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- Almqvist, Helena, et al.
(författare)
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CETSA screening identifies known and novel thymidylate synthase inhibitors and slow intracellular activation of 5-fluorouracil
- 2016
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Target engagement is a critical factor for therapeutic efficacy. Assessment of compound binding to native target proteins in live cells is therefore highly desirable in all stages of drug discovery. We report here the first compound library screen based on biophysical measurements of intracellular target binding, exemplified by human thymidylate synthase (TS). The screen selected accurately for all the tested known drugs acting on TS. We also identified TS inhibitors with novel chemistry and marketed drugs that were not previously known to target TS, including the DNA methyltransferase inhibitor decitabine. By following the cellular uptake and enzymatic conversion of known drugs we correlated the appearance of active metabolites over time with intracellular target engagement. These data distinguished a much slower activation of 5-fluorouracil when compared with nucleoside-based drugs. The approach establishes efficient means to associate drug uptake and activation with target binding during drug discovery.
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| 7. |
- Jafari, Rozbeh, 1977-
(författare)
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Construction, expression and evaluation of anti-keratin 8 single-chain antibody fragments
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Antibodies are glycoproteins specifically binding to a variety of antigens and today extensively used in prevention, diagnosis and treatment of diseases. Carcinomas originate from epithelial tissues and are the most common forms of human malignancies. In the necrotic areas of carcinomas significant amounts of keratins (K) are found extracellularly. They remain there due to their low solubility and can be used as targets in immuno-targeting and -therapy. Single-chain fragment variable (scFv) may display several advantages over intact IgGs in various applications but their use in immunotherapy of tumors could be limited. Their small size and monovalency may result in low accumulation of the scFv in the tumors. The targeting efficiency of antibodies needs to be evaluated in vitro prior to in vivo studies in order to exclude poor candidates. In vitro models should preferably resemble the in vivo situation as much as possible. In the present study a scFv variant (TS1-218) of the anti-keratin 8 (K 8) monoclonal antibody TS1 (mAb TS1) was constructed and characterized using site-directed mutagenesis. In addition, the valency of the TS1-218 and one of its mutants, HE1-Q, were increased by construction of a covalently linked divalent single-chain fragment variable (sc(Fv)2). To improve the yield of the antibody fragments, the expression host was changed from E. coli to P. pastoris and culture conditions were optimized using Design of Experiments (DoE). Furthermore, a HeLa HEp-2 multicellular tumor spheroid (MCTS) in vitro model was established. The functionality of the radiolabeled TS1-218 alone and in immunocomplex with its anti-idiotype scFv, αTS1 scFv, were evaluated in the MCTS model and compared to a tumor xenograft nude mouse model. The targeting efficiency of the scFv and sc(Fv)2s were also investigated using MCTS. TS1-218 in immunocomplex with αTS1 scFv displayed a significantly higher uptake than the TS1-218 alone in both MCTS and tumor xenografts. The sc(Fv)2s, DiTS1-218 and DiHE1-Q demonstrated a higher functional affinity to K 8 in ELISA and MCTS and were retained to a larger extent in the MCTS than their scFv counterparts, with a 3.9 and 9.4-fold longer half-life, respectively. Furthermore, the yield of the antibody fragments were improved after expression in P. pastoris with an 86-fold improvement for the TS1-218 following optimization using DoE.
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| 8. |
- Jafari, Rozbeh, 1977-, et al.
(författare)
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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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Ingår i: JIM - Journal of Immunological Methods. - : Elsevier BV. - 0022-1759 .- 1872-7905. ; 364:1-2, s. 65-76
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Tidskriftsartikel (refereegranskat)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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- Jafari, Rozbeh, 1977-, et al.
(författare)
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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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Ingår i: Cancer Biotherapy and Radiopharmaceuticals. - : Mary Ann Liebert Inc. - 1084-9785 .- 1557-8852. ; 25:4, s. 455-463
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Tidskriftsartikel (refereegranskat)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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| 10. |
- Leo, Isabelle Rose, et al.
(författare)
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Integrative multi-omics and drug response profiling of childhood acute lymphoblastic leukemia cell lines
- 2022
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Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 13:1
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Tidskriftsartikel (refereegranskat)abstract
- Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. Although standard-of-care chemotherapeutics are sufficient for most ALL cases, there are subsets of patients with poor response who relapse in disease. The biology underlying differences between subtypes and their response to therapy has only partially been explained by genetic and transcriptomic profiling. Here, we perform comprehensive multi-omic analyses of 49 readily available childhood ALL cell lines, using proteomics, transcriptomics, and pharmacoproteomic characterization. We connect the molecular phenotypes with drug responses to 528 oncology drugs, identifying drug correlations as well as lineage-dependent correlations. We also identify the diacylglycerol-analog bryostatin-1 as a therapeutic candidate in the MEF2D-HNRNPUL1 fusion high-risk subtype, for which this drug activates pro-apoptotic ERK signaling associated with molecular mediators of pre-B cell negative selection. Our data is the foundation for the interactive online Functional Omics Resource of ALL (FORALL) with navigable proteomics, transcriptomics, and drug sensitivity profiles at https://proteomics.se/fora.
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