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.
[!IMPORTANT] Integrated Immunogenicity Assessment: Sapiens, OASign, and RPEMHC must be evaluated in conjunction for a complete developability profile. OASign and Sapiens quantify sequence humanness (which corresponds to T-cell receptor self-tolerance), whereas RPEMHC predicts physical peptide presentation to human MHC-II. A therapeutic candidate is only safe if it scores well on both metrics: a "Good" (Green) RPEMHC score on a sequence with low humanness (such as a mouse sequence) is still highly immunogenic in patients because there is no self-tolerance for the presented peptides.
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. Mode Selection (OASign vs. RPEMHC)
The Humanness Analysis workspace can toggle between two primary metrics using the Mode dropdown selector on the toolbar:
- OASign Mode (Repertoire Similarity): Evaluates sequences against human antibody repertoires.
- RPEMHC Mode (MHC-II Immunogenicity): Predicts peptide presentation to Major Histocompatibility Complex Class II (MHC-II) alleles to identify helper T-cell epitopes. When enabled, a dropdown menu allows selecting the target HLA-DRB1 allele (HLA-DRB1*0101, *0301, *0401, or *1501).
2. Aggregate Scoring
- VL/VH OASign: The fraction of 9-mer peptides in the variable region that appear in the human repertoire.
- VL/VH RPEMHC: The sum of 15-mer MHC-II binding scores above
0.5across the variable domain. - Fv Score: The peptide-weighted average of both chains (OASign) or the combined VL + VH score (RPEMHC).
- Gene Assignment: Automatically assigns the closest human V/J germlines to support PFA and germline comparisons.
3. Germline Heatmap
Visualizes the alignment of your antibody against the top 5 most similar human germlines.
- Antibody Row: Colored based on OASign human repertoire similarity or RPEMHC binding affinity depending on the active mode (red indicates high immunogenicity / low humanness).
- Region Bars: CDRs and Frameworks (FMWK) are annotated above the sequence, restricted strictly to the Fv region.
- Peptide Tooltips: Hover over the Antibody row to see overlapping 9-mers (OASign) or 15-mer binding scores (RPEMHC).
4. Residue-Level Analysis (Dual-Pane)
The table at the bottom provides a comprehensive diagnostic view for every residue in the Fv.
- 9-mer Human / MHC-II Max: Displays the percentage of human peptides (OASign mode) or the maximum predicted 15-mer binding affinity score (RPEMHC mode) 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 Modal: Clicking any residue opens the Engineering modal, which includes advanced de-immunization options:
- Predictive De-Immunization Scan: Runs an in silico mutational scan for all 20 amino acids at the selected position. Recommendations are sorted dynamically based on the active workspace mode:
- OASign Mode: Prioritizes the highest predicted 9-mer human coverage.
- RPEMHC Mode: Prioritizes the lowest predicted MHC-II binding affinity score (disrupting helper T-cell epitopes).
- PFA Integration: Displays the human germline frequency for each substitution. Clicking any row in the scan table instantly auto-populates the mutation input.
- Live Score Preview: As you type or select mutations, the modal performs a live evaluation on the mutated sequence, displaying a "Before vs. After" preview of your Fv RPEMHC and OASign scores to let you confirm de-risking before saving.
- Predictive De-Immunization Scan: Runs an in silico mutational scan for all 20 amino acids at the selected position. Recommendations are sorted dynamically based on the active workspace mode:
- 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: Triage positions using the Heatmap. Use PFA and AbLang scores to find weak spots. Open the Engineering modal on high-risk residues to inspect the predictive scans and select human-favored substitutions that lower predicted MHC-II binding scores without disrupting structural properties.
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. |
| RPEMHC Fv Score | Category | UI Color | Description |
|---|---|---|---|
| ≤ 24.0 | Good | Green | Highly de-immunized / low immunogenic risk profile. |
| 24.0 – 28.5 | Warning | Yellow | Moderate clinical risk range. |
| > 28.5 | Severe | Red | High helper T-cell epitope density / immunogenic risk. |
| 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).
- RPEMHC (MHC-II binding prediction) is based on the DeepMHCII / RPEMHC ensemble deep learning model for predicting class II peptide-MHC binding affinity. Described in Wang et al., Bioinformatics (2024).
- 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.