WO2016095812A1 - 人工多抗原融合蛋白及其制备和应用 - Google Patents

人工多抗原融合蛋白及其制备和应用 Download PDF

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WO2016095812A1
WO2016095812A1 PCT/CN2015/097470 CN2015097470W WO2016095812A1 WO 2016095812 A1 WO2016095812 A1 WO 2016095812A1 CN 2015097470 W CN2015097470 W CN 2015097470W WO 2016095812 A1 WO2016095812 A1 WO 2016095812A1
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antigen
fusion protein
cells
antigen fusion
sequence
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PCT/CN2015/097470
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English (en)
French (fr)
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姜石松
夏小兵
丁航海
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牛津疫苗医学生物科技（英国）有限公司
姜石松
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Priority to EP21181903.2A priority Critical patent/EP3971215A3/en
Priority to EP15869319.2A priority patent/EP3235831A4/en
Priority to US15/536,428 priority patent/US11833220B2/en
Priority to JP2017533496A priority patent/JP6917303B2/ja
Priority to CN201580068647.0A priority patent/CN107207617A/zh
Publication of WO2016095812A1 publication Critical patent/WO2016095812A1/zh

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    • A61K47/6855—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from breast cancer cell
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    • A61K49/0006—Skin tests, e.g. intradermal testing, test strips, delayed hypersensitivity
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Definitions

  • the present invention relates to the field of medicine, and more particularly to an artificial multi-antigen fusion protein capable of simultaneously stimulating CD4 + and CD8 + T cell immune responses, and a preparation and application thereof.
  • the artificial multi-antigen fusion protein of the present invention can be directly used for immunodiagnosis or used as a prophylactic or therapeutic vaccine after being purified and expressed.
  • cellular immunity is important for tumor cells and bacteria and viruses that have been infected into cells. After the human body is first stimulated by the above-mentioned exogenous, it will sensitize T lymphocytes and transform into memory T lymphocytes. When the human body is exposed to the same antigen again, it will rapidly produce a specific immune response, including an effector T lymphocyte reaction, and produce a variety of cytokines to exert an immunological effect (cellular immunity). Since cellular immunity plays a non-negligible role in controlling and eliminating bacteria, viruses and tumors, cellular immune vaccines have a role in preventing bacterial viral infections and tumor treatment.
  • a commonly used method for detecting cellular immunity is the enzyme linked immunospot assay (ELISPOT), which detects T cell secreting cytokines from a single cell level. For example, cellular immune function can be evaluated by detecting the number of T cells secreting cytokines after tuberculosis (TB)-specific antigen stimulation. For tuberculosis, detecting cellular immunity is one of the ways to diagnose whether or not to infect TB.
  • the US FDA approved the ELISPOT commercialization kit T-SPOT for the diagnosis of tuberculosis infection. These diagnostic kits basically use a mixture of 18-20 amino acids as a source of stimulation.
  • polypeptides are phagocytosed by antigen-presenting cells of peripheral blood, processed and presented to the cell surface together with their own HLA antigen, and then recognized by T cell receptors of memory T lymphocytes. After stimulation of T cells, cytokines represented by gamma interferon are detected and detected.
  • T cells There are many different cloned T cells in the human body. Each clone needs to be stimulated by different short peptides plus HLA. Different humans also have different T cell clones, and different human HLA antigens are different. To select T cells from a certain HLA population, it is necessary to identify and select T cell epitope antigens of this population. Identifying HLA phenotypes and identifying T cell epitope sequences are time consuming and labor intensive. The most common method for identifying all T cell recognition epitopes in all populations and a stimulus (protein) is to synthesize peptides that are 18-20 amino acids in length, or even longer. However, for clinical use, each peptide needs to establish strict production processes and quality testing standards.
  • ESAT6 96 amino acids
  • CFP10 100 amino acids
  • ESAT6 96 amino acids
  • CFP10 100 amino acids
  • An alternative is to select a limited number of peptides as a source of stimulation, but because of the HLA polymorphisms in different populations, the epitopes recognized by T cells are different, so this approach reduces the coverage of epitopes despite cost savings. rate.
  • Prophylactic and therapeutic vaccines are delivered to antigen-presenting cells, especially dendritic cells (DC), to stimulate and promote antigen-specific cytotoxic CD8 + (CTL) and helper CD4 + T lymphocytes.
  • Helper CD4 + T cells are effective in stimulating and amplifying cytotoxic CD8 + T cells and helping B cells to produce antibodies.
  • CD8 + T cells specifically recognize and lyse target cells containing the target antigen. Activation of specific CD8 + T cells depends on the ability of the antigen to be efficiently presented to MHC class I molecules (in humans, ie HLA class I antigens).
  • CD8 + cytotoxic T lymphocytes are the main active components of prophylactic and therapeutic cellular immune vaccines because they can directly recognize and destroy infected cells such as tumor cells or viruses. Therefore, the vast majority of current therapeutic vaccines are designed around how to stimulate the body to specifically recognize tumor or viral antigen CD8 + T cells.
  • the current mainstream view is that only antigens in the antigen-presenting cell (APC) pulp can be degraded into small peptides by the proteasome, transported into the endoplasmic reticulum via the TAP protein, in which the CTL epitope polypeptide and MHC-I (HLA-I)
  • the class binds to the surface of the antigen-presenting cell and specifically activates the latter after binding to a specific receptor of CD8 + T cells.
  • the traditional view also believes that only the protein antigen produced after transcriptional translation in the cytoplasm can enter the presentation pathway of MHC-I (HLA-I).
  • This is also the core design strategy for current therapeutic vaccines, namely the development of vaccines that replicate transcriptional translation in the cytoplasm. If a protein antigen is engulfed by a cell, the antigen does not enter the cytosol but enters the endocytosis and lysosomes, so this protein antigen cannot effectively stimulate CD8 + T cells.
  • Vaccines that currently stimulate CD8 + T cell immunity are mainly DNA vaccines, epitope peptide vaccines, and bacterial or viral vector vaccines. These vaccines have some obvious drawbacks: DNA vaccines are less efficient to enter cells and have a certain risk of integration into the genome; single epitope polypeptide vaccines cover a very small population and reduce their range of applications; bacterial/viral vector vaccine side effects Large, and easy to make the body immune to the carrier itself to reduce immunity to the target antigen. In addition, many people may have immunity to these bacteria/viruses before immunization, so the vaccine has been destroyed without functioning, thus greatly reducing its use.
  • Protein vaccines are a safe vaccine because proteins cannot replicate themselves.
  • proteins are engulfed into endocytic and lysosomes by antigen presentation cells (APCs) so that they do not enter the cytosol and are not degraded by the cytoplasmic proteasome and are presented to MHC-I.
  • APCs antigen presentation cells
  • the protein antigen vaccine therefore does not stimulate the CTL response.
  • the protein After being phagocytosed by APC cells, the protein enters the lysosome and is degraded into small peptides. Then some of the peptides (CD4 epitope) bind to the MHC class II molecules on the lysosomal membrane and are presented to the cell surface. Stimulate CD4 + T cells.
  • CD4 + can help B cells produce antibodies.
  • protein vaccines can stimulate B cells. Therefore, protein antigens have been used mainly to stimulate the body to produce antibodies. Studies have found that APC can also take antigen from the outside and present it to MHC class I molecules to stimulate CTL. This process is called antigen cross-presentation. But the process is so inefficient that no one can design a protein vaccine that stimulates CD8 + T cell immunity.
  • the object of the present invention is to provide a protein vaccine which is capable of effectively stimulating CD8 + T cell immunity, easy quality control and high safety.
  • an artificial multi-antigen fusion protein which, when administered to a mammalian subject, simultaneously stimulates CD4 + and CD8 + T cell immunity in said subject Answer.
  • the CD4 + T cell immune response and the CD8 + T cell immune response result in the T cell recognizing a target cell carrying the antigen on the cell surface.
  • the antigen in the artificial multi-antigen fusion protein is derived from a virus, a bacterium, a parasite, a chlamydial, a tumor cell, or a combination thereof.
  • the artificial multi-antigen fusion protein contains ⁇ 3, preferably ⁇ 5, more preferably ⁇ 10 antigenic segments.
  • the upper limit of the number of antigenic segments is ⁇ 200, preferably ⁇ 100, more preferably ⁇ 50.
  • each of the antigenic segments may be different, partially identical, or identical.
  • each antigen segment is from the same or a different pathogen, or from the same or a different species, or each antigen segment is an artificial sequence (ie, artificially designed, does not exist in nature) the sequence of).
  • each antigen segment is from a protein or a plurality of different proteins.
  • each of the antigenic segments is from a viral antigen, a bacterial antigen, a parasitic antigen, a Chlamydia antigen, a tumor antigen, or a combination thereof.
  • the antigenic segment comprises at least one (preferably at least 2) CD8 + epitope or a motif sequence capable of stimulating a CD8 + T cell immune response; and at least one (preferably At least 2) CD4 + epitopes or motif sequences capable of stimulating a CD4 + T cell immune response.
  • the at least one (preferably at least 2) amino acid sequences which are simultaneously a CD8 + epitope and a CD4 + epitope are included in each of the antigenic segments.
  • each of the antigenic segments is 8 to 50 amino acids in length, preferably 10 to 40 amino acids, more preferably 15 to 35 amino acids.
  • the artificial multi-antigen fusion protein further comprises a cleavage site sequence located between each antigenic segment.
  • the cleavage site sequence comprises a cleavage site for cathepsin.
  • the cathepsin cleavage site is selected from the group consisting of a cathepsin S cleavage site (such as Leu-Arg-Met-Lys or a similar cleavage site), and a cathepsin B enzyme.
  • a cleavage site (such as a Met-Lys-Arg-Leu or similar cleavage site), a cleavage site for cathepsin K (such as His-Pro-Gly-Gly or similar cleavage site), or a combination thereof.
  • the cathepsin S cleavage site is selected from the group consisting of 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 each X is independently any natural amino acid, and ⁇ indicates enzymatic cleavage position.
  • the cathepsin S cleavage site is X-Arg-Met-Lys (such as Leu-Arg-Met-Lys), X-Ile-Val-Gln, or a combination thereof.
  • each antigen segment is directly linked by the cleavage site sequence.
  • sequence of the cleavage site for ligation of each antigenic segment is the same or different.
  • sequence of the cleavage site for ligation of each antigenic segment is the same.
  • the antigenic segment itself does not contain the restriction enzyme site sequence; or the antigenic segment itself contains the restriction enzyme site sequence, but the antigenic segment At least one of the digested products (or some or all of the digested products) formed after digestion may still serve as a CD8 + epitope or a CD4 + epitope.
  • the artificial multi-antigen fusion protein further comprises a sequence selected from one or more optional elements of the group consisting of:
  • a tag sequence eg for purification, such as 6His
  • an adjuvant element sequence eg LRMK
  • the artificial multi-antigen fusion protein is from 100 to 2000 amino acids in length, preferably from 150 to 1500 amino acids, more preferably from 200 to 1000 amino acids or from 300 to 800 amino acids.
  • the mammal comprises: a human, a domestic animal (such as a cow, a sheep, a pig), a pet (such as a dog, a cat), a rodent, a rabbit, a monkey, and the like.
  • the antigenic segment of the antigen fusion protein covers ⁇ 10%, ⁇ 20%, ⁇ 30%, ⁇ 40%, ⁇ 50 of one or two or more target proteins %, ⁇ 60%, ⁇ 70%, ⁇ 80%, ⁇ 90%, more preferably 100% of the target protein amino acid sequence.
  • the fusion protein has the structure of Formula I:
  • A is an antigenic segment
  • C is a cathepsin cleavage site sequence
  • n is a positive integer ⁇ 3;
  • Y is a sequence represented by no or "Y0-B", wherein Y0 is a signal peptide sequence, a tag sequence, a transmembrane element sequence, an adjuvant element sequence, a cell necrosis inducing factor element sequence, or any combination of the above sequences, and B a sequence of no or cleavage sites;
  • Z is none, or a tag sequence, a transmembrane element sequence, an adjuvant element sequence, a cell necrosis inducing factor element sequence, or any combination of the above sequences;
  • the cleavage site sequence is different from C (ie, B ⁇ C).
  • said n is any integer from 5 to 100, preferably from 6 to 50, more preferably from 7 to 30.
  • composition comprising the artificial multi-antigen fusion protein of the first aspect of the invention and a pharmaceutically acceptable carrier.
  • the composition includes a pharmaceutical composition and a vaccine composition.
  • the dosage form of the composition comprises an injection, a lyophilized powder, a liquid preparation, an oral preparation, or a transdermal preparation.
  • a third aspect of the invention there is provided the use of the artificial multi-antigen fusion protein of the first aspect of the invention for the preparation of a prophylactic and/or therapeutic vaccine composition or pharmaceutical composition.
  • the vaccine composition comprises a vaccine composition against a pathogen (e.g., a virus), an anti-tumor vaccine composition.
  • a pathogen e.g., a virus
  • the pharmaceutical composition is for treating and/or preventing a disease selected from the group consisting of a bacterial-related disease, a virus-related disease, an autoimmune disease, a tumor-related disease, or a combination thereof.
  • the use of the artificial multi-antigen fusion protein of the first aspect of the invention characterized in that it is used for the preparation of a reagent or kit for detecting specific T cell immunity.
  • the specific T cell immunity comprises CD4 + T cell immunity and CD8 + T cell immunity.
  • such an agent or kit includes a skin test.
  • a method such as a method of treatment or prophylaxis, comprising the steps of: administering to a subject in need of treatment an artificial multi-antigen fusion protein according to the first aspect of the invention or a second aspect of the invention The composition of the aspect.
  • the method is for treating and/or preventing a pathogen infection or tumor.
  • the subject comprises a human and a non-human mammal.
  • the manner of administration includes intravenous injection, subcutaneous injection, oral administration, transdermal administration, and the like.
  • Figure 1 shows a schematic representation of the cloning site of the pNIC28a-Bsa4 expression vector.
  • Figure 2 shows electrophoretic identification of PCR products of E. coli containing the ESAT6/CFP10 gene (left) and SDS-PAGE electrophoresis analysis of the purified ESAT6/CFP10 multi-antigen fusion protein (right).
  • Figure 3 shows electrophoretic identification of PCR products of E. coli containing OVA: 242-352 gene (left) and SDS-PAGE electrophoresis analysis of purified OVA: 242-352 multi-antigen fusion protein (right).
  • Figure 4 shows electrophoretic identification of PCR products of E. coli containing HPV16-E7 gene (left) and SDS-PAGE electrophoresis analysis of purified HPV16-E7 multi-antigen fusion protein (right).
  • Figure 5 shows flow cytometry analysis of normal human PBMCs after treatment with HPV16-E7 multi-antigen fusion protein, a two-parameter scatter plot showing the distribution of CD54 and antigen in PBMC.
  • CD54 is labeled with PE and antigen is labeled with FITC.
  • Figure 6 shows the spots formed by the ESAT6/CFP10 multi-antigen fusion protein, the ESAT6/CFP10 mature protein and the commercially available T-SPOT.TB, respectively.
  • the results showed that the stimulation of total T lymphocytes, ESAT6/CFP10 multi-antigen fusion protein and the effect of antigen in commercial T-SPOT.TB were comparable (upper), while stimulation of CD8 + lymphocytes, ESAT6/CFP10 multi-antigen fusion The protein is significantly better than the ESAT6/CFP10 mature protein (bottom).
  • FIG. 7 shows the results of skin testing of the ESAT6/CFP10 multi-antigen fusion protein.
  • a positive control (PPD) a negative control
  • a recombinant ESAT6/CFP10 multi-antigen fusion protein denoted as "LRMK”
  • E6-C10 recombinant ESAT6-CFP10 mature protein
  • Figure 8 shows the distribution of SIINFEKL-MHC-I on DC2.4 cells by flow cytometry.
  • the histogram shows that the SIINFEKL peptide in the OVA (242-252) multi-antigen fusion protein was efficiently presented by DC cells at a concentration of 100 micrograms per ml (presentation efficiency 17.6%).
  • the original protein fragment containing the SIINFEKL peptide was not presented to the MHC class I molecule.
  • Figure 9 shows the survival-time curve of B16-HPV-E7 tumor-bearing mice (tumor cell inoculation day is day 0).
  • Figure 10 shows the survival-time relationship of B16-survivin mice (day of tumor cell inoculation is day 0).
  • the inventors After extensive and intensive research, the inventors have for the first time developed a protein vaccine having the advantages of being able to stimulate CD8 + T cell immunity, low production cost, and convenient quality control, and excellent comprehensive performance.
  • the vaccine of the present invention can simultaneously effectively stimulate stimulation of CD4 + T cell immunity as compared with existing vaccines.
  • the protein vaccine of the present invention is effective in stimulating CD8 + cells through the MHC-I antigen presenting pathway. The present invention has been completed on this basis.
  • the inventors have unexpectedly discovered through repeated practice that a multi-antigen fusion protein vaccine linked by a cathepsin cleavage site can be extremely efficiently presented by antigen-presenting cells via the MHC-I pathway.
  • the protein vaccine is very effective in stimulating CD8 + T cells and at the same time has the ability to stimulate CD4 + T cells.
  • the terms "protein of the invention”, “fusion protein of the invention”, “artificial multi-antigen fusion protein”, “antigen fusion protein”, “fusion protein of the invention for stimulating CD4 + and CD8 + T cell immune responses” Interchangeable use refers to artificially recombinant proteins having the structure of Formula I that are effective for simultaneously activating CD4 + and CD8 + T cell immune responses.
  • Short-segment polypeptide antigens can be presented to cells in addition to being able to enter the lysosomal enzyme to be presented to the MHC-II molecule. It is absorbed into the cytoplasm and processed and presented to MHC class I molecules. Protein-like antigens are first endocytosed by antigen-presenting cells, then passed through early phagosomes, late phagosomes, and finally into lysosomes. Typically, proteins that enter the lysosome are completely degraded to amino acids.
  • the protein will gradually degrade, and the resulting polypeptide can be bound by the lysosomal MHC-II molecule to form a relatively stable complex, which is presented to the cell surface and stimulates CD4 + T cell immunity.
  • protein antigens do not leak from the lysosome into the cytosol and therefore do not enter the MHC-I pathway to stimulate CD8 + cells.
  • the inventors have unexpectedly discovered that the antigen fusion protein specifically designed by the present invention not only stimulates CD8 + T cells, but also retains the original ability to stimulate CD4 + T cells.
  • the major protease in the lysosome of antigen presenting cells is cathepsin S.
  • Leu-Arg-Met-Lys is a preferred sequence for cathepsin S.
  • Leu-Arg-Met-Lys it is preferred to use Leu-Arg-Met-Lys to join a set of antigenic segments to form a novel artificial multi-antigen fusion protein and to stimulate CD4 + and CD8 + T cells.
  • Ordinary short-fragment antigen polypeptides can be directly absorbed by antigen-presenting cells into the cytoplasm and presented to MHC class-1 molecules.
  • Ordinary protein antigens are only phagocytized into lysosomes by antigen-presenting cells, and the common mature protein in lysosomes only binds to MHC-II, but only stimulates CD4+ T cells.
  • the cleavage first occurs at the ligation site in the lysosome due to the presence of a cathepsin cleavage site such as cathepsin S.
  • a set of polypeptide(s) is rapidly produced to achieve and facilitate cross-presentation, thereby simultaneously stimulating CD8 + T cells and CD4 + T cells.
  • the test of the present invention shows that when the fusion protein of the present invention enters the cell lysosome, it inhibits lysosomal function. In the case of the fusion protein, the fusion protein still disappears quickly, indicating that the protein of the present invention or its digested product can leak into the cytosol in the lysosome.
  • Fusion protein that stimulates CD4 + and CD8 + T cell immune responses
  • a single protein, a multi-antigen fusion protein, for the specific cellular immunoassay and as a prophylactic and therapeutic vaccine is formed by ligating a plurality of antigenic epitopes (or antigenic segments).
  • At least one, more preferably 2, 3, 4, 5 or more (eg, any positive integer of 6-20) CD8+ T cell table is included. Bit.
  • At least one, more preferably 2, 3, 4, 5 or more (eg, any positive integer of 6-20) CD4+ T cell table is included. Bit.
  • the sequence of the antigenic segment may be from any polypeptide having a stimulatory immune response, preferably a sequence (i.e., a T cell epitope) capable of stimulating a CD4 + or CD8 + T cell response.
  • Cathepsins are a group of cysteine proteases whose main function is to degrade proteins in lysosomes.
  • the known major function of cathepsin-S (cathepsin-S) is to participate in the antigen presentation process of MHC-2.
  • these polypeptides containing a CD8+ T cell epitope and a CD4+ T cell epitope may be ligated by any antigen presenting intracellular cathepsin recognition site, preferably using The Leu-Arg-Met-Lys sequence favored by cathepsin Cathepsin S.
  • cleavage sites for the expression of cathepsins B and K in the presenting cells may be employed; for example; Met-Lys-Arg ⁇ -Leu is the cleavage site of cathepsin B , and His-Pro-Gly ⁇ -Gly is a restriction site for cathepsin K, but the effect is particularly excellent is the cathepsin S restriction site.
  • the artificial multi-antigen fusion protein may employ different cathepsin S cleavage sites, 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(R) and X-Asn-Leu-Arg(R) are preferred ligase cleavage sites, wherein X is any amino acid and ⁇ represents the cleavage position.
  • the multi-antigen fusion protein covers more than 40% of the protein.
  • the antigen fusion protein covers the protein antigen sequence. At least 50%, 60%, 70%, 80%, 90%; optimally 100%.
  • the coverage of the HPV16-E7 multi-antigen fusion protein is 100%.
  • the coverage of OVA is 40% because OVA has only a single MHC-I epitope and the selected fragment includes this epitope.
  • the multi-antigen fusion protein may cover more than one antigenic protein.
  • the antigen fusion protein of the tuberculosis of the present invention covers the two protein sequences of ESAT6 and CFP10, which can reduce the cost of production and facilitate quality control. .
  • the artificial multi-antigen fusion protein of the present invention is different from a natural protein such as a virus, a bacterium, a parasite protein or a tumor antigen.
  • a natural protein such as a virus, a bacterium, a parasite protein or a tumor antigen.
  • One of the main differences is the loss of the spatial structure of the native protein and therefore the function of the native protein, thereby avoiding potential hazards such as pathogen proteins such as viral capsid proteins.
  • the artificial multi-antigen fusion protein of the present invention is also different from a short-fragment antigen polypeptide (e.g., an antigen peptide of ⁇ 70 or ⁇ 60 amino acids and an epitope polypeptide).
  • a short-fragment antigen polypeptide e.g., an antigen peptide of ⁇ 70 or ⁇ 60 amino acids and an epitope polypeptide.
  • the protein of the present invention has a large molecular weight and belongs to a macromolecular protein (rather than a small molecule polypeptide), and thus the pathway of entering the cell is different, thereby causing the antigen presentation pathway, the stimulation T cell mode and the mechanism to be different from the short fragment.
  • Antigenic polypeptide e.g., an antigen peptide of ⁇ 70 or ⁇ 60 amino acids and an epitope polypeptide.
  • the protein of the present invention can be obtained by mass production by recombinant methods. This is usually the cloning of the synthetic coding DNA into an expression vector, which is then transferred into a cell and then isolated from the host cell or fermentation product by conventional methods.
  • a method for preparing a typical antigen fusion protein includes the following steps:
  • an artificial multi-antigen fusion protein comprising or consisting of a series of antigenic segments of a specific length, wherein the respective antigenic segments are cleaved by the same or different cathepsins, based on the amino acid sequence of the protein antigen Points (such as leu-Arg-Met-Lys) are connected.
  • telomeres include prokaryotic cells and eukaryotic cells.
  • a commonly used prokaryotic cell is Escherichia coli.
  • Commonly used eukaryotic cells include, but are not limited to, yeast cells, insect cells, and mammalian cells.
  • the host cells used are E. coli, such as BL21 (DE3) and yeast, and CHO cells.
  • the invention also provides the use of a fusion protein of the invention, for example for immunodiagnosis and preparation of a vaccine.
  • Immunodiagnosis includes skin test and T cell spot enzyme-linked immunosorbent assay.
  • the skin test is to immunize the test subject with the multi-antigen fusion protein of the present invention, and then observe the skin reaction of the individual to determine whether the individual is exposed to a similar antigen.
  • the immunodiagnosis is a fusion protein of the present invention as a stimulation source for detecting an individual's T cell immune response. For example, by observing the production of gamma interferon by the T cells of an individual after being stimulated by the ESAT6-CFP10 multi-antigen fusion protein of the present invention, it is possible to determine whether the patient is infected with tuberculosis or the stage, or for prognosis.
  • Vaccines include preventive and therapeutic vaccines. The latter is primarily involved in the activation of T cells, particularly CD8 + killer T cell (CTL) cells. Activation of CTL cells is restricted by MHC-I molecules.
  • CTL CD8 + killer T cell
  • MHC-I molecules MHC-I molecules.
  • the HPV16-E7 multi-antigen fusion protein of the present invention can be phagocytosed by human antigen-presenting cells and activate antigen-presenting cells.
  • 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.
  • the fusion protein of the present invention utilizes the endocytosis of the antigen-presenting cell itself and the cathepsin recognition sequence in the lysosome, and the plurality of epitope peptides containing the CD4 + and CD8 + epitopes are linked into a single protein and expressed. After purification, it can be directly applied to immunodiagnosis or as a preventive and therapeutic vaccine.
  • the protein or composition of the present invention when used as a prophylactic and therapeutic vaccine, it can be injected directly into the animal either alone or in combination with an adjuvant, or used in vitro with a cell therapy method.
  • a cell therapy method For example, DC/NK cell culture is added to stimulate production of specific antigen-presenting cells, and then the antigen-presenting cells are used to stimulate production of specific CD8 + T cells (i.e., CTLs) in vivo/in vitro.
  • these in vitro sensitized specific antigen-presenting cells and/or CTL antigens produced in vitro can be returned to the body of the corresponding subject for various purposes such as antiviral, antitumor and the like.
  • the protein of the present invention or the in vitro sensitized cells are preferably administered to an individual by the following administration from the group consisting of intravenous injection, perfusion, subcutaneous injection, transdermal administration, and the like.
  • the antigen fusion protein of the present invention can be phagocytosed by antigen-presenting cells and activates CD8 + cells.
  • Reagents and techniques for immunodiagnosis can be developed using the fusion protein of the present invention. For example, specific cellular immunity is detected by a skin test, or specific cellular immunity is detected by an ELISPOT test.
  • a therapeutic or prophylactic drug or vaccine can be developed, including DC cells and/or CTL cell preparations prepared by the fusion protein sensitization of the present invention, and the industrial application value is immeasurable.
  • the cost of the method and product of the present invention is significantly reduced, typically by at least 80% or more, compared to conventional protocols using a short set of polypeptides; and quality control is easy, saving a lot of manpower, material and time.
  • the antigen fusion protein is designed based on the amino acid sequence of the protein.
  • the antigen fusion protein is composed of a series of 25-35 amino acid polypeptide fragments (short fragment antigen peptides) linked by the same tissue-cutting S-preferred sequence site (Leu-Arg-Met-Lys).
  • the DNA encoding the antigen fusion protein is codon optimized to a codon that is suitable for E. coli preference.
  • the DNA coding sequence was prepared by full artificial synthesis and added at its 5' end. (SEQ ID NO.: 7) and 3' end addition (SEQ ID NO.: 8) sequence.
  • the synthesized DNA molecule was treated with T4 DNA polymerase and dCTP for 30 minutes.
  • a conventional pNIC28-Bsa4 vector (obtained from Oxford University; US 8,148,100B2; GenBank ID: EF198106) was digested with BsaI for 1 hour.
  • An important component of this vector that is relevant for clonal expression is shown in Figure 1.
  • the linearized vector was separated by 1% agarose gel electrophoresis and treated with T4 DNA polymerase and dGTP for 30 minutes. After mixing the two T4 DNA polymerase-treated products, the conventional competent E. coli DN5 ⁇ was transformed, and the monoclonal culture was picked up by plating. Bacterial fluid PCR identified positive colonies.
  • LC-MS analysis also showed that the molecular weight measurements of the purified TB-antigen fusion protein (30974 Da), the OVA antigen fusion protein (16589 Da), and the HPV 16-E7 antigen fusion protein (16231 Da) were all consistent with the predicted values.
  • Plasmid DNA of a positive colony containing the coding sequence of the artificial multi-antigen fusion protein was extracted, and Escherichia coli BL21 (DE3) was transformed by a conventional method.
  • the transformed single colonies were inoculated into low-salt LB medium and cultured overnight at 37 ° C.
  • Induction of protein expression was carried out by adding 0.2 mM IPTG, and incubation was continued for 16 hours at 18 ° C, and then centrifuged at 4000 rpm to collect the cells and resuspend in phosphate buffer.
  • the cells were lysed by sonication and the inclusion bodies were collected by centrifugation.
  • the inclusion body was dissolved in denaturing buffer (8M urea in HEPES buffer at pH 7.4), passed through a Ni-NTA column, and the histidine-labeled antigen fusion protein was bound to the column and removed by thorough washing. After the heteroprotein, the antigen fusion protein is eluted with a urea elution buffer.
  • the eluted antigen fusion protein was first diluted 8 times with PBS (pH 9.5) containing 0.5 M arginine, and then urea was removed by dialysis against PBS (Fig. 2 right and Fig. 3 right).
  • the cells were lysed by sonication, and the supernatant containing the protein of interest was collected by centrifugation, passed through a Ni-NTA column, and the histidine-labeled antigen fusion protein was bound to the column, and after extensive washing to remove the heteroprotein, the HPV16-E7 antigen was used.
  • the fusion protein was eluted with an imidazole-containing elution buffer ( Figure 4 right).
  • the cells were harvested by EDTA treatment and washed twice with 1 ml of ice-cold FACS buffer (2% FCS in PBS). Then, PE-labeled commercial monoclonal antibody 25.D1-16 was added to a concentration of 0.6 ⁇ g/ml. Incubation was carried out for 30 minutes at 4 ° C, the cells were washed three times with FACS buffer, and finally the cells were resuspended in 0.2 ml of FACS fixation buffer (BD) and subjected to flow cytometry analysis.
  • FACS fixation buffer BD
  • the cell concentration was adjusted to 5 ⁇ 10 6 viable cells/ml.
  • the .TB kit comes with the antigen T-spot A and T-spot B as the stimulus source; or the mature CFP10-ESAT6 fusion native protein (denoted as TB-ESAT6-CFP10) as the control stimulus).
  • the specific anti-CD4 magnetic beads were used to clear the patient's CD4 + T cells, and the residual T cells were mainly CD8 + T cells. On the basis of this, the difference between the multi-antigen fusion protein and the CFP10 mature protein was compared.
  • the TB-antigen fusion protein of the present invention has a significant stimulating effect on CD8 + T cells.
  • the wild-type sequence of ESAT6-CFP10 mature protein has almost no stimulating effect on CD8 + T cells (Fig. 6 ,under).
  • Antigen fusion protein-loaded DC induces specific CD8 + T cells in vitro
  • Preparation of antigen-loaded DC cells After isolation of the obtained single nuclear peripheral blood cells (PBMC), partial cryopreservation, the remaining cells were adjusted to a cell concentration of 1 ⁇ 10 7 cells / mL, put into the conventional AIM-V After culturing for 2 hours in the medium, the suspension cells were removed (further frozen), and AIM-V medium containing GM-CSF (1000 IU/ml) and IL-4 (50 IU/ml) was added to continue at 37 ° C, 5% CO. The culture was carried out under 2 , and the HPV16-E7 antigen fusion protein (50 ⁇ g/ml) or recombinant HPV16-E7 protein (50 ⁇ g/ml) was added on the third day.
  • PBMC peripheral blood cells
  • Antigen-loaded DCs sensitized CD8 + T cells in vitro On the fifth day, adherent DC cells were harvested while cryopreserved PBMCs were resected, CD8 positive cells were isolated with CD8 magnetic beads, and CD8 negative cells were cryopreserved.
  • CD8 positive cells DC cells were co-cultured in a ratio of 5:1, and IL-2 100 IU/ml, IL-7 25 IU/ml was added. In addition, IL-2 (100 IU/ml) and IL-7 (25 IU/ml) or half-time replacement were added every 2 days.
  • CD8-negative cells were resuscitated, and after treatment with mitomycin, CD8-negative cells were added to the CD8-negative cells at a ratio of 1/10 and subjected to secondary stimulation, supplemented with corresponding protein 50 ⁇ g/ml, and supplemented with IL.
  • IL-2 100 IU/ml, IL-7 25 IU/ml.
  • IL-2 100 IU/ml and IL-7 25 IU/ml were added every two days.
  • Preparation of antigen-presenting cells The suspension cells which were additionally frozen on the first day were taken out and resuscitated, cultured in a plate coated with CD19 monoclonal antibody for 2 hours, and the non-adherent cells were discarded, and the CD19-positive cells were suspended by washing with the medium. The cell concentration was adjusted to 5 ⁇ 10 5 /ml, and 2 ml was taken. After incubation at 37 ° C, 5% CO 2 for 48 h, IL-4 was added to a concentration of 100 ⁇ g/ml.
  • ⁇ -interferon release assay evaluates the effect of multi-antigen fusion protein-loaded DCs in inducing specific CD8 + T in vitro: in 96-well plates, add CD ⁇ positive cells 5 ⁇ 10 4 /50 ⁇ l, and add the corresponding protein (HPV16-E7)
  • the antigen fusion protein (SEQ ID NO.: 5) or the conventional recombinant HPV 16-E7 protein) was incubated for 2 h at 50 ⁇ g/ml, 37 ° C, 5% CO 2 .
  • Table 1 ⁇ -interferon release assay HPV16-E7 antigen fusion protein and recombinant HPV16-E7 protein stimulate DC-induced specific CD8 + T cells in vitro
  • Guinea pigs weighing about 250 g were intradermally injected with PPD 0.1 ml, and the skin test reaction was observed at 24 hours.
  • the guinea pigs with negative skin test were randomly divided into 5 groups.
  • 0.2 ml of 40 mg/ml M. tuberculosis H37Rv suspension was injected subcutaneously, and immunized once a week for 5 times.
  • the guinea pigs were randomly divided into 5 groups, the guinea pigs were plucked from the back, and the positive control (PPD), the negative control (recombinant protein irrelevant to nodules), and the recombinant ESAT6-CFP10 protein were injected into the rim.
  • PPD positive control
  • the negative control negative control
  • the recombinant ESAT6-CFP10 protein were injected into the rim.
  • a mixture of recombinant ESAT6-CFP10-antigen fusion protein (1 mg/ml, 0.1 mg/ml) was 0.1 ml each, and the longitudinal and transverse diameters (mm) of the skin redness at each injection point on the back of the guinea pig were measured 24 hours after the injection, and recorded. average value.
  • the results of the 24-hour skin reaction are shown in Figure 7.
  • the average skin induration diameter of the TB-antigen fusion protein group (SEQ ID NO.: 1) is greater than the PPD and ESAT6-CFP10 native protein.
  • the control group there was no redness and induration in the local skin of guinea pigs. This indicates that the TB-antigen fusion protein can be used in skin tests to effectively detect tuberculosis infection.
  • E6-C10 represents ESAT6-CFP10-native protein
  • LRMK represents a TB-antigen fusion protein in which each antigenic segment is linked by a cathepsin S cleavage site, and the sequence is as shown in SEQ ID NO.: TEV indicates a control protein formed by replacing all of the cathepsin S cleavage sites in SEQ ID NO.: 1 with a TEV cleavage site (a non-cathepsin cleavage site).
  • T cell epitopes can be efficiently presented to MHC-I molecules (only MHC-I molecules can be expressed to activate CD8 + T cells, resulting in cytotoxic reactions, Kill bacterial/viral infected cells or cancerous cells).
  • the OVA antigen fusion protein was designed and expressed according to the amino acid sequence of 242-352 of chicken ovalbumin (OVA), which contains the MHC-1 epitope SIINFKL.
  • OVA protein fragment OVA 255-340 containing this epitope was also used as a control.
  • a mouse dendritic cell line (DC2.4) was used as an antigen presenting cell to study the presentation of SIINFEKL in the OVA antigen fusion protein. Finally, detection was performed with a T cell receptor-like antibody (identifying the SIINFEKL/MHC-I complex).
  • OVA protein fragment 100 ⁇ g/ml (or OVA antigen fusion protein (SEQ ID NO.: 3) 30 ⁇ g/ml) was mixed with DC2.4 cells with 10% heat-inactivated fetal bovine serum (sigma), 2 mM L- Glutamine RPMI1640 (Sigma) was cultured for 13 hours and stained with a PE-labeled commercially available 25.D1-16 antibody (this monoclonal antibody 25.D1-16 specifically recognizes MHC-1 molecules that bind to SIINFEKL). ). 1 ml of ice-cold FACS buffer (2% FCS in PBS) was washed twice.
  • PE-labeled monoclonal antibody 25.D1-16 was added to a concentration of 0.6 ⁇ g/ml. Incubation was carried out for 30 minutes at 4 ° C, the cells were washed three times with FACS buffer, and finally the cells were resuspended in 0.2 ml of FACS fixation buffer (BD) and subjected to flow cytometry analysis.
  • FACS fixation buffer BD
  • the 8 peptide (SIINFEKL) binds directly to the MHC-1 molecule on the cell surface, so more than 80% of the cells stain positive (C1 and C2 in Fig. 8).
  • the SIINFEKL peptide in the OVA native protein cannot be processed by DC cell processing onto the MHC-1 molecule (A1 and A2 in Figure 8). (Note: The blue and red peaks in Figure 8A2 overlap almost.)
  • SIINFIKL peptide in the OVA-antigen fusion protein (SEQ ID NO.: 3) is efficiently processed by the mouse dendritic cell line DC2.4 and presented to the cell. The surface, resulting in up to about 17.6% of cells stained positive for the 25.D1-16 antibody (B1 and B2 in Figure 8).
  • Antigen solution 2 mg/ml (control group: recombinant HPV-E7 protein, recombinant Survivin protein; experimental group: HPV-E7 antigen fusion protein (SEQ ID NO.: 5), Survivin antigen fusion protein) mixed with 1 mg/ml MPL in equal volume
  • control group recombinant HPV-E7 protein, recombinant Survivin protein
  • experimental group HPV-E7 antigen fusion protein (SEQ ID NO.: 5), Survivin antigen fusion protein
  • 10 randomly divided C57/B6 female mice with a weekly age of 7-8 weeks and a body weight of 25 g were injected subcutaneously in the neck on day 0, 21 and 42 days. Immune suspension.
  • mice For each immunized mouse, 100 ⁇ l of mouse B16 cells stably transfected with human HPV-E7 or Survivin were subcutaneously inoculated into the right side of the mouse with a syringe (BD), and the amount of cells injected was 7 ⁇ 10 5 cells, and began to count and evaluate the survival of the mice. The results are shown in Figures 9 and 10.
  • HPV-E7 antigen fusion protein of the present invention significantly prolonged the survival rate of mice compared with the recombinant HPV-E7 protein control group (Fig. 9), and 50% of the mice survived on the 25th day; The protein control group died all.
  • the Survivin antigen fusion protein of the present invention significantly prolonged the survival rate of the mice (Fig. 10), and 70% of the mice survived on the 25th day; while the recombinant protein control group only survived 40%.
  • protein antigens were not clinically used as vaccines for stimulating CD8 + cellular immunity.
  • Protein vaccine production and quality control are mature technologies and many clinical drugs and vaccines (stimulating antibodies) are proteins. If a mature method for producing proteins can be used, it would be ideal to produce a vaccine that stimulates CD8 + T cell immunity.
  • a mature method for producing proteins it would be ideal to produce a vaccine that stimulates CD8 + T cell immunity.
  • how to transfer protein antigens from the endocytosis-lysosomal-MHC-II pathway to the cytoplasmic-MHC-I pathway is a very challenging subject. Many immunologists and vaccinologists are trying but there is no breakthrough yet.
  • the main obstacles include:
  • the lysosomal membrane Since the role of the lysosomal membrane is to separate the acidic substance in the lysosome from the cytoplasm, the lysosome is not an easily leaking organelle. Whether a conventional expressed protein or recombinant protein escapes or leaks out of lysosomes even if it is not completely degraded is an unknown number.
  • the present inventors designed a plurality of antigen fusion proteins based on enzymatic principles and biorecombination techniques, and after extensive screening, determined a novel antigen fusion protein structure: in a series of antigen-containing segments.
  • the peptides are linked by a cleavage site of cathepsin.
  • the cathepsin in the lysosome degrades the antigen fusion protein into a polypeptide containing a different epitope.
  • the experiment of the present invention unexpectedly confirmed that after the fusion protein of the specific structure of the present invention is degraded, the degraded polypeptide can leak out of the lysosome and enter the cytoplasm, and is processed and presented to the MHC class I molecule, thereby The MHC-I antigen presentation pathway is initiated to stimulate CD8 + cells; and the originally stimulated CD4 + T cell function can be retained or enhanced.

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Abstract

提供了一种人工多抗原融合蛋白及其制备法，所述融合蛋白能够有效刺激CD8+T细胞免疫和CD4+T细胞免疫，可应用于免疫诊断或者作为预防性或治疗性疫苗。

Description

人工多抗原融合蛋白及其制备和应用 技术领域
本发明涉及医药领域，更具体地涉及能同时刺激CD4+和CD8+T细胞免疫应答的人工多抗原融合蛋白及其制法和应用。本发明的人工多抗原融合蛋白，经表达纯化后可直接用于免疫诊断或者用作预防性或治疗性疫苗。
背景技术
人体感染细菌病毒或者受到癌细胞刺激后会产生一系列体液免疫和细胞免疫变化。细胞免疫对肿瘤细胞和已感染入细胞内的细菌及病毒非常重要。人体初次受到上述外源刺激后，会使T淋巴细胞致敏，转化为记忆T淋巴细胞。当人体再次接触同一种抗原后，会迅速产生特异性免疫反应，包括效应T淋巴细胞反应，产生多种细胞因子发挥免疫学效应(细胞免疫)。由于细胞免疫在控制和清除细菌、病毒及肿瘤中起着不可忽视的作用，因此细胞免疫的疫苗对预防细菌病毒感染和肿瘤治疗有一定作用。
目前临床上对于肿瘤及一些细胞内感染的细菌及病毒，尚无有效的化学药物。因此，刺激细胞免疫的治疗性疫苗对控制肿瘤及细菌病毒感染起着非常重要的作用。目前，美国FDA已经批准治疗前列腺癌的治疗疫苗上市。
另外对细胞免疫的监测对于疾病的治疗、预后以及评价疫苗的有效性至关重要。
常用的检测细胞免疫的方法是酶联免疫斑点法(enzyme linked immunospot assay，ELISPOT)，该方法可以从单细胞水平检测分泌细胞因子的T细胞。例如，可以通过检测结核(TB)特异性抗原剌激后分泌细胞因子的T细胞数量，来评价细胞免疫功能。对结核来说，检测细胞免疫是诊断是否感染TB的方法之一。2008年美国FDA己批准ELISPOT商品化试剂盒T-SPOT用于结核感染的诊断。这些诊断试剂盒基本都是使用18-20个氨基酸的多肽混合物作为刺激源。这些多肽被外周血的抗原递呈细胞吞噬后，加工递呈后与自身HLA抗原一起到细胞表面，然后被记忆T淋巴细胞的T细胞受体识别。T细胞被刺激后，产生以伽马干扰素为代表的细胞因子而被检测到。
人体内有许多不同克隆的T细胞，每个克隆都需要被不同的短肽加HLA刺激；不同人体也有不同的T细胞克隆，不同人体的HLA抗原也不同。如要选择某个HLA人群的T细胞，就要鉴定、选择该类人群的T细胞表位抗原。鉴定HLA表型及鉴定T细胞表位序列都是费时费力的工作。如要覆盖所有人群及某个刺激源(蛋白质)的所有T细胞识别表位最常见的方法就是合成长度18-20个氨基酸，甚至更长的多肽。然而要在临床上使用，每种多肽都需要建立严格的生产工艺和质量检测标准。以用于TB检测的ESAT6(96个氨基酸)和CFP10(100个氨基酸)为例，通常需要20个以上的多肽混合物才能覆盖两个蛋白抗原的全部表位，导致成本很高，试剂盒价格昂贵，因而在一定 程度上限制了其广泛应用。一种替代方案是选择有限的几个多肽作为刺激源，但由于不同人群的HLA多态性，T细胞识别的抗原表位有差异，因此这种方案尽管节约成本但是也降低了表位的覆盖率。
预防与治疗性疫苗被送到抗原递呈细胞尤其树突状细胞(DC)处理提呈后，能刺激和促使抗原特异性的细胞毒CD8+(CTL)和辅助性CD4+T淋巴细胞。辅助性CD4+T细胞能够有效刺激和放大细胞毒CD8+T细胞及帮助B细胞产生抗体。CD8+T细胞能特异识别并溶解含靶抗原的靶细胞。特异性CD8+T细胞的激活依赖于抗原能被有效递呈到MHC-I类分子(在人类即HLA-I类抗原)。CD8+细胞毒性T淋巴细胞由于能够直接识别和破坏肿瘤细胞或者病毒等感染的细胞，是预防及治疗性细胞免疫疫苗的主要有效成分。因此，目前绝大多数治疗性疫苗的设计都围绕在如何刺激机体产生特异性的识别肿瘤或者病毒抗原CD8+T细胞。
目前主流的观点是只有在抗原呈递细胞(APC)浆内的抗原才能被蛋白酶体降解成小分子肽，通过TAP蛋白运输进入内质网，其中CTL表位多肽与MHC-I(HLA-I)类结合并呈送到抗原递呈细胞表面，与CD8+T细胞的特异性受体结合后，特异性激活后者。同时传统观点还认为，只有在细胞浆内转录翻译后产生的蛋白抗原才能进入MHC-I(HLA-I)的提呈途径。这也是目前治疗性疫苗的核心设计策略，即开发能在细胞浆内复制转录翻译的疫苗。如果一个蛋白质抗原是被细胞吞噬进来的，那么这个抗原就不会进入细胞浆，而是进入内吞体及溶酶体，因此这个蛋白质抗原就无法有效刺激CD8+T细胞。
目前能有效刺激CD8+T细胞免疫的疫苗主要包括DNA疫苗、表位多肽疫苗及细菌或病毒载体疫苗。这些疫苗具有一些明显的缺点：DNA疫苗进入细胞的效率低，而且有一定整合入基因组中的危险；单一表位多肽疫苗覆盖的人群非常小而使其应用范围减小；细菌/病毒载体疫苗副作用大，而且易使机体产生针对载体本身的免疫从而降低对靶抗原的免疫力。另外，许多人群可能在免疫前已有针对这些细菌/病毒的免疫能力，因而疫苗还没起作用就已被销毁，从而大大降低了其使用范围。
蛋白质疫苗是一种安全的疫苗，因为蛋白质不能自我复制。一般，蛋白质都是被抗原递呈细胞(antigen presentation cells,APC)吞噬进内吞体及溶酶体，这样就不会进入胞浆也不能被胞浆蛋白酶体降解并被递呈到MHC-I类分子上，因此蛋白抗原疫苗不会刺激CTL反应。蛋白质被APC细胞内吞噬后进入溶酶体，被降解成小分子肽，然后其中的某些多肽(CD4表位)与溶酶体膜上的的MHC-II类分子结合，递呈到细胞表面刺激CD4+T细胞。CD4+可以帮助B细胞产生抗体。另外，蛋白质疫苗可以刺激B细胞。因此一直以来蛋白质抗原主要用于激发机体产生抗体。有研究发现，APC也可以从外部摄取抗原，并递呈到MHC-I类分子上，刺激CTL。这个过程被称为抗原交叉呈递。但是这个过程的效率非常之低，以至于没有人能够设计出能够刺激CD8+T细胞免疫的蛋白质疫苗。
同样由于人的HLA的限制性，要制备广泛的多肽类疫苗，最佳的方法是制备一系 列的多肽，使得其序列覆盖蛋白质所有CD4与CD8表位，然而每种多肽都需要建立严格的成产工艺和质量检测标准，导致成本极高，无法产业化。
综上所述，目前本领域尚缺乏令人满意的能够刺激CD8+T细胞免疫的疫苗。因此，本领域迫切需要开发新的能够有效刺激CD8+T细胞免疫、质控容易且安全性高的疫苗。
发明内容
本发明的目的就是提供一种具有能够有效刺激CD8+T细胞免疫、质控容易且安全性高等特点的蛋白质疫苗。
在本发明的第一方面，提供了一种人工多抗原融合蛋白，所述的人工多抗原融合蛋白被施用于哺乳动物对象时，可在所述对象中同时刺激CD4+和CD8+T细胞免疫应答。
在另一优选例中，所述的CD4+T细胞免疫应答和CD8+T细胞免疫应答导致所述的T细胞识别在细胞表面携带所述抗原的靶细胞。
在另一优选例中，所述的人工多抗原融合蛋白中的抗原来源于病毒、细菌、寄生虫、衣原体、肿瘤细胞、或其组合。
在另一优选例中，所述的人工多抗原融合蛋白含有≥3个，较佳地≥5个，更佳地≥10个抗原区段。
在另一优选例中，所述的抗原区段的数量上限≤200个，较佳地≤100个，更佳地≤50个。
在另一优选例中，所述的各抗原区段可各不相同、部分相同、或完全相同。
在另一优选例中，所述的各抗原区段来自相同或不同的病原体、或来自相同或不同的物种、或者所述的各抗原区段是人工序列(即人工设计的、在自然界不存在的序列)。
在另一优选例中，所述的各抗原区段来自一个蛋白或多个不同蛋白。
在另一优选例中，所述的各抗原区段来自病毒抗原、细菌抗原、寄生虫抗原、衣原体抗原、肿瘤抗原、或其组合。
在另一优选例中，所述各抗原区段中包括至少一个(较佳地至少2个)CD8+表位或能够刺激CD8+T细胞免疫应答的基序序列；和至少一个(较佳地至少2个)CD4+表位或能够刺激CD4+T细胞免疫应答的基序序列。
在另一优选例中，所述的各抗原区段中包括至少一个(较佳地至少2个)同时作为CD8+表位和CD4+表位的氨基酸序列。
在另一优选例中，所述的各抗原区段的长度为8-50个氨基酸，较佳地为10-40个氨基酸，更佳地为15-35个氨基酸。
在另一优选例中，所述的人工多抗原融合蛋白还包括位于各抗原区段之间的酶切位点序列。
在另一优选例中，所述的酶切位点序列包括组织蛋白酶的酶切位点。
在另一优选例中，所述的组织蛋白酶酶切位点选自下组：组织蛋白酶S酶切位点(如Leu-Arg-Met-Lys或类似酶切位点)、组织蛋白酶B的酶切位点(如Met-Lys-Arg-Leu或类似酶切位点)、组织蛋白酶K的酶切位点(如His-Pro-Gly-Gly或类似酶切位点)、或其组合。
在另一优选例中，所述的组织蛋白酶S酶切位点选自下组：Arg-Cys-Gly↓-Leu、Thr-Val-Gly↓-Leu、Thr-Val-Gln↓-Leu、X-Asn-Leu-Arg↓、X-Pro-Leu-Arg↓、X-Ile-Val-Gln↓和X-Arg-Met-Lys↓；其中各X独立地为任一天然氨基酸,↓表示酶切位置。
在另一优选例中，所述的组织蛋白酶S酶切位点为X-Arg-Met-Lys(如Leu-Arg-Met-Lys)、X-Ile-Val-Gln、或其组合。
在另一优选例中，所述的人工多抗原融合蛋白中，通过所述的酶切位点序列将各抗原区段直接相连。
在另一优选例中，所述的用于连接各抗原区段的酶切位点序列是相同的或不同的。
在另一优选例中，所述的用于连接各抗原区段的酶切位点序列是相同的。
在另一优选例中，所述的各抗原区段自身不含有所述的酶切位点序列；或者所述的抗原区段自身含有所述的酶切位点序列，但所述抗原区段酶切后形成的至少一个酶切产物(或部分或全部酶切产物)仍可作为CD8+表位或CD4+表位。
在另一优选例中，所述的人工多抗原融合蛋白还含有选自下组的一个或多个任选元件的序列：
(a)标签序列(如用于纯化，如6His)；
(b)信号肽序列；
(c)穿膜元件序列(如CPP)
(d)佐剂元件序列(如LRMK)；
(e)细胞坏死诱导因子元件序列。
在另一优选例中，所述的人工多抗原融合蛋白的长度为100-2000个氨基酸，较佳地为150-1500个氨基酸，更佳地为200-1000个氨基酸或300-800个氨基酸。
在另一优选例中，所述的哺乳动物包括：人、家畜(如牛、羊、猪)、宠物(如狗、猫)、啮齿动物、兔、猴等。
在另一优选例中，所述抗原融合蛋白中的所述抗原区段覆盖了一个或二个、或更多靶蛋白的≥10％、≥20％、≥30％、≥40％、≥50％、≥60％、≥70％、≥80％、≥90％，更佳地100％的靶蛋白氨基酸序列。
在另一优选例中，所述融合蛋白具有式I结构：
Y-(A-C)n-Z  (I)
式中，
A为抗原区段；
C为组织蛋白酶酶切位点序列；
n为≥3的正整数；
Y为无或“Y0-B”所示的序列，其中，Y0为信号肽序列、标签序列、穿膜元件序列、佐剂元件序列、细胞坏死诱导因子元件序列、或上述序列任意组合，而B为无或切割位点(cleavage site)序列；
Z为无，或标签序列、穿膜元件序列、佐剂元件序列、细胞坏死诱导因子元件序列、或上述序列任意组合；
附加条件是，当Z为无时，最后一个“A-C”中的C可以为无。
在另一优选例中，所述的切割位点(cleavage site)序列不同于C(即B≠C)。
在另一优选例中，所述的切割位点序列与C相同(即B＝C)。
在另一优选例中，所述的n为5-100，较佳地为6-50，更佳地7-30的任一整数。
在本发明的第二方面，提供了一种组合物，所述的组合物含有本发明第一方面所述的人工多抗原融合蛋白和药学上可接受的载体。
在另一优选例中，所述的组合物包括药物组合物和疫苗组合物。
在另一优选例中，所述的组合物的剂型包括：注射剂、冻干粉末、液体制剂、口服制剂、或透皮制剂。
在本发明的第三方面，提供了本发明第一方面所述的人工多抗原融合蛋白的用途，它被用于制备预防性和/或治疗性的疫苗组合物或药物组合物。
在另一优选例中，所述的疫苗组合物包括抗病原体(如病毒)的疫苗组合物、抗肿瘤的疫苗组合物。
在另一优选例中，所述药物组合物用于治疗和/或预防选自下组的疾病：细菌相关疾病、病毒相关疾病、自身免疫病、肿瘤相关疾病或其组合。
在本发明的第四方面，提供了本发明第一方面所述的人工多抗原融合蛋白的用途，其特征在于，用于制备检测特异性T细胞免疫的试剂或试剂盒。
在另一优选例中，所述的特异性T细胞免疫包括CD4+T细胞免疫和CD8+T细胞免疫。
在另一优选例中，这种试剂或试剂盒包括用于皮肤试验。
在本发明的第五方面，提供了一种方法(如治疗或预防方法)，该方法包括步骤：给需要治疗的对象施用本发明第一方面所述的人工多抗原融合蛋白或本发明第二方面所述的组合物。
在另一优选例中，所述的方法用于治疗和/或预防病原体感染或肿瘤。
在另一优选例中，所述的对象包括人和非人哺乳动物。
在另一优选例中，所述的施用的方式包括：静脉注射、皮下注射、口服、透皮给药等。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了pNIC28a-Bsa4表达载体克隆位点示意图。
图2显示了含有ESAT6/CFP10基因的大肠杆菌的PCR产物电泳鉴定(左)以及纯化的ESAT6/CFP10多抗原融合蛋白的SDS-PAGE电泳分析(右)。
图3显示了含有OVA：242-352基因的大肠杆菌的PCR产物电泳鉴定(左)以及纯化的OVA：242-352多抗原融合蛋白的SDS-PAGE电泳分析(右)。
图4显示了含有HPV16-E7基因的大肠杆菌的PCR产物电泳鉴定(左)以及纯化的HPV16-E7多抗原融合蛋白的SDS-PAGE电泳分析(右)。
图5显示了流式细胞术分析正常人PBMC经过含有HPV16-E7多抗原融合蛋白处理后，双参数散点图显示PBMC中CD54和抗原的分布。CD54为PE标记而抗原用FITC标记。
图6显示了ESAT6/CFP10多抗原融合蛋白，ESAT6/CFP10成熟蛋白和商业化T-SPOT.TB中抗原分别刺激后形成的斑点。结果显示，对总T淋巴细胞的刺激，ESAT6/CFP10多抗原融合蛋白和商业化T-SPOT.TB中抗原的效果相当(上)，而对CD8+淋巴细胞的刺激，ESAT6/CFP10多抗原融合蛋白的效果显著优于ESAT6/CFP10成熟蛋白(下)。
图7显示了ESAT6/CFP10多抗原融合蛋白的皮肤测试结果。其中显示了四组由灭活结核分枝杆菌H37Rv来源的蛋白/多肽致敏豚鼠的皮肤测试结果。以轮圈法皮内注射阳性对照(PPD)、阴性对照，重组ESAT6/CFP10多抗原融合蛋白(记为“LRMK”)，重组ESAT6-CFP10成熟蛋白(记为“E6-C10”)各0.1ml，于注射后24小时分别测量豚鼠各注射点皮肤红肿的面积。
图8显示了流式细胞术分析DC2.4细胞上SIINFEKL-MHC-I的分布。直方图显示在100微克每毫升的浓度时OVA(242-252)多抗原融合蛋白中SIINFEKL肽能有效地被DC细胞递呈(递呈效率17.6％)。而同样含有SIINFEKL肽的原始蛋白质片段却不能递呈到MHC-I类分子上。
图9显示了B16-HPV-E7荷瘤小鼠的存活率-时间曲线(肿瘤细胞接种日为第0天)。
图10显示了B16-survivin小鼠的存活率-时间关系(肿瘤细胞接种日为第0天)。
具体实施方式
本发明人经过广泛而深入的研究，经过不断尝试和反复摸索，首次开发了一种具有能够刺激CD8+T细胞免疫、生产成本低且质控方便等优点、综合性能优异的蛋白质 疫苗。此外，与现有的疫苗相比，本发明疫苗还可同时有效刺激刺激CD4+T细胞免疫。实验证明，本发明的蛋白质疫苗能够有效地通过MHC-I抗原提呈途径而刺激CD8+细胞。在此基础上完成了本发明。
具体地，本发明人经过反复实践意外发现，基于用组织蛋白酶酶切位点连接的多抗原融合蛋白疫苗，可以极其有效地被抗原提呈细胞经MHC-I途径提呈。体外实验和体内实验表明，该蛋白质疫苗能够很有效地刺激CD8+T细胞，并且同时具有刺激CD4+T细胞的能力。
术语
如本文所用，术语“本发明蛋白”、“本发明融合蛋白”、“人工多抗原融合蛋白”、“抗原融合蛋白”、“本发明的刺激CD4+和CD8+T细胞免疫应答的融合蛋白”可互换使用，指具有式I结构的、可以有效同时激活CD4+和CD8+T细胞免疫应答的人工重组蛋白。
溶酶体和MHC-I途径
短片段多肽抗原(比如商业化的T.SPOT.TB试剂盒用于刺激人抗原递呈细胞的抗原)除了能进入溶酶体酶被递呈到MHC-II分子上，也能被递呈细胞吸收到细胞浆中，被加工递呈到MHC-I类分子上。而蛋白质类抗原则首先被抗原递呈细胞内吞，然后经过早期吞噬体，晚期吞噬体，最后进入溶酶体。通常，进入溶酶体的蛋白会被完全降解为氨基酸。但在某些情况下，蛋白会逐渐降解，产生的多肽能被位于溶酶体的MHC-II分子结合形成相对稳定的复合物，递呈到细胞表面，刺激CD4+T细胞免疫。通常情况下，蛋白抗原不会从溶酶体漏到细胞浆中，因此不会进入MHC-I途径刺激CD8+细胞。本发明人意外的发现，本发明特别设计的抗原融合蛋白不仅能够刺激CD8+T细胞，而且还仍然保持原来刺激CD4+T细胞功能。
抗原递呈细胞的溶酶体中主要的蛋白酶是组织蛋白酶S。Leu-Arg-Met-Lys是组织蛋白酶S的偏爱序列。在发明中，可优选用Leu-Arg-Met-Lys连接一组抗原区段构成新的人工多抗原融合蛋白，并用于刺激CD4+以及CD8+T细胞。
普通的短片段抗原多肽(一般≤70aa、≤60aa或更短)能直接被抗原递呈细胞吸收到细胞浆，从而递呈到MHC-1类分子上。普通蛋白质抗原只是被递抗原递呈细胞吞噬进入溶酶体，并且在溶酶体内的普通成熟蛋白只是和MHC-II结合，只是刺激CD4+T细胞。
与此相反，本发明的人工多抗原融合蛋白被抗原递呈细胞吞噬进入溶酶体后，由于组织蛋白酶S等组织蛋白酶酶切位点的存在，切割首先发生在连接序列处，在溶酶体内快速产生一组(多个)多肽，从而实现和促进交叉递呈，从而同时刺激CD8+T细胞和CD4+T细胞。
本发明的试验表明，当本发明融合蛋白进入细胞溶酶体中后，在抑制溶酶体功能 的情况下，融合蛋白依旧很快消失，这表明，在溶酶体中本发明蛋白或其酶切产物能够泄漏进入细胞浆。
刺激CD4+和CD8+T细胞免疫应答的融合蛋白
本发明中，提供了将多个抗原表位(或抗原区段)连接形成的单个用于特异性细胞免疫检测和作为预防与治疗性疫苗的单一蛋白，即多抗原融合蛋白。
在本发明的多抗原融合蛋白中，包括至少1个，更佳地包括2个、3个、4个、5个或更多个(如6-20的任一正整数)CD8+T细胞表位。
在本发明的多抗原融合蛋白中，包括至少1个，更佳地包括2个、3个、4个、5个或更多个(如6-20的任一正整数)CD4+T细胞表位。
在本发明中，抗原区段的序列可以是来自任何具有刺激免疫反应的多肽，优选是能刺激CD4+或者CD8+T细胞反应的序列(也即T细胞表位)。
组织蛋白酶是一组半胱氨酸蛋白酶，主要作用是降解溶酶体内的蛋白。组织蛋白酶-S(cathepsin-S)的已知的主要功能是参与MHC-2的抗原递呈过程。
在本发明的多抗原融合蛋白中，这些含有CD8+T细胞表位和CD4+T细胞表位的多肽的连接方式可以是通过任何抗原递呈细胞内的组织蛋白酶识别位点，优选使用的是组织蛋白酶Cathepsin S偏爱的Leu-Arg-Met-Lys序列。
在另一优选例中，可采用其他几个在递呈细胞表达比较多的组织蛋白酶B和K的酶切位点；譬如；Met-Lys-Arg↓-Leu为组织蛋白酶B的酶切位点，和His-Pro-Gly↓-Gly为组织蛋白酶K的酶切位点，但效果特别优异的是组织蛋白酶S酶切位点。
在另一优选例中，所述人工多抗原融合蛋白可以采用不同的组织蛋白酶S酶切位点，其中包括(但并不限于)；Arg-Cys-Gly↓-Leu、Thr-Val-Gly↓-Leu、Thr-Val-Gln↓-Leu、X-Asn-Leu-Arg↓、X-Pro-Leu-Arg↓、X-Ile-Val-Gln↓和X-Arg-Met-Lys↓；其中X-Arg-Met-Lys↓和X-Asn-Leu-Arg↓为优选的连接酶切位点，式中，X为任何氨基酸,↓表示酶切位置。
任何一种蛋白质都可以应用到本发明中，通常情况下，多抗原融合蛋白覆盖的序列要占蛋白质的40％以上，在更优的情况下，抗原融合蛋白的覆盖范围要占覆盖蛋白质抗原序列的至少50％、60％、70％、80％、90％；最佳地是100％。
作为本发明的优选例，HPV16-E7多抗原融合蛋白的覆盖范围是100％。当然，覆盖率即使降低，有时也是可行的，尤其是在目标蛋白中的MHC-I抗原表位较少且明确的情况下。例如，在本发明的一个优选例中，OVA的覆盖范围是40％，这是因为OVA只有单一的MHC-I抗原表位，而所选的片段包括了这个抗原表位。
多抗原融合蛋白可以覆盖一个以上的抗原蛋白质，在另一个优选例中，本发明的结核菌的抗原融合蛋白就覆盖了ESAT6以及CFP10两个蛋白序列，这样做可以减少成产成本，利于质量控制。
本发明的人工多抗原融合蛋白与天然蛋白(如病毒、细菌、寄生虫的蛋白或肿瘤抗原)不同。主要区别之一是失去了天然蛋白的空间结构，因此不具备天然蛋白的功能，从而避免了例如病原体蛋白(如病毒衣壳蛋白)的潜在危害。
本发明的人工多抗原融合蛋白与短片段抗原多肽(如≤70或≤60个氨基酸的抗原肽及抗原表位多肽)也不同。一个主要区别是本发明蛋白的分子量大，属于大分子蛋白质(而不是小分子多肽)，因而进入细胞的途径不一样，从而导致抗原提呈的途径、刺激T细胞方式及机制都不同于短片段抗原多肽。
制备方法和工程细胞
一旦确定了抗原蛋白序列，本发明蛋白就可以用重组法大量生产获得。这通常是将人工合成的编码DNA克隆到表达载体，再转入细胞，然后用常规方法从宿主细胞或发酵产物中分离得到。
一种典型的抗原融合蛋白的制备方法包括以下步骤:
根据蛋白质抗原的氨基酸顺序构建一个人工多抗原融合蛋白，该多抗原肽融合蛋白含有或由一系列特定长度的抗原区段组成，其中各抗原区段之间由相同或不同的组织蛋白酶酶切位点(如leu-Arg-Met-Lys)连接。
此外，用于表达本发明融合蛋白的细胞也包括在本发明中。“宿主细胞”包括原核细胞和真核细胞。常用的原核细胞为大肠杆菌。常用的真核细胞包括(但并不限于)：酵母细胞，昆虫细胞和哺乳动物细胞。作为本发明的优选方式，所用的宿主细胞是大肠杆菌，如BL21(DE3)和酵母，以及CHO细胞。
疫苗组合物和药物组合物
本发明还提供了本发明融合蛋白的用途，例如用于免疫诊断和制备疫苗。
免疫诊断包括皮试和T细胞斑点酶联免疫。皮试就是向受试个体免疫本发明多抗原融合蛋白，然后观察该个体的皮肤反应，从而判断该个体是否接触感染过类似抗原。作为本发明的优选方式，所述的免疫诊断是本发明融合蛋白作为刺激源来检测个体的T细胞免疫反应。例如，可通过观察个体的T细胞在受到本发明ESAT6-CFP10多抗原融合蛋白的刺激后，伽马干扰素的产生情况，从而判断病人是否感染过结核或所处阶段，或用于预后。
疫苗包括预防性疫苗和治疗性疫苗。后者主要与T细胞尤其是CD8+杀伤性T细胞(CTL)细胞的激活有关。而CTL细胞的激活受MHC-I分子限制。作为本发明的一个优选例，在体外证明了本发明HPV16-E7多抗原融合蛋白能被人抗原递呈细胞吞噬并激活抗原递呈细胞。而在另一优选例中，以本发明OVA多抗原融合蛋白为模型，证明了其能被抗原递呈细胞加工并递呈到MHC-I类分子上。
应用
本发明的融合蛋白利用抗原递呈细胞本身内吞体和溶酶体内的组织蛋白酶(Cathepsin)识别序列，将含有CD4+以及CD8+表位的多个抗原表位肽连接成单个蛋白，经表达纯化后，可直接应用于免疫诊断或者作为预防及治疗性疫苗。
当本发明蛋白或组合物作为预防与治疗性疫苗时，可以直接单独或与佐剂共同注射入动物体内，或者在体外与细胞治疗方法同时使用。例如，在DC/NK细胞培养时加入来刺激生产特异性抗原递呈细胞，然后用所述抗原递呈细胞在体内/体外刺激生产特异性CD8+T细胞(即CTL)。
在本发明中，这些体外致敏的特异性抗原递呈细胞和/或体外产生的CTL抗原可以被回输到相应对象的体内，用于抗病毒、抗肿瘤等各种不同的用途。
在本发明中，本发明蛋白或所述体外致敏的细胞(或相应制剂)，优选通过以下选自下组的施用的方式给予个体：静脉注射、灌注、皮下注射、透皮给药等。
本发明的主要优点包括：
(1)首次发现，本发明的人工多抗原融合蛋白能有效被递呈到MHC-I分子上。
(2)首次发现，单一的多抗原融合蛋白代替多个抗原短肽，可特异性刺激人T细胞释放伽马干扰素。
(3)首次证明了，本发明抗原融合蛋白能被抗原递呈细胞吞噬并激活CD8+细胞。
(4)利用本发明融合蛋白，可以开发用于免疫诊断的试剂和技术。例如，通过皮肤试验检测特异性细胞免疫，或通过ELISPOT试验检测特异性细胞免疫。
(5)利用本发明融合蛋白，可以开发治疗性或预防性的药物或疫苗，包括经本发明融合蛋白致敏所制备的DC细胞和/或CTL细胞制剂，产业应用价值不可估量。
(6)与常规的使用一组短片段多肽的方案相比，本发明方法和产品的成本显著下降，通常至少下降80％或更多；并且质控容易，可节省大量人力、物力和时间。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
实施例1
多抗原融合蛋白的克隆
根据蛋白的氨基酸顺序设计抗原融合蛋白。抗原融合蛋白由一系列长度为25-35个氨基酸多肽片段(短片段抗原肽)相连组成，抗原肽之间由相同的组织酶切S偏爱序列位点(Leu-Arg-Met-Lys)连接。
编码抗原融合蛋白的DNA进行密码子优化成适合大肠杆菌偏爱的密码子后。通过全人工合成方式制备DNA编码序列，并在其5’端添加
Figure PCTCN2015097470-appb-000001
(SEQ ID NO.:7)以及3’端添加
Figure PCTCN2015097470-appb-000002
(SEQ ID NO.:8)序列。将合成的DNA分子，用T4DNA聚合酶和dCTP处理30分钟。
将常规的pNIC28-Bsa4载体(获自牛津大学；US8,148,100B2；GenBank ID:EF198106)用BsaI酶切1小时后。该载体中与克隆可表达相关的重要组成部分见图1。用1％琼脂糖凝胶电泳分离线性化的载体，并用T4DNA聚合酶和dGTP处理30分钟。将两种T4DNA聚合酶处理产物混合后，转化常规的感受态大肠杆菌DN5α，铺板挑去单克隆培养。菌液PCR鉴定阳性菌落。
结果如图2、图3和图4所示，表明所获得的融合蛋白的编码序列和蛋白分子量的大小与设计或预测值相符。
各个多抗原融合蛋白的氨基酸和核苷酸编码如下所示。
Figure PCTCN2015097470-appb-000003
SEQ ID NO.:1
Figure PCTCN2015097470-appb-000004
SEQ ID NO.:2
Figure PCTCN2015097470-appb-000005
SEQ ID NO.:3
Figure PCTCN2015097470-appb-000006
Figure PCTCN2015097470-appb-000007
SEQ ID NO.:4
Figure PCTCN2015097470-appb-000008
SEQ ID NO.:5
Figure PCTCN2015097470-appb-000009
SEQ ID NO.:6
Figure PCTCN2015097470-appb-000010
此外，LC-MS分析也表明，纯化的TB-抗原融合蛋白的分子量测量值(30974Da)、OVA抗原融合蛋白(16589Da)和HPV16-E7抗原融合蛋白(16231Da)都与预测值相符。
实施例2
抗原融合蛋白的表达
抽取经测定含人工多抗原融合蛋白编码序列的阳性菌落的质粒DNA，用常规方法转化大肠杆菌BL21(DE3)。
将经转化的单菌落接种到低盐LB培养液中并于37℃过夜培养，在低盐LB培养液中1:100稀释培养物并37℃震荡培养至OD600＝1.0后，降温到18℃并加入0.2mM的IPTG进行蛋白的诱导表达，继续于18℃震荡培养16小时后，于4000rpm离心，收集菌体，并在磷酸缓冲液中重悬。
实施例3
抗原融合蛋白的纯化
(a)ESAT6-CFP10和OVA抗原融合蛋白的纯化
用超声法裂解细胞，离心收集包涵体。包涵体用变性缓冲液(8M尿素,在pH7.4的HEPES缓冲液中)溶解后，使其通过Ni-NTA柱，含有组氨酸标记抗原融合蛋白被结合到柱子上，经过充分的洗涤去掉杂蛋白后，抗原融合蛋白被含有尿素洗脱缓冲液洗脱下来。洗脱的抗原融合蛋白首先用含有0.5M精氨酸的PBS(pH9.5)稀释8倍，然后对PBS透析充分去掉尿素(图2右和图3右)。
(b)HPV16-E7抗原融合蛋白的纯化
用超声法裂解细胞，离心收集含目的蛋白的上清，使其通过Ni-NTA柱，含有组氨酸标记抗原融合蛋白被结合到柱子上，经过充分的洗涤去掉杂蛋白后，HPV16-E7抗原融合蛋白质用含有咪唑的洗脱缓冲液洗脱下来(图4右)。
实施例4
抗原递呈细胞对抗原融合蛋白的摄取
从来自于自愿捐血者的血液中用Ficoll分离人外周血淋巴细胞PBMC后，洗涤并用细胞培养液调节细胞浓度在2×106/ml，取5ml细胞悬浮液置细胞培养瓶中，加入HPV16-E7抗原融合蛋白(50μg/ml)或者同时加入FITC-HPV16-E7(5μg/ml),在37C，5％C02下培养24小时后继续培养12小时。细胞用抗人CD54抗体在4℃染色30分钟后用流式细胞仪分析。处理13小时后的细胞经EDTA处理收获，1ml冰冷的FACS缓冲液(PBS中2％FCS)洗涤两次。然后加入PE标记的市售单克隆抗体25.D1–16到浓度为0.6μg/ml。4℃孵育30分钟，细胞用FACS缓冲液洗涤三次，最后细胞重新悬浮在0.2ml的FACS固定缓冲液(BD)和并进行流式细胞仪分析。
结果显示，吞噬多抗原融合蛋白的主要细胞为CD54阳性细胞(图5)。
实施例5
抗原融合蛋白检测病人的γ干扰素分泌T细胞
在本实施例中，通过γ-干扰素释放检测，测试TB-抗原融合蛋白对CD8+T细胞的刺激能力。方法如下：
分离到结核菌痰涂片阳性病人的外周血单个核细胞后，将细胞浓度调整为5×106个活细胞/毫升。分别取50ul上述细胞悬液至阴性孔、阳性孔(以CONA为刺激源)、检测孔(以TB-抗原融合蛋白作为刺激源)以及对照孔(以市售的牛津免疫技术公司的T-SPOT.TB试剂盒自带抗原T-spot A和T-spot B为刺激源；或以成熟的CFP10-ESAT6融合天然蛋白质(记为TB-ESAT6-CFP10)为对照刺激源)。
在37℃，5％CO2孵育16-20小时后洗板，再加50μl标记抗体工作液，2-8℃孵育60分钟后洗板，加入50μl BCIP INBT底物溶液，室温避光孵育，干燥，计数。结果判断根据T-SPOT.TB试剂盒说明书进行：空白对照孔斑点为0-5个时，(有抗原孔斑点数-空白对照孔斑点数)≥6；或空白对照孔斑点数为6-10个时，有抗原 孔斑点数斑点数≥2倍空白对照孔斑点数。
检测结果显示，用ESAT6/CFP10抗原融合蛋白的刺激和用T-SPOT.TB试剂盒自带的抗原刺激效果一致(图6，上)。
此外，利用特异性抗CD4的磁珠清除了病人的CD4+T细胞后，使得残留的T细胞主要为CD8+T细胞，在此基础上比较了多抗原融合蛋白和CFP10成熟蛋白的差别。
结果显示，本发明的TB-抗原融合蛋白对CD8+T细胞有明显的刺激效果，与之相反，野生型序列的ESAT6-CFP10成熟蛋白则几乎对CD8+T细胞无明显的刺激效果(图6，下)。
实施例6
抗原融合蛋白负载DC体外诱导特异性CD8+T细胞
制备抗原负载DC细胞：对于分离获得的单个核外周核细胞(PBMC)后，取部分冻存，其余的细胞被调整至细胞浓度为1×107个细胞/mL，放入常规的AIM-V培养基中培养2小时后，去除悬浮细胞(另外冻存)，加入含有GM-CSF(1000IU/ml)，IL-4(50IU/ml)的AIM-V培养基继续在37℃，5％CO2下培养，第三天加入HPV16-E7抗原融合蛋白(50μg/ml)或者重组HPV16-E7蛋白(50μg/ml)。
抗原负载的DC体外致敏CD8+T细胞：第五天收集贴壁DC细胞，同时复苏冻存的PBMC，其中的CD8阳性细胞用CD8磁珠分离，并冻存CD8阴性细胞。将CD8阳性细胞：DC细胞按照5:1的比例共培养，并补加IL-2 100IU/ml,IL-7 25IU/ml。此外,每隔2天补加一次IL-2(100IU/ml)以及IL-7(25IU/ml)或者半量换液。第7天将冻存的CD8阴性细胞复苏，用丝裂霉素处理后按1/10的CD8+T细胞数加入CD8阴性细胞并进行二次刺激，补加对应蛋白50μg/ml，补加IL-2 100IU/ml,IL-7 25IU/ml。每隔两天补加IL-2 100IU/ml和IL-7 25IU/ml。
制备抗原递呈细胞：将第一天另外冻存的悬浮细胞取出复苏，在包被了CD19单抗的板中培养2h,弃去非贴壁细胞后，用培养基吹洗使CD19阳性细胞悬浮，并调整细胞浓度为5x105/ml,取2ml,于37℃，5％CO2培养48h后，补加IL-4到浓度为100μg/ml。
γ-干扰素释放检测评价多抗原融合蛋白负载DC体外诱导特异性CD8+T的效果：在96孔板中，加入CD19阳性细胞5×104个/50μl,同时加入相应的蛋白(HPV16-E7抗原融合蛋白(SEQ ID NO.:5)或者常规的重组HPV16-E7蛋白)到50μg/ml，37℃，5％CO2继续孵育2h。离心去上清后，加入新鲜培养基50μl重悬细胞，并加入CD8阳性细胞5x104个/50μl，37℃，5％CO2孵育18h。用ELISA检测上清中IFN-γ的浓度。
结果如表1所示：抗原融合蛋白HPV16-E7负载DC后，能在体外致敏并再次激活CD8+T细胞，该抗原融合蛋白的效果要远远高于对照HPV-E7蛋白，即与作为对照的重组HPV16-E7蛋白相比，本发明融合蛋白的γ-干扰素释放量提高了372.5％。((152.7-58.2)/(2.5-(-17.5))-1)*100％＝372.5％)。
表1：γ-干扰素释放检测HPV16-E7抗原融合蛋白与重组HPV16-E7蛋白在体外刺激DC诱导特异性CD8+T细胞的效果
Figure PCTCN2015097470-appb-000011
实施例7
结核病皮肤敏感测试
体重为250g左右的豚鼠，用PPD 0.1ml皮内注射，于24小时观察皮试反应。皮试反应阴性的豚鼠随机分成5组，于后肢腹股沟处，皮下注射40mg/ml的结核分枝杆菌H37Rv混悬液0.2ml，每周免疫1次，共免疫5次。第5次免疫1周后，将豚鼠随机分成5组，豚鼠背部拔毛，以轮圈法皮内注射阳性对照(PPD)、阴性对照(与结核无关的重组蛋白)、重组ESAT6-CFP10蛋白及重组ESAT6-CFP10-抗原融合蛋白的混合液(1mg/ml,0.1mg/ml)各0.1ml，于注射后24小时测量豚鼠背部各注射点皮肤红肿的纵、横直径大小(mm)，记录其平均值。
24小时皮肤反应结果见图7，TB-抗原融合蛋白组(SEQ ID NO.:1)的平均皮肤硬结直径大于PPD以及ESAT6-CFP10天然蛋白。对照组，豚鼠局部皮肤无红肿、硬结反应。这表明，TB-抗原融合蛋白能用于皮肤试验有效检测结核感染。
图中，E6-C10表示ESAT6-CFP10-天然蛋白；LRMK表示TB-抗原融合蛋白，其中各抗原区段之间用组织蛋白酶S酶切位点相连，序列如SEQ ID NO.:1所示；TEV表示与用TEV酶切位点(一种非组织蛋白酶的酶切位点)替换SEQ ID NO.:1中所有的组织蛋白酶S酶切位点所形成的对照蛋白)。
实施例8
抗原递呈细胞对抗原融合蛋白和普通抗原的处理与递呈机制
为了确定抗原递呈细胞摄取并被抗原融合蛋白激活后，能有效递呈T细胞表位到MHC-I分子上(只有递呈MHC-I分子上才能激活CD8+T细胞，产生细胞毒反应，杀死细菌/病毒感染细胞或者癌变细胞)。在本实施例中，根据鸡卵清蛋白(OVA)的242-352位氨基酸序列(该序列含有MHC-1表位SIINFKL)设计表达纯化了OVA抗原融合蛋白。同时用含有该表位的OVA蛋白质片段OVA 255-340作为对照。将小鼠树突状细胞系(DC2.4)作为抗原递呈细胞用来研究OVA抗原融合蛋白中的SIINFEKL的递呈情况。最后以T细胞受体样抗体(识别SIINFEKL/MHC-I复合物)检测。
将OVA蛋白质片段100μg/ml(或OVA抗原融合蛋白(SEQ ID NO.:3)30μg/ml)与DC2.4细胞混合，用含10％热灭活胎牛血清(sigma)，2mM的L-谷氨酰胺的RPMI1640(Sigma)进行培养13小时后，用PE标记的市售的25.D1-16抗体染色(该单克隆抗体25.D1-16能特异性识别结合了SIINFEKL的MHC-1分子)。1ml冰冷的FACS缓冲液(PBS中2％FCS)洗涤两次。然后加入PE标记的单克隆抗体25.D1–16至浓度为0.6μg/ml。4℃孵育30分钟，细胞用FACS缓冲液洗涤三次，最后细胞重新悬浮在0.2ml的FACS固定缓冲液(BD)和并进行流式细胞仪分析。
结果如图8显示。图中，蓝色峰(箭头所指)为DC+PBS。
作为阳性对照，8肽(SIINFEKL)能直接和细胞表面的MHC-1分子结合，故80％以上的细胞染色阳性(图8中的C1和C2)。
OVA天然蛋白质中的SIINFEKL肽不能被DC细胞加工递呈到MHC-1分子上(图8中的A1和A2)。(注：图8A2中蓝色峰和红色峰几乎重叠。)
令人意外的是，同样是大分子量的蛋白，但OVA-抗原融合蛋白(SEQ ID NO.:3)中的SIINFIKL肽能有效被小鼠树突状细胞系DC2.4加工并递呈到细胞表面，从而导致高达约17.6％的细胞为25.D1–16抗体染色阳性(图8中的B1和B2)。
实施例9
抗原融合蛋白免疫的C57/BL6小鼠对于过表达Survivin或HPV-E7的小鼠黑色素瘤细胞B16的抗肿瘤作用。
抗原溶液2mg/ml(对照组：重组HPV-E7蛋白,重组Survivin蛋白；实验组：HPV-E7抗原融合蛋白(SEQ ID NO.:5)，Survivin抗原融合蛋白)与1mg/ml MPL等体积混合制成免疫悬液后，分别对10只随机分组的周龄为7-8周，体重为25克的C57/B6雌性小鼠，在第0天，21天和42天于颈部皮下注射100μl免疫悬液。
对于每只免疫后小鼠，用注射器(BD)在小鼠右侧侧腹部皮下接种100μl混匀后的稳定转染了人HPV-E7或者Survivin)的小鼠B16细胞，注射的细胞量为7×105个细胞，并开始统计和评价小鼠的存活状况。结果如图9和图10显示。
结果显示，与重组HPV-E7蛋白对照组相比，本发明的HPV-E7抗原融合蛋白可显著延长小鼠的存活率(图9)，在第25天仍有50％小鼠存活；而重组蛋白对照组则全死亡。
与重组Survivin蛋白相比，本发明的Survivin抗原融合蛋白可显著延长小鼠的存活率(图10)，在第25天仍有70％小鼠存活；而重组蛋白对照组仅40％存活。
讨论
在本发明之前，在临床上没有成功将蛋白抗原用作刺激CD8+细胞免疫的疫苗。
蛋白质类疫苗生产和质量控制都已是成熟技术而且许多临床用药及疫苗(刺激抗体)都是蛋白质。如果能用生产蛋白质的成熟方法，产生能够刺激CD8+T细胞免疫的 疫苗将会非常理想。但是如何能将蛋白质抗原提呈从内吞体-溶酶体-MHC-II途径转成细胞浆-MHC-I途径是一个非常有挑战的课题。许多免疫学家及疫苗学家都在尝试但目前尚未有突破。
主要的障碍包括：
1.溶酶体中有很多的酶，它们功能一般是将蛋白质完全降解，会将蛋白降解成氨基酸而不是多肽，一般的重组蛋白抗原就会被完全降解而不能刺激T细胞。
2.由于溶酶体膜的作用是将溶酶体内酸性物质与细胞质隔开，所以溶酶体不是一个容易漏的细胞器。常规的表达蛋白或重组蛋白即使没有被完全降解，是否能够从溶酶体中逃逸或泄漏出来，是一个未知数。
3.对于可以刺激CD4+T细胞的蛋白质抗原，如果添加了CD8+抗原后的融合蛋白被降解并漏到细胞浆中(刺激CD8+T细胞)，是否会导致原本刺激CD4+T细胞功能下降或消失，这也是未知的。
在本发明中，本发明人基于酶学原理及生物重组技术设计了多种抗原融合蛋白，并经过大量筛选后，确定了一种新颖的抗原融合蛋白结构：在一系列含抗原区段的长肽之间由组织蛋白酶的酶切位点连接。当细胞吞噬抗原融合蛋白进入溶酶体后，溶酶体中的组织蛋白酶会将抗原融合蛋白降解成含不同抗原表位的多肽。
本发明实验出乎意料地证实了，本发明这种特定结构的融合蛋白被降解后，降解后的多肽居然可以漏出溶酶体而进入细胞浆，被加工递呈到MHC-I类分子，从而启动MHC-I抗原提呈途径进而而刺激CD8+细胞；并且原本刺激CD4+T细胞功能可以保留或得以提高。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种人工多抗原融合蛋白，其特征在于，所述的人工多抗原融合蛋白被施用于哺乳动物对象时，可在所述对象中同时刺激CD4+和CD8+T细胞免疫应答。
  2. 如权利要求1所述的人工多抗原融合蛋白，其特征在于，所述人工多抗原融合蛋白含有≥3个，较佳地≥5个，更佳地≥10个抗原区段。
  3. 如权利要求2所述的人工多抗原融合蛋白，其特征在于，所述的各抗原区段来自病毒抗原、细菌抗原、寄生虫抗原、衣原体抗原、肿瘤抗原、或其组合。
  4. 如权利要求1所述的人工多抗原融合蛋白，其特征在于，所述的人工多抗原融合蛋白还包括位于各抗原区段之间的酶切位点序列。
  5. 如权利要求4所述的人工多抗原融合蛋白，其特征在于，所述的酶切位点序列包括组织蛋白酶的酶切位点；以及
    较佳地，所述的组织蛋白酶酶切位点选自下组：组织蛋白酶S酶切位点(如Leu-Arg-Met-Lys或类似酶切位点)、组织蛋白酶B的酶切位点(如Met-Lys-Arg-Leu或类似酶切位点)、组织蛋白酶K的酶切位点(如His-Pro-Gly-Gly或类似酶切位点)、或其组合。
  6. 如权利要求1所述的人工多抗原融合蛋白，其特征在于，所述融合蛋白具有式I结构：
    Y-(A-C)n-Z   (I)
    式中，
    A为抗原区段；
    C为组织蛋白酶酶切位点序列；
    n为≥3的正整数；
    Y为无或“Y0-B”所示的序列，其中，Y0为信号肽序列、标签序列、穿膜元件序列、佐剂元件序列、细胞坏死诱导因子元件序列、或上述序列任意组合，而B为无或切割位点(cleavage site)序列；
    Z为无，或标签序列、穿膜元件序列、佐剂元件序列、细胞坏死诱导因子元件序列、或上述序列任意组合；
    附加条件是，当Z为无时，最后一个“A-C”中的C可以为无。
  7. 一种组合物，其特征在于，所述的组合物含有权利要求1-6中任一所述的人工多抗原融合蛋白和药学上可接受的载体。
  8. 如权利要求1所述的人工多抗原融合蛋白的用途，其特征在于，用于制备预防性和/或治疗性的疫苗组合物或药物组合物。
  9. 如权利要求1所述的人工多抗原融合蛋白的用途，其特征在于，用于制备检测特异性T细胞免疫的试剂或试剂盒。
  10. 一种方法，其特征在于，包括步骤：给需要治疗的对象施用权利要求1所述的人工多抗原融合蛋白、或权利要求7所述的组合物。
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022090679A1 (en) 2020-10-28 2022-05-05 Oxford Vacmedix UK Limited Coronavirus polypeptide
WO2022090716A1 (en) 2020-10-28 2022-05-05 Oxford Vacmedix UK Limited Polypeptides for cancer treatment
WO2022129881A1 (en) * 2020-12-15 2022-06-23 Chain Biotechnology Limited Compositions and methods
WO2022238689A1 (en) 2021-05-11 2022-11-17 Oxford Vacmedix UK Limited Vaccine formulation comprising recombinant overlapping peptides and native prtoeins

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB202209115D0 (en) 2022-06-21 2022-08-10 Chain Biotechnology Ltd Compositions and methods

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101745104A (zh) * 2010-02-09 2010-06-23 中国药品生物制品检定所 含有复合佐剂的结核亚单位疫苗
CN102343103A (zh) * 2011-07-26 2012-02-08 马丁 人乳头状瘤病毒16型三肽疫苗的筛选和验证及持续表达hpv16 e5,e6,e7的肿瘤动物模型的构建
CN103180343A (zh) * 2010-08-13 2013-06-26 株式会社吉耐森 用于预防或治疗宫颈癌的、包含人乳头瘤病毒变形体及免疫增强剂的组合物

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5693325A (en) * 1994-03-15 1997-12-02 Molecumetics, Ltd. Peptide vaccines and methods relating thereto
GB0023203D0 (en) * 2000-09-21 2000-11-01 Isis Innovation Vaccination method
US6432409B1 (en) * 1999-09-14 2002-08-13 Antigen Express, Inc. Hybrid peptides modulate the immune response
US7179645B2 (en) * 2002-09-24 2007-02-20 Antigen Express, Inc. Ii-Key/antigenic epitope hybrid peptide vaccines
GB0519605D0 (en) 2005-09-26 2005-11-02 Isis Innovation Assay
GB0608368D0 (en) * 2006-04-28 2006-06-07 Isis Innovation Process for making Oligopeptides
CN101920009B (zh) * 2010-03-06 2012-08-08 河北医科大学 一种用于预防和治疗肿瘤的疫苗
WO2011146559A1 (en) 2010-05-19 2011-11-24 The Henry M. Jackson Foundation For The Advancement Of Military Medicine, Inc. Combination therapy with ae37 peptide and an antibody that binds to her2/neu protein
CN103266119B (zh) * 2013-06-03 2014-10-29 苏州大学 一种结核杆菌三抗原融合基因疫苗及其制备方法和应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101745104A (zh) * 2010-02-09 2010-06-23 中国药品生物制品检定所 含有复合佐剂的结核亚单位疫苗
CN103180343A (zh) * 2010-08-13 2013-06-26 株式会社吉耐森 用于预防或治疗宫颈癌的、包含人乳头瘤病毒变形体及免疫增强剂的组合物
CN102343103A (zh) * 2011-07-26 2012-02-08 马丁 人乳头状瘤病毒16型三肽疫苗的筛选和验证及持续表达hpv16 e5,e6,e7的肿瘤动物模型的构建

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP3235831A4 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022090679A1 (en) 2020-10-28 2022-05-05 Oxford Vacmedix UK Limited Coronavirus polypeptide
WO2022090716A1 (en) 2020-10-28 2022-05-05 Oxford Vacmedix UK Limited Polypeptides for cancer treatment
WO2022129881A1 (en) * 2020-12-15 2022-06-23 Chain Biotechnology Limited Compositions and methods
WO2022238689A1 (en) 2021-05-11 2022-11-17 Oxford Vacmedix UK Limited Vaccine formulation comprising recombinant overlapping peptides and native prtoeins

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