<i>In vitro</i>affinity optimization of an anti-BDNF monoclonal antibody translates to improved potency in targeting chronic pain states<i>in vivo</i>

Stack, Edwina; McMurray, Sheridan; McMurray, Gordon; Wade, Jason; Clark, Mark M.; Young, Gareth T.; Marquette, Kim; Jain, Sadhana; Kelleher, Kerry; Chen, Ting; Lin, Qingcong; Bloom, Laird; Lin, Laura; Finlay, William J. J.; Suzuki, Rie; Cunningham, Orla

doi:10.1080/19420862.2020.1755000

Cited by 9 publications

(5 citation statements)

References 63 publications

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“…Nonetheless, we were able to get 12 pTyr binders with enhanced binding affinities, likely due to the bias design of this library (see Section 4). The soft randomization method, in which the wild type residues have the dominant proportion, is widely used in in vitro affinity maturation, especially for antibody engineering 17,27,28 . The rationale is that if a residue in the binding pocket contributes less to the binding affinity, the other one(s) will show up during the biopanning by affinity selection, even if this/these residue has/have less proportion at this position in the library.…”

Section: Discussionmentioning

confidence: 99%

Revisiting the phosphotyrosine binding pocket of Fyn SH2 domain led to the identification of novel SH2 superbinders.

Zou

Zhao

et al. 2020

Protein Science

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Protein engineering through directed evolution is an effective way to obtain proteins with novel functions with the potential applications as tools for diagnosis or therapeutics. Many natural proteins have undergone directed evolution in vitro in the test tubes in the laboratories worldwide, resulting in the numerous protein variants with novel or enhanced functions. we constructed here an SH2 variant library by randomizing 8 variable residues in its phosphotyrosine (pTyr) binding pocket. Selection of this library by a pTyr peptide led to the identification of SH2 variants with enhanced affinities measured by EC50. Fluorescent polarization was then applied to quantify the binding affinities of the newly identified SH2 variants. As a result, three SH2 variants, named V3, V13 and V24, have comparable binding affinities with the previously identified SH2 triple‐mutant superbinder. Biolayer Interferometry assay was employed to disclose the kinetics of the binding of these SH2 superbinders to the phosphotyrosine peptide. The results indicated that all the SH2 superbinders have two‐orders increase of the dissociation rate when binding the pTyr peptide while there was no significant change in their associate rates. Intriguingly, though binding the pTyr peptide with comparable affinity with other SH2 superbinders, the V3 does not bind to the sTyr peptide. However, variant V13 and V24 have cross‐reactivity with both pTyr and sTyr peptides. The newly identified superbinders could be utilized as tools for the identification of pTyr‐containing proteins from tissues under different physiological or pathophysiological conditions and may have the potential in the therapeutics.

show abstract

Section: Discussionmentioning

confidence: 99%

Revisiting the phosphotyrosine binding pocket of Fyn SH2 domain led to the identification of novel SH2 superbinders.

Zou

Zhao

et al. 2020

Protein Science

View full text Add to dashboard Cite

show abstract

“…Nonetheless, we were able to got 12 pTyr binders with enhanced binding affinities, likely due to the bias design of this library (See Methods). Soft randomization method, in which the wild type residues have the dominant proportion, is widely used in in vitro affinity maturation, especially for antibody engineering [17, 27, 28]. The rationale is that if a residue in the binding pocket contributes less to the binding affinity, the other one(s) will show up during the biopanning by affinity selection, even if this/these residue has/have fewer proportion at this position in the library.…”

Section: Discussionmentioning

confidence: 99%

Revisiting the phosphotyrosine binding pocket of Fyn SH2 domain led to the Identification of novel SH2 superbinders

Zou

Zhao

et al. 2020

Preprint

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Protein engineering through directed evolution is an effective way to obtain proteins with novel functions with the potential applications as tools for diagnosis or therapeutics. Many natural proteins, largely antibodies as well as some non-antibody proteins, have undergone directed evolution in vitro in the test tubes in the laboratories around the world, resulted in the numerous protein variants with novel or enhanced functions. In this study, we constructed a Fyn SH2 variant library by randomizing the 8 variable residues in its phosphotyrosine (pTyr) binding pocket. Selection of this library by a pTyr peptide from MidT antigen led to the identification of SH2 variants with enhanced affinities to the peptide, compared to the wild type SH2, by EC50 assay. Fluorescent polarization (FP) was then applied to quantify the binding affinity of the newly identified SH2 variants. As a result, three SH2 variants, named V3, V13 and V24, have comparable binding affinities with the previously identified SH2 triple-mutant superbinder (refer to Trm). Biolayer Interferometry (BLI) assay was employed to disclose the kinetics of the binding of these SH2 superbinders, in addition to the wild type SH2, to the phosphotyrosine peptide. The results indicated that all the SH2 superbinders have two-orders increase of the dissociation rate when binding the pTyr peptide while there was no significant change in their associate rates. The previously identified SH2 superbinder Trm as well as the V13 and V24 discovered in this study have cross-reactivity with the sulfotyrosine (sTyr) containing peptide while the wild type SH2 does not. Intriguingly, though binding the pTyr peptide with comparable affinity with other SH2 superbinders, the V3 does not bind to the sTyr peptide, implying it binds to the pTyr peptide with a different pattern from the other superbinders. The newly identified superbinders could be utilized as tools for the identification of pTyr-containing proteins from tissues under different physiological or pathophysiological conditions and may have the potential in the therapeutics.

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“… 765 BDNF R3bH01 Peripheral nerve injury SNI and tibial nerve transection in male SD rats R3bH01 inhibits hyperactivity to heat and mechanical stimuli of sensory neurons in a dose-dependent manner. 766 HMGB1 anti-HMGB1 nAb Trigeminal neuralgia Constriction of distal infraorbital nerve in male ddY mice Anti-HMGB1 nAb mitigates neuropathic pain through blocking activation of immune cells. 720 TrkA MNAC13 Inflammation and nerve injury Treatment with formalin and CCI in male CD1 mice MNAC13 alleviates hyperalgesia and has synergistic effects in combination with opioids.…”

Section: Intervention Methods For Pain Reliefmentioning

confidence: 99%

Pathology of pain and its implications for therapeutic interventions

Cao,

Xu,

Shi

et al. 2024

Sig Transduct Target Ther

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Pain is estimated to affect more than 20% of the global population, imposing incalculable health and economic burdens. Effective pain management is crucial for individuals suffering from pain. However, the current methods for pain assessment and treatment fall short of clinical needs. Benefiting from advances in neuroscience and biotechnology, the neuronal circuits and molecular mechanisms critically involved in pain modulation have been elucidated. These research achievements have incited progress in identifying new diagnostic and therapeutic targets. In this review, we first introduce fundamental knowledge about pain, setting the stage for the subsequent contents. The review next delves into the molecular mechanisms underlying pain disorders, including gene mutation, epigenetic modification, posttranslational modification, inflammasome, signaling pathways and microbiota. To better present a comprehensive view of pain research, two prominent issues, sexual dimorphism and pain comorbidities, are discussed in detail based on current findings. The status quo of pain evaluation and manipulation is summarized. A series of improved and innovative pain management strategies, such as gene therapy, monoclonal antibody, brain-computer interface and microbial intervention, are making strides towards clinical application. We highlight existing limitations and future directions for enhancing the quality of preclinical and clinical research. Efforts to decipher the complexities of pain pathology will be instrumental in translating scientific discoveries into clinical practice, thereby improving pain management from bench to bedside.

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In vitroaffinity optimization of an anti-BDNF monoclonal antibody translates to improved potency in targeting chronic pain statesin vivo

Abstract: Cunningham (2020) Invitro affinity optimization of an anti-BDNF monoclonal antibody translates to improved potency in targeting chronic pain states invivo, mAbs, 12:1, 1755000,

Cited by 9 publications

References 63 publications

Revisiting the phosphotyrosine binding pocket of Fyn SH2 domain led to the identification of novel SH2 superbinders.

Revisiting the phosphotyrosine binding pocket of Fyn SH2 domain led to the identification of novel SH2 superbinders.

Revisiting the phosphotyrosine binding pocket of Fyn SH2 domain led to the Identification of novel SH2 superbinders

Pathology of pain and its implications for therapeutic interventions

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