Impact of Drug Conjugation and Loading on Target Antigen Binding and Cytotoxicity in Cysteine Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs) consist of a target-specific antibody that is covalently conjugated to a drug via a linker. ADCs are designed to deliver cytotoxic drugs (payloads), specifically to cancer cells, while minimizing systemic toxicity. Conventional cysteine conjugation typically results in the formation of ADC molecules containing a heterogeneous mixture of 2, 4, 6, and 8 drug-loaded species. The drug-to-antibody ratio (DAR) of the mixture represents the weighted average of these species. In this report, we have investigated the impact of the hydrophobicity of payloads and the overall drug loading on the in vitro binding and cytotoxicity of ADC species. Several ADCs were prepared by conventional cysteine conjugation using different payloads. ADC species with different DAR values were purified from the ADC mixture and characterized by standard analytical techniques. These ADC species were evaluated for target antigen binding using an immunoassay, enzyme-linked immunosorbent assay (ELISA). The potency was assessed using a cell-based cytotoxicity assay. These structure-function studies lead to a better understanding of factors that impact the in vitro target binding and cytotoxicity of ADC species. ADC species containing hydrophobic payloads with high DAR were found to have lower target binding by ELISA compared to that of the unconjugated antibody or the heterogeneous reference ADC with DAR ∼4. Under similar assay conditions, the ADCs conjugated to hydrophilic payloads did not show a significant impact on the target binding. The cytotoxic potency of ADC species increased with increasing level of drug loading in the cell-based cytotoxicity assay.

Molecular imaging of VEGF receptors in angiogenic vasculature with single-chain VEGF-based probes.

We describe a new generation of protein-targeted contrast agents for multimodal imaging of the cell-surface receptors for vascular endothelial growth factor (VEGF). These receptors have a key role in angiogenesis and are important targets for drug development. Our probes are based on a single-chain recombinant VEGF expressed with a cysteine-containing tag that allows site-specific labeling with contrast agents for near-infrared fluorescence imaging, single-photon emission computed tomography or positron emission tomography. These probes retain VEGF activities in vitro and undergo selective and highly specific focal uptake into the vasculature of tumors and surrounding host tissue in vivo. The fluorescence contrast agent shows long-term persistence and co-localizes with endothelial cell markers, indicating that internalization is mediated by the receptors. We expect that multimodal imaging of VEGF receptors with these probes will be useful for clinical diagnosis and therapeutic monitoring, and will help to accelerate the development of new angiogenesis-directed drugs and treatments.

Receptor-targeted liposomal delivery of boron-containing cholesterol mimics for boron neutron capture therapy (BNCT).

Liposomes have been a main focus of tumor-selective boron delivery strategies in boron neutron capture therapy (BNCT), a binary method for the treatment of cancer that is based on the nuclear reaction between boron atoms and low-energy thermal neutrons. Three novel carboranyl cholesterol derivatives were prepared as lipid bilayer components for the construction of nontargeted and receptor-targeted boronated liposomes for BNCT. A major structural feature of these novel boronated cholesterol mimics is the replacement of the B and the C ring of cholesterol with a carborane cluster. Computational analyses indicated that all three boronated compounds have structural features and physicochemical properties that are very similar to those of cholesterol. One of the synthesized boronated cholesterol mimics was stably incorporated into non-, folate receptor (FR)-, and vascular endothelial growth factor receptor-2 (VEGFR-2)-targeted liposomes. No major differences were found in appearance, size distribution, and lamellarity between conventional dipalmitoylphosphatidylcholine (DPPC)/cholesterol liposomes, nontargeted, and FR-targeted liposomal formulations of this carboranyl cholesterol derivative. FR-targeted boronated liposomes were taken up extensively in FR overexpressing KB cells in vitro, and the uptake was effectively blocked in the presence of free folate. In contrast, a boronated cholesterol mimic incorporated into nontargeted liposomes showed significantly lower cellular uptake. There was no apparent in vitro cytotoxicity in FR overexpressing KB cells and VEGFR-2 overexpressing 293/KDR cells when these were incubated with boronated FR- and (VEGFR-2)-targeted liposomes, respectively, although the former accumulated extensively in KB cells and the latter effectively interacted with VEGFR-2 by causing autophosphorylation and protecting 293/KDR cells from SLT (Shiga-like toxin)-VEGF cytotoxicity.

In vivo tumor angiogenesis imaging with site-specific labeled (99m)Tc-HYNIC-VEGF.

PURPOSE: We recently developed a cysteine-containing peptide tag (C-tag) that allows for site-specific modification of C-tag-containing fusion proteins with a bifunctional chelator, HYNIC (hydrazine nicotinamide)-maleimide. We then constructed and expressed C-tagged vascular endothelial growth factor (VEGF) and labeled it with HYNIC. We wished to test (99m)Tc-HYNIC-C-tagged VEGF ((99m)Tc-HYNIC-VEGF) for the imaging of tumor vasculature before and after antiangiogenic (low continuous dosing, metronomic) and tumoricidal (high-dose) cyclophosphamide treatment. METHODS: HYNIC-maleimide was reacted with the two thiol groups of C-tagged VEGF without any effect on biologic activity in vitro. (99m)Tc-HYNIC-VEGF was prepared using tin/tricine as an exchange reagent, and injected via the tail vein (200-300 microCi, 1-2 microg protein) followed by microSPECT imaging 1 h later. RESULTS: Sequencing analysis of HYNIC-containing peptides obtained after digestion confirmed the site-specific labeling of the two accessible thiol groups of C-tagged VEGF. Tumor vascularity was easily visualized with (99m)Tc/VEGF in Balb/c mice with 4T1 murine mammary carcinoma 10 days after implantation into the left axillary fat pad in controls (12.3+/-5.0 tumor/bkg, n=27) along with its decrease following treatment with high (150 mg/kg q.o.d. x 4; 1.14+/-0.48 tumor/bkg, n=9) or low (25 mg/kg q.d. x 7; 1.03+/-0.18 tumor/bkg, n=9) dose cyclophosphamide. Binding specificity was confirmed by observing a 75% decrease in tumor uptake of (99m)Tc/biotin-inactivated VEGF, as compared with (99m)Tc-HYNIC-VEGF. CONCLUSION: (99m)Tc can be loaded onto C-tagged VEGF in a site-specific fashion without reducing its bioactivity. (99m)Tc-HYNIC-VEGF can be rapidly prepared for the imaging of tumor vasculature and its response to different types of chemotherapy.