15 research outputs found
Germline-encoded neutralization of a Staphylococcus aureus virulence factor by the human antibody repertoire.
Staphylococcus aureus is both an important pathogen and a human commensal. To explore this ambivalent relationship between host and microbe, we analysed the memory humoral response against IsdB, a protein involved in iron acquisition, in four healthy donors. Here we show that in all donors a heavily biased use of two immunoglobulin heavy chain germlines generated high affinity (pM) antibodies that neutralize the two IsdB NEAT domains, IGHV4-39 for NEAT1 and IGHV1-69 for NEAT2. In contrast to the typical antibody/antigen interactions, the binding is primarily driven by the germline-encoded hydrophobic CDRH-2 motifs of IGHV1-69 and IGHV4-39, with a binding mechanism nearly identical for each antibody derived from different donors. Our results suggest that IGHV1-69 and IGHV4-39, while part of the adaptive immune system, may have evolved under selection pressure to encode a binding motif innately capable of recognizing and neutralizing a structurally conserved protein domain involved in pathogen iron acquisition
Modulation of P2X3 and P2X2/3 Receptors by Monoclonal Antibodies*
Purinergic homomeric P2X3 and heteromeric P2X2/3 receptors are ligand-gated cation channels activated by ATP. Both receptors are predominantly expressed in nociceptive sensory neurons, and an increase in extracellular ATP concentration under pathological conditions, such as tissue damage or visceral distension, induces channel opening, membrane depolarization, and initiation of pain signaling. Hence, these receptors are considered important therapeutic targets for pain management, and development of selective antagonists is currently progressing. To advance the search for novel analgesics, we have generated a panel of monoclonal antibodies directed against human P2X3 (hP2X3). We have found that these antibodies produce distinct functional effects, depending on the homomeric or heteromeric composition of the target, its kinetic state, and the duration of antibody exposure. The most potent antibody, 12D4, showed an estimated IC50 of 16 nm on hP2X3 after short term exposure (up to 18 min), binding to the inactivated state of the channel to inhibit activity. By contrast, with the same short term application, 12D4 potentiated the slow inactivating current mediated by the heteromeric hP2X2/3 channel. Extending the duration of exposure to ∼20 h resulted in a profound inhibition of both homomeric hP2X3 and heteromeric hP2X2/3 receptors, an effect mediated by efficient antibody-induced internalization of the channel from the plasma membrane. The therapeutic potential of mAb12D4 was assessed in the formalin, complete Freund's adjuvant, and visceral pain models. The efficacy of 12D4 in the visceral hypersensitivity model indicates that antibodies against P2X3 may have therapeutic potential in visceral pain indications
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Germline-encoded neutralization of a Staphylococcus aureus virulence factor by the human antibody repertoire.
Staphylococcus aureus is both an important pathogen and a human commensal. To explore this ambivalent relationship between host and microbe, we analysed the memory humoral response against IsdB, a protein involved in iron acquisition, in four healthy donors. Here we show that in all donors a heavily biased use of two immunoglobulin heavy chain germlines generated high affinity (pM) antibodies that neutralize the two IsdB NEAT domains, IGHV4-39 for NEAT1 and IGHV1-69 for NEAT2. In contrast to the typical antibody/antigen interactions, the binding is primarily driven by the germline-encoded hydrophobic CDRH-2 motifs of IGHV1-69 and IGHV4-39, with a binding mechanism nearly identical for each antibody derived from different donors. Our results suggest that IGHV1-69 and IGHV4-39, while part of the adaptive immune system, may have evolved under selection pressure to encode a binding motif innately capable of recognizing and neutralizing a structurally conserved protein domain involved in pathogen iron acquisition
Effectiveness of Alpha-toxin Fab Monoclonal Antibody Therapy in Limiting the Pathology of Staphylococcus aureus
Site-Dependent Degradation of a Non-Cleavable Auristatin-Based Linker-Payload in Rodent Plasma and Its Effect on ADC Efficacy
<div><p>The efficacy of an antibody-drug conjugate (ADC) is dependent on the properties of its linker-payload which must remain stable while in systemic circulation but undergo efficient processing upon internalization into target cells. Here, we examine the stability of a non-cleavable Amino-PEG6-based linker bearing the monomethyl auristatin D (MMAD) payload site-specifically conjugated at multiple positions on an antibody. Enzymatic conjugation with transglutaminase allows us to create a stable amide linkage that remains intact across all tested conjugation sites on the antibody, and provides us with an opportunity to examine the stability of the auristatin payload itself. We report a position-dependent degradation of the C terminus of MMAD in rodent plasma that has a detrimental effect on its potency. The MMAD cleavage can be eliminated by either modifying the C terminus of the toxin, or by selection of conjugation site. Both approaches result in improved stability and potency <i>in vitro</i> and <i>in vivo</i>. Furthermore, we show that the MMAD metabolism in mouse plasma is likely mediated by a serine-based hydrolase, appears much less pronounced in rat, and was not detected in cynomolgus monkey or human plasma. Clarifying these species differences and controlling toxin degradation to optimize ADC stability in rodents is essential to make the best ADC selection from preclinical models. The data presented here demonstrate that site selection and toxin susceptibility to mouse plasma degradation are important considerations in the design of non-cleavable ADCs, and further highlight the benefits of site-specific conjugation methods.</p></div
Protease inhibition studies of the PEG6-C2-MMAD degradation in mouse plasma.
<p>“Yes” indicates the same extent of cleavage as observed in plasma without inhibitors, “partial” indicates reduced cleavage compared to uninhibited plasma, while “no” indicates that no degradation was observed. All assays were carried out at pH 7.4.</p><p>Protease inhibition studies of the PEG6-C2-MMAD degradation in mouse plasma.</p
Stability studies of site-specific non-cleavable ADCs.
<p>a) Positions of conjugation sites on an antibody. b) Structure of the PEG6-C2-MMAD non-cleavable payload conjugated to the glutamine tag on the antibody, and its cleavage product. The glutamine residue is shown in blue. c) Structure of the PEG6-C2-Aur3377 non-cleavable payload conjugated to the glutamine tag shown in blue.</p
Effect of Attachment Site on Stability of Cleavable Antibody Drug Conjugates
The
systemic stability of the antibody–drug linker is crucial
for delivery of an intact antibody–drug conjugate (ADC) to
target-expressing tumors. Linkers stable in circulation but readily
processed in the target cell are necessary for both safety and potency
of the delivered conjugate. Here, we report a range of stabilities
for an auristatin-based payload site-specifically attached through a
cleavable valine-citrulline-<i>p</i>-aminobenzylcarbamate
(VC-PABC) linker across various sites on an antibody. We demonstrate
that the conjugation site plays an important role in determining VC-PABC
linker stability in mouse plasma, and that the stability of the linker
positively correlates with ADC cytotoxic potency both in vitro and
in vivo. Furthermore, we show that the VC-PABC cleavage in mouse plasma
is not mediated by Cathepsin B, the protease thought to be primarily
responsible for linker processing in the lysosomal degradation pathway.
Although the VC-PABC cleavage is not detected in primate plasma in
vitro, linker stabilization in the mouse is an essential prerequisite
for designing successful efficacy and safety studies in rodents during
preclinical stages of ADC programs. The divergence of linker metabolism
in mouse plasma and its intracellular cleavage offers an opportunity
for linker optimization in the circulation without compromising its
efficient payload release in the target cell
Degradation of the C-terminal portion of the PEG6-C2-MMAD payload in the plasma of different species.
<p>Degradation is calculated as percentage of payload cleaved. Calculations are based on DAR values obtained from HIC analysis of the Site A-PEG6-2-MMAD conjugate before and after incubation in plasma for 4.5 days in three independent experiments.</p
Comparative efficacy studies of non-cleavable ADCs.
<p>Comparison of <i>in vitro</i> cytotoxic activities of untreated and <i>in vivo</i>-exposed non-cleavable conjugates against BxPC3 cells (M1S1+++). a) C16 Site A-PEG6-C2-MMAD conjugate. b) C16 Site I-PEG6-C2-MMAD conjugate. c) C16 Site A-PEG6-C2-Aur3377 conjugate. d) <i>In vivo</i> comparison of the three conjugates in the BxPC3 xenograft model, along with a negative control conjugate NCC Site F-PEG6-C2-MMAD. All compounds were given as a single dose at 10 mg/kg.</p