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. 2025 Dec;17(1):2451296.
doi: 10.1080/19420862.2025.2451296. Epub 2025 Jan 24.

Engineered ipilimumab variants that bind human and mouse CTLA-4

Brett Robison  1 , S J Diong  1 , Anusha Kumar  1 , Thomas M Moon  1 , Olin Chang  1 , Bryant Chau  2 , Christine Bee  3 , Ishita Barman  4 , Arvind Rajpal  5 , Alan J Korman  6 , Sean West  1 , Pavel Strop  7 , Peter S Lee  8
Affiliations

Engineered ipilimumab variants that bind human and mouse CTLA-4

Brett Robison et al. MAbs. 2025 Dec.

Abstract

Testing 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.

Keywords: Antibody engineering; CTLA-4; immune checkpoint; phage display; species cross reactivity; yeast display.

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Conflict of interest statement

The sequences of the mipi variants are provided in this work.

Figures

(a) SPR equilibrium binding constants as function of pH for ipi and 9D9 binding showing less binding of 9D9 at low pH and uniform binding of ipi. (b) Beta sheet fold diagram of CTLA-4. (c) Crystal structure of mouse CTLA-4 bound by 9D9 Fab on its membrane-distal end. (d) Crystal structure of human CTLA-4 bound on its side by ipi Fab and crystal structure of human CTLA-4 bound on its side by treme Fab. (e) The epitopes of human and mouse CTLA-4 binders compared to B7 ligand interfaces.
Figure 1.
Characterization of 9D9 binding to mouse CTLA-4.
(a) Phage display process producing mipi.1 followed by yeast display process using mipi.1 as a starting point. (b) Yeast display selection round conditions for mipi engineering. (c) Amino acid sequence depiction of parental ipi and engineered mipi variants with up to nine mutations. (d) Deep mutational scanning heatmaps showing the enrichment for each possible mutation to the ipi complementarity determining regions following mipi selections.
Figure 2.
Engineering mouse CTLA-4 binding into ipilimumab.
Surface plasmon resonance binding sensorgrams showing improved binding of four mipi variants to mouse CTLA-4 and human CTLA-4.
Figure 3.
Measurement of fab – CTLA-4 interactions at pH 6.0 and 7.4 by SPR.
(a) Increased binding of mipi IgGs and Fabs to human CTLA-4 expressing cells. (b) Increased binding of mipi IgGs and Fabs to mouse CTLA-4 expressing cells. (c) Increased human CTLA-4 checkpoint blockade reporter cell-line activity with mipi variant IgG and Fab molecules.
Figure 4.
Mipi variant cell binding and CTLA-4 blockade.
(a) Structural alignment of human and mouse CTLA-4 bound by mipi.4 variable domains showing similarity of the complex structures. (b) Ipi epitope overlaid on human CTLA-4. (c) mipi.4 epitope overlaid on human CTLA-4 showing similarity to ipi. (d) mipi.4 epitope overlaid on mouse CTLA-4 showing similarity to ipi-hCTLA-4. (e) Alignment of human and mouse CTLA-4 extracellular domain amino acid sequences with epitope residues highlighted for ipi and mipi.4.
Figure 5.
Structural comparison of ipilimumab and mipi.4 binding to human and mouse CTLA-4.
(a) Surface representations of human and mouse CTLA-4 with ipi and mipi.4 complementarity determining regions overlaid to show similarity. (b) Alignment of ipi and mipi.4 variable fragment structures and complementarity determining regions showing similarity of crystal structure conformations. (c) Lists of paratope residues from crystal structures of ipi and mipi.4. (d) Buried surface area for key paratope residues for ipi and mipi.4 from their respective complex structures showing similarity.
Figure 6.
Structural comparison of ipilimumab and mipi.4 paratopes.
An overlay of ipi and mipi.4 paratopes on human and mouse CTLA-4 showing key interactions generated by engineered mutations. (a) Two key ipi paratope residues showing one electrostatic interaction with hCTLA-4. (b) Two key mipi.4 paratope residues showing four electrostatic interactions to hCTLA-4. (c) Two key mipi.4 paratope residues showing three electrostatic interactions with mCTLA-4. (d) A different key ipi residue showing no electrostatic interactions with a different region of hCTLA-4. (e) A different key mipi.4 paratope residue gaining two electrostatic interactions with hCTLA-4. (f) The same mipi.4 paratope residue showing interaction with mouse CTLA-4.
Figure 7.
Electrostatic interactions guide mipi.4 cross-reactive binding to human and mouse CTLA-4.
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