Biophysical characterization of antibodies with isothermal titration calorimetry

Frasca, Verna

doi:10.17145/jab.16.013

Cited by 9 publications

(5 citation statements)

References 47 publications

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“…The binding of H C B ΔH C loop mutant to dual-receptor nanodiscs revealed the same thermodynamic reaction profile. In contrast, a large and favorable binding entropy, which is typically associated with the burial of hydrophobic groups away from the solvent [ 58 ], was measured for H C B wild-type bound to dual-receptor nanodiscs, which indicates an integration of the H C loop into the membrane bilayer. Indeed, the BoNT/B H C loop is solvated by up to 20 molecules of water [ 55 ] which are released upon membrane integration of the H C loop.…”

Section: Discussionmentioning

confidence: 99%

A lipid-binding loop of botulinum neurotoxin serotypes B, DC and G is an essential feature to confer their exquisite potency

et al. 2018

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The exceptional toxicity of botulinum neurotoxins (BoNTs) is mediated by high avidity binding to complex polysialogangliosides and intraluminal segments of synaptic vesicle proteins embedded in the presynaptic membrane. One peculiarity is an exposed hydrophobic loop in the toxin’s cell binding domain HC, which is located between the ganglioside- and protein receptor-binding sites, and that is particularly pronounced in the serotypes BoNT/B, DC, and G sharing synaptotagmin as protein receptor. Here, we provide evidence that this HC loop is a critical component of their tripartite receptor recognition complex. Binding to nanodisc-embedded receptors and toxicity were virtually abolished in BoNT mutants lacking residues at the tip of the HC loop. Surface plasmon resonance experiments revealed that only insertion of the HC loop into the lipid-bilayer compensates for the entropic penalty inflicted by the dual-receptor binding. Our results represent a new paradigm of how BoNT/B, DC, and G employ ternary interactions with a protein, ganglioside, and lipids to mediate their extraordinary neurotoxicity.

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Section: Discussionmentioning

confidence: 99%

A lipid-binding loop of botulinum neurotoxin serotypes B, DC and G is an essential feature to confer their exquisite potency

et al. 2018

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“…Antibody-antigen binding is a product of noncovalent interactions including electrostatic, van der Waals, hydrogen bonds, and hydrophobic effects [30]. ITC is a useful tool to investigate the thermodynamics controlling an antibody-antigen interaction and calculate crucial parameters such as the K d , stoichiometry, ΔH, and ΔS [31]. Previous reports show that DM1 conjugation does not significantly affect the binding affinity (measured here as K d ) of trastuzumab [23,24,25].…”

Section: Discussionmentioning

confidence: 99%

In-Depth Comparison of Lysine-Based Antibody-Drug Conjugates Prepared on Solid Support Versus in Solution

et al. 2018

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Antibody drug conjugates are a rapidly growing form of targeted chemotherapeutics. As companies and researchers move to develop new antibody-drug conjugate (ADC) candidates, high-throughput methods will become increasingly common. Here we use advanced characterization techniques to assess two trastuzumab-DM1 (T-DM1) ADCs; one produced using Protein A immobilization and the other produced in solution. Following determination of payload site and distribution with liquid chromatography-mass spectrometry (LC/MS), thermal stability, heat-induced aggregation, tertiary structure, and binding affinity were characterized using differential scanning calorimetry (DSC), dynamic light scattering (DLS), Raman spectroscopy, and isothermal titration calorimetry (ITC), respectively. Small differences in the thermal stability of the C H 2 domain of the antibody as well as aggregation onset temperatures were observed from DSC and DLS, respectively. However, no significant differences in secondary and tertiary structure were observed with Raman spectroscopy, or binding affinity as measured by ITC. Lysine-based ADC conjugation produces an innately heterogeneous population that can generate significant variability in the results of sensitive characterization techniques. Characterization of these ADCs indicated nominal differences in thermal stability but not in tertiary structure or binding affinity. Our results lead us to conclude that lysine-based ADCs synthesized following Protein A immobilization, common in small-scale conjugations, are highly similar to equivalent ADCs produced in larger scale, solution-based methods.

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“…Furthermore, this tag was a marker for purifying the soluble ScFv-tagged with anti-DYKDDDDK affinity tag resin beads. The potential of purified ScFv revealed strong binding affinity ( K d ) based on favorable enthalpy and unfavorable entropy [ 34 ].…”

Section: Discussionmentioning

confidence: 99%

A novel anti-membrane CD30 single-chain variable fragment discovered from the human phage library: A potential targeted immunotherapy

et al. 2023

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Hodgkin’s lymphoma and anaplastic large cell lymphoma, especially relapsed or refractory diseases, could recently be cured by CD30-targeted immunotherapy. However, the CD30 antigen releases the soluble ectodomain of CD30, which might obscure the targeted therapy. Therefore, the membrane epitope of CD30 (mCD30), left on the cancer cells, might be a prospective target for lymphoma treatment. The discovery of novel mCD30 monoclonal antibodies (mAbs) using phage technology yielded 59 potential human single-chain variable fragments (HuscFvs). Ten candidate HuscFv clones have been selected based on various methods, i.e., direct PCR, ELISA and western blot assays, and nucleotide sequencing techniques. Fortunately, only one potential HuscFv clone, clone #A4, was determined by the prediction of HuscFv-peptide molecular docking and the binding affinity test using isothermal titration calorimetry. Finally, we proved that the HuscFv #A4, which had a binding affinity (Kd) of 421e-9 ± 2.76e-6 M, might be the novel mCD30 mAb. We generated chimeric antigen receptor-modified T lymphocytes using HuscFv #A4 as an antigen detection part (anti-mCD30-H4CART). The cytotoxicity assay of anti-mCD30-H4CART cells showed significant eradication of the CD30-expressing cell line, K562 (p = 0.0378). We found a novel mCD30 HuscFv using human phage technology. We systematically examined and proved that our HuscFv #A4 could specifically eradicate CD30-expressing cancers.

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Biophysical characterization of antibodies with isothermal titration calorimetry

Cited by 9 publications

References 47 publications

A lipid-binding loop of botulinum neurotoxin serotypes B, DC and G is an essential feature to confer their exquisite potency

A lipid-binding loop of botulinum neurotoxin serotypes B, DC and G is an essential feature to confer their exquisite potency

In-Depth Comparison of Lysine-Based Antibody-Drug Conjugates Prepared on Solid Support Versus in Solution

A novel anti-membrane CD30 single-chain variable fragment discovered from the human phage library: A potential targeted immunotherapy

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