Stability of the recombinant anti‑erbB2 scFv‑Fc‑interleukin‑2 fusion protein and its inhibition of HER2‑overexpressing tumor cells

  • Authors:
    • Yu‑Jia Du
    • Ze‑Min Lin
    • Ying‑Hua Zhao
    • Xiu‑Ping Feng
    • Chang‑Qing Wang
    • Gang Wang
    • Chun‑Di Wang
    • Wei Shi
    • Jian‑Ping Zuo
    • Fan Li
    • Cheng‑Zhong Wang
  • View Affiliations

  • Published online on: December 20, 2012     https://doi.org/10.3892/ijo.2012.1747
  • Pages: 507-516
Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )


Abstract

The anti‑erbB2 scFv‑Fc‑IL‑2 fusion protein (HFI) is the basis for development of a novel targeted anticancer drug, in particular for the treatment of HER2‑positive cancer patients. HFI was fused with the anti‑erbB2 antibody and human IL‑2 by genetic engineering technology and by antibody targeting characteristics of HFI. IL‑2 was recruited to target cells to block HER2 signaling, inhibit or kill tumor cells, improve the immune capacity, reduce the dose of antibody and IL‑2 synergy. In order to analyse HFI drug ability, HFI plasmid stability was verified by HFI expression of the trend of volume changes. Additionally, HFI could easily precipitate and had progressive characteristics and thus, the buffer system of the additive phosphate‑citric acid buffer, arginine, Triton X‑100 or Tween‑80, the establishment of a microfiltration, ion exchange, affinity chromatography and gel filtration chromatography‑based purification process were explored. HFI samples were obtained according to the requirements of purity, activity and homogeneity. In vivo, HFI significantly delayed HER2 overexpression of non‑small cell lung cancer (Calu‑3) in human non‑small cell lung cancer xenografts in nude mice, and the inhibition rate was more than 60% (P<0.05) in the group treated with 1 mg/kg the HFI dose; HFI significantly inhibited HER2 expression of breast cancer (FVB/neu) transgenic mouse tumor growth in 1 mg/kg of the HFI dose group, and in the following treatment the 400 mm3 tumors disappeared completely. Combined with other HFI test data analysis, HFI not only has good prospects, but also laid the foundation for the development of antibody‑cytokine fusion protein‑like drugs.

Introduction

The HER2 (ErbB2/neu) proto-oncogene encodes a transmembrane receptor protein of 185 kDa, which is structurally related to the epidermal growth factor receptor. ErbB2 is usually expressed at a very low level in adult tissues, but it is overexpressed in a variety of human tumors, such as breast cancer (25–30%), ovarian cancer (25–32%), lung cancer (30–35%), and primary renal cell carcinoma (30–40%) (14).

A recombinant humanized monoclonal antibody, Herceptin (trastuzumab) has shown good response in clinical trials, in the treatment of HER2-overexpressing metastatic breast cancers (58). The objective response rates of Herceptin monotherapy were lower than its combination therapies. Current treatment regimens which combine Herceptin with the taxane, paclitaxel or docetaxel have increased the response rates, time to progression, and survival (9,10). The humanized Herceptin suppressed the growth of HER2-overexpressing tumor cells by inhibiting HER2-driven signaling, inhibiting VEGF secretion or activity and mediating antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (1114), but the humanized Herceptin did not induce apoptosis when it is used alone.

Interleukin-2 (IL-2) is an immunoregulatory protein with a wide range of immune effects. IL-2 exerts its biological effects by binding with the IL-2 receptor on the surface of target cells. The activities of IL-2 include stimulating T cells to proliferate and become cytotoxic, augment NK cytotoxicity, enhancing macrophages cytotoxicity, giving rise to lymphokine-activated killer (LAK) cells (1520). In tumor therapy, IL-2 has already shown some efficacy in the treatment of metastatic melanoma, kidney cancer or non-Hodgkin’s lymphoma when used alone or combined with other anticancer drugs. However, the antitumor efficiency of IL-2 is often associated with unacceptable toxicity. HFI ensures both the prevention of the systemic side-effects and the locally restricted enrichment activity to the tumor sites with decreased IL-2 toxicity (2123).

Despite the success of the Herceptin in clinical trials for the treatment of ErbB2-expressing metastatic breast cancer, the fusion of IL-2 to an ErbB2-specific antibody may enhance its antitumor efficacy. Firstly, the presence of the antibody variable region should target the tumor cells, allowing for higher doses of IL-2 at HER2 antigen located in tumor tissue, where they elicit an immune response and thus destroy tumors. Secondly, IL-2 is unstable in vivo and it will be quickly eliminated by the kidney due to limiting quantities of the protein. The half-life of IL-2 may be extended by MAb-conjugation due to both the size and stability of the MAb. Thirdly, an antibody-IL2 fusion protein may increase immune access to the antibody’s target ligand by increasing permeability of blood vessels near the tumor nidus (2430). In preliminary studies, it was observed that the HFI expression of clonal cells strained between 60–80 mg/l; the in vitro experiments proved that the HFI contained dual activity of antibodies and IL-2; the in vivo tests showed that the HFI inhibited HER2 expression of ovarian cancer; in (SKOV3) transgenic mouse tumor growth, the HFI antibody ADCC activity and IL-2-mediated killing activity of effector cells; to stimulate effector cells to secrete perforin, and the adhesion factor. However, many aspects of HFI has not been verified, including whether the cell lines is stable, whether the HFI purification technology is mature, or the HFI sample is stable, whether the HFI inhibits the role of HER2 overexpression in other cell lines, safety and so on. In this study, the HFI drug ability for some of these issues were analyzed and discussed.

Materials and methods

Expression and purification of the HFI

The highest-producing of HFI fusion clone was harvested after the HFI expression vector had been transfected into adherent Chinese Hamster Ovary (CHO-K1) cells (ATCC No. CRL-9096), incubated with the selection medium containing 300 μg/ml G418 (also known as geneticin) (31,32). The primary cell bank (PCB) of the HFI engineering cells was built when the adherent CHO cells was domesticated into suspension cells in tissue culture with HyClone SFM4CHO (HyClone) or EX-CELL™325 (Sigma-Aldrich) cell culture medium, which is a protein-free formulation that contained no components of bovine origin. The master cell bank (MCB) and the working cell bank (WCB) with the stable expressing HFI were obtained from the PCB in tissue culture roller bottles with the cell culture medium, as described above. The supernatant containing fusion protein of HFI was harvested as the cell culture process of the Cell Bioreactor (BIOSTAT® Bplus), which was determined through the batch culture or the continuous flow culture. The fusion protein of HFI was purified over the following sophisticated purification processes, including microfiltration, ion-exchange, affinity and molecular sieve chromatography.

Enzyme-linked immunosorbent assay (ELISA)

Quantification of the HFI was assayed by ELISA. Goat anti-human (100 μl) IgG (1:200 dilution) per well was coated onto 96-well ELISA plates overnight at 4°C. After washing the plates, serial dilution of the samples, which were maintained in PBS with 3% bovine serum albumin (BSA) were added to the plates and incubated at 37°C for 2 h. Then after washing the plates, 100 μl per well of horse-radish peroxidase-conjugated goat anti-human IgG (1:1,000 dilution) was bound and incubated at 37°C for 1 h. Color was generated by adding peroxidase substrate and absorbance was measured on a plate reader at 492 nm. Dilutions of the HFI, for which the concentration was calibrated by the Kjeldahl method and the Lowry method, were used to generate a standard curve to estimate the concentration of unknown samples.

Bioactivity assay

The cytokine activity of the HFI was determined by IL-2 dependent T cell proliferation assay. Recombinant human IL-2 (rhIL-2) standard was obtained from National Institute for the control of Pharmaceutical and Biological products. The mouse CTLL-2 cell line expressing high-affinity IL-2 receptor was used to determine the IL-2 bioactivity by the T cell proliferation assay. Serially diluted samples and IL-2 standards were incubated with 4×104 cells per well in triplicate in 96-well flat bottom plates for 24 h at 37°C in a humidified atmosphere of 5% CO2. MTT (5 mg/ml) reagent was added 4 h before the end of culture, and then cells were lysed with 10% SDS and 50% N,N-dimethyl formamide, pH was maintained at 7.2. The OD values were read at 570 nm. The mouse CTLL-2 cell was provided by Professor N. Guo (Institute of Basic Medical Sciences, China).

Antigen binding assay

About 1×106 cells per well were incubated with varying concentrations of the HFI or the same molar concentrations of the different proteins in a final volume of 0.2 ml. After incubation on ice for 30 min, cells were washed twice with PBS-BSA, and a fluorescein isothiocyanate (FITC)-conjugated goat anti-human IgG antibody (Beijing Dingguo Biotechnology Co. Ltd.) was added. Incubation was continued for an additional 30 min on ice. After two washes with PBS-BSA, the cells were analyzed by flow cytometry (FACSCalibur, Becton Dickinson).

Animal experiments

The three types of mice including female FVB/neu mice, BALB/c mice and female Balb/C athymic nude mice of 7 to 10-weeks-age, were obtained from Shanghai Laboratory Animal Center of the Chinese Academy of Sciences. All mice were housed under specific pathogen-free conditions. Experiments were carried out according to the National Institute of Healthy Guide for Care and Use of Laboratory Animals, and were approved by the Institutional Animal Care and Use Committee at the Shanghai Institute of Materia Medica, Chinese Academy of Sciences.

Pharmacokinetic analysis

BALB/c mice (48 animals per group) were injected with 25 or 100 μg of the purified HFI sample in a volume of 0.2 ml in the tail vein using a slow push, respectively. At various time points, small blood samples (6 animals per time point) were taken by retro-orbital bleeding and collected in tubes coated with heparin to prevent clotting. After centrifugation, the plasma was assayed by ELISA with goat anti-human IgG and goat anti-human IL-2 antibody. The results were normalized to the initial concentration in the serum of each mouse taken immediately after injection.

In vitro antitumor activity

When the signals of HER2 were blocked on the surface of the HER2 overexpression cells, the growth of cells was inhibited or the cells entered apoptosis. The three types of cells including SKBR3 cells (HER2+++), SKOV3 cells (HER2++) and MCF7 cells (HER2±) were provided by Professor N. Guo (Institute of Basic Medical Sciences, China), and were chosen to evaluate HFI antitumor activity in vitro(33). Firstly, the cells were placed on 96-well cell plates, then, serial dilution of the samples were added to the plates and incubated at 37°C in a humidified atmosphere of 5% CO2. Apoptosis of the cells were assayed for different time points by MTT cell proliferation assay methods.

In vivo antitumor activity

i) Six to seven-week-old female Balb/C athymic nude mice were subcutaneously injected 1×107 Calu-3 cells (human non-small cell lung cancer) into the right flank. The mice were treated with PBS (iv: 0.2 ml per mice) (control) or HFI (iv: 1.0, 0.5 and 0.25 mg/kg mice weight, respectively). Seven or eight mice were used in each group. ii) The antitumor activity of HFI was evaluated in FVB/neu transgenic mouse model, which expressed the ErbB2/neu proto-oncogene and closely recapitulated the ontogeny and progression of human breast cancer. When tumors reached a size of more than 300 mm3, the mice were grouped (n=5) and PBS or HFI (1.0 mg/kg mice weight) or Herceptin (3.0 mg/kg mice weight) in a volume of 0.2 ml, was injected i.v. into the lateral tail vein. Dosage regimen: the first week for five continuous days, then once every other day. The mice weight were monitored daily and tumor volume was measured with a caliper once per two days, using the formula volume = length × width2/2.

Splenocyte culture

Splenocytes were harvested on day 20 after HFI administration and were cultured in triplicate (5×105/well) in 96-well culture plates (Corning) for 24 h with 0.5 μg/ml of concanavalin A (Con A) or 1 μg/ml of lipopolysaccharide (LPS). The cells were pulsed with 0.5 μCi/well of [3H]thymidine for 8 h and harvested onto glass fiber filters. The incorporated radioactivity was then counted using a Beta Scintillation Counter (MicroBeta Trilux, PerkinElmer Life Sciences, Boston, MA).

ELISA detection of cytokines

Splenocytes were cultured in 24-wells culture plates (Corning) for 24 and 48 h with 0.5 μg/ml of Con A or 1 μg/ml of LPS. Culture supernatants were harvested and submitted to assays for the determination of cytokine concentrations. Cytokines in culture supernatant was assayed using mouse IFN-γ ELISA kit (BD Pharmingen), according to the manufacturer’s instructions.

Cytotoxicity assays

Murine YAC-1 cells were used as targets in the NK assay. Target tumor cells were labeled with 250 kBq [3H]thymidine by incubating overnight in a culture dish (Corning) containing 10 ml of culture medium. Targeted cells (2×106) were cultured in triplicate with different numbers of effector cells (effector: Target ratio = 100:1, 50:1 or 12.5:1) for 4, 8 and 12 h in 96-wells flat-bottomed plates. Cells were harvested onto glass fiber filters. The incorporated radioactivity was then counted using a Beta Scintillation Counter (MicroBeta Trilux, PerkinElmer Life Sciences, Boston, MA). Cytotoxic activity was calculated and expressed as the mean of percent cytotoxicity. Percent cytotoxicity = (cpm in effector-free wells − cpm in effector positive wells)/(cpm in effector-free wells) ×100.

Flow cytometric analysis

A single-cell suspension of spleen cells harvested on day 20 after HFI administration were blocked with anti-mCD16/CD32 (2.4G2, purified inhouse), stained with fluorescein isothiocyanate (FITC)-conjugated, phycoerythrin (PE)-conjugated, (PerCPCy5.5)-conjugated, (APC)-conjugated, and analyzed using a FACSCalibur system with Cell Quest software (Becton Dickinson). (FITC), (PE), (PerCPCy5.5) or (APC)-conjugated mAb against CD3 (145-2c11), CD4 (Gk1.5), CD8 (Ly-2) (53-6.7), CD1 (1d3), TCR-γ/δ (GL3) were purchased from BD Pharmingen. PerCPCy5.5-conjugated anti-mouse/rat FoxP3 (FJK-16s), PerCPCy5.5-conjugated rat IgG2a and the FoxP3 staining buffer set were purchased from eBioscience.

Statistics

The statistical significance of the time course data was determined by two-way analysis of variance testing and Student’s t-test was used to compare two treatment groups. Statistical significance was set at p<0.05.

Results

HFI protein expression and its characterization

The stable HFI expression was first assayed in the differential course of cell passage by using a pair of goat anti-human IgG antibodies in a ‘sandwich ELISA’, SDS-PAGE and IL-2 dependent T cell proliferation. The fusion proteins of HFI were stably expressed in different cell lines, such as the primary cell line (PCL), the master cell line (MCL), the working cell line (WCL) and the production of end-stage cell line (ECL) in 1,000 ml tissue culture roller bottles with a culture volume of 200–300 ml and at (24)×106 cells/ml density at 30°C for 7 days. The culture supernatants were harvested following 1,500 rpm for 5 min to 8,000 rpm for 30 min. The molecular weight of HFI was found in a bivalent form of 140 kDa (Fig. 1A) and a monovalent form of 70 kDa (Fig. 1B) on 8% SDS-PAGE stained with Coomassie Brilliant Blue. The HFI expression of the four kinds of cells cultured for 7 days was examined at 100–150 mg/l by ELISA (Fig. 1C). The IL-2 bioactivity of HFI was assessed at (2–2.5) ×106 IU/mg by the standard IL-2 dependent T cell proliferation assay and ELISA calibration concentration (data not shown). Fig. 1 shows that the HFI gene copy number and HFI expression were relatively stable in the differential course of cell passage.

Since the expression of HFI in the cell lines was stable, the purification and stability of HFI was crucial to the ability of this fusion protein. The supernatant of HFI reaction liquid was sampled at 30°C and assayed for one month by the Cell Bioreactor under a continuous flow mode. In the HFI purification process, insoluble cell debris and other irrelevant substances were first removed from the HFI supernatant by 0.2 or 0.45 μm microfiltration membrane bag (Millipore), then the microfiltration sample was concentrated with the 30–50 kD nitrocellulose membrane bag (Millipore) to a proper volume. This concentration step was critical for HFI purification, as it could not only remove a large number of cellular components, but also deplete certain host proteins and small molecules in culture medium. In order to protect the affinity chromatography media from erosion, the ultra-filtration sample was purified by DEAE ion exchange in 0.2–0.4 M NaCl, 40 mM phosphate-citrate buffer and pH 7.2 was maintained, this process was mainly to remove endotoxin, nucleic acids and other substances. Purity of antibody protein reached 97% or higher after affinity chromatography purification. Therefore, the HFI was purified over Mabselect Sure (GE Healthcare) affinity chromatography, eluting with acidic conditions. The purified solution of the HFI was obtained by Superdex 200 (GE Healthcare) chromatography media to remove HFI monomer and polymer (Fig. 2). After the protein purification process, the HFI yield was measured by a microplate spectrophotometer at 490 nm, and the final HFI sample was more than 40% yield, the HFI purity was examined by reducing and non-reducing SDS-PAGE (Fig. 2A), the quantity of HFI monomers and polymers were less than 5% of HFI dimers (Fig. 2A; lanes 7 and 8), more than 98% purity of the HFI sample was determined by HPLC (Fig. 2B), fluorescent PCR, and host protein ELISA. Endotoxin was monitored by LAL agglutination. The HFI bioactivity was consistent with IL-2 activity, measured by a standard IL-2-dependent T cell proliferation assay (Fig. 2C). Meanwhile, the stability of the purified solution of the HFI was also analyzed at 4°C for 2 months. These results suggested that the fusion protein of the HFI was relatively stable, and could be suitable for further drug development.

HFI binds specifically to HER2

The HFI built targeting was based on the binding properties of the ant-ErbB2 antibody and interleukin-2 bioactivity. The biological activity of IL-2 of the HFI was based on the ability of the HFI to support IL-2 dependent T cell proliferation (Fig. 2C). Human CD3+ and CD16+ cells were detected from the peripheral blood of mice at several doses of the HFI treated groups (33). The binding characteristics of the HFI was identified, and then three HER2 expressing cells, SKBR3 (HER2+++), SKOV3 (HER2++) and MCF7 cells (HER2±) were used to test HFI binding activity in different tumor cell lines. In the same molar concentration, HFI, Herceptin (Trastuzumab Injection, Roche Pharma Ltd.) and HF (antibody part of HFI, self-made sample) showed the same binding ability (Fig. 3). The HFI binding capacity was not affected by IL-2 fusion with HFI as compared to HF (Fig. 3A–D). This result demonstrated that the HFI binding ErbB2 protein characteristics depended on the expression of the ErbB2 protein (Fig. 3A–D). HFI showed a similar characteristic of binding HER2 with the Herceptin (Fig. 3A–D).

Pharmacokinetic properties

The pharmacokinetic properties of immunocytokines were reported to be dependent on their uptake by the FcR-bearing cells and the intracellular proteolysis (28). Half-life of drugs was related to their molecular size. The structure of HFI was determined by the in vivo stability, and properties of IL-2 and anti-ErbB2 protein. The half-life of HFI was detected in the mouse serum at different time points by ELISA. The results showed that the concentration of HFI decreased quickly during the first 4 h, and showed a relatively stable plateau following 64 h (Fig. 4). At 100 μg HFI maintained relatively higher levels than 25 μg HFI, demonstrating that the higher levels were maintained with higher concentrations (Fig. 4). The results indicated that the HFI structural design was conducive to recruitment around the cells, and also extended the half-life of IL-2.

In vitro antitumor activity

The HFI has a combination of activities, i.e., binding properties of HER2 receptor and interleukin-2 bioactivity. The ability of the HFI binding HER2 receptor depends on the HER2 overexpression on the cell surface. In this study, whether the HFI inhibited the cell proliferation when the HFI was bound to HER2 receptor was evaluated. The three cell types, breast cancer (SKBR-3, HER2+++), breast cancer (MCF-7, HER2±) and ovarian cancer cells (SKOV-3, HER2++) were incubated with the different concentrations of HFI for 72 h. The results showed that the HFI did not inhibit cells proliferation, and proved that the HFI did not have chemical toxicity. In vitro, the role of HFI was to inhibit tumor cell growth by activating the effector cells.

The in vivo antitumor activity was investigated using a Calu-3 xenograft model. Calu-3 is known to be a HER2-overpressing tumor. A total of 1×107 Calu-3 cells suspended in 0.15 ml PBS were injected subcutaneously into the right flank of the mice. Normally, 9–12 days after tumor cell implantation, when tumors were clearly palpable and reached a size of 200 to 350 mm3, the mice were grouped (n=7) and PBS or the HFI was administered intravenously into the lateral tail vein, respectively (Fig. 5). To determine the antitumor activity of HFI at the different doses and the effect of treatment, therapy was started in groups of mice treated with PBS or the HFI (1.0, 0.5 or 0.25 mg/kg mice weight), respectively. The HFI was administered intravenously once daily for the first 5 days (0–4 day). At day 20 of the treatment, the therapeutic effect observed in the mice treated with the 1.0 mg/kg dose was considerably better than that observed with the 0.5 mg/kg dose (P<0.01), and the 0.25 mg/kg dose (P<0.01). Fig. 6 demonstrates that the HFI had a significant antitumor activity on models expressing high levels of HER2 in vivo.

The fusion proteins (HFI) retained the activities of both HER2 binding and IL-2. The role of IL-2 was mainly to activate lymphocytes to release cytokines and other cells which indirectly killed tumor cells. Thus, nude mice could not fully reflect the HFI antitumor activity in vivo. In order to verify the integrity of antitumor activity of HFI, the high expression of HER2 transgenic mouse model (immunocompetent) of spontaneous tumor formation was chosen, and the mice were treated with HFI (Fig. 6). After 4-day treatment, HFI (1.0 mg/kg mouse weight) caused a dramatic suppression of FVB/neu in the transgenic mouse model. Moreover, after treatment for 15 days, the tumor volume which was initially <400 mm3, was undetectable.

HFI promoted mitogen-induced splenocyte proliferation and natural killer (NK) cell-mediated cytotoxicity

The ex vivo immunoenhancement of HFI was evaluated on splenocyte proliferation induced by Con A and LPS. The results showed that the splenocytes of FVB/neu tumor bearing mice treated with 1 mg/kg HFI for 3 weeks had significant proliferation activity compared to those treated with PBS induced by LPS (Fig. 7A) or Con A (Fig. 7B). The production of IFN-γ was induced by FVB/neu in mice treated with HFI (Fig. 7C) or Herceptin (Fig. 7D). HFI also increased the NK cell-mediated cytotoxicity (Fig. 7E–G).

Effect of HFI treatment on regulatory T cells and T cell percentage

The therapeutic mechanisms of HFI were investigated ex vivo to determine whether its effects were attributable to influenced FoxP3+ Treg cells, and T cells. Intracellular staining of transcription factors showed that HFI treatment significantly reduced the expression of FoxP3 (Fig. 8A and C). HFI also increased the T cell population (Fig. 8B and D).

Discussion

Proteins are marginally stable and readily denatured by various stresses encountered in solution or in the frozen or dried states. Various additives are known to minimize the damage and to enhance the stability of proteins (34). The recombinant anti-ErbB2 scFv-Fc-IL-2 fusion gene was transfected into CHO dhfr-cells and selected for the dhfr-phenotype (31,32,35). The CHO cells stably expressing the fusion proteins (HFI) were cultured in serum-free medium, so that HFI stability was guaranteed when the protein purification process was downstream. HFI purified liquid was stable at 4°C for two months (Fig. 2), indicating that HFI can be produced in big batches. Stability data of freeze-dried HFI products were obtained after one year’s experiment, including quality analysis and activity detection, there was no stability data of the HFI available for more than three years, yet the current process and the data showed that the stability was not a problem for HFI, in drug development.

IL-2 is an important pleiotrophic cytokine and exhibits a wide variety of biological activities, including the stimulation of antitumor effector cells (18,36,37). IL-2 treatment can augment the activation of pre-existing antigen-specific T cells, enhance their recognition and destruction of neoplastic tissue, and activate NK cells (33,38,39). However, systemic cytokine therapy frequently caused severe problems with toxicity, and made it impossible to achieve an effective dose at the tumor sites. The recombinant human IL-2 gene is fused with the anti-ErbB2 scFv-Fc gene at the C-terminal (31,32,35). The biological activities of both antibody and IL-2 were maintained. The specific local activation of IL-2 at tumor sites provided more effective tumor destruction (33,40,41). HFI not only extended IL-2 half-life in vivo (Fig. 4), but also reduced the retention time of IL-2 in the blood because of HFI targeted activity (Fig. 3), and thus reduced the systemic toxicity of IL-2.

Patients with ErbB2-overexpressing tumors have shown a significantly lower overall survival rate and shorter relapse time than those with ErbB2-negative tumors (33,42,43). In therapy, ErbB2 overexpression is a significant negative prognostic indicator for a variety of therapies (33,44). In the pre-clinical trials using murine models, antibody-IL-2 fusion proteins have shown to be very effective antitumor agents (11,4548). HFI was effective in killing SKBR3 and SKOV3 cells, which expressed ErbB2 at higher levels, than MCF7 cells (33,49), suggesting that the killing effect mediated by HFI was dependent on the expression level of ErbB2 molecules on the target cells. To further determine the efficacy of HFI in vivo, the xenograft models of human non-small cell lung cancer (Calu-3) cells (HER2+++) in nude mice were treated with different concentrations of HFI (Fig. 5). The 1.0 mg/kg dose of HFI showed that the HFI had more than 60% of antitumor activity in vivo. These data suggested that HFI had significant antitumor activity on HER2-overexpressing tumor cells. Moreover, HFI could also prevent the progression of tumor development. In order to verify whether the HFI could inhibit tumor growth, FVB/neu transgenic mouse model (50) was used. In Fig. 6, results indicate that HFI significantly inhibited tumor growth activity in vivo. In ex vivo, HFI increased mitogen-induced splenocyte proliferation, IFN-γ production and cytotoxic responses to tumor cells (Fig. 7). Moreover, with early or post-operative treatment, the tumor could be completely suppressed or treated. IL-2 is critical for Treg cell growth, survival, and activity. Treg cells are inhibitors of antitumor immunity and considered as immunotherapy target (51). It is reported that daily low-dose of IL-2 increased numbers of Treg cells (52). Interestingly, in this study, the CD4+ population expressed relatively low FoxP3 levels in FVB/neu mice treated with HFI for 21 days, but the number of T cells increased. NK cells, NKT cells, T cells along with cytokines such as IFN-γ have been implicated in the processes of elimination and immuno-editing during cancer immunosurveillance (53). In this study, significant changes of the population of NK cells or NKT cells were not observed.

In this study, it was observed that HFI had a similar binding capacity with HER2 by equimolar concentration of Herceptin (Fig. 3). Moreover, the capacity of HFI binding HER2 did not receive its structural effects. HFI retained IL-2 biological activity (Fig. 2A), with efficient stimulation by targeted IL-2, and lymphocytes elicited an antitumor response in patients who are deficient in lymphocytes following high-dose chemotherapy.

In conclusion, HFI purified solution was stable, and could be prepared in mass for drug development. Fusion protein of the HFI retained ErbB2 specificity and IL-2 biological activity. Pharmacokinetics of HFI was not only associated with its molecular weight, but also positively correlated with HFI concentration. In vivo, HFI had significant activity on inhibiting HER2-overexpressing tumor growth. HFI demonstrated potency of initiating a cytotoxic activity on unstimulated PBMC against human breast and ovarian cancer cells at low effector-to-target ratios in vitro experiments. In this study, it was observed that HFI promoted apoptosis had no impact on HER2 expression, indicating HFI had potential toxicity. There are many issues to be resolved during the process of HFI drug development, including the long-term stability, mechanism of action, toxicological effects as well as the immunogenic effects.

Acknowledgements

This study was supported by ‘The Key New Drug Creation and Manufacturing’ (2009ZX09103-695) of the Eleventh Five-Year Plan. We thank Professor Ning Guo and Dr Ming Shi for excellent technical assistance.

References

1. 

Jallal B, Schlessinger J and Ullrich A: Tyrosine phosphatase inhibition permits analysis of signal transduction complexes in p185HER2/neu-overexpressing human tumor cells. J Biol Chem. 267:4357–4363. 1992.

2. 

Rusnak DW, Lackey K, Affleck K, et al: The effects of the novel, reversible epidermal growth factor receptor/ErbB-2 tyrosine kinase inhibitor, GW2016, on the growth of human normal and tumor-derived cell lines in vitro and in vivo. Mol Cancer Ther. 1:85–94. 2001.

3. 

Agus DB, Akita RW, Fox WD, et al: Targeting ligand-activated ErbB2 signaling inhibits breast and prostate tumor growth. Cancer Cell. 2:127–137. 2002. View Article : Google Scholar : PubMed/NCBI

4. 

Konopleva M, Zhang W, Shi YX, et al: Synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid induces growth arrest in HER2-overexpressing breast cancer cells. Mol Cancer Ther. 5:317–328. 2006. View Article : Google Scholar : PubMed/NCBI

5. 

Bergman I, Barmada MA, Griffin JA and Slamon DJ: Treatment of meningeal breast cancer xenografts in the rat using an anti-p185/HER2 antibody. Clin Cancer Res. 7:2050–2056. 2001.PubMed/NCBI

6. 

Spiridon CI, Ghetie MA, Uhr J, et al: Targeting multiple Her-2 epitopes with monoclonal antibodies results in improved anti-growth activity of a human breast cancer cell line in vitro and in vivo. Clin Cancer Res. 8:1720–1730. 2002.PubMed/NCBI

7. 

Meng S, Tripathy D, Shete S, et al: HER-2 gene amplification can be acquired as breast cancer progresses. Proc Natl Acad Sci USA. 101:9393–9398. 2004. View Article : Google Scholar : PubMed/NCBI

8. 

Jerome L, Alami N, Belanger S, et al: Recombinant human insulin-like growth factor binding protein 3 inhibits growth of human epidermal growth factor receptor-2-overexpressing breast tumors and potentiates herceptin activity in vivo. Cancer Res. 66:7245–7252. 2006. View Article : Google Scholar

9. 

Merlin JL, Barberi-Heyob M and Bachmann N: In vitro comparative evaluation of trastuzumab (Herceptin) combined with paclitaxel (Taxol) or docetaxel (Taxotere) in HER2-expressing human breast cancer cell lines. Ann Oncol. 13:1743–1748. 2002. View Article : Google Scholar

10. 

Nahta R, Hung MC and Esteva FJ: The HER-2-targeting antibodies trastuzumab and pertuzumab synergistically inhibit the survival of breast cancer cells. Cancer Res. 64:2343–2346. 2004. View Article : Google Scholar

11. 

Moulder SL, Yakes FM, Muthuswamy SK, Bianco R, Simpson JF and Arteaga CL: Epidermal growth factor receptor (HER1) tyrosine kinase inhibitor ZD1839 (Iressa) inhibits HER2/neu (erbB2)-overexpressing breast cancer cells in vitro and in vivo. Cancer Res. 61:8887–8895. 2001.PubMed/NCBI

12. 

Liang K, Lu Y, Jin W, Ang KK, Milas L and Fan Z: Sensitization of breast cancer cells to radiation by trastuzumab. Mol Cancer Ther. 2:1113–1120. 2003.PubMed/NCBI

13. 

Tseng PH, Wang YC, Weng SC, et al: Overcoming trastuzumab resistance in HER2-overexpressing breast cancer cells by using a novel celecoxib-derived phosphoinositide-dependent kinase-1 inhibitor. Mol Pharmacol. 70:1534–1541. 2006. View Article : Google Scholar

14. 

Arpino G, Gutierrez C, Weiss H, et al: Treatment of human epidermal growth factor receptor 2-overexpressing breast cancer xenografts with multiagent HER-targeted therapy. J Natl Cancer Inst. 99:694–705. 2007. View Article : Google Scholar : PubMed/NCBI

15. 

Talmadge JE, Phillips H, Schindler J, Tribble H and Pennington R: Systematic preclinical study on the therapeutic properties of recombinant human interleukin 2 for the treatment of metastatic disease. Cancer Res. 47:5725–5732. 1987.PubMed/NCBI

16. 

Kovacs JA, Vogel S, Albert JM, et al: Controlled trial of interleukin-2 infusions in patients infected with the human immunodeficiency virus. N Engl J Med. 335:1350–1356. 1996. View Article : Google Scholar : PubMed/NCBI

17. 

Kozlowski T, Sablinski T, Basker M, et al: Decreased graft-versus-host disease after haplotype mismatched bone marrow allografts in miniature swine following interleukin-2 treatment. Bone Marrow Transplant. 25:47–52. 2000. View Article : Google Scholar

18. 

Gaffen SL and Liu KD: Overview of interleukin-2 function, production and clinical applications. Cytokine. 28:109–123. 2004. View Article : Google Scholar : PubMed/NCBI

19. 

Malek TR and Bayer AL: Tolerance, not immunity, crucially depends on IL-2. Nat Rev Immunol. 4:665–674. 2004. View Article : Google Scholar : PubMed/NCBI

20. 

Cesana GC, DeRaffele G, Cohen S, et al: Characterization of CD4+CD25+ regulatory T cells in patients treated with high-dose interleukin-2 for metastatic melanoma or renal cell carcinoma. J Clin Oncol. 24:1169–1177. 2006.PubMed/NCBI

21. 

Goldsmith MA, Lai SY, Xu W, et al: Growth signal transduction by the human interleukin-2 receptor requires cytoplasmic tyrosines of the beta chain and non-tyrosine residues of the gamma c chain. J Biol Chem. 270:21729–21737. 1995. View Article : Google Scholar

22. 

Hughes-Fulford M, Sugano E, Schopper T, Li CF, Boonyaratanakornkit JB and Cogoli A: Early immune response and regulation of IL-2 receptor subunits. Cell Signal. 17:1111–1124. 2005. View Article : Google Scholar : PubMed/NCBI

23. 

Dybkaer K, Iqbal J, Zhou G, et al: Genome wide transcriptional analysis of resting and IL2 activated human natural killer cells: gene expression signatures indicative of novel molecular signaling pathways. BMC Genomics. 8:2302007. View Article : Google Scholar

24. 

Harvill ET and Morrison SL: An IgG3-IL2 fusion protein activates complement, binds Fc gamma RI, generates LAK activity and shows enhanced binding to the high affinity IL-2R. Immunotechnology. 1:95–105. 1995. View Article : Google Scholar : PubMed/NCBI

25. 

Budagian V, Nanni P, Lollini PL, et al: Enhanced inhibition of tumour growth and metastasis, and induction of antitumour immunity by IL-2-IgG2b fusion protein. Scand J Immunol. 55:484–492. 2002. View Article : Google Scholar : PubMed/NCBI

26. 

Heuser C, Ganser M, Hombach A, et al: An anti-MUC1-antibody-interleukin-2 fusion protein that activates resting NK cells to lysis of MUC1-positive tumour cells. Br J Cancer. 89:1130–1139. 2003. View Article : Google Scholar : PubMed/NCBI

27. 

Neal ZC, Yang JC, Rakhmilevich AL, et al: Enhanced activity of hu14.18-IL2 immunocytokine against murine NXS2 neuroblastoma when combined with interleukin 2 therapy. Clin Cancer Res. 10:4839–4847. 2004. View Article : Google Scholar : PubMed/NCBI

28. 

Gillies SD, Lan Y, Williams S, et al: An anti-CD20-IL-2 immunocytokine is highly efficacious in a SCID mouse model of established human B lymphoma. Blood. 105:3972–3978. 2005. View Article : Google Scholar : PubMed/NCBI

29. 

Wagner K, Schulz P, Scholz A, Wiedenmann B and Menrad A: The targeted immunocytokine L19-IL2 efficiently inhibits the growth of orthotopic pancreatic cancer. Clin Cancer Res. 14:4951–4960. 2008. View Article : Google Scholar : PubMed/NCBI

30. 

Marlind J, Kaspar M, Trachsel E, et al: Antibody-mediated delivery of interleukin-2 to the stroma of breast cancer strongly enhances the potency of chemotherapy. Clin Cancer Res. 14:6515–6524. 2008. View Article : Google Scholar : PubMed/NCBI

31. 

Shi M, Xie Z, Feng J, et al: A recombinant anti-erbB2, scFv-Fc-IL-2 fusion protein retains antigen specificity and cytokine function. Biotechnol Lett. 25:815–819. 2003. View Article : Google Scholar : PubMed/NCBI

32. 

Shi M, Xie Z, Yu M, Shen B and Guo N: Controlled growth of Chinese hamster ovary cells and high expression of antibody-IL-2 fusion proteins by temperature manipulation. Biotechnol Lett. 27:1879–1884. 2005. View Article : Google Scholar : PubMed/NCBI

33. 

Shi M, Zhang L, Gu HT, et al: Efficient growth inhibition of ErbB2-overexpressing tumor cells by anti-ErbB2 ScFv-Fc-IL-2 fusion protein in vitro and in vivo. Acta Pharmacol Sin. 28:1611–1620. 2007. View Article : Google Scholar : PubMed/NCBI

34. 

Arakawa T, Prestrelski SJ, Kenney WC and Carpenter JF: Factors affecting short-term and long-term stabilities of proteins. Adv Drug Deliv Rev. 46:307–326. 2001. View Article : Google Scholar : PubMed/NCBI

35. 

Chen L, Xie Z, Teng Y, et al: Highly efficient selection of the stable clones expressing antibody-IL-2 fusion protein by a dicistronic expression vector containing a mutant neo gene. J Immunol Methods. 295:49–56. 2004. View Article : Google Scholar

36. 

Lin JX and Leonard WJ: Signaling from the IL-2 receptor to the nucleus. Cytokine Growth Factor Rev. 8:313–332. 1997. View Article : Google Scholar : PubMed/NCBI

37. 

Lacosta S, Merali Z and Anisman H: Central monoamine activity following acute and repeated systemic interleukin-2 administration. Neuroimmunomodulation. 8:83–90. 2000. View Article : Google Scholar : PubMed/NCBI

38. 

Sereti I, Anthony KB, Martinez-Wilson H, et al: IL-2-induced CD4+ T-cell expansion in HIV-infected patients is associated with long-term decreases in T-cell proliferation. Blood. 104:775–780. 2004.PubMed/NCBI

39. 

Jo D, Liu D, Yao S, Collins RD and Hawiger J: Intracellular protein therapy with SOCS3 inhibits inflammation and apoptosis. Nat Med. 11:892–898. 2005. View Article : Google Scholar : PubMed/NCBI

40. 

Lewis GD, Figari I, Fendly B, et al: Differential responses of human tumor cell lines to anti-p185HER2 monoclonal antibodies. Cancer Immunol Immunother. 37:255–263. 1993. View Article : Google Scholar : PubMed/NCBI

41. 

Smith KA and Griffin JD: Following the cytokine signaling pathway to leukemogenesis: a chronology. J Clin Invest. 118:3564–3573. 2008. View Article : Google Scholar : PubMed/NCBI

42. 

Friess T, Scheuer W and Hasmann M: Combination treatment with erlotinib and pertuzumab against human tumor xenografts is superior to monotherapy. Clin Cancer Res. 11:5300–5309. 2005. View Article : Google Scholar : PubMed/NCBI

43. 

Johnson BE and Janne PA: Rationale for a phase II trial of pertuzumab, a HER-2 dimerization inhibitor, in patients with non-small cell lung cancer. Clin Cancer Res. 12:4436s–4440s. 2006. View Article : Google Scholar : PubMed/NCBI

44. 

Konecny GE, Pegram MD, Venkatesan N, et al: Activity of the dual kinase inhibitor lapatinib (GW572016) against HER-2-overexpressing and trastuzumab-treated breast cancer cells. Cancer Res. 66:1630–1639. 2006. View Article : Google Scholar : PubMed/NCBI

45. 

King DM, Albertini MR, Schalch H, et al: Phase I clinical trial of the immunocytokine EMD 273063 in melanoma patients. J Clin Oncol. 22:4463–4473. 2004. View Article : Google Scholar : PubMed/NCBI

46. 

Zhang X, Feng J, Ye X, Yao Y, Zhou P and Chen X: Development of an immunocytokine, IL-2-183B2scFv, for targeted immunotherapy of ovarian cancer. Gynecol Oncol. 103:848–852. 2006. View Article : Google Scholar : PubMed/NCBI

47. 

Johnson EE, Lum HD, Rakhmilevich AL, et al: Intratumoral immunocytokine treatment results in enhanced antitumor effects. Cancer Immunol Immunother. 57:1891–1902. 2008. View Article : Google Scholar : PubMed/NCBI

48. 

Singh H, Serrano LM, Pfeiffer T, et al: Combining adoptive cellular and immunocytokine therapies to improve treatment of B-lineage malignancy. Cancer Res. 67:2872–2880. 2007. View Article : Google Scholar : PubMed/NCBI

49. 

Spiridon CI, Guinn S and Vitetta ES: A comparison of the in vitro and in vivo activities of IgG and F(ab’)2 fragments of a mixture of three monoclonal anti-Her-2 antibodies. Clin Cancer Res. 10:3542–3551. 2004.PubMed/NCBI

50. 

Xie H, Lin L, Tong L, et al: AST1306, a novel irreversible inhibitor of the epidermal growth factor receptor 1 and 2, exhibits antitumor activity both in vitro and in vivo. PLoS One. 6:e214872011. View Article : Google Scholar

51. 

Zou W: Regulatory T cells, tumour immunity and immunotherapy. Nat Rev Immunol. 6:295–307. 2006. View Article : Google Scholar : PubMed/NCBI

52. 

Koreth J, Matsuoka K, Kim HT, et al: Interleukin-2 and regulatory T cells in graft-versus-host disease. N Engl J Med. 365:2055–2066. 2011. View Article : Google Scholar : PubMed/NCBI

53. 

Vesely MD, Kershaw MH, Schreiber RD and Smyth MJ: Natural innate and adaptive immunity to cancer. Annu Rev Immunol. 29:235–271. 2011. View Article : Google Scholar : PubMed/NCBI

Related Articles

Journal Cover

February 2013
Volume 42 Issue 2

Print ISSN: 1019-6439
Online ISSN:1791-2423

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
APA
Du, Y., Lin, Z., Zhao, Y., Feng, X., Wang, C., Wang, G. ... Wang, C. (2013). Stability of the recombinant anti‑erbB2 scFv‑Fc‑interleukin‑2 fusion protein and its inhibition of HER2‑overexpressing tumor cells. International Journal of Oncology, 42, 507-516. https://doi.org/10.3892/ijo.2012.1747
MLA
Du, Y., Lin, Z., Zhao, Y., Feng, X., Wang, C., Wang, G., Wang, C., Shi, W., Zuo, J., Li, F., Wang, C."Stability of the recombinant anti‑erbB2 scFv‑Fc‑interleukin‑2 fusion protein and its inhibition of HER2‑overexpressing tumor cells". International Journal of Oncology 42.2 (2013): 507-516.
Chicago
Du, Y., Lin, Z., Zhao, Y., Feng, X., Wang, C., Wang, G., Wang, C., Shi, W., Zuo, J., Li, F., Wang, C."Stability of the recombinant anti‑erbB2 scFv‑Fc‑interleukin‑2 fusion protein and its inhibition of HER2‑overexpressing tumor cells". International Journal of Oncology 42, no. 2 (2013): 507-516. https://doi.org/10.3892/ijo.2012.1747