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An Open-Source Framework for Mass Calculation of Antibody-Based Therapeutic Molecules
Published on: June 16, 2023
An experimentally validated structure-based computational framework for humanisation of anti-orthopoxvirus antibodies
Xuehua Yang1, Xuemeng Dong2, Jiahan Lu3
1Key Laboratory of Pathogen Infection Prevention and Control (Ministry of Education), National Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 102629, China; NHC Key Laboratory of Systems Biology of Pathogens, National Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 102629, China; State Key Laboratory of Respiratory Health and Multimorbidity, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 102629, China; Suzhou Centre for Disease Control and Prevention, Suzhou, Jiangsu 215004, China.
Background:
The re-emergence of orthopoxviruses, most notably mpox virus (MPXV), poses a growing global public health threat. Well-characterised murine anti-orthopoxvirus antibodies are clinically limited by anti-mouse antibody responses, while traditional sequence-based humanisation often impairs antigen-binding activity.
Methods:
We developed an experimentally validated structure-guided computational humanisation framework prioritising 3D architectural congruence over sequence identity, integrating Foldseek-based structural alignment and interface-residue constraints. We applied this framework to humanise two murine anti-orthopoxvirus antibodies (7D11, A27D7), with comprehensive in vitro and in vivo validation.
Findings:
Structural superimposition confirmed high conformational conservation between the humanised variants (POX1.1 and POX2.1) and their parental mAbs, with root mean square deviation (RMSD) values below 0.6 Å for all variable domains. Both humanised variants retained full epitope specificity with natural humanness profiles. POX1.1 showed enhanced neutralisation potency against vaccinia virus (VACV) and MPXV, compared with the parental 7D11. POX2.1 preserved the broad cross-reactive binding and the extracellular enveloped virion neutralising activity of the parental A27D7. In the lethal VACV mouse model, both monotherapies conferred significant prophylactic and therapeutic protection, reducing pulmonary viral loads and improving survival. The dual-targeting combination of POX1.1 and POX2.1 achieved markedly improved in vivo efficacy compared with individual antibodies, delivering 100% survival even when administered 2 days post-challenge. In the MPXV CAST/EiJ mouse model, the combination significantly reduced splenomegaly and MPXV DNA loads in plasma, spleen and lung tissues, effectively suppressing systemic viral dissemination.
Interpretation:
These findings establish that the structure-centric workflow enables efficient humanisation of well-characterised murine anti-orthopoxvirus antibodies, providing a validated framework to support the development of countermeasures for orthopoxvirus pandemic.
Funding:
This work was supported by the National Natural Science Foundation of China, the Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences, the Scientific Research Innovation Capability Support Project for Young Faculty and the National Science and Technology Major Project.
Insights
A new structure-guided method efficiently humanizes mouse antibodies against mpox virus (MPXV). This approach overcomes limitations of traditional methods, leading to potent antibody therapies for orthopoxvirus infections.
Area of Science:
- Immunology
- Virology
- Structural Biology
Background:
- Orthopoxviruses, including mpox virus (MPXV), represent a significant global health concern.
- Murine antibodies are limited by anti-mouse responses, and sequence-based humanization can reduce efficacy.
Purpose of the Study:
- To develop and validate a structure-guided computational framework for humanizing murine anti-orthopoxvirus antibodies.
- To assess the efficacy of humanized antibodies against orthopoxvirus infections.
Main Methods:
- A novel framework prioritizing 3D structural congruence over sequence identity was developed.
- The framework integrated Foldseek structural alignment and interface-residue constraints.
- Two murine antibodies (7D11, A27D7) were humanized (POX1.1, POX2.1) and extensively validated in vitro and in vivo.
Main Results:
- Humanized variants (POX1.1, POX2.1) maintained high conformational similarity to parental antibodies.
- Both variants demonstrated full epitope specificity and natural humanness.
- POX1.1 enhanced neutralization of vaccinia virus (VACV) and MPXV; POX2.1 retained broad cross-reactivity.
- Monotherapies provided significant protection in lethal VACV and MPXV models.
- Combination therapy achieved 100% survival in VACV models and suppressed MPXV dissemination.
Conclusions:
- The structure-centric workflow enables efficient humanization of murine anti-orthopoxvirus antibodies.
- This validated framework supports the development of countermeasures against orthopoxvirus pandemics.
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