Synthesis of the next-generation therapeutic antibodies that combine cell targeting and antibody-catalyzed prodrug activation

Abstract

An obstacle in the utilization of catalytic Abs for selective prodrug activation in cancer therapy has been systemic tumor targeting. Here we report the generation of catalytic Abs that effectively target tumor cells with undiminished prodrug activation capability. Ab conjugates were prepared by covalent conjugation of an integrin alpha(v)beta(3)-targeting antagonist to catalytic Ab 38C2 through either sulfide groups of cysteine residues generated by reduction of the disulfide bridges in the hinge region or surface lysine residues not involved in the catalytic activity. Using flow cytometry, the Ab conjugates were shown to bind efficiently to integrin alpha(v)beta(3)-expressing human breast cancer cells. The Ab conjugates also retained the retro-aldol activity of their parental catalytic Ab 38C2, as measured by methodol and doxorubicin (dox) prodrug activation. Complementing these Ab conjugates, an evolved set of dox prodrugs was designed and synthesized. Dox prodrugs that showed higher stability and lower toxicity were evaluated both in the presence and absence of the integrin alpha(v)beta(3)-targeting 38C2 conjugates for cell-killing efficacy by using human breast cancer cells. Our study reveals that cell targeting and prodrug activation capabilities can be efficiently combined for selective chemotherapy with novel dox prodrugs.

Fig. 1.

Production of catalytic Ab conjugates. (A) Structure of small-molecule arginine-glycine-aspartic acid peptidomimetic antagonists equipped with a linker. (B) Schematic drawing of a catalytic cell-targeting antagonist–38C2 conjugate for targeting cells that express integrin α_vβ₃. Mal, maleimide; NHS, N-hydroxysuccinimide.

Fig. 2.

Analysis of the catalytic Ab conjugates. (A) Comparison of 38C2-2 (M_{r avg} = 153,758) and 38C2-3 (M_{r avg} = 153,046) conjugates to untreated 38C2 (M_{r avg} = 151,997) by MALDI-MS analysis. (B) Flow cytometry histogram showing the binding of 38C2 conjugates, 38C2-2 and 38C2-3, to integrin α_vβ₃ expressing human breast cancer cell line MDA-MB-231. Conjugates 38C2-2 and 38C2-3, 38C2 alone, and the previously described chemically programmed 38C2 (cp38C2) construct (17) obtained from a diketone derivative of 1 were used at 5 μg/ml concentration. In all experiments, FITC-conjugated goat anti-mouse secondary Abs were used for detection. The y axis gives the number of events in linear scale, and the x axis gives the fluorescence intensity in logarithmic scale.

Fig. 3.

Catalytic activity of 38C2-2 and 38C2-3 in comparison to free 38C2. Shown are rate of the retro-aldol reaction of aldol 4 to produce aldehyde 5 in the presence of the catalytic amounts of 38C2-2, 38C2-3, and free 38C2. The negative control reaction was in PBS buffer (50 mM).

Fig. 4.

Synthesis and activation of prodoxs. (A) Structure of dox and its prodrugs (prodoxs). (B and C) Structure of prodoxs and schemes showing 38C2-catalyzed activation of these prodoxs to produce dox via the labile intermediates. DA, dox aglycon.

Scheme 1.

Synthesis of prodoxs 13-14 and epirubicin prodrugs 22 (A) and prodoxs 9–12 (B). DA, dox aglycon; PNP, 4-nitrophenyl; RT, room temperature. Yields are given for compounds leading to 9, 11, and 13. Comparable yields were obtained for their analogs. (a) 4-Nitrophenyl chloroformate, Py, CH₂Cl₂, 0°C; (b) (i) OsO₄ (cat), NMO, citric acid, CH₂Cl₂-H₂O (10:1), RT, (ii) NaIO₄, THF- H₂O (1:1), RT; (c) Et₃N, DMF; (d) DIPEA, HOBT, DMF, RT; (e) (i) DIBAL-H, CH₂Cl₂, −10°C, (ii) step a, and (iii) Dess Martin periodinane, CH₂Cl₂, RT.

Fig. 5.

Effect of dox and prodoxs 10 and 12 (A), dox and prodox 9 (B), prodoxs 7 and 11 (C), and dox and prodoxs 7, 9, and 11 (D) on human breast cancer cells, MDA-MB-231, in vitro, in the absence or presence of a catalytic amount of Ab 38C2. Ab 38C2 was used at a 1 μM concentration in B and C. Cells (20,000) were used and developed by using the Cell Titer 96 AQ_ueous One Solution Cell Proliferation Assay kit after 72 h of incubation with drug, prodrug, or prodrug/38C2 combination. In D, dox was used at a 10 μM concentration. The y axis shows cell density in a linear scale, and the x axis shows the dox or prodox concentration in a logarithmic scale in A–C and Ab concentration in a linear scale in D.

Fig. 6.

Effect of dox and prodoxs 7, 9, and 11 on human breast cancer cells, MDA-MB-231, in vitro, in the absence and/or presence of 38C2 (0.1 μM concentration) or 38C2 conjugates (0.2 μM concentration). Dox and prodoxs were used at 5 μM (A) and 10 μM (B) concentrations. The experiments were conducted as described earlier, except that cells were used at a lower number (3,000 cells per well) and developed after 120 h of incubation with dox, prodox/38C2, prodox/38C2-2, or prodox/38C2-3 combinations. The y axis shows cell density in a linear scale, and the x axis shows the buffer or catalyst used.