| Mucin 1(MUC1) is a transmembrane glycoprotein that belongs to mucins family, it consists of core peptides and carbohydrate chain. It is expressed on the apical surface of epithelial cells and is aberrantly overexpressed on most epithelial malignant tumors and several malignant hematological tumors, and plays a key role in the development of various adenocarcinoma. The expression of MUC1 is different in normal and tumor tissues, the former is glycosylated and is distributed in the surface of ductal epithelial cells, while the latter is widely distributed and aberrantly overexpressed in the surface of cancer cells, the glycosylation is not completely, and it can expose the normally hidden form, of which becomes the attack targets of the immune cells, and making MUC1 as an attractive targets for anti-tumor vaccine research. To date, numerous anti-tumor vaccine formulations targeting MUC1 have been developed, including carbohydrate chain vaccines, protein and peptide vaccines, and DNA vaccines. Most importantly, recombinant protein vaccines have been widely studied in animals and humans because of their relative safety and simplicity of production. Our previous studies showed that maltose binding protein(MBP) induced the activation of Th1 cells via TLR2/4, and induced macrophages polarizated to M1. In order to generate an anti-tumor vaccine, we generated a recombinant MUC1-MBP fusion protein(MUC1-MBP) expression plasmid by inserting seven tandem repeats encoding the human MUC1 gene into an efficient expression MBP vector, and constructed an engineering bacteria that stably expressing recombinant MUC1-MBP fusion protein, as well as pilot project bacteria fermentation and protein production technology. Our previous results showed that the recombinant MUC1-MBP anti-tumor vaccine induced a Th1-dominant immune response and NK cell activity in addition to stimulating MUC1-specific cytotoxic T lymphocyte killing activity. Furthermore, the recombinant MUC1-MBP anti-tumor vaccine significantly inhibited MUC1-expression B16 cell growth in mice.In the present study, to move the recombinant MUC1-MBP anti-tumor vaccine into a Phase I clinical trial, a comprehensive pre-clinical toxicity and immunobiological evaluation was conducted. Our studies include the following contents. The qualityanalysis of recombinant MUC1-MBP fusion proteinThe recombinant MUC1-MBP anti-tumor vaccine was produced in our lab. The purity of recombinant MUC1-MBP was analyzed by SDS-PAGE and high performance liquid chromatography, and the results showed that the purity was above 95%; the residual of endotoxin in 1 mg/ml recombinant MUC1-MBP liquid was detected by limulus reagent gel method, and the results showed that the content of endotoxin was less than 5EU/ml.The concentration of recombinant MUC1-MBP solution was 391.23 ?g/ml and the accuracy was 97.81%, of which fit with the experimental requirements. Acute toxicity study of recombinant MUC1-MBP anti-tumor vaccine in miceTo evaluate the acute toxicity of a single-dose recombinant MUC1-MBP anti-tumor vaccine on ICR mice when administrated by subcutaneous(s.c.) injection, forty ICR mice were randomized into control and experimental groups. A single human test dose of recombinant MUC1-MBP anti-tumor vaccine was s.c. Injected into the experiment groups, while the control group was given equal volumes of saline. The general clinical signs of the mice were observed for 4 h after immunization, and then there was continuous observation twice per day for 14 days. Body weight was measured on day1, 8 and 15. Food consumption was evaluated on day8 and 15. All of the animals were sacrificed on day 14, and each received a complete necropsy.During the experiment, the result showed that the mental status and behavior activity of all the animals were good, and there were no dead or dying mice. The recombinant MUC1-MBP anti-tumor vaccine did not cause any significant differernces in body weight or food consumption between the experimental and control groups(P> 0.05). In addition, exposing the mice to a single human test dose of MUC1-MBP anti-tumor vaccine did not cause any abnormalities in necropsy findings when compared with the controls.All these results indicate that treatment with the MUC1-MBP/BCG anti-tumor vaccine produces no observable toxicity when a single human test dose of the vaccine is s.c. injected into ICR mice, and the maximum tolerated dose is greater than a single human test dose. Repeat-dose chronic toxicity and immunogenicity study of recombinant MUC1-MBP anti-tumor vaccine in ratsTo further investigate the chronic toxicity and immunogenicity of the recombinant MUC1-MBP anti-tumor vaccine on rats, 168 rats were randomized into 4 groups based on individual body weight: negative control group, BCG control group, low-dose group and high-dose group(15 rats per sex as experimental animals, while 6 rats per sex as satellite animals. In this study, rats were immunized with the recombinant MUC1-MBP anti-tumor vaccine at either a single dose or 3 human test doses once per week for 4 times, followed by a 4-week recovery period. The experimental animals were allocated to investigate general toxicity, to assess anti-MUC1 specific Ig G antibody production and NK cells ratio, and to monitor the distribution of T lymphocyte subsets. The satellite animals were allocated to test cytokine production by spleen lymphocytes.1. The effects of repeat-dose recombinant MUC1-MBP anti-tumor vaccine was s.c. in SD rats onthe general conditionof these ratsDuring the experiment, there were no dead or dying rats in any of the groups during the experiment. There were no abnormalities of the rats in the control group. Several rats in the BCG control group(4/30), low-dose group(2/30) and high-dose group(1/30) exhibited abnormal symptoms of swelling around the ankles. In addition, after the first immunization, the rats in the BCG control and high-dose groups showed obvious callosity and escharosis at their injection sites, and as additional immunizations were adminstered over time, many additional rats in the BCG control(30/30), low-dose(19/30) and high-dose(27/30) groups appeared with the same symptoms by the end of the recovery period. These result indicating that recombinant MUC1-MBP anti-tumor vaccine2. The effects of repeat-dose recombinant MUC1-MBP anti-tumor vaccine was s.c. in SD rats on the body weight, food consumption and temperature of these ratsThe results showed that exposing the rats to repeat-dose of MUC1-MBP anti-tumor vaccine did not show any significant differernces in body weight, food consumption or temperature between the experimental and control groups(P> 0.05).3. The effects of repeat-dose recombinant MUC1-MBP anti-tumor vaccine was s.c. in SD rats on the ophthalmic features of these ratsThere were no abnormalities of eyelids, conjunctiva, cornea, iris, pupil, lens, retina, sclera, vitreous and retinal of those rats in all the groups.4. The effects on the of repeat-dose recombinant MUC1-MBP anti-tumor vaccine was s.c. to SD rats on the haematological index of these rats(1) Blood cell countThreedays after the first immunization, the white blood cell(WBC), neutrophil(Neut), and monocyte(Mono) levels in BCG control group and both low- and high-dose groups increased, and the WBC, Neut and Mono levels were higher while the blood cell count(RBC), hemoglobin concentration(HGB), and hematocrit(HCT) levelswere lower at 3 days after the last immunization; the WBC level did not return to the original level by the end of the recovery period.All these result indicating that recombinant MUC1-MBP anti-tumor vaccine induces the immune response.(2) Blood coagulation functionThe blood coagulation function indexes in all groups fluctuated within reference ranges. Although PT in the BCG control, low-dose and high-dose groups was extended compared with the negative control group(P<0.05), and APTT in the male rats in the BCG control group was shortened compared to the negative control group(P<0.05) on day 31, there was no toxicological significance because of a lack of correlation between changes in PT or APTT and vaccine administration.(3) Clinical chemistry analysisThe clinical chemistry analysis showed that both albumin(ALb) and albumin/globulin ratio(A/G) decreased in the BCG control, low-dose and high-dose groups when detected on day 31 and returned to original levels by the end of the recovery period. Whereas, changes in glucose(Glu), phosphorus(P) and total bilirubin(TBil) varied within a normal range in several rats at some time points, but these changes were not dose- or time-related and therefore were not considered biologically significant.5. The effects of repeat-doserecombinant MUC1-MBP anti-tumor vaccine was s.c. to SD rats on the organs of these ratsThe results showed that the absolute and relative weights of the spleens in the BCG control, low-dose and high-dose groups increased by 25.8%/64.3%, 13.5%/42.2%, and 51.8%/75.8% on day 31, and the weight of spleen in the high-dose group was higher than BCG control and low-dose groups. There was an significant difference of the weight of spleen in the high-dose group compared to BCG control or low-dose groups at the end of the recovery period. 3 days after the last immunization and at the end of the recovery, there were no obvious gross anatomy and the microscopic observation systemic toxicity of pathological changes associated with drug delivery. The increasing of spleen weight(absolute weight and relative weight), spleen red pulp medullary hematopoiesis, and the bone marrow granulocyte/red blood cells rate are associated with inflammatory reaction caused by BCG.6. The immunogenicity analysis by s.c. with repeat-dose MUC1-MBP anti-tumor vaccine to SD rats(1) T lymphocyte subsets and NK cellsThe results showed that MUC1-MBP treatment did not change the distribution of T lymphocyte subsets and NK cells compared with the control group in rats(P>0.05).(2) MUC1-specific Ig G antibodies in the serumThe results showed that there were no specific Ig G antibodies in the negative and BCG control groups, while all of the rats in the low-dose and high-dose groups produced detectable levels of MUC1-specific Ig G antibodies, and the highest antibody titers of the two groups reached 1:1600 without any obvious difference. As the immunization period increased, antibody titers also increased, reaching a peak(1:6400) on day 14. By the end of the recovery period, the positive rate and levels of MUC1-specific Ig G antibodies were decreased, and the levels in the low-dose group were much lower. These results indicating that recombinant MUC1-MBP anti-tumor vaccine induces MUC1-specfic humoral immune response in rats.(3) The cytokines secreted by spleen lymphocytesAt the end of the 4-week recovery period, there was a significant increase in IFN-? and TNF-? secretionin the low- and high-dose groups, and a decrease in IL-4 secretion, suggesting that the recombinant MUC1-MBP anti-tumor vaccine induced Th1 cell immune response. Pilot general toxicity and immunogenicity study of MUC1-MBP anti-tumor vaccine in cynomolgus monkeysTo evaluate the toxicity and immunogenicity of the recombinant MUC1-MBP anti-tumor vaccine on cynomolgus monkeys, five monkeys were randomized into an experimental group(2 males and 1 female) and a control group(1 male and 1 female). The experimental group received s.c. immunizations of recombinant MUC1-MBP anti-tumor vaccine at a single human test dose once per week for 4 times, followed by an 8-week recovery period. The control group did not receive immunization and was subjected to blood collection for the following test at the same time point as the experimental group. During the experiment time, the general condition, body weight, lymphocyte subsets and serum MUC1-specific antibodies of these animals were detected.1. The effects of repeat-dose MUC1-MBP anti-tumor vaccine was s.c. in cynomolgus monkeys on the general condition of these cynomolgus monkeysThe results showed that there were no dead or dying rats in any of the groups during the experiment. One week after the first immunization, 3 cynomolgus monkeys exhibited the evidence of erythema or induration at the injection sites, which was caused by BCG.2. The effects of repeat-dose MUC1-MBP anti-tumor vaccine was s.c. in cynomolgus monkeys on the body weights of these cynomolgus monkeysThe results showed that there were no abnormalities in body weight that associated with MUC1-MBP anti-tumor vaccine, except for a little fluctuation within a narrow range.3. The effects of repeat-dose MUC1-MBP anti-tumor vaccine was s.c. in cynomolgus monkeys on the lymphocyte subsets of these cynomolgus monkeysThe results showed that MUC1-MBP treatment did not change the percentages of CD3+T, CD3+CD4+T, or CD3+CD8+T cells in cynomolgus monkeys, indicating that the MUC1-MBP anti-tumor vaccine did not affect the distribution of T lymphocyte subsets in cynomolgus monkeys.4. The effects of repeat-dose MUC1-MBP anti-tumor vaccine was s.c. in cynomolgus monkeys on the serum MUC1-specific antibodies of these cynomolgus monkeysThe specific Ig G and Ig G1 antibodies in the experimental group was detected on day 8, and antibody titers increased as the immunization period increased, reaching a peak(1:3200; 1:6400) on day 29. Eight weeks after the fourth immunization, the Ig G antibody titer decreased to 26% of the titer peak. Moreover, Ig G1 first appeared and was the main Ig G antibody found, while Ig G2, Ig G3 and Ig G4 appeared on day 11, and the titers of these subtype antibodies were lower than that of Ig G1. These results indicating that recombinant MUC1-MBP anti-tumor vaccine induces the specific humoral immune response; the production of Ig G2 and Ig G3 antibodies indicating that a specific cellular immune response may be induced.Conclusion: The data from these studies indicate that treatment with the MUC1-MBP anti-tumor vaccine causes no organ toxicity, except for the presence of arthritis or local nodules induced by BCG in serveral rats. Furthermore, MUC1-MBP anti-tumor vaccine significantly induces the secretion of IFN-?, suggesting that Th1 cellular immune response are activated. MUC1-MBP anti-tumor vaccine induces MUC1-specific Ig G antibodies or Ig G1, Ig G2, Ig G3, and Ig G4 in rats and cynomolgus monkeys, indicating that recombinant MUC1-MBP anti-tumor vaccine not only inducesa specific,but also induces anti-MUC1 specific humoral immune response.In summary, all the results are beneficial to move the recombinant MUC1-MBP/BCG anti-tumor vaccine into a Phase I clinical trial. |
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