| 1. |
- Jafari, Rozbeh, 1977-, et al.
(author)
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Construction of divalent anti-keratin 8 single-chain antibodies (sc(FV)2), expression in Pichia Pastoris and their reactivity with multicellular tumor spheroids
- 2011
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In: JIM - Journal of Immunological Methods. - : Elsevier BV. - 0022-1759 .- 1872-7905. ; 364:1-2, s. 65-76
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Journal article (peer-reviewed)abstract
- Single-chain variable fragments (scFvs) are small monovalent recombinant antibody fragments that retain the specificity of their parent immunoglobulins. ScFvs are excellent building blocks for new and improved immunodiagnostic and therapeutic proteins. However, the monovalency and the rapid renal elimination of scFvs result in poor tumor accumulation and retention. Engineering divalent antibody fragments is an excellent way to address these shortcomings. In this study, covalent divalent single-chain variable fragments (sc(Fv)2s), were constructed from the monovalent anti-keratin 8 scFvs, TS1-218 and its mutant, HE1-Q. The scFvs and sc(Fv)2s were expressed in the methylotrophic yeast Pichia pastoris, utilizing the alpha-factor secretion signal (α-factor) for extracellular secretion. The immunoreactivity and specificity of the antibody fragments were analyzed with enzyme-linked immunosorbent assay (ELISA) and the uptake and retention of the 125I labeled antibody fragments were evaluated using HeLa HEp-2 multicellular tumor spheroids (MCTSs). Analysis of the antibody fragments demonstrated that parts of the α-factor remained at the N-terminal of the antibody fragments. Despite incomplete processing of the α-factor, the antibody fragments were functional where the sc(Fv)2s gave a three-fold stronger signal in ELISA compared to their scFv counterparts and the mutant antibodies demonstrated a stronger signal than their initial wild types. In addition, the sc(Fv)2s DiTS1-218 and DiHE1-Q displayed an approximately two-fold higher uptake and were retained to a larger extent in the MCTS, demonstrating a 3.9 and 9.4-fold increase in half-life respectively compared to their corresponding scFvs. In conclusion, expression in P. pastoris improved the yield 20-fold and facilitated the purification of the antibody fragments. Furthermore, the sc(Fv)2s presented a higher functional affinity to K 8 both in ELISA and MCTS compared to the scFvs with DiHE1-Q being the best candidate for further studies.
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| 2. |
- Jafari, Rozbeh, 1977-, et al.
(author)
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Localization of complexed anticytokeratin 8 scFv TS1-218 to HeLa HEp-2 multicellular tumor spheroids and experimental tumors
- 2010
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In: Cancer Biotherapy and Radiopharmaceuticals. - : Mary Ann Liebert Inc. - 1084-9785 .- 1557-8852. ; 25:4, s. 455-463
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Journal article (peer-reviewed)abstract
- Recombinant single-chain fragment variable (scFv) antibodies with specificity to tumor antigens can be used to target tumors in vivo. The approach to use administration of complexes of idiotypic-anti-idiotypic scFvs when targeting tumors has not been tested earlier, and from a theoretical point it could contribute to longer in vivo circulation and improved targeting efficiency by dissociation, when in contact with the target antigen. In this study two models to evaluate the targeting efficiency of such complexes were used. HeLa HEp-2 tumor cells were grown as multicellular tumor spheroids (MCTS) and exposed to the antibody constructs in vitro. The behavior in vivo was tested in an in vivo tumor xenograft model. To increase the size of the anticytokeratin 8 scFv, TS1-218, complexes were formed between TS1-218 and its anti-idiotype, alphaTS1 scFv. The functionality of (125)I-labeled TS1-218 alone and in complex was studied in both models. The uptake patterns were similar in both models. The idiotypic TS1-218 was able to localize to the MCTS and xenografted tumors, both alone and in complex with alphaTS1 scFv. TS1-218 in complex, however, demonstrated a significantly higher uptake than the monomeric TS1-218 in both models (p < 0.0005 and p < 0.0089, respectively). When complexes were administered in vivo, a slower clearance and an increased tumor half-life could be observed. The present investigation indicates that administration of targeting antibodies, with initially blocked antigen-binding sites by complex formation with their anti-idiotypes, may improve targeting efficiency.
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| 3. |
- Rendel, Filip, et al.
(author)
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Effects of Di-Isononyl Phthalate on Neuropeptide Y Expression in Differentiating Human Neuronal Cells
- 2017
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In: Basic & Clinical Pharmacology & Toxicology. - : Blackwell Publishing. - 1742-7835 .- 1742-7843. ; 120:3, s. 218-323
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Journal article (peer-reviewed)abstract
- Neuropeptide Y (NPY) is an abundant neuropeptide in the mammalian brain important for behavioural consequences of stress and energy metabolism. We have addressed possible effects of the phthalate DiNP on NPY expression in human SH‐SY5Y cells, a neuronal in vitro differentiation model. Pico‐ to nanomolar doses of DiNP and its metabolite MiNP resulted in decreased NPY mRNA and peptide expression in retinoid‐differentiated cells. Thus, dys‐regulated NPY may be an adverse outcome for exposure to low doses of DiNP in human beings.
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