Engineering of binding functions into proteins

Gebauer, Michaela; Skerra, Arne

doi:10.1016/j.copbio.2019.05.007

Cited by 30 publications

(25 citation statements)

References 109 publications

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“…Gastrointestinal transit is particularly challenging for therapeutic proteins because of the presence of proteases whose mission is to degrade proteins to ensure proper digestion of food and provide tissues and cells throughout the body with essential nutrients. The goal of this study was first to evaluate the stability of Affitins, a new class of specific affinity binders 12,13 , in simulated gastric (FaSSGF) and intestinal (FaSSIF) fluids. For this purpose, FaSSGF and FaSSIF were prepared based on the updated composition proposed by Biorelevant.com.…”

Section: Discussionmentioning

confidence: 99%

Characterization of Affitin proteolytic digestion in biorelevant media and improvement of their stabilities via protein engineering

Loussouarn

Béhar

Pecorari

et al. 2020

Sci Rep

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Affitins are a novel class of small 7 kDa artificial proteins which can be used as antibody substitutes in therapeutic, diagnostic and biotechnological applications. One challenge for this type of protein agent is their behaviour in the context of oral administration. The digestive system is central, and biorelevant media have fast emerged as relevant and reliable tools for evaluating the bioavailability of drugs. This study describes, for the first time, the stability of Affitins under simulated gastric and intestinal digestion conditions. Affitins appear to be degraded into stable fragments in in vitro gastric medium. We identified cleavage sites generated by pepsin that were silenced by site-directed mutagenesis. This protein engineering allowed us to enhance Affitin properties. We showed that a mutant M1 containing a double mutation of amino acid residues 6 and 7 in H4 and C3 Affitins acquired a resistance against proteolytic digestion. In addition, these mutations were beneficial for target affinity, as well as for production yield. Finally, we found that the mutated residues kept or increased the important pH and temperature stabilities of Affitins. These improvements are particularly sought after in the development of engineered binding proteins for research tools, preclinical studies and clinical applications.

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Section: Discussionmentioning

confidence: 99%

Characterization of Affitin proteolytic digestion in biorelevant media and improvement of their stabilities via protein engineering

Loussouarn

Béhar

Pecorari

et al. 2020

Sci Rep

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“…It is an ideal compound for recognizing the desired targets due to ease of chemical synthesis, quick tumor accumulation, rapid blood clearance, etc. [21,22]. ZHER 2:342 affibody binds explicitly to HER2, and its derivatives have been labeled with PET nuclides ([ 18 F], [ 68 Ga], etc) [23][24][25].…”

Section: Introductionmentioning

confidence: 99%

Synthesis of a novel 89Zr-labeled HER2 affibody and its application study in tumor PET imaging

Wang

Pan

et al. 2020

EJNMMI Res

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Background: Human epidermal growth factor receptor-2 (HER2) is an essential biomarker for tumor treatment. Affibody is an ideal vector for preparing HER2 specific probes because of high affinity and rapid clearance from normal tissues, etc. Zirconium-89 is a PET imaging isotope with a long half-life and suitable for monitoring biological processes for more extended periods. In this study, a novel 89 Zr-labeled HER2 affibody, [ 89 Zr]Zr-DFO-MAL-Cys-MZHER2, was synthesized, and its imaging characters were also assessed. Results: The precursor, DFO-MAL-Cys-MZHER2, was obtained with a yield of nearly 50%. The radiochemical yield of [ 89 Zr]Zr-DFO-MAL-Cys-MZHER2 was 90.2 ± 1.9%, and the radiochemical purity was higher than 95%. The total synthesis time was only 30 min. The probe was stable in PBS and serum. The tracer accumulated in HER2 overexpressing human ovarian cancer SKOV-3 cells. In vivo studies in mice bearing tumors showed that the probe was highly retained in SKOV-3 xenografts even for 48 h. The tumors were visualized with good contrast to normal tissues. ROI analysis revealed that the average uptake values in the tumor were greater than 5% IA/g during 48 h postinjection. On the contrary, the counterparts of MCF-7 tumors kept low levels (~1% IA/g). The outcome was consistent with the immunohistochemical analysis and ex vivo autoradiography. The probe quickly cleared from the normal organs except kidneys and mainly excreted through the urinary system. Conclusion: The novel HER2 affibody for PET imaging was easily prepared with satisfactory labeling yield and radiochemical purity. [ 89 Zr]Zr-DFO-MAL-Cys-MZHER2 is a potential candidate for detecting HER2 expression. It may play specific roles in clinical cancer theranostics.

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“…It is an ideal compound for recognizing the desired targets due to ease of chemical synthesis, quick tumor accumulation and rapid blood clearance, etc. [21,22] ZHER 2:342 affibody specifically binds to HER2 and its derivatives have been labeled with PET nuclides ( 18 F, 68 Ga,etc). [23][24][25]18 F labeled ZHER 2:342 analog, 18 F-FBEM-ZHER 2:342 showed specific binding towards HER2-positive tumors and might be benefit for monitoring status of the receptor in response to therapeutic interventions.…”

Section: Introductionmentioning

confidence: 99%

Synthesize of a novel 89Zr labeled HER2 affibody and its application study in tumor PET imaging

Xu¹,

Wang²,

Pan³

et al. 2020

Preprint

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Background: Human epidermal growth factor receptor-2 (HER2) is an important biomarker for tumor diagnosis and therapy. Affibody is an ideal vector for preparing HER2 specific probes due to the advantages such as high affinity and rapid blood clearance etc. 89Zr is a novel PET imaging isotope with long half-lives and suitable for tracking biological processes for longer periods. In this study, a novel 89Zr labeled HER2 affibody, 89Zr-DFO-MAL-Cys-MZHER2, was synthesized and its imaging properties were also evaluated. Results: The precursors, DFO-MAL-Cys-MZHER2, were obtained with the yields of nearly 50%. The yields of 89Zr -DFO-MAL-Cys-MZHER2 were 90.2±1.9% and the radiopurities were more than 95%. The total synthesis time was only 30 minutes. The probes were stable in PBS and serum. The tracer accumulated in HER2 overexpression human ovarian cancer SKOV-3 cells. In vivo studies in mice bearing tumors showed that the probe highly retained in SKOV-3 xenografts even for 48 hours. The tumors were visualized with good contrast to normal tissue. ROI analysis revealed that the average uptakes in the tumor were greater than 5 %ID/g. On the contrary, the counterparts of MCF-7 tumors kept low levels of （~1%ID/g）. The outcome was consistent with the immunohistochemical analysis and ex vivo autoradiography. The probe quickly cleared from the blood and normal organs, and mainly excreted through the urinary system. Conclusion: The novel HER2 affibody for PET imaging was easily prepared with satisfactory labeling yield and radiochemical purity. 89Zr-DFO-MAL-MZHER is a potential candidate for monitoring HER2 expression and may play specific roles in clinical cancer theranostics.

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Engineering of binding functions into proteins

Cited by 30 publications

References 109 publications

Characterization of Affitin proteolytic digestion in biorelevant media and improvement of their stabilities via protein engineering

Characterization of Affitin proteolytic digestion in biorelevant media and improvement of their stabilities via protein engineering

Synthesis of a novel 89Zr-labeled HER2 affibody and its application study in tumor PET imaging

Synthesize of a novel 89Zr labeled HER2 affibody and its application study in tumor PET imaging

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