Reference
Reference TypeLiterature
IEDB_Reference:1045650
TitleEngineered ipilimumab variants that bind human and mouse CTLA-4.
AuthorsBrett Robison; S J Diong; Anusha Kumar; Thomas M Moon; Olin Chang; Bryant Chau; Christine Bee; Ishita Barman; Arvind Rajpal; Alan J Korman; Sean West; Pavel Strop; Peter S Lee
AffiliationsDiscovery Biotherapeutics, Bristol Myers Squibb, Redwood City, CA, USA; Large Molecule Drug Discovery, Genentech, Research and Early Development, South San Francisco, CA, USA; Biochemistry and Biophysics, Merck, South San Francisco, CA, USA; Protein and Antibody Portfolio, Genscript, South San Francisco, CA, USA; Research, Xaira Therapeutics, Brisbane, CA, USA; Research, Vir Biotechnology, San Francisco, CA, USA; Research, Tallac Therapeutics, Burlingame, CA, USA; Biotherapeutics and Genetic Medicine, AbbVie, South San Francisco, CA, USA.
JournalMAbs
PMID:39849917
Year2025
AbstractTesting of candidate monoclonal antibody therapeutics in preclinical models is an essential step in drug development. Identification of antibody therapeutic candidates that bind their human targets and cross-react to mouse orthologs is often challenging, especially for targets with low sequence homology. In such cases, surrogate antibodies that bind mouse orthologs must be used. The antibody 9D9, which binds mouse CTLA-4, is a commonly used surrogate for CTLA-4 checkpoint blockade studies in mouse cancer models. In this work, we reveal that 9D9 has significant biophysical dissimilarities to therapeutic CTLA-4 antibodies. The 9D9-mCTLA4 complex crystal structure was determined and shows that the surrogate antibody binds an epitope distinct from ipilimumab and tremelimumab. In addition, while ipilimumab has pH-independent binding to hCTLA-4, 9D9 loses binding to mCTLA-4 at physiologically relevant acidic pH ranges. We used phage and yeast display to engineer ipilimumab to bind mouse CTLA-4 with single-digit nM affinity from an initial state with no apparent binding. The engineered variants showed pH-independent and cross-reactive binding to both mouse and human CTLA-4. Crystal structures of a variant in complex with both mouse and human CTLA-4 confirmed that it targets an equivalent epitope as ipilimumab. These cross-reactive ipilimumab variants may facilitate improved translatability and future mechanism-of-action studies for anti-CTLA-4 targeting in murine models.
Curation Last Updated2025-03-14 20:01:13
Epitope
Epitope ID2275083
IEDB_epitope:2275083
Chemical TypeDiscontinuous peptide
Source NameCytotoxic T-lymphocyte protein 4
UniProt:P09793
Source OrganismMus musculus (mouse)
NCBITaxon:10090
Discontinuous ResiduesI38, Q39, E68, R70, L74, M81, E83, T88, Y100, K130, E132, M134, Y135, P138, Y139, F140, V141, G142, M143
Epitope Reference Details
Epitope Structure DefinesExact Epitope
Epitope NameEpitope of Fab mipi.4 on mCTLA4
Reference RegionI1, Q2, E31, R33, L37, M44, E46, T51, Y63, K93, E95, M97, Y98, P101, Y102, F103, V104, G105, M106
CommentsThe epitope residues were calculated from [PDB: 9DQ4] as the antigen residues at 4Å atomic distance from the antibody.
Location of Data in ReferencePDB 9DQ4
Immunization
Host OrganismMus musculus HuMAb (Medarex)
ONTIE:0000525
1st In Vivo Process
In Vivo Process TypeAdministration in vivo
OBI:0600007
1st Immunogen
Epitope RelationStructurally Related
Chemical TypeProtein
Molecule NameCytotoxic T-lymphocyte protein 4
UniProt:P16410.3
OrganismHomo sapiens (human)
NCBITaxon:9606
Immunogen Details
Immunogen Reference NamehCTLA-4
Immunization Comments
Immunization CommentsIpilimumab, described in cited reference [PMID: 28978021] is the fully humanized version of the parent monoclonal antibody (10D1) originally described in reference [PMID: 14634142]. Ipilimumab was engineered to bind both human and mouse CTLA-4 using yeast and phage display of mutated VH and VL resulting in the final selection of mipi.4.
B Cell Assay
Qualitative MeasurementPositive
Method/Techniquex-ray crystallography
OBI:0001738
Measurement of3D structure
Assay Type Unitsangstroms
Measurement Details
Measurement Inequality=
Quantitative measurement1.57
Assayed Antibody
Assayed Antibody Source MaterialAntibody construct
Assayed Antibody Immunoglobulin DomainFab
Assayed Antibody Purification StatusDisplay Library (monoclonal)
Assayed Antibody Namemipi.4
Assayed Antibody Heavy Chain TypeIgG
Assayed Antibody Light Chain TypeKappa
Assayed Antibody Object
Chemical TypeMulti-Chain protein
Chain 1 Accession NameChain H, mipi.4 heavy chain
GenPept:9DQ3_H
Source OrganismHomo sapiens (human)
NCBITaxon:9606
Molecule Namemipi4
Chain 2 NameChain L, mipi.4 light chain
GenPept:9DQ3_L
Source OrganismHomo sapiens (human)
NCBITaxon:9606
Antigen
Epitope RelationSource Antigen
Chemical TypeProtein
Molecule NameCytotoxic T-lymphocyte protein 4
UniProt:P09793
OrganismMus musculus (mouse)
NCBITaxon:10090
3D Structure of Complex
Complex PDB ID9DQ4
PDB:9DQ4
Antibody Chain 1 PDB ChainH
Antibody Chain 2 PDB ChainL
Antigen PDB ChainA
CommentsThe crystal structure showed the binding of mipi.4 to mCTLA-4 (this structure) mirrors its interaction with hCTLA-4 [PDB: 9DQ3]. mipi.4 residues that contact mCTLA-4 are nearly identical to those that contact hCTLA-4 with heavy and light chains contributing to the buried surface area in the same proportions as observed with hCTLA-4 (60% and 40%, respectively).
Calculated Contacts
Epitope ResiduesA: I1, Q2, E31, R33, L37, M44, E46, T51, Y63, K93, E95, M97, Y98, P101, Y102, F103, V104, G105, M106
Antibody Residues Interacting with AntigenH: K31, V33, S52, Y53, R56, H57, K58, Y59, W101, L102; L: V29, G30, W31, Y92, G93, Q94, S95, W97
Contact Area for Antibody 928
View 3D Structure
[View 3D Structure]
Assay Reference Details
Assay Comments by IEDB CuratorThe epitope for Fab mipi.4 on mCTLA4 was determined by the crystal structure of the complex.
Location of Assay Data in ReferenceFigures 5 and 6, Supplementary Table 2, and PDB 9DQ4