Folate-immunoglobulin G as an anticancer therapeutic antibody

Abstract

Folate receptor-alpha (FR) is a promising cellular marker for tumor-specific drug delivery. Conjugation of folic acid to therapeutic and imaging agents has been shown to enhance their delivery to FR (+) cancer cells in vitro and in tumor-bearing mice via an FR-mediated cellular uptake mechanism. In this study, immunoglobulin G (IgG) was conjugated to folate and evaluated as a therapeutic antibody against folate receptor (FR)-positive tumors. Murine IgG (mIgG) was conjugated to folate via an amide bond to yield folate-conjugated mIgG (f-mIgG) that contained an average of approximately 2.6 folates per molecule. Selective uptake of f-IgG by FR (+) tumor cells was determined by fluorescence microscopy and by flow cytometry. Lysis of L1210JF cells by NK cells from murine donors was increased 1.4-9.0-fold at the effector:target (E:T) ratio of 25:1, relative to control mIgG. In mice bearing L1210JF tumors, f-mIgG was found to significantly inhibit tumor growth and to have prolonged the median survival time (MeST). Significantly, the antitumor efficacy of f-mIgG was greatly increased when combined with liposomal G3139, an 18-mer phosphorothioate oligonucleotide. In fact, the combination resulted in a 100% cure rate among the tumor-bearing mice. Injection of f-mIgG significantly increased serum INF-gamma and IL-6 level in mice compared with mIgG and dramatically increased serum INF-gamma and IL-6 level when combined with liposomal G3139. These results suggested that f-IgG, a novel immunotherapy agent, has potent activity as a therapeutic antibody to the FR-positive cancer, and the therapeutic activity is enhanced by immunomodulatory agents.

Figure 1

Synthesis of f-IgG conjugate. Conjugation to γ-carboxylic acid in folate is shown in this figure. However, the α-carboxylic acid group can also be conjugated.

Figure 2

Uptake of f-IgG-FITC in KB (A), L1210JF (B), and K562 (C) cells. Cells were treated with 200 nM FITC labeled IgG or f-IgG in folate-free RPMI1640 at 37 °C for 30 min. For the receptor blocking study, 200 μM of free folate was added to media during f-IgG-FITC exposure. f-IgG-FITC binding in KB cells by fluorescence microscopy. After treatment, KB cells were photographed in both phase-contrast (upper panels) and fluorescence (lower panels) mode on a Nikon Eclipse 800 fluorescence microscopy. Uptake of f-IgG-FITC in L1210JF and K562 cells by flow cytometry. (1) Untreated cells. (2) Cells were treated with IgG-FITC. (3) Cells were treated with f-IgG-FITC. (4) Cells were treated with f-IgG-FITC and free folate.

Figure 3

Effect of f-IgG on ADCC activity against FR-positive L1210JF cells at different effector:target ratio (A) and different donor from different mice (B). The NK cell lytic activity was assessed in a standard 4 h chromium release assay using f-mIgG-coated L1210JF cancer cells. The percentage of lysis was calculated as described in Experimental Section. Data represent the mean ± SD (n = 3).

Figure 4

In vivo efficacy of folate receptor mediated immunotherapy in L1210JF (A,B) and L1210 (C,D) bearing DBA/2 mice. Mice (5–6 per group) were implanted subcutaneously on day 0 with 1 × 10⁶ L1210JF cells. On day 5 or 7, animals were treated with 100 mg/kg of mIgG or f-mIgG. The treatment was continued every 4 days for four times. (A,C) Inhibition of tumor growth. Tumor dimensions were measured every 2 days using calipers. (B,D) Survival of L1210JF and L1210 tumor-bearing mice after treatment with mIgG or f-mIgG. PBS was injected as a vehicle control. (*, P < 0.01 compared with PBS group.)

Figure 5

In vivo antitumor efficacy of G3139 in L1210JF bearing DBA/2 mice. Mice (6 per group) were implanted subcutaneously on day 0 with 1 × 10⁶ L1210JF cells. On day 5, animals were treated with with 1 mg/kg of free G3139 or liposomal G3139. The treatment was continued every 4 days for four times. (A) Inhibition of tumor growth. Tumor dimensions were measured every 2 days using caliper. (B) Survival of L1210JF bearing mice after treatment with free G3139 or liposomal G3139. PBS was injected as a vehicle control. (*; P < 0.05 compared with PBS group.)

Figure 6

In vivo antitumor efficacy of f-mIgG combined with free G3139 or liposomal G3139 in L1210JF bearing DBA/2 mice. Mice (6 per group) were implanted subcutaneously on day 0 with 1 × 10⁶ L1210JF cells. On day 5, animals were treated with 100 mg/kg of f-mIgG alone or combined with 1 mg/kg of free G3139 or liposomal G3139. The treatment was continued every 4 days for four times. (A) Inhibition of tumor growth. Tumor dimensions were measured every 2 days using caliper. (B) Survival of L1210JF bearing mice after treatment with free G3139 or liposomal G3139. PBS was injected as a vehicle control. (*; P < 0.01 compared with PBS group.)

Figure 7

In vivo serum INF-γ (A) and IL-6 (B) production in L1210JF tumor-bearing mice. The tumor-bearing mice were treated with mIgG, f-mIgG alone or in combination with free or liposomal G3139. Blood samples were collected at 2, 6, and 24 h after injection. Serum was obtained by centrifugation and analyzed for INF-γ and IL-6 content by ELISA.