Design, Synthesis, and Biological Evaluation of Polyaminocarboxylate Ligand-Based Theranostic Conjugates for Antibody-Targeted Cancer Therapy and Near-Infrared Optical Imaging

Abstract

We report the design, synthesis, and evaluation of polyaminocarboxylate ligand-based antibody conjugates for potential application in targeted cancer therapy and near-infrared (NIR) fluorescence imaging. We synthesized a new polyaminocarboxylate chelate (CAB-NE3TA) as a potential anticancer agent. CAB-NE3TA displayed potent inhibitory activities against various cancer cell lines. We then designed a multifunctional theranostic platform (CAB-NE3TA-PAN-IR800) constructed on an epidermal growth factor receptor (EGFR)-targeted antibody (panitumumab, PAN) labeled with a NIR fluorescent dye. We also built the first atomistic model of the EGFR-PAN complex and loaded it with the cytotoxic CAB-NE3TA and the NIR dye. The therapeutic (CAB-NE3TA-PAN) and theranostic (CAB-NE3TA-PAN-IR800) conjugates were evaluated using an EGFR-positive A431 (human skin cancer) cell xenograft mouse model. Biodistribution studies using NIR fluorescence imaging demonstrated that the CAB-NE3TA-PAN labeled with the IR800 dye selectively targeted the A431 tumors in mice and resulted in prolonged retention in the tumor tissue and displayed excellent clearance in blood and normal organs. The therapeutic conjugate was capable of significantly inhibiting tumor growth, leading to nearly complete disappearance of tumors in the mice. The results of our pilot in vivo studies support further evaluation of the novel ligand-based therapeutic and theranostic conjugates for targeted iron chelation cancer therapy and imaging applications.

Keywords: antibody-drug conjugates; anticancer agents; fluorescence imaging; theranostic agents.

Figure 1.

Iron-Chelating Antitumor Agents in clinical and preclinical evaluations

Figure 2.

Anti-proliferative activity of CAB-NE3TA (10 μM) and DFO (10 μM) against cancer cells. The compound was incubated with the cells for 72 h, and cell viability was determined using MTS assay. The reported data on the anti-proliferative activity of the known compound DFO against HeLa, HT29, and PC3 are included for comparison [17,22].

Figure 3.

Anti-proliferative activity of CAB-NE3TA on cancer cells. CAB-NE3TA was incubated with the cells for 72 h, and cell viability was determined using MTS assay.

Figure 4.

UV-Vis and fluorescence emission spectra of CAB-NE3TA-PAN and CAB-NE3TA- PAN-IR800 conjugates.

Figure 5.

Pilot in vivo anti-cancer therapy study. A431 (human skin cancer)-bearing mice received IP injections of the therapeutic conjugate (CAB-NE3TA-PAN, L/P = 5) or vehicle (PBS only) four times for a period of 8 days and were sacrificed at 21 days post-treatment. (A) Median volumes of tumors in the treated and untreated mice measured during the treatment period. (B) Kaplan-Meier survival curves of the treated and untreated mice bearing A431 tumors. (C) Images of representative A431-bearing mice and tumors removed from the mice sacrificed at 11–21 days post-treatment. (D) Median volumes of tumors removed from the treated and untreated mice. (E) Median weight of tumors removed from the treated and untreated mice.

Figure 6.

In vivo NIR fluorescence images of A431-bearing male nude mice intraperitoneally (IP) injected with the theranostic conjugate (CAB-NE3TA-PAN-IR800, 5 μM).

Figure 7.

A proposed docking model for a complex of EGFR and Panitumumab conjugated with CAB-NE3TA (green) and IR800CW (cyan).

Figure 8.

(A) The panitumumab binding surface of EGFR obtained by docking. Amino acid residues critical for panitumumab binding (P349, F352, D355, W386, P387, E388, N389, R390, T391)^[38] are shown in red. The EGF binding residues (V350, D355, F357, L382, Q384, F412, I438)^[40] are shown in green. The CDR loops of panitumumab are shown in orange ribbons. (B) Superposition of the panitumumab binding surface obtained by docking (red) with the cetuximab binding surface (blue) obtained from the X-ray crystal structure^[39] of cetuximab-EGFR complex.

Scheme 1.

Synthesis of CAB-NE3TA analogues. Reagents and Conditions: a) H₂ (15 psi), 10% Pd/C, EtOH, RT, 5.5 h, 97%; b) CBZ-Cl, K₂CO₃, CH₃CN, RT, 24 h, 56%; c) TFA, CHCl₃, RT, 3 h, 87%; d) BrCH₂CO₂tBu, K₂CO₃, CH₃CN, RT, 18 h, 72%; e) p-NO₂-BnBr, K₂CO₃, CH₃CN, RT, 18 h, 53%; f) NBS or I₂, Imidazole, CH₂Cl₂, 0 °C 4 h, RT, 1 h, 44% (8a), 86% (8b); g) AgClO₄, CH₃CN, - 5 °C, 15 min; h) 10, DIPEA, CH₃CN, 0 °C to RT, 89 h, 70% (from 8a), 48 h, 76% (from 8b), 70–76%; i) 4M HCl/1,4-dioxane, RT, 18 h, 95%; j) CSCl₂, CHCl₃/H₂ O, RT, 4 h, 93%.

Scheme 2.

Construction of CAB-NE3TA-Based Therapeutic and Theranostic ADCs.