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Single cycle structure-based humanization of an anti-nerve growth factor therapeutic antibody.

Covaceuszach S, Marinelli S, Krastanova I, Ugolini G, Pavone F, Lamba D, Cattaneo A - PLoS ONE (2012)

Bottom Line: Here we show that mAb αD11 displays a significant analgesic effect in two different models of persistent pain in mice, with a remarkable long-lasting activity.The humanized antibody (hum-αD11) was tested in vitro and in vivo, showing that the binding mode and the NGF neutralizing biological activities of the parental antibody are fully preserved, with even a significant affinity improvement.The results firmly establish hum-αD11 as a lead candidate for clinical applications in a therapeutic area with a severe unmet medical need.

View Article: PubMed Central - PubMed

Affiliation: Lay Line Genomics, SpA, Roma, Italy.

ABSTRACT
Most forms of chronic pain are inadequately treated by present therapeutic options. Compelling evidence has accumulated, demonstrating that Nerve Growth Factor (NGF) is a key modulator of inflammatory and nociceptive responses, and is a promising target for the treatment of human pathologies linked to chronic and inflammatory pain. There is therefore a growing interest in the development of therapeutic molecules antagonising the NGF pathway and its nociceptor sensitization actions, among which function-blocking anti-NGF antibodies are particularly relevant candidates.In this respect, the rat anti-NGF αD11 monoclonal antibody (mAb) is a potent antagonist, able to effectively antagonize rodent and human NGF in a variety of in vitro and in vivo systems. Here we show that mAb αD11 displays a significant analgesic effect in two different models of persistent pain in mice, with a remarkable long-lasting activity. In order to advance αD11 mAb towards its clinical application in man, anti-NGF αD11 mAb was humanized by applying a novel single cycle strategy based on the a priori experimental determination of the crystal and molecular structure of the parental Fragment antigen-binding (Fab). The humanized antibody (hum-αD11) was tested in vitro and in vivo, showing that the binding mode and the NGF neutralizing biological activities of the parental antibody are fully preserved, with even a significant affinity improvement. The results firmly establish hum-αD11 as a lead candidate for clinical applications in a therapeutic area with a severe unmet medical need. More generally, the single-cycle structure-based humanization method represents a considerable improvement over the standard humanization methods, which are intrinsically empirical and require several refinement cycles.

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Hum-αD11 retains the biological activity of the parental mAb αD11 in vitro.(a–d) mNGF induced differentiation of PC-12 cells. Neurite outgrowth inhibition assay: photomicrographs of PC-12 cells, treated with (a) mNGF 100 ng/ml alone or preincubated (b) with mAb αD11 (5 µg/ml) or (c) with IgG1 hum-αD11 (5 µg/ml), concentrated from stable cotransfected CHO cells supernatants. (d) Negative control: untreated PC-12 cells. (e) TrkA phosphorylation inhibition assay in 3T3 TrkA cells. 3T3 TrkA cells were incubated with the indicated combinations of mNGF, parental mAb αD11, IgG1 hum-αD11 (purified by Protein A-Sepharose) and the irrelevant mAb SV5 as a negative control. Cell lysates were separated on a 10% SDS gel and phosphorylated TrkA detected using anti-phospho-Y490 TrkA Ab. The ubiquitous band of tubulin served as gel loading control.
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pone-0032212-g007: Hum-αD11 retains the biological activity of the parental mAb αD11 in vitro.(a–d) mNGF induced differentiation of PC-12 cells. Neurite outgrowth inhibition assay: photomicrographs of PC-12 cells, treated with (a) mNGF 100 ng/ml alone or preincubated (b) with mAb αD11 (5 µg/ml) or (c) with IgG1 hum-αD11 (5 µg/ml), concentrated from stable cotransfected CHO cells supernatants. (d) Negative control: untreated PC-12 cells. (e) TrkA phosphorylation inhibition assay in 3T3 TrkA cells. 3T3 TrkA cells were incubated with the indicated combinations of mNGF, parental mAb αD11, IgG1 hum-αD11 (purified by Protein A-Sepharose) and the irrelevant mAb SV5 as a negative control. Cell lysates were separated on a 10% SDS gel and phosphorylated TrkA detected using anti-phospho-Y490 TrkA Ab. The ubiquitous band of tubulin served as gel loading control.

Mentions: First, in an NGF-induced neurite outgrowth bioassay on rat PC-12 cells [43] (Figure 7A to Figure 7D) both the parental mAb and the IgG1 hum-αD11 exerted identical effects. Indeed, mNGF treated PC-12 cells (Figure 7A), preincubated with either the parental mAb or the IgG1 hum-αD11, failed to show any neurite outgrowth (Figure 7B and Figure 7C), as in the absence of mNGF (Figure 7D). In control experiments NGF-induced differentiation occurs normally (Figure 7A), even if mNGF was preincubated with a non relevant mAb or with the concentrated untransfected CHO cell supernatants (data not shown). The nuclear morphology of IgG1 hum-αD11 treated PC-12, stained with 4′,6-diamidino-2-phenylindole (DAPI), under the different conditions of the assays, was normal (data not shown), underlining that the failure of cells treated with mNGF and IgG1 hum-αD11 to differentiate was not due to a non-specific toxic effect, but indeed to the neutralization of NGF binding and of the ensuing differentiation.


Single cycle structure-based humanization of an anti-nerve growth factor therapeutic antibody.

Covaceuszach S, Marinelli S, Krastanova I, Ugolini G, Pavone F, Lamba D, Cattaneo A - PLoS ONE (2012)

Hum-αD11 retains the biological activity of the parental mAb αD11 in vitro.(a–d) mNGF induced differentiation of PC-12 cells. Neurite outgrowth inhibition assay: photomicrographs of PC-12 cells, treated with (a) mNGF 100 ng/ml alone or preincubated (b) with mAb αD11 (5 µg/ml) or (c) with IgG1 hum-αD11 (5 µg/ml), concentrated from stable cotransfected CHO cells supernatants. (d) Negative control: untreated PC-12 cells. (e) TrkA phosphorylation inhibition assay in 3T3 TrkA cells. 3T3 TrkA cells were incubated with the indicated combinations of mNGF, parental mAb αD11, IgG1 hum-αD11 (purified by Protein A-Sepharose) and the irrelevant mAb SV5 as a negative control. Cell lysates were separated on a 10% SDS gel and phosphorylated TrkA detected using anti-phospho-Y490 TrkA Ab. The ubiquitous band of tubulin served as gel loading control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3293900&req=5

pone-0032212-g007: Hum-αD11 retains the biological activity of the parental mAb αD11 in vitro.(a–d) mNGF induced differentiation of PC-12 cells. Neurite outgrowth inhibition assay: photomicrographs of PC-12 cells, treated with (a) mNGF 100 ng/ml alone or preincubated (b) with mAb αD11 (5 µg/ml) or (c) with IgG1 hum-αD11 (5 µg/ml), concentrated from stable cotransfected CHO cells supernatants. (d) Negative control: untreated PC-12 cells. (e) TrkA phosphorylation inhibition assay in 3T3 TrkA cells. 3T3 TrkA cells were incubated with the indicated combinations of mNGF, parental mAb αD11, IgG1 hum-αD11 (purified by Protein A-Sepharose) and the irrelevant mAb SV5 as a negative control. Cell lysates were separated on a 10% SDS gel and phosphorylated TrkA detected using anti-phospho-Y490 TrkA Ab. The ubiquitous band of tubulin served as gel loading control.
Mentions: First, in an NGF-induced neurite outgrowth bioassay on rat PC-12 cells [43] (Figure 7A to Figure 7D) both the parental mAb and the IgG1 hum-αD11 exerted identical effects. Indeed, mNGF treated PC-12 cells (Figure 7A), preincubated with either the parental mAb or the IgG1 hum-αD11, failed to show any neurite outgrowth (Figure 7B and Figure 7C), as in the absence of mNGF (Figure 7D). In control experiments NGF-induced differentiation occurs normally (Figure 7A), even if mNGF was preincubated with a non relevant mAb or with the concentrated untransfected CHO cell supernatants (data not shown). The nuclear morphology of IgG1 hum-αD11 treated PC-12, stained with 4′,6-diamidino-2-phenylindole (DAPI), under the different conditions of the assays, was normal (data not shown), underlining that the failure of cells treated with mNGF and IgG1 hum-αD11 to differentiate was not due to a non-specific toxic effect, but indeed to the neutralization of NGF binding and of the ensuing differentiation.

Bottom Line: Here we show that mAb αD11 displays a significant analgesic effect in two different models of persistent pain in mice, with a remarkable long-lasting activity.The humanized antibody (hum-αD11) was tested in vitro and in vivo, showing that the binding mode and the NGF neutralizing biological activities of the parental antibody are fully preserved, with even a significant affinity improvement.The results firmly establish hum-αD11 as a lead candidate for clinical applications in a therapeutic area with a severe unmet medical need.

View Article: PubMed Central - PubMed

Affiliation: Lay Line Genomics, SpA, Roma, Italy.

ABSTRACT
Most forms of chronic pain are inadequately treated by present therapeutic options. Compelling evidence has accumulated, demonstrating that Nerve Growth Factor (NGF) is a key modulator of inflammatory and nociceptive responses, and is a promising target for the treatment of human pathologies linked to chronic and inflammatory pain. There is therefore a growing interest in the development of therapeutic molecules antagonising the NGF pathway and its nociceptor sensitization actions, among which function-blocking anti-NGF antibodies are particularly relevant candidates.In this respect, the rat anti-NGF αD11 monoclonal antibody (mAb) is a potent antagonist, able to effectively antagonize rodent and human NGF in a variety of in vitro and in vivo systems. Here we show that mAb αD11 displays a significant analgesic effect in two different models of persistent pain in mice, with a remarkable long-lasting activity. In order to advance αD11 mAb towards its clinical application in man, anti-NGF αD11 mAb was humanized by applying a novel single cycle strategy based on the a priori experimental determination of the crystal and molecular structure of the parental Fragment antigen-binding (Fab). The humanized antibody (hum-αD11) was tested in vitro and in vivo, showing that the binding mode and the NGF neutralizing biological activities of the parental antibody are fully preserved, with even a significant affinity improvement. The results firmly establish hum-αD11 as a lead candidate for clinical applications in a therapeutic area with a severe unmet medical need. More generally, the single-cycle structure-based humanization method represents a considerable improvement over the standard humanization methods, which are intrinsically empirical and require several refinement cycles.

Show MeSH
Related in: MedlinePlus