# Supplementary Information

**Manuscript:** Steering into competitive whitespace — a public-data, computation-driven campaign nominates GUCY2C and DKK1 and delivers de novo binder leads across the esophageal adenocarcinoma trajectory

*All results are computational (in-silico). No experimental validation has been performed. Every value below is reproduced from the upstream program artifacts named in each section.*

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## Table S1 — Complete de novo binder design set (all 16 designs)
Source: `design_leads.csv`. Pipeline: RFdiffusion (Complex_base, noise 0) -> ProteinMPNN (v_48_020, binder chain, 8 seq/backbone, T=0.1) -> Boltz-2 fold-back (target `--use_msa_server`, binder single-sequence, 5 diffusion samples). `iptm` = interface predicted TM (pass > 0.5); `complex_plddt` = fold confidence (>0.7). Sequences are the ProteinMPNN binder-chain designs.

| design | target | binder_len | mpnn_score | iptm | complex_plddt | confidence | pass | seq |
|---|---|---|---|---|---|---|---|---|
| dkk1_bb1 | DKK1 | 63 | 1.232 | 0.858 | 0.908 | 0.898 | Y | SSEEKEKLAKEYEEKAKKAEELAKQAKEKAKISSPEDKPGYEALAKEAEKGAKEYKEKAEKLK |
| dkk1_bb4 | DKK1 | 63 | 1.248 | 0.844 | 0.846 | 0.846 | Y | STAEGLRKGREELLKKAEELRAKAEKLEAEAKTEEQKKLAEEYRAAAEHAEAGAKALEARLAA |
| dkk1_bb5 | DKK1 | 60 | 1.275 | 0.821 | 0.777 | 0.785 | Y | AAAAAAAAAAERAAAAANSEEIAARIKAEETARAYALNPALAALVAAGCEATLAEARARL |
| dkk1_bb2 | DKK1 | 62 | 1.044 | 0.745 | 0.743 | 0.743 | Y | MTREELIAAAGAAGLAYGAALTAALAAAAAAAGADTAAVLALGAAGAAAAAALAAAAAAAAA |
| dkk1_bb6 | DKK1 | 89 | 1.315 | 0.743 | 0.837 | 0.818 | Y | ALAVLLLALLAAALTALLLGACTAKVAELAAERERYRALAEAEKDNPELKAELLAKAEEADKAAAEARAAGRAALELEAAVRAAAAALL |
| dkk1_bb7 | DKK1 | 85 | 1.172 | 0.709 | 0.899 | 0.861 | Y | MEKKKKAEELLKLAEEYLKKAEEVAREAGERFAELEAAAAASAANPERQAALLEEARRVAAEAAKKVEELKKKAEEARKKAEELL |
| dkk1_bb3 | DKK1 | 76 | 1.008 | 0.571 | 0.88 | 0.818 | Y | SAALAAAAAAAGLAALAVAAAGTAAMTALRAAAAAAASDPALQAAYLAAAAAVRAATSAAVAALRAAAAALRAAAA |
| dkk1_bb0 | DKK1 | 89 | 1.189 | 0.524 | 0.887 | 0.815 | Y | EELEKKKKEAEEKLKKAFEETGKKLGEYLKKYEEYRKKAEEALKKGDLEEAKKYIEKAKEAAKKGEEELKKLKEVKEEYEKIKKEIEEK |
| gucy2c_bb2 | GUCY2C | 71 | 0.95 | 0.915 | 0.896 | 0.9 | Y | TPEDIFAAGKAAADAAAGDVAALEALRNEYIEKAAKSTPELANAYGKAASYAGLVAAQARAAEAAAAAAAA |
| gucy2c_bb5 | GUCY2C | 80 | 0.834 | 0.861 | 0.863 | 0.863 | Y | AELAALAAEAEALAAAVLAADPAAAGAAEALLAAARALREGDTDAAIAALNEAAAYLEALGLTDFAAQARALAARVKAAA |
| gucy2c_bb4 | GUCY2C | 90 | 0.872 | 0.811 | 0.878 | 0.865 | Y | MKEQAKKALEAAKKAAEEALKAAKKAIELAKEGKMEEAKKELEKAKAYENAVRGLAEALEELDKEEAKEAKKLAEKAAKAVKEAQKLLEA |
| gucy2c_bb1 | GUCY2C | 73 | 1.065 | 0.715 | 0.878 | 0.845 | Y | AEEELKKKLEEEAKKKEEEAKKLGTEAYKLSEEADKALEESEEKYKEVAKKLSEVNKKAGEALGEAQKLEKEL |
| gucy2c_bb3 | GUCY2C | 84 | 0.842 | 0.619 | 0.865 | 0.816 | Y | AEEKAKEKIEKALELAKKIAEKTKLGEGIVKLLELAKEGDKETRLEALRAVEGGARVCAELYPEVKELAEELAELAREAAKLVE |
| gucy2c_bb0 | GUCY2C | 87 | 0.909 | 0.536 | 0.865 | 0.8 | Y | ELEEAGKKAEELMEKIKKELKEAKEEWAEGALNWTKGEAAANPENVEGYAENARKRLEEAIKSEEEAKRLIGAGKGGKRTYEGWLKK |
| gucy2c_bb7 | GUCY2C | 81 | 0.881 | 0.521 | 0.862 | 0.793 | Y | REAVAKRIEEALKKAEAGDAKAAITAAAEGAGQCLQLGLEKVAKLLNEALAKAAELALAGKEEEAKKVAVEALKKALKEVK |
| gucy2c_bb6 | GUCY2C | 72 | 1.005 | 0.401 | 0.852 | 0.762 | n | SEEVEKKKEELLKEADEYLKKSGEEINKIEEEADKAEKENKEKAKELYDKGSKKIKETGKKVKEMREKAEKL |

**Leads:** gucy2c_bb2 (treatment arm; ipTM 0.915) and dkk1_bb1 (interception arm; ipTM 0.858). 15/16 designs pass the ipTM>0.5 interface line (only gucy2c_bb6 fails at 0.401).

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## Table S2 — Target nomination and honest druggability triage (16 candidates)
Source: `target_nomination.csv`. Lane: T = treatment (surface antigens), I = interception (Barrett's->dysplasia). `composite` = raw nomination score (association + selectivity + tractability + novelty). `biologic_druggability` is the explicit filter applied over the raw score: STRONG/GOOD/MODERATE = addressable by an engineered biologic; CROWDED = deprioritized under the novelty steer; WEAK-BIOMARKER / NOT-A-BINDER-TARGET = down-weighted. The highest raw composite (GPX7, 13.86) is a lost tumor suppressor and is *not* carried forward.

| target | lane | composite | tumor_selectivity | novelty | biologic_druggability | trajectory | druggability_note |
|---|---|---|---|---|---|---|---|
| GPX7 | I | 13.86 | 4 | 5 | NOT-A-BINDER-TARGET | Barrett's (early) | LOST tumor suppressor -> restoration/synthetic-lethal problem, NOT antibody-addressable |
| GUCY2C | T | 13.0 | 5 | 5 | STRONG | advanced + adjuvant | Surface, luminal-restricted; antibody/ADC/bispecific/CAR all viable; therapeutic window is the key asset |
| REG4 | I | 12.47 | 4 | 5 | WEAK-BIOMARKER | Barrett's->dysplasia | Secreted lectin; trap-able BUT therapeutic efficacy unproven; mainly progression biomarker |
| CDH17 | T | 12.38 | 4 | 5 | STRONG | advanced | GI-restricted surface cadherin; antibody/ADC/CAR viable; emerging validation |
| ERBB2 | T | 12.29 | 4 | 1 | CROWDED | advanced (crowded) | Deprioritized per steer (T-DXd approved) |
| CEACAM5 | T | 12.0 | 3 | 4 | STRONG | advanced | Surface; ADC precedent (tusamitamab); moderate window |
| CD276 | T | 12.0 | 3 | 5 | STRONG | advanced | B7-H3 surface; ADC clinical-stage; antibody-tractable but broader normal expression |
| MET | T | 11.55 | 3 | 2 | GOOD | advanced | Surface RTK; antibody/ADC viable; lower amp frequency |
| ERBB3 | T | 11.49 | 3 | 3 | GOOD | advanced | HER3 surface receptor; antibody/bispecific viable; less crowded than HER2 |
| DKK1 | I | 11.37 | 3 | 4 | GOOD | advanced + progression | Secreted -> ligand-trap/neutralizing antibody viable; DKN-01 precedent |
| TFF3 | I | 10.51 | 3 | 4 | WEAK-BIOMARKER | Barrett's | Secreted trefoil; strong Cytosponge biomarker; not a validated therapeutic target |
| OLFM4 | I | 10.46 | 3 | 4 | WEAK-BIOMARKER | Barrett's->dysplasia | Secreted; interception biomarker; therapeutic role unproven |
| TACSTD2 | T | 10.0 | 3 | 4 | GOOD | advanced | TROP2 surface; ADC-tractable; pan-epithelial (window caution) |
| MUC1 | T | 10.0 | 3 | 3 | MODERATE | advanced | Tumor-glyco-MUC1 epitope needed; complex but antibody-addressable |
| EGFR | T | 9.88 | 2 | 1 | CROWDED/POOR-WINDOW | advanced (crowded) | Deprioritized; broad normal expression |
| CLDN18 | T | 9.36 | 4 | 1 | CROWDED | advanced (crowded) | Deprioritized per steer (zolbetuximab approved) |

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## Table S3 — Competitive landscape (EAC target axes)
Source: `competitive_landscape.csv`. Crowding is the qualitative active-EAC-trial density; approved/lead anchors confirmed against Drugs@FDA. Whitespace verdict drove the novelty steer.

| Target/axis | Mechanism | Approved/lead anchor | Crowding (active EAC) | Trajectory position | Whitespace verdict |
|---|---|---|---|---|---|
| PD-1 / PD-L1 | Immune checkpoint | pembrolizumab, nivolumab (approved, incl. adjuvant CheckMate-577) | VERY HIGH (41 active mentions) | Advanced + adjuvant | CROWDED — backbone, combination-only entry |
| HER2 / ERBB2 | Amplified RTK | trastuzumab, T-DXd (approved GEJ/gastric) | HIGH (12) | Advanced (HER2+ ~15%) | CROWDED — but ADC/bispecific still active |
| VEGF / VEGFR2 | Angiogenesis | ramucirumab (approved gastric/GEJ) | MED (8) | Advanced | MATURE — incremental |
| Claudin-18.2 | Tight-junction surface Ag | zolbetuximab (approved 2024, CLDN18.2+ G/GEJ) | MED (3, fast-growing) | Advanced | RECENTLY VALIDATED — ADC/CAR-T next wave |
| FGFR2b | Amplified RTK | bemarituzumab (Ph3) | LOW (2) | Advanced (FGFR2b+ subset) | EMERGING — near-term |
| TIGIT | Immune checkpoint | domvanalimab (Ph2/3) | LOW (1-2) | Advanced | EMERGING IO — crowded pan-tumor |
| B7-H3 (CD276) | Surface Ag / ADC | ifinatamab deruxtecan (Ph1/2) | VERY LOW (1) | Advanced | WHITESPACE — novel surface ADC target |
| TROP2 | Surface Ag / ADC | sacituzumab tirumotecan (Ph1/2) | VERY LOW (1) | Advanced | WHITESPACE — ADC expanding into GI |
| GUCY2C | GI-restricted surface Ag | Ad5-GUCY2C-PADRE vaccine (Ph1/2) | VERY LOW (1) | Advanced/adjuvant | WHITESPACE — GI-selective, vaccine/CAR/ADC |
| CD73 / adenosine | Immunosuppressive axis | quemliclustat (Ph1) | VERY LOW (1) | Advanced | WHITESPACE — TME immuno-metabolic |
| DKK1 | Wnt modulator (secreted) | DKN-01 (Ph2) | VERY LOW (1) | Advanced | WHITESPACE — secreted, low-DKK1 vs high |
| GATA6 / MYC / CCNE1 amplicons | Amplified TFs / cell-cycle | none (undrugged TFs) | NONE | All | HARD — transcription-factor/undruggable |
| TP53 / CDKN2A loss | Tumor-suppressor loss | none directly | NONE | Barrett's->EAC (early) | HARD — synthetic-lethal / interception only |
| Barrett's/dysplasia interception | Progression biology | none approved (chemoprevention: PPI+aspirin AspECT) | LOW (203 trials, mostly ablation/screening) | Precursor | WHITESPACE — molecular interception unexploited |

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## S4 — Detailed computational methods

### Target structure preparation
- **GUCY2C:** AlphaFold DB UniProt P25092 (model v6). Extracellular domain isolated (residues 24-430; pLDDT>70 across ~50-425). Intracellular kinase/cyclase domain (~490-1073) excluded.
- **DKK1:** AlphaFold DB UniProt O94907 (model v6). CRD2 domain isolated (residues 178-256), the LRP6-binding functional region; disordered N-terminus and linkers excluded.
- **Hotspot definition (design input):** surface-exposed, high-confidence residue patches by neighbor-count + pLDDT heuristic - GUCY2C ECD [239,240,267,422,424,425]; DKK1 CRD2 [181,182,183,194,195] (isolated-domain numbering).

### Design and validation
- **RFdiffusion:** Complex_base checkpoint, noise 0, 8 backbones/target, binder length 70-100 aa (GUCY2C) / 60-90 aa (DKK1).
- **ProteinMPNN:** v_48_020, binder chain only, 8 sequences/backbone, sampling T=0.1; best per backbone carried forward.
- **Boltz-2 fold-back:** target chain with `--use_msa_server`, binder as single sequence, 5 diffusion samples. Metrics: interface ipTM (pass > 0.5), complex pLDDT (fold > 0.7).
- **Interface residues (Fig 4):** computed post-hoc as target Ca within 8 A of any binder Ca in the predicted complex - GUCY2C 13 residues [130,132,146,192,325,326,327,330,347,348,349,378,379]; DKK1 8 residues [10,11,12,13,28,29,30,31] (complex-file numbering).
- **Hardware:** cloud A100 GPU.

### Data sources
- Genomics: cBioPortal `esca_tcga_pan_can_atlas_2018` (n=182, adenocarcinoma).
- Target association/tractability: Open Targets (EAC MONDO_0005028; Barrett's MONDO_0013662).
- Landscape: ClinicalTrials.gov (351 EAC interventional / 106 active; 203 Barrett's/dysplasia); Open Targets known-drug sets (106 agents); Drugs@FDA for approved-agent confirmation.

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## S5 — Epitope patches and cross-reactivity
- Design hotspots were chosen by structural heuristic (exposure + confidence), **not** by experimental functional-site mapping. It remains to be confirmed that the DKK1 binder occludes the LRP6 interface and that the GUCY2C binder engages a tumor-exposed epitope (open question O-5).
- Cross-reactivity screen (`epitope_cross_reactivity.csv`): **no high-similarity binder pairs detected** across the design set, indicating diverse solutions rather than a single repeated motif.

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## S6 — Development gates and open questions
Reproduced from the scientific development plan and program dossier.

### Go/no-go decision gates
| Gate | Criterion |
|---|---|
| G1 developability | Leads pass ESM / biophysics liability screen -> else redesign |
| G2 binding | KD <= 50 nM + confirmed epitope -> else affinity maturation |
| G3 function + selectivity | Potent in disease-relevant assay + >=10x normal-tissue window -> else kill/reformat |
| G4 in-vivo PoC | Tumor control (treatment) / dysplasia regression (interception) at tolerated dose |
| G5 tox | Acceptable GI (GUCY2C) / bone-GI (DKK1) safety margin -> IND |

### Open questions
| ID | Question | Kind |
|---|---|---|
| O-1 | ESM developability/liability screen + orthogonal fold-back (Chai-1/AF2) on leads | computational (near-term) |
| O-2 | GUCY2C normal-gut therapeutic window (human IHC + TDCC selectivity; mouse cross-reactivity) | preclinical - make-or-break |
| O-3 | GUCY2C T-cell-engager GI toxicity manageable at efficacious dose | clinical |
| O-4 | DKK1 interception surrogate endpoint (dysplasia regression) acceptance + payer | clinical/regulatory - make-or-break |
| O-5 | Epitope hotspots validated as functional (LRP6-block for DKK1; tumor-exposed for GUCY2C) | computational/experimental |

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## S7 — Strategy briefs (accompanying documents)
The following program documents accompany this manuscript as separate files and contain the clinical/regulatory/commercial reasoning, whose forward-looking statements are flagged there as assumptions requiring expert confirmation:
- `scientific_development_plan.md` - dual-arm development plan, assay cascade, gates
- `regulatory_brief.md` - EAC precedent, treatment/interception pathways, assumptions A-1..A-4
- `commercial_financing_brief.md` - epidemiology, market thesis, staged financing, assumptions C-1..C-4