Breaking the law: unconventional strategies for antibody diversification

Kanyavuz, Alexia; Marey-Jarossay, Annaelle; Lacroix‐Desmazes, Sébastien; Dimitrov, Jordan D.

doi:10.1038/s41577-019-0126-7

Cited by 61 publications

(77 citation statements)

References 127 publications

(148 reference statements)

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“…There were many Abs that bind to heme but did not acquire antigen-binding promiscuity and vice versa. This result can be explained by the fact that for the acquisition of polyreactivity, the heme molecule should be displayed in an appropriate manner on the antigen-binding site and most probably to be itself engaged in interactions with antigen (33). If heme binding results in burial of the macrocyclic molecule in the protein interior, this most probably would not cause acquisition of binding polyreactivity.…”

Section: Discussionmentioning

confidence: 99%

Interaction of clinical-stage antibodies with heme predicts their physiochemical and binding qualities

Lecerf

Kanyavuz

Rossini

et al. 2020

Preprint

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Development of monoclonal antibody therapeutics is a sophisticated process. Early stage detection of negative traits of antibodies, which can impact the production, storage stability or therapeutic efficacy is of critical importance for saving time and resources in drug development programs. Since the developability issues of antibodies are diverse, including expression yield, chemical and physical stability, propensity for aggregation, antigen-binding polyreactivity, etc., a battery of experimental and in silico assays have been developed for thorough examination of drug candidates. Here, by using a set of 113 samples with variable region sequences matching clinical-stage therapeutic antibodies, we demonstrated that unique physicochemical properties of heme allow this cofactor molecule to be applied as a probe for simultaneous detection of different deleterious characteristics of antibodies i.e., antigen-binding polyreactivity, self-association, aggregation, hydrophobicity and expression yield. Moreover, our study demonstrates that similarly as antibody repertoires from healthy individuals, a fraction of therapeutic antibodies acquires use heme as cofactor for recognition of diverse antigens. These results reveal presence of a hidden vulnerability issue for some antibodies, which would not be detectable by standard analytical assays. This liability may be of concern for monoclonal antibodies intended to be use in patients with pathophysiological conditions where extracellular heme is present e.g. hemolysis or tissue damage. We provided evidence about the mechanistic basis of the unique features of heme and offer a powerful analytical tool for rapid and simple detection of important negative traits in candidate therapeutic antibodies.

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Section: Discussionmentioning

confidence: 99%

Interaction of clinical-stage antibodies with heme predicts their physiochemical and binding qualities

Lecerf

Kanyavuz

Rossini

et al. 2020

Preprint

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“…or rhabdomyolysis (crush syndrome, muscle damage as in car accidents, natural cataclysms, or military trauma) (1). Heme triggers inflammation by activating immune and endothelial cells (EC) or plasma systems, such as the complement cascade, coagulation, or inducing antibody polyreactivity (2)(3)(4). In physiological conditions excess of cell-free heme is scavenged by its natural binding protein hemopexin (Hpx).…”

Section: Introductionmentioning

confidence: 99%

Hemopexin as an Inhibitor of Hemolysis-Induced Complement Activation

et al. 2020

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Hemopexin is the main plasmatic scavenger of cell-free heme, released in the context of intravascular hemolysis or major cell injury. Heme is indispensable for the oxygen transport by hemoglobin but when released outside of the erythrocytes it becomes a danger-associated molecular pattern, contributing to tissue injury. One of the mechanisms of pro-inflammatory action of heme is to activate the innate immune complement cascade. Therefore, we hypothesized that injection of hemopexin will prevent hemolysis-induced complement activation. Human plasma-derived hemopexin is compatible with the heme clearance machinery of the mice. 100 or 500 mg/kg of hemopexin was injected in C57Bl/6 mice before treatment with phenylhydrazine (inducer of erythrocytes lysis) or with PBS as a control. Blood was taken at different timepoints to determine the pharmacokinetic of injected hemopexin in presence and absence of hemolysis. Complement activation was determined in plasma, by the C3 cleavage (western blot) and in the kidneys (immunofluorescence). Kidney injury was evaluated by urea and creatinine in plasma and renal NGAL and HO-1 gene expression were measured. The pharmacokinetic properties of hemopexin (mass spectrometry) in the hemolytic mice were affected by the target-mediated drug disposition phenomenon due to the high affinity of binding of hemopexin to heme. Hemolysis induced complement overactivation and signs of mild renal dysfunction at 6 h, which were prevented by hemopexin, except for the NGAL upregulation. The heme-degrading capacity of the kidney, measured by the HO-1 expression, was not affected by the treatment. These results encourage further studies of hemopexin as a therapeutic agent in models of diseases with heme overload.

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“…Different strategies have been tested to enhance the therapeutic potential of IVIG and polyclonal immunoglobulin preparations, including diversification of the repertoire of antigen specificities by mild chemical modification (3,17). As also evidenced by crystallography, a single antibody can adopt different binding-site conformations (conformational diversity), spontaneously in absence of antigen or by an inducedfit mechanism, eventually resulting in the capacity to bind unrelated antigens (multispecificity) (18).…”

Section: Introductionmentioning

confidence: 99%

Enhanced Pro-apoptotic Effects of Fe(II)-Modified IVIG on Human Neutrophils

et al. 2020

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Mild modification of intravenous immunoglobulin (IVIG) has been reported to result in enhanced polyspecificity and leveraged therapeutic effects in animal models of inflammation. Here, we observed that IVIG modification by ferrous ions, heme or low pH exposure, shifted the repertoires of specificities in different directions. Ferrous ions exposed Fe(II)-IVIG, but not heme or low pH exposed IVIG, showed increased pro-apoptotic effects on neutrophil granulocytes that relied on a FAS-dependent mechanism. These effects were also observed in human neutrophils primed by inflammatory mediators or rheumatoid arthritis joint fluid in vitro, or patient neutrophils ex vivo from acute Crohn's disease. These observations indicate that IVIG-mediated effects on cells can be enhanced by IVIG modification, yet specific modification conditions may be required to target specific molecular pathways and eventually to enhance the therapeutic potential.

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Breaking the law: unconventional strategies for antibody diversification

Cited by 61 publications

References 127 publications

Interaction of clinical-stage antibodies with heme predicts their physiochemical and binding qualities

Interaction of clinical-stage antibodies with heme predicts their physiochemical and binding qualities

Hemopexin as an Inhibitor of Hemolysis-Induced Complement Activation

Enhanced Pro-apoptotic Effects of Fe(II)-Modified IVIG on Human Neutrophils

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