Anti-tumor effect against human cancer xenografts by a fully human monoclonal antibody to a variant 8-epitope of CD44R1 expressed on cancer stem cells

Abstract

Background: CD44 is a major cellular receptor for hyaluronic acids. The stem structure of CD44 encoded by ten normal exons can be enlarged by ten variant exons (v1-v10) by alternative splicing. We have succeeded in preparing MV5 fully human IgM and its class-switched GV5 IgG monoclonal antibody (mAb) recognizing the extracellular domain of a CD44R1 isoform that contains the inserted region coded by variant (v8, v9 and v10) exons and is expressed on the surface of various human epithelial cancer cells.

Methods and principal findings: We demonstrated the growth inhibition of human cancer xenografts by a GV5 IgG mAb reshaped from an MV5 IgM. The epitope recognized by MV5 and GV5 was identified to a v8-coding region by the analysis of mAb binding to various recombinant CD44 proteins by enzyme-linked immunosorbent assay. GV5 showed preferential reactivity against various malignant human cells versus normal human cells assessed by flow cytometry and immunohistological analysis. When ME180 human uterine cervix carcinoma cells were subcutaneously inoculated to athymic mice with GV5, significant inhibition of tumor formation was observed. Furthermore, intraperitoneal injections of GV5markedly inhibited the growth of visible established tumors from HSC-3 human larynx carcinoma cells that had been subcutaneously transplanted one week before the first treatment with GV5. From in vitro experiments, antibody-dependent cellular cytotoxicity and internalization of CD44R1 seemed to be possible mechanisms for in vivo anti-tumor activity by GV5.

Conclusions: CD44R1 is an excellent molecular target for mAb therapy of cancer, possibly superior to molecules targeted by existing therapeutic mAb, such as Trastuzumab and Cetuximab recognizing human epidermal growth factor receptor family.

Figure 1. Immunoperoxidase staining of human xenografts developed in athymic mice with anti-CD44 fully human IgM mAb.

Tissue sections of human ME180 tumors developed in athymic mice were fixed with PFA, and were sequentially incubated with human mAb, species-specific biotinylated anti-human IgG + IgM (H+L), ABC reagent and substrate solution containing DAB and H₂O₂. Nuclei were stained with hematoxylin.

Figure 2. Immunoperoxidase staining of human xenografts developed in athymic mice with an anti-CD44R1 fully human IgG mAb (GV5).

Tissue sections of human ME180 tumors developed in athymic mice were fixed with cold acetone, and were sequentially incubated with primary mAb, species-specific biotinylated anti-human IgG Fcγ, ABC reagent and substrate solution containing DAB and H₂O₂. Nuclei were stained with hematoxylin. Upper or lower panels respectively show low or high magnification of the stained tissues.

Figure 3. Specificity and epitope of anti-CD44 fully human mAb.

(A) MV1 (left), MV5 (middle) and GV5 (right) were compared for the reactivity with HEK293F cells expressing human CD44R1-GFP (upper) or human CD44s-GFP (lower) by FCM. (B) Reactivity of antibodies against various GST-fused recombinant CD44 proteins (R1a and R1b; Δex5-v8-v9-v10-Δex16, v8, v9, v10 and ex5) fused to GST was determined by ELISA. Difference in the length of Δex5 and Δex16 between R1a and R1b recombinant proteins is described in “Materials and Methods”.

Figure 4. Fine specificity of a class-switched anti-CD44R1 fully human IgG mAb (GV5) revealed by FCM.

GV5, Cetuximab and Trastuzumab were compared for the reactivity with HUVEC, NHEK and HCT116 by FCM (histograms) with an Accuri C6 flow cytometer (A). Reactivity of GV5 with various human cells was analyzed by FCM, and depicted as histograms (B) using a BD-LSR flow cytometer and as ΔMFI (C) using an Accuri C6 flow cytometer.

Figure 5. Immunoperoxidase staining of human breast and colon tissues with anti-human CD44 rat mAb.

Reactivity of anti-human CD44s or anti-human CD44R1 (v8) rat mAb with breast and colon tissue sections from human surgical specimens was analyzed with ABC immunoperoxidase staining. Sections from PFA-fixed and paraffin-embedded human breast and colon tissues were treated with microwaves in citrate buffer for the retrieval of antigens, and incubated with RV7 or RV9 rat mAb. After being rinsed in PBS, tissue sections were sequentially incubated with species-specific biotinylated donkey anti-rat IgG (H + L), ABC reagent and substrate solution containing DAB and H₂O₂. Nuclei were stained with hematoxylin. Non-Cancer or Cancer tissues mean specimens from an identical patient.

Figure 6. Immunoperoxidase staining of human tissues with anti-human CD44 fully human IgG mAb (GV5).

Sections from PFA-fixed and paraffin-embedded human tissues were treated with microwaves in citrate buffer for the retrieval of antigens, and incubated with biotinylated GV5. After being rinsed in PBS, tissue sections were incubated with ABC reagent and substrate solution containing DAB and H₂O₂. Nuclei were stained with hematoxylin.

Figure 7. Therapeutic effect of anti-CD44R1 fully human mAb in the tumor neutralization model.

Tumor neutralization model was adopted. ME180 human tumor cells (1.0×10⁶) with or without mAb (50 µg/site) in 200 µl of PBS were subcutaneously inoculated into the right dorsal flank of each animal. The size of each tumor formed was periodically measured, and tumor volume (mm³) was calculated by the formula 0.4×(length)×(width)². Results were analyzed statistically by two-way ANOVA tests with repeated measures.

Figure 8. Therapeutic effect of anti-CD44R1 fully human mAb in the established tumor model.

Established tumor model was adopted. Seven days after human larynx carcinoma-derived HSC-3 tumor cells (1.0×10⁶) in 200 µl of PBS were inoculated subcutaneously to athymic mice and visible tumor in each mouse was confirmed, 500 µl of PBS with or without GV5 (100 µg) was intraperitoneally inoculated at day 7 and day 14 (vertical arrows). The size of each tumor formed was periodically measured, and tumor volume (mm³) was calculated by the formula 0.4×(length)×(width)². Results were analyzed statistically by two-sided Student's t tests (upper), and by two-way ANOVA tests with repeated measures (lower). Vertical bars show standard deviations.

Figure 9. Internalization of CD44R1, CDC and ADCC by GV5 fully human mAb.

(A) HSC3 cells were cultured with or without GV5 (10 µg/ml) for 12 h, and were immunostained with GV5 followed by FITC-conjugated donkey anti-human IgG (H+L). Cell-surface CD44R1 proteins in these cells were detected by FCM. Experiments were repeated three times, and similar results were obtained. (B) After HSC-3 cells and sera for complement and mAb were mixed and incubated in each well of U-bottomed 96-well plate for 1 h, DAPI was added to each well. Percentages of DAPI-stained cells (dead cells) were calculated by FCM. Experiments were repeated three times, and similar results were obtained. (C) HSC-3 cells (1.5×10⁵) were mixed with effector cells of splenocytes (3×10⁶ or 9×10⁶) from KNS nude mice, which were pre-cultured overnight with lL-2, with or without mAb, in each well of U-bottomed 96-well plate for 5 h, and PI was added to each well. Cytotoxicity was analyzed using an Accuri flow cytometer. R1, HSC3 human target cells; R2, mouse effector cells; R3, PI-stained dead cells in R1; R4, PI-unstained living cells in R1. (D) Cell death (%) of HSC-3 cells by mouse effector cells (E/T = 0, 20 or 60) with or without GV5 was plotted. E/T means effector to target ratio.

Figure 10. ADCC activity of anti-CD44R1 fully human mAb against human tumor cells using human lymphocytes.

HSC-3 cells were labeled with Calcein-AM, and cells (2×10⁵) were mixed with effector cells of human PBL (5×10⁵ or 2×10⁶), which were pre-cultured overnight with IL-2 with or without mAb in each well of U-bottomed 96-well plate for 7 h. Cytotoxicity was evaluated by the release of Calcein-AM by dead tumor cells into the medium, and results were automatically recorded using a Terascan VP microfluorocytometer at 1 h intervals for 7 h. Results were analyzed statistically by two-way ANOVA tests with repeated measures (A) and by two-sided Student's t tests (A and B). Spots and vertical bars respectively show means and standard errors.