Tumour imaging by the detection of fibrin clots in tumour stroma using an anti-fibrin Fab fragment.
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ABSTRACT:
SUBMITTER: Obonai T
PROVIDER: S-EPMC4806360 | biostudies-other | 2016 Mar
REPOSITORIES: biostudies-other
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Project description:UnlabelledThe critical challenge in abdominal aortic aneurysm (AAA) research is the accurate diagnosis and assessment of AAA progression. Angiogenesis is a pathologic hallmark of AAA, and CD105 is highly expressed on newly formed vessels. Our goal was to use (64)Cu-labeled anti-CD105 antibody Fab fragment for noninvasive assessment of angiogenesis in the aortic wall in a murine model of AAA.MethodsFab fragment of TRC105, a mAb that specifically binds to CD105, was generated by enzymatic papain digestion and conjugated to NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) for (64)Cu labeling. The binding affinity/specificity of NOTA-TRC105-Fab was evaluated by flow cytometry and various ex vivo studies. BALB/c mice were anesthetized and treated with calcium phosphate to induce AAA and underwent weekly PET scans using (64)Cu-NOTA-TRC105-Fab. Biodistribution and autoradiography studies were also performed to confirm the accuracy of PET results.ResultsNOTA-TRC105-Fab exhibited high purity and specifically bound to CD105 in vitro. Uptake of (64)Cu-NOTA-TRC105-Fab increased from a control level of 3.4 ± 0.1 to 9.5 ± 0.4 percentage injected dose per gram (%ID/g) at 6 h after injection on day 5 and decreased to 7.2 ± 1.4 %ID/g on day 12, which correlated well with biodistribution and autoradiography studies (i.e., much higher tracer uptake in AAA than normal aorta). Of note, enhanced AAA contrast was achieved, due to the minimal background in the abdominal area of mice. Degradation of elastic fibers and highly expressed CD105 were observed in ex vivo studies.Conclusion(64)Cu-NOTA-TRC105-Fab cleared rapidly through the kidneys, which enabled noninvasive PET imaging of the aorta with enhanced contrast and showed increased angiogenesis (CD105 expression) during AAA. (64)Cu-NOTA-TRC105-Fab PET may potentially be used for future diagnosis and prognosis of AAA.
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Project description:Immunoglobulin E (IgE) plays a central role in the allergic response, in which cross-linking of allergen by FcεRI-bound IgE triggers mast cell and basophil degranulation and the release of inflammatory mediators. The high-affinity interaction between IgE and FcεRI is a long-standing target for therapeutic intervention in allergic disease. Omalizumab is a clinically approved anti-IgE monoclonal antibody that binds to free IgE, also with high affinity, preventing its interaction with FcεRI. All attempts to crystallize the pre-formed complex between the omalizumab Fab and the Fc region of IgE (IgE-Fc), to understand the structural basis for its mechanism of action, surprisingly failed. Instead, the Fab alone selectively crystallized in different crystal forms, but their structures revealed intermolecular Fab/Fab interactions that were clearly strong enough to disrupt the Fab/IgE-Fc complexes. Some of these interactions were common to other Fab crystal structures. Mutations were therefore designed to disrupt two recurring packing interactions observed in the omalizumab Fab crystal structures without interfering with the ability of the omalizumab Fab to recognize IgE-Fc; this led to the successful crystallization and subsequent structure determination of the Fab/IgE-Fc complex. The mutagenesis strategy adopted to achieve this result is applicable to other intractable Fab/antigen complexes or systems in which Fabs are used as crystallization chaperones.
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Project description:The structure of the anti-C60 fullerene antibody Fab fragment (FabC60) was solved by X-ray crystallography. The computer-aided docking of C60 into the antigen-binding pocket of FabC60 showed that binding of C60 to FabC60 is governed by the enthalpy and entropy; namely, by π-π stacking interactions with aromatic residues of the antigen-binding site and reduction of the solvent-accessible area of the hydrophobic surface of C60. A fragment of the mobile CDR H3 loop located on the surface of FabC60 interferes with C60 binding in the antigen-binding site, thereby resulting in low antibody affinity for C60. The structure of apo-FabC60 has been deposited with pdbid 6H3H.
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Project description:Dynamic contrast agent-enhanced magnetic resonance imaging measurements of the perfusion of an immunogenic murine tumour showed that immune rejection was preceded by an increase in the apparent vascular volume of the tumour. This increase in vascularity, which has been observed previously in other tumours undergoing immune rejection, was confirmed by histological analysis of tumour sections obtained postmortem. Magnetic resonance imaging measurements similar to this could be used in the clinic to monitor the early responses of tumours to immunotherapy, before there is any change in tumour growth rate or volume.
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Project description:Thyroid hormones are important regulatory hormones, acting on nearly every cell in the body. The two main thyroid hormones are l-thyroxine (tetraiodo-l-thyronine, T4) and 3,3',5-triiodo-l-thyronine (T3), which are produced in the thyroid gland and secreted into the blood stream. Other important thyroid hormone metabolites are 3,3'-diiodo-l-thyronine (T2) and l-thyronine (T0), which may show increased levels in circulation due to dietary iodine deficiency or other medical disorders. Owing to their central role in cellular functions, sensitive and specific detection methods for thyroid hormones are needed. In this work, native mass spectrometry (MS) was used to quantitate thyroid hormone binding to the anti-T4 antibody Fab fragment. First, the binding affinity for T2 was determined via direct ligand titration experiments. Then, the affinities for the other ligands were determined by competition experiments using T2 as the "low-affinity" reference ligand. The highest affinity was measured for T3, followed by T4, T2, and T0 (K d = 29, 3.4, and 260 nM and 130 ?M, respectively). Thus, it is evident that the number and positions of the iodine substituents within the thyronine rings are important for the ligand binding affinity of anti-T4 Fab. Surprisingly, structurally related tetrahalogen bisphenols were also able to bind to anti-T4 Fab with nanomolar affinities.
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Project description:P-glycoprotein (Pgp) has been considered as a major cause of cancer multidrug resistance; however, clinical solutions to overcome this drug resistance do not exist despite the tremendous endeavors. The lack of cancer specificity is a main reason for clinical failure of conventional approaches. Targeted photodynamic therapy (PDT) is highly cancer specific by combining antibody targeting and locoregional light irradiation. We aimed to develop Pgp-targeted PDT using antibody-photosensitizer conjugates made of a recombinant Fab fragment. We prepared the photosensitizer conjugates by expressing a recombinant Fab fragment and specifically linking IR700-maleimide at the C-terminal of the Fab heavy chain. In vitro studies showed that the Fab conjugates specifically bind to Pgp. Their phototoxicity was comparable to full antibody conjugates when assayed with conventional 2-D cell culture, but they outperformed the full antibody conjugates in a 3-D tumor spheroid model. In a mouse xenograft model of chemoresistant tumors, Fab conjugates showed Pgp specific delivery to chemoresistant tumors. Upon irradiation with near-infrared light, they caused rapid tumor shrinkage and significantly prolonged the survival of tumor-bearing mice. Compared to the full antibody conjugates, Fab conjugates took a shorter time to reach peak tumor levels and achieved a more homogeneous tumor distribution. This allows light irradiation to be initiated at a shorter time interval after the conjugate injection, and thus may facilitate clinical translation. We conclude that our targeted PDT approach provides a highly cancer-specific approach to combat chemoresistant tumors, and that the conjugates made of recombinant antibody fragments are superior to full antibody conjugates for targeted PDT.
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Project description:We have prepared a monoclonal Buckminsterfullerene specific antibody and report the sequences of its light and heavy chains. We also show, by x-ray crystallographic analysis of the Fab fragment and by model building, that the fullerene binding site is formed by the interface of the antibody light and heavy chains. Shape-complementary clustering of hydrophobic amino acids, several of which participate in putative stacking interactions with fullerene, form the binding site. Moreover, an induced fit mechanism appears to participate in the fullerene binding process. Affinity of the antibody-fullerene complex is 22 nM as measured by competitive binding. These findings should be applicable not only to the use of antibodies to assay and direct potential fullerene-based drug design but could also lead to new methodologies for the production of fullerene derivatives and nanotubes as well.
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Project description:BackgroundMany antibody crystal structures have been solved. Structural modeling programs have been developed that utilize this information to predict 3-D structures of an antibody based upon its sequence. Because of the problem of self-reference, the accuracy and utility of these predictions can only be tested when a new structure has not yet been deposited in the Protein Data Bank.MethodsWe have solved the crystal structure of the Fab fragment of RAC18, a protective anti-ricin mAb, to 1.9 Å resolution. We have also modeled the Fv structure of RAC18 using publicly available Ab modeling tools Prediction of Immunoglobulin Structures (PIGS), RosettaAntibody, and Web Antibody Modeling (WAM). The model structures underwent energy minimization. We compared results to the crystal structure on the basis of root-mean-square deviation (RMSD), template modeling score (TM-score), Z-score, and MolProbity analysis.FindingsThe crystal structure showed a pocket formed mainly by AA residues in each of the heavy chain complementarity determining regions (CDRs). There were differences between the crystal structure and structures predicted by the modeling tools, particularly in the CDRs. There were also differences among the predicted models, although the differences were small and within experimental error. No one modeling program was clearly superior to the others. In some cases, choosing structures based only on sequence homology to the crystallized Ab yielded RMSDs comparable to the models.ConclusionsMolecular modeling programs accurately predict the structure of most regions of antibody variable domains of RAC18. The hypervariable CDRs proved most difficult to model, particularly H chain CDR3. Because CDR3 is most often involved in contact with antigen, this defect must be considered when using models to identify potential contacts between antibody and antigen. Because this study represents only a single case, the results cannot be generalized. Rather they highlight the utility and limitations of modeling programs.
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Project description:Intercellular communication between cancer cells themselves or with healthy cells in the tumor microenvironment and/or pre-metastatic sites plays an important role in cancer progression and metastasis. In addition to ligand-receptor signaling complexes, extracellular vesicles (EVs) are emerging as novel mediators of intercellular communication both in tissue homeostasis and in diseases such as cancer. EV-mediated transfer of molecular activities impacting morphological features and cell motility from highly metastatic SW620 cells to non-metastatic SW480 cells is a good in vitro example to illustrate the increased malignancy of colorectal cancer leading to its transformation and aggressive behavior. In an attempt to intercept the intercellular communication promoted by EVs, we recently developed a monovalent Fab fragment antibody directed against human CD9 tetraspanin and showed its effectiveness in blocking the internalization of melanoma cell-derived EVs and the nuclear transfer of their cargo proteins into recipient cells. Here, we employed the SW480/SW620 model to investigate the anti-cancer potential of the anti-CD9 Fab antibody. We first demonstrated that most EVs derived from SW620 cells contain CD9, making them potential targets. We then found that the anti-CD9 Fab antibody, but not the corresponding divalent antibody, prevented internalization of EVs from SW620 cells into SW480 cells, thereby inhibiting their phenotypic transformation, i.e., the change from a mesenchymal-like morphology to a rounded amoeboid-like shape with membrane blebbing, and thus preventing increased cell migration. Intercepting EV-mediated intercellular communication in the tumor niche with an anti-CD9 Fab antibody, combined with direct targeting of cancer cells, could lead to the development of new anti-cancer therapeutic strategies.
| S-EPMC9406449 | biostudies-literature