ARTIFICIAL MULTI-ANTIGEN FUSION PROTEIN AND PREPARATION AND USE THEREOF

Abstract

Provided are an artificial multi-antigen fusion protein and a preparation method thereof. The fusion protein can effectively stimulate CD8+T and CD4+ T cell immunities, and can be applied to immunodiagnostics or serve as a prophylactic or therapeutic vaccine.

Claims

1. An artificial multi-antigen fusion protein, wherein, when administered to a mammalian subject, the artificial multi-antigen fusion protein simultaneously stimulates CD4.sup.+ and CD8.sup.+ T cell immune responses in said subject.

2. The artificial multi-antigen fusion protein of claim 1, wherein the artificial multi-antigen fusion protein comprises ≧3, preferably ≧5, more preferably ≧10 antigen segments.

3. The artificial multi-antigen fusion protein of claim 2, wherein each antigen segment is from a viral antigen, a bacterial antigen, a parasite antigen, a chlamydial antigen, a tumor antigen, or a combination thereof.

4. The artificial multi-antigen fusion protein of claim 1, wherein the artificial multi-antigen fusion protein further comprises sequences of cleavage site located between antigen segments.

5. The artificial multi-antigen fusion protein of claim 4, wherein the sequence of cleavage site comprises a cleavage site of cathepsin; and Preferably, the cleavage site of cathepsin is selected from a group consisting of a cleavage site of cathepsin S (e.g., Leu-Arg-Met-Lys or a similar cleavage site), a cleavage site of cathepsin B (e.g., Met-Lys-Arg-Leu or a similar cleavage site), a cleavage site of cathepsin K (e.g., His-Pro-Gly-Gly or a similar restriction site), and combinations thereof.

6. The artificial multi-antigen fusion protein of claim 1, wherein the fusion protein is shown in the structure of formula I:
Y-(A-C)n-Z  (I) wherein, A is an antigen segment; C is a sequence of cleavage site of cathepsin; n is a positive integer ≧3; Y is absent or is a sequence represented by “Y0-B”, wherein Y0 is a signal peptide sequence, a tag sequence, a membrane-penetrating element sequence, an adjuvant element sequence, a cell necrosis-inductive element sequence, or any combination of the above sequences, and B is absent or a sequence of cleavage site; Z is absent, or a tag sequence, a membrane-penetrating element sequence, an adjuvant element sequence, a cell necrosis-inductive element sequence, or any combination of the above sequences; provided that when Z is absent, C in the last “A-C” can be absent.

7. A composition, wherein the composition comprises the artificial multi-antigen fusion protein of claim 1 and a pharmaceutically acceptable carrier.

8. Use of the artificial multi-antigen fusion protein of claim 1 used for the preparation of a prophylactic and/or therapeutic vaccine composition or pharmaceutical composition.

9. Use of the artificial multi-antigen fusion protein of claim 1 used for preparing an agent or a kit for detecting specific T cell immunity.

10. A method, comprising administering the artificial multi-antigen fusion protein of claim 1.

11. A method, comprising administering the composition of claim 7 to a subject in need thereof.

Description

DESCRIPTION OF DRAWINGS

[0069] FIG. 1 shows cloning sites of pNIC28a-Bsa4 expression vector.

[0070] FIG. 2 shows electrophoresis identification of PCR products of Escherichia coli containing ESAT6/CFP10 gene (left) and SDS-PAGE electrophoresis analysis of purified ESAT6/CFP10 multi-antigen fusion protein (right).

[0071] FIG. 3 shows electrophoresis identification of PCR products of Escherichia coli containing OVA: 242-352 gene (left) and SDS-PAGE electrophoresis analysis of purified OVA: 242-352 multi-antigen fusion protein (right).

[0072] FIG. 4 shows electrophoresis identification of PCR products of Escherichia coli containing HPV16-E7 gene (left) and SDS-PAGE electrophoresis analysis of purified HPV16-E7 multi-antigen fusion protein (right).

[0073] FIG. 5 shows the flow cytometry analysis of normal human PBMC which is treated by HPV16-E7 multi-antigen fusion protein, and the two-parameter scatter plot shows distribution of CD54 and antigen in PBMC. CD54 is labeled with PE and the antigen is labeled with FITC.

[0074] FIG. 6 shows the spots formed after stimulated by ESAT6/CFP10 multi-antigen fusion protein, ESAT6/CFP10 mature protein, and antigens in a commercially-available T-SPOT.TB kit, respectively. The results shows that for stimulation of total T lymphocytes, effects of ESAT6/CFP10 multi-antigen fusion protein and antigens in the commercially-available T-SPOT.TB kit are equivalent (above panel), while for the stimulation of CD8.sup.+ lymphocytes, effects of ESAT6/CFP10 multi-antigen fusion protein are significantly better than those of ESAT6/CFP10 mature protein (below panel).

[0075] FIG. 7 shows results of skin test for ESAT6/CFP10 multi-antigen fusion protein. In the figure, results of skin test for four groups of guinea pigs sensitized by proteins/polypeptides derived from inactivated Mycobacterium tuberculosis H37Rv are shown. 0.1 ml of positive control (PPD), negative control, recombinant ESAT6/CFP10 multi-antigen fusion protein (marked as “LRMK”), recombinant ESAT6-CFP10 mature protein (marked as “E6-C10”) were intradermally injected by alternative skin test, and the area of red and swollen skin at each injection site of guinea pigs was measured 24 hours after injection.

[0076] FIG. 8 shows flow cytometry analysis of distribution of SIINFEKL-MHC-I on DC2.4 cells. Histograms show that SIINFEKL peptide in OVA (242-252) multi-antigen fusion protein can be efficiently presented by DC cells at a concentration of 100 μg/ml (delivery efficiency is 17.6%). Whereas original protein fragments containing SIINFEKL peptide can not be presented to MHC class I molecule.

[0077] FIG. 9 shows survival-time curve for B16-HPV-E7 tumor-bearing mice (the date of inoculation of tumor cell is deemed as day 0).

[0078] FIG. 10 shows survival-time curve for B16-survivin mice (the date of inoculation of tumor cell is deemed as day 0).

MODES FOR CARRYING OUT THE INVENTION

[0079] After extensive and in-depth research as well as continuous and repeated exploration, the present inventors have developed a protein vaccine capable of stimulating CD8.sup.+ T cell immunity for the first time, and the protein vaccine possesses advantages, such as low production cost and convenient control. Additionally, compared with existing vaccines, the vaccine of the present invention can also simultaneously stimulate CD4.sup.+ T cell immunity. Experiments demonstrate that the protein vaccine of the present invention is effective in stimulating CD8.sup.+ cells via MHC-I antigen presenting pathway. The present invention has been completed based on the above findings.

[0080] In particular, the present inventors have unexpectedly found that a multi-antigen fusion protein vaccine connected through cleavage site of cathepsin can be effectively presented by antigen-presenting cells via MHC-I pathway. In vitro and in vivo experiments have shown that the protein vaccine can effectively stimulate CD8.sup.+ T cells, and also have the ability to stimulate CD4.sup.+ T cells.

Terms

[0081] As used herein, the terms “protein of the present inventive”, “fusion protein of the present invention”, “artificial multi-antigen fusion protein”, “antigen fusion protein”, “fusion protein of the present invention stimulating CD4.sup.+ and CD8.sup.+ T cell immune responses” can be interchangeably used, and refer to an artificial recombinant protein having a structure of Formula I and capable of effectively and simultaneously activating CD4.sup.+ and CD8.sup.+ T cell immune responses.

[0082] Lysosome and MHC-I Pathway

[0083] Polypeptide antigens of short fragment (such as commercially-available T.SPOT.TB kit used to stimulate antigens of human antigen presenting cells) can not only enter lysosome and can be presented to MHC-II molecules, but also can be absorbed into cytoplasm by presenting cells, processed and presented to MHC-I class molecules. Protein antigens are firstly endocytosed by antigen-presenting cells, and then pass through early phagosome, late phagosome, and finally enter into lysosome. In general, proteins that enter lysosomes are completely degraded to amino acids. However, under certain situation, a protein will gradually degrade, and the resulting polypeptide can bind to MHC-II molecules located at lysosomal to form a relatively stable complex, presented to the cell surface, and stimulate CD4.sup.+ T cell immunity. In general, protein antigens do not leak from lysosomes into cytoplasm and therefore do not enter MHC-I pathway to stimulate CD8.sup.+ cells. The present inventors have unexpectedly found that the antigen fusion protein specially designed in the present invention can not only stimulate CD8.sup.+ T cells, but also retain the original function of stimulating CD4.sup.+ T cell.

[0084] The major protease in lysosomes of antigen-presenting cells is cathepsin S. Leu-Arg-Met-Lys is a preference sequence for cathepsin S. In the present invention, Leu-Arg-Met-Lys is preferably used to connect a group of antigen segments to form a new artificial multi-antigen fusion protein and stimulate CD4.sub.+ and CD8.sup.+ T cells.

[0085] Common antigen polypeptides of short fragment (generally ≦70aa, ≦60aa or shorter) can be directly absorbed into cytoplasm by antigen-presenting cells, and thus presented to MHC-class 1 molecules. Common protein antigens can only be endocytosed into lysosomes by antigen-presenting cells, and the common mature protein in lysosome only binds to MHC-II, and only stimulates CD4.sup.+ T cells.

[0086] In contrast, the artificial multi-antigen fusion protein of the present invention is endocytosed into lysosomes by antigen presenting cells, and then cleavage occurs firstly in the ligation sequence in the presence of cleavage sites of cathepsin, such as cathepsin S, thereby rapidly generating a group (plurality) of polypeptides as well as achieving and facilitating cross-presentation, and simultaneously stimulating CD8.sup.+ T cells and CD4.sup.+ T cells.

[0087] Experiments of the present invention show that after the fusion protein of the present invention enters lysosomes, the fusion protein still disappears rapidly even lysosomal function is inhibited, indicating that the protein of the present invention or cleavage produces thereof in lysosomes can leak into cytoplasm.

[0088] Fusion Protein that Stimulates CD4.sup.+ and CD8.sup.+ T Cell Immune Response

[0089] In the present invention, a single protein, i.e., a multi-antigen fusion protein, is provided, which is formed by connecting a plurality of antigen epitopes (or antigen segments) and used for specific cellular immunoassays and as a prophylactic and therapeutic vaccine.

[0090] In the multi-antigen fusion protein of the present invention, there are at least one, more preferably 2, 3, 4, 5 or more (e.g., any positive integer of 6-20) CD8.sup.+ T cell epitopes.

[0091] In the multi-antigen fusion protein of the present invention, at least one, more preferably two, three, four, five or more (e.g., any positive integer of 6-20) CD4.sup.+ T cell epitopes are included.

[0092] In the present invention, the sequence of antigen segment may be from any polypeptide capable of stimulating immune response, preferably a sequence capable of stimulating CD4.sup.+ or CD8.sup.+ T cell responses (i.e., T cell epitopes).

[0093] Cathepsin is a group of cysteine proteases, the main role of which is to degrade lysosomal proteins. The known primary function of cathepsin-S is to participate in antigen presentation of MHC-2.

[0094] In the multi-antigen fusion protein of the present invention, these polypeptides containing CD8.sup.+ T cell epitope and CD4.sup.+ T cell epitope may be joined by recognition sites of cathepsin in any antigen presenting cell, and preferred Leu-Arg-Met-Lys sequence for cathepsin S is preferably used.

[0095] In another preferred embodiment, several other cleavage sites of cathepsin B and K, which are relatively more expressed in presenting cells, may be used; for example, Met-Lys-Arg ↓-Leu is the cleavage site of cathepsin B, and His-Pro-Gly ↓-Gly is the cleavage site of cathepsin K. However, the effect of cleavage site of cathepsin S is particularly excellent.

[0096] In another preferred embodiment, different cleavage sites of cathepsin S can be used in the artificial multi-antigen fusion protein, including, but not limited to, Arg-Cys-Gly↓-Leu, Thr-Val-Gly↓-Leu, Thr-Val-Gln↓-Leu, X-Asn-Leu-Arg↓, X-Pro-Leu-Arg↓, X-Ile-Val-Gln↓ and X-Arg-Met-Lys↓; wherein X-Arg-Met-Lys ↓ and X-Asn-Leu-Arg ↓ are preferred linking cleavage sites, where X is any amino acid and ↓ is the cleavage site.

[0097] Any protein may be used into the present invention. In general, the sequence covered by the multi-antigen fusion protein accounts for more than 40% of the protein, and in a preferred embodiment, at least 50%, 60%, 70%, 80%, 90%, preferably 100% of the sequence of protein antigen is covered by the antigen fusion protein.

[0098] As a preferred example of the present invention, the coverage of HPV16-E7 multi-antigen fusion protein is 100%. Of course, even if the coverage is reduced, it is sometimes feasible, especially when the MHC-I antigen epitope in a target protein is less and distinct. For example, in a preferred embodiment of the present invention, the coverage of OVA is 40% because OVA has only a single MHC-I antigen epitope and the selected fragment includes the epitope.

[0099] The multi-antigen fusion protein may cover more than one antigenic protein. In another preferred embodiment, the antigen fusion protein of tubercule bacillus of the present invention covers both ESAT6 and CFP10 protein sequences, which may reduce the cost of production and facilitate quality control.

[0100] The artificial multi-antigen fusion protein of the present invention is different from natural proteins (such as proteins or tumor antigens from viruses, bacteria, parasites). One of the main differences is the loss of spatial structure of the natural protein and therefore does not possess the function of a natural protein, thus avoiding the potential hazards of, for example, pathogen proteins (such as viral capsid proteins).

[0101] The artificial multi-antigen fusion protein of the present invention is also different from the antigen polypeptide of short fragment (e.g., antigen peptide and epitope polypeptide of ≦70 or ≦60 amino acids). One of the main differences is that the protein of the invention has a large molecular weight and belongs to a macromolecule protein (rather than a small molecule polypeptide), and thus the pathway into a cell is not the same, resulting in that the pathway through which the antigen is presented, and the mode and mechanism for stimulating T cells are different from those of the antigen polypeptides of short fragment.

[0102] Preparation Method and Engineered Cell

[0103] Once the antigen protein sequence is determined, the protein of the present invention can be obtained by mass production using recombinant method. Generally, encoding DNAs artificially synthesized are cloned into an expression vector, transferred into a cell and then isolated from a host cell or a fermentation product by conventional methods.

[0104] A typical method for preparing an antigen fusion protein comprises steps of:

[0105] Constructing an artificial multi-antigen fusion protein according to the amino acid sequence of the protein antigen, and the multi-antigen fusion protein comprises or consists of a series of antigen segments of a specific length, wherein each antigen segment is connected by the same or different cleavage sites of cathepsin (e.g., leu-Arg-Met-Lys).

[0106] In addition, the cells used to express the fusion protein of the present invention are also included in the present invention. “Host cells” include prokaryotic cells and eukaryotic cells. Commonly used prokaryotic cells are E. coli. Commonly used eukaryotic cells include, but are not limited to, yeast cells, insect cells, and mammalian cells. As a preferred embodiment of the present invention, the used host cells are Escherichia coli, such as BL21 (DE3) and yeast, and CHO cells.

[0107] Vaccine Composition and Pharmaceutical Composition

[0108] In the present invention, the use of the fusion proteins of the invention is provided, for example, for immuno-diagnosis and preparation of vaccines.

[0109] Immunological diagnosis includes skin test and T cell dot enzyme immunoassay. The skin test is to immunize a subject with the multi-antigen fusion protein of the present invention, and then the skin reaction of the individual is observed to determine whether the individual has been exposed or infected with similar antigens. As a preferred embodiment of the present invention, the immuno-diagnosis is to use the fusion protein of the present invention as a source of stimulation to detect T cell immune response of an individual. For example, generation of gamma interferon after stimulating T cells of an individual with ESAT6-CFP10 multi-antigen fusion protein of the present invention can be observed to determine whether a patient is infected with tuberculosis or infection stage or prognosis.

[0110] Vaccines include prophylactic vaccines and therapeutic vaccines. The latter is mainly related to the activation of T cells, especially CD8.sup.+ killer T cells (CTL). The activation of CTL cells is limited by MHC-I molecules. As a preferred embodiment of the present invention, it has been demonstrated in vitro that HPV16-E7 multi-antigen fusion protein of the present invention can be endocytosed by human antigen presenting cells and activate antigen presenting cells. In another preferred embodiment, the OVA multi-antigen fusion protein of the present invention is used as a model to demonstrate that it can be processed by antigen presenting cells and presented to MHC class I molecules.

[0111] Uses

[0112] In the fusion protein of the present invention, recognition sequences of cathepsin in the endosome and lysosome of antigen presenting cells are used to connect a plurality of antigen epitope peptides containing CD4.sup.+ and CD8.sup.+ epitopes into a single protein, which, after expression and purification, can be directly used in immune diagnosis or as a prophylactic and therapeutic vaccine.

[0113] When used as a prophylactic and therapeutic vaccine, the protein or composition of the present invention can be injected into an animal directly or with an adjuvant or used in vitro with a cell therapy. For example, it is added into DC/NK cell culture to stimulate the production of specific antigen presenting cells and then to stimulate the production of specific CD8.sup.+ T cells (CTL) in vivo/in vitro with the antigen presenting cells.

[0114] In the present invention, these externally sensitized specific antigen presenting cells and/or in vitro produced CTL antigens can be returned to the body of a corresponding subject for antiviral, antitumor and other different uses.

[0115] In the present invention, the protein or in vitro sensitized cells (or corresponding formulation) of the invention are preferably administered to a subject by the following administration mode: intravenous injection, perfusion, subcutaneous injection, transdermal administration, and the like.

[0116] Main advantages of the present invention include:

[0117] (1) It has been found for the first time that the artificial multi-antigen fusion protein of the present invention can be efficiently presented onto MHC-I molecules.

[0118] (2) It has been found for the first time that a single multi-antigen fusion protein instead of several antigen short peptides, can specifically stimulate human T cells to release gamma interferon.

[0119] (3) It has been demonstrated for the first time that the antigen fusion protein of the present invention can be phagocytosed by antigen-presenting cells and activate CD8.sup.+ cells.

[0120] (4) The fusion protein of the present invention can be used in the development of reagents and techniques for immuno-diagnosis. For example, specific cellular immunity is detected by skin tests, or specific cellular immunity is detected by ELISPOT assays.

[0121] (5) The fusion protein of the present invention can be used in the development of therapeutic or prophylactic drugs or vaccines, including DC cells sensitized by the fusion protein of the present invention and/or CTL cell preparation, and the industrial application value is immeasurable.

[0122] (6) Compared with a conventional solution using a group of short fragment polypeptides, the cost of the method and product of the present invention is significantly reduced (typically at least 80% or more); and quality control is easy and huge amount of manpower, material and time can be saved.

[0123] The invention will be further illustrated with reference to the following specific examples. It is to be understood that these examples are only intended to illustrate the invention, but not to limit the scope of the invention. For the experimental methods in the following examples without particular conditions, they are performed under routine conditions, such as conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 1989, or as instructed by the manufacturer. Unless otherwise stated, the percentages and parts are percentages by weight and parts by weight.

Example 1

[0124] Clone of Multi-Antigen Fusion Protein

[0125] The antigen fusion protein was designed according to the amino acid sequence of the protein. The antigen fusion protein was formed by connecting a series of polypeptide fragments (short fragment antigen peptide) of 25-35 amino acids in length, and the antigen peptides are ligated by the same preferred sequence of cathepsin S (Leu-Arg-Met-Lys).

[0126] Codons in the DNA encoding the antigen fusion protein are optimized into E. coli preferred codons. DNA coding sequences were prepared by whole artificial synthesis and TACTCCCATATATAT (SEQ ID NO.: 7) was added at 5′-end and TATCCACCTTTACTGTTA (SEQ ID NO.: 8) was added at 3′-end. Synthesized DNA molecules were treated with T4 DNA polymerase and dCTP for 30 minutes.

[0127] A conventional pNIC28-Bsa4 vector (obtained from Oxford University; U.S. Pat. No. 8,148,100 B2; GenBank ID: EF198106) was digested with BsaI for 1 hour. The important components involved in the expression of the clone in this vector are shown in FIG. 1. The linearized vector was isolated by 1% agarose gel electrophoresis and treated with T4 DNA polymerase and dGTP for 30 minutes. After two products from T4 DNA polymerase treatment were mixed, regular competent E. coli DN5α was transformed, and inoculated into a plate, and mono colony was picked up for culture. Positive colonies were identified by PCR performed on the culture.

[0128] Results are shown in FIGS. 2, 3 and 4, indicating that the size of the coding sequence of the obtained fusion protein and molecular weight of the protein are consistent with the design or predicted value.

[0129] The amino acid and nucleotide sequences of each multi-antigen fusion protein are shown as below.

TABLE-US-00001 Amino acid Nucleotide Name of Protein sequence sequence ESAT6-CFP10 antigen fusion protein SEQ ID NO.: 1 SEQ ID NO.: 2 (also named as ″TB-antigen fusion protein″) OVA antigen fusion protein SEQ ID NO.: 3 SEQ ID NO.: 4 HPV16-E7 antigen fusion protein SEQ ID NO.: 5 SEQ ID NO.: 6 SEQ ID NO.: 1 MAEMKTDAAT LAQEAGNFER ISGDLKTQID QVESTLRMKT QIDQVESTAG SLQGQWRGAA  60 GTAAQAAVVR FQELRMKAQA AVVRFQEAAN KQKQELDEIS TNIRQAGVQY SRLRMKIRQA 120 GVQYSRADEE QQQALSSQMG FLRMKMTEQQ WNFAGIEAAA SAIQGNVTSI HSLLDEGKQS 180 LRMKHSLLDE GKQSLTKLAA AWGGSGSEAY QGVQQKWDAL RMKYQGVQQK WDATATELNN 240 ALQNLARTIS EAGQAMASLR MKISEAGQAM ASTEGNVTGM FA 282 SEQ ID NO.: 2 caaatcgatc aagtggaaag taccgcaggt agcctgcagg gtcagtggcg tggtgcagca 180 ggcaccgcag cacaggcagc agttgttcgt tttcaagaac tgcgcatgaa agcccaggca 240 gccgtggtgc gcttccaaga agccgcaaat aaacagaaac aagagctgga tgaaatcagc 300 accaatattc gtcaggcagg cgttcagtat agccgtttac ggatgaaaat tcgtcaagcc 360 ggtgtgcagt attcacgtgc agatgaagaa cagcagcaag cactgagcag ccagatgggt 420 tttttaagaa tgaaaatgac cgagcagcag tggaattttg caggtattga agcagccgca 480 agcgcaattc agggtaatgt taccagcatt catagcctgc tggacgaagg taaacagagc 540 ctgcggatga agcatagtct gttagatgaa ggcaaacagt cactgaccaa actggcagca 600 gcatggggtg gtagcggtag cgaagcatat cagggtgttc agcagaaatg ggatgcatta 660 cgtatgaagt atcagggcgt gcaacaaaag tgggacgcaa ccgcaaccga actgaataat 720 gcactgcaga atctggcacg taccattagt gaagccggtc aggcaatggc cagcttacgc 780 atgaagattt ctgaagcagg ccaagctatg gcaagcaccg aaggcaatgt gaccggtatg 840 tttgcataa 849 SEQ ID NO.: 3 MLVLLPDEVS GLEQLESIIN FEKLTEWTSS LRMKLESIIN FEKLTEWTSS NVMEERKIKV  60 YLPRMKMEEK YNLTSVLMAM GITDVFSSSA NLSGISSAES LKISQAVHAA HAEINEAGRL 120 RMKISQAVHA AHAEINEAGR EVVGSAEAGV DA 152 SEQ ID NO.: 4 atgctggttc tgctgccgga tgaagttagc ggtctggaac agctggaaag cattatcaat  60 tttgaaaaac tgaccgaatg gaccagcagc ctgcgtatga aactggaatc catcattaac 120 ttcgagaaac tgacagagtg gacaagcagc aatgttatgg aagaacgtaa aatcaaagtg 180 tacctgcctc gcatgaaaat ggaagagaaa tataacctga ccagcgttct gatggcaatg 240 ggtattaccg atgtttttag cagcagcgca aatctgagcg gtattagcag cgcagaaagc 300 ctgaaaatta gccaggcagt tcatgcagca catgccgaaa ttaatgaagc aggtcgtctg 360 cggatgaaaa tttcacaggc cgtgcatgct gcccatgcag aaatcaacga agctggccgt 420 gaagttgttg gtagtgccga agccggtgtt gatgcataa 459 SEQ ID NO.: 5 MHGDTPTLHE YMLDLQPETT DLYCYEQLND SSEEELRMKE QLNDSSEEED EIDGPAGQAE  60 PDRAHYNIVT FCCKLRMKHY NIVTFCCKCD STLRLCVQST HVDIRTLEDL LMGLRMKIRT 120 LEDLLMGTLG IVCPICSQKP 140 SEQ ID NO.: 6 atgcatggtg ataccccgac cctgcatgaa tatatgctgg atctgcaacc ggaaaccacc  60 gatctgtatt gttatgagca gctgaatgat agcagcgaag aggaattacg catgaaggaa 120 cagctgaacg attcaagcga agaagaggac gaaattgacg gtccggcagg tcaggcagaa 180 ccggatcgtg cacattacaa cattgttacc ttttgttgca aactgagaat gaaacactac 240 aatatcgtga ccttctgctg taaatgtgat agcaccctgc gtctgtgtgt tcagagcacc 300 catgttgata ttcgtacatt agaggacctg ctgatgggcc tgcggatgaa aattcgtacc 360 ctggaagacc tgttaatggg caccctgggt attgtttgtc cgatttgtag ccagaaaccg 420 taa 423

[0130] In addition, LC-MS analysis also showed that the measures molecular weight of purified TB-antigen fusion protein (30974 Da), OVA antigen fusion protein (16589 Da) and HPV16-E7 antigen fusion protein (16231 Da) were consistent with the predicted values.

Example 2

[0131] Expression of Antigen Fusion Protein

[0132] Plasmid DNA of positive colonies containing encoding sequence of the artificial multi-antigen fusion protein was extracted and transformed into Escherichia coli BL21 (DE3).

[0133] Transformed single colonies were inoculated into low salt LB broth and incubated overnight at 37° C. The culture was diluted at 1:100 in low salt LB broth, incubated at 37° C. with shaking until OD600=1.0 and cooled to 18° C. 0.2 mM IPTG was added for inducing expression of protein. Cells were incubated at 18° C. for another 16 hours and then centrifuged at 4000 rpm. The cells were collected and re-suspended in phosphate buffer.

Example 3

[0134] Purification of Antigen Fusion Protein

[0135] (a) Purification of ESAT6-CFP10 and OVA Antigen Fusion Protein

[0136] Cells were lysed by ultrasonic method and inclusion bodies were collected by centrifugation. The inclusion bodies were dissolved in denaturation buffer (8 M urea in HEPES buffer at pH 7.4) and passed through a Ni-NTA column, and the antigen fusion protein containing histidine label bound to the column. After impurity proteins were removed by sufficient washing, the antigen fusion protein was eluted with urea elution buffer. The eluted antigen fusion protein was firstly diluted 8-fold with PBS containing 0.5 M arginine (pH 9.5) and the urea was removed through dialysis against PBS (right panel in FIG. 2 and right panel in FIG. 3).

[0137] (b) Purification of HPV16-E7 Antigen Fusion Protein

[0138] The cells were lysed by ultrasonic method. The supernatant containing target proteins was collected by centrifugation and passed through a Ni-NTA column. The histidine-labeled antigen fusion protein was bound to the column. After impurity proteins were removed by sufficient washing, HPV16-E7 antigen fusion protein was eluted with an elution buffer containing imidazole (right panel in FIG. 4).

Example 4

[0139] Intake of Antigen Fusion Protein by Antigen Presenting Cells

[0140] Human peripheral blood lymphocytes PBMCs were isolated from blood of voluntary blood donors by Ficoll and washed. The cell concentration was adjusted to 2×10.sup.6/ml in cell culture medium and 5 ml of cell suspension was placed in a cell culture flask. HPV16-E7 antigen fusion protein (50 μg/ml) was added or FITC-HPV16-E7 (5 μg/ml) was added at the same time, and cultured hours at 37° C. and 5% CO.sub.2 for 24 hours and for another 12 hours. Cells were stained with anti-human CD54 antibody at 4° C. for 30 minutes and analyzed by flow cytometry. After treated for 13 hours, the cells were treated by EDTA, harvested and washed with 1 ml of ice-cold FACS buffer (2% FCS in PBS) for two times. And then a commercially available PE-tagged monoclonal antibody 25.D1-16 was added to a concentration of 0.6 μg/ml, and incubate at 4° C. for 30 min. Cells were washed for three times with FACS buffer and the cells were re-suspended in 0.2 ml of FACS fixation buffer (BD) and analyzed by flow cytometry.

[0141] Results showed that the major cells which phagocytose multi-antigen fusion protein were CD54 positive cells (FIG. 5).

Example 5

[0142] Detection of γ-Interferon Secreting T Cells in Patients with Antigen Fusion Protein

[0143] In this example, the ability of TB-antigen fusion protein to stimulate CD8+ T cells was tested by γ-interferon release assay. The method is described as below:

[0144] After peripheral blood mononuclear cells were isolated from tuberculosis sputum smear positive patients, the cell concentration was adjusted to 5×10.sup.6 live cells/ml. 50 μl of the above mentioned cell suspension was added into negative wells, positive wells (with CONA as stimulating source), detection wells (With TB-antigen fusion protein as stimulating source), and control wells (with antigen T-spot A and T-spot B in the commercially available T-SPOT.TB kit from Oxford Immunology Technology Ltd., as stimulating source; or with mature CFP10-ESAT6 fusion natural protein (marked as TB-ESAT6-CFP10) as control stimulating source)) respectively.

[0145] Cells were incubated at 37° C., 5% CO.sub.2 for 16-20 hours, plates were washed, and 50 μl of labeled antibody working solution was added, and incubated at 2-8° C. for 60 minutes. Afterwards, the plates were washed, and 50 μl of BCIP INBT substrate solution was added, incubated at room temperature in darkness, dried, and counted. Results were judged according to the instruction of T-SPOT.TB kit: when the number of spots in a blank control well is 0-5, the number of spots in an antigen-containing well−the number of spots in a blank control well ≧6; or when the number of spots in a blank control well is 6-10, the number of spots in an antigen-containing well ≧2 times of the number of spots in a blank control well.

[0146] The results showed that effects obtained from stimulation with ESAT6/CFP10 antigen fusion protein was consistent with those obtained from stimulation with antigens in T-SPOT.TB kit (FIG. 6).

[0147] In addition, after CD4.sup.+ T cells of the patient were removed by using specific anti-CD4 magnetic beads, remaining T cells were mainly CD8.sup.+ T cells, and multi-antigen fusion protein and CFP10 mature protein were compared on this basis.

[0148] Results showed that TB-antigen fusion protein of the present invention exerted a significant stimulating effect on CD8.sup.+ T cells. In contrast, the wild-type sequence of ESAT6-CFP10 mature protein exerted little effect on CD8.sup.+ T cells (FIG. 6).

Example 6

[0149] In Vitro Induction of Specific CD8.sup.+ T Cells by Antigen Fusion Protein-Loaded DCs

[0150] Preparation of antigen-loaded DC cells: After mononuclear peripheral cells (PBMC) were isolated, part of the cells were frozen and the remaining cells were adjusted to a cell concentration of 1×10.sup.7 cells/mL and incubated in conventional AIM-V culture for 2 hours. Suspended cells were removed (cryo-preserved) and AIM-V medium containing GM-CSF (1000 IU/ml) and IL-4 (50 IU/ml) was added and incubated at 37° C., 5% CO.sub.2. HPV16-E7 antigen fusion protein (50 μg/ml) or recombinant HPV16-E7 protein (50 μg/ml) was added at day 3.

[0151] CD8.sup.+ T cells were sensitized in vitro by antigen-loaded DCs: At day 5, adherent DC cells were collected, cryo-preserved PBMCs were thawed, in which CD8-positive cells were separated with CD8 beads, and CD8-negative cells were frozen. CD8 positive cells: DC cells were co-cultured in a 5:1 ratio and IL-2 100 IU/ml, IL-7 25 IU/ml were added. In addition, IL-2 (100 IU/ml) and IL-7 (25 IU/ml) were added every 2 days or half-volume of fluid was changed. On day 7, the frozen CD8-negative cells were thawed and treated with mitomycin. Afterwards, CD8-negative cells were added according to 1/10 of the number of CD8+T cells for secondary stimulation. 50 μg/ml of corresponding protein was supplemented, and IL-2 100 IU/ml, IL-7 25 IU/ml were supplemented. IL-2 100 IU/ml and IL-7 25 IU/ml were supplemented every two days.

[0152] Preparation of antigen presenting cells: the suspended cells frozen at day 1 were thawed, and incubated in a plate coated with CD19 monoclonal antibody for 2 h. Non-adherent cells were discarded, CD19 positive cells were suspended in the culture medium. The cell concentration was adjusted to 5×10.sup.5/ml, 2 ml of the cell suspension was cultured at 37° C., 5% CO.sub.2 for 48 h, and IL-4 was added to a concentration of 100 μg/ml.

[0153] Effects of in vitro induction of specific CD8.sup.+ T by multi-antigen fusion protein-loaded DCs evaluated through γ-interferon release assay: 5×10.sup.4 cells/50 μl of CD19 positive cells were added to a 96-well plate, and corresponding proteins (HPV16-E7 antigen fusion Protein (SEQ ID NO.: 5) or conventional recombinant HPV16-E7 protein) were added to 50 μg/ml, and incubated at 37° C., 5% CO.sub.2 for 2 h. After centrifugation, supernatant was discarded, and cells were re-suspended in 50 μl of fresh medium. 5×10.sup.4 cells/50 μl of CD8 positive cells were added and incubated at 37° C. for 18 hr. The concentration of IFN-γ in the supernatant was measured by ELISA.

[0154] Results are shown in Table 1: CD8.sup.+ T cells can be sensitized in vitro and re-activated by antigen fusion protein HPV16-E7-loaded DC, effects of the antigen fusion protein effect is much higher than that of the control protein HPV-E7, i.e., compared with the recombinant HPV16-E7 protein as the control, the release of gamma-interferon caused by the fusion protein of the present invention was increased by 372.5%. (152.7−58.2)/(2.5−(−17.5))−1)*100%=372.5%).

TABLE-US-00002 TABLE 1 Effects of HPV16-E7 antigen fusion protein and recombinant HPV16-E7 protein on in vitro stimulating DC to induce specific CD8.sup.+ T cells detected through γ-interferon release assay Concentration of IFN-γ (ng/l) CD8.sup.+ CD8.sup.+ CD19 CD19 + (negative Net release antigen control) of IFN-γ HPV16-E7-antigen 152.7 58.2 94.5 fusion protein HPV16-E7-natural 2.5 −17.5 20.0 protein Non-antigen −29.0 −27.3 −1.7

Example 7

[0155] Skin Sensitive Test for Tuberculosis

[0156] A guinea pig weighing about 250 g was intradermally injected with 0.1 ml of PPD and the skin reaction was observed at 24 hours. Skin reaction-negative guinea pigs were randomly divided into 5 groups, subcutaneously injected with 0.2 ml of 40 mg/ml of Mycobacterium tuberculosis H37Rv suspension at the inguinal inguinal, and immunized once every week for five times. One week after the fifth immunization, the guinea pigs were randomly divided into five groups. Furs on the back of guinea pig were plucked, 0.1 ml of positive control (PPD), negative control (tuberculosis-irrelevant recombinant protein), recombinant ESAT6-CFP10 protein, a mixture liquid of recombinant ESAT6-CFP10-antigen fusion protein (1 mg/ml, 0.1 mg/ml) were intradermally injected by alternative skin test respectively. The vertical diameter and horizontal diameter (mm) of skin redness at each injection spot on the back of a guinea pig were measured 24 hours after the injection, and average value was recorded.

[0157] Results of 24-hour skin reaction are shown in FIG. 7. In TB-antigen fusion protein group (SEQ ID NO: 1), the average diameter of skin induration is greater than that of PPD group and ESAT6-CFP10 natyral protein group. In the control group, no swelling, or induration reaction was observed on local skin of a guinea pig. This suggests that TB-antigen fusion proteins can be used in a skin test to effectively detect tuberculosis infections.

[0158] In the figure, E6-C10 represents ESAT6-CFP10-natural protein; LRMK represents TB-antigen fusion protein, in which cleavage site of cathepsin S is used to connect each antigen segment, and sequence of which is shown in SEQ ID NO: 1; and TEV represents a control protein formed by replacing all cleavage sites of cathepsin S in SEQ ID NO: 1 with TEV cleavage site (cleavage site of non-cathepsin).

Example 8

[0159] Treatment and Delivery Mechanism of Antigen Presenting Cells on Antigen Fusion Proteins and Common Antigens

[0160] In order to determine after antigen presenting cells intake antigen fusion proteins and are activated by antigen fusion proteins, whether T cell epitopes can be efficiently presented to MHC-I molecule (CD8.sup.+ T cells can be only activated by presenting on MHC-I molecule, thereby producing cytotoxic reaction, and killing bacteria/virus-infected cells or cancerous cells). In the present example, OVA antigen fusion protein was designed, expressed and purified according to positions 242-352 of amino acid sequence of chicken ovalbumin (OVA), which contains MHC-1 epitope SIINFKL. And the OVA protein fragment OVA 255-340 containing the epitope was used as a control. The mouse dendritic cell line (DC2.4) was used as antigen presenting cells to study the presentation of SIINFEKL in OVA antigen fusion protein. Finally, T cell receptor-like antibodies (identifying SIINFEKL/MHC-I complex) were detected.

[0161] 100 μg/ml of OVA protein fragment (or 30 μg/ml of OVA antigen fusion protein (SEQ ID NO: 3)) was mixed with DC2.4 cells, cultured in RPMI 1640(Sigma) containing 10% heat-inactivated fetal bovine serum (sigma), 2 mM L-glutamine (Sigma) for 13 hours, then stained with a commercially available 25.D1-16 antibody labeled with PE (the monoclonal antibody 25. D1-16 specifically recognizes MHC-1 molecule binding to SIINFEKL), and washed by 1 ml of ice-cold FACS buffer (2% FCS in PBS) for two times. Afterwards, PE-labeled monoclonal antibody 25.D1-16 was added to a concentration of 0.6 μg/ml, and incubate at 4° C. for 30 min. Cells were washed for three times with FACS buffer, re-suspended in 0.2 ml of FACS fixation buffer (BD) and analyzed by flow cytometry.

[0162] Results are shown in FIG. 8. In the figure, the blue peak (indicated by the arrow) is DC+PBS.

[0163] As a positive control, 8 peptide (SIINFEKL) can directly bind to MHC-1 molecule on cell surface, so that more than 80% of the cells were positively stained (C1 and C2 in FIG. 8).

[0164] SIINFEKL peptide in OVA natural protein can not be processed by DC cells and presented on MHC-1 molecule (A1 and A2 in FIG. 8). (Note: The blue and red peaks in FIG. 8A2 almost overlap).

[0165] Surprisingly, as a protein with large molecular weight, SIINFIKL peptide in OVA-antigen fusion protein (SEQ ID NO: 3) can be efficiently processed by mouse dendritic cell line DC2.4 and presented to the cell surface, resulting in up to about 17.6% of cells being 25.D1-16 antibody positively-stained (B1 and B2 in FIG. 8).

Example 9

[0166] Anti-Tumor Effects of C57/BL6 Mice Immunized with Antigen Fusion Protein on Murine Melanoma Cell B16 Overexpressing Survivin or HPV-E7

[0167] 2 mg/ml of antigen solution (control group: recombinant HPV-E7 protein, recombinant Survivin protein; experimental group: HPV-E7 antigen fusion protein (SEQ ID NO: 5), Survivin antigen fusion protein) was mixed with 1 mg/ml of MPL at equal volume to prepare an immunization suspension. Afterwards, 10 randomly assigned C57/B6 female mice aged 7-8 weeks and weighing 25 grams were subcutaneously injected at neck with 100 μl of immunization suspension on the 0 day, 21 day and 42 day.

[0168] For each immunized mouse, 100 μl of homogeneously mixed mice B16 cells stably transfected with human HPV-E7 or Survivin were subcutaneously inoculated at right flank of the mice, and the amount of injected cells was 7×10.sup.5 cells. The survival of mice was recorded and evaluated. Results are shown in FIG. 9 and FIG. 10.

[0169] The results showed that compared with the recombinant HPV-E7 protein control group, HPV-E7 antigen fusion protein of the present invention can significantly prolong the survival rate of mice (FIG. 9) and 50% of mice still survived on day 25; while all mice in recombinant protein control group were dead.

[0170] Compared with the recombinant Survivin protein, the Survivin antigen fusion protein of the present invention can significantly prolong the survival of mice (FIG. 10), and 70% of mice still survived on day 25; while only 40% of mice in the recombinant protein control group survived.

DISCUSSION

[0171] Prior to the present invention, clinically protein antigens were not successfully used as vaccines to stimulate CD8.sup.+ cell immunity.

[0172] Production and quality control of protein-like vaccine are mature technology and many clinical drugs and vaccines (stimulating antibodies) are proteins. It would be ideal if vaccines that could stimulate CD8.sup.+ T cell immunity can be produced by the proven method of producing protein. However, how to convert protein antigen presentation from endosome-lysosome-MHC-II pathway to cytoplasm-MHC-I pathway is a very challenging subject. Many immunologists and vaccine scientists are making efforts but there is no breakthrough yet.

[0173] Main obstacles include:

[0174] 1. There are a lot of enzymes in Lysosomes, their function is generally to completely degrade a protein, and a protein will be degraded into amino acids instead of peptides. Therefore, a general recombinant protein antigen will be completely degraded and can not stimulate T cells.

[0175] 2. Since lysosomal membrane is to isolate acid substances and cytoplasm in lysosome, lysosome is not an organelle which is easy to leak. Even a conventionally expressed protein or recombinant protein is not completely degraded, whether it can escape from or leak out of lysosome is unknown.

[0176] 3. For protein antigens that can stimulate CD4.sup.+ T cells, if a fusion protein, after CD8.sup.+ antigen is added, is degraded and leaks into cytoplasm (stimulating CD8.sup.+ T cells), whether the function of stimulating CD4.sup.+ T cell will decrease or disappear, is also unknown.

[0177] In the present invention, the present inventors have designed a variety of antigen fusion proteins based on enzymatic principles and biological recombination techniques, and identified a novel structure of antigen fusion protein, after a huge amount of screening: a series of long peptides containing antigen segment are ligated by cleavage sites of cathepsin. When the antigen fusion protein is phagocytosed in a cell and enters into lysosome, cathepsin in lysosome will degrade the antigen fusion protein into polypeptides containing different antigen epitopes.

[0178] The experiments of the present invention have surprisingly confirmed that after the fusion protein with particular structure of the present invention is degraded, degraded polypeptides are capable of leaking out of lysosomes and entering into cytoplasm and being processed to MHC class I molecule, thereby initiating MHC-I antigen presenting pathway and in turn stimulating CD8.sup.+ cells; and the original function of stimulating CD4.sup.+ T cell can be retained or improved.

[0179] All documents mentioned in the present invention are incorporated herein by reference, as if each document were individually recited for reference. It is to be understood that those skilled in the art will be able to make various changes or modifications to the present invention after reading the teachings of the present invention, which also fall within the scope of the claims appended hereto.