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Humanization

Modify specific residues to humanize the sequence.

This tool is useful for humanizing antibodies (or converting to other species) by evaluating germlines and selecting, via templates or via BioPhi Sapiens, the residues to mutate to target germline residues. Structural position alignment and numbering are powered by the ANARCI engine, providing industry-standard precision for antibody annotation, while closest germline identification and V/J gene assignments are determined by sequence identity matching against the platform's embedded germlines database. The title bar for each chain displays the standard V/J assignment, including the detected parent species. The goal is to improve the humanness score while maintaining the binding affinity of the antibody, with the hope of reducing the potential of ADA (anti-drug antibodies).

Reflecting my core work principle to "Be Inherently Lazy", allow a computer with set exclusions and methods help automate, simplify, and lend reproducability to humanization decisions, while tracking designs in an Engineering tool for easy combinatorial variant generation. All at the click of a few buttons.

Accessing the Tool

Select exactly one antibody in the Project View. Go to the Edit menu and select Humanization. This will open the Humanization workspace in a new tab.

Humanization Humanization Engineering

Using the Tool

  • Convert to Species: Select the target species for the germline templates (e.g., Human, Mouse, Alpaca, Cat, Dog, Pig, Rabbit, Rat, Rhesus, Chicken). The BioPhi Sapiens section will be disabled when a species other than Human is selected.
  • Number of Germlines: Set the maximum number of germlines to view for potential humanization.
  • Sort By: Sort the germlines by modifiable positions (Modifiable Residues Identity) or all positions (Full Sequence Identity).
  • Exclusions: User defined list of amino acid positions to exclude from humanization. This is useful for positions that are important for antibody activity, if known, or to include or exclude positions based on user knowledge or preference. Be carefule with selection/deselection. Make sure you have selected the intended residues if you use manual exclusions!
    • Honegger: Uses the Honegger exclusions. See reference.
    • CDRs: Excludes the CDR residues using the definition in the associated menu.
    • Selected: Excludes the residues selected by the column highlighter. These may be selected using the buttons below and/or by selecting columns directly. Us the "Clear" link to clear the column seelctions entirely.
    • Select on Grid:
      • CDRs: Selects the CDR residues based on the CDRs menu setting above to exclude from humanization.
      • Honegger: Selects the Honegger residues to exclude from humanization. These are the standard exclusions used in the humanization pipeline.
      • Vernier: Selects the Vernier residues to exclude from humanization.
      • VHH: Selects the VHH interface residues to exclude from humanization to maintain solubility. These are IMGT positions 42, 49, 50, and 52.
  • The germline position colors reflect which positions will be mutated:
    • Magenta: Parent Position Kept
    • Cyan: Germline Position Used
    • Green: Germline same as Parent
  • The Vernier residues are indicated by a 'V' and the VHH interface positions are indicated with a 'VHH' in the Honegger Template row.
  • Germline Selection: For each of the Light Chain and Heavy Chain V and J regions, select the desired germline using the wand icon to the left of the germline name. This will populate the Engineering mutation sets with the germline residues which are in the mutation template regions and which are different from the parent antibody in non-exclusion positions. The generated mutations will automatically have their Notes set to [Parent Species] to [Target Species] (e.g., "Human to Mouse").
  • BioPhi Sapiens Humanization: An alternate method to germline picking is to let BioPhi Sapiens humanize the antibody. This is done by selecting the Suggest Mutations button. This will populate the Engineering mutation set with BioPhi Sapiens-defined mutations to optimize the humanness score while not mutating the Parent Position set as shown in Honegger Template, following the same Exclusion definition as the template method. See the reference below for more details.
  • Confidence (p): The minimum probability (0.0 to 1.0) required to suggest a mutation. A value of 0.1 is recommended for high-sensitivity humanization.
  • Build New Antibodies: After defining the mutations, go to the Engineering tool. Mutation design may be edited at this point to optimize the humanization design. Once the final design is entered, select the Mutation Designs, Generate Combinations, select the Variant Design, and Build Selected. The newly built antibody is a humanized version of the parent antibody.

Exclusion Example: Let us suppose the user wants to use standard Honegger exclusions and to add the Vernier residues as excluded posotions.

  • Set the Exclusions to Selected.
  • In the Select on Grid section click the Honegger button, then click the Vernier button.
  • Click the wand icon next to each desired germline or click the Sapiens Suggest Mutations button to enter those mutations in the Engineering tool.

3D Structure Visualization & Residue Selection

The Humanization workspace includes an interactive PDBe Mol* 3D structure viewer positioned below the alignment grids, allowing real-time mapping of sequence changes to the three-dimensional antibody structure.

  • Interactive Resizer: Drag the horizontal divider bar directly above the 3D window up or down to adjust the vertical height of the viewer pane.

  • Cartoon Region Coloring: The ribbon structures of the light and heavy chains are colored by region to match the centralized color schemes:

    • Variable Domain (Fv): Frameworks (dim gray/silver) and CDRs (blues, purples, cyans) are colored according to domain region standards.
    • Constant Domains: Reconstructed constant domains (CL, CH1, hinge, CH2, CH3) are automatically rendered and colored by their specific sub-domain region colors.

  • Selected Residue Highlighting:

    • Clicking column headers in the alignment grids (scheme position or linear number) highlights those columns in blue and adds them to the selection set.
    • The corresponding residues on the 3D structure are instantly highlighted in CPK element coloring (with position number labels), regardless of which exclusion method is active.
    • Residue Style: Toggle the dropdown under the BioPhi Sapiens configurations panel in the sidebar to change the rendering style of selected residues between Spacefill (default) and Ball-and-Stick.

Supported Reference Species

The Humanization tool supports conversion templates and germline analysis using reference sequences from the following species:

Scientific Name Common Name (AbLead Badge)
Homo sapiens Human (H)
Macaca mulatta Rhesus (Rh)
Mus musculus Mouse (M)
Rattus norvegicus Rat (R)
Oryctolagus cuniculus Rabbit (Rb)
Vicugna pacos Alpaca (Al)
Canis lupus familiaris Dog (Dg)
Felis catus Cat (Ct)
Gallus gallus Chicken (Ck)
Sus scrofa Pig (Pg)

Bulk Humanization

For humanizing multiple antibodies in a single batch, see the Bulk Humanize documentation.

References