Figure 1: Molecular design and chemical composition of covalent gemcitabine and epirubicin immunochemotherapeutics.

Legends: (Panel 1) gemcitabine-(C4 -amide)-[anti-EGFR]; and (Panel 2) epirubicin-(C3 -amide)-[anti-HER2/neu]. Both covalent immunochemotherapeutics were synthesized utilizing a 2-stage organic chemistry reaction scheme that initially produces a chemotherapeutic analog that is a UV-photoactivated intermediate. Covalent bonds are formed at the monoamine groups of gemcitabine or epirubicin and the side chains of amino acid residues within the sequence of immunoglobulin fractions.

Figure 2: Characterization of the molecular weight profile for the covalent immunochemotherapeutics, epirubicin-(C3 -amide)-[anti-HER2/neu] and gemcitabine-(C4 -amide)-[anti-EGFR] relative to reference control antiEGFR and anti-HER2/neu monoclonal immunoglobulin fractions.

Legends: (Lane-1) murine anti-human EGFR monoclonal immunoglobulin; (Lane-2) gemcitabine-(C4 -amide)-[anti-EGFR]; (Lane-3) murine antihuman HER2/neu monoclonal immunoglobulin; and (Lane-4) epirubicin- (C3 -amide)-[anti-HER2/neu]; Covalent immunochemotherapeutics and monoclonal immunoglobulin fractions were size-separated by non-reducing SDS-PAGE followed by lateral transfer onto sheets of nitrocellulose membrane to facilitate detection with biotinylated goat anti-mouse IgG immunoglobulin. Subsequent analysis entailed incubation of membranes with strepavidin-HRPO in combination with the use of a HRPO chemiluminescent substrate and the acquisition of autoradiography images.

Figure 3: Detection of total immunoglobulin in the form of gemcitabine- (C4 -amide)-[anti-EGFR] or epirubicin-(C3 -amide)-[anti-HER2/neu] selectively bound to the exterior surface membrane of chemotherapeuticresistant mammary adenocarcinoma. Legends: (◆) gemcitabine-(C4 -amide)-[anti-EGFR]; and (■) epirubicin- (C3 -amide)-[anti-HER2/neu]. Covalent gemcitabine-(C4 -amide)-[antiEGFR] or epirubicin-(C3 -amide)-[anti-HER2/neu] immunochemotherapeutics formulated at gradient concentrations were incubated with triplicate monolayer populations of chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) over a 4-hour period and total immunoglobulin bound to the exterior surface membrane was then measured by cellELISA.

Figure 4: Relative anti-neoplastic cytotoxicity for the covalent immunochemotherapeutic dual-combination of gemcitabine-(C4 -amide)-[antiEGFR] and epirubicin-(C3 -amide)-[anti-HER2/neu] compared to gemcitabine-(C4 -amide)-[anti-EGFR] alone against chemotherapeutic-resistant human mammary adenocarcinoma.

Figure 4gemcitabine-(C4 -amide)-[anti-EGFR] in dual-combination with epirubicin-(C3 -amide)-[anti-HER2/neu] at 182-hours; (▲) gemcitabine-(C4 -amide)-[anti-EGFR] at 182-hours; and (◆) epirubicin-(C3 - amide)-[anti-HER2/neu] at 96-hours; (●) epirubicin-(C3 -amide)-[antiHER2/neu] at 182-hours. Individual covalent immunochemotherapeutics or the dual 50/50 combination of gemcitabine-(C4 -amide)-[anti-EGFR] with epirubicin-(C3 -amide)-[anti-HER2/neu] were formulated at gradient chemotherapeutic-equivalent concentrations and incubated in direct contact with triplicate monolayer populations of chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) for period of 182-hours. Anti-neoplastic cytotoxicity was detected and measured using a MTT cell vitality assay and values reported as a percentage of matched negative reference controls (100%).

Figure 5: Relative anti-neoplastic cytotoxicity for the dual-combination of covalent gemcitabine and epirubicin immunochemotherapeutics against chemotherapeutic-resistant human mammary adenocarcinoma

Legends:Left-Panel (■) gemcitabine-(C4 -amide)-[anti-EGFR], and (◆) gemcitabine-(C4 -amide)-[anti-EGFR] with epirubicin-(C3 -amide)-[anti-HER2/neu]. Right-Panel (■) gemcitabine-(C4 -amide)-[anti-EGFR] with epirubicin-(C3 -amide)-[anti-HER2/neu]; and (◆) gemcitabine-(C4 -amide)-[anti-EGFR] with gemcitabine-(C4 -amide)-[anti-HER2/neu]. Dual-combinations of covalent immunochemotherapeutics were formulated at gradient 50/50 chemotherapeutic-equivalent concentrations. Both individual and dual covalent immunochemotherapeutic combinations were incubated in direct contact with triplicate monolayer populations of chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) over a period of 182-hours. Anti-neoplastic cytotoxicity was detected and measured using a MTT cell vitality assay relative to matched negative reference controls.

Figure 6: Relative anti-neoplastic cytotoxicity for gemcitabine and epirubicin against chemotherapeutic-resistant human mammary adenocarcinoma as a function of challenge duration.

Legend:Legend: (..●..) gemcitabine-(C4-amide)-[anti-EGFR] following a 182-hour incubation period;[98] (--◆--) gemcitabine-(C4-amide)-[anti-HER2/neu] following a 96-hour incubation period;[98] (--☐--)* gemcitabine-(C5- carbonate)-[thiolated anti-HER2/neu] following a 182-hour incubation period;[97] (∆) gemcitabine-(C4-amide)-[anti-HER2/neu] following a 96- hour incubation period;[99] (-.-◆-.-) epirubicin-(C3 -amide)-[anti-HER2/ neu] following a 96-hour incubation period;[102] (●) epirubicin-(C3 - amide)-SS-[anti-HER2/neu] following a 96-hour incubation period;[160] (∆)* epirubicin-(C13-imino)-[thiolated-anti-HER2/neu] following a 96- hour incubation period;[85] and (❍)* epirubicin-(C3 -amide)-[thiolatedanti-HER2/neu] following a 96-hour incubation period.[66] Covalent gemcitabine or epirubicin immunochemotherapeutics were formulated at gradient chemotherapeutic-equivalent concentrations and incubated with triplicate monolayer populations of chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3). Anti-neoplastic cytotoxicity was detected and measured using a MTT cell vitality assay relative to matched negative reference controls. Note: (*) = incorporation of aeromatic ring structure into synthetic bond structure of covalent immunochemotherapeutic; and (-SS- ) designates incorporation of a potentially cleavable disulfide bond into the structure of covalent immunochemotherapeutic

Figure 7: Relative anti-neoplastic cytotoxicity for gemcitabine and epirubicin against chemotherapeutic-resistant human mammary adenocarcinoma as a function of challenge duration.

Legend: (◆) gemcitabine following a 96-hour incubation period; (■) gemcitabine following a 182-hour incubation period; and (▲) epirubicin following a 96-hour incubation period. Gemcitabine or epirubicin formulated at gradient chemotherapeutic-equivalent concentrations were incubated in direct contact with triplicate monolayer populations of chemotherapeuticresistant mammary adenocarcinoma (SKBr-3). Anti-neoplastic cytotoxicity was detected and measured using a MTT cell vitality assay relative to matched negative reference controls.

Figure 8: Relative anti-neoplastic cytotoxicity of gemcitabine-(C4 -amide)- [anti-EGFR] in dual-combination with epirubicin-(C3 -amide)-[anti-HER2/ neu] with and without mebendazole.

Legends: (◆) gemcitabine-(C4 -amide)-[anti-EGFR] with epirubicin-(C3 - amide)-[anti-HER2/neu] following a 182-hour incubation period; (▲) gemcitabine-(C4 -amide)-[anti-EGFR] and epirubicin-(C3 -amide)-[anti-HER2/ neu] in combination with mebendazole following a 182-hour incubation period; and (■) gemcitabine-(C4 -amide)-[anti-EGFR] and epirubicin-(C3 - amide)-[anti-HER2/neu] in combination with mebendazole following a 96- hour incubation period. Mean numerical results for (■) and (▲) are nearly identical so their marker legends are visually superimposed in the figure illustration. The covalent immunochemotherapeutic dual-combination formulated at gradient 50/50 chemotherapeutic-equivalent concentrations (+/- mebendazole 0.15µM fixed concentration) were incubated in direct contact with triplicate monolayer populations of chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) over a period of either 96-hours or 182-hours. Anti-neoplastic cytotoxicity was detected and measured using a MTT cell vitality assay relative to matched negative reference controls

Figure 9: Relative anti-neoplastic cytotoxicity of benzimidazoles against chemotherapeutic-resistant human mammary adenocarcinoma.

Legends: (◆) (◆) albendazole; (▲) flubendazole; and (■) mebendazole. Benzimidazole tubulin/microtubule inhibitors formulated at gradient molarequivalent concentrations were incubated in direct contact with triplicate monolayer populations of chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) over a period of 96-hours. Anti-neoplastic cytotoxicity was detected and measured using a MTT cell vitality assay relative to matched negative reference controls.

Figure 10: Relative anti-neoplastic cytotoxicity of mebendazole against chemotherapeutic-resistant mammary adenocarcinoma as a function of challenge duration (incubation period).

Legends: (◆) (■) mebendazole following a 96-hour incubation period; and (◆) mebendazole following a 182-hour incubation period. Mebendazole formulated at gradient molar-equivalent concentrations was incubated in direct contact with triplicate monolayer populations of chemotherapeutic-resistant mammary adenocarcinoma (SKBr-3) over a period of either 96-hours or 182-hours. Anti-neoplastic cytotoxicity was detected and measured using a MTT cell vitality assay relative to matched negative reference controls.