High Specificity of Germline Antibodies Targeting Hapten: A Universally Intrinsic Feature
Abstract
Polyspecificity has been widely regarded as a fundamental feature of germline antibodies that enables broad antigen recognition with the limited size of the germline antibody repertoire. However, accumulating evidence indicates that germline antibodies against carbohydrate antigens can exhibit high specificity, thereby challenging this traditional paradigm. To investigate whether the polyspecificity paradigm applies to haptens (single-epitope antigens), we inferred and identified 14 germline antibodies targeting distinct haptens; unexpectedly, all exhibited intrinsically high specificity. To explore the underlying mechanisms, we constructed a comprehensive sequence-structure database of germline antibodies recognizing haptens, peptides, and proteins. Our results reveal that hapten-binding germline antibodies are characterized by shorter CDRH3 regions, enrichment of aromatic residues in CDRs, and fewer CDR-localized surface patches—features that may underlie their intrinsically high specificity. Conversely, germline antibodies recognizing peptides and proteins display longer CDRH3 regions, increased enrichment of polar and charged residues, and denser CDR-localized surface patches, providing structural determinants of their greater polyspecificity. In addition, germline gene usage biases—particularly V-gene selection and IGHV–IGKV/IGLV pairing patterns—collectively shape the antigen-binding properties of germline antibodies. Together, these findings represent the first large-scale, systematic analysis of germline antibody polyspecificity and have broad implications for fundamental immunology, vaccine development, and antibody discovery.
References
1. H. W. Schroeder, L. Cavacini, Structure and function of immunoglobulins. Journal of Allergy and Clinical Immunology 125, S41–S52 (2010).
2. Y. Zhang, Q. Li, L. Luo, C. Duan, J. Shen, Z. Wang, Application of germline antibody features to vaccine development, antibody discovery, antibody optimization and disease diagnosis. Biotechnology Advances 65, 108143 (2023).
3. S. Vajda, K. A. Porter, D. Kozakov, Progress toward improved understanding of antibody maturation. Curr Opin Struct Biol 67, 226–231 (2021).
4. J. R. Willis, B. S. Briney, S. L. DeLuca, J. E. Crowe, J. Meiler, Human germline antibody gene segments encode polyspecific antibodies. PLoS Comput Biol 9, e1003045 (2013).
5. A. T. DeLaitsch, J. R. Pridgen, A. Tytla, M. L. Peach, R. Hu, D. W. Farnsworth, A. K. McMillan, N. Flanagan, J. S. Temme, M. C. Nicklaus, J. C. Gildersleeve, Selective Recognition of Carbohydrate Antigens by Germline Antibodies Isolated from AID Knockout Mice. J. Am. Chem. Soc. 144, 4925–4941 (2022).
6. E. Sterner, M. L. Peach, M. C. Nicklaus, J. C. Gildersleeve, Therapeutic Antibodies to Ganglioside GD2 Evolved from Highly Selective Germline Antibodies. Cell Reports 20, 1681–1691 (2017).
7. M. Babor, T. Kortemme, Multi-constraint computational design suggests that native sequences of germline antibody H3 loops are nearly optimal for conformational flexibility. Proteins 75, 846–858 (2009).
8. D. W. Evans, S. Muller-Loennies, C. L. Brooks, L. Brade, P. Kosma, H. Brade, S. V. Evans, Structural insights into parallel strategies for germline antibody recognition of lipopolysaccharide from Chlamydia. Glycobiology 21, 1049–1059 (2011).
9. L. Scharf, A. P. West, S. A. Sievers, C. Chen, S. Jiang, H. Gao, M. D. Gray, A. T. McGuire, J. F. Scheid, M. C. Nussenzweig, L. Stamatatos, P. J. Bjorkman, Structural basis for germline antibody recognition of HIV-1 immunogens. eLife 5, e13783 (2016).
10. L. Scharf, A. P. West, H. Gao, T. Lee, J. F. Scheid, M. C. Nussenzweig, P. J. Bjorkman, R. Diskin, Structural basis for HIV-1 gp120 recognition by a germ-line version of a broadly neutralizing antibody. PNAS 110, 6049–6054 (2013).
11. S. Hoot, A. T. McGuire, K. W. Cohen, R. K. Strong, L. Hangartner, F. Klein, R. Diskin, J. F. Scheid, D. N. Sather, D. R. Burton, L. Stamatatos, Recombinant HIV Envelope Proteins Fail to Engage Germline Versions of Anti-CD4bs bNAbs. PLOS Pathogens 9, e1003106 (2013).
12. D. K. Sethi, A. Agarwal, V. Manivel, K. V. S. Rao, D. M. Salunke, Differential Epitope Positioning within the Germline Antibody Paratope Enhances Promiscuity in the Primary Immune Response. Immunity 24, 429–438 (2006).
13. F. E. Romesberg, B. Spiller, P. G. Schultz, R. C. Stevens, Immunological origins of binding and catalysis in a Diels-Alderase antibody. Science 279, 1929–1933 (1998).
14. G. J. Wedemayer, P. A. Patten, L. H. Wang, P. G. Schultz, R. C. Stevens, Structural Insights into the Evolution of an Antibody Combining Site. Science, New Series 276, 1665–1669 (1997).
15. P. A. Patten, N. S. Gray, P. L. Yang, C. B. Marks, G. J. Wedemayer, J. J. Boniface, R. C. Stevens, P. G. Schultz, The Immunological Evolution of Catalysis. Science 271, 1086–1091 (1996).
16. H. P. Nguyen, N. O. L. Seto, C. R. MacKenzie, L. Brade, P. Kosma, H. Brade, S. V. Evans, Germline antibody recognition of distinct carbohydrate epitopes. Nat Struct Mol Biol 10, 1019–1025 (2003).
17. R. Jimenez, G. Salazar, J. Yin, T. Joo, F. E. Romesberg, Protein dynamics and the immunological evolution of molecular recognition. Proc Natl Acad Sci U S A 101, 3803–3808 (2004).
18. S. Éliás, C. Wrzodek, C. M. Deane, A. C. Tissot, S. Klostermann, F. Ros, Prediction of polyspecificity from antibody sequence data by machine learning. Front. Bioinform. 3 (2024).
19. K. A. K. Finton, D. Friend, J. Jaffe, M. Gewe, M. A. Holmes, H. B. Larman, A. Stuart, K. Larimore, P. D. Greenberg, S. J. Elledge, L. Stamatatos, R. K. Strong, Ontogeny of Recognition Specificity and Functionality for the Broadly Neutralizing Anti-HIV Antibody 4E10. PLOS Pathogens 10, e1004403 (2014).
20. X. Xiao, W. Chen, Y. Feng, Z. Zhu, P. Prabakaran, Y. Wang, M.-Y. Zhang, N. S. Longo, D. S. Dimitrov, Germline-like predecessors of broadly neutralizing antibodies lack measurable binding to HIV-1 envelope glycoproteins: Implications for evasion of immune responses and design of vaccine immunogens. Biochemical and Biophysical Research Communications 390, 404–409 (2009).
21. V. Manivel, N. C. Sahoo, D. M. Salunke, K. V. S. Rao, Maturation of an Antibody Response Is Governed by Modulations in Flexibility of the Antigen-Combining Site. Immunity 13, 611–620 (2000).
22. H. P. Nguyen, N. O. L. Seto, C. R. MacKenzie, L. Brade, P. Kosma, H. Brade, S. V. Evans, Germline antibody recognition of distinct carbohydrate epitopes. Nat Struct Mol Biol 10, 1019–1025 (2003).
23. R. Adhikary, W. Yu, M. Oda, R. C. Walker, T. Chen, R. L. Stanfield, I. A. Wilson, J. Zimmermann, F. E. Romesberg, Adaptive Mutations Alter Antibody Structure and Dynamics during Affinity Maturation. Biochemistry 54, 2085–2093 (2015).
24. J. Yin, S. E. Andryski, A. E. Beuscher, R. C. Stevens, P. G. Schultz, Structural evidence for substrate strain in antibody catalysis. Proc Natl Acad Sci U S A 100, 856–861 (2003).
25. L. C. James, P. Roversi, D. S. Tawfik, Antibody multispecificity mediated by conformational diversity. Science 299, 1362–1367 (2003).
26. E. A. Padlan, C. Abergel, J. P. Tipper, Identification of specificity-determining residues in antibodies. The FASEB Journal 9, 133–139 (1995).
27. G. Raghunathan, J. Smart, J. Williams, J. C. Almagro, Antigen-binding site anatomy and somatic mutations in antibodies that recognize different types of antigens. J Mol Recognit 25, 103–113 (2012).
28. T. Khan, D. M. Salunke, Structural elucidation of the mechanistic basis of degeneracy in the primary humoral response. J Immunol 188, 1819–1827 (2012).
29. J. Yin, A. E. Beuscher, S. E. Andryski, R. C. Stevens, P. G. Schultz, Structural Plasticity and the Evolution of Antibody Affinity and Specificity. Journal of Molecular Biology 330, 651–656 (2003).
30. J. Zimmermann, E. L. Oakman, I. F. Thorpe, X. Shi, P. Abbyad, C. L. Brooks, S. G. Boxer, F. E. Romesberg, Antibody evolution constrains conformational heterogeneity by tailoring protein dynamics. Proceedings of the National Academy of Sciences 103, 13722–13727 (2006).
31. G. J. Wedemayer, L. H. Wang, P. A. Patten, P. G. Schultz, R. C. Stevens, Crystal structures of the free and liganded form of an esterolytic catalytic antibody1. Journal of Molecular Biology 268, 390–400 (1997).
32. I. F. Thorpe, C. L. Brooks, Molecular evolution of affinity and flexibility in the immune system. Proc. Natl. Acad. Sci. U.S.A. 104, 8821–8826 (2007).
33. T. Li, M. B. Tracka, S. Uddin, J. Casas-Finet, D. J. Jacobs, D. R. Livesay, Rigidity Emerges during Antibody Evolution in Three Distinct Antibody Systems: Evidence from QSFR Analysis of Fab Fragments. PLOS Computational Biology 11, e1004327 (2015).
34. S. Alberti, A. Gladfelter, T. Mittag, Considerations and Challenges in Studying Liquid-Liquid Phase Separation and Biomolecular Condensates. Cell 176, 419–434 (2019).
35. H. Ausserwöger, M. M. Schneider, T. W. Herling, P. Arosio, G. Invernizzi, T. P. J. Knowles, N. Lorenzen, Non-specificity as the sticky problem in therapeutic antibody development. Nat Rev Chem 6, 844–861 (2022).
36. N. E. Kaleli, M. Karadag, S. Kalyoncu, Phage display derived therapeutic antibodies have enriched aliphatic content: Insights for developability issues. Proteins 87, 607–618 (2019).
37. M. I. J. Raybould, C. Marks, K. Krawczyk, B. Taddese, J. Nowak, A. P. Lewis, A. Bujotzek, J. Shi, C. M. Deane, Five computational developability guidelines for therapeutic antibody profiling. Proc Natl Acad Sci U S A 116, 4025–4030 (2019).
38. Y. Zhang, C. Duan, Q. Li, Y. Bai, B. Dong, Y. Tang, M. He, C. Hao, K. Wen, J. Shen, Z. Wang, Fluorescence polarization immunoassay based on fragmentary hapten for rapid and sensitive screening of polymyxins in human serum. Sensors and Actuators B: Chemical 370, 132404 (2022).
39. Z. Wang, R. C. Beier, Y. Sheng, S. Zhang, W. Jiang, Z. Wang, J. Wang, J. Shen, Monoclonal antibodies with group specificity toward sulfonamides: selection of hapten and antibody selectivity. Anal Bioanal Chem 405, 4027–4037 (2013).
40. H. Li, B. Dong, L. Dou, W. Yu, X. Yu, K. Wen, Y. Ke, J. Shen, Z. Wang, Fluorescent lateral flow immunoassay for highly sensitive detection of eight anticoagulant rodenticides based on cadmium-free quantum dot-encapsulated nanospheres. Sensors and Actuators B: Chemical 324, 128771 (2020).
41. Y. Zhang, J. Mi, W. Wu, J. Fei, B. Lv, X. Yu, K. Wen, J. Shen, Z. Wang, Investigation of Antibody Tolerance in Methanol for Analytical Purposes: Methanol Effect Patterns and Molecular Mechanisms. Advanced Science 11, 2402050 (2024).
42. J. Dunbar, K. Krawczyk, J. Leem, T. Baker, A. Fuchs, G. Georges, J. Shi, C. M. Deane, SAbDab: the structural antibody database. Nucleic Acids Research 42, D1140–D1146 (2014).
43. Y. Barrios, P. Jirholt, M. Ohlin, Length of the antibody heavy chain complementarity determining region 3 as a specificity-determining factor. Journal of Molecular Recognition 17, 332–338 (2004).
44. A. V. J. Collis, A. P. Brouwer, A. C. R. Martin, Analysis of the Antigen Combining Site: Correlations Between Length and Sequence Composition of the Hypervariable Loops and the Nature of the Antigen. Journal of Molecular Biology 325, 337–354 (2003).
45. M. L. Chiu, G. L. Gilliland, Engineering antibody therapeutics. Curr Opin Struct Biol 38, 163–173 (2016).
46. J. Abramson, J. Adler, J. Dunger, R. Evans, T. Green, A. Pritzel, O. Ronneberger, L. Willmore, A. J. Ballard, J. Bambrick, S. W. Bodenstein, D. A. Evans, C.-C. Hung, M. O’Neill, D. Reiman, K. Tunyasuvunakool, Z. Wu, A. Žemgulytė, E. Arvaniti, C. Beattie, O. Bertolli, A. Bridgland, A. Cherepanov, M. Congreve, A. I. Cowen-Rivers, A. Cowie, M. Figurnov, F. B. Fuchs, H. Gladman, R. Jain, Y. A. Khan, C. M. R. Low, K. Perlin, A. Potapenko, P. Savy, S. Singh, A. Stecula, A. Thillaisundaram, C. Tong, S. Yakneen, E. D. Zhong, M. Zielinski, A. Žídek, V. Bapst, P. Kohli, M. Jaderberg, D. Hassabis, J. M. Jumper, Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature, 1–3 (2024).
47. D. Kuroda, H. Shirai, M. Kobori, H. Nakamura, Structural classification of CDR-H3 revisited: a lesson in antibody modeling. Proteins 73, 608–620 (2008).
48. B. North, A. Lehmann, R. L. Dunbrack, A new clustering of antibody CDR loop conformations. J Mol Biol 406, 228–256 (2011).
49. Y. Tsuchiya, K. Mizuguchi, The diversity of H3 loops determines the antigen-binding tendencies of antibody CDR loops. Protein Sci 25, 815–825 (2016).
50. K.-M. A. Dam, H. B. Gristick, Y. E. Li, Z. Yang, P. N. P. Gnanapragasam, A. P. West, M. S. Seaman, P. J. Bjorkman, Mapping essential somatic hypermutations in a CD4-binding site bNAb informs HIV-1 vaccine design. Cell Reports 44 (2025).
51. S. Birtalan, Y. Zhang, F. A. Fellouse, L. Shao, G. Schaefer, S. S. Sidhu, The Intrinsic Contributions of Tyrosine, Serine, Glycine and Arginine to the Affinity and Specificity of Antibodies. Journal of Molecular Biology 377, 1518–1528 (2008).
52. L. Lo Conte, C. Chothia, J. Janin, The atomic structure of protein-protein recognition sites. J Mol Biol 285, 2177–2198 (1999).
53. I. S. Mian, A. R. Bradwell, A. J. Olson, Structure, function and properties of antibody binding sites. J Mol Biol 217, 133–151 (1991).
54. C. R. Rupakheti, B. Roux, F. Dehez, C. Chipot, Modeling induction phenomena in amino acid cation– $$pi $$ interactions. Theor Chem Acc 137, 1–6 (2018).
55. R. L. Kelly, D. Le, J. Zhao, K. D. Wittrup, Reduction of Nonspecificity Motifs in Synthetic Antibody Libraries. J Mol Biol 430, 119–130 (2018).
56. C.-H. Luan, T. M. Parker, D. C. Gowda, D. W. Urry, Hydrophobicity of amino acid residues: Differential scanning calorimetry and synthesis of the aromatic analogues of the polypentapeptide of elastin. Biopolymers 32, 1251–1261 (1992).
57. B. S, F. Rd, S. Ss, The functional capacity of the natural amino acids for molecular recognition. Molecular bioSystems 6 (2010).
58. D. Jain, D. M. Salunke, Antibody specificity and promiscuity. Biochem J 476, 433–447 (2019).
59. Z.-H. Zhou, Y. Zhang, Y.-F. Hu, L. M. Wahl, J. O. Cisar, A. L. Notkins, The broad antibacterial activity of the natural antibody repertoire is due to polyreactive antibodies. Cell Host Microbe 1, 51–61 (2007).
60. H. Mouquet, M. C. Nussenzweig, Polyreactive antibodies in adaptive immune responses to viruses. Cell. Mol. Life Sci. 69, 1435–1445 (2012).
61. A. G. Schmidt, H. Xu, A. R. Khan, T. O’Donnell, S. Khurana, L. R. King, J. Manischewitz, H. Golding, P. Suphaphiphat, A. Carfi, E. C. Settembre, P. R. Dormitzer, T. B. Kepler, R. Zhang, M. A. Moody, B. F. Haynes, H.-X. Liao, D. E. Shaw, S. C. Harrison, Preconfiguration of the antigen-binding site during affinity maturation of a broadly neutralizing influenza virus antibody. Proceedings of the National Academy of Sciences 110, 264–269 (2013).
62. H. Ausserwöger, G. Krainer, T. J. Welsh, N. Thorsteinson, E. de Csilléry, T. Sneideris, M. M. Schneider, T. Egebjerg, G. Invernizzi, T. W. Herling, N. Lorenzen, T. P. J. Knowles, Surface patches induce nonspecific binding and phase separation of antibodies. Proceedings of the National Academy of Sciences 120, e2210332120 (2023).
63. M. Sangesland, A. S. Yousif, L. Ronsard, S. W. Kazer, A. L. Zhu, G. J. Gatter, M. R. Hayward, R. M. Barnes, M. Quirindongo-Crespo, D. Rohrer, N. Lonberg, D. Kwon, A. K. Shalek, D. Lingwood, A Single Human VH-gene Allows for a Broad-Spectrum Antibody Response Targeting Bacterial Lipopolysaccharides in the Blood. Cell Rep 32, 108065 (2020).
64. J. Jardine, J.-P. Julien, S. Menis, T. Ota, O. Kalyuzhniy, A. McGuire, D. Sok, P.-S. Huang, S. MacPherson, M. Jones, T. Nieusma, J. Mathison, D. Baker, A. B. Ward, D. R. Burton, L. Stamatatos, D. Nemazee, I. A. Wilson, W. R. Schief, Rational HIV Immunogen Design to Target Specific Germline B Cell Receptors. Science 340, 711–716 (2013).
65. X. Brochet, M.-P. Lefranc, V. Giudicelli, IMGT/V-QUEST: the highly customized and integrated system for IG and TR standardized V-J and V-D-J sequence analysis. Nucleic Acids Research 36, W503–W508 (2008).
66. M. Baek, F. DiMaio, I. Anishchenko, J. Dauparas, S. Ovchinnikov, G. R. Lee, J. Wang, Q. Cong, L. N. Kinch, R. D. Schaeffer, C. Millán, H. Park, C. Adams, C. R. Glassman, A. DeGiovanni, J. H. Pereira, A. V. Rodrigues, A. A. van Dijk, A. C. Ebrecht, D. J. Opperman, T. Sagmeister, C. Buhlheller, T. Pavkov-Keller, M. K. Rathinaswamy, U. Dalwadi, C. K. Yip, J. E. Burke, K. C. Garcia, N. V. Grishin, P. D. Adams, R. J. Read, D. Baker, Accurate prediction of protein structures and interactions using a three-track neural network. Science, eabj8754 (2021).
67. W. Kabsch, C. Sander, Dictionary of protein secondary structure: Pattern recognition of hydrogen-bonded and geometrical features. Biopolymers 22, 2577–2637 (1983).
68. P. Labute, LowModeMD—Implicit Low-Mode Velocity Filtering Applied to Conformational Search of Macrocycles and Protein Loops. J. Chem. Inf. Model. 50, 792–800 (2010).
69. Protonate 3D: Assignment of Macromolecular Protonation State and Geometry. https://server.ccl.net/cca/documents/proton/proton.htm.
Metrics
DOI:
Submission ID:
Downloads
Posted
How to Cite
Download Citation
Declaration of Competing Interests
The authors declare no competing interests to disclose.
Copyright
The copyright holder for this preprint is the author/funder.
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.