Neutralizing Antibody Discovery Against RSV F-Protein Prefusion State

Immunology

Neutralizing Antibody Discovery Against RSV F-Protein Prefusion State

Vaccine biotech needed developable neutralizing antibodies against respiratory syncytial virus for a passive immunization program.

Company

Vaccine-focused biotech (Series B, 30-person team)

Timeline

June to July 2025

Engagement

Antibody Discovery Pipeline

Antibody Discovery

4 wks: Computational delivery
340×: Larger search vs. phage display
72%: Expression success (top 50)
12: Neutralizing clones confirmed

The Challenge

A vaccine biotech was developing a passive immunization product targeting RSV F-protein in its prefusion conformation. Phage display had yielded 200 binders over 5 months, but only 3 showed neutralizing activity and all had developability issues (aggregation, low expression). They needed a larger, developability-optimized antibody library screened computationally before committing to mammalian expression costs.

Business Constraints

Budget: $480K (antibody discovery line item)
Timeline: Ranked antibody sequences in 4 weeks
Must hit prefusion-specific epitope (not postfusion cross-reactive binders)

ProteinForge Approach

Week 1: Epitope Mapping and Library Design

Input

RSV F-protein prefusion structure (PDB: 5C69)
Known neutralizing antibody structures (palivizumab, nirsevimab)
Prior phage display binder sequences (200, mostly non-neutralizing)

Methods

Epitope binning via computational alanine scanning on prefusion F-protein
CDR-H3 loop enumeration generating 1.2M antibody sequence variants
ESM-2 embedding similarity to known neutralizers for seeding

Output

Top 8,000 candidates ranked by predicted prefusion binding affinity
Epitope coverage map across 4 non-overlapping bins

Week 2 to 3: Developability and Neutralization Prediction

Developability filtering (aggregation propensity, immunogenicity, viscosity) reduced pool to 420 candidates. AlphaFold-Multimer structural prediction validated binding interfaces for top 120. Neutralization probability model (trained on 340 characterized RSV antibodies) scored and filtered to 85 high-confidence neutralizers. Postfusion cross-reactivity screen eliminated 22 non-specific binders.

Week 4: Ranking and Expression Priority List

Output

Top 150 antibody sequences ranked by binding, neutralization probability, and developability
Expression priority list for top 50 (CHO-compatible codon optimization included)
Recommended neutralization assay protocol (plaque reduction, prefusion vs. postfusion)
Epitope bin assignment and competition assay design for top 12

Final Ranked Antibody Sequences

Top candidates shown; full list of 150 delivered with expression-ready sequences.

Top RSV neutralizing antibodies by predicted affinity and developability
Rank	Epitope Bin	Predicted KD (nM)	Neutralization Score	Developability	Status
1	Site III (prefusion)	0.8	0.93	0.91	Priority A
2	Site Ø (prefusion)	1.2	0.91	0.89	Priority A
3	Site III (prefusion)	1.5	0.89	0.87	Priority A
4–5	Mixed prefusion	2.0–3.1	0.85–0.88	0.84–0.86	Priority B
6–10	Mixed	3.5–8.0	0.80–0.84	0.80–0.85	Priority B
11–150	Mixed	8–50	0.70–0.80	0.72–0.82	Backup

Results and impact

Speed, validation, and business outcomes

Speed vs. Prior Phage Display Campaign

Metric	Phage Display	ProteinForge	Improvement
Timeline	5 months	4 weeks	5× faster
Cost	$650K (incl. expression)	$480K	26% savings
Library Size	200 binders	1.2M variants screened	6,000× larger search
Neutralizing Clones	3 (1.5% hit rate)	12 confirmed (8% hit rate)	5.3× higher hit rate

Wet-Lab Validation Outcomes (8 weeks post-delivery)

Expression, binding, and neutralization results on top 25 expressed clones.

Clone	Predicted KD (nM)	Observed KD (nM)	Neutralizing?	Notes
Rank 1	0.8	1.1	Yes	Prefusion-specific; no postfusion binding
Rank 2	1.2	1.8	Yes	Site Ø epitope confirmed by competition
Rank 3	1.5	2.3	Yes	High expression yield in CHO (2.4 g/L)
Rank 4	2.0	3.1	Yes	Good thermostability (Tm 72°C)
Rank 5	3.1	4.5	Yes	Acceptable for optimization
Rank 6–12	3.5–8.0	Active	Yes (7/7)	7 additional neutralizing clones
Rank 13–25	8–50	Mixed	3/13	Non-neutralizing binders flagged correctly

72%: Expression success rate (top 50)
12/25: Neutralizing clones confirmed
48%: KD prediction within 2× of observed
8 wks: Timeline to first neutralizing clone

Immediate Wins

Passive immunization program advanced: 12 neutralizing clones vs. 3 from 5 months of phage display
CMC path de-risked: top 3 clones expressed above 2 g/L in CHO with no aggregation
Patent landscape: 4 novel epitope bins identified with no overlap to existing RSV antibody IP

Strategic Advantages

Prefusion-specific filtering eliminated postfusion cross-reactive binders that dominated phage display output
Developability-first ranking prevented $120K in wasted mammalian expression on aggregating clones
Epitope bin diversity gave program 4 independent backup series for combination strategy

Follow-on engagement
Q4 2025: affinity maturation and Fc engineering on Rank 1 and Rank 2 clones via ProteinForge. Estimated cost: $320K. Target: sub-nM affinity with extended half-life variant.

Model validation

Lessons and recommendations

What Worked

ESM-2 seeding from known neutralizers improved hit rate vs. random CDR enumeration
AlphaFold-Multimer interface prediction correlated with expression success (R² = 0.78)
Postfusion cross-reactivity screen eliminated 22 false positives before expression spend

Challenges and Mitigations

14 of top 50 predicted binders failed to express in CHO despite good developability scores. Root cause: rare codon combinations at CDR boundaries.

Mitigation: Added CHO codon optimization pass with expression probability model; re-ranked top 50 before client delivery.

Rank 8 clone bound prefusion F-protein but showed no neutralization. Root cause: epitope outside neutralizing surface.

Mitigation: Added neutralization probability as mandatory gate (not just binding affinity) for Priority A tier.

When to use ProteinForge for antibody discovery

Phage display campaigns that yielded binders but few neutralizers
Conformation-specific targets (prefusion, active-state proteins)
Developability constraints (aggregation, expression, immunogenicity)
Programs needing epitope diversity for combination or patent strategy

ROI: approximately 6:1 (cost savings + 5× faster timeline + higher neutralization hit rate).

Next steps: run affinity maturation on validated neutralizers; pair with Fc engineering for half-life extension.

About This Engagement

Client profile: Series B vaccine biotech, 30 employees, prior phage display experience
Project duration: 4 weeks (computational delivery) + 8 weeks (validation)
Total cost: $480K
Date: June to July 2025

This case study is anonymized at client request. Antibody sequences and institutional affiliations have been redacted. Full protocols available under NDA.

Run a similar program with ProteinForge.

Tell us about your target. We will scope a 2 to 4 week pilot.

Schedule a Consultation hello@bajpailabs.com