Paradoxical concentration effect of a homodimerizing antibody against a human non-small cell lung cancer cell line

Abstract

Drug dosing is commonly based on the dogma that, increasing the dose maximizes the therapeutic response until a dose level that is prohibitively toxic is reached. This doctrine also applies to antibody therapy, as several protocols have explored dose escalation. We have analyzed the effect of different amounts of a homophilic Herceptin targeting a human lung tumor cell line, and discovered that the normal dose-potency relationship does not apply. To study this paradoxical effect of antibody concentration on potency, we examined the molecular species of the homophilic Herceptin under different concentrations using size exclusion chromatography and gel electrophoresis. We also varied experimental conditions in FACS tumor targeting, such as concentration of antibody, membrane immobilization, temperature, and antibody homo/dimer immobilization. We observed that high concentrations of homophilic Herceptin reduce targeting, and also noted the tumor growth arrest in the xenograft mice after the tumor reached a critical size. The therapeutic window appears to be defined by tumor size and antibody concentration. Since the concentration of this homophilic antibody defines the optimum targeting window, our data suggest the therapeutic dose of antibody should be matched with the tumor burden.

Fig. 1

Photo-affinity conjugation of Herceptin with homophilic peptide at different ratios. Tryptophan containing homophilic peptide was added to Herceptin at different amounts and photo-crosslinked as described [17]. The conjugate was applied to Sephacryl S300 in PBS containing 2% PEG. Collected fractions were monitored at 280 nm

Fig. 2

Conjugated Herceptin (homophilic Herceptin) at different amounts, 3.4 and 340 μg, was chromatographed on Sephacryl S300 in PBS. Overlay chromatograms with 3.4 and 340 μg, monitored with Ig-capture ELISA

Fig. 3

Conjugated Herceptin, 340 and 34 μg, was chromatographed on Sephadex G25 in PBS. Eluted antibody was detected by IG-ELISA

Fig. 4

Native gel electrophoresis with a non-denaturing detergent as described. Left lane 3.4 μg Homophilic Herceptin, right lane 3.4 μg of Herceptin

Fig. 5

Comparison of FACS intensity using Herceptin and homophilic Herceptin on live NSCLC H1650 cells. Solid and the dashed lines show Herceptin- and homophilic Herceptin-treated cells, respectively. a 0.05 μg/ml, b 0.1 μg/ml, c 0.5 μg/ml, d 1 μg/ml antibody

Fig. 6

Comparison of FACS staining with homophilic Herceptin on fixed and live H1650 cells (dashed line and solid line, respectively). a FACS using secondary FITC antibody only. b Using 1 μg/ml of homophilic antibody. c Using 5 μg/ml of homophilic antibody

Fig. 7

Comparison of FACS staining of live H1650 cells at 4 and 37°C [open circle (cold) and solid circle (warm), respectively]. a, b FACS gated at low-fluorescence intensity a using dilutions of homophilic Herceptin, and b dilutions of Herceptin. c, d FACS gated at high-fluorescence intensity c using dilutions of homophilic Herceptin, and d dilutions of Herceptin

Fig. 8

FACS of live H1650 cells using untreated homophilic Herceptin or treated with gluteraldehyde (solid line and dotted line, respectively). a 1 μg/ml, b 5 μg/ml, and c 10 μg/ml

Fig. 9

Induction of apoptosis in H1650 cells using different concentrations of homophilic Herceptin. H1650 cells were incubated overnight at 37°C with either no antibody or homophilic Herceptin. Cells were stained with Annexin V and PI, and analyzed on FACS. a No antibody. b 5 μg/ml antibody. c 10 μg/ml antibody. d 20 μg/ml antibody

Fig. 10

Induction of apoptosis in H1650 cells using 2 μg/ml of homophilic Herceptin (a) or Herceptin (b)

Fig. 11

Xenograft using H1650 cells in nude mice. 5 × 10⁶ H1650 cells were injected into the flank of each mouse. Groups (12 mice each) were untreated (diamond), treated with Herceptin (solid box), or were treated with homophilic Herceptin (open box). Tumor growth was monitored three times weekly using a caliper and is shown on the y axis as cm³. Mice were treated twice weekly with antibody (110°μg/mouse) starting 24 h post-tumor inoculation. All groups of mice were euthanized at day 32. Error bars indicate the standard deviation of each group of 12 mice