Humanizing Antibody Structures with BioLuminate

Tutorial Created with Software Release: 2026-1
Topics: Antibody Design, Biologics Drug Discovery
Products Used: BioLuminate

Tutorial files

1.4 MB
This tutorial is written for use with a 3-button mouse with a scroll wheel.
Words found in the Glossary of Terms are shown like this: Workspacethe 3D display area in the center of the main window, where molecular structures are displayed

 

Tip: You can hover over a glossary term to display its definition. You can click on an image to expand it in the page.
Abstract:

 

This tutorial provides a guide to the humanization of antibody structures, typically derived from non-human sources like mice, through the widely used CDR (Complementarity Determining Regions) grafting approach.

 

Tutorial Content

  1. Introduction

  1. Creating Projects and Importing Structures

  1. Humanizing an Antibody Framework

  1. Conclusion and References

  1. Glossary of Terms

1. Introduction

Humanization refers to strategies used to modify a non-human (commonly murine or mouse-derived) antibody sequence so that it more closely resembles a human antibody while preserving its antigen-binding specificity and affinity. Antibody therapeutics targeting diseases in humans must not trigger an anti-drug immune response in order to be effective. As most antibody drugs are derived from non-human sources, humanization eliminates or reduces the risk of immunogenicity by maximizing the human-likeness of the antibody drug.

The first-developed humanization strategy is chimerization, in which non-human variable regions are grafted onto human constant domains. The most widely used strategy is CDR grafting, in which the CDRs (Complementarity Determining Regions) from a non-human source are grafted onto a human framework. This approach has three steps:

  • Determining non-human CDR sequences binding to a specific antigen target
  • Selecting a compatible human framework to graft the non-human CDRs
  • Analyzing humanized antibody for steric clashes between non-human CDRs and human framework regions to make informed decisions for back-mutations to resolve clashes or restore binding affinity in the humanized antibody

A related strategy is SDR (Specificity Determining Residues) grafting, which transfers only key antigen-binding residues rather than entire CDRs, aiming to maximize human-likeness while maintaining function. To know more about antibody humanization strategies, please watch this video.

This tutorial uses the 2VXT structure, which is an antigen-bound murine antibody. In this tutorial, you will learn to humanize antibody structures from non-human sources by CDR grafting. You will also examine and resolve clashes in the resulting humanized structure.

2. Creating Projects and Importing Structures

  1. Open BioLuminate and create a new project named antibody_humanization.prj for this tutorial.
  2. Download the tutorial zip file including input files and reference outputs here: https://www.schrodinger.com/sites/default/files/s3/release/current/Tutorials/zip/antibody_humanization.zip
  3. After downloading the zip file, unzip the contents in your Working Directorythe location where files are saved for ease of access throughout the tutorial.

3. Humanizing an Antibody Framework

In this section, you will humanize the 2VXT murine antibody by grafting its CDRs onto a human framework. To accomplish this, an antibody structure database is queried to identify human frameworks that have a high level of sequence and geometrical similarity to the murine antibody. The returned results will have a composite score based on the sequence similarities of the human heavy and light chain sequences to the murine sequences, as well as a CDR stem geometry similarity score. In choosing the best framework, we recommend that you choose a template that maximizes stem geometry similarity, while minimizing sequence similarity, to avoid steric clashes. Higher resolution methods are preferred when deciding between frameworks with similar properties (e.g. x-ray diffraction over solution scattering). After a suitable framework is found, the CDR regions of the murine antibody will be grafted onto that framework. The resulting humanized structure will be examined for clashes introduced.

3.1 Graft murine CDRs onto human framework

Figure 3-1. Importing the prepared murine antibody structure into the Workspace.

  1. Go to File > Import Structures.
  2. Navigate to and choose proteinprep_2VXT-out.maegz in the tutorial files.
  3. Click Open.
    • Structure is added to Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion and included in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
  4. Find and open Humanization - CDR Grafting from Tasks.
    • Antibody Humanization:CDR grafting panel opens.

Figure 3-2. Importing the prepared murine antibody structure for humanization.

  1. For Import antibody structure to humanize, click From Workspace.
    • A green box indicates the import process was successful.

Figure 3-3. Searching the database for compatible human frameworks.

  1. For Use framework from, choose Structural database.
  2. Click Load.
    • A curated database is searched for compatible human frameworks.
    • This takes ~ 1 minute.
    • The Framework table populates with the templates that were found for the framework regions, along with sequence identity and stem geometry scores.

 

Note: The table rows are ordered by Weighted Geom+Sim score, which is the weighted combination of the stem geometry fitness score and the sequence similarity score. The lower the score, the better.

 

Note: You can choose a default library of human germline as the database for selecting a framework. Additionally, you can click Choose Databases in the Options menu to load in custom databases that you may want to use.

The following non-CDR residues are grafted along with the CDRs by default to maintain the conformations of CDRs and retain binding affinity (see this publication):-

  1. Vernier zone – layer of residues that lies between the CDR and FR
  2. Canonical structural residues –  framework residues that are important in determining CDR conformations
  3. Interface residues – residues at the heavy chain-light chain interface

 

The residue identity and structures of these non-CDR residues are preserved from the mouse structure. By clicking Advanced Options in the Options menu, you can see information regarding these non-CDR regions. These regions can be adjusted manually if so desired.

Figure 3-4. Replacing the query framework with the framework selected in the table.

When selecting a framework for grafting CDRs, we recommend choosing the framework on the top of the generated list, as it has the best combination of geometric fitness and sequence similarity (determined by its Weighted Geom+Sim score). You should also take into account the PDB resolution and the Heavy and Light Germline identities.

 

Now, you will choose the template with the lowest Weighted Geom+Sim score.

 

  1. Confirm that the 6E4X row is selected in the table.
  2. Optional: Click View Alignment.
    • The Multiple Sequence Viewer/Editor (MSV) panel opens, displaying the sequence identity between the selected human framework and the murine antibody (query).
  3. Click Replace Framework button.
    • The humanized structure appears in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
    • Residues with atomic clashes, if any, are displayed as thin tubes in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
    • The Define back mutation from human framework to query section in the panel updates.

 

Note: The human percentage is above 85% for both the heavy chain and light chain.

Figure 3-5. Humanized antibody as a result of framework replacement.

Note: The humanized antibody structure is annotated as follows:

  • CDR: Pink
  • Vernier Zone (VZ): Purple
  • Canonical Structure Residues (CSR): Green
  • VZ+CSR: Red
  • Interface Residues: Orange
  • Framework: Tan

Figure 3-6. Viewing framework residues that clash with CDR residues.

  1. In the Humanization:CDR grafting panel, confirm that Show residues with CDR/framework vdW clash option is checked.
    • This option lists residues in the table with clashes between the grafted CDR regions and the human framework region (based on van der Waals interactions).

Two residues L:71 (TYR) and L:46 (ARG) clash with CDR sidechains. Since these vernier zone residues are grafted along with the murine CDRs (see the query sequence in MSV panel) to maintain conformation and retain binding affinity, we will not back-mutate these residues. To resolve the clashes, we first recommend carrying out a restrained minimization of the humanized antibody structure using Protein Preparation Workflow. If the clashes still persist, we then recommend using advanced structure refinement tools like Prime.

 

In scenarios where a vernier zone residue is inherited from the selected human framework and differs from the corresponding residue in the original murine (query) antibody, a back-mutation to the murine amino acid is strongly recommended to maintain the structural integrity and function of humanized antibody.

3.2 Resolve steric clashes in the humanized structure

Figure 3-7. Running a restrained minimization to resolve clashes.

  1. Confirm that Grafted model on 6E4X is includedthe entry is represented in the Workspace, the circle in the In column is blue in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.
  2. Click Protein Preparation in the Favorites toolbar.
  3. Switch to INTERACTIVE mode.
  4. Under Minimize and Delete Waters, click Settings and confirm Distant from ligands (hets) is unchecked.
  5. Click Clean Up to run a restrained minimization.
    • The minimized structure is added to the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion and includedthe entry is represented in the Workspace, the circle in the In column is blue in the Workspacethe 3D display area in the center of the main window, where molecular structures are displayed.

Figure 3-8. Assessing the structure quality for minimized structure.

  1. Go to Diagnostics > Reports.
  2. For View, choose Protein Reliability Report.
  3. In the Protein Reliability Report, change the Job name to prot_rel_minimzed_humanized_model.
  4. Click Run.
    • This job takes ~ 1 minute.
    • Once the job is completed, a banner appears and a new entry is added to the Entriesa simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion table.

Figure 3-9. Viewing structure reliability report.

  1. Click Protein Reliability Report in the banner.
    • The Protein Reliability Report Panel updates to show the report.
    • The steric clashes are resolved in the minimized structure.

You can also perform antibody humanization via residue mutation in BioLuminate using the Antibody Humanization: Residue Mutation panel. This panel allows you to humanize an antibody by mutating residues to create a human-compatible protein. Appropriate sites for mutation can be found by comparing the antibody to homologs and identifying residues that satisfy various criteria, such as solvent accessibility, maximum number of side-chain interactions with either the antigen or with the antibody itself.

4. Conclusion and References

In this tutorial, you learned how to humanize antibodies using CDR grafting. You also learned how to resolve steric clashes in the humanized structure and identify key residues for back-mutation to restore binding affinity in the humanized structure. For additional resources, please consult the Antibody Modeling learning path or further learning section below.

5. Glossary of Terms

Entries - a simplified view of the Project Table that allows you to perform basic operations such as selection and inclusion

Included - the entry is represented in the Workspace, the circle in the In column is blue

Project Table - displays the contents of a project and is also an interface for performing operations on selected entries, viewing properties, and organizing structures and data

Recent actions - This is a list of your recent actions, which you can use to reopen a panel, displayed below the Browse row. (Right-click to delete.)

Scratch Project - a temporary project in which work is not saved, closing a scratch project removes all current work and begins a new scratch project

Selected - (1) the atoms are chosen in the Workspace. These atoms are referred to as "the selection" or "the atom selection". Workspace operations are performed on the selected atoms. (2) The entry is chosen in the Entries (and Project Table) and the row for the entry is highlighted. Project operations are performed on all selected entries

Working Directory - the location where files are saved

Workspace - the 3D display area in the center of the main window, where molecular structures are displayed