This is the first known high-resolution structure of the A3-B3 domains of CEACAM5, including Nlinked glycans, and the first detailed epitope mapping of a monoclonal antibody against CEACAM5. Four other human CEACAM structures have been solved, but they are all limited to the Nterminal IgV-like domain (CEACAM1 [PDB: 6XNO], CEACAM5 [PDB: 2QSQ], CEACAM6 [PDB: 4WHC], and CEACAM8 [PDB: 4YIQ]).
The epitopes and paratopes identified by HDX-MS, SPR, and cryo-EM in this study were in general agreement. Although there were some differences, this is not unexpected given the differences in how these techniques probe protein structure and macromolecular interactions. The identification of a set of residues in both the hCEACAM5A3 − B3 epitope and tusa Fab paratope by multiple methods provides greater confidence that these amino acids play a critical role in antibody recognition and specific binding between hCEACAM5 and tusa Fab, compared with epitope/paratope residues that were only identified by a single method (Fig. 3)
Antibody paratopes preferentially involve aromatic residues (Tyr, Trp, and Phe), amino acids with short hydrophilic side chains (Ser, Thr, Asp, and Asn), and glycine 19,20. Consistent with these propensities, 13 of 15 (87%) of the amino acids in the tusa Fab paratope that were identified by 2 + methods (8 of 9 in the heavy chain and 5 of 6 in the light chain) are one of these amino acids. These residues tend to be enriched in antibody paratopes because they can form multiple, diverse interactions, including hydrogen bonds, hydrophobic interactions, and van der Waals interactions, that stabilize the paratope-epitope interface. In addition, the clustering of these residues in the heavy chain, particularly CDR3, is consistent with the importance of this region in antibody-binding specificity. 21 Furthermore, the large number of hydrogen bonds, predominantly in the tusa Fab heavy chain, is in agreement with the mean (standard deviation) hydrogen bonds that occur in the VH (5.2 ± 3.0) and VL (2.4 ± 2.0) domains 22. These findings suggest that these 13 amino acids in the tusa Fab paratope that were identified by multiple methods contribute to the specificity and affinity of tusamitamab for hCEACAM5. Of these, 6 residues (heavy chain: Y32, D33, Y100, and F101; light chain: Y32 and N50) that completely eliminated binding to hCEACAM5A3 − B3 when mutated to alanine and 2 residues in the light chain (F30 and Y92) that had weaker binding and ΔΔG > − 2.5 kcal/mol when mutated to alanine (Table 1) are likely to be key.
The hCEACAM5 epitope involves residues in both the A3 and B3 domains; however, the latter provides a greater overall contribution to binding. Most epitope interactions within the B3 domain involve 6 residues (S622, Q624, Q635, H636, T637, and V639) that account for all of the hydrogen bonds with the tusa Fab heavy chain and 2 of the 3 hydrogen bonds with the light chain (Fig. 3). One amino acid in particular, Q624, hydrogen bonds with both the heavy and light chains. In addition, 3 hydrophobic residues (F641, L660, and A661) may contribute hydrophobic interactions. Four of the 6 hydrogen-bonding residues (Q635, H636, T637, and V639) and 1 of the 3 hydrophobic residues (F641) were identified by multiple methods and are likely to be critical to the hCEACAM5 epitope.
In addition, an N-linked mannose at residue 612 in the B3 domain forms a hydrogen bond with the tusa Fab heavy chain. Because N-glycosylation tends to decrease protein flexibility and increase protein stability globally 23,24, all Nlinked glycans in hCEACAM5A3 − B3, including the one linked to N612, may play an important role in shaping the overall conformation that is recognized by the tusa Fab and by other proteins involved in the cellular function of hCEACAM5. The role of glycans in CEACAM5 is also important due to the absence of salt bridges in the hCEACAM5-tusa Fab binding interface. Salt bridges play an important role in antibody-antigen interactions by imposing geometric constraints that limit conformational flexibility 25–27. It is possible that the N-linked mannose at residue 612 may have a compensatory, stabilizing function, similar to critical anchor residues in other protein complexes without salt bridges28. This would increase the rigidity of the epitope-paratope interface and, consequently, the specificity of Fab796 for hCEACAM5A3 − B3.
We hypothesize that the specificity of the tusa Fab for the A3-B3 domains of hCEACAM5 is due to diverse types of interactions between the tusa Fab heavy and light chains and hCEACAM5A3 − B3 combined with conformational constraints on the epitope-paratope interface (shape complementarity), which is known to play an important role in molecular recognition 22. This binding mechanism may enable tusamitamab to differentiate hCEACAM5 from other human CEACAMs (61–80% identity between the ECDs of CEACAM5 and CEACAMs 1, 6, and 8; Supplementary Fig. S1) and differentiate the A3B3 domain from the A1-B1 and A2-B2 domains in hCEACAM5, which are 72% and 67% identical, respectively (Supplementary Fig. S7).
Human CEACAM5A3 − B3 epitopes such as the one reported here may not be unique. Recently, Baek et al. 29 isolated from a human Fab phage-displayed library a monoclonal antibody, 1G9, that also specifically binds the A3B3 domains of hCEACAM5. Initial epitope-mapping experiments using negative-stain transmission electron microscopy and site-directed mutagenesis suggested that the 1G9 epitope involves glycans at residues 612 and 650 in the B3 domain. Assuming that the epitope is located between these residues, it may partially overlap with the epitope recognized by tusamitamab but is likely to be distinct (Fig. 4). In agreement with our findings, the 1G9 epitope supports the importance of the B3 domain in specific antibody recognition of hCEACAM5.
The main strengths of this study were the use of multiple epitope mapping methods. The high quality of the cryo-EM structure enabled the determination of a high-resolution structure of hCEACAM5A3 − B3 including linked glycans. In addition, the high sequence coverage of hCEACAM5 peptides that were identified by HDX-MS provides confidence that other possible epitopes were not missed in that analysis.
Our findings support ongoing clinical development of tusamitamab ravtansine as well as future identification and/or design of next-generation antibody therapeutics targeting hCEACAM5. Currently, clinical trials of tusamitamab ravtansine are underway in patients with several types of cancer, including NSCLC (CARMEN-LC03, NCT04154956) 30, gastric cancer (CARMEN-GC01, NCT05071053) 31, and breast and pancreatic cancer (CARMEN-BT01, NCT04659603) 32.