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Workflows

The analysis and engineering tools can be integrated into workflows for lead selection and developability engineering. Below are examples.

Lead Selection

The Project View overviews the attributes of individual entries and is highly effective for lead selection by ranking entries based on the KBC score. The weights used for the KBC score can be adjusted in the user settings section, as well as in the Excel export. If leads with the desired attributes already exist in the project, no further engineering may be required. However, an evaluation of the clades should be performed to ensure sufficient diversity exists among the selected leads, in case related sequences are later found to have developability issues.

Humanization

If humanization is required, the Humanization tool can be used to humanize the selected leads. Multiple different humanizations can be generated and further evaluated for developability. It is often helpful to test multiple humanization strategies using different germlines or methods. This helps to develop leads with high humanness scores while maintaining target binding. Once humanization designs are entered in the Engineering tool, the humanized versions can be exported as a FASTA file or added as new project entries using 'Build Selected'. To build multiple humanized versions, either duplicate the entry to preserve its existing design or clear the Engineering designs to configure a new one.

  1. Select the lead to humanize in the Project View.
  2. Select Edit > Engineering and clear any existing designs from both the Mutation Designs and the Variant Designs.
  3. In the Project View, select Edit > Humanization.
  4. For a germline template-based humanization, evaluate the residue positions for each germline which are colored cyan, as these will be the mutations made for that germline selection. A good initial method is to choose germlines which are most similar to the parental sequence.
  5. Click the wand icon to the left of the desired germline to open a dialog showing the mutation sites. Click Apply Mutations. This adds the mutation designs to the Engineering tool.
  6. Repeat this process for each V and J region of each chain.
  7. This builds the mutation designs for a single humanized sequence.
  8. Click on the open Engineering tab and click the refresh button in the Mutation Designs table to update the table with the new mutation designs.
  9. Select all Mutation Designs, or exclude positions if so desired based on paratope knowledge, and click Generate > Generate Combinations. This will build a single variant which is shown in the Variant Designs tab.
  10. Select this variant and click Build Selected to build the humanized sequence into the Project.
  11. Repeat this process for each humanization strategy to build multiple humanized versions.
  12. Review the humanized versions in the Project View for overall developability features. At this point further engineering for developability can be performed if needed.

For an AI-based method, after clearing the Engineering Mutation Designs for the entry, set the Threshold and Exclusions in the Humanization tool and click Define Mutations. This will build the mutation designs for a single humanized sequence. Then proceed with the Engineering tool steps above.

Stability Optimization

If high AbLang scores are observed, particularly for AbLang FR, the specific residues causing the violations can be identified and optimized. Another critical stability issue is a missing HC-CDR3 salt bridge, which is highlighted in the Project View and Liabilities tool.

  • Liability Tool: Starting with the Liabilities tool, identify the specific residues causing the violations. Pay attention to those with large likelihood differences between the parental and most likely residues. If a PDB file is associated with the entry, use the 3D View to evaluate the spatial positions of these violations. Alternatively, for more advanced visualization, use the Export SVL function in the Liabilities tool to export the violations to a file and view them in MOE. A likelihood difference of ≥ 3.0 is a practical threshold for selecting residues to optimize. The Liabilities tool displays the likelihood difference and optimal residue in the liability table. Clicking a liability in the grid opens the Engineering modal to enter desired mutations, which are then tracked in the Engineering tool.

  • PFA Tool: The PFA tool can be used to evaluate individual positions for mutations to optimize AbLang scores. The data grid for AbLang, AbLang2, and human reference statistics provides a clear view of the most likely residue at any given position. Clicking on a residue in this tool opens the Engineering modal to enter desired mutations, which are also tracked in the Engineering tool.

    Note: If a residue is determined to potentially be necessary for function, use "Include Parental" to ensure the parental residue is retained in combinatorial variant designs.

  • Engineering Tool: After entering mutations via the Liabilities and PFA tools, the Engineering tool can be used to edit mutation designs or manually enter new ones, as well as to generate combinatorial variants. After generating variant designs, they can be selected for FASTA export or automatically generated as new project entries using 'Build Selected'.

PTM Optimization

If potential Post-Translational Modifications (PTMs) are found that could interfere with activity, they can be addressed in much the same way as stability optimizations discussed above. These corrections can also be combined with stability repairs to create comprehensive variant designs. Once the designs are built, evaluate them for further developability issues in the Project View and Liabilities tools.

Isoelectric Point Optimization

If the isoelectric point (pI) is outside of the desired range, the Engineer pI tool can be used to shift the pI while minimizing stability violations and impact on humanness. See the Engineer pI tool documentation for more details.

Assembler

Often, the initial sequence dataset does not contain optimal leader or constant domain sequences. The Assembler tool can be used to efficiently generate new versions across groups of entries, avoiding manual copy/paste errors. See the Assembler tool documentation for more details.

These workflow examples illustrate how AbLead's analysis and engineering tools provide a powerful and intuitive approach to antibody design and optimization.