Cancer Chemotherapy and Pharmacology

, Volume 71, Issue 3, pp 799–807

Immunogenicity and toxicity of transferrin receptor-targeted hybrid peptide as a potent anticancer agent

Authors

  • Megumi Kawamoto
    • Department of Pharmacoepidemiology, Graduate School of Medicine and Public HealthKyoto University
  • Masayuki Kohno
    • Department of Pharmacoepidemiology, Graduate School of Medicine and Public HealthKyoto University
  • Tomohisa Horibe
    • Department of Pharmacoepidemiology, Graduate School of Medicine and Public HealthKyoto University
    • Department of Pharmacoepidemiology, Graduate School of Medicine and Public HealthKyoto University
Original Article

DOI: 10.1007/s00280-013-2074-4

Cite this article as:
Kawamoto, M., Kohno, M., Horibe, T. et al. Cancer Chemother Pharmacol (2013) 71: 799. doi:10.1007/s00280-013-2074-4

Abstract

Purpose

Transferrin receptor (TfR) is a cell membrane-associated glycoprotein involved in the cellular uptake of iron and the regulation of cell growth. Recent studies have shown elevated expression levels of TfR on cancer cells compared with normal cells. We previously designed a TfR-lytic hybrid peptide, which combines the TfR-binding peptide and a lytic peptide, and reported that it bound specifically to TfR and selectively killed cancer cells. Furthermore, the intravenous administration of TfR-lytic peptide in an athymic mouse model significantly inhibited tumor progression. To evaluate the immunogenicity of this peptide as a novel and potent anticancer agent, we investigated whether TfR-lytic hybrid peptide elicits cellular and humoral immune responses to produce antibodies. We also examined the toxicity of this peptide in syngeneic mice.

Methods

We performed hematologic and blood chemistry test and histological analysis and assessed hemolytic activity to check toxicity. To evaluate the immunogenicity, measurement of murine interferon-gamma and detection of TfR-lytic-specific antibody by ELISA were demonstrated.

Results

No T cell immune response or antibodies were detected in the group treated with TfR-lytic hybrid peptide. No hematologic toxicity, except for a decrease in leukocytes, was observed, and no remarkable influence on metabolic parameters and organs (liver, kidney, and spleen) was noted.

Conclusions

Therefore, TfR-lytic hybrid peptide might provide an alternative therapeutic option for patients with cancer.

Keywords

Transferrin receptorImmunogenicityToxicityPeptide drugMolecular target drug for cancer treatmentImmunotoxin

Introduction

Immunotoxins are chimeric proteins with a cell-selective ligand chemically linked or genetically fused to a toxin moiety. They can target cancer cells overexpressing tumor-associated antigens, membrane receptors, or carbohydrate antigens [1, 2]. Generally, ligands for these receptors, monoclonal antibodies, and single-chain variable fragments directed against these antigens fuse with bacterial or plant toxins to generate immunotoxins. Several such fusion proteins including Pseudomonas exotoxin-based interleukin (IL)-4-Pseudomonas exotoxin (IL4(38-37)-PE38KDEL) and IL-13-Pseudomonas exotoxin (IL13-PE38QQR) fusion proteins have been tested in clinical trials [3, 4] IL-2-diphtheria toxin fusion protein (IL2-DT; Ontak™) is an FDA-approved fusion protein [5, 6]. However, bacterial or plant toxin-based chimeric proteins pose several problems that limit their clinical application [7]. Because Pseudomonas exotoxin and diphtheria toxin are large molecules with high immunogenicity, they elicit a high degree of humoral response including neutralization antibodies in humans [8, 9]. Besides, at sufficiently high doses, these fusion proteins also cause vascular leak syndrome.

Transferrin receptor (TfR) is a cell membrane-associated glycoprotein involved in the cellular uptake of iron and the regulation of cell growth [10]. Various studies have shown elevated levels of TfR expression on cancer cells when compared with their normal counterparts, and TfR expression correlated with tumor grade and stage or prognosis [1116]. As a new generation of immunotoxins, we have recently designed a novel class of drug termed “hybrid peptide,” which is chemically synthesized and comprise a target-binding peptide and a lytic peptide containing cationic-rich amino acid components that disrupt the cell membrane leading to cancer cell death via lysis [1719]. We previously designed the TfR-lytic hybrid peptide, which is a combination of the TfR-binding peptide and a lytic peptide, and showed that this hybrid peptide bounds specifically to TfR and selectively killed cancer cells. Furthermore, the intravenous administration of TfR-lytic peptide in an athymic mice model significantly inhibited tumor progression [18].

Therapeutic peptides are increasingly gaining popularity for clinical use in a variety of applications [20], including as tumor vaccines [21], for antimicrobial therapy [22], and for nucleic acid delivery [23]. It is also known that peptide therapeutic agents are generated relatively easily using solid-phase chemical synthesis techniques and are generally less expensive than antibody-based therapeutics. In addition, in contrast to immunotoxins, the risk of immune system mobilization and toxicity after treatment may be minimized because these peptides have lower molecular weights than proteins. However, it remains necessary to examine whether TfR-lytic hybrid peptide induces an immune response when considering its potential as an anticancer therapeutic agent. In the present study, we investigated whether the TfR-lytic hybrid peptide induces cellular and humoral immune responses leading to antibody production. In addition, we evaluated the toxicity of this peptide in syngeneic mice.

Materials and methods

Cell lines

Mouse (GL261) and human glioblastoma (U251) cells were purchased from the American Type Culture Collection (Manassas, VA). Human normal pancreatic epithelial (PE; ACBRI 515) cells were purchased from DS Pharma Biomedical (Tokyo, Japan). Cells were cultured in RPMI-1640 (U251 and GL261) and CS-C medium (PE) with 10 % fetal bovine serum (BioWest, Miami, FL), 100 μg/ml penicillin, and 100 μg/ml streptomycin (Nacalai Tesque, Kyoto, Japan) under 5 % CO2.

Preparation and synthesis of peptides

The following peptides were purchased from Invitrogen (Carlsbad, CA) or Sigma (St Louis, MO):
  1. 1.

    Lytic peptide: KLLLKLLKKLLKLLKKK

     
  2. 2.

    TfR-lytic hybrid peptide: THRPPMWSPVWPGGGKLLLKLLKKLLKLLKKK

     

Note that in both cases, bold and underlined letters indicate d-amino acids. Both peptides were synthesized using solid-phase chemical techniques, purified to homogeneity (i.e., >80 %) by reversed-phase high-pressure liquid chromatography, and assessed by mass spectrometry. Peptides were dissolved in water.

Binding assay

A binding assay was performed as previously described [9]. Briefly, TfR-lytic peptide labeled with CF488A (Biotium, CA) was incubated with U251, GL261, and PE cells for 30 min, and then binding peptides were detected using a FACSCalibur (Becton–Dickinson, Mountain View, CA). Binding activity was calculated from the mean fluorescence intensity (MFI). MFI was determined using WinMDI version 2.9 software (The Scripps Research Institute, La Jolla, CA).

Anti-tumor effect in syngeneic mouse model

Animal experiments were carried out in accordance with the guidelines of Kyoto University School of Medicine. Murine glioblastoma GL261 cells of 1 × 106 cells/100 μl in phosphate-buffered saline (PBS) were implanted subcutaneously into the flank region of 7-week-old C57BL/6 female mice weighing 17–21 g. After 7 days of the implantation, these animals were randomly assigned to two groups receiving either saline (control) or TfR-lytic peptide (3 mg/kg) as intravenous injections (50 μl/injection). Tumors were measured with a caliper, and the tumor volume (in mm3) was calculated using the following formula: length × width2 × 0.5. All values are expressed as the mean ± SD.

Hemolytic activity

The hemolytic activity of TfR-lytic peptide was determined using murine erythrocytes. Whole blood was removed and transferred into EDTA-containing tubes, rinsed three times with PBS, centrifuged for 15 min at 900 g, and resuspended in PBS. A 4 % (v/v) erythrocyte suspension was prepared and incubated with different concentrations of the peptide (0–100 μM) for different lengths of time (30 min, 2, 24 h). The absorbance of the supernatants was measured at 540 nm. Correlating the measured values of treated (Asample) and untreated (Acontrol) erythrocytes led to the percentage of hemolytic activity. Total hemolysis (Atotal) was obtained by treating erythrocytes with 0.1 % Triton-X 100 (Merck, Darmstadt, Germany). Percent hemolytic activity was calculated using the following equation: (Asample  Acontrol)/(Atotal  Acontrol).

Flow cytometry

Murine lymph node cells were prepared from lymph node sample from mice treated with either TfR-lytic peptide or saline for 12, 19, 26, or 33 days after tumor implantation and isolated using ACCUMAX (Innovative Cell Technology, Inc., San Diego) solution according to the manufacturer’s instructions. Populations of CD3, CD4, and CD8 expressed in lymph node cells were determined using flow cytometry by incubating 1 × 106 cells with a mouse antibody to phycoerythrin-Cy5-conjugated CD3, phycoerythrin-conjugated CD4, and FITC-conjugated CD8, respectively (eBioscience San Jose, CA). All staining was performed at room temperature for 20 min. The cell fluorescence was measured by flow cytometry (FACS caliber, Becton–Dickinson). Data were analyzed using WinMDI version 2.9 software.

Measurement of murine interferon (IFN)-γ

Murine splenocytes were prepared from spleen samples from mice treated with either TfR-lytic peptide or saline for 12, 19, 26, or 33 days after tumor implanting and isolated using ACCUMAX solution according to the manufacturer’s instructions. These cells were maintained in RPMI1640 medium supplemented with 10 % fetal bovine serum and 50 μM of 2-mercaptoethanol, and they were pulsed with none, 1 μM of TfR-lytic peptide, or 1 μg/ml of ionomycin and 25 ng/mol of phorbol myristate acetate (PMA) as a positive control. The splenocyte supernatant was collected after 5 h. The concentration of murine IFN-γ in the culture supernatants was determined using an enzyme-linked immunosorbent assay (ELISA) kit (eBioscience San Jose, CA) according to the manufacturer’s instructions. The absorbance of the test sample was converted to picograms per milliliter based on a standard curve.

Serum sample preparation

Murine glioblastoma GL261 cells of 1 × 106 cells/100 μl in phosphate-buffered saline (PBS) were implanted subcutaneously into the flank region of 7-week-old C57BL/6 female mice weighing 17–21 g. After 7 days of the implantation, these animals were divided into three groups treated with saline, TfR-lytic peptide, or keyhole limpet hemocyanin (KLH)-conjugated TfR-lytic peptide (TfR-lytic–KLH) as a positive control. The saline and TfR-lytic peptide (3 mg/kg) were injected intravenously (50 μl/injection) three times a week. TfR-lytic–KLH was administered intraperitoneally (100 μg) with Freund’s complete adjuvant (only first injection) and Freund’s incomplete adjuvant once a week for a total of three doses. Serum samples were obtained from mice treated with saline, TfR-lytic, or TfR-lytic–KLH on days 12, 19, 26, or 33.

Antibody binding to TfR-lytic hybrid peptide

To measure antibody binding, 96-well plates were coated overnight at 4 °C with 37 μg/ml of TfR-lytic peptide or TfR-lytic–KLH. The plates were washed with PBS-T buffer and incubated for 1 h at 37 °C with PBS containing 1 % bovine serum albumin. Serial twofold dilutions of mouse serum were tested in duplicate. Mouse immunoglobulin (Ig) G was detected using an anti-mouse IgG-alkaline phosphatase conjugate (Sigma) followed by substrate (1 mg p-nitrophenyl phosphate). Absorbance at 405 nm was measured. Serum samples from mice injected with TfR-lytic–KLH were used for positive controls.

Statistical analysis

Data are expressed as the mean ± SD of triplicate determinations. Statistical difference was determined using Student’s t test, and P values less than 0.05 were considered statistically significant.

Results

Cytotoxic and binding activity of the TfR-lytic hybrid peptide in murine glioma GL261 cells

To evaluate the immunogenicity and toxicity of TfR-lytic peptide in the syngeneic mouse model, we examined cytotoxic activity and binding to mouse glioma GL261 cells when compared with human glioma U251 cells in vitro. The IC50 of TfR-lytic peptide binding to U251 and GL261 cells was 7.0 and 3.0 μM, and the IC50 of lytic peptide alone was 17.0 and 19.1 μM, respectively (Supplementary Table 1 and Fig 1A). TfR-lytic peptide also showed high sensitivity to and cytotoxicity against murine glioma GL261 cells compared with human glioma U251 cells. Checking the binding activation, TfR-lytic peptide exhibited an almost equivalent binding activity to U251 and GL261 (Fig. 1b). Human PE cells, which expressed low levels of human TfR, were used as a negative control.
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Fig. 1

Cytotoxic activity against murine glioma GL261 cells and binding activity of TfR-lytic hybrid peptide. a GL261 murine glioma cells were cultured with various concentrations of TfR-lytic hybrid peptide or lytic peptide (0–20 μM) for 72 h, and cytotoxic activity was assessed using WST-8 reagent. Black and white squares indicate TfR-lytic hybrid peptide and lytic peptide, respectively. b Binding activity of TfR-lytic peptide to human glioma U251, murine glioma GL261, and human pancreatic epithelia (PE) cells treated with CF488A-labeled TfR-lytic peptide (20 μM). Fold changes in fluorescence intensity show the extent of binding of CF488A-labeled TfR-lytic peptide to each cell, where the fluorescence intensity for non-treated cells was set as the control

Anti-tumor activity of TfR-lytic hybrid peptide in xenograft and syngeneic mouse model

We previously demonstrated that the intravenous administration of TfR-lytic peptide in athymic mice after implantation of MDA-MB-231 human breast cancer cells significantly inhibited tumor progression at a dose of 3 mg/kg, three times a week for a total of nine doses [9]. In the present study, we also assessed the in vivo anti-tumor effect of TfR-lytic peptide in athymic mice following implantation of U251 cells. The TfR-lytic peptide significantly inhibited tumor growth in this athymic model (Supplementary Fig 1). To evaluate the anti-tumor effect of TfR-lytic peptide in a syngeneic model, murine glioma GL261 cells were implanted subcutaneously. TfR-lytic peptide was injected intravenously at a dose of 3 mg/kg, three times a week for a total of nine doses. The tumor volume was inhibited significantly (P < 0.05) (Fig 2a). The tumor volume of GL261 on day 28 in the 3 mg/kg dosage group was reduced to 53 % that of the control group with saline. On histological examination, no abnormalities were observed in organs such as liver, kidney, and spleen (Fig 2b), and there were no differences in body weight (Supplementary Table 2) or serum chemistry between the saline-treated and TfR-lytic peptide-treated groups, including no differences in serum iron, total iron binding capacity, or transferrin in the serum sample of day 26, which was prepared 24 h after the ninth administration (Table 1). Significant leucopenia was observed (Table 1); however, there were no differences in the other hematology parameters.
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Fig. 2

Anti-tumor activity of TfR-lytic hybrid peptide in syngeneic mouse model in vivo: a GL261 murine glioma cells were implanted subcutaneously into syngeneic mice. Intravenous injection of either saline (control) or TfR-lytic peptide (3 mg/kg) as indicated by the arrows. Data are expressed as mean ± SD, n = 6 animals in each group. b Histological examination after treatment with TfR-lytic peptide. Images (×400 magnification) of liver, kidney, and spleen from mice after all nine treatments (as indicated by the arrows in a) with saline (control) or TfR-lytic peptide (3 mg/kg) were obtained by staining with hematoxylin and eosin

Table 1

Hematologic and serum metabolic characteristics

24 h after ninth administration (day 26)

Treatment group

 

Unit

Saline

3 mg/kg

P value

Leukocyte

1,000/μl

4.5 ± 1.0

1.2 ± 0.2

0.006

Red blood cell

10,000/μl

473.3 ± 86.3

465.3 ± 109.8

0.478

Hemoglobin

g/dl

7.6 ± 1.3

7.9 ± 0.6

0.447

Hematocrit

%

27.3 ± 4.8

25.5 ± 7.5

0.405

Platelet

10,000/μl

63.6 ± 8.0

85.8 ± 50.4

0.313

Leukocyte classification

 Neutrophil

%

45.7 ± 19.8

50.4 ± 9.2

0.329

 Eosinocyte

%

0.0 ± 0.0

0.0 ± 0.0

N/A

 Basophil

%

0.0 ± 0.0

0.0 ± 0.0

N/A

 Lymphocyte

%

50.7 ± 19.8

34.3 ± 18.0

0.144

 Monocyte

%

3.7 ± 0.0

3.7 ± 2.1

0.500

AST

IU/l

351.0 ± 38.2

304.0 ± 169.1

0.406

ALT

IU/l

86.7 ± 1.4

82.0 ± 61.7

0.470

ALP

IU/l

75.0 ± 60.8

52.3 ± 8.6

0.237

LDH

IU/l

5452.7 ± 871.2

2285.3 ± 272.7

0.216

CPK

IU/l

2603.7 ± 85.6

857.0 ± 465.7

0.239

Uric acid

mg/dl

4.6 ± 1.1

6.4 ± 1.4

0.186

Urea nitrogen

mg/dl

23.0 ± 2.1

20.3 ± 2.1

0.312

Albumin

g/dl

2.3 ± 0.4

2.1 ± 0.1

0.213

Creatinine

mg/dl

0.1 ± 0.0

0.1 ± 0.0

0.425

Na

mEq/l

147.7 ± 2.1

148.7 ± 6.4

0.409

Cl

mEq/l

103.3 ± 3.5

106.3 ± 5.8

0.240

K

mEq/l

6.3 ± 0.8

6.6 ± 0.4

0.288

Ca

mg/dl

9.4 ± 1.1

9.9 ± 0.4

0.213

Mg

mg/dl

3.6 ± 0.4

3.4 ± 0.4

0.291

Fe

μg/dl

91.7 ± 14.8

108.0 ± 12.8

0.131

TIBC

μg/dl

494.7 ± 164.0

649.0 ± 99.7

0.085

Transferrin

mg/dl

134.3 ± 26.9

142.0 ± 13.9

0.381

Mean ± SD (n = 3 mice/group). Statistical values are between the 2 groups

AST aspartate aminotransferase, ALT alanine aminotransferase, ALP alkaline phosphatase, LDH lactate dehydrogenase, CPK creatine phosphokinase, TIBC total iron binding capacity

Hemolytic activity of TfR-lytic hybrid peptide

The influence of TfR-lytic peptide on murine erythrocytes was measured using a hemolytic assay. Figure 3 shows the hemolytic activity of murine erythrocytes after incubation with different concentrations of TfR-lytic peptide. After incubation periods of 30 min and 2 h, both saline-treated and TfR-lytic peptide-treated erythrocytes showed less than 5 % hemolysis compared with the positive control, even at the highest peptide concentration (100 μM). A 24-h incubation period with the peptide increased hemolytic activity in human erythrocytes by 17 %.
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Fig. 3

Hemolytic activity of isolated murine erythrocytes after incubation with different concentrations of TfR-lytic hybrid peptide. Murine erythrocytes were incubated with different concentrations of TfR-lytic hybrid peptide (0–100 μM) for different periods of time (30 min, 2, 24 h). The absorbance of the supernatants was measured at 540 nm. Erythrocytes were treated with 0.1 % Triton-X 100 as positive control, and the activity was set to 100 % hemolytic activity. The values are mean ± SD from triplicate determination, and the assay was performed twice

Evaluation of cellular immune responses

To investigate the cellular immune responses, we assessed the phenotype of T cells from regional lymph nodes using anti-mouse monoclonal antibodies against CD3, CD4, CD8 by flow cytometry (Fig 4a). We also determined IFN-γ production at weekly intervals from splenocytes of mice treated with TfR-lytic peptide or saline using an ELISA (Fig 4b). Splenocytes were primed with TfR-lytic peptide (1 μM) or PMA and ionomycin (positive control). Treatment of splenocytes with TfR-lytic peptide for 5 h did not induce any cytotoxic effect (data not shown). There were no differences in CD3+CD4+, CD3+CD8+ double-positive T cell population, and IFN-γ production between the saline- and TfR-lytic peptide-treated groups.
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Fig. 4

Population of CD3+CD4+ and CD3+CD8+ lymphocytes from regional lymph nodes and IFN-γ production from spleen in TfR-lytic peptide- or saline-treated mice. GL261 murine glioma cells were implanted subcutaneously into syngeneic mice, and TfR-lytic peptide or saline was administered intravenously from day 7. Lymph node cells and splenocytes were prepared from lymph node and spleen samples from mice on days 12, 19, 26, and 33 after implanting GL261 cells. Mean ± SD (n = 3 mice/group). a Populations of CD3+CD4+ and CD3+CD8+ lymphocytes were determined by flow cytometry. b Splenocytes stimulated with TfR-lytic hybrid peptide at 1 μM. PMA/ionomycin was used as a positive control for IFN-γ production

Evaluation of humoral immune responses

To confirm whether TfR-lytic peptide elicited humoral immune responses, we attempted to detect TfR-lytic-specific antibody by ELISA. Mice treated with TfR-lytic–KLH were used as a positive control. Although the IgG antibody titer of serum samples treated with TfR-lytic–KLH was dramatically increased, the antibody titer of serum samples treated with TfR-lytic peptide was no different compare with saline in all serum dilution (Fig 5a, b). TfR-lytic peptide-specific IgG antibody was not detected in mice treated with TfR-lytic peptide. A similar result was obtained using immunoglobulin M (Supplementary Fig 2).
https://static-content.springer.com/image/art%3A10.1007%2Fs00280-013-2074-4/MediaObjects/280_2013_2074_Fig5_HTML.gif
Fig. 5

Specific immunoglobulin G for TfR-lytic hybrid peptide or KLH-conjugated TfR-lytic peptide in the serum of mice treated with these peptides. Serum samples were collected on days 12, 19, 26, and 33 after implanting GL261 cells, and a used in a serial dilutions, b assessed to determine the optical density of serum specimens diluted 1:16. Mean ± SD (n = 3 mice/group)

Discussion

It is advised by the ICH S8 guidelines that potential adverse effects of pharmaceutical products on the immune system should be evaluated. Influence on the immune system elicits a variety of adverse effects, including suppression or enhancement of the immune response. Suppression of the immune response can lead to decreased host resistance to tumor cells. Enhancing the immune response can exaggerate autoimmune diseases or hypersensitivity. Protein drugs such as immunotoxins are recognized as foreign and, as a result, stimulate an anti-drug response including neutralization antibodies.

In this study, we investigated whether TfR-lytic peptide induces cellular and humoral immune responses using a syngeneic model of mouse glioma GL261 cells in C57BL/6 mice that do not require a deficient immune system.

First, we evaluated the binding and cytotoxic activity of TfR-lytic peptide to mouse glioma GL261 cells because, using phage display, the TfR-binding sequence was identified as the peptide capable of binding to human TfR [24]. Glioma cells are known to overexpress TfR, and the level of human TfR expression positively correlates with tumor grade [25]. It has also been confirmed that mouse TfR is highly expressed in GL261 cells [26]. When we compared the extracellular domain of TfR between human and mouse, we found 76 % homology. Our results (Supplementary Table 1 and Fig. 1) are in agreement with the above findings where TfR-lytic peptide showed high binding activity to mouse glioma GL261. For this reason, the peptide might result in a significant anti-tumor effect in syngeneic mouse following implantation of GL261 cells as well as in the U251 cell xenograft model. The only adverse effect observed in the TfR-lytic peptide-treated group was a decrease in the leukocyte count in serum chemistry and hematology tests, which suggests that TfR-lytic peptide may have a high affinity for leukocyte membranes; additional study is needed to elucidate the toxicity of leukocytes. Next, we examined the hemolytic activity of the TfR-lytic peptide since cationic-rich peptides disrupt the cell membrane and induce cell death. Due to this mechanism of action, several cationic-rich peptides often have hemolytic activity [27, 28]. Given that the total blood volume of the nude mice (body weight ~20 g) is 1.5 ml, the dose of 3 mg/kg that is effective in the in vivo mouse model is approximately 10 μM; at this concentration, TfR-lytic peptide showed almost no hemolytic activity. In addition, there was no toxicity related to hemolysis parameters such as red blood cell count, hemoglobin, and hematocrit in mice that received repeated doses of TfR-lytic peptide (Table 1).

As utilized in cancer immunotherapy, a short amino acid sequence elicits a cellular immune response. Therefore, we examined whether TfR-lytic peptide induced a cellular immune response, but no remarkable immune response was observed. A decrease in the population of CD3+CD4+ and CD3+CD8+ double-positive T cells was observed week by week in both groups, likely caused by the disappearance of expression of MHC with tumor progression [29]. Essentially, small compounds (MW <6,000) are not immunogenic. Although compounds smaller than this can often be bound by membrane IgM on the surface of the B cell, they are not large enough to facilitate crosslinking of the membrane IgM molecules. However, peptides may have the complexity necessary to be antigenic; therefore, it is important to investigate whether TfR-lytic peptide induces the production of antibodies. No increase in IgG or IgM antibody titer was observed in the TfR-lytic peptide-treated group.

In conclusion, the results of the present study indicate that TfR-lytic peptide repeatedly administered at an effective dose of 3 mg/kg is not immunogenic in syngeneic mouse. No hematologic toxicity, except for a decrease in leukocytes, was observed, and no remarkable influence on metabolic parameters and organs such as liver, kidney, and spleen was noted. Therefore, TfR-lytic peptide may provide an alternative therapeutic option for patients with cancer.

Acknowledgments

We thank Ms. Nana Kawaguchi, Ms. Kumi Kodama, Ms. Aya Torisawa, Ms. Keiko Shimoura, and Ms. Maiko Yamada of the Department of Pharmacoepidemiology, Kyoto University, for technical assistance with cell culturing and animal care. This study was supported by a grant-in-aid for Young Scientists (A) (grant no. 23680089) from the Japan Society for the Promotion of Science.

Conflict of interest

None.

Supplementary material

280_2013_2074_MOESM1_ESM.ppt (60 kb)
Supplementary Fig. 1 Anti-tumor activity of TfR-lytic hybrid peptide in athymic mouse model in vivo. U251 human glioma cells were implanted subcutaneously into athymic mice. Intravenous injection of either saline (control) or TfR-lytic peptide (3 mg/kg) as indicated by the arrows. Data are expressed as mean ± SD, n = 6 animals in each group. Supplementary material 1 (PPT 60 kb)
280_2013_2074_MOESM2_ESM.ppt (87 kb)
Supplementary Fig. 2 Specific immunoglobulin M for TfR-lytic hybrid peptide or KLH-conjugated TfR-lytic peptide in the serum of treated mice with these peptides. Serum samples were collected on days 12, 19, 26, and 33 in GL261 cells implanting, and (A) used in a serial dilutions, (B) assessed to determine the optical density of serum specimens diluted 1:16. Mean ± SD (n = 3 mice/group). Supplementary material 2 (PPT 87 kb)
280_2013_2074_MOESM3_ESM.doc (41 kb)
Supplementary material 3 (DOC 41 kb)

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