Chemical control over immune recognition: a class of antibody-recruiting small molecules that target prostate cancer

Abstract

Prostate cancer is the second leading cause of cancer-related death among the American male population, and society is in dire need of new approaches to treat this disease. Here we report the design, synthesis, and biological evaluation of a class of bifunctional small molecules called antibody-recruiting molecules targeting prostate cancer (ARM-Ps) that enhance the recognition of prostate cancer cells by the human immune system. ARM-P derivatives were designed rationally via the computational analysis of crystallographic data, and we demonstrate here that these materials are able to (1) bind prostate-specific membrane antigen (PSMA) with high affinity (high pM to low nM), (2) template the formation of ternary complexes of anti-DNP antibodies, ARM-P, and LNCaP human prostate cancer cells, and (3) mediate the antibody-dependent killing of LNCaP cells in the presence of human effector cells. This manuscript describes the application of fundamental chemical principles to the design of a novel class of molecules with high therapeutic potential. We believe that this general small-molecule-based strategy could give rise to novel directions in treating cancer and other diseases.

Figure 1

Schematic depiction of the reported approach to prostate cancer targeting. An antibody-recruiting small molecule (ARM) binds the cell-surface prostate cancer marker prostate-specific membrane antigen (PSMA), thus recruiting antibodies to these cells for recognition and targeted killing by the immune system. Bifunctional ARMs are composed of an antibody binding terminus (ABT), a linker region, and a cell-binding terminus (CBT).

Figure 2

Structure-based design studies. (A) Modeled complex illustrating the design of a CBT for use in ARM-Ps. (B) Structural model of the ternary complex between the Fv region of an anti-DNP antibody, ARM-P, and the PSMA dimer. (C) Known PSMA-binding small molecules and structures of ARM-P derivatives utilized in this study. Figures 2A and B were created with the program VMD.

Figure 3

Representative PSMA inhibition curves for ARM-Ps. K_i values were calculated from measured IC₅₀ and K_M values through the Cheng-Prusoff equation and are reported as the average of 3 runs ± standard deviation.

Figure 4

Evaluating ternary complex formation. (A) and (B) Representative traces from flow cytometry experiments. (C) Dose dependence of competitor concentration on ternary complex. (D) Epifluorescence (Fluor) and brightfield (BF) microscopy experiments performed in the presence of ARM-P8 at 37 °C and 4 °C.

Figure 5

Antibody-Dependent Cellular Cytotoxicity (ADCC) Assays. LNCaP (PSMA-positive) and DU145 (PSMA-negative) cells were treated with the ARM-P derivatives at the indicated concentrations, and cell death was measured with and without exposure to anti-DNP antibody (Ab, 24 µg/mL) and peripheral blood mononuclear cells (PBMC). Points represent the average of 4 measurements ± standard deviation. All depicted trends were observed on at least three separate occasions.