TWI670280B - Lipidated antigen of human papillomavirus and immunotherapeutic composition against hpv associated diseases - Google Patents

Abstract

The present invention relates to a lipidated recombinant fusion protein comprising a lipidated sequence derived from a natural lipoprotein, such as an N-terminal portion of Ag473, and a tumor associated antigen, such as an inactivated human papillomavirus oncoprotein E6/ E7 (E6mE7m), and the tumor-associated antigen is located downstream of the lipidation sequence; the invention further provides an immunotherapeutic composition and method thereof, which are capable of inducing toxic T lymphocytes to protect mammals from cancer diseases (eg HPV) Invasion of related tumors).

Description

Lipid antigen of human papillomavirus and its use in human papillomavirus-related diseases Epidemic treatment composition

The present invention relates to a lipidated recombinant fusion protein comprising a lipidated sequence and an inactivated tumor-associated antigen of human papillomavirus, which can activate dendritic cells and helper T lymphocytes to produce immunity. The reaction, in turn, protects mammals from human papillomavirus (HPV)-related diseases, including cancer.

Human papillomavirus (HPV) infection has been shown to be associated with the development of many cancers, particularly cervical cancer, which is the world's second-largest type of female lethal cancer and has established its pathogen HPV (Schwarz, TF, Expert Rev Vaccines 7 : 1465-73, 2008), a 2002 study estimated that there will be 493,000 cases of cervical cancer each year (Parkin, DM et al., CA Cancer J Clin 55 : 74-108, 2005), in cervical cancer In the case of the case, 50% of HPV16 were detected, and 18% of cases of cervical cancer were associated with HPV18; in adenocarcinoma and adenosquamous-carcinoma, HPV infection rate (76.4%) was significant. Less than squamous cell carcinoma (87.3%), and HPV18 is the main type (about 40%) (Clifford, GM et al., Br J Cancer 89 : 101-5, 2003), therefore, most of them are used to control the cervix Cancer treatment strategies focus more on HPV16 (Kanodia, S. et al., Int J Cancer 122 :247-59, 2008).

Several clinical trials have been conducted for the treatment of HPV-associated tumors. Since E6 and E7 oncoproteins are persistently present in precancerous lesions and malignancies, most of the vaccine is based on the oncogenic protein E7 or E7. /E6 is designed. US Patent No. 8,658,176 discloses an isolated fusion protein comprising a first lipidation sequence, and the first lipidation sequence comprises at least 40 amino acid residues (D1) at the N-terminus of Ag473 and The second inactivated HPV oncoprotein E7 (E7m), which protects the mouse against the inoculated tumor cells. In order to provide immunotherapy for HPV-related diseases, the present invention has developed a lipidated recombinant protein lipo-E6mE7m produced in an E. coli expression system.

The present invention is based on the discovery that the lipidated recombinant protein E6mE7m (rlipo-E6mE7m) is capable of inducing poisonous T lymphocytes and thereby responding to HPV-associated tumors to protect mice from HPV-associated tumors.

Accordingly, the present invention, in one aspect, discloses a recombinant fusion protein comprising: a first fragment comprising a lipidated sequence; and a second fragment comprising a different genotype of human papillomavirus (HPV) a sequence of inactivated oncoproteins E6 and E7 (E6mE7m); wherein the first fragment is located at the N-terminus of the second fragment.

In some embodiments of the invention, the second fragment has the sequence of the HPV oncoprotein E6mE7m. In another embodiment of the present invention, the lipidated sequence of the lipidated recombinant protein is derived from a native lipoprotein leader sequence, and the leader sequence is derived from a lipid of N. meningococcal group B. protein.

In a particular embodiment of the invention, the lipidation leader sequence is derived from lipoprotein Ag473. In one embodiment, the lipidation leader sequence comprises at least 40 amine groups in the N-terminus (D1 domain) of lipoprotein Ag473. Acid residue.

In another aspect, the invention features a method of preparing a lipidated recombinant fusion protein E6mE7m (rlipo-E6mE7m), the method comprising the steps of: (1) providing a host E. coli cell, the host E. coli cell line An expression plastid comprising a first nucleotide sequence encoding one of a lipidated sequence, and a second nucleotide sequence encoding one of the inactivated oncoprotein (E6mE7m) proteins; and (2) culturing The transgenic strain of Escherichia coli expresses a fusion protein comprising the lipidated sequence and the E6mE7m protein sequence; in one embodiment, the N-terminal to C-terminal of the lipidated fusion protein comprises a lipidated sequence A23 (MKKLLIAAMMAAALAACSQEAKQEVKEAVQAVESDVKDTA SEQ ID No. 1) and the inactivated oncoprotein E6mE7m (SEQ ID No. 2) of HPV16, the fusion protein isolated from the E. coli cells, whose lipid state can be verified by the prior art.

The lipid fusion protein rlipo-E6mE7m produced by the method is used for inducing an immune reaction in an animal without adding an adjuvant, and the lipidated fusion protein can also be prepared as a drug for immunomodulation.

In another aspect, the present invention relates to an immunotherapeutic composition for human cancer diseases caused by HPV, the immunotherapeutic composition comprising a lipidated recombinant fusion protein, A lipidated sequence derived from one of the native lipoproteins and one of the HPV antigens located downstream of the lipidated sequence.

In one embodiment of the invention, the HPV antigen is derived from one of human glandular viruses (HPV) of different genotypes, and the inactive oncoproteins E6 and E7 are bound to each other.

Other features or advantages of the present invention will be more fully understood from the following description of the appended claims.

Fig. 1 is an amino acid sequence of the HPV16 oncoprotein E6mE7m of the present invention, and the E6 protein and E7 protein are not carcinogenic.

Fig. 2 is a schematic diagram showing the construction of the E6mE7m gene of the present invention in the pET-22b vector to express the recombinant proteins rE6mE7 and rlipoE6mE7m; the second layer is the plastid map of the vector pET-22b; the second layer is constructed by the A23E6mE7m The plastid map of the vector pET-22b.

Fig. 3 is a representation and purification of the lipidated recombinant protein rE6mE7m of the present invention; the first column is a lipidated recombinant protein rE6mE7m induced by isopropyl β-D-thiogalactoside (IPTG); The column was not expressed by IPTG to express the lipidated recombinant protein rE6mE7m; the third column was the isolated lipidated recombinant protein rE6mE7m; the fourth column was the purified lipidated recombinant protein rE6mE7m; the 3A and 3B lines were respectively twelve Sodium sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting analysis results; The recombinant protein rE6mE7m was expressed in E. coli strain BL21(DE3)star strain, wherein the arrow indicates the position of the recombinant protein rE6mE7m at the electrophoresis gel.

Figure 4: The performance and purification of the lipidated recombinant protein rlipo-rE6mE7m of the present invention. The first column is the lipidated recombinant protein rlipo-rE6mE7m induced by IPTG; the second column is not lipidated by IPTG induction. The recombinant protein rlipo-rE6mE7m; the third column is the isolated lipidated recombinant protein rlipo-rE6mE7m; the fourth column is the purified lipidated recombinant protein rlipo-rE6mE7m; the 4A and 4B lines are analyzed by SDS-PAGE and Western blotting, respectively. The results showed that the lipoprotein recombinant rlipo-rE6mE7m was expressed by E. coli BL21 (C43) strain, and the arrow indicated that the lipidated recombinant protein rlipo-rE6mE7m was in the position of the electrophoresis gel.

Figure 5: The recombinant protein of the present invention is confirmed by an antibody specific for HPV16 oncoprotein E7; the 1-4 column is a recombinant protein rE6mE7m; and the fifth column is a lipidated recombinant protein rlipo-rE6mE7m; The first and fifth columns are recombinant proteins expressed after IPTG induction, the second and sixth columns are recombinant proteins that are not expressed under the induction of IPTG, and the third and seventh columns are isolated recombinant proteins, 4th and Column 8 is the purified recombinant protein.

Fig. 6 is a mass spectrometry analysis of the lipidated recombinant protein rlipo-rE6mE7m of the present invention; the lipoprotein recombinant rlipo-rE6mE7m is subjected to trypsin splicing to obtain an N-terminal fragment, followed by mass spectrometry (Waters ^® MALDI) Micro MX ^TM ) was analyzed and analyzed to obtain three sets of peaks with mass-to-charge ratios (m/z) of 1453, 1467 and 1480, respectively.

Figure 7 is a diagram showing the results of dendritic cells activated by the lipidated recombinant protein rlipo-rE6mE7m of the present invention; mouse bone marrow-derived dendritic cells obtained from wild-type mice (Bone marrow-derived dendritic) cells, BM-DCs) in the culture medium, and each additional recombinant lipidated protein rlipo-rE6mE7m 0.01 μ g / ml of lipopolysaccharide (LPS), 10 μ g / ml of recombinant protein rE6mE7m or 10 μ g / ml, the of the experimental group were more or without added 20 μ g / ml polymyxin much B (polymyxin B), after 24 hours of incubation by flow cytometry analysis based dendritic cell surface marker protein CD40 and CD80, experimental Three replicates were performed, and mouse bone marrow-derived dendritic cells were cultured in a medium without the addition of the aforementioned lipopolysaccharide, recombinant protein, and polymyxin B as a control group, and the mean value (MFI) of the fluorescence density was calculated.

Figure 8 is a graph showing the results of secretion of tumor necrosis factor- α (TNF- α ) by dendritic cells stimulated by the lipidated recombinant protein rlipo-rE6mE7m of the present invention.

Fig. 9 is a graph showing the results of the TRP-proliferating protein rlipo-rE6mE7m and the recombinant protein rE6mE7m inducing T cell proliferation in mice after induction of an immune reaction, and the results show that the lipidated recombinant protein rlipo-rE6mE7m is compared. The recombinant protein rE6mE7m has a better ability to induce T cell proliferation, and the results of the experiment are shown by mean ± standard deviation (means ± SD) after three repetitions.

Figure 10: Department of lipidated recombinant proteins thereof of the present invention and the recombinant protein rlipo-rE6mE7m rE6mE7m respectively eliciting an immune response in mice induced by cytokines (cytokines) secretion after the results shown in FIG, 10 μ g of recombinant lipidated protein rlipo-rE6mE7m rE6mE7m based or recombinant proteins were injected into mice subcutaneously twice at two weeks interval between the two injections and at seven days after the second injection of mouse spleen cells and obtaining respectively 10 μ g / ml of lipidated The recombinant protein rlipo-rE6mE7m or recombinant protein rE6mE7m was stimulated for four to five days, and then the supernatant was taken and analyzed by interferon-γ (IFN-γ) and interleukin-5 (IL-5) by enzyme immunoassay (ELISA). , the content of interleukin-10 (IL-10) or interleukin-17 (IL-17), the experimental system showed the mean ± standard deviation (means ± SD) of three groups of samples (n = 3) result.

Figure 11 is a diagram showing the results of the lipid-producing recombinant protein rlipo-rE6mE7m and the recombinant protein rE6mE7m of the present invention, which induce E7-specific IFN-γ-secreting cells after induction of an immune response in mice, and the results show that lipidation recombination The protein rlipo-rE6mE7m has a better ability to induce E7-specific IFN-γ secreting cells than the recombinant protein rE6mE7m.

Fig. 12 is a diagram showing the results of the production of E6-specific IFN-γ-secreting cells by the lipidated recombinant protein rlipo-rE6mE7m of the present invention after induction of an immune response in mice, with 2*10 ⁵ cells/well of mouse spleen cells Cultured in 96-well plate (ELISPOT plate) coated with identifiable IFN-γ, with or without HPV16 E6 peptide (KQQLLRREVYDFAFRDLCIVYRDGN; abbreviated as KQQ) or (EVYDFAFRDL; EVY for short) The cells were cultured for 48 hours, and then the IFN-γ secreting spots were measured by an ELISPOT reader. Each experimental group was tested in three animals, and the mean value of the three animals was measured ± standard deviation. (means±SD) shows the result.

Fig. 13 is a graph showing the comparison of the lipidated recombinant protein rlipo-rE6mE7m and the recombinant protein rE6mE7m of the present invention for inhibiting tumor cell growth; each mouse was subcutaneously inoculated into mice with 2*10 ⁵ TC-1 cells, each and each group comprises 5 to 10 mice, the tumor-bearing mice each were injected with cells after seven days of RAH 10 μ g / IFA, rE6mE7m 30 μ g's, 30 μ g of phosphoric acid or rlipo-E6mE7m Buffer solution (PBS), tumor volume is shown as the mean ± standard error of the mean ± mean of the tumor length * length * width / 2 (mm ³ ).

Disclosed herein is a method for producing a lipidated recombinant fusion protein rlipo-E6mE7m in Escherichia coli, the lipidated recombinant fusion protein comprising a lipidated sequence and a polypeptide of the inactivated HPV16 oncoprotein E6 and E7, the lipidated sequence It can be derived from Ag473 and is located at the N-terminus of the recombinant fusion protein.

This fusion sequence has been disclosed in other literature (Chen, HW et al. Vaccine 27: 1400-9, 2009); the technical term "lipidation sequence" as used herein refers to a non-naturally occurring amino acid sequence, which comprises There is (a) a first fragment which is at least 80% (including 85%, 90%, 95% or 99%) similarity to Ag473's signal peptide (SP), and a second fragment, It has at least 80% (including 85%, 90%, 95% or 99%) similarity to Domain 1 of Ag473, the first fragment is located at the N-terminus of the lipidation sequence, and (b) a multi-peptide having a lipidation sequence at the N-terminus which promotes a lipidation reaction in E. coli; in the lipidation sequence, the first fragment is directly linked to the second fragment or passed through a peptide and The second fragment is ligated; preferably, the lipidation sequence has a longest length of 40 to 100 amino acids (eg, 40 to 80 amino acids; in one embodiment, the lipidation sequence described herein) Contains the signal peptide of Ag473 and the sequence of domain 1.

The "% homology" of the amino acid sequence as described herein is based on the algorithm disclosed in Karlin and Altschul, Proc, Natl. Acad. Sci. USA 87: 2264-2268, 1990. The algorithm is disclosed in Karlin and Altschul, Proc, Natl. Acad. Sci. USA 90:5873-5877, 1993. The algorithm is applied to Altschul et al., J. Mol. Biol. 215: 403-410, 1990. NBLAST and XBLAST alignment programs disclosed; wherein the XBLAST alignment program is used for BLAST protein search with a score of 50 and a length of 3 to obtain a reference peptide a homologous amino acid sequence; the literature of Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997. The Gapped BLAST system is used to obtain gapped alignments for comparison. When performing BLAST and Gapped BLAST programs, the default parameters of each program are used for comparison (for example, XBLAST and NBLAST), which can be found at www.ncbi.nlm.nih.gov .

In the present invention, the lipidation sequence is linked to a viral antigen derived from human papillomavirus (HPV), such as HPV16, to form a fusion protein, and when the fusion protein is expressed in Escherichia coli by using existing recombinant technology, The lipidation pattern is shown; the following is an example: a DNA fragment encoding a lipid sequence and a DNA fragment encoding E6m and E7m (inactivated HPV oncoproteins E6 and E7) are inserted into an expression. The vector is constructed to form an expression plastid. Preferably, the expression vector carries a strong promoter (for example, T7, T5, T3 or SP6), and the strong promoter can be inducible, for example, isopropyl. The β-D-thiogalactoside (IPTG) was induced.

Next, the expression plastid is transfected into an E. coli host strain, and the transgenic strain carrying the expression plastid is cultured under suitable culture conditions for protein expression; preferably, the E. coli host strain is Exogenous protein over-expression The toxicity induced by the time is resistant, and such E. coli strains can be produced and confirmed by the method disclosed in U.S. Patent No. 6,361,966. For example, such E. coli strains may comprise C43 (DE3) (ECCC B96070445), C41 ( DE3) (ECCC B96070444), CO214 (DE3), DK8 (DE3) S (NCIMB 40885) and C2014 (DE3) (NCIMB 40884), but not limited thereto.

Preferably, after the fusion protein expressed by the E. coli host cell is isolated, the lipid state can be confirmed by techniques well known in the art, for example, immunoblotting using an anti-lipoprotein antibody or Analysis by mass spectrometer.

The lipidated fusion protein may be mixed with a pharmacologically suitable carrier such as monophosphate buffered saline (PBS), bicarbonate solution or adjuvant to form a pharmaceutical composition; the so-called suitable carrier means The carrier must be compatible with the active ingredient in the composition. Preferably, the carrier is stable to the active ingredient and is not deleterious to the agent; the carrier is made according to the form and route of administration and standard pharmaceutical practice Selection, suitable pharmaceutical carriers, diluents, and pharmaceutical necessities for use thereof are disclosed in Remington's Pharmaceutical Sciences.

In one embodiment, the fusion protein is mixed with an adjuvant to form a composition that can be used for immunomodulation; as described in US Pat. Or an emulsion as an injection, for example, the adjuvant may comprise cholera toxin, E. coli heat-labile enterotoxin, liposome, immunostimulating complex (ISCOM), immunostimulatory sequences oligodeoxynucleotide and aluminum hydroxide; such as Audran R. et al. Vaccine 21:1250-5, 2003; and Denis-Mize et al. Cell Immunol. , 225: 12-20, 2003. The composition may also comprise a polymer that promotes in vivo delivery; in addition, the rlipo-E6mE7m fusion protein disclosed herein may be used in existing HPV vaccines without Additional adjuvant is added.

Any of the above pharmaceutical compositions can be administered parenterally, for example, by subcutaneous injection or intramuscular injection, and other forms of administration, including suppositories and oral preparations, can also be administered; in the case of suppositories, Containing a binder and a carrier, for example: polyalkalene glycols or triglycerides; and oral preparations may contain conventional excipients such as pharmaceutical grade saccharin, cellulose or magnesium carbonate. Etc. These pharmaceutical compositions may be in the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders.

Based on the above description, those skilled in the art can expand the scope of the present invention from the technical features disclosed in the present invention; the following description will be made in the specific embodiments, and not to limit other embodiments not illustrated in the present invention; In addition, the publications cited in this article are used as literature.

The following examples are the expression and characteristics of a fusion protein lipo-Em6E7m, the sequence of which is shown in Figure 1 and the SEQ ID No. 3 of the Sequence Listing, which comprises a lipidation sequence derived from Ag473. And inactivated HPV16 oncoproteins E6 and E7 and Em6E7m.

Example

Cloning and expression of recombinant proteins rE6mE7m and rlipo-E6mE7m

The synthetic nucleotide sequence of the HPV16 E6mE7m or HPV16 A23E6mE7m gene in the pBR322 vector was purchased from Invitrogen (CA, USA), and the Nde I and Xho I restriction enzyme sites were located at the 5' end and the 3' end of the nucleotide sequence, respectively; The plastids were purified and cloned into the expression vector pET-22b(+) (Novagen, Madison, WI) to produce plastids pET-22b E6mE7m and pET-22b A23E6mE7m; and six groups attached to the C-terminus of the recombinant protein. The plastid map of the amino acid tag (HisTag); pET-22b E6mE7m and pET-22b A23E6mE7m is shown in Fig. 2.

The expressed plastid pET-22b E6mE7m line was transfected into E. coli strain BL21Star (DE3) (Invitrogen, USA) to express rE6mE7m, and the transgenic strain was cultured in LB medium at 37 ° C, followed by addition of 0.1 mM (mM). The IPTG was induced, and the cells were harvested at a cell concentration of OD=0.6, and resuspended in 20 mM Tris-Cl, 500 mM sodium chloride (NaCl), 50 mM sucrose. And the pH value is in a buffer of pH 8.0 for subsequent purification reaction.

The expressed plastid pET-22b A23E6mE7m was transfected into E. coli strain CD43 (DE3) (Chen HW, Liu SJ, Liu HH, et al. Vaccine 2009; 27: 1400-9) to express rlipo-E6mE7m, a transgenic strain. The cells were cultured in M9 medium at 37 ° C, followed by the addition of 1 mM IPTG and induced at 12 ° C. After three days, the cells were harvested and resuspended in a buffer containing 20 mM Tris-Cl and having a pH of 8.9. A subsequent purification reaction is carried out.

Purification of recombinant protein

The recombinant protein rE6mE7m was purified by the following procedure: The collected cells were dissolved in a homogenization buffer (500 mM NaCl; 50 mM sucrose; 20 mM Tris-HCl; pH 8.0) and disrupted by French cell at a pressure of 27 Kpsi. The machine (French Press, Constant Systems, Daventry, UK) was sterilized, and then the cell lysate was centrifuged (80,000 × g; 40 minutes), followed by urea buffer (8 M urea; 50 mM sucrose; 20 mM Tris-HCl; PH8) .0) Dissolve the deposit and homogenize it with a Dounce homogenizer, then centrifuge the mixture again (80,000 x g; 40 minutes), followed by GdnHCl buffer (6M GdnHCl; 0.1% mannitol; 20 mM) The recombinant protein rE6mE7m was extracted by Tris-HCl; pH 8.0. After centrifugation, the supernatant was statically placed in a 5 ml Ni-NTA resin column (1.6 cm id × 2.5 cm), which was firstly 50 mM imidazole. (imidazole) extraction buffer washing, followed by washing with 0.1% Triton X-114 buffer to remove lipopolysaccharide (LPS), and finally eluting the recombinant protein rE6mE7m with 3M GdNHCl extraction buffer containing 500 mM imidazole, and then 1.8 mM Dialysis was carried out with potassium dihydrogen phosphate (KH ₂ PO ₄ ) (pH 2.7).

The expression and purification of the recombinant protein rE6mE7m were analyzed by SDS-PAGE and Western blot. The anti-Histidine was used in Western blot. (anti-His) antibody (Santa) and mouse anti-Human Papilloma virus 16 E7 (VALDOSPAN GmbH, cat: VS13004) detected the recombinant protein rE6mE7m, and the results are shown in Fig. 3 and Fig. 5. It is shown that it obtains the high-purity recombinant protein rE6mE7m; in addition, lipopolysaccharide (LPS) is further removed by immobilized metal affinity chromatography (IMAC) coupled with copper ions, and washed with 1000 mL of elution buffer and 300 mL. The buffer (100 mM imidazole; 1% Triton X-100; 50 mM Tris; pH 8.9) was washed thoroughly to obtain a recombinant protein solution having a lipopolysaccharide content of less than 0.03 EU/mg.

The lipidated recombinant protein rlipoE6mE7m was purified by the following procedure, and the collected cells were dissolved in a homogenization buffer (0.1% Triton X-100; 20 mM Tris-HCl; pH 8.9) and passed through a pressure of 27 Kpsi. The cells were sterilized by French Press (Constant Systems, Daventry, UK), followed by centrifugation of the cell lysate (80,000 x g; 40 minutes) with urea buffer (8 M urea; 50 mM sucrose; 20 mM Tris- HCl; pH 8.0) The deposit was dissolved and homogenized by a Dounce homogenizer, and the mixture was again centrifuged (80,000 x g; 40 minutes), followed by GdnHCl buffer (6M GdnHCl; 0.1% Triton X) -100; 20 mM β-mercaptoethanol; 20 mM Tris-HCl; pH 8.9) The lipidated recombinant protein rlipoE6mE7m was extracted, and after centrifugation, the supernatant was statically placed on a 5 ml Ni-NTA resin column (1.6 cm id x 2.5). In cm), the column was first washed with 20 mM imidazole extraction buffer, followed by 0.1% Triton X-114 buffer to remove lipopolysaccharide, and finally with 20 mM sodium hydrogen phosphate containing 500 mM imidazole ( na ₂ HPO ₄₎ eluting the recombinant lipidated protein rlipoE6mE7m, 20mM of potassium phosphate and then (PH2.7) dialysis.

Then the expression and purification of the lipoprotein recombinant rlipoE6mE7m were analyzed by SDS-PAGE and Western blot. In Western blot, anti-His antibody (Santa) and anti-human papilloma virus 16E7 were small. The mouse anti-Human Papilloma virus 16 E7 (VALDOSPAN GmbH, cat: VS13004) detected the recombinant protein rE6mE7m, and as shown in Fig. 4 and Fig. 5, it obtained a high-purity lipidated recombinant protein rlipoE6mE7m; Further removing lipopolysaccharide (LPS) by immobilized metal affinity chromatography (IMAC) coupled with copper ions, and using 1000 mL of elution buffer and 300 mL of washing buffer (100 mM imidazole; 1% Triton X-100) 50 mM Tris; pH 8.9) was washed thoroughly to obtain a recombinant protein solution having a lipopolysaccharide content of less than 0.03 EU/mg.

Qualitative of recombinant protein

To identify the N-terminal fragment of the lipidated recombinant fusion protein rlipo-E6mE7m, it was dialyzed against 5 mM ammonium bicarbonate at pH 8.5, followed by cutting rlipo-E6mE7m with trypsin (Promega Co., Madison, WI). Wherein the ratio of rlipo-E6mE7m to trypsin was 50:1 (Wt/Wt), and after reacting for 5 minutes at room temperature with 25 mM ammonium hydrogencarbonate (pH 8.5), a final concentration of 1.2% formic acid was added ( Formic acid) terminates the digestion reaction, and then removes the salt in the reaction mixture with Ziptip (Millipore), and each 1 μl (μl) of trypsin cleavage protein and 1 μl of the saturated solution α -cyano-4- Alpha-ciano-4-hydrozycinnamic acid (Sigma) was mixed, and the saturated solution was dissolved in acetonitrile/0.1% trifluoroacetic acid (1:3 (vol/vol)). In the mixed solution, 1 μl of the mixture was placed in a sample disk of a mass-assisted laser desorption ionization time-of-flight (MALDI-TOF; Bruker), and the result obtained by MALDI-TOF analysis was as above. Said that the trypsin-cleaved line corresponds to the N-terminal fragment of rlipo-E6mE7m, and these peptides are lipidated. Peptide (see Figure 6).

Activation of bone marrow-derived dendritic cells (BMDC) via rlipo-E6mE7m

The BMDCs obtained from C57BL/6 mice were evaluated by the method described previously (Chen HW, Liu SJ, Liu HH, et al. Vaccine 2009; 27: 1400-9); in short, mouse bone marrow cells were per ml 2 × 10 ⁶ cells were cultured in a density of 10ml complete medium (CM) of the culture dish, the complete medium was added with the 10% FCS RPMI 1640,1% of the P / S, 50 μ M of 2 -ME, 2 mM glutamine (Glutamine), 25 mM HEPES, 1 mM sodium pyruvate, and 20 ng/ml of recombinant mouse granule mononuclear ball stimulating growth factor (GM-CSF), followed by On the third day after the culture, 10 ml of CM medium containing 20 ng/ml of GM-CSF was additionally added, and on the 6th day after the culture, non-adherent cells were collected and transferred to a 24-well plate, followed by 1*10 ⁶ cells. / BMDC ml of lines at 0.1 μ g / ml of LPS or as shown in FIG. 7 or concentrations rE6mE7m rlipo-E6mE7m 24 hours of stimulation, supernatants were collected and this time point from the different conditions of culture, the supernatant Stored at -20 °C for cytokine analysis; then analyzed by cell flow cytometry (FACSCalibur, BD bioscience, San Jose, CA) for cell surface marker protein (CD40) CD80), anti-mouse anti-mouse CD80 antibody (BD) with PE fluorescence and anti-mouse CD40 antibody (PE anti-mouse CD40) with PE fluorescence (BD) As shown in Figure 7, rlipo-E6mE7m up-regulated the co-stimulatory molecule of dendritic cells.

Next, the cytokine (TNF- α ) produced by BMDC was measured using an ELISA kit (Mouse TNF- α ELISA Ready-SET-Go!, eBioscience), and the BMDCs were cultured in the following culture conditions: (1) 0.01 μ g / ml of LPS; (2) plus 0.625-10 μ g / rE6mE7m ml; and (3) plus 0.625-10 μ g / rlipo-E6mE7m ml of the aforementioned culture conditions further divided into three groups added 20 μ g / ml polymyxin B as much or without 20 μ g / ml polymyxin B as much as two groups, after 24 hours of incubation, supernatants were collected and analyzed through ELISA TNF- α production amount; as a result of As shown in Figure 8, rlipo-E6mE7m does stimulate dendritic cells to secrete TNF- α .

Lymphocyte proliferation test

Spleen and lymph nodes from C57BL / 6 mice to obtain the lymphocytes, based seeded at a density of 2 * 10 ⁵ cells / well in a flat bottom 96 well plate, respectively, 10 μ g / mL of rE6mE7m stimulate lymphocytes or rlipo-E6mE7m And cultured in a wet incubator at 37 ° C and 5% carbon dioxide for 48 hours. In the last 24 hours of culture, 1 millicurie (mCi) of [3H]-thymidine was added to each well. 5 mCi/mmol; Amersham, Arlington Heights. IL), and the plate was again placed overnight in a humidified environment at 37 ° C and CO ₂ , and then the supernatant was aspirated and an automated cell harvester (FilterMate, Packard, Meriden, CT) cells were harvested, and then the radioactivity of the cells was determined using a microplate scintillation counter (TopCount, Packard, Meriden, CT); all results were averaged ± standard deviations per minute (mean cpm ± standard) Deviation (SD)) to indicate.

T cell response test

Lipidated recombinant protein or recombinant protein rlipo-rE6mE7 rE6mE7 lines of 10 μ g were injected subcutaneously into C57 / BL6 mice twice, two weeks apart, and between the two injections at seven days after the second injection the mice from the spleen cells and lymph node acquired lymphocytes, respectively, and then to 10 μ g / lipidated recombinant protein or recombinant protein rlipo-rE6mE7 rE6mE7 ml four to five days of stimulation, the supernatant was then whichever enzyme immunoassay (ELISA Analysis of interferon-γ (IFN-γ), interleukin-5 (IL-5), interleukin-10 (IL-10) or interleukin-17 (IL-17); IFN- The γ-enzyme-linked immunosorbent assay (IFN-γ ELISPOT assay) analyzes the response of cells to stimulation as previously described (Huang et al PLoS One. 2012; 7(7): e40970). In short, the spleen cell line The density of 5*10 ⁵ /well was cultured in a microplate coated with IFN-γ, and the peptide derived from HPV E7 (RAHYNIVTF) and HPV16 E6 peptide (KQQLLRREVYDFAFRDLCIVYRDGN; KQQ for short) were added or not. Or (EVYDFAFRDL; EVY for short) was cultured in a final medium of 200 μl complete medium, and the cells were removed after 48 hours of culture and dissolved in PBS. .05% (w/v) Tween 20 washed microplates, followed by addition of biotin-containing anti-IFN-γ antibody to each well for 2 hours, followed by removal of the antibody solution after antibody binding reaction was completed. The diluted streptavidin-HRP solution was added to each well for 1 hour, and then the enzyme was subjected to 3-amine-9-ethylcarbazole (3). -amine-9-ethyl carbazole) (Sigma, St. Louis, MO) to form spots and count spots by ELISPOT reader (Cellular Technology Ltd.); results are average ± standard deviation (means ± SD) (sample number n = 3-6).

In vivo tumor protection experiment

The 6 to 12 weeks of female C57 / BL6 mice were inoculated subcutaneously with TC-1 cells ^{(2 * 10 5 cells / mice} ), 7 days after the tumor cells per mouse were injected with 10 μ g of RAH / IFA, 30 μ g of rE6mE7m, 30 μ g of rlipo-E6mE7m or PBS (5-10 mice for each injection condition); every two days in a two-dimensional caliper measurements of tumor length amounts to obtain the size of the tumor, and to Formula [width * width * length / 2] cubic millimeter (mm ³ ) to calculate tumor volume; according to the "National Institutes of Health Laboratory Animal Care and Use Committee Guidelines", when the tumor size reaches 2000mm ³ or the mouse loses weight is greater than the initial At 20% of body weight, the mouse is sacrificed, and the experimental method is approved by the National Institutes of Health Laboratory Animal Care and Use Committee Guidelines (license number: NHRI-IACUC-103020-A), mouse in antigen Tumor growth was recorded for 60 days after immunotherapy.

As shown in Figure 13, the mice were first immunized with the immunogen, and then the TC-1 tumor cells were inoculated into the mice; after immunization with rlipo-E6mE7m, 100% of the mice were swollen. Tumor growth was maintained within 55 days after tumor inoculation, and tumor growth was observed within 21 days in mice inoculated with PBS and rE6mE7m, respectively. These results indicated that rlipo-E6mE7m was used on the 7th day after tumor inoculation of the tumor in mice. Treatment of mice inhibited tumor growth, but treatment of mice with rE6mE7m did not inhibit tumor growth; compared with mice immunized with rE6mE7m, immunization with rlipo-E6mE7m, resistance to TC-1 in mice Cell attack provides significant protection.

Other embodiments

All the features disclosed in the present specification may be combined in any form, and each of the technical features disclosed in the specification may be replaced by alternative technical features of the same or similar purposes, and therefore, unless otherwise explicitly stated, Each feature is only one particular embodiment that is common to equivalent or similar features.

In view of the above, those skilled in the art can easily devise the necessary technical features of the present invention, and various changes and modifications can be made to the various uses and conditions without departing from the spirit and scope of the invention. Therefore, other embodiments are included in the scope of the patent application.

<110> National Institute of Health Research

<120> Lipid antigen of human papillomavirus and immunotherapy composition for human papillomavirus-related diseases

<160> 3

<170> PatentIn version 3.5

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<211> 40

<212> PRO

<213> Artificial sequence

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<223> Lipidation sequence

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<210> 2

<211> 251

<212> PRO

<213> Human papillomavirus (HPV)

<400> 2

<210> 3

<211> 291

<212> PRO

<213> Artificial sequence

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<223> rlipo-E6mE7m

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Claims (14)

An immunotherapeutic composition for human papillomavirus (HPV)-related diseases, comprising: a lipidated recombinant fusion protein comprising a lipoprotein derived from N. meningococcal group B a lipid sequence, and a sequence derived from the inactivated human papillomavirus oncoproteins E6 and E7 (E6mE7m) of different genotypes (the sequence shown in SEQ ID No. 2), the inactivated human mastoid The sequences of the viral oncoproteins E6 and E7 are located downstream of the lipidated sequence derived from the native lipoprotein. The immunotherapeutic composition according to claim 1, wherein the sequence of the inactivated human papillomavirus oncoprotein E6 and E7 is a sequence of human papillomavirus E6mE7m having the sequence shown in SEQ ID No. 2. sequence. The immunotherapeutic composition of claim 3, wherein the lipidation sequence is Ag473. A lipidated recombinant fusion protein rlipo-E6mE7m comprising: a first fragment comprising a lipidated sequence derived from a lipoprotein of N. meningococcal group B; and a second fragment, It comprises a sequence derived from the inactivated human papillomavirus oncoproteins E6 and E7 (E6mE7m) of different genotypes (SEQ ID No. 2); wherein the inactivated human papillomavirus The sequences of the oncogenic proteins E6 and E7 are located downstream of the lipidation sequence. The lipidated recombinant fusion protein of claim 4, wherein the lipidated sequence is 40 amino acid residues comprising the sequence of SEQ ID No. 1. The lipidated recombinant fusion protein according to claim 4, wherein the sequence of the inactivated human papillomavirus oncoproteins E6 and E7 consists of the sequence of human papillomavirus 16E6mE7m, having SEQ ID No. 2 The sequence shown. A method for preparing a lipidated recombinant fusion protein according to claim 4, which comprises the steps of: providing an E. coli host cell strain, and transfecting an expression plastid into the E. coli host cell strain, the expression The system consists of a first nucleotide sequence encoding a lipoprotein derived from a lipoprotein of N. meningococcal group B, encoding an inactivated human papillomavirus oncoprotein E6mE7m (for SEQ a second nucleotide sequence of one of the sequences shown in ID No. 2; and culturing the E. coli host cell strain transfected with the expression plastid to express the lipid comprising the lipidated sequence and the oncogenic protein E6mE7m Recombinant fusion protein. The preparation method according to claim 7, wherein the N-terminal to C-terminal of the lipidated recombinant fusion protein comprises a lipidated sequence Ag473 and the oncoprotein E6mE7m. A use of a lipidated recombinant fusion protein according to claim 4 for the preparation of a pharmaceutical composition for producing an immune response against a human papillomavirus antigen, wherein the lipidated recombinant fusion protein comprises a B-type Lipid sequence of lipoproteins of N. meningococcal group B, and sequence derived from inactivated human papillomavirus oncoproteins E6 and E7 of different genotypes (shown as SEQ ID No. 2) The sequence of the inactivated human papillomavirus oncoproteins E6 and E7 is located downstream of the lipidated sequence derived from the native lipoprotein. The use according to the invention of claim 9, wherein the sequence of the inactivated human papillomavirus oncoproteins E6 and E7 is a sequence of human papillomavirus E6mE7m having the sequence shown in SEQ ID No. 2. The use of claim 9, wherein the lipidated recombinant fusion protein is formulated with an adjuvant. A pharmaceutical composition for use in a human papillomavirus (HPV)-associated disease based on a lipidated recombinant fusion protein, which comprises the lipidated recombinant fusion protein rlipo-E6mE7m as described in claim 4 of the patent application. The pharmaceutical composition according to claim 12, which further comprises a pharmacologically acceptable adjuvant. The pharmaceutical composition according to claim 12, wherein the human papillomavirus (HPV)-related disease is cervical cancer.