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Hydrogen-deuterium exchange mass spectrometry reveals three unique binding responses of mAbs directed to the catalytic domain of hCAIX.

ABSTRACT: Human carbonic anhydrase (hCAIX), an extracellular enzyme that catalyzes the reversible hydration of CO₂, is often overexpressed in solid tumors. This enzyme is instrumental in maintaining the survival of cancer cells in a hypoxic and acidic tumor microenvironment. Absent in most normal tissues, hCAIX is a promising therapeutic target for detection and treatment of solid tumors. Screening of a library of anti-hCAIX monoclonal antibodies (mAbs) previously identified three therapeutic candidates (mAb c2C7, m4A2 and m9B6) with distinct biophysical and functional characteristics. Selective binding to the catalytic domain was confirmed by yeast surface display and isothermal calorimetry, and deeper insight into the dynamic binding profiles of these mAbs upon binding were highlighted by bottom-up hydrogen-deuterium exchange mass spectrometry (HDX-MS). Here, a conformational and allosterically silent epitope was identified for the antibody-drug conjugate candidate c2C7. Unique binding profiles are described for both inhibitory antibodies, m4A2 and m9B6. M4A2 reduces the ability of the enzyme to hydrate CO₂ by steric gating at the entrance of the catalytic cavity. Conversely, m9B6 disrupts the secondary structure that is necessary for substrate binding and hydration. The synergy of these two inhibitory mechanisms is demonstrated in in vitro activity assays and HDX-MS. Finally, the ability of m4A2 to modulate extracellular pH and intracellular metabolism is reported. By highlighting three unique modes by which hCAIX can be targeted, this study demonstrates both the utility of HDX-MS as an important tool in the characterization of anti-cancer biotherapeutics, and the underlying value of CAIX as a therapeutic target.

SUBMITTER: Sheff JG

PROVIDER: S-EPMC8632303 | biostudies-literature | 2021 Jan-Dec

REPOSITORIES: biostudies-literature

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Hydrogen-deuterium exchange mass spectrometry reveals three unique binding responses of mAbs directed to the catalytic domain of hCAIX.

Sheff Joey G JG Kelly John F JF Robotham Anna A Sulea Traian T Malenfant Félix F L'Abbé Denis D Duchesne Mélanie M Pelletier Alex A Lefebvre Jean J Acel Andrea A Parat Marie M Gosselin Mylene M Wu Cunle C Fortin Yves Y Baardsnes Jason J Van Faassen Henk H Awrey Shannon S Chafe Shawn C SC McDonald Paul C PC Dedhar Shoukat S Lenferink Anne E G AEG

mAbs 20210101 1

Human carbonic anhydrase (hCAIX), an extracellular enzyme that catalyzes the reversible hydration of CO<sub>2</sub>, is often overexpressed in solid tumors. This enzyme is instrumental in maintaining the survival of cancer cells in a hypoxic and acidic tumor microenvironment. Absent in most normal tissues, hCAIX is a promising therapeutic target for detection and treatment of solid tumors. Screening of a library of anti-hCAIX monoclonal antibodies (mAbs) previously identified three therapeutic c ...[more]

PMID: 34812124

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Project description:Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a powerful technique to interrogate protein structure and dynamics. With the ability to study almost any protein without a size limit, including intrinsically disordered ones, HDX-MS has shown fast growing importance as a complement to structural elucidation techniques. Current experiments compare two or more related conditions (sequences, interaction partners, excipients, conformational states, etc.) to determine statistically significant differences at a number of fixed time points and highlight areas of changed structural dynamics in the protein. The work presented here builds on the fundamental research performed in the early days of the technique and re-examines exchange rate calculations with the aim of establishing HDX-MS as an absolute and quantitative, rather than relative and qualitative, measurement. We performed millisecond HDX-MS experiments on a mixture of three unstructured peptides (angiotensin, bradykinin, and atrial natriuretic peptide amide rat) and compared experimental deuterium uptake curves with theoretical ones predicted using established exchange rate calculations. With poly-dl-alanine (PDLA) commonly used as a reference,, we find that experimental rates are sometimes faster than theoretically possible, while they agree much better, and are never faster, with the fully unstructured trialanine peptide (3-Ala). Molecular dynamics (MD) simulations confirm the high helical propensity of the longer and partially structured PDLA peptides, which need as few as 15 residues to form a stable helix and are therefore not suitable as an unstructured reference. Reanalysis of previously published data by Weis et al. at 100 mM NaCl however still shows a discrepancy with predictions based on 3-Ala in the absence of salt, highlighting the need for a better understanding of salt effects on exchange rates. Such currently unquantifiable salt effects prevent us from proposing a comprehensive, universal calibration framework at the moment. Nevertheless, an accurate recalibration of intrinsic exchange rate calculations is crucial to enable kinetic modeling of the exchange process and to ultimately allow HDX-MS to move toward a direct link with atomistic structural models.

Dataset Information

Hydrogen-deuterium exchange mass spectrometry reveals three unique binding responses of mAbs directed to the catalytic domain of hCAIX.

Publications

Hydrogen-deuterium exchange mass spectrometry reveals three unique binding responses of mAbs directed to the catalytic domain of hCAIX.

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