Team:GreatBay SCIE/Contribution
Contribution
Description
As GreatBay_SCIE 2021’s is focusing on aptamer-based targeted drugs delivery systems(seen in Project: Description; issues with antibody-based targeted drugs), we try to broaden our project by finding out other possibilities in cancer therapy.
After browsing large amount of previous teams’ wiki and research papers, our team concentrated contributions to two registry pages of two parts- Part:BBa_K1781002 and Part:BBa_K1694005, therefore our Contribution was made up of detailing scFvs and ZHER2 comprehensively and presenting new research direction of future IGEM team.
To work efficiently in E.coli—scFvs Improvement of solubility and biological activity of the inclusion bodies of scfvs(Part:BBa_K1694005)
When preparing scFvs in E.coli, it usually leads to formation of inclusion bodies (IBs). The aim of this research was to improve solubilizing and refolding conditions for IBs of scFv version of pertuzumab (anti-human epidermal growth factor receptor 2 (HER2) antibody). After protein overexpression in E. coli BL21 (DE3), bacterial cells were lysed and IBs were extracted via repeated washing and centrifugation. The effect of different types, concentrations, pHs, and additive of denaturing agents on IBs solubility were evaluated. More than 40 refolding additives were screened and combinations of 10 of the best additives were check out using Plackett-Burman design to choose three refolding additives with the most positive effect on refolding of the scFv. Response surface methodology (RSM) was used to optimize the concentration of adopted additives. The most efficient buffer to solubilize IBs was a buffer containing 6 M urea with 6 mM beta mercaptoethanol, pH 11. The optimum concentration of three buffer additives for refolding of the scFv was 23 mM tricine, 0.55 mM arginine, and 14.3 mM imidazole. The bioactivity of the refolded scFv was confirmed by immunohistochemical staining of breast cancer tissue, a specific binding based method. The systematic optimization of refolding buffer developed in the present work will contribute to improve the refolding of other scFv fragments.
Plackett-Burman experimental design of 11 factors at 2 levels and effect of these factors on refolding of anti-HER2 scFv.
Box-Behnken experimental design of 3 factors (refolding additive) at 3 levels (concentration).
Solubilizing of anti-human epidermal growth factor receptor 2 single chain variable fragment antibody inclusion bodies (HER2 scFv IBs) using (a) different concentrations of urea, guanidine hydrochloride (GdnHCl), and their combinations. Lane 1, urea 6 M; Lane 2, urea 4 M; Lane 4, urea 2 M; Lane 5, GdnHCl 2 M and urea 2 M; Lane 6, GdnHCl 4 M and urea 4 M; Lane 7, GdnHCl 2 M; lane 8, GdnHCl 4 M; Lane 9, GdnHCl 6 M; Lane 10, urea 8 M; and Lane 11, GdnHCl 8 M; (b) Urea 6 M at different pH. Lane 1-4, pH 5, pH 7, pH 9, and pH 11, (C) urea 6 M at pH 11 supplemented with different additives. Lane 2, beta mercaptoethanol (BME) 4 mM; Lane 3, n-Propanol 5%; Lane 4, dithiothreitol (DTT) 4 mM; and Lane 5, no additive; (d) Urea (6 M) at pH 11 supplemented with different concentrations of BME. Lane 1: BME 2 mM; Lane 2: BME 4 mM; Lane 3: BME 6 mM and Lane 4: BME 8 mM. Lanes 3a, 5b, 1c, and 5d are protein marker.
scFvs with nanoparticles
https://www.nature.com/articles/s41467-018-06271-5
https://link.springer.com/article/10.1186/s12951-018-0341-6
scFvs with liposome
https://www.sciencedirect.com/science/article/abs/pii/S014181301940408X
https://sci.bban.top/pdf/10.1016/j.actbio.2020.04.029.pdf?download=true
Improvement and New Directions for ZHER2(Part:BBa_K1694005)
In the first set of experiments, the Affibody molecule, ZHER2:4, was dimerized by head-to-tail fusion to gain avidity effects, and the resulting dimer (ZHER2:4)2, showed an improved apparent affinity by one order of magnitude to 3 nM.34 The use of the dimeric Affibody molecule for tumor targeting and imaging was then analyzed using different radionuclides and labeling chemistries. Comparison of the cellular-retained radioactivity of the iodinated monomeric and dimeric forms, denoted as 125I-PIB-ZHER2:4 and 125I-PIB-(ZHER2:4)2, respectively, confirmed that the dimerized molecule was superior. Later, both monomeric and dimeric ZHER2:4 were shown to specifically target HER2-expressing xenografts in vivo. A tumor-to-blood ratio of 10 was achieved 8 hours post injection (p.i.) of the dimeric form in a murine model, and a clear vi- sualization of SKOV-3 xenografts was obtained using a clinical gamma camera. The specificity of HER2 targeting in vivo was proven by the pre- saturation of HER2 receptors with an excess of unlabeled (ZHER2:4)2, which led to a decreased tu- mor uptake of radiolabeled (ZHER2:4)2. Further- more, no specific tumor accumulation could be seen after the administration of a non relevant Affibody molecule, (125I-PIB-ZTaq, binding a bac- terial protein).35 As an alternative to radioio- dination, 99mTc (I)-tricarbonyl labeling of the His-tagged Affibody molecule His6-(ZHER2:4)2 was evaluated. 99mTc-His6-(ZHER2:4)2 showed a specific tumor uptake nearly equal to the uptake of 125I-PIB-His6-(ZHER2:4)2 and enabled the gamma-camera imaging of HER2-expressing xenografts in mice. However, hepatic uptake of 99mTc-labeled His6-(ZHER2:4)2 was higher than for radioiodinated (ZHER2:4)2 .
To facilitate site-specific labeling, a unique cysteine residue was introduced at the C-termi- nus of the dimeric Affibody molecule. Radio- bromination of (ZHER2:4)2-Cys using (4-hydrox- yphenyl)ethyl)maleimide (HPEM) at slightly acidic conditions (pH 6.0) provided a conjugate with preserved specific binding to HER2-expres- sing cells. In a murine xenograft model, 76/77Br- HPEM-His6-(ZHER2:4)2-Cys demonstrated a tu- mor-to-blood ratio of nine at 4 hours p.i. This method is sufficiently rapid to be used for label- ing with the positron-emitting isotope, 76Br (T1/2 16 hours), to obtain a tracer for the PET imaging of HER2-expressing tumors. Another in- teresting observation was that radio bromination using the HPEM linker resulted in less renal ac- cumulation of radioactivity, compared with the 4-bromo-benzoate linker. The same observation was done with radioiodinated conjugates,38 indi- cating that the biodistribution of radiolabeled Affibody molecules could be modified by the use of an appropriate labeling technique.
The second strategy to improve the target bind- ing affinity of HER2-specific Affibody mole- cules was to perform an affinity maturation based on sequence analysis of first-generation HER2- binders, followed by the directed combinatorial mutagenesis of selected positions in the binding site. The most favorable Affibody molecule, ZHER2:342, selected by this strategy showed a two thousand two hundred fold increased HER2- binding affinity; from 50 nM to 22 pM.27 ZHER2:342 was radioiodinated and demonstrated efficient, selective targeting of HER2-expressing xenografts in vivo, yielding a tumor-to-blood ra- tio of 38 at 4 hours p.i. A comparative biodistri- bution experiment demonstrated that 125I-PIB- ZHER2:342 was superior to the monomeric, as well as the dimeric form of the parental molecule ZHER2:4.
Indirect radioiodination using PIB is a two-step process that not readily converts into the user- friendly kit-formulation required for a smooth in- troduction into the clinical routine. Furthermore, radiometals, such as indium-111, have an appar- ent advantage, since the logistics of radiometal- labeling in the clinic is appreciably better than for radiohalogens, such as 123I. Labeling of re- combinant ZHER2:342 with 111In was done using the chelators benzyl-DTPA,39 CHX-A-DTPA,40 or benzyl-DOTA.41 Isothiocyanate derivatives of these chelators were coupled to Affibody mole- functional Affibody molecules. In addition, chem- ical synthesis allows for the site-specific modifi- cation of Affibody molecules as an intrinsic part of the synthesis process. For example, it is possi- ble to couple a chelator at a specifically protected side group of the peptide chain. Thus, homoge- nous, well-defined tracers are obtained. Several different HER2-specific Affibody-DOTA deriva- tives were synthesized in this way, and DOTA- ZHER2:342-pep2 (ABY-002) with a DOTA chelator coupled to the N-terminus was selected as the most promising candidate and analyzed in more detail.28 After synthesis, deprotection, and conventional reversed-phase high-performance liquid chromatog- raphy (HPLC) purification, DOTA-ZHER2:342-pep2 folds readily into its active three-dimensional conformation and binds HER2 with 65 pM affinity, as measured by plasmon resonance. Biodistribu- tion experiments in a murine SKOV-3 xenograft model with 111In-labled DOTA-ZHER2:342-pep2 demonstrated a tumor uptake as high as 23% IA/g 1 hour p.i., and high-contrast images were obtained already 1 hour p.i., using the same xenograft model. In vivo, the HER2-binding specificity of
111In-DOTA-ZHER2:342-pep2 was demonstrated by the successful receptor blocking with an excess of cold tracer, by the absence of radioactivity accu- mulation in tumors of non-HER2-expressing lymphoma xenografts, and supported by the absence of the accumulation of a negative control Affibody molecule (111In-DOTA-ZTaq4:5) in SKOV-3 xenograft tumors. It was also shown in vivo that trastuzumab (Herceptin®) does not interfere with the targeting of 111In-DOTA-ZHER2:342-pep2 to HER2-expressing xenografts. Thus, monitoring the HER2 status also of patients undergoing trastuzumab therapy seems possible. A first clini- cal investigation in a limited number of patients with recurrent HER2-expressing breast cancer demonstrated the utility of both 111In-DOTA- ZHER2:342-pep2 and 68Ga-DOTA-ZHER2:342-pep2 to visualize HER2-expressing metastases.
Reference
- Javad Salehinia, Hamid Mir Mohammad Sadeghi, Daryoush Abedi, and Vajihe Akbari/ Improvement of solubility and refolding of an anti-human epidermal growth factor receptor 2 single-chain antibody fragment inclusion bodies/ Res Pharm Sci. 2018 Dec; 13(6): 566–574.
- Anna Orlova, Joachim Feldwisch, Lars Abrahmsén, and Vladimir Tolmachev /Affibody Molecules for Molecular Imaging and Therapy for Cancer /CANCER BIOTHERAPY & RADIOPHARMACEUTICALS Volume 22, Number 5, 2007