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Targeting of human squamous carcinomas by SPA470-doxorubicin immunoconjugates
1Department of Dignostic Sciences and pathology University of Maryland, Baltimore, MD 21201, USA.
Abstract:
In a quest to identify a favorable target for head and neck cancers and squamous cell carcinoma, we sought to determine if Hsp47/CBP2 could be used as a target and whether the expression of this target was influenced by hypoxia. Moreover, we determined if doxorubicin (DOX) immunoconjugates directed against Hsp47/CBP2 that linked monoclonal antibodies (MAbs) to the 13-keto position of the drug possessed high cytotoxic drug activity and antibody-directed killing of antigen bearing tumor target cells. Experiments were performed using established cell lines of human oral squamous carcinoma cells (SCCs) (SCC-4, -9, -15 and -25) obtained from American Type Culture Collection (ATCC) (Manassas, VA). In addition, the UMB2 cell line is a spontaneous mutant of SCC-9 that does not express Hsp47/CBP2 was also used. Synthesis of the immunoconjugates was accomplished by thiolating the MAbs with 2 IT and reacting the MAbs with the DOX-hydrazone. The binding of MAb-DOX conjugates to SCC cells was determined by indirect immunofluorescence and analyzed using a Becton Dickinson FACS scan with Cell Quest software. Comparison of the cytotoxicity of DOX, MAb-DOX conjugates and MAb+DOX were determined using a limited dilution assay and colony survival assays during normoxia and hypoxia. These studies revealed that SCC cells treated with the SPA470-DOX conjugate for 2 h retained the original binding activity for targeted SCC cells and was significantly more potent that unconjugated DOX, DOX-hydrazone or equivalent MAb protein+DOX. Also, SPA47-DOX produced equal to and at lower concentrations greater cell killing than equivalent dose of free DOX. During hypoxia cells treated with SPA470-DOX demonstrated a small increase in colony survival and a diminishment in cytotoxicity. SPA470-DOX conjugates target SCC cells that express Hsp47/CBP2. The demonstration that SPA470-DOX is effective during hypoxia or conditions that mimic hypoxia presumes the further utility of SPA470-DOX in treating head and neck cancers.
Insights
Researchers explored Hsp47/CBP2 as a target for head and neck cancers. Doxorubicin (DOX) immunoconjugates targeting Hsp47/CBP2 showed potent cancer cell killing, even under hypoxic conditions.
Area of Science:
- Oncology
- Cancer Biology
- Immunotherapy
Background:
- Head and neck cancers and squamous cell carcinoma (SCC) require novel therapeutic targets.
- Hypoxia is a common feature in solid tumors, influencing treatment efficacy.
- Hsp47/CBP2 is investigated as a potential therapeutic target in SCC.
Purpose of the Study:
- To evaluate Hsp47/CBP2 as a target for head and neck cancers.
- To assess the influence of hypoxia on Hsp47/CBP2 expression.
- To determine the efficacy of doxorubicin (DOX) immunoconjugates targeting Hsp47/CBP2.
Main Methods:
- Utilized human oral squamous carcinoma cell lines (SCCs) and a Hsp47/CBP2-negative mutant.
- Synthesized monoclonal antibody (MAb)-DOX immunoconjugates.
- Assessed conjugate binding via indirect immunofluorescence and flow cytometry.
- Compared cytotoxicity of conjugates versus free DOX under normoxia and hypoxia using colony survival assays.
Main Results:
- SPA470-DOX conjugates demonstrated potent cytotoxic activity against Hsp47/CBP2-expressing SCC cells.
- Conjugates were significantly more potent than unconjugated DOX or MAb+DOX mixtures.
- SPA470-DOX exhibited comparable or greater cell killing than free DOX at lower concentrations.
- Hypoxia slightly reduced SPA470-DOX cytotoxicity but maintained efficacy.
Conclusions:
- Hsp47/CBP2 is a viable target for SCC therapy.
- SPA470-DOX immunoconjugates show significant anti-cancer activity.
- These conjugates hold promise for treating head and neck cancers, including under hypoxic conditions.