Preferential light-chain labeling of native monoclonal antibodies improves the properties of fluorophore conjugates

Luciano, Michael; Dingle, Ivan; Nourian, Saghar; Schnermann, Martin J.

doi:10.1016/j.tetlet.2021.153211

Cited by 3 publications

(5 citation statements)

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“…This strategy, which maintains the original charge profile of the parent protein, while simultaneously introducing a highly charged functional group, may represent a promising approach to mask the undesirable hydrophobicity of drug conjugates in the circulation. Future studies will investigate the potential impact of these modifications in homogeneous mAb conjugates, where we hypothesize that charge will also play a significant role. , Additionally, we suggest that the incorporation of zwitterionic functional groups into otherwise hydrophobic therapeutic payloads may have significant promise. Studies toward these goals are underway in our group and will be reported in due course.…”

Section: Discussionmentioning

confidence: 95%

“…Future studies will investigate the potential impact of these modifications in homogeneous mAb conjugates, where we hypothesize that charge will also play a significant role. 61,62 Additionally, we suggest that the incorporation of zwitterionic functional groups into otherwise hydrophobic therapeutic payloads may have significant promise. Studies toward these goals are underway in our group and will be reported in due course.…”

Section: ■ Conclusionmentioning

confidence: 95%

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Cyanine Masking: A Strategy to Test Functional Group Effects on Antibody Conjugate Targeting

et al. 2022

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Conjugates of small molecules and antibodies are broadly employed diagnostic and therapeutic agents. Appending a small molecule to an antibody often significantly impacts the properties of the resulting conjugate. Here, we detail a systematic study investigating the effect of various functional groups on the properties of antibody−fluorophore conjugates. This was done through the preparation and analysis of a series of masked heptamethine cyanines (CyMasks)-bearing amides with varied functional groups. These were designed to exhibit a broad range of physical properties, and include hydrophobic (−NMe 2 ), pegylated (NH-PEG-8 or NH-PEG-24), cationic (NH-(CH 2 ) 2 NMe 3 + ), anionic (NH-(CH 2 ) 2 SO 3 − ), and zwitterionic (N-(CH 2 ) 2 NMe 3 + )-(CH 2 ) 3 SO 3 − ) variants. The CyMask series was appended to monoclonal antibodies (mAbs) and analyzed for the effects on tumor targeting, clearance, and non-specific organ uptake. Among the series, zwitterionic and pegylated dye conjugates had the highest tumor-to-background ratio (TBR) and a low liver-to-background ratio. By contrast, the cationic and zwitterionic probes had high tumor signal and high TBR, although the latter also exhibited an elevated liver-to-background ratio (LBR). Overall, these studies provide a strategy to test the functional group effects and suggest that zwitterionic substituents possess an optimal combination of high tumor signal, TBR, and low LBR. These results suggest an appealing strategy to mask hydrophobic payloads, with the potential to improve the properties of bioconjugates in vivo.

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Section: Discussionmentioning

confidence: 95%

Section: ■ Conclusionmentioning

confidence: 95%

Cyanine Masking: A Strategy to Test Functional Group Effects on Antibody Conjugate Targeting

et al. 2022

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“…36 More recently, Luciano et al used perfluorophenyl esters of the near-infrared fluorophore diSulfo-FNIR to create site-selectively modified antibody-fluorophore conjugates that displayed improved biological photophysical properties compared to randomly labeled analogues. 45 Here, and in these previous studies, the key to this bioconjugation approach lies in the selective reactivity of PFP esters with the primary amine of K188. Mechanistic experiments eschew explanations related to the pK a of K188 or the binding of the PFP moiety to nearby residues.…”

Section: ■ Discussionmentioning

confidence: 99%

“…This chemistry was first described in a 2011 patent from Bhat et al, but the earliest literature report arose in 2018, when Pham et al compared the site-selective reactivity of N -hydroxysuccinimidyl and fluorophenyl esters of biotin . More recently, Luciano et al used perfluorophenyl esters of the near-infrared fluorophore diSulfo-FNIR to create site-selectively modified antibody-fluorophore conjugates that displayed improved biological photophysical properties compared to randomly labeled analogues . Here, and in these previous studies, the key to this bioconjugation approach lies in the selective reactivity of PFP esters with the primary amine of K188.…”

Section: Discussionmentioning

confidence: 99%

Lysine-Directed Site-Selective Bioconjugation for the Creation of Radioimmunoconjugates

et al. 2022

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The synthesis of radioimmunoconjugates via the stochastic attachment of bifunctional chelators to lysines can yield heterogeneous products with suboptimal in vitro and in vivo behavior. In response to this, several site-selective approaches to bioconjugation have been developed, yet each has intrinsic drawbacks, such as the need for expensive reagents or the complexity of incorporating unnatural amino acids into IgGs. Herein, we describe the use of a simple and facile approach to lysine-directed site-selective bioconjugation for the generation of radioimmunoconjugates. This strategy relies upon on the selective modification of single lysine residues within each light chain of the monoclonal antibody (mAb) with a branched azide-bearing perfluorophenyl ester (PFP-bisN3) followed by the ligation of dibenzocyclooctyne (DBCO)-bearing payloads to these bioorthogonal handles via the strain-promoted azide–alkyne cycloaddition. This methodology was used to create [89Zr]Zr-SSKDFO-pertuzumab, a radioimmunoconjugate of the HER2-targeting mAb pertuzumab labeled with desferrioxamine (DFO) and the positron-emitting radiometal zirconium-89 (89Zr). [89Zr]Zr-SSKDFO-pertuzumab was compared to a pair of analogous probes: one synthesized via random lysine modification ([89Zr]Zr-DFO-pertuzumab) and another via thiol-maleimide chemistry ([89Zr]Zr-malDFO-pertuzumab). The bioconjugation strategy was assessed using ESI mass spectrometry, SDS-PAGE, and autoradiography. All three immunoconjugates demonstrated comparable binding to HER2 via flow cytometry and surface plasmon resonance (SPR), and 89Zr-labeled variants of each were synthesized in >99% radiochemical yield and molar activities of up to ∼55.5 GBq/μmol (10 mCi/mg). Subsequently, the in vivo behavior of this trio of 89Zr-immunoPET probes was interrogated in athymic nude mice bearing subcutaneous HER2-expressing BT-474 human breast cancer xenografts. [89Zr]Zr-SSKDFO-pertuzumab, [89Zr]Zr-malDFO-pertuzumab, and [89Zr]Zr-DFO-pertuzumab produced positron emission tomography (PET) images with high tumoral uptake and high tumor-to-healthy organ activity concentration ratios. A terminal biodistribution study complemented the PET results, revealing tumoral activity concentrations of 126.9 ± 50.3%ID/g, 86.9 ± 53.2%ID/g, and 92.5 ± 27.2%ID/g at 144 h post-injection for [89Zr]Zr-SSKDFO-pertuzumab, [89Zr]Zr-malDFO-pertuzumab, and [89Zr]Zr-DFO-pertuzumab, respectively. Taken together, the data clearly illustrate that this highly modular and facile approach to site-selective bioconjugation produces radioimmunoconjugates that are better-defined and more homogeneous than stochastically modified constructs and also exhibit excellent in vitro and in vivo performance. Furthermore, we contend that this lysine-directed strategy holds several key advantages over extant approaches to site-selective bioconjugation, especially in the context of production for the clinic.

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“…In this perspective, pentafluorophenyl ester (120a) was shown to render improved selectivity. 335,336 While the overall conversions are relatively lower, such a homogeneous antibody conjugate exhibits better solubility and reduced aggregation. It leads to improved distribution with better pharmacokinetics and tissue availability.…”

Section: Asparagine (N-glycan)mentioning

confidence: 99%

Chemical technology principles for selective bioconjugation of proteins and antibodies

Chauhan,

V.,

Kumar

et al. 2024

Chem. Soc. Rev.

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Preferential light-chain labeling of native monoclonal antibodies improves the properties of fluorophore conjugates

Cited by 3 publications

References 35 publications

Cyanine Masking: A Strategy to Test Functional Group Effects on Antibody Conjugate Targeting

Cyanine Masking: A Strategy to Test Functional Group Effects on Antibody Conjugate Targeting

Lysine-Directed Site-Selective Bioconjugation for the Creation of Radioimmunoconjugates

Chemical technology principles for selective bioconjugation of proteins and antibodies

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