Antibody Humanization: Residue Mutation Panel

This panel allows you to humanize an antibody by mutating residues to create a more 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.

To open this panel, click the Tasks button and browse to Biologics → Humanization - Residue Mutation.

Using
Features
Additional Resources

Using the Antibody Humanization: Residue Mutation Panel

The basic workflow in this panel is:

Import a structure into the workflow, either from the Workspace or from a Maestro file (Import structure from).
Select the chains to be treated as the "ligand".
Click Homology Criteria.
(Optional) Choose the antibody databases that are used to search for homologs.
Run the search (Search Antibody Database for Homologs) and align the homologs (Align Homologs).
(Optional) Select an option for the chain to show, and examine the alignment for that chain.
Choose the regions that you want to substitute residues in.
Specify the criteria for automatic selection of residues, in the Selection by homology criteria and Parent structure 3D criteria sections.
Click Save.

The Homology Suggestions dialog box closes, and the mutations identified from the homology model and the applied criteria are set in the residues table. The mutants are set to the variations seen in the homologs.
Make any changes to the residues marked for mutation, by clicking in the Mutations column of the table and selecting from the popup that is displayed. You can also use the tools above the table.
Choose the properties to calculate. Affinity and pKa are not selected by default as they take longer than the other properties.
Choose Job Settings from the Settings button menu,

set the job parameters in the Job Settings dialog box, and click Run to run the job.

The mutated structures are incorporated into the project as new entries, and the Humanize Antibody Viewer opens (see MM-GBSA Residue Scanning/Humanize Antibody Viewer Panel for details).

Only one site is mutated at a time to produce a mutated structure: multiple simultaneous mutations are not performed. The number of structures is linear in both the number of sites mutated and the number of mutations at each site (it is in fact the sum of the number of mutations at each site). The number of mutation sites and the total number of mutations is reported below the table.

If you want to perform mutations on other parts of the antibody, for example to mutate the antigen and the antibody at the same time, you can use the MM-GBSA Residue Scanning Panel.

To run a residue mutation job for humanization from the command line, you can use the following command. Run the command with -h for more information.

$SCHRODINGER/run mut_pred.py

To write out input files, click the arrow next to the Settings button,

and choose Write (more...).

For information on command options, see mut_pred.py Command Help.

Antibody Humanization: Residue Mutation Panel Features

Import tools
Homology Criteria button
Residues tab
Options tab
Job toolbar
Status bar

Import tools

The import tools allow you to import a protein structure into the workflow for residue mutation. After import, the antibody is analyzed to locate the antibody regions and calculate solvent-accessible surface areas (SASA).

Choose an option from the Import structure from option menu to use the entire structure in the Workspace, the selected residues in the Workspace, or a structure from a Maestro file. For the first two options, click the Import button to import and analyze the structure. If importing from a file, the button changes to Browse, and you can click this button to open a file selector to locate and open the file.

If you want to analyze the effect of the mutations on the binding of the antibody to an antigen, you can include the entire complex in the Workspace and select only the antibody chains you want to analyze, then choose Workspace (selected residues) from the option menu to limit the analysis to the Workspace selection. If you want to examine binding, you must include the entire structure in the Workspace at this stage (or read it from a file).

Once the analysis is complete, you are prompted to choose chains for binding affinity calculations. One set of chains is used as the “ligand”, the remaining chains are the “receptor”. These labels are arbitrary, so it does not matter if the antigen is treated as the ligand or the receptor.

Homology Criteria button

Select residues to mutate based on suggestions from homologous proteins. When you click this button, the Antibody Humanization: Homology Suggestions Dialog Box opens, in which you can build a homology model for a chain and select residues by conservation or structural criteria. Clicking Save in this dialog box selects the suggested residues in the residue table of the Residues tab and sets the mutations for the residue to the residues in the homologs that differ from the query.

Residues tab

In this tab you select the residues for mutation, and choose the mutations for each of these residues.

Set mutations for option menu

Select the residues whose mutations you want to set up, from this option menu. The mutations are chosen from the to option menu, and are applied to all selected residues. The choices are:

All residues—Set the mutations for all residues in the table. When you select this option, all residues are selected in the residues table.
Selected residues—Set the mutations for the residues that are selected in the residues table.
Residue selection—Set the mutations for selected residue types. Opens the Residue Scanning - Select Residues dialog box, in which you can limit the selection of residues by chain, solvent exposure, or residue type (including nonstandard residues). All residues that meet the criteria are selected in the residues table when you click OK in the dialog box. The setting on the Mutate option menu is switched to Selected residues, as the dialog box is simply a means of selecting residues.
Atom selection—Select residues for setting up mutations by general atom selection procedures. Opens the Atom Selection Dialog Box, in which you can make selections by combinations of criteria. All residues that meet the criteria are selected in the residues table when you click OK in the dialog box. The setting on the Mutate option menu is switched to Selected residues, as use of the dialog box is simply a means of selecting residues.

to option menu and Apply button

Select a set of mutations from the popup that appears when you click on the menu, and click Apply to set these mutations for the residues that are selected in the table. The popup allows selection of individual residues, selection by type, and the selection can include nonstandard residues defined by the current nonstandard residues database.

Residues table

Table containing a list of all the residues in the Workspace that were included in the analysis, and so can be considered for mutation. This table can be used to define and list the mutations for each residue. You can select multiple rows in the table, for example to set a common set of mutations. The number of residues selected and the total are reported below the table. Residues that are selected in the table are also selected in the Workspace. The table lists properties of the residues that may be useful for choosing residues to mutate.

Chain	Chain name. When mutations are applied symmetrically to multiple chains, the chains are listed here, e.g. A,B,C. If one of these chains does not have a particular residue, it is not included in the list, e.g. if only chain B has residues 1 and 2 and chains A and C start at residue 3, this column would contain only B for residues 1 and 2.
Residue	Residue number and insertion code, and 3-letter residue name.
Mutations	List of mutations that will be applied to the residue. If you click in the column, a small pane is displayed, from which you can choose the mutations. In this pane you can select multiple mutations, either by individual residue or by residue type (click Select to choose the residue type). To mutate to nonstandard residues, select them first in the Nonstandard Residues Panel; they are listed below the standard residues. You can use the search field to filter the list of nonstandard residues.The check boxes for some residues may be disabled if they may not be selected as valid mutations for the current residue: this is the case for mutations between proline and charged residues. You can select all of the standard or nonstandard residues (or both) by checking the relevant box. Click Clear All to clear the selection. The small pane closes when you click Close, click elsewhere in the main panel, press Esc or Enter, or move the pointer out of the main panel.
Disulfide	Indication of whether the residue forms a disulfide bond with another residue.
#Pi-pi	Number of pi-pi interactions formed by the residue.
#H-bonds	Number of hydrogen bonds formed by the residue with other molecules (other chains, ligands, waters, cofactors).
Buried	Percentage of the solvent-accessible surface area of the residue that is buried due to the presence of other chains. If the residue is not at the interface or there is only one chain, the column contains the text "NA", for "not applicable".
Surface Complementarity	If the residue is at the interface between two chains, the surface complementarity of the residue to residues in the other chain is evaluated and displayed in this column. The surface complementarity measures how well the shape of one surface matches the shape of the other. It is calculated from the scalar product of the normal to one surface at each surface point and the normal to the other surface at the point nearest to the first surface, with a weighting factor for the distance between the points. The score is the normalized sum over surface points, such that perfect complementarity (parallel normals) has a value of 1, and no complementarity has a value of 0. It is evaluated over the buried solvent-accessible surface area. (See Lawrence, M. C.; Colman, P. M. J. Mol. Biol., 1993, 234, 946–950.) If the residue is not at the interface or there is only one chain, the column contains the text "NA", for "not applicable".

Fit on select option

Fit the selected residues in the Workspace to the screen size, when residues are selected in the table. The selected residues are colored with green carbons, and all other residues are colored with a darker color. The color scheme remains in effect when you deselect this option.

Calculate options

Calculate the changes in the selected properties due to mutation. The change in property value that is displayed is the value for the mutated structure minus the value for the parent structure, so a positive value for the change means that the mutated structure has a larger (more positive) value than the parent structure. The change in stability is always calculated. The properties are described in more detail in Table 1 The available properties are:

Affinity—Change in binding affinity of the antibody to the rest of the system (e.g. an antigen). A negative value means that the mutant binds better than the parent antibody.
pKa—Change in acidity of the mutated residue, calculated using PROPKA. This is the most time-consuming property, so it is not selected by default.
SASA—Change in solvent-accessible surface area on mutation. This option represents three properties, total SASA, polar SASA, and nonpolar SASA. A positive value indicates an increase in SASA on mutation.
Hydropathy—Change in hydrophobic or hydrophilic nature of the mutated residue, as defined on the Kyte-Doolittle scale. A positive value indicates a more hydrophobic residue and a negative value indicates a less hydrophobic residue in the mutant.
Rot. bonds—Change in the total number of rotatable bonds.
Complementarity—Change in the van der Waals surface complementarity of residues at chain interfaces due to the mutation.

Options tab

In this tab you can set options for refinement of the residues adjacent to the mutations.

Refinement option menu

Choose the method for refinement of the mutated residues and optionally of nearby residues as well.

When residues are mutated, the structure around the mutation site should be allowed to relax in response to the mutation. Before the minimization of the region around the mutation site, a side-chain prediction of the mutated residues is performed, which does a thorough exploration of possible side-chain conformations.

Side-chain prediction—Run a Prime side-chain prediction, adjusting only the side-chain rotamers (repacking), then minimizing the side-chain atoms.
Side-chain prediction with backbone minimization—Run a Prime side-chain prediction first, adjusting only the side chains, followed by a minimization of the entire residue. This allows relaxation of the entire residue.
Side-chain prediction with Cbeta sampling—Run a Prime side-chain prediction that includes variation of the CA-CB direction as well as side-chain rotamer adjustment, followed by minimization of the entire residue.
Side-chain prediction with backbone sampling—Run a Prime side-chain prediction that includes backbone adjustments of the atoms between the CA atoms on the adjacent residues, as well as CA-CB adjustments and side-chain rotamer adjustments, followed by minimization.

Cutoff text box

Specify the cutoff distance for including residues in the refinement that are near to the mutated residues. Any residue that has an atom within the specified distance of a hypothetical Arg residue at the mutation site is included in the refinement. A hypothetical Arg residue is used to ensure that the set of residues refined is identical regardless of the initial or mutated residue identities.

Define Nonstandard Residues button

Define nonstandard residues to be used for mutating the structure. Opens the Nonstandard Residues Panel.

Job toolbar

Manage job submission and settings. See Job Toolbar for a description of this toolbar.

The Job Settings button opens the Antibody Humanization: Residue Mutation - Job Settings Dialog Box, where you can make settings for running the job.

Status bar

The status bar displays information about the current job settings and status for the panel. The settings includes the job name, task name and task settings (if any), number of subjobs (if any) and the host name and job incorporation setting. The job status can include messages about job start, job completion and incorporation.

Use the Reset button to reset the panel to its default settings and clear any data from the panel. You can also reset the panel from the Job toolbar.

The status bar also contains the Help button , which opens the help topic for the panel in your browser. If the panel is used by one or more tutorials, hovering over the Help button displays a button, which you can click to display a list of tutorials (or you can right-click the Help button instead). Choosing a tutorial opens the tutorial topic.

Tutorials