Humanness Analysis
Evaluates antibody humanness using OASign (Observed Antibody Space Immunogenicity) and PFA (Positional Frequency Analysis) to quantify how "human-like" a sequence is based on natural antibody repertoires.
This tool provides a granular, residue-level view of humanness, helping identify specific non-human regions that may require engineering or humanization.
Accessing the Tool
Select an antibody in the Project View. Go to the Analysis menu and select Humanness. This will open the Humanness Analysis workspace.
Using the Tool
The Humanness Analysis workspace provides an aggregate view of antibody humanness metrics alongside granular residue-level diagnostics.
1. Aggregate Scoring
- VL/VH OASign: The fraction of 9-mer peptides in the variable region that appear in the human repertoire.
- Fv Score: The peptide-weighted average of both chains.
- Gene Assignment: Automatically assigns the closest human V/J germlines to support PFA and germline comparisons.
2. Germline Heatmap
Visualizes the alignment of your antibody against the top 5 most similar human germlines.
- Antibody Row: Colored based on OASign humanness. Darker red indicates a high density of non-human peptides overlapping that position.
- Region Bars: CDRs and Frameworks are annotated above the sequence.
- Peptide Tooltips: Hover over the Antibody row to see overlapping 9-mers and their "muts to human" distance.
3. Residue-Level Analysis (Dual-Pane)
The table at the bottom provides a comprehensive diagnostic view for every residue in the Fv.
- OASign Score: The percentage of human peptides overlapping this residue.
- AbLang Diff: The difference between the actual residue's AbLang likelihood and the highest-scoring alternative at that position. Shown as
Diff, HighestResidue(e.g.,1.2, T). - PFA [Highest]: The Positional Frequency Analysis score. Displays the frequency of the query residue and, if it is not the highest, the frequency of the most common human residue at that position (e.g.,
0.5% [90.7% T]). - Engineering: Click on an amino acid to open the Engineering modal to enter mutations or observations.
- Export Excel: Click on the Export Excel button to export the humanness analysis to an Excel file.
- Indicator Dots: Dots on a residue position indicate that there is an associated engineering design or observation at that position. Engineering mutations will appear as a small eggshell square while observations will appear as a small orange circle.
Strategy: Evaluate the positions which are in peptide regions flagged as not represented in OAS. For these positions, identify the positions with poor AbLang and PFA scores. Use these data to propose mutations to improve the humanness of the antibody using the Engineering modal by clicking on the query residue at that position. Once the mutations are proposed, use the Engineering tool to build variants and evaluate their overall results.
Scoring Thresholds
AbLead uses standardized clinical thresholds to categorize OASign results:
| OASign Score | Category | UI Color | Description |
|---|---|---|---|
| 85% – 100% | High | Green | Matches the mean score of natural human antibodies. |
| 75% – 84% | Medium | Orange | Typical range for clinically successful humanized antibodies. |
| < 75% | Low | Red | Below standard clinical humanization norms. |
| PFA Score | Category | UI Color | Description |
|---|---|---|---|
| ≥ 90% | Very High | Green | Highly conserved human germline residue. |
| ≥ 80% | High | Orange | Common human germline residue. |
| ≥ 70% | Medium | Yellow | Moderately common human germline residue. |
| < 70% | Unusual | Red | Infrequent or unusual residue at this position. |
| AbLang Diff | Category | UI Color | Description |
|---|---|---|---|
| < 3 | Good | Green | High naturalness; residue matches or is close to optimal. |
| 3 – 5 | Medium | Orange | Moderate divergence from natural antibody profiles. |
| ≥ 5 | High | Red | Significant divergence; residue may be unstable or non-natural. |
Methodology
- OASign (OAS Identity Search): Segments the variable region into overlapping 9-mers and queries them against a curated database of human sequences from the Observed Antibody Space (OAS).
- Trimming: Sequences are automatically trimmed to the Variable Region (Fv).
- Numbering Integration: Supports all project numbering schemes (IMGT, Kabat, AHo, etc.) while maintaining structural parity for PFA frequency lookups.
References
- The closest germlines are from the IMGT database. See IMGT.
- Humanness is determined using OASign against curated human 9-mer sequences from the Observed Antibody Space (OAS).
- OASign is based on the OASis method in BioPhi. See David Prihoda, Jad Maamary, Andrew Waight, Veronica Juan, Laurence Fayadat-Dilman, Daniel Svozil & Danny A. Bitton (2022) BioPhi: A platform for antibody design, humanization, and humanness evaluation based on natural antibody repertoires and deep learning, mAbs, 14:1, DOI: https://doi.org/10.1080/19420862.2021.2020203.