In vitro affinity maturation to improve the efficacy of a hypoxia-inducible factor 1α single-domain intrabody

Hu, Min; Kang, Guangbo; Cheng, Xin; Wang, Jiewen; Li, Ruowei; Bai, Zixuan; Yang, Dong; Huang, He

doi:10.1016/j.bbrc.2020.06.097

Cited by 19 publications

(18 citation statements)

References 35 publications

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“…There are several examples demonstrating the feasibility of this approach, but these are still the results of case-by-case projects that require a major input from specialized researchers and often require access to structural information [9][10][11][12]. On the other hand, we recently confirmed that publicly accessible algorithms that require minimal data inputs can rapidly and effectively help in the identification of protein variants that exhibit astonishingly higher yields than the original sequences [13].…”

Section: Introductionmentioning

confidence: 79%

“…The possibility of improving clone stability, increasing their yields, and maintaining or improving the sequence humanization by rational mutagenesis would represent a substantial advancement, and community efforts have already resulted in the development of successful approaches. There are several examples demonstrating the feasibility of this approach, but these are still the results of case-by-case projects that require a major input from specialized researchers and often require access to structural information [ 9 , 10 , 11 , 12 ]. On the other hand, we recently confirmed that publicly accessible algorithms that require minimal data inputs can rapidly and effectively help in the identification of protein variants that exhibit astonishingly higher yields than the original sequences [ 13 ].…”

Section: Introductionmentioning

confidence: 99%

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CDR1 Composition Can Affect Nanobody Recombinant Expression Yields

et al. 2021

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The isolation of nanobodies from pre-immune libraries by means of biopanning is a straightforward process. Nevertheless, the recovered candidates often require optimization to improve some of their biophysical characteristics. In principle, CDRs are not mutated because they are likely to be part of the antibody paratope, but in this work, we describe a mutagenesis strategy that specifically addresses CDR1. Its sequence was identified as an instability hot spot by the PROSS program, and the available structural information indicated that four CDR1 residues bound directly to the antigen. We therefore modified the loop flexibility with the addition of an extra glycine rather than by mutating single amino acids. This approach significantly increased the nanobody yields but traded-off with moderate affinity loss. Accurate modeling coupled with atomistic molecular dynamics simulations enabled the modifications induced by the glycine insertion and the rationale behind the engineering design to be described in detail.

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Section: Introductionmentioning

confidence: 79%

Section: Introductionmentioning

confidence: 99%

CDR1 Composition Can Affect Nanobody Recombinant Expression Yields

et al. 2021

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“…However, several studies are required to improve the application of VHH212 in pancreatic cancer treatments. For example, with the development of data accumulation and algorithm optimization, high affinity nanobodies can be explored using virtual mutation screening technology 28 , 60 . In addition, synthetic biology is driving a new era of nanobody-based drug delivery systems for digestive system diseases 61 .…”

Section: Discussionmentioning

confidence: 99%

“…The recombinant protein was produced in Rosetta (DE3) (Novagen, Darmstadt, Germany) and purified by affinity chromatography using a GSTrapFF column and ÄKTA primer plus (GE Healthcare, Chicago, IL, USA). The amino acid sequence of anti-HIF-1α-PAS-B-VHH212 ( Figure 2B ) (GenBank accession number: MK978327) was previously selected from a novel naive nanobody library by ribosome display technology and kept in our laboratory 28 . The VHH212 sequence was codon-optimized, synthesized, and subcloned into a pET-32a + expression plasmid, with RBS/TATA box at the N-terminal and 6*His tag at the C-terminal, and was subsequently expressed in Origami™ B (DE3) (Novagen).…”

Section: Methodsmentioning

confidence: 99%

VHH212 nanobody targeting the hypoxia-inducible factor 1α suppresses angiogenesis and potentiates gemcitabine therapy in pancreatic cancer <i>in vivo</i>

Kang

Ren

et al. 2021

Cancer Biol. Med.

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Objective: We aimed to develop a novel anti-HIF-1α intrabody to decrease gemcitabine resistance in pancreatic cancer patients. Methods: Surface plasmon resonance and glutathione S-transferase pull-down assays were conducted to identify the binding affinity and specificity of anti-HIF-1α VHH212 [a single-domain antibody (nanobody)]. Molecular dynamics simulation was used to determine the protein-protein interactions between hypoxia-inducible factor-1α (HIF-1α) and VHH212. The real-time polymerase chain reaction (PCR) and Western blot analyses were performed to identify the expressions of HIF-1α and VEGF-A in pancreatic ductal adenocarcinoma cell lines. The efficiency of the VHH212 nanobody in inhibiting the HIF-1 signaling pathway was measured using a dual-luciferase reporter assay. Finally, a PANC-1 xenograft model was developed to evaluate the anti-tumor efficiency of combined treatment. Immunohistochemistry analysis was conducted to detect the expressions of HIF-1α and VEGF-A in tumor tissues. Results: VHH212 was stably expressed in tumor cells with low cytotoxicity, high affinity, specific subcellular localization, and neutralization of HIF-1α in the cytoplasm or nucleus. The binding affinity between VHH212 and the HIF-1α PAS-B domain was 42.7 nM. Intrabody competitive inhibition of the HIF-1α heterodimer with an aryl hydrocarbon receptor nuclear translocator was used to inhibit the HIF-1/VEGF pathway in vitro. Compared with single agent gemcitabine, co-treatment with gemcitabine and a VHH212-encoding adenovirus significantly suppressed tumor growth in the xenograft model with 80.44% tumor inhibition. Conclusions:We developed an anti-HIF-1α nanobody and showed the function of VHH212 in a preclinical murine model of PANC-1 pancreatic cancer. The combination of VHH212 and gemcitabine significantly inhibited tumor development. These results suggested that combined use of anti-HIF-1α nanobodies with first-line treatment may in the future be an effective treatment for pancreatic cancer.

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“…The tumor microenvironment plays a vital role in the development of tumors and is closely related to the e cacy of tumor treatment. Targeting adaptive response for tumor environment may be a therapeutic strategy for PC [3]. Hypoxic microenvironment induces the up-regulation of a series of hypoxic-responsive genes, and then induce the proliferation, migration, drug resistance and various other biological events in cancer cells [4].…”

Section: Introductionmentioning

confidence: 99%

Hypoxia-Related Gene FUT11 Promoted Pancreatic Cancer Progression Via Maintaining The Stabilization of PDK1

Cao

Zeng

et al. 2021

Preprint

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Background: Hypoxia participated in the occurrence and development of pancreatic cancer (PC). However, genes associated with hypoxia respond and their regulated mechanism in PC cells were unclear. The current research was aimed to illuminate the role and hypoxia regulated mechanism of fucosyltransferase 11 (FUT11) in the progression of PC.Methods: After predicting FUT11 as a key hypoxia associated gene in PC using bioinformatics analysis. The expression of FUT11 in PC using quantitative real-time fluorescent PCR, western blot and immunohistochemistry. The effects of FUT11 on PC cells proliferation, migration and invasion under normoxia and hypoxia were detected using Cell Counting Kit 8, 5-ethynyl-2’-deoxyuridine assay, colony formation assay and transwell assay. Spleen capsule injected liver metastasis and subcutaneously injected model were performed to confirm the effects of FUT11 in vivo. Furthermore, western blot, luciferase assay and immunoprecipitation were performed to explore the regulated relationship among FUT11, hypoxia-inducible factor 1α (HIF1α) and pyruvate dehydrogenase kinase 1 (PDK1) in PC.Results: FUT11 was markedly increased of PC cells in hypoxia, up-regulated in the PC clinical tissues, and predicted a poor outcome. Inhibition of FUT11 reduced PC cell growth and mobility of PC cells under normoxia and hypoxia conditions in vitro, and growth and mobility in vivo. FUT11 bind with PDK1 and regulated the expression PDK1 under normoxia and hypoxia. FUT11 knockdown significantly increased the degradation rate of PDK1 under hypoxia, while treatment with MG132 can relieve the degradation of PDK1 induced by FUT11 knockdown. Overexpression of PDK1 in PC cells under hypoxia conditions reversed the suppressiv impacts of FUT11 knockdown on PC cell growth and mobility. In addition, HIF1α bound to the enhancer of FUT11 and increased its expression, as well as co-expressing with FUT11 in PC tissues. Furthermore, overexpress of FUT11 partially rescued the suppressiv effects of HIF1α knockdown on PC cell growth and mobility in hypoxia conditions.Conclusion: Our data further implicate that hypoxia-induced FUT11 in PC contributes to proliferation and metastasis by maintaining the stability of PDK1, and suggest FUT11 maybe a novel and effective target for treatment of pancreatic cancer.

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In vitro affinity maturation to improve the efficacy of a hypoxia-inducible factor 1α single-domain intrabody

Cited by 19 publications

References 35 publications

CDR1 Composition Can Affect Nanobody Recombinant Expression Yields

CDR1 Composition Can Affect Nanobody Recombinant Expression Yields

VHH212 nanobody targeting the hypoxia-inducible factor 1α suppresses angiogenesis and potentiates gemcitabine therapy in pancreatic cancer <i>in vivo</i>

Hypoxia-Related Gene FUT11 Promoted Pancreatic Cancer Progression Via Maintaining The Stabilization of PDK1

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