Ubi Titer Issue #3
Why Target Expression Fails to Predict ADC Response
Target expression doesn't predict ADC response — two independent papers point to payload sensitivity as the real driver
- ADC biomarkers
- payload sensitivity
- circulating tumor cells
- biparatopic antibodies
- afucosylation
What this issue covers
- 1.Dynamic monitoring of antibody drug conjugates targeting TROP2 or HER2 in breast cancer using circulating tumor cells
Prospective cohort (n=35): baseline TROP2/HER2 expression on CTCs does not predict sacituzumab govitecan or T-DXd response. >80% CTC reduction at week 3 does (TROP2: HR 5.15; HER2: HR 6.01). Cross-ADC switching (same TOP1i payload class) rarely produces second-line response — implicating payload resistance.
- 2.Telisotuzumab Adizutecan (ABBV-400) in Gastric/Gastroesophageal Junction Adenocarcinoma
ABBV-400 (c-Met ADC, TOP1i payload, DAR 8) in 42 GEA patients: ORR 29%, CBR 71%, DOR 4.2 mo, PFS 4.0 mo, OS 5.8 mo. Responses in c-Met-low patients. Grade ≥3 TEAEs 88%. Independent clinical signal that payload sensitivity — not c-Met expression — drives response.
- 3.Biparatopic targeting of IL-23 enables dual-epitope engagement and enhanced neutralization potency
Ustekinumab + guselkumab variable regions combined into biparatopic 1+1 IgG and 2+2 Fab-IgG formats. Picomolar SPR affinities with avidity enhancement; dual-epitope engagement confirmed by bridging SPR and mass photometry. Functional neutralization equivalent to equimolar antibody mixture — a clean engineering framework without synergistic potency gain.
- 4.miRNA-Mediated FUT8 Knockdown Enables Complete Afucosylation of Therapeutic Antibodies in CHO Without Titer Loss
RNA Pol II miRNA targeting FUT8 in CHO: >90% afucosylation (HILIC-HPLC), no fucosylated product by intact MS, no titer loss. Beats shRNA (U6 promoter) which causes Dicer saturation → residual fucosylation + reduced titers. Clean solution to the afucosylation-vs-yield tradeoff in antibody manufacturing.
Paper 1 · PNAS
Dynamic monitoring of antibody drug conjugates targeting TROP2 or HER2 in breast cancer using circulating tumor cells
Prospective cohort (n=35): baseline TROP2/HER2 expression on CTCs does not predict sacituzumab govitecan or T-DXd response. >80% CTC reduction at week 3 does (TROP2: HR 5.15; HER2: HR 6.01). Cross-ADC switching (same TOP1i payload class) rarely produces second-line response — implicating payload resistance.
Core finding
In 35 metastatic breast cancer patients treated with sacituzumab govitecan (TROP2) or trastuzumab deruxtecan (HER2), baseline TROP2 and HER2 expression on CTCs was highly heterogeneous even within individual patients and did not predict depth of clinical response. By contrast, absence of CTCs or >80% CTC count reduction at three weeks of treatment strongly predicted durable benefit — HR 5.15 (p=0.012) for TROP2 ADC and HR 6.01 (p<0.001) for HER2 ADC. At acquired resistance, TROP2/HER2 expression was maintained on CTCs (not downregulated), and switching between TROP2 and HER2 ADCs sharing TOP1i payloads rarely yielded second-line responses.
What is novel
First prospective clinical study demonstrating that on-treatment CTC burden change at three weeks, not pre-treatment receptor expression, is the functionally predictive ADC biomarker. The cross-ADC switching failure data — both agents use TOP1i payloads — provides direct evidence that acquired resistance operates at the payload level rather than the target level.
Limitations
Small n=35 single-centre cohort at MGH. Both ADCs tested use topoisomerase I inhibitor payloads, so findings may not generalise to MMAE, MMAF, or other payload classes. CTC capture relies on MGH's proprietary microfluidics platform. Follow-up duration not uniformly reported.
Why it matters in context
Patient selection for ADCs has historically relied on target expression (HER2 IHC/FISH for T-DXd, TROP2 expression for SG). This study challenges that directly, showing receptor heterogeneity makes pre-treatment expression a poor predictor while pharmacodynamic response (CTC clearance) is informative. Two clinical trials now specifically studying CTC-based monitoring for ADC response are underway. Together with the ABBV-400 GEA data, this week produced two independent clinical datasets pointing to payload sensitivity as the dominant mechanism.
Paper 2 · Clinical Cancer Research
Telisotuzumab Adizutecan (ABBV-400) in Gastric/Gastroesophageal Junction Adenocarcinoma
ABBV-400 (c-Met ADC, TOP1i payload, DAR 8) in 42 GEA patients: ORR 29%, CBR 71%, DOR 4.2 mo, PFS 4.0 mo, OS 5.8 mo. Responses in c-Met-low patients. Grade ≥3 TEAEs 88%. Independent clinical signal that payload sensitivity — not c-Met expression — drives response.
Core finding
ABBV-400 (telisotuzumab adizutecan — anti-c-Met IgG1 + adizutecan TOP1i payload, DAR 8, 3 mg/kg Q3W) achieved ORR 29%, clinical benefit rate 71%, median DOR 4.2 months, PFS 4.0 months, OS 5.8 months in 42 heavily pre-treated GEA patients (data cutoff September 25, 2024). Critically, responses occurred in c-Met-low patients (IHC score <25%), decoupling efficacy from target receptor expression level. Grade ≥3 TEAEs were seen in 88% of patients; anemia in 67%, nausea in 52%.
What is novel
Demonstration of anti-tumor activity in c-Met-low GEA patients by a c-Met-targeted ADC challenges the receptor-expression-as-predictor paradigm for this target, consistent with the contemporaneous PNAS CTC paper showing the same in TROP2/HER2 breast cancer ADCs. Taken together, these independent datasets suggest payload class resistance — not target loss or low expression — is the operative mechanism for TOP1i ADC response and resistance.
Limitations
Phase I/Ib dose-expansion; heavily pre-treated, performance-status-selected population; no comparator arm; c-Met IHC cut-off for 'low' is not standardized; 88% Grade ≥3 TEAE rate is substantial. Short follow-up.
Why it matters in context
GEA has few approved second-line options and c-Met amplification or overexpression occurs in 15-20% of cases. ABBV-400 is also being developed in EGFR-mutant NSCLC (Phase II results 2025). The TOP1i payload class (DXd, adizutecan, SN-38) now dominates ADC development and payload-class resistance is becoming a clinical reality that trial design and biomarker strategy need to address.
Paper 3 · mAbs
Biparatopic targeting of IL-23 enables dual-epitope engagement and enhanced neutralization potency
Ustekinumab + guselkumab variable regions combined into biparatopic 1+1 IgG and 2+2 Fab-IgG formats. Picomolar SPR affinities with avidity enhancement; dual-epitope engagement confirmed by bridging SPR and mass photometry. Functional neutralization equivalent to equimolar antibody mixture — a clean engineering framework without synergistic potency gain.
Core finding
Variable regions from the clinically validated IL-23/IL-12 dual inhibitor ustekinumab (anti-p40) and the IL-23-specific inhibitor guselkumab (anti-p19) were combined into biparatopic constructs using heterodimer-promoting C3 mutations and redox-repair half-antibody assembly in 1+1 IgG-like and 2+2 Fab-IgG extended formats. SPR kinetics confirmed picomolar affinities with avidity-driven enhancement. A dual-engagement bridging SPR assay and mass photometry both validated simultaneous IL-23A and IL-12B binding and stoichiometry-dependent multimerisation. A two-parameter mechanistic model accurately predicted the EC shifts seen across formats in an IL-23 luciferase reporter assay. Functional neutralization was equivalent to equimolar mixtures of the parent antibodies.
What is novel
First systematic application of biparatopic engineering to a soluble cytokine target using clinically validated variable regions. The 2+2 Fab-IgG extended format is a distinctive biparatopic architecture. The two-parameter mechanistic neutralization model is a new analytical tool for predicting potency in soluble cytokine-targeting biparatopics. Mass photometry for biparatopic immune complex stoichiometry characterisation is an emerging application.
Limitations
Functional potency is equivalent to, not greater than, equimolar monospecific mixtures — no synergistic advantage. Assembly uses heterodimer-promoting C3 mutations and redox repair, which may complicate manufacturing. No in vivo data.
Why it matters in context
Biparatopic antibodies against membrane-bound targets (HER2, DR5, EGFR) are well established and often show clustering-driven efficacy gains. Extension to soluble cytokines is less explored. IL-23 is one of the most validated immunology targets with five approved antibodies; using existing VRs in a biparatopic format offers a de-risked path to combination IL-23/IL-12 blockade. The paper's honest reporting that potency matches the mixture — rather than exceeding it — is scientifically important even without a functional upside.
Paper 4 · mAbs
miRNA-Mediated FUT8 Knockdown Enables Complete Afucosylation of Therapeutic Antibodies in CHO Without Titer Loss
RNA Pol II miRNA targeting FUT8 in CHO: >90% afucosylation (HILIC-HPLC), no fucosylated product by intact MS, no titer loss. Beats shRNA (U6 promoter) which causes Dicer saturation → residual fucosylation + reduced titers. Clean solution to the afucosylation-vs-yield tradeoff in antibody manufacturing.
Core finding
An RNA Pol II promoter-driven miRNA construct targeting FUT8 in CHO cells achieves >90% antibody afucosylation by HILIC-HPLC with no detectable fucosylated product by intact mass spectrometry, maintaining mAb production titers equivalent to unmodified CHO. In head-to-head comparison, U6 promoter-driven shRNA targeting FUT8 produced higher residual fucosylation and reduced mAb titers, attributable to Dicer saturation by the exogenous shRNA. Afucosylated antibodies from the miRNA system showed enhanced ADCC in functional assays.
What is novel
Direct head-to-head comparison showing RNA Pol II miRNA avoids Dicer saturation that limits U6-shRNA, achieving simultaneously higher afucosylation completeness and preserved titer. Use of intact mass spectrometry as a zero-fucosylation detection method (more sensitive than HILIC-HPLC alone) strengthens the complete-afucosylation claim. First system reported achieving both benchmarks simultaneously in CHO.
Limitations
CHO cell line-specific; long-term transgene stability across manufacturing-scale passages not reported; no clinical validation; ADCC enhancement is in vitro only.
Why it matters in context
Afucosylated antibodies have 5-100× enhanced ADCC via improved FcγRIIIa binding. Approved examples: obinutuzumab, mogamulizumab. Kyowa Kirin's POTELLIGENT (FUT8 KO) is the commercial benchmark. Prior shRNA approaches have long suffered from Dicer saturation (documented in RNAi literature since ~2010). This paper applies that insight directly to manufacturing, from IGI Therapeutics (ETH Zurich spin-out).
Primary papers
- [1] Dynamic monitoring of antibody drug conjugates targeting TROP2 or HER2 in breast cancer using circulating tumor cells (PNAS)
- [2] Telisotuzumab Adizutecan (ABBV-400) in Gastric/Gastroesophageal Junction Adenocarcinoma (Clinical Cancer Research)
- [3] Biparatopic targeting of IL-23 enables dual-epitope engagement and enhanced neutralization potency (mAbs)
- [4] miRNA-Mediated FUT8 Knockdown Enables Complete Afucosylation of Therapeutic Antibodies in CHO Without Titer Loss (mAbs)