# 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.*

---

## Table S0 — Triage scoring rubric, all component scores, and rank-robustness

**Rubric (exact):** `composite = tumor_selectivity + novelty + tractability_score + 5 × disease_association`
- `tumor_selectivity` — analyst ordinal 1–5: 5 = tissue-restricted with a strong therapeutic window; 3 = moderate window / some normal expression; 1–2 = broad normal expression / poor window.
- `novelty` — analyst ordinal 1–5: 5 = essentially unexploited in EAC; 3–4 = clinical-stage elsewhere / emerging; 1 = approved/crowded in EAC.
- `tractability_score` — antibody-tractability tier mapped to integer: surface-confirmed = 3, clinical-stage = 4, clinical-validated = 5.
- `disease_association` — Open Targets association score (EAC for treatment lane, Barrett's for interception lane; 0 if unavailable), weighted ×5.

This rubric reproduces all 16 published composite values (max residual 0.005).

**Component scores:**

| target | lane | tumor_selectivity | novelty | ab_tractability | eac_assoc | barretts_assoc | composite | biologic_druggability |
|---|---|---|---|---|---|---|---|---|
| GPX7 | I | 4 | 5 | surface-confirmed | nan | 0.372 | 13.86 | NOT-A-BINDER-TARGET |
| GUCY2C | T | 5 | 5 | surface-confirmed | nan | nan | 13.0 | STRONG |
| REG4 | I | 4 | 5 | surface-confirmed | nan | 0.094 | 12.47 | WEAK-BIOMARKER |
| CDH17 | T | 4 | 5 | surface-confirmed | nan | 0.076 | 12.38 | STRONG |
| ERBB2 | T | 4 | 1 | clinical-validated | 0.457 | nan | 12.29 | CROWDED |
| CEACAM5 | T | 3 | 4 | clinical-validated | nan | nan | 12.0 | STRONG |
| CD276 | T | 3 | 5 | clinical-stage | nan | nan | 12.0 | STRONG |
| MET | T | 3 | 2 | clinical-validated | 0.31 | nan | 11.55 | GOOD |
| ERBB3 | T | 3 | 3 | clinical-stage | 0.298 | nan | 11.49 | GOOD |
| DKK1 | I | 3 | 4 | clinical-stage | nan | 0.074 | 11.37 | GOOD |
| TFF3 | I | 3 | 4 | surface-confirmed | nan | 0.102 | 10.51 | WEAK-BIOMARKER |
| OLFM4 | I | 3 | 4 | surface-confirmed | nan | 0.091 | 10.46 | WEAK-BIOMARKER |
| TACSTD2 | T | 3 | 4 | surface-confirmed | nan | nan | 10.0 | GOOD |
| MUC1 | T | 3 | 3 | clinical-stage | nan | nan | 10.0 | MODERATE |
| EGFR | T | 2 | 1 | clinical-validated | 0.377 | nan | 9.88 | CROWDED/POOR-WINDOW |
| CLDN18 | T | 4 | 1 | clinical-stage | nan | 0.072 | 9.36 | CROWDED |

**Rank robustness (Monte Carlo, 20,000 draws; each of the four weights independently scaled — selectivity/novelty/tractability by U(0.5,1.5), association by U(2.5,7.5) — restricted to biologic-addressable targets per lane):**

| Lane | Lead | # addressable targets | % draws lead ranks #1 | % draws lead in top-3 | Nearest competitor |
|---|---|---|---|---|---|
| Treatment (T) | GUCY2C | 8 | 80.7% | 94.2% | CDH17 |
| Interception (I) | DKK1 | 1 | 100%* | 100%* | (none addressable) |

*DKK1 is the only biologic-addressable interception target after the druggability filter; its rank-1 result is therefore trivial and reflects the thinness of the addressable interception space rather than out-competing a field.

**Selectivity recalibration (important sensitivity).** GUCY2C's tumor_selectivity was recorded as 5 ("strong therapeutic window"); because that window is in fact unresolved (O-2), a more honest anchor is 4 ("tissue-restricted, window pending"). Re-running the triage with GUCY2C selectivity = 4 lowers its composite from 13.00 to 12.00 and **does change the lane ranking**: GUCY2C is overtaken by CDH17 (12.38) and falls into a tie with CEACAM5 and CD276 (all 12.00), and its Monte-Carlo rank-1 frequency drops from 80.7% to ~0% (top-three ~48%). In other words, GUCY2C's position as the single top treatment-lane target is contingent on the optimistic selectivity score, which is precisely what open question O-2 tests. This does not disqualify GUCY2C — it remains a strong, biologic-addressable, novelty-consistent candidate for which we have a designed binder — but it means (i) resolving O-2 is decisive for lead choice as well as for safety, and (ii) **CDH17 is a credible backup treatment-lane target** that should be carried forward in parallel.

---

## 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).

---

## 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) |

---

## 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 |

---

## 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.

---

## 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.

---

## Table S4 / Figure S1 — Normal-tissue selectivity (GTEx v8)
Median TPM profiling of the two leads and four comparators across GTEx v8 tissues. GI fraction = share of summed median expression in intestine/colon/stomach/esophagus tissues. **This is normal-tissue data and establishes tissue selectivity only — it does NOT establish EAC tumor-cell expression** (a flagged gap for GUCY2C). GUCY2C and CDH17 are sharply GI-restricted; TROP2 (TACSTD2) is pan-epithelial (high in skin), illustrating the poor-window designation. See Figure S1.

*Footnote:* DKK1's top GTEx row, `Cells_Cultured_fibroblasts` (852.8 TPM), is a cell-culture artifact and not a physiological normal-tissue value; DKK1's solid-tissue levels are low, consistent with a stromal/paracrine source. In Barrett's tissue the biologically relevant DKK1 is likely stromal/paracrine — a pattern compatible with (and arguably favorable to) a secreted-ligand neutralizing-trap strategy.

| gene | top_tissue | top_median_TPM | GI_fraction | top_nonGI_tissue | top_nonGI_TPM | esophagus_mucosa_TPM | esophagus_gej_TPM |
|---|---|---|---|---|---|---|---|
| GUCY2C | Small_Intestine_Terminal_Ileum | 28.2 | 0.85 | Skin_Sun_Exposed_Lower_leg | 1.7 | 0.68 | 0.08 |
| DKK1 | Cells_Cultured_fibroblasts | 852.8 | 0.0 | Cells_Cultured_fibroblasts | 852.8 | 1.19 | 0.39 |
| CDH17 | Colon_Transverse | 141.1 | 0.98 | Artery_Tibial | 0.4 | 0.3 | 0.35 |
| CEACAM5 | Esophagus_Mucosa | 293.4 | 0.84 | Vagina | 78.9 | 293.42 | 0.13 |
| TACSTD2 | Esophagus_Mucosa | 1419.1 | 0.29 | Skin_Not_Sun_Exposed_Suprapubic | 708.7 | 1419.1 | 1.97 |
| EGFR | Skin_Sun_Exposed_Lower_leg | 78.3 | 0.15 | Skin_Sun_Exposed_Lower_leg | 78.3 | 29.91 | 34.63 |
| ERBB2 | Nerve_Tibial | 130.1 | 0.17 | Nerve_Tibial | 130.1 | 113.1 | 35.77 |

---

## Table S5 — Binder sequence-composition liability screen
Computed directly from the ProteinMPNN designed sequences. `pctA` = % alanine; high alanine content and long homopolymer runs are the signature of a generic low-complexity scaffold that can fold with spuriously high ipTM. The top GUCY2C design by ipTM (gucy2c_bb2) carries a high-alanine liability (44% Ala, 8-residue run); the DKK1 lead (dkk1_bb1) is clean on both interface confidence and composition. Orthogonal fold-back and monomer/decoy specificity controls (O-1) remain the decisive next check and have not been run.

**Binder diversity (length-penalized pairwise sequence identity, all 16 designs):** maximum pairwise identity 21.4% within the GUCY2C set (top pair gucy2c_bb5/bb3) and 32.9% within the DKK1 set (top pair dkk1_bb2/bb3, both alanine-rich); set means ≈15%. No pair approaches a high-similarity threshold; the designs are independent solutions. *Note:* %alanine is reported against no external benchmark distribution here — designs are flagged relative to an internal 15%-alanine caution line, and benchmarked calibration against published RFdiffusion/ProteinMPNN composition distributions (e.g. Cao et al. 2022; Bennett et al. 2023) is recommended before external submission.

| design | target | len | iptm | pctA | pct_hydrophobic | max_homopolymer_run | frac_charged |
|---|---|---|---|---|---|---|---|
| dkk1_bb1 | DKK1 | 63 | 0.858 | 17.5 | 25.4 | 2 | 55.6 |
| dkk1_bb4 | DKK1 | 63 | 0.844 | 27.0 | 39.7 | 3 | 46.0 |
| dkk1_bb5 | DKK1 | 60 | 0.821 | 50.0 | 63.3 | 10 | 21.7 |
| dkk1_bb2 | DKK1 | 62 | 0.745 | 61.3 | 77.4 | 9 | 6.5 |
| dkk1_bb6 | DKK1 | 89 | 0.743 | 38.2 | 62.9 | 5 | 28.1 |
| dkk1_bb7 | DKK1 | 85 | 0.709 | 27.1 | 44.7 | 5 | 48.2 |
| dkk1_bb3 | DKK1 | 76 | 0.571 | 63.2 | 78.9 | 7 | 6.6 |
| dkk1_bb0 | DKK1 | 89 | 0.524 | 9.0 | 23.6 | 4 | 65.2 |
| gucy2c_bb2 | GUCY2C | 71 | 0.915 | 43.7 | 56.3 | 8 | 21.1 |
| gucy2c_bb5 | GUCY2C | 80 | 0.861 | 48.8 | 68.8 | 3 | 20.0 |
| gucy2c_bb4 | GUCY2C | 90 | 0.811 | 27.8 | 44.4 | 2 | 48.9 |
| gucy2c_bb1 | GUCY2C | 73 | 0.715 | 13.7 | 28.8 | 3 | 56.2 |
| gucy2c_bb3 | GUCY2C | 84 | 0.619 | 17.9 | 42.9 | 2 | 45.2 |
| gucy2c_bb0 | GUCY2C | 87 | 0.536 | 14.9 | 32.2 | 3 | 46.0 |
| gucy2c_bb7 | GUCY2C | 81 | 0.521 | 28.4 | 50.6 | 3 | 37.0 |
| gucy2c_bb6 | GUCY2C | 72 | 0.401 | 6.9 | 22.2 | 3 | 62.5 |

---

## Table S6 — Interface-residue mapping to canonical UniProt positions
Interface residues (target Cα within 8 Å of any binder Cα) in complex-file numbering and their canonical UniProt positions. Offsets: GUCY2C complex residue + 23 = UniProt P25092 position; DKK1 complex residue + 177 = UniProt O94907 position.

| Target | Complex-file residues | UniProt positions | Domain (range) | Within domain? |
|---|---|---|---|---|
| GUCY2C | 130,132,146,192,325,326,327,330,347,348,349,378,379 | 153,155,169,215,348,349,350,353,370,371,372,401,402 | ECD (P25092 24–430) | yes |
| DKK1 | 10,11,12,13,28,29,30,31 | 187,188,189,190,205,206,207,208 | CRD2 (O94907 178–256) | yes |

---

## 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) / skeletal+bone-GI systemic (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 |

---

## 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