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. 2024 Sep;102(9):1151-1161.
doi: 10.1007/s00109-024-02470-4. Epub 2024 Jul 25.

Structure-based humanization of a therapeutic antibody for multiple myeloma

Stephen F Marino  1   2 , Oliver Daumke  3
Affiliations

Structure-based humanization of a therapeutic antibody for multiple myeloma

Stephen F Marino et al. J Mol Med (Berl). 2024 Sep.

Abstract

The optimal efficacy of xenogeneically generated proteins intended for application in humans requires that their own antigenicity be minimized. This necessary adaptation of antibodies to a humanized version poses challenges since modifications even distant from the binding sites can greatly influence antigen recognition and this is the primary feature that must be maintained during all modifications. Current strategies often rely on grafting and/or randomization/selection to arrive at a humanized variant retaining the binding properties of the original molecule. However, in terms of speed and efficiency, rationally directed approaches can be superior, provided the requisite structural information is available. We present here a humanization procedure based on the high-resolution X-ray structure of a chimaeric IgG against a marker for multiple myeloma. Based on in silico modelling of humanizing amino acid substitutions identified from sequence alignments, we devised a straightforward cloning procedure to rapidly evaluate the proposed sequence changes. Careful inspection of the structure allowed the identification of a potentially problematic amino acid change that indeed disrupted antigen binding. Subsequent optimization of the antigen binding loop sequences resulted in substantial recovery of binding affinity lost in the completely humanized antibody. X-ray structures of the humanized and optimized variants demonstrate that the antigen binding mode is preserved, with surprisingly few direct contacts to antibody atoms. These results underline the importance of structural information for the efficient optimization of protein therapeutics. KEY MESSAGES: Structure-based humanization of an IgG against BCMA, a marker for Multiple Myeloma. Identification of problematic mutations and unexpected modification sites. Structures of the modified IgG-antigen complexes verified predictions. Provision of humanized high-affinity IgGs against BCMA for therapeutic applications.

Keywords: Humanization; Monoclonal antibody; Multiple myeloma; X-ray structure.

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Conflict of interest statement

SFM and OD are inventors on multiple pending and granted patents derived from the international patent applications PCT/EP2013/072857, PCT/EP2015/059562, and PCT/EP2017/063862 concerning J22.9-xi and variants.

Figures

Fig. 1
Fig. 1
Humanizing mutations in J22.9-xi. A Protein sequences of the mouse variable region heavy (mVH) and light (mVL) chains aligned with the corresponding human germline sequences (hVH and hVL). CDRs 1, 2, and 3 from each chain are underlined. Humanizing mutations are highlighted in magenta, proposed CDR positions for stabilizing PTM mutations are highlighted in red and the complex disrupting A46 indicated in green. Vertical dashed lines indicate the corresponding boundaries of the cloning cassettes. B Space-filling model of the J22.9-xi variable domain showing the positions of the proposed mutations on the structure (PDB ID: 4ZFO). As in A, the VH is depicted in blue, the VL in yellow, humanizing mutation positions in magenta, stabilizing CDR PTM positions in red, and A46 in green. The right panel view depicts a 180° rotation along the vertical axis of the left panel view
Fig. 2
Fig. 2
The A46 hydrophobic pocket of J22.9-xi. Two views of the A46 binding environment with corresponding residues in space-filling representation and labeled. VH is in blue, VL in yellow, and BCMA in violet. A46 (VL) is seen in the center in stick representation with color-coding of individual atoms (nitrogen blue, carbon green, and oxygen red) and the beta carbon atom in the pocket represented as a green sphere. The right panel view depicts a 180° rotation along the vertical axis of the left panel view. The close packing around the A46 sidechain is clear from both panels as are the interactions with L17 and L18 of BCMA
Fig. 3
Fig. 3
Measurement of BCMA binding affinities of J22.9 variants. SPR sensorgrams of J22.9 variants (labeled) binding to hBCMA and cynoBCMA. The colored traces correspond to increasing concentrations of BCMA in the mobile phase binding to immobilized IgG. The leftmost vertical dashed line indicates the start of ligand flow over the immobilized antibodies, during which association is measured, and the rightmost indicates the switch to BCMA-free buffer, initiating the dissociation phase
Fig. 4
Fig. 4
Structural alignments of J22.9-xi with humanized and optimized variants. A View into the binding pocket of all four J22.9 aligned variants with BCMA removed. Only the backbone atoms are shown as lines, with the CDRs depicted as sticks. The VH CDRs of J22.9-xi are colored blue, and the VL CDRs are colored yellow with J22.9-H shown in magenta, J22.9-ISY in light blue, and J22.9-FNY in light green. The positions of all 4 PTM modifications are indicated. B Separate views of the VL (left panel) and VH (right panel) domains with CDR depictions and coloring as in A. The conformations of all VL CDR loops are nearly identical, as are those of the VH CDRs of the humanized variants that all show a slight shift from the corresponding positions in J22.9-xi. C Overlay of BCMA from all J22.9 variants generated by alignment of all 4 complete complex structures. BCMA bound to J22.9-xi is shown in violet and BCMA molecules from the humanized structures are colored as per their corresponding VL and VH chains in A. The DxL loop residues are indicated
Fig. 5
Fig. 5
J22.9-H and J22.9-FNY binding site comparison. Structural alignment of J22.9-H:BCMA and J22.9-FNY:BCMA showing the interaction of BCMA with CDR2 and the position of the NAG modification. The J22.9-H VH is colored blue, and the J22.9-FNY VH is colored light blue. The N-acetylglucosamine (NAG) modification on J22.9-FNY is colored orange. BCMA bound to J22.9-H is shown in violet and BCMA bound to J22.9-FNY in magenta. The NAG modification in J22.9-FNY induces no substantial changes in the CDR2 loop. The most important features of both binding partners are labeled, including the DxL loop of BCMA, showing that the central L17 residue occupies a nearly identical position in both structures relative to the antibody. The single most extensive interaction in the complex, between W33 in VH CDR1 and H19 in BCMA, is also explicitly shown and is unchanged between the two structures. The electron density (contoured at 1.0σ) for the NAG modification of J22.9-FNY is depicted as a grey mesh.
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References

    1. Martin KP, Grimaldi C, Grempler R, Hansel S, Kumar S (2023) Trends in industrialization of biotherapeutics: a survey of product characteristics of 89 antibody-based therapeutics. MAbs 15:2191301. 10.1080/19420862.2023.2191301 10.1080/19420862.2023.2191301 - DOI - PMC - PubMed
    1. Lo K-M, Leger O, Hock B. Antibody engineering. Microbiol Spectrum 2(1):AID-0007–2012 (2014). 10.1128/microbiolspec.AID-0007-12 - PubMed
    1. Morrison SL, Johnson MJ, Herzenberg LA, Ol VT (1984) Chimaeric human antibody molecules: mouse antigen-binding domains with human constant region domains. PNAS 81:6851–6855. 10.1073/pnas.81.21.6851 10.1073/pnas.81.21.6851 - DOI - PMC - PubMed
    1. Boulianne GL, Hozumi N, Shulman MJ (1984) Production of functional chimaeric mouse/human antibody. Nature 312:643–646. 10.1038/312643a0 10.1038/312643a0 - DOI - PubMed
    1. Neuberger MS et al (1985) A hapten-specific chimaeric IgE antibody with human physiological effector function. Nature 314:268–270. 10.1038/314268a0 10.1038/314268a0 - DOI - PubMed

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