• Impact on safety: injection-site reactions were the main drug-attributable finding but were not associated with ADA status; hypersensitivity events were rare, mild-to-moderate and ADA-independent, with no anaphylaxis or immune-complex disease.

The tiered assay strategy — statistically defined screening, confirmatory and titre tiers with adequate sensitivity (22.8 ng/mL) and drug tolerance (25 µg/mL), complemented by arm-resolved neutralising assays — meets current FDA and EMA expectations and reliably supports these conclusions. These interpretations are appropriately qualified by the small size of the ADA-positive subgroups and the induction-only (12-week) observation window. Overall, immunogenicity does not materially alter the benefit-risk profile of TILA-278 over the induction period. Continued ADA and NAb monitoring, with the same domain-resolved framework, is planned for the maintenance and long-term phases to characterise persistence and any late boosting under chronic dosing.

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Appendix A. Abbreviations

ADA, anti-drug antibody; ANCOVA, analysis of covariance; CHO, Chinese hamster ovary; CI, confidence interval; CLB, competitive ligand binding; Ctrough, pre-dose (trough) concentration; ECL, electrochemiluminescence; FAS, full analysis set; IgG1, immunoglobulin G1; IL-22R, interleukin-22 receptor; ISR, injection-site reaction; LS-mean, least-squares mean; mAb, monoclonal antibody; MSD, Meso Scale Discovery; NAb, neutralising antibody; PC, positive control; PK, pharmacokinetics; RF, rheumatoid factor; SC, subcutaneous; SCP, screening cut point; STAT3, signal transducer and activator of transcription 3; TE-ADA, treatment-emergent anti-drug antibody; TEAE, treatment-emergent adverse event; TL1A/TNFSF15, TNF-like ligand 1A; TMDD, target-mediated drug disposition; UC, ulcerative colitis; USP, United States Pharmacopeia.

Parameters annotated are bioanalytical validation and derived ADA-subgroup values reported to the conventions of the cited FDA and EMA guidances; the primary efficacy and safety results (including the Section 5.2 reference figures) are as reported for study TILA278-201.