Simultaneous binding of the anti-cancer IgM monoclonal antibody PAT-SM6 to low density lipoproteins and GRP78

Abstract

The tumour-derived monoclonal IgM antibody PAT-SM6 specifically kills malignant cells by an apoptotic mechanism linked to the excessive uptake of plasma lipids. The mechanism is postulated to occur via the multi-point attachment of PAT-SM6 to the unfolded protein response regulator GRP78, located on the surface of tumour cells, coupled to the simultaneous binding of plasma low density lipoprotein (LDL). We prepared and characterised LDL and oxidized LDL using sedimentation velocity and small-angle X-ray scattering (SAXS) analysis. Enzyme-linked immunosorbent (ELISA) techniques indicated apparent dissociation constants of approximately 20 nM for the binding of LDL or oxidized LDL to PAT-SM6. ELISA experiments showed cross competition with LDL inhibiting PAT-SM6 binding to immobilised GRP78, while, in the reverse experiment, GRP78 inhibited PAT-SM6 binding to immobilized LDL. In contrast to the results of the ELISA experiments, sedimentation velocity experiments indicated relatively weak interactions between LDL and PAT-SM6, suggesting immunoabsorbance to the microtiter plate is driven by an avidity-based binding mechanism. The importance of avidity and the multipoint attachment of antigens to PAT-SM6 was further investigated using antigen-coated polystyrene beads. Absorption of GRP78 or LDL to polystyrene microspheres led to an increase in the inhibition of PAT-SM6 binding to microtiter plates coated with GRP78 or LDL, respectively. These results support the hypothesis that the biological action of PAT-SM6 in tumour cell apoptosis depends on the multivalent nature of PAT-SM6 and the ability to interact simultaneously with LDL and multiple GRP78 molecules clustered on the tumour cell surface.

Figure 1. Cu²⁺-induced oxidation of LDL.

(A) Change in absorbance at 234 nm as a function of incubation time. (B) Time course for the change in ThT fluorescence of LDL (solid line), LDL incubated with 20 mM CuSO₄ (dashed line) or PBS alone (dotted line). (C) Sedimentation coefficient distribution obtained from sedimentation velocity analysis of LDL (dashed line) and oxidized LDL (solid line).

Figure 2. SAXS analysis of LDL and oxidized LDL.

A. The SAXS data are shown as mean intensity I(q) ±1 standard deviation, as a function of momentum transfer q for LDL (solid circles) and oxidized LDL (open circles). (B) Guinier plots of the SAXS data at low q for LDL (solid circles) and oxidized LDL (open circles). The lower and upper q.R _g limits for the Guinier analyses are indicated. (C) Pair distance distribution function P(r) as a function of radial distance r for LDL (closed circles) and oxidized LDL (open circles).

Figure 3. ELISA analysis of PAT-SM6 interactions.

Assays were performed using (A) LDL or (B) oxidized LDL at coating concentrations of 0 µg/mL (open circles), 1 µg/mL (closed triangles), 2 µg/mL (open diamonds), 3 µg/mL (closed squares), 4 µg/mL (open inverted triangles), and 5 µg/mL (closed circles). (C) The data were fitted to a simple binding isotherm to obtain apparent binding constants (K_a) for PAT-SM6 binding as a function of the coating concentration of LDL (open circles) or oxidized LDL (closed circles). Error bars represent the 95% confidence intervals from the fitting of the ELISA data to a Langmuir binding curve.

Figure 4. Competition ELISA data for PAT-SM6 obtained using (A) soluble LDL or (B) GRP78 as competitors.

Wells were coated with 7.5 mg/mL GRP78 (circles), 7.5 mg/mL LDL (triangles) or were left uncoated (squares). PAT-SM6 (5 mg/mL) was incubated with various concentrations of competitor prior to application to the microtiter plate.

Figure 5. Sedimentation velocity analysis of the PAT-SM6 interaction with LDL.

(A) Sedimentation coefficient distributions obtained using absorbtion optics at 280 nm for PAT-SM6 (0.3 µM, black), LDL (1.8 µM, red) and a mixture of PAT-SM6 and LDL (0.3 µM and 1.8 µM, respectively (green). (B) Sedimentation coefficient distributions obtained using fluorescence optics for FITC-labelled PAT-SM6 (40 nM) in the presence (solid line) and absence (broken line) of LDL (1.8 µM). (C) Sedimentation coefficient distributions obtained in the presence of BSA (50 mg/mL) using fluorescence optics for FITC-labelled PAT-SM6 (40 nM) in the presence (solid line) and absence (broken line) of LDL (1.8 µM).

Figure 6. Binding of GRP78 to polystyrene microspheres.

(A) Amount of protein bound per gram of microspheres as a function of GRP78 (closed circles) or BSA concentration (open circles). (B) Timecourse of GRP78 (solid circles) and LDL (open triangles) binding to polystyrene microspheres.

Figure 7. Competition ELISA data for the binding of PAT-SM6 to microtiter plates coated with (A) GRP78 (30 µg/mL) or (B) LDL (30 µg/mL).

PAT-SM6 (5 mg/mL) was incubated with GRP78-coated microspheres (circles), soluble GRP78 (triangles) or BSA coated microspheres (squares) prior to addition to the microtiter plates.

Figure 8. Competition ELISA data for the binding of PAT-SM6 to microtiter plates coated with (A) LDL (30 µg/mL) or (B) GRP78 (30 µg/mL).

PAT-SM6 (5 mg/mL) was incubated with LDL-coated microspheres (circles), soluble LDL (triangles) or BSA coated microspheres (squares) prior to addition to the microtiter plates.