We have developed a technetium labeling technology based on a new organometallic chemistry, which involves simple mixing of the novel reagent, a 99m Tc(I)-carbonyl compound, with a His-tagged recombinant protein. This method obviates the labeling of unpaired engineered cysteines, which frequently create problems in large-scale expression and storage of disulfide-containing proteins. In this study, we labeled antibody single-chain Fv fragments to high specific activities (90 mCi/mg), and the label was very stable to serum and all other challenges tested. The pharmacokinetic characteristics were indistinguishable from iodinated scFv fragments, and thus scFV fragments labeled by the new method will be suitable for biodistribution studies. This novel labeling method should be applicable not only to diagnostic imaging with 99mTc, but also to radioimmunotherapy approaches with 186/188 Re, and its use can be easily extended to almost any recombinant protein or synthetic peptide.
Multimerization of antibody fragments increases the valency and the molecular weight, both identified as key features in the design of the optimal targeting molecule. Here, we report the construction of mono-, di-, and tetrameric variants of the anti-tumor p185 HER-2 single chain Fv fragment 4D5 by fusion of self-associating peptides to the carboxyl terminus. Dimeric miniantibodies with a synthetic helix-turn-helix domain and tetrameric ones with the multimerization domain of the human p53 protein were produced in functional form in the periplasm of Escherichia coli. We have directly compared these molecules and the single-chain Fv fragment in the targeting of SK-OV-3 xenografts. Tetramerization of the 4D5 antibody fragment resulted in increased serum persistence, significantly reduced off-rate, due to the avidity effect, both in surface plasmon resonance measurements on purified p185 HER-2 and on SK-OV-3 cells. The 99m technetium-tricarbonyl-labeled tetrameric 4D5-p53 miniantibody localized with the highest dose at the tumor and remained stably bound for at least 72 h. The highest total dose was 4.3% injected dose/g after 24 h, whereas the highest tumor-to-blood ratio was found to be 13.5:1 after 48 h, with a total dose of 3.2% injected dose/g. The tetramer shows no higher avidity than the dimer, presumably since the simultaneous binding to more than two antigen molecules on the surface of cells is not possible, and the improvement in performance over the dimer must at least be due in part to the molecular weight. These results demonstrate that multimerization by self-associating peptides can be used for the development of more effective targeting molecules for medical diagnostics and therapy.
The most advanced oncolytic Newcastle disease virus (NDV) strains that are used in clinical trials for the treatment of cancer are wild-type mesogenic strains. These virus strains have an inherent, nongenetically engineered, oncolytic activity and selectively replicate in tumor cells but not in normal human cells. To date no investigations have been performed with genetically engineered mesogenic NDV regarding the oncolytic activity. We describe here the generation of recombinant viruses of the mesogenic naturally oncolytic NDV strain MTH68. We show that not only one, but also two additional transgenes coding for amino-acid chains with a molecular weight of 25 and 50 kDa can be inserted into the viral genome without affecting viral growth, oncolytic potency or tumor-selective replication of the virus. Transgenic expression of the heavy and light chains of a monoclonal antibody, as separate additional transcriptional cassettes, leads to the expression of full immunoglobulin G (IgG) monoclonal antibody by recombinant NDV. Infection of tumor cells with antibody-transgenic viruses results in the efficient production and secretion of a functional full size IgG antibody by the tumor cells, that specifically binds to its target-antigen in tumor tissue. This approach will allow to combine the advantages of oncolytic RNA viruses and monoclonal antibodies in a single powerful anticancer agent with improved or even new therapeutic properties.
The EphA2 receptor tyrosine kinase has been shown to be over-expressed in cancer and a monoclonal antibody (mAb) that activates and down-modulates EphA2 was reported to inhibit the growth of human breast and lung tumor xenografts in nude mice. Reduction of EphA2 levels by treatment with anti-EphA2 siRNA also inhibited tumor growth, suggesting that the anti-tumor effects of these agents are mediated by decreasing the levels of EphA2. As these studies employed human tumor xenograft models in nude mice with reagents whose cross reactivity with murine EphA2 is unknown, we generated a mAb (Ab20) that preferentially binds, activates, and induces the degradation of murine EphA2. Treatment of established murine CT26 colorectal tumors with Ab20 reduced EphA2 protein levels to approximately 12% of control tumor levels, yet had no effect on tumor growth. CT26 tumor cell colonization of the lung was also not affected by Ab20 administration despite having barely detectable levels of EphA2. We also generated and tested a potent agonistic mAb against human EphA2 (1G9-H7). No inhibition of humanMDA-231 breast tumor xenograft growth was observed despite evidence for >85% reduction of EphA2 protein levels in the tumors. These results suggest that molecular characteristics of the tumors in addition to EphA2 over-expression may be important for predicting responsiveness to EphA2-directed therapies.
C4.4A (LYPD3) has been identified as a cancer-and metastasisassociated internalizing cell surface protein that is expressed in non-small cell lung cancer (NSCLC), with particularly high prevalence in the squamous cell carcinoma (SCC) subtype. With
To improve tumor selectivity of cytotoxic agents, we designed VIP236, a small molecule–drug conjugate consisting of an αVβ3 integrin binder linked to a modified camptothecin payload (VIP126), which is released by the enzyme neutrophil elastase (NE) in the tumor microenvironment (TME). The tumor targeting and pharmacokinetics of VIP236 were studied in tumor-bearing mice by in vivo near-infrared imaging and by analyzing tumor and plasma samples. The efficacy of VIP236 was investigated in a panel of cancer cell lines in vitro, and in MX-1, NCI-H69, and SW480 murine xenograft models. Imaging studies with the αVβ3 binder demonstrated efficient tumor targeting. Administration of VIP126 via VIP236 resulted in a 10-fold improvement in the tumor/plasma ratio of VIP126 compared with VIP126 administered alone. Unlike SN38, VIP126 is not a substrate of P-gp and BCRP drug transporters. VIP236 presented strong cytotoxic activity in the presence of NE. VIP236 treatment resulted in tumor regressions and very good tolerability in all in vivo models tested. VIP236 represents a novel approach for delivering a potent cytotoxic agent by utilizing αVβ3 as a targeting moiety and NE in the TME to release the VIP126 payload—designed for high permeability and low efflux—directly into the tumor stroma.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.