Immunogenicity and toxicity of transferrin receptor-targeted hybrid peptide as a potent anticancer agent
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- Kawamoto, M., Kohno, M., Horibe, T. et al. Cancer Chemother Pharmacol (2013) 71: 799. doi:10.1007/s00280-013-2074-4
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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.
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.
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.
Therefore, TfR-lytic hybrid peptide might provide an alternative therapeutic option for patients with cancer.
KeywordsTransferrin receptorImmunogenicityToxicityPeptide drugMolecular target drug for cancer treatmentImmunotoxin
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 . 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 . 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 [11–16]. 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 [17–19]. 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 .
Therapeutic peptides are increasingly gaining popularity for clinical use in a variety of applications , including as tumor vaccines , for antimicrobial therapy , and for nucleic acid delivery . 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
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
Lytic peptide: KLLLKLLKKLLKLLKKK
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.
A binding assay was performed as previously described . 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.
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).
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.
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.
Cytotoxic and binding activity of the TfR-lytic hybrid peptide in murine glioma GL261 cells
Anti-tumor activity of TfR-lytic hybrid peptide in xenograft and syngeneic mouse model
Hematologic and serum metabolic characteristics
24 h after ninth administration (day 26)
4.5 ± 1.0
1.2 ± 0.2
Red blood cell
473.3 ± 86.3
465.3 ± 109.8
7.6 ± 1.3
7.9 ± 0.6
27.3 ± 4.8
25.5 ± 7.5
63.6 ± 8.0
85.8 ± 50.4
45.7 ± 19.8
50.4 ± 9.2
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
50.7 ± 19.8
34.3 ± 18.0
3.7 ± 0.0
3.7 ± 2.1
351.0 ± 38.2
304.0 ± 169.1
86.7 ± 1.4
82.0 ± 61.7
75.0 ± 60.8
52.3 ± 8.6
5452.7 ± 871.2
2285.3 ± 272.7
2603.7 ± 85.6
857.0 ± 465.7
4.6 ± 1.1
6.4 ± 1.4
23.0 ± 2.1
20.3 ± 2.1
2.3 ± 0.4
2.1 ± 0.1
0.1 ± 0.0
0.1 ± 0.0
147.7 ± 2.1
148.7 ± 6.4
103.3 ± 3.5
106.3 ± 5.8
6.3 ± 0.8
6.6 ± 0.4
9.4 ± 1.1
9.9 ± 0.4
3.6 ± 0.4
3.4 ± 0.4
91.7 ± 14.8
108.0 ± 12.8
494.7 ± 164.0
649.0 ± 99.7
134.3 ± 26.9
142.0 ± 13.9
Hemolytic activity of TfR-lytic hybrid peptide
Evaluation of cellular immune responses
Evaluation of humoral immune responses
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 . Glioma cells are known to overexpress TfR, and the level of human TfR expression positively correlates with tumor grade . It has also been confirmed that mouse TfR is highly expressed in GL261 cells . 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 . 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.
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