WO2017177908A1 - Pd-l1 and pd-l2 recombinant proteins and uses thereof - Google Patents
Pd-l1 and pd-l2 recombinant proteins and uses thereof Download PDFInfo
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- WO2017177908A1 WO2017177908A1 PCT/CN2017/080144 CN2017080144W WO2017177908A1 WO 2017177908 A1 WO2017177908 A1 WO 2017177908A1 CN 2017080144 W CN2017080144 W CN 2017080144W WO 2017177908 A1 WO2017177908 A1 WO 2017177908A1
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Definitions
- the present invention relates to the field of bioengineering, and in particular, to recombinant proteins and uses thereof.
- Targeted therapies mainly include monoclonal antibodies (sometimes classified as passive cell transfusion and tumor vaccines, immunotherapy through the immune system of the motivational body, enhanced tumor microenvironment anti-tumor immunotherapy) and small molecule targeted drugs, while immunotherapy It mainly includes cytokine therapy, immunoassay monoclonal antibody, and adoptive immunotherapy to control and kill tumor cells. Therefore, it has the advantages of high efficiency, high specificity and good tolerance, and has broad prospects in cancer therapy.
- Tumor immunotherapy vaccines mainly include tumor cell vaccines, dendritic cell (DC cell) vaccines, protein & peptide vaccines, nucleic acid vaccines, genetically engineered vaccines and anti-idiotype tumor vaccines.
- the main mechanism by which these vaccines can kill tumors is through The patient is directed against a tumor-specific antigen immune response, including antibody response and cytotoxic T lymphocyte (CTL) specific killing.
- CTL cytotoxic T lymphocyte
- tumor vaccines target tumor-associated antigens, and their clinical efficacy is weak, and further research and development are needed to enhance clinical efficacy.
- Tumor cells highly express immune checkpoint molecules PD-L1 or PD-L2, and activated cytotoxic T lymphocytes on the surface
- PD-1 which inhibits the T lymphocyte response of the tumor, escapes the immune killing of cytotoxic T lymphocytes.
- CTL immune cells-cytotoxic T lymphocytes
- an object of the present invention is to provide a recombinant PD-L1 protein having an immune response which causes a tumor-specific antigen, which is actively stimulated to produce an anti-PD-L1 antibody in a patient, and the patient has been mobilized.
- the presence of spontaneously induced immune cell CTLs is induced and stimulates the production of new anti-PD-L1 CTLs, which in turn specifically kill tumor cells.
- the active immune killing effect on tumor cells caused by the recombinant protein proposed by the invention is remarkable.
- the invention proposes a recombinant protein.
- the recombinant protein comprises: an immunological checkpoint molecule fragment; a helper T cell epitope fragment; and an immunostimulatory molecule fragment.
- the recombinant protein proposed in the embodiments of the present invention can continuously stimulate the production of anti-immunization checkpoint antibodies in vivo, mobilize the spontaneously induced immune cell CTLs existing in the body, and stimulate the production of CTLs against the immune checkpoints, thereby specifically killing the tumor cells. .
- the active immune killing effect on tumor cells caused by the recombinant protein proposed by the embodiments of the present invention is remarkable.
- the recombinant protein may further comprise at least one of the following additional technical features:
- the immune checkpoint molecule is PD-L1 or PD-L2.
- PD-L1 or PD-L2 is specifically expressed in tumor cells, and the specificity of the tumor antigen immune response caused by the recombinant protein proposed in the examples of the present invention is stronger.
- the immunological checkpoint molecule fragment is an extracellular molecular fragment of the PD-L1 or PD-L2 removal transmembrane region.
- the extracellular molecular fragment of PD-L1 or PD-L2 in the transmembrane region has only tumor antigenicity and does not have tumor immunosuppressive function, and thus the extracellular molecular fragment of PD-L1 or PD-L2 in the transmembrane region can be further removed.
- the specificity is further improved.
- the helper T cell epitope is a broad spectrum PADRE helper T cell epitope.
- Broad-spectrum PADRE-assisted T cell epitopes can effectively activate helper T cells, thereby further enhancing the specific killing of cytotoxic T lymphocytes (CTLs) caused by recombinant proteins.
- CTLs cytotoxic T lymphocytes
- the immunostimulatory molecule is a granulocyte colony-stimulating biological factor, interleukin-12 or a chemokine.
- the above immunostimulatory molecule has biological activity, can significantly enhance the antigen presenting function of dendritic cells (DC cells) and enhance the activity of cytotoxic T lymphocytes (CTLs) and B lymphocytes, and the recombinant protein of the embodiments of the present invention can be further To effectively cause tumor antigen immune response.
- the N-terminus of the helper T cell epitope fragment is linked to the C-terminus of the immunological checkpoint molecule fragment, and the C-terminus of the helper T cell epitope fragment and the immunostimulatory molecule fragment N ends are connected.
- the related molecular fragment in the recombinant protein of the present invention facilitates the presentation of the tumor antigen PD-L1 or PD-L2 fragment in DC cells, and is beneficial to the help of T cell epitopes and immunostimulatory molecules.
- the corresponding function of activating immune cells, and thus the recombinant protein of the examples of the present invention can more effectively cause a tumor antigen immune response.
- the invention proposes a recombinant protein.
- the recombinant protein has the amino acid sequences shown in SEQ ID NOS: 1 to 9.
- the recombinant protein proposed in the embodiment of the present invention can cause a tumor-specific antigen immune response, and the protein stimulates the production of anti-PD-L1 or PD-L2 antibody in the patient by active immunization, and mobilizes the spontaneous induction of the existing in the patient.
- the cells are immunized with CTL and stimulated to produce anti-PD-L1 or PD-L2 CTL, thereby specifically killing tumor cells.
- the active immune killing effect on tumor cells caused by the recombinant protein proposed by the invention is remarkable.
- the invention proposes a nucleic acid.
- the nucleic acid encodes the recombinant protein described above, and the nucleic acid has the nucleotide sequence shown in SEQ ID NOS: 10 to 18.
- the recombinant protein encoded by the nucleic acid according to the embodiment of the present invention stimulates the production of anti-PD-L1 or PD-L2 antibody in the patient by active immunization, mobilizes the spontaneously induced immune cell CTL which has already existed in the patient, and stimulates the production of anti-drug.
- PD-L1 or PD-L2CTL which specifically kills tumor cells.
- the active immune killing effect on tumor cells caused by the recombinant protein proposed by the invention is remarkable.
- the invention proposes a construct.
- the construct Carry the nucleic acid described above.
- the construct introduced into the recipient cell of the present invention can achieve high-efficiency expression of the nucleic acid described above, and thereby efficiently express the recombinant protein described above in the recipient cell.
- the construct may further comprise at least one of the following additional technical features:
- the vector of the construct is a pET series vector, a pPIC series vector, a BacPAK, a pSV series vector or a pCMV series vector.
- the above vector of the embodiment of the present invention can achieve further efficient expression of the above recombinant protein in prokaryotic cells or eukaryotic cells.
- the invention proposes a construct.
- the construct carries the following nucleic acid molecule: (1) a nucleic acid molecule encoding a fragment of an immunological checkpoint molecule having the amino acid sequence set forth in SEQ ID NOS: 19-21,
- the nucleic acid molecule encoding the immunological checkpoint molecule fragment has the nucleotide sequence shown in SEQ ID NOS: 22-24; (2) the nucleic acid molecule encoding the helper T cell epitope fragment, the helper T cell epitope
- the fragment has the amino acid sequence set forth in SEQ ID NO: 25, the nucleic acid molecule encoding the helper T cell epitope fragment has the nucleotide sequence set forth in SEQ ID NO: 26; and (3) the fragment encoding the immunostimulatory molecule
- the nucleic acid molecule having the amino acid sequence of SEQ ID NOS: 27 to 29, and the nucleic acid molecule encoding the immunostimulatory molecule fragment has the following nucleic acid molecule: (1)
- the construct according to the embodiment of the present invention efficiently expresses a recombinant protein containing an immunological checkpoint molecular fragment, a helper T cell epitope fragment and an immunostimulatory molecule fragment in a recipient cell, and the recombinant protein is actively immunized.
- the patient stimulates the production of anti-PD-L1 antibody, mobilizes the spontaneously induced immune cell CTL already present in the patient, and stimulates the production of anti-PD-L1CTL, thereby specifically killing the tumor cells.
- the invention provides a transgenic cell.
- the transgenic cell carries a construct as described above.
- the transgenic cells proposed in the embodiments of the present invention can express the recombinant protein as described above, and the obtained recombinant protein stimulates an anti-immunological checkpoint, such as PD-L1 or PD-L2 antibody, by active immunization, and mobilizes.
- an anti-immunological checkpoint such as PD-L1 or PD-L2 antibody
- Spontaneously induced immune cell CTLs already present in the patient and stimulate the production of anti-immunological checkpoints, such as PD-L1 or PD-L2 CTL, which specifically kill tumor cells.
- the transgenic cell may further comprise at least one of the following additional technical features:
- the transgenic cells are BL21, BL21 (DE3), BL21 (DE3) pLysS, DH10B, XL1-Blue, Pichia pastors, Kluyveromyces lactis, Sf9, Sf21, High-Five T, CHO cell line, HEK cell line, Hela cell line or COS cell line.
- the transgenic cell can efficiently express the recombinant protein described above, and then the recombinant protein obtained by protein purification can be administered to a patient, and the patient can be further effectively stimulated to produce anti-PD in the patient by active immunization.
- the -L1 or PD-L2 antibody mobilizes spontaneously induced immune cell CTLs already present in the patient and stimulates the production of anti-PD-L1 or PD-L2CTL, thereby specifically killing the tumor cells.
- the transgenic cell is an antigen presenting cell.
- the antigen presenting cell is derived from a patient, and the antigen presenting cell carrying the aforementioned construct can be further input into the patient, thereby realizing the continuous expression of the recombinant protein described above in the patient, and further Actively immunizing in the body to produce anti-PD-L1 or PD-L2 antibodies in vivo, mobilizing the spontaneously induced immune cell CTLs already present in the patient, and stimulating the production of anti-PD-L1 or PD-L2CTL, Specific killing of tumor cells.
- the transgenic cell is a DC cell.
- the DC cells have an antigen-presenting function, and the DC cells derived from the patient themselves carry the aforementioned construct and are returned to the patient, thereby realizing the high-efficiency expression of the recombinant protein described above in the patient and the tumor antigen PD-L1.
- PD-L2 is efficiently presented on the surface of DC cells, thereby further effectively stimulating the production of anti-PD-L1 or PD-L2 antibodies, mobilizing the spontaneously induced immune cell CTLs already present in the patient, and stimulating the production of anti-PD-L1 or PD -L2CTL, which further effectively kills tumor cells specifically.
- the invention provides the use of a recombinant protein as described above for the preparation of a medicament for the prevention or treatment of a tumor.
- the recombinant protein proposed by the embodiments of the present invention can cause a significant tumor-specific antigen immune response in a tumor patient, and effectively stimulates the production of an anti-immunological checkpoint, such as PD-L1 or PD-L2 antibody, to mobilize the spontaneous induction already existing in the patient.
- the resulting immune cell CTL is stimulated to produce an anti-immunological checkpoint, such as PD-L1 or PD-L2CTL, which effectively kills the tumor cells specifically.
- the inventors further verified through experiments that The recombinant protein proposed in the examples of the present invention has utility in the preparation of a medicament effective for preventing or treating a tumor.
- the invention provides the use of a recombinant protein as described above for the preparation of a vaccine for the prevention or treatment of a tumor.
- the recombinant protein proposed by the embodiments of the present invention can cause a significant tumor-specific antigen immune response in a tumor patient, and effectively stimulates the production of an anti-immunological checkpoint, such as PD-L1 or PD-L2 antibody, to mobilize the spontaneous induction already existing in the patient.
- the resulting immune cell CTL is stimulated to produce an anti-immunological checkpoint, such as PD-L1 or PD-L2CTL, which effectively kills the tumor cells specifically.
- the recombinant protein proposed in the examples of the present invention has a use in preparing a vaccine effective for preventing or treating a tumor.
- the invention provides the use of a recombinant protein as described above for the preparation of a vaccine for the treatment of a viral infection.
- the inventors have found that HBV, HCV, HIV, EBV virus-infected cells express PD-L1, and the vaccine prepared by the recombinant protein of the present invention can stimulate anti-PD-L1 in a patient.
- the antibody mobilizes spontaneously induced immune cell CTLs already present in the patient and stimulates the production of anti-immunological checkpoints, such as PD-L1 CTL, to effectively kill cells infected with the above virus.
- the invention provides a pharmaceutical composition.
- the pharmaceutical composition comprises: the recombinant protein described above; and a pharmaceutically acceptable adjuvant.
- the recombinant protein in the pharmaceutical composition proposed in the examples of the present invention can cause a significant specific antigen immune response, and the function of the adjuvant to enhance the immune response.
- the pharmaceutical composition provided by the embodiment of the present invention effectively stimulates an anti-immunization checkpoint, such as a PD-L1 or PD-L2 antibody, to mobilize an already existing spontaneously induced immune cell in a patient. CTL, and stimulate the production of anti-immunological checkpoints, such as PD-L1 or PD-L2CTL, to effectively kill tumor cells or cells infected with viruses (HBV, HCV, HIV, EBV).
- viruses HBV, HCV, HIV, EBV
- the invention proposes a DC cell.
- the DC cells are loaded with the recombinant protein described above.
- the DC cells proposed in the embodiments of the present invention can present antigens in the recombinant protein (such as the immunological checkpoint molecular fragment described above), helper T cell epitope fragments, and immunostimulatory molecule fragments, respectively.
- anti-immunological checkpoints such as PD-L1 or PD-L2 antibodies
- mobilizes spontaneously induced immune cell CTLs already present in patients and stimulates the production of anti-immune checkpoints such as PD-L1 or PD-L2CTL, in turn, effectively kills tumor cells or cells infected with viruses (HBV, HCV, HIV, EBV).
- viruses HBV, HCV, HIV, EBV
- the invention provides a targeted immune cell population.
- the targeted immune cell population is obtained by co-culture of DC cells with lymphocytes as described above.
- the targeted immune cell population proposed in the embodiments of the present invention can specifically kill tumor cells, secrete antibodies that specifically bind tumor antigens, and achieve specific removal of tumor cells.
- the invention proposes a vaccine.
- the vaccine comprises a recombinant protein as described above, a DC cell as described above or a targeted immune cell population as described above.
- the recombinant protein, DC cell and targeted immune cell population proposed by the embodiments of the present invention can cause a significant specific antigen immune response in a patient.
- the vaccine according to the embodiment of the present invention can effectively stimulate the production of an anti-immunization checkpoint, such as a PD-L1 or PD-L2 antibody, mobilize the spontaneously induced immune cell CTL already existing in the patient, and stimulate An anti-immunization checkpoint, such as PD-L1 or PD-L2CTL, is generated to effectively kill tumor cells or cells infected with viruses (HBV, HCV, HIV, EBV).
- an anti-immunization checkpoint such as a PD-L1 or PD-L2 antibody
- the invention provides an antibody.
- the antibody specifically recognizes the recombinant protein described above, and the antibody proposed in the embodiment of the present invention specifically recognizes a tumor antigen.
- the invention finds that the antibody specifically recognizes an antigen and specifically binds to a tumor cell or a cell infected by a virus (H BV, HCV, HIV, EBV), thereby causing the tumor cell or the virus to be infected ( Cells infected with HBV, HCV, HIV, EBV) are engulfed by phagocytic cells to achieve specific clearance of tumor cells or cells infected with viruses (HBV, HCV, HIV, EBV).
- the invention provides a method of preparing an antibody.
- the method comprises: immunizing an animal with the recombinant protein described above; collecting serum of the immunized animal; and purifying the antibody of interest from the serum.
- the method for preparing an antibody proposed in the embodiments of the present invention is simple and convenient, and the antibody can specifically recognize the recombinant protein.
- the invention provides a therapeutic composition.
- the therapeutic composition comprises: a recombinant protein as described above, a nucleic acid as described above, a construct as described above, a transgenic cell as described above, a pharmaceutical composition as described above, a front The DC cells, the aforementioned targeted immune cell population, the vaccine described above or the antibodies described above.
- the therapeutic composition proposed by the embodiments of the present invention can directly or indirectly cause a specific antigen immune response, and achieve specificity to tumor cells or cells infected by viruses (HBV, HCV, HIV, EBV). Kill and clear.
- the invention provides a method of stimulating anti-PD-L1 antibody production or cytotoxic T lymphocyte response in a patient.
- the method is achieved by at least one of the following: 1) the recombinant protein described above is co-cultured with DC cells taken from a patient, and the DC of the recombinant protein described above is loaded The cells are returned to the patient; 2) the patient is administered the pharmaceutical composition described above; 3) the previously described construct is introduced into the DC cells taken from the patient, and the DC cells introduced into the construct are returned to the patient. In vivo; and 4) administering to the patient a construct as described above.
- the manner proposed in the examples of the present invention can significantly stimulate anti-PD-L1 antibody production or cytotoxic T lymphocyte reaction in a patient.
- the invention provides a method of treating cancer. According to an embodiment of the present invention, it is achieved by at least one of the following methods: 1) the recombinant protein described above is co-cultured with DC cells taken from a cancer patient, and the DC cells loaded with the recombinant protein described above are returned. Loss to a cancer patient; 2) administration of the aforementioned pharmaceutical composition to a cancer patient; 3) introduction of the aforementioned construct into DC cells taken from a cancer patient, and introduction into the body The DC cells of the construct are returned to the cancer patient; and 4) the cancer patient is administered the construct described above.
- the method proposed by the embodiments of the present invention can directly or indirectly cause a specific antigen immune response, and achieve specific killing and elimination of tumor cells.
- the invention provides a method of treating a patient infected with a virus.
- it is achieved by at least one of the following methods: 1) the recombinant protein described above is co-cultured with DC cells taken from a patient, and the DC cells loaded with the recombinant protein described above are returned.
- the patient is administered the construct described above.
- the method proposed by the embodiments of the present invention can directly or indirectly cause a specific antigen immune reaction, and achieve specific killing and elimination of cells infected by viruses (HBV, HCV, HIV, EBV), thereby effectively treating patients infected with the virus.
- the above method may further include at least one of the following additional technical features:
- the virus comprises at least one selected from the group consisting of HBV, HCV, HIV and EBV. Further effective treatment of patients infected with HBV, HCV, HIV or EBV can be achieved using the above method according to an embodiment of the invention.
- FIG. 1 is a schematic view showing the structure of a fusion protein according to an embodiment of the present invention.
- PD-L1 ⁇ -PADRE Th-GM-CSF fusion protein
- PD-L1 ⁇ -PADRE Th-GM-CSF fusion protein
- PD-L1 ⁇ -PADRE Th-GM-CSF a fusion protein capable of significantly controlling the growth of PD-L1 + lung cancer according to an embodiment of the present invention.
- first and second are used for descriptive purposes only, and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, features defining “first” and “second” may include one or more of the features either explicitly or implicitly. Further, in the description of the present invention, the meaning of "a plurality" is two or more unless otherwise specified.
- the invention proposes a recombinant protein.
- the recombinant protein comprises: an immunological checkpoint molecule fragment; a helper T cell epitope fragment; and an immunostimulatory molecule fragment.
- the recombinant protein proposed in the embodiments of the present invention stimulates the production of anti-immunological checkpoint antibodies in the patient, mobilizes the spontaneously induced immune cell CTLs already existing in the body, and stimulates the production of CTLs against the immune checkpoints, thereby specifically killing the tumor cells.
- the active immune killing effect on tumor cells caused by the recombinant protein proposed by the embodiments of the present invention is remarkable.
- the immune checkpoint molecule may be selected from, but not limited to, at least one of PD-L1 and PD-L2.
- PD-L1 or PD-L2 is specifically expressed in tumor cells, and the specificity of the tumor antigen immune response caused by the recombinant protein proposed in the examples of the present invention is stronger.
- the immunological checkpoint molecule fragment is an extracellular molecular fragment (PD-L1 ⁇ /PD-L2 ⁇ ) of the transmembrane region of the PD-L1 or PD-L2.
- the extracellular molecular fragment of the PD-L1 or PD-L2 removal transmembrane region has only tumor antigenicity and does not have tumor immunosuppressive function, and then the extracellular molecular fragment of the transmembrane region of PD-L1 or PD-L2 is antigen-removed.
- Presenting cells such as DC cells are presented on the surface of the cells, which can effectively elicit a tumor antigen immune response, and the specificity is further improved.
- the helper T cell epitope can be a broad spectrum PADRE helper T cell epitope (PADRE Th).
- PADRE Th PADRE helper T cell epitope
- the broad-spectrum PADRE helper T cell epitope is an epitope peptide that binds to a broad spectrum of human leukocyte antigen DR (HLA-DR) with high affinity or intermediate affinity and 16 of the most prevalent HLA- 15 of the DR types are combined. Because of its universal binding, PADRE needs to overcome the problems caused by the diversity of HLA-DR molecules in the population. PADRE acts as a helper T cell epitope and binds to the antigen. After binding, it activates the Ag-specific antigen reaction efficiently and for a long time.
- HLA-DR human leukocyte antigen DR
- the PADRE peptide is specifically processed to immunologically activate helper T lymphocyte 1 (Th1) in humans to assist in the activation of killer immune T cells and to activate helper T lymphocyte 2 (Th2) to assist B lymphocytes to secrete antibodies, thereby Further enhancing the antigenic immune response caused by the recombinant protein.
- Th1 helper T lymphocyte 1
- Th2 helper T lymphocyte 2
- the immunostimulatory molecule may be selected from the group consisting of granulocyte colony stimulating biological factor (GM-CSF), interleukin-12 (IL-12) or chemokine (RANTES).
- GM-CSF granulocyte colony stimulating biological factor
- IL-12 interleukin-12
- RANTES chemokine
- GM-CSF is used to enhance the immune response in animal models and clinical trials.
- immunotherapy GM-CSF is also widely used as an adjuvant to enhance the immune response.
- tumor cells are irradiated by radiation, which secretes GM-CSF to stimulate a potent, specific, and prolonged anti-tumor immune response. This immunization induces infiltration of CD4+ and CD8+ T lymphocytes and plasma cells in metastatic lesions of advanced melanoma, which in turn causes necrosis of a large number of tumor cells.
- GM-CSF The immunopotentiating effect of secreting tumor cells and GM-CSF are capable of recruiting DC cells and maturation and activation of DC cells, thereby activating the role of immune killing of T lymphocytes and B lymphocytes.
- the GM-CSF in the recombinant protein proposed by the embodiment of the present invention can significantly enhance the antigen presentation of dendritic cells (DC cells).
- DC cells dendritic cells
- CTL cytotoxic T lymphocytes
- B lymphocytes are enhanced, and the recombinant protein of the embodiment of the present invention can more effectively induce a tumor antigen immune response.
- Chemokines control the migration of specific leukocyte populations in immune responses, hematopoiesis, and routine immune surveillance.
- RANTES regulated upon activation normal T-cell expressed, CCL5
- CCL5 normal T-cell expressed, CCL5
- CCL5 regulated upon activation normal T-cell expressed
- IL-12 is a pleiotropic cytokine that activates the association between autoimmune and adaptive immunity. Based on its response, IL-12 is considered to be "cytotoxic lymphocyte maturation factor" and "natural killer cell stimulating factor". Since IL-12 can establish the correlation between autoimmune and adaptive immunity, it can strongly stimulate the production of IFN- ⁇ -a, thereby coordinating the body's own anti-cancer mechanism. IL-12 has been used in the human body for immunotherapy of tumors. IL-12 plays a role in a variety of immune cells, including T lymphocytes and B lymphocytes. IL-12 plays a key role in promoting the anti-tumor immune response of Th1 cells.
- the ligation sequence of the helper T cell epitope fragment and the immunostimulatory molecule fragment is as follows: the N-terminus of the helper T cell epitope is linked to the C-terminus of the immunological checkpoint molecule fragment, and the helper T cell epitope fragment C-end and The N-terminus of the immunostimulatory molecule fragment is linked.
- the corresponding molecular fragment in the recombinant protein of the present invention can be presented on the surface of the DC cell, respectively.
- APCs main antigen-presenting cells
- the recombinant protein proposed by the embodiments of the present invention allows DC cells to present intracellular antigens to the cell surface, which is presented to the MHC class II by endocytic pathway, and is presented to the MHC class I by a cross-priming pathway, thereby leading to the production of antigen-specific Th cells. Reacts with CTL cells.
- the recombinant protein presented by the embodiments of the present invention causes a strong antibody response on the surface of DC cells. It has long been believed that the activation of humoral responses by DC cells is achieved by the interaction of T lymphocytes and B lymphocytes by CD4+ Th cells. However, existing in vitro and in vivo experiments have demonstrated that DC cells activate a humoral response as a direct mode of action.
- DC cells have been shown to strongly promote cell differentiation and production of antibodies to CD40-activated B lymphocytes. Inoculation of antigen-loaded DC cells is capable of inducing a protective humoral immune response.
- the recombinant protein of the embodiments of the present invention can more effectively cause a tumor antigen immune response.
- the recombinant protein has the amino acid sequences shown in SEQ ID NOS: 1 to 9.
- SEQ ID NO: 1 is the amino acid sequence of human PD-L1 ⁇ -PADRE Th-human GM-CSF recombinant protein
- SEQ ID NO: 2 is the amino acid sequence of human PD-L1 ⁇ -PADRE Th-human RANTES recombinant protein
- SEQ ID NO:3 is the amino acid sequence of human PD-L1 ⁇ -PADRE Th-IL-12 recombinant protein
- SEQ ID NO: 4 is the amino acid sequence of human PD-L2 ⁇ -PADRE Th-human GM-CSF recombinant protein
- SEQ ID NO: 5 is Human PD-L2 ⁇ -PADRE
- SEQ ID NO: 6 is the amino acid sequence of the human PD-L2 ⁇ -PADRE Th-human IL-12 recombinant
- the present invention proposes a nucleic acid encoding the recombinant protein described above, and according to an embodiment of the present invention, the nucleic acid encoding the recombinant protein described above has the nucleotide sequences shown in SEQ ID NOS: 10 to 18.
- SEQ ID NO: 10 is the nucleotide sequence of a nucleic acid encoding a human PD-L1 ⁇ -PADRE Th-human GM-CSF recombinant protein
- SEQ ID NO: 11 is a recombinant protein encoding human PD-L1 ⁇ -PADRE Th-human RANTES
- the nucleotide sequence of the nucleic acid, SEQ ID NO: 12 is the nucleotide sequence of the nucleic acid encoding the human PD-L1 ⁇ -PADRE Th-IL-12 recombinant protein
- SEQ ID NO: 13 is the encoding human PD-L2 ⁇ -PADRE Th-
- SEQ ID NO: 14 is the nucleotide sequence of the nucleic acid encoding the human PD-L2 ⁇ -PADRE Th-human RANTES recombinant protein
- SEQ ID NO: 11 is a recombinant protein encoding human PD-L1 ⁇ -PAD
- the recombinant protein encoded by the nucleic acid proposed in the embodiments of the present invention can cause tumor-specific antigen immune response, cause specific killing of cytotoxic T lymphocytes (CTL) and secrete specific antibodies of B cells, and achieve specificity to tumor cells. Killing.
- CTL cytotoxic T lymphocytes
- the present invention proposes a construct which carries the nucleic acid described above.
- the construct introduced into the recipient cell of the embodiment of the present invention can achieve high-efficiency expression of the nucleic acid described above, thereby efficiently expressing the recombinant protein described above in the recipient cell.
- the vector of the construct is a pET series vector, a pGEX series vector, a pPIC series vector, a BacPAK, a pSV series vector or a pCMV series vector, wherein the pET series vector is in the T7 promoter in E.
- coli Down-regulated expression of recombinant protein pGEX series vectors were used to regulate the expression of recombinant proteins in E. coli under the tac promoter, pPIC series vectors were used to regulate the expression of recombinant proteins in yeast under the AOX1 promoter, and BacPAK vectors were used in Recombinant proteins are expressed in baculovirus under the control of the pPolh promoter, and the pSV series vector or pCMV series vectors are used to express recombinant proteins in mammalian cells under the control of the CMV, SV40 and (EF)-1 promoters.
- the above vector of the embodiment of the present invention can achieve further efficient expression of the above recombinant protein in prokaryotic cells or eukaryotic cells.
- the above construct carries the following nucleic acid molecules: (1) encoding an immunological checkpoint molecule A nucleic acid molecule of the fragment having the amino acid sequence of SEQ ID NOS: 19 to 21, wherein SEQ ID NO: 19 is the amino acid sequence of human PD-L1 ⁇ , and SEQ ID NO: 20 is human PD-L2 ⁇
- the amino acid sequence of SEQ ID NO: 21 is the amino acid sequence of human PD-L1-PD-L2 ⁇
- the nucleic acid molecule encoding the immunological checkpoint molecule fragment has the nucleotide sequence shown by SEQ ID NOs: 22-24, wherein SEQ ID NO: 22 is the nucleotide sequence of a nucleic acid molecule encoding human PD-L1 ⁇ , SEQ ID NO: 23 is the nucleotide sequence of a nucleic acid molecule encoding human PD-L2 ⁇ , and SEQ ID NO: 24 is encoding a human PD- a
- the vector of the construct is a prokaryotic or eukaryotic protein expression vector.
- the construct according to the embodiment of the present invention efficiently expresses a recombinant protein containing an immunological checkpoint molecular fragment, a helper T cell epitope fragment and an immunostimulatory molecule fragment in a recipient cell, and the recombinant protein can be significantly caused in a tumor patient.
- the tumor-specific antigen immunoreaction which in turn causes specific killing of cytotoxic T lymphocytes (CTL) and specific antibodies against B lymphocyte secretion, achieves specific killing of tumor cells.
- CTL cytotoxic T lymphocytes
- the present invention also proposes a transgenic cell.
- the transgenic cell carries the construct described above, and the transgenic cell proposed in the embodiment of the present invention can express the recombinant protein as described above to actively stimulate the anti-immunoassay in the patient body by active immunization.
- Point such as antibodies to PD-L1 or PD-L2, mobilize spontaneously induced immune cell CTLs already present in the patient, and stimulate the production of anti-immunological checkpoints, such as PD-L1 or PD-L2 CTL, thereby specifically killing Tumor cells.
- the transgenic cells are BL21, BL21 (DE3), BL21 (DE3) pLysS, DH10B, XL1-Blue, Pichia pastors, Kluyveromyces lactis, Sf9, Sf21, High-Five T, CHO cell line , HEK cell line, Hela cell line or COS cell line.
- BL21, BL21 (DE3), BL21 (DE3) pLysS, DH10B and XL1-Blue are E. coli cells
- Pichia pastors and Kluyveromyces lactis are yeast cells
- Sf9, Sf21, and High-Five T are used for baculovirus.
- CHO cell line, HEK cell line, Hela cell line or COS cell line is a mammalian cell line.
- the transgenic cell described above can efficiently express the recombinant protein described above, and then the recombinant protein obtained by protein purification can be administered to a patient.
- the patient actively stimulates the production of anti-PD-L1 or PD-L2 antibodies in the patient's body, mobilizes the spontaneously induced immune cell CTLs already present in the patient, and stimulates the production of anti-PD-L1 or PD-L2CTL. Specific killing of tumor cells.
- the transgenic cell can be an antigen presenting cell and the transgenic cell is a DC cell.
- the antigen presenting cell is derived from a patient, and the antigen presenting cell carrying the aforementioned construct can be further input into the patient, thereby realizing the continuous expression of the recombinant protein described above in the patient, and further Actively immunizing in the body to produce anti-PD-L1 or PD-L2 antibodies in vivo, mobilizing the spontaneously induced immune cell CTLs already present in the patient, and stimulating the production of anti-PD-L1 or PD-L2CTL, Specific killing of tumor cells.
- the inventors have proposed the use of the aforementioned recombinant protein for the preparation of a medicament or a vaccine for preventing or treating a tumor.
- the recombinant protein proposed by the embodiments of the present invention can cause a significant tumor-specific antigen immune response in a tumor patient, and effectively stimulates the production of an anti-immunological checkpoint, such as PD-L1 or PD-L2 antibody, to mobilize the spontaneous induction already existing in the patient.
- the resulting immune cell CTL is stimulated to produce an anti-immunological checkpoint, such as PD-L1 or PD-L2CTL, which effectively kills the tumor cells specifically.
- the recombinant protein proposed in the examples of the present invention has a use in the preparation of a medicament or vaccine effective for preventing or treating a tumor.
- the present invention also proposes the use of the aforementioned recombinant protein for the preparation of a vaccine for the treatment of a viral infection.
- the inventors have found that HBV, HCV, HIV, EBV virus-infected cells express PD-L1, and the vaccine prepared by the recombinant protein of the present invention can stimulate anti-PD-L1 in a patient.
- the antibody mobilizes spontaneously induced immune cell CTLs already present in the patient and stimulates the production of anti-immunological checkpoints, such as PD-L1 CTL, to effectively kill cells infected with the above virus.
- the invention provides a pharmaceutical composition.
- the pharmaceutical composition comprises: the recombinant protein described above; and a pharmaceutically acceptable adjuvant.
- the recombinant protein in the pharmaceutical composition proposed in the examples of the present invention can cause a significant specific antigen immune response, and the function of the adjuvant to enhance the immune response.
- the pharmaceutical composition provided by the embodiment of the present invention effectively stimulates an anti-immunological checkpoint, such as a PD-L1 or PD-L2 antibody, in a tumor patient, and mobilizes the patient already.
- viruses HBV, HCV, HIV, EBV
- the invention provides a DC cell.
- the DC cells are loaded with the recombinant protein described above.
- the DC cells proposed in the embodiments of the present invention can treat tumor antigens in the recombinant protein (such as the immunological checkpoint molecular fragment described above), helper T cell epitope fragments, and immunospins.
- the excimer fragments are presented to the cell surface, respectively, thereby effectively stimulating the production of anti-immunological checkpoints, such as PD-L1 or PD-L2 antibodies, mobilizing the spontaneously induced immune cell CTLs already present in the patient, and stimulating the production of anti-immune checkpoints, For example, PD-L1 or PD-L2CTL, thereby effectively killing tumor cells or cells infected with viruses (HBV, HCV, HIV, EBV). .
- viruses HBV, HCV, HIV, EBV
- the invention provides a targeted immune cell population.
- the targeted immune cell population is obtained by co-culture of DC cells with lymphocytes as described above.
- the targeted immune cell population proposed in the embodiments of the present invention can specifically kill tumor cells, secrete antibodies that specifically bind tumor antigens, and achieve specific removal of tumor cells.
- the invention provides a vaccine.
- the vaccine comprises a recombinant protein as described above, a DC cell as described above or a targeted immune cell population as described above.
- the recombinant protein, DC cell, and targeted immune cell population proposed in the examples of the present invention can cause a significant specific antigen immune response in a patient.
- the vaccine according to the embodiment of the present invention can effectively stimulate the production of an anti-immunization checkpoint, such as a PD-L1 or PD-L2 antibody, mobilize the spontaneously induced immune cell CTL already existing in the patient, and stimulate An anti-immunization checkpoint, such as PD-L1 or PD-L2CTL, is generated to effectively kill tumor cells or cells infected with viruses (HBV, HCV, HIV, EBV).
- an anti-immunization checkpoint such as a PD-L1 or PD-L2 antibody
- the invention provides an antibody.
- the antibody specifically recognizes the recombinant protein described above, and the antibody proposed in the embodiment of the present invention specifically recognizes a tumor antigen.
- the present invention finds that the antibody specifically recognizes an antigen, specifically binds to a tumor cell or a cell infected by a virus (HBV, HCV, HIV, EBV), thereby causing a tumor cell or a virus (HBV) Cells infected with HCV, HIV, EBV) are engulfed by phagocytic cells to achieve specific clearance of tumor cells or cells infected with viruses (HBV, HCV, HIV, EBV).
- a virus HCV, HCV, HIV, EBV
- the present invention proposes a method of preparing an antibody.
- the method comprises: immunizing an animal with the recombinant protein described above; collecting serum of the immunized animal; and purifying the antibody of interest from the serum.
- the method for preparing an antibody proposed in the embodiments of the present invention is simple and convenient, and the antibody can specifically recognize the recombinant protein.
- the therapeutic composition proposed by the present invention may comprise the recombinant protein described above, the nucleic acid described above, the aforementioned construct, the above-described transgenic cell, the aforementioned pharmaceutical composition
- the therapeutic composition proposed by the embodiments of the present invention can directly or indirectly cause a specific antigen immune response, and achieve specificity to tumor cells or cells infected by viruses (HBV, HCV, HIV, EBV). Kill and clear.
- the patient is administered a therapeutically effective amount of the recombinant protein described above, a pharmaceutical composition as described above, a DC cell as described above, a previously described targeted immune cell population, a vaccine as described above, or a front
- the antibodies can be Effectively treat or prevent tumors that express PD-L1 or PD-L2.
- administering refers to introducing a predetermined amount of a substance into a patient in some suitable manner.
- the recombinant protein, pharmaceutical composition, DC cell, targeted immune cell population, vaccine or antibody in the embodiments of the present invention can be administered by any common route as long as it can reach the intended tissue.
- Various modes of administration are contemplated, including peritoneal, venous, muscular, subcutaneous, cortical, oral, topical, nasal, pulmonary, and rectal, but the invention is not limited to these exemplary modes of administration.
- the active ingredient of the orally administered composition should be coated or formulated to prevent its degradation in the stomach.
- the compositions of the invention may be administered as an injectable preparation.
- the pharmaceutical compositions of the invention may be administered using a particular device that delivers the active ingredient to the target cells.
- the frequency and dose of administration of the recombinant protein, pharmaceutical composition, DC cell, targeted immune cell population, vaccine or antibody in the examples of the present invention can be determined by a plurality of related factors including the type of disease to be treated. , route of administration, patient age, sex, weight and severity of the disease, and the type of drug as the active ingredient.
- the daily dose may be divided into 1 dose, 2 doses or multiple doses in a suitable form for administration once, twice or more times throughout the time period, as long as a therapeutically effective amount is achieved. .
- terapéuticaally effective amount refers to an amount sufficient to significantly ameliorate certain symptoms associated with a disease or condition, that is, an amount that provides a therapeutic effect for a given condition and dosage regimen.
- treatment is used to mean obtaining the desired pharmacological and/or physiological effect.
- treatment encompasses administration of a recombinant protein, pharmaceutical composition, DC cell, targeted immune cell population, vaccine or antibody in an embodiment of the invention to a subject, including, but not limited to, administration comprising the agents described herein. The individual in need.
- the recombinant protein and the use thereof, the pharmaceutical composition, the DC cell, the targeted immune cell population, the vaccine, the antibody, the method and system for treating and diagnosing cancer according to the embodiment of the present invention are the inventors of the present application. Hard creative labor and optimization work were discovered and completed.
- the method of isolating DC cells from mouse bone marrow is as follows: bone marrow is punched out from the limbs of the mouse, and the bone marrow is passed through a nylon mesh, and red blood cells are removed with ammonium chloride. The cells were then thoroughly washed with RPMI-1640 medium, and then cultured in 2.5 ml of RPMI-1640 medium containing 10% FBS and 20 ng/ml recombinant mouse GM-CSF (rmGM-CSF). And 20 ng/ml recombinant mouse IL-4 (rmIL-4) (available from PeproTech, Inc., Rocky Hill, NJ).
- the cell supernatant was removed and fresh medium was replaced with 20 ng/ml of rmGM-CSF and 20 ng/ml of rmIL-4.
- the cells were cultured in an incubator at 37 ° C in a 5% CO 2 atmosphere. At 48 hours of the culture process, non-adherent granulocytes were removed and fresh medium was replaced. After 7 days of cell culture, about 80% or more of the cells expressed DC cell-specific markers by FACS analysis.
- Recombinant protein was added to bone marrow-derived DC cells (obtained after 5-7 days of bone marrow cell culture) to activate DC cells, and after 8 hours, the cells were washed with PBS three times, and after further culture for 136 hours, DC cells were used as Immunization model.
- antigen-activated DC cells were stimulated with 100 ng/ml LPS (Sigma, St. Louis, MO) for 24 hours, and the cells were washed with PBS and injected into mice via the soles of mice (C57BL/6, Jackson). Laboratory) In vivo.
- rodent lung cancer cells CMT167 (C57BL) (purchased from the European Collection of Authenticated Cell Cultures (ECAC)) were introduced into the right abdominal cavity of syngeneic C57BL/6 mice by subcutaneous injection. After tumor inoculation, mice were randomized and injected into antigen-activated DC cells or PBS in different groups on different days. Tumor volume was measured 2 or 3 times a week using a caliper.
- the CD8+ CTL response was assessed by standard chromium release assays.
- Standard chromium release assessment experiments were performed by measuring the ability of spleen cells to lyse target cells in vitro. Splenocytes from immunized mice were restimulated in vitro with RPMI containing the polypeptide for 4 to 6 days. Target cells and control cells were labeled with 51Cr sodium chromate solution for 90 min. Different numbers of effector cells were co-cultured with a certain number (1 ⁇ 104/well) of target cells for 3 hours at 37° C. in a 96-well plate v-bottom plate (200 microliters of medium per well). 100 microliters of supernatant was collected from every 3 wells.
- the dissolution rate is calculated by the following formula: (experimental chromium release amount - spontaneous chromium release amount) / (maximum chromium release amount - spontaneous chromium release amount) X 100.
- the amount of chromium released was achieved by co-cultivation, placing the wells on a centrifuge and centrifuging, and calculating the radiation activity in the supernatant by a gamma counter (purchased from Beckman Coulter, Inc., Fullerton, CA) ( Chromium release).
- Synthesis of fusion gene 1 contains part of human PD-L1 sequence or PD-L2 sequence (Accession number GenBank: AF177937.1), intact PADRE helper T cell epitope sequence and complete GM-CSF, IL-12 or RANTES sequence (Accession number GenBank: M11734.1) and flanking cloning site sequences (fusion gene-structure shown in Figure 1) (synthesized by GENEWIZ, South Plainfield, NJ, USA).
- Synthetic Fusion Gene 2 Contains a partial human PD-L1 sequence or a PD-L2 sequence (GenBank: AF177937.1) and an intact helper T cell PADRE epitope sequence.
- the target recombinant plasmid was electroporated into Escherichia coli BL21(DE3) (Novagen) competent cells, and then Escherichia coli BL21 (DE3) was seeded on LB agar plates (containing 50 ⁇ g/ml of ampicillin). Amplification culture is carried out.
- the method of recombinant protein expression described below is one of a series of experiments under different experimental conditions.
- the frozen cell pellet was resuspended in lysate (50 mM Tris, pH 8.0, 1 mM EDTA and 1 mM PMSF) with a mass to volume ratio of cell pellet to lysate of 1:10.
- the inclusion bodies containing the recombinant protein were restored to activity under the conditions of French Pressure (Constant Systems LTD) of 137.9 MPa.
- French Pressure Constant Systems LTD
- an equal volume of lysate is added to dilute to reduce viscosity, which is more advantageous for obtaining inclusion bodies.
- the lysed solution was centrifuged at 48,000 x g for 30 min to cause inclusion bodies to precipitate.
- the supernatant was discarded and the pellet was washed three times to remove endotoxin, protein and DNA from the host cells.
- the solution used for the first pass of cleaning contained 50 mM Tris, pH 8.0, 5 mM EDTA and 2% Triton x-100.
- the solution used for the second pass of cleaning contained 50 mM Tris, pH 8.0, 5 mM EDTA, 1% sodium deoxycholate.
- the solution used for the third cleaning consisted of 50 mM Tris, pH 8.0, 5 mM EDTA, and 1 M NaCl. After washing, the pellet was resuspended at room temperature with a special lysate (mass to volume ratio of 1:40), stirred for 30 min and centrifuged.
- the solubilized protein was further purified by a Ni-NTA Fast Start Kit (Qiagen).
- the eluted proteins were analyzed by 12% SDS-PAGE gel electrophoresis, and the protein concentration was determined by Bradford et al. (Bio-Rad Laboratories). Recombinant proteins with a purity greater than 90% were stored at -20 °C for subsequent studies.
- Example 4 DC cells loaded with a fusion protein comprising human PD-L1 were effective in inducing anti-PD-L1 antibody production and CTL responses in mice
- DC cells loaded with the PD-L1 fusion protein were able to induce anti-PD-L1 antibody production and CTL responses in mice.
- DC cells loaded with PD-L1 fusion protein in mice The ability of the body to elicit a PD-L1-specific response was verified by immunizing mice with DC cells.
- PD-L1 ⁇ -PADRE Th-GM-CSF recombinant protein
- PD-L1 ⁇ protein
- immunostimulatory factor recombinant GM-CSF, Genzyme, Tarzana, CA
- the levels of PD-L1-specific IgG in the serum of each group of rats were determined by ELISA, and the recombinant PD-L1 protein (Abeam, Cambridge, MA, USA) was plated every 3 wells of the ELISA plate, and the ELISA value was passed through serum (1). : 100 times dilution) The mean value of OD450nm values ⁇ SD was obtained.
- Figure 2 shows that iPD-L1-Vax DC cells can induce significant anti-PD-L1 antibody responses, whereas PD-L1 ⁇ protein-loaded DC cells can only induce weak anti-PD- L1 antibody reaction.
- spleen cells were isolated from tumor cell suspensions vaccinated with immunized mice.
- the isolated T cells were re-stimulated by PD-L1 recombinant protein-activated DC cells (10 ⁇ g/ml), and then the in vitro 51Cr release test was performed, and the 51Cr release test was performed according to the specified T/E (target cell: effector cell) ratio.
- the target cell was the PD-L1+ murine lung cancer cell line CMT167 (C57BL) (available from the European Collection of Authenticated Cell Cultures (ECACC)).
- Figure 3 shows that iPD-L1-Vax DC cells were able to induce significant anti-PD-L1 CTL responses, whereas DC cells loaded with PD-L1 ⁇ protein induced only weak anti-PD-L1 CTLs. reaction.
- DC cells DC cells were loaded with recombinant protein (PD-L1 ⁇ -PADRE Th-GM-CSF) (iPD-L1-Vax), protein PD-L1 ⁇ , immunostimulatory factor (GMCSF), or PBS, and DC cells were pre-treated Maturation was stimulated twice in vitro using LPS in vitro. Tumor growth was measured every 3 to 4 days after immunization.
- Figure 4 shows that the immunization of mice with iPD-L1-Vax DC can significantly inhibit the growth of PD-L1+ lung cancer, but with DC immunization with protein PD-L1 ⁇ or PBS. Mice did not inhibit the growth of PD-L1+ lung cancer.
- the inventors examined the loading of recombinant protein (PD-L1 ⁇ -PADRE Th-IL-12 or PD-L1 ⁇ -PADRE Th-RANTES or PD-L2 ⁇ -PADRE Th-GM-CSF or PD-L2 ⁇ - PADRE Th-IL-12 or DC cells of PD-L1 ⁇ -PADRE Th-RANTES or PD-L1/L2 ⁇ -PADRE Th-GM-CSF or PD-L1/L2 ⁇ -PADRE Th-IL-12 or PD-L1/L2 ⁇ -PADRE Th-RANTES)
- the condition of inducing anti-PD-L1 or anti-PD-L2 antibody production and CTL reaction in mice and the case of vaccination controlling the growth of PD-L1+ or PD-L2+ lung cancer, the experimental methods are as described in Example 4 and Example 5.
Abstract
A recombinant protein and uses thereof. The recombinant protein comprises an immune checkpoint molecule segment, an auxiliary T cell epitope segment, and an immunostimulatory molecule segment.
Description
优先权信息Priority information
本申请请求2016年04月11日向中国国家知识产权局提交的、专利申请号为201610222458.0的专利申请的优先权和权益，并且通过参照将其全文并入此处。Priority is claimed on Japanese Patent Application No. 201610222458.0, the entire disclosure of which is hereby incorporated by reference.
本发明涉及生物工程领域，具体地，本发明涉及重组蛋白及其用途。The present invention relates to the field of bioengineering, and in particular, to recombinant proteins and uses thereof.
癌症，由于细胞内基因突变导致细胞增殖失控的一种疾病。目前已成为人类健康的重大威胁，是导致人类死亡的主要原因之一。世界卫生组织(WHO)在发表的《全球癌症报告2014》中指出，2012年全球癌症患者和死亡病例都在迅速增加，而新增癌症病例有近一半出现在亚洲，其中大部分在中国，中国新增癌症病例高居第一位。《2012年中国肿瘤登记年报》数据显示，中国每年新增癌症病例约350万，约有250万人因此死亡。因此，寻找高效特异的癌症治疗方法具有重大的临床价值。Cancer, a disease in which cell proliferation is out of control due to genetic mutations in cells. It has become a major threat to human health and one of the main causes of human death. According to the World Health Organization (WHO) published in the Global Cancer Report 2014, global cancer patients and deaths are increasing rapidly in 2012, and nearly half of new cancer cases occur in Asia, most of which are in China, China. New cancer cases ranked first. According to the 2012 China Cancer Registration Annual Report, about 3.5 million new cancer cases are reported each year in China, and about 2.5 million people die. Therefore, finding a highly effective and specific cancer treatment method has great clinical value.
传统的肿瘤治疗方法主要包括手术、放疗和化疗，但这几种方法都具有较大的局限性，比如由于癌细胞的近端入侵或远端转移，手术切除后的肿瘤转移复发率较高，而放疗和化疗对于机体自身的正常细胞尤其是造血系统和免疫系统会造成严重的损害，因此对于已发生肿瘤转移的患者也很难达到较好的远期疗效。随着肿瘤分子机制的深入研究和生物技术的进一步发展，靶向药物治疗和免疫治疗在肿瘤的综合治疗中发挥着愈来愈大的作用。靶向疗法主要包括单克隆抗体(有时归为被动胞回输和肿瘤疫苗等。免疫疗法通过调动机体的免疫系统，增强肿瘤微环境抗肿瘤免免疫疗法)和小分子靶向药物，而免疫疗法主要包括细胞因子疗法、免疫检验点单抗、过继免疫疗法，从而控制和杀伤肿瘤细胞，因此有效率高，特异性强，耐受性好的优点，在肿瘤治疗中具有广阔的前景。Traditional methods of cancer treatment mainly include surgery, radiotherapy and chemotherapy, but these methods have great limitations. For example, due to proximal invasion or distant metastasis of cancer cells, the tumor metastasis recurrence rate after surgical resection is high. Radiotherapy and chemotherapy can cause serious damage to the body's own normal cells, especially the hematopoietic system and immune system. Therefore, it is difficult to achieve better long-term results for patients who have already had tumor metastasis. With the in-depth study of the molecular mechanism of tumors and the further development of biotechnology, targeted drug therapy and immunotherapy play an increasingly important role in the comprehensive treatment of tumors. Targeted therapies mainly include monoclonal antibodies (sometimes classified as passive cell transfusion and tumor vaccines, immunotherapy through the immune system of the motivational body, enhanced tumor microenvironment anti-tumor immunotherapy) and small molecule targeted drugs, while immunotherapy It mainly includes cytokine therapy, immunoassay monoclonal antibody, and adoptive immunotherapy to control and kill tumor cells. Therefore, it has the advantages of high efficiency, high specificity and good tolerance, and has broad prospects in cancer therapy.
肿瘤免疫治疗疫苗主要包括肿瘤细胞疫苗、树突状细胞(DC细胞)疫苗、蛋白&多肽疫苗、核酸疫苗、基因工程疫苗和抗独特型肿瘤疫苗.这些疫苗能够杀伤肿瘤的主要机制即是通过引起患者针对于肿瘤特异性抗原免疫反应，包括抗体反应和细胞毒性T淋巴细胞(CTL)特异性杀伤等。Tumor immunotherapy vaccines mainly include tumor cell vaccines, dendritic cell (DC cell) vaccines, protein & peptide vaccines, nucleic acid vaccines, genetically engineered vaccines and anti-idiotype tumor vaccines. The main mechanism by which these vaccines can kill tumors is through The patient is directed against a tumor-specific antigen immune response, including antibody response and cytotoxic T lymphocyte (CTL) specific killing.
然而，这些肿瘤疫苗针对肿瘤相关抗原，临床疗效弱，仍有待进一步深入研究和开发.来增强临床疗效。However, these tumor vaccines target tumor-associated antigens, and their clinical efficacy is weak, and further research and development are needed to enhance clinical efficacy.
发明内容Summary of the invention
本发明是发明人基于以下问题和事实的发现而提出的：The present invention has been made by the inventors based on the findings of the following problems and facts:
肿瘤细胞高表达免疫检查点分子PD-L1或PD-L2，与活化的细胞毒性T淋巴细胞上表
达的PD-1相结合，进而抑制肿瘤的T淋巴细胞反应，来逃避细胞毒性T淋巴细胞的免疫杀伤。目前，通过体外生产抗PD－L1/PD抗体，再注射病人体内的被动免疫疗法，虽可短暂激活病人体内已经存在自发诱导产生的免疫细胞-细胞毒性T淋巴细胞(CTL)，来杀伤肿瘤细胞，但临床效果持续时间短，需不断重复注射抗PD－L1/PD抗体，来维持体内抗体浓度。Tumor cells highly express immune checkpoint molecules PD-L1 or PD-L2, and activated cytotoxic T lymphocytes on the surface
The combination of PD-1, which inhibits the T lymphocyte response of the tumor, escapes the immune killing of cytotoxic T lymphocytes. At present, by in vitro production of anti-PD-L1/PD antibodies, re-injection of passive immunotherapy in patients, although transient activation of immune cells-cytotoxic T lymphocytes (CTL) that have been spontaneously induced in patients, to kill tumor cells However, the clinical effect lasts for a short time, and it is necessary to continuously inject anti-PD-L1/PD antibody to maintain the antibody concentration in the body.
本发明旨在至少在一定程度上解决相关技术中的技术问题之一。为此，本发明的一个目的在于提出了一种具有引起肿瘤特异性抗原免疫反应的重组PD-L1蛋白，该蛋白通过主动免疫，在病人体内持续刺激产生抗PD－L1抗体，调动病人体内已经存在的自发诱导产生的免疫细胞CTL，并且刺激产生新的抗PD-L1CTL，进而特异性杀伤肿瘤细胞。本发明所提出的重组蛋白引起的对肿瘤细胞的主动免疫杀伤效果显著。The present invention aims to solve at least one of the technical problems in the related art to some extent. To this end, an object of the present invention is to provide a recombinant PD-L1 protein having an immune response which causes a tumor-specific antigen, which is actively stimulated to produce an anti-PD-L1 antibody in a patient, and the patient has been mobilized. The presence of spontaneously induced immune cell CTLs is induced and stimulates the production of new anti-PD-L1 CTLs, which in turn specifically kill tumor cells. The active immune killing effect on tumor cells caused by the recombinant protein proposed by the invention is remarkable.
在本发明的第一方面，本发明提出了一种重组蛋白。根据本发明的实施例，所述重组蛋白包括：免疫检查点分子片段；辅助T细胞抗原决定基片段；以及免疫刺激分子片段。本发明实施例所提出的重组蛋白可在体内持续刺激产生抗免疫检查点抗体，调动体内已经存在的自发诱导产生的免疫细胞CTL，并且刺激产生抗免疫检查点的CTL，进而特异性杀伤肿瘤细胞。本发明实施例所提出的重组蛋白引起的对肿瘤细胞的主动免疫杀伤效果显著。In a first aspect of the invention, the invention proposes a recombinant protein. According to an embodiment of the invention, the recombinant protein comprises: an immunological checkpoint molecule fragment; a helper T cell epitope fragment; and an immunostimulatory molecule fragment. The recombinant protein proposed in the embodiments of the present invention can continuously stimulate the production of anti-immunization checkpoint antibodies in vivo, mobilize the spontaneously induced immune cell CTLs existing in the body, and stimulate the production of CTLs against the immune checkpoints, thereby specifically killing the tumor cells. . The active immune killing effect on tumor cells caused by the recombinant protein proposed by the embodiments of the present invention is remarkable.
根据本发明的实施例，所述重组蛋白还可以及进一步包括如下附加技术特征至少之一：According to an embodiment of the invention, the recombinant protein may further comprise at least one of the following additional technical features:
根据本发明的实施例，所述免疫检查点分子为PD-L1或PD-L2。PD-L1或PD-L2在肿瘤细胞中特异性表达，进而本发明实施例所提出的重组蛋白可引起的肿瘤抗原免疫反应的特异性更强。According to an embodiment of the invention, the immune checkpoint molecule is PD-L1 or PD-L2. PD-L1 or PD-L2 is specifically expressed in tumor cells, and the specificity of the tumor antigen immune response caused by the recombinant protein proposed in the examples of the present invention is stronger.
根据本发明的实施例，所述免疫检查点分子片段为所述PD-L1或PD-L2的去除跨膜区的细胞外分子片段。PD-L1或PD-L2的去除跨膜区的细胞外分子片段仅具有肿瘤抗原性，不具有肿瘤免疫抑制功能，进而PD-L1或PD-L2的去除跨膜区的细胞外分子片段可更为有效地引起肿瘤抗原免疫反应，且特异性进一步提高。According to an embodiment of the invention, the immunological checkpoint molecule fragment is an extracellular molecular fragment of the PD-L1 or PD-L2 removal transmembrane region. The extracellular molecular fragment of PD-L1 or PD-L2 in the transmembrane region has only tumor antigenicity and does not have tumor immunosuppressive function, and thus the extracellular molecular fragment of PD-L1 or PD-L2 in the transmembrane region can be further removed. In order to effectively cause a tumor antigen immune response, the specificity is further improved.
根据本发明的实施例，所述辅助T细胞抗原决定基为广谱PADRE辅助T细胞抗原决定基。广谱PADRE辅助T细胞抗原决定基可有效激活辅助T细胞，进而进一步增强重组蛋白所引起的细胞毒性T淋巴细胞(CTL)的特异性杀伤。According to an embodiment of the invention, the helper T cell epitope is a broad spectrum PADRE helper T cell epitope. Broad-spectrum PADRE-assisted T cell epitopes can effectively activate helper T cells, thereby further enhancing the specific killing of cytotoxic T lymphocytes (CTLs) caused by recombinant proteins.
根据本发明的实施例，所述免疫刺激分子为粒细胞集落刺激生物因子、白细胞介素-12或趋化因子。上述免疫刺激分子具有生物活性，可显著增强树突状细胞(DC细胞)的抗原呈递功能和增强细胞毒性T淋巴细胞(CTL)和B淋巴细胞的活性，进而本发明实施例的重组蛋白可更为有效地引起肿瘤抗原免疫反应。According to an embodiment of the invention, the immunostimulatory molecule is a granulocyte colony-stimulating biological factor, interleukin-12 or a chemokine. The above immunostimulatory molecule has biological activity, can significantly enhance the antigen presenting function of dendritic cells (DC cells) and enhance the activity of cytotoxic T lymphocytes (CTLs) and B lymphocytes, and the recombinant protein of the embodiments of the present invention can be further To effectively cause tumor antigen immune response.
根据本发明的实施例，所述辅助T细胞抗原决定基片段N端与所述免疫检查点分子片段的C端相连，所述辅助T细胞抗原决定基片段C端与所述免疫刺激分子片段的N端相连。
本发明实施例的重组蛋白中的相关分子片段在上述连接状态下，有利于肿瘤抗原PD-L1或PD-L2片段在DC细胞中的呈递，有利于辅助T细胞抗原决定基和免疫刺激分子发挥相应的激活免疫细胞的功能，进而本发明实施例的重组蛋白可更为有效地引起肿瘤抗原免疫反应。According to an embodiment of the present invention, the N-terminus of the helper T cell epitope fragment is linked to the C-terminus of the immunological checkpoint molecule fragment, and the C-terminus of the helper T cell epitope fragment and the immunostimulatory molecule fragment N ends are connected.
In the above-mentioned ligation state, the related molecular fragment in the recombinant protein of the present invention facilitates the presentation of the tumor antigen PD-L1 or PD-L2 fragment in DC cells, and is beneficial to the help of T cell epitopes and immunostimulatory molecules. The corresponding function of activating immune cells, and thus the recombinant protein of the examples of the present invention, can more effectively cause a tumor antigen immune response.
在本发明的第二方面，本发明提出了一种重组蛋白。根据本发明的实施例，所述重组蛋白具有SEQ ID NO：1～9所示的氨基酸序列。In a second aspect of the invention, the invention proposes a recombinant protein. According to an embodiment of the present invention, the recombinant protein has the amino acid sequences shown in SEQ ID NOS: 1 to 9.
本发明实施例所述提出的重组蛋白可引起肿瘤特异性抗原免疫反应，该蛋白通过主动免疫，在病人体内刺激产生抗PD－L1或PD-L2抗体，调动病人体内已经存在的自发诱导产生的免疫细胞CTL，并且刺激产生抗PD-L1或PD-L2CTL，进而特异性杀伤肿瘤细胞。本发明所提出的重组蛋白引起的对肿瘤细胞的主动免疫杀伤效果显著。The recombinant protein proposed in the embodiment of the present invention can cause a tumor-specific antigen immune response, and the protein stimulates the production of anti-PD-L1 or PD-L2 antibody in the patient by active immunization, and mobilizes the spontaneous induction of the existing in the patient. The cells are immunized with CTL and stimulated to produce anti-PD-L1 or PD-L2 CTL, thereby specifically killing tumor cells. The active immune killing effect on tumor cells caused by the recombinant protein proposed by the invention is remarkable.
在本发明的第三方面，本发明提出了一种核酸。根据本发明的实施例，所述核酸编码前面所述的重组蛋白，并且所述核酸具有SEQ ID NO：10～18所示的核苷酸序列。In a third aspect of the invention, the invention proposes a nucleic acid. According to an embodiment of the present invention, the nucleic acid encodes the recombinant protein described above, and the nucleic acid has the nucleotide sequence shown in SEQ ID NOS: 10 to 18.
本发明实施例所述提出的核酸编码的重组蛋白通过主动免疫，在病人体内刺激产生抗PD－L1或PD-L2抗体，调动病人体内已经存在的自发诱导产生的免疫细胞CTL，并且刺激产生抗PD-L1或PD-L2CTL，进而特异性杀伤肿瘤细胞。本发明所提出的重组蛋白引起的对肿瘤细胞的主动免疫杀伤效果显著。The recombinant protein encoded by the nucleic acid according to the embodiment of the present invention stimulates the production of anti-PD-L1 or PD-L2 antibody in the patient by active immunization, mobilizes the spontaneously induced immune cell CTL which has already existed in the patient, and stimulates the production of anti-drug. PD-L1 or PD-L2CTL, which specifically kills tumor cells. The active immune killing effect on tumor cells caused by the recombinant protein proposed by the invention is remarkable.
在本发明的第四方面，本发明提出了一种构建体。根据本发明的实施例，所述构建体
携带前面所述的核酸。本发明实施例所提出的构建体导入受体细胞，可实现前面所述的核酸的高效表达，进而在受体细胞中高效表达前面所述的重组蛋白。In a fourth aspect of the invention, the invention proposes a construct. According to an embodiment of the invention, the construct
Carry the nucleic acid described above. The construct introduced into the recipient cell of the present invention can achieve high-efficiency expression of the nucleic acid described above, and thereby efficiently express the recombinant protein described above in the recipient cell.
根据本发明的实施例，所述的构建体还可以进一步包括如下附加技术特征至少之一：According to an embodiment of the invention, the construct may further comprise at least one of the following additional technical features:
根据本发明的实施例，所述构建体的载体为pET系列载体、pPIC系列载体、BacPAK、pSV系列载体或pCMV系列载体。本发明实施例的上述载体可实现在原核细胞或真核细胞中进一步高效表达上述重组蛋白。According to an embodiment of the invention, the vector of the construct is a pET series vector, a pPIC series vector, a BacPAK, a pSV series vector or a pCMV series vector. The above vector of the embodiment of the present invention can achieve further efficient expression of the above recombinant protein in prokaryotic cells or eukaryotic cells.
在本发明的第五方面，本发明提出了一种构建体。根据本发明的实施例，所述构建体携带下列核酸分子:(1)编码免疫检查点分子片段的核酸分子，所述免疫检查点分子片段具有SEQ ID NO：19～21所示的氨基酸序列，所述编码免疫检查点分子片段的核酸分子具有SEQ ID NO：22～24所示的核苷酸序列；(2)编码辅助T细胞抗原决定基片段的核酸分子，所述辅助T细胞抗原决定基片段具有SEQ ID NO：25所示的氨基酸序列，所述编码辅助T细胞抗原决定基片段的核酸分子具有SEQ ID NO：26所示的核苷酸序列；以及(3)编码免疫刺激分子片段的核酸分子，所述免疫刺激分子片段具有SEQ ID NO：27～29所示的氨基酸序列，所述编码免疫刺激分子片段的核酸分子具有SEQ ID NO：30～32所示的核苷酸序列。任选地，所述构建体的载体为pET系列载体、pPIC系列载体、BacPAK、pSV系列载体或pCMV系列载体。In a fifth aspect of the invention, the invention proposes a construct. According to an embodiment of the present invention, the construct carries the following nucleic acid molecule: (1) a nucleic acid molecule encoding a fragment of an immunological checkpoint molecule having the amino acid sequence set forth in SEQ ID NOS: 19-21, The nucleic acid molecule encoding the immunological checkpoint molecule fragment has the nucleotide sequence shown in SEQ ID NOS: 22-24; (2) the nucleic acid molecule encoding the helper T cell epitope fragment, the helper T cell epitope The fragment has the amino acid sequence set forth in SEQ ID NO: 25, the nucleic acid molecule encoding the helper T cell epitope fragment has the nucleotide sequence set forth in SEQ ID NO: 26; and (3) the fragment encoding the immunostimulatory molecule The nucleic acid molecule having the amino acid sequence of SEQ ID NOS: 27 to 29, and the nucleic acid molecule encoding the immunostimulatory molecule fragment has the nucleotide sequence shown by SEQ ID NOS: 30 to 32. Optionally, the vector of the construct is a pET series vector, a pPIC series vector, a BacPAK, a pSV series vector or a pCMV series vector.
本发明实施例所述提出的构建体在受体细胞中高效表达含有免疫检查点分子片段、辅助T细胞抗原决定基片段和免疫刺激分子片段的重组蛋白，该重组蛋白通过主动免疫，在
病人体内刺激产生抗PD－L1抗体，调动病人体内已经存在的自发诱导产生的免疫细胞CTL，并且刺激产生抗PD-L1CTL，进而特异性杀伤肿瘤细胞。The construct according to the embodiment of the present invention efficiently expresses a recombinant protein containing an immunological checkpoint molecular fragment, a helper T cell epitope fragment and an immunostimulatory molecule fragment in a recipient cell, and the recombinant protein is actively immunized.
The patient stimulates the production of anti-PD-L1 antibody, mobilizes the spontaneously induced immune cell CTL already present in the patient, and stimulates the production of anti-PD-L1CTL, thereby specifically killing the tumor cells.
在本发明的第六方面，本发明提出了一种转基因细胞。根据本发明的实施例，所述转基因细胞携带前面所述的构建体。本发明实施例所提出的转基因细胞可高表达前面所述的重组蛋白，所获得的重组蛋白通过主动免疫，在病人体内刺激产生抗免疫检查点，如PD－L1或PD-L2的抗体，调动病人体内已经存在的自发诱导产生的免疫细胞CTL，并且刺激产生抗免疫检查点，如PD-L1或PD-L2的CTL，进而特异性杀伤肿瘤细胞。In a sixth aspect of the invention, the invention provides a transgenic cell. According to an embodiment of the invention, the transgenic cell carries a construct as described above. The transgenic cells proposed in the embodiments of the present invention can express the recombinant protein as described above, and the obtained recombinant protein stimulates an anti-immunological checkpoint, such as PD-L1 or PD-L2 antibody, by active immunization, and mobilizes. Spontaneously induced immune cell CTLs already present in the patient and stimulate the production of anti-immunological checkpoints, such as PD-L1 or PD-L2 CTL, which specifically kill tumor cells.
根据本发明的实施例，所述转基因细胞还可以进一步包括如下附加技术特征至少之一：According to an embodiment of the invention, the transgenic cell may further comprise at least one of the following additional technical features:
根据本发明的实施例，所述转基因细胞为BL21，BL21(DE3)，BL21(DE3)pLysS，DH10B，XL1-Blue，Pichia pastors，Kluyveromyces lactis，Sf9，Sf21，High-Five T，CHO细胞系，HEK细胞系，Hela细胞系或COS细胞系。根据本发明的实施例，上述的转基因细胞可高效表达前面所述的重组蛋白，进而通过蛋白纯化，获得的重组蛋白给予患者，可在患者体内通过主动免疫，在病人体内进一步有效刺激产生抗PD－L1或PD-L2抗体，调动病人体内已经存在的自发诱导产生的免疫细胞CTL，并且刺激产生抗PD-L1或PD-L2CTL，进而特异性杀伤肿瘤细胞。According to an embodiment of the present invention, the transgenic cells are BL21, BL21 (DE3), BL21 (DE3) pLysS, DH10B, XL1-Blue, Pichia pastors, Kluyveromyces lactis, Sf9, Sf21, High-Five T, CHO cell line, HEK cell line, Hela cell line or COS cell line. According to an embodiment of the present invention, the transgenic cell can efficiently express the recombinant protein described above, and then the recombinant protein obtained by protein purification can be administered to a patient, and the patient can be further effectively stimulated to produce anti-PD in the patient by active immunization. The -L1 or PD-L2 antibody mobilizes spontaneously induced immune cell CTLs already present in the patient and stimulates the production of anti-PD-L1 or PD-L2CTL, thereby specifically killing the tumor cells.
根据本发明的实施例，所述转基因细胞为抗原呈递细胞。根据本发明的实施例，所述抗原呈递细胞来源于病人，进而携带前面所述构建体的抗原呈递细胞可进一步回输入病人体内，进而在病人体内实现前面所述重组蛋白的持续表达，进而可在患者体内通过主动免疫，在体内进一步有效刺激产生抗PD－L1或PD-L2抗体，调动病人体内已经存在的自发诱导产生的免疫细胞CTL，并且刺激产生抗PD-L1或PD-L2CTL，进而特异性杀伤肿瘤细胞。According to an embodiment of the invention, the transgenic cell is an antigen presenting cell. According to an embodiment of the present invention, the antigen presenting cell is derived from a patient, and the antigen presenting cell carrying the aforementioned construct can be further input into the patient, thereby realizing the continuous expression of the recombinant protein described above in the patient, and further Actively immunizing in the body to produce anti-PD-L1 or PD-L2 antibodies in vivo, mobilizing the spontaneously induced immune cell CTLs already present in the patient, and stimulating the production of anti-PD-L1 or PD-L2CTL, Specific killing of tumor cells.
根据本发明的实施例，所述转基因细胞为DC细胞。DC细胞具有抗原递呈功能，来源于病人自身的DC细胞携带前面所述的构建体，回输回病人体内，可实现前面所述重组蛋白在并病人体内的高效表达并将肿瘤抗原PD-L1或PD-L2高效呈递在DC细胞的表面，进而进一步有效刺激产生抗PD－L1或PD-L2抗体，调动病人体内已经存在的自发诱导产生的免疫细胞CTL，并且刺激产生抗PD-L1或PD-L2CTL，进而进一步有效地特异性杀伤肿瘤细胞。According to an embodiment of the invention, the transgenic cell is a DC cell. The DC cells have an antigen-presenting function, and the DC cells derived from the patient themselves carry the aforementioned construct and are returned to the patient, thereby realizing the high-efficiency expression of the recombinant protein described above in the patient and the tumor antigen PD-L1. Or PD-L2 is efficiently presented on the surface of DC cells, thereby further effectively stimulating the production of anti-PD-L1 or PD-L2 antibodies, mobilizing the spontaneously induced immune cell CTLs already present in the patient, and stimulating the production of anti-PD-L1 or PD -L2CTL, which further effectively kills tumor cells specifically.
在本发明的第七方面，本发明提出了前面所述的重组蛋白在制备药物中的用途，所述药物用于预防或治疗肿瘤。本发明实施例所提出的重组蛋白在肿瘤患者体内可引起显著的肿瘤特异性抗原免疫反应，有效刺激产生抗免疫检查点，如PD－L1或PD-L2抗体，调动病人体内已经存在的自发诱导产生的免疫细胞CTL，并且刺激产生抗免疫检查点，如PD-L1或PD-L2CTL，进而有效地特异性杀伤肿瘤细胞。进而发明人进一步经过实验验证发现，
本发明实施例所提出的重组蛋白具有在制备有效用于预防或治疗肿瘤的药物中的用途。In a seventh aspect of the invention, the invention provides the use of a recombinant protein as described above for the preparation of a medicament for the prevention or treatment of a tumor. The recombinant protein proposed by the embodiments of the present invention can cause a significant tumor-specific antigen immune response in a tumor patient, and effectively stimulates the production of an anti-immunological checkpoint, such as PD-L1 or PD-L2 antibody, to mobilize the spontaneous induction already existing in the patient. The resulting immune cell CTL is stimulated to produce an anti-immunological checkpoint, such as PD-L1 or PD-L2CTL, which effectively kills the tumor cells specifically. Furthermore, the inventors further verified through experiments that
The recombinant protein proposed in the examples of the present invention has utility in the preparation of a medicament effective for preventing or treating a tumor.
在本发明的第八方面，本发明提出了前面所述的重组蛋白在制备疫苗中的用途，所述疫苗用于预防或治疗肿瘤。本发明实施例所提出的重组蛋白在肿瘤患者体内可引起显著的肿瘤特异性抗原免疫反应，有效刺激产生抗免疫检查点，如PD－L1或PD-L2抗体，调动病人体内已经存在的自发诱导产生的免疫细胞CTL，并且刺激产生抗免疫检查点，如PD-L1或PD-L2CTL，进而有效地特异性杀伤肿瘤细胞。进而发明人进一步经过实验验证发现，本发明实施例所提出的重组蛋白具有在制备有效用于预防或治疗肿瘤的疫苗中的用途。In an eighth aspect of the invention, the invention provides the use of a recombinant protein as described above for the preparation of a vaccine for the prevention or treatment of a tumor. The recombinant protein proposed by the embodiments of the present invention can cause a significant tumor-specific antigen immune response in a tumor patient, and effectively stimulates the production of an anti-immunological checkpoint, such as PD-L1 or PD-L2 antibody, to mobilize the spontaneous induction already existing in the patient. The resulting immune cell CTL is stimulated to produce an anti-immunological checkpoint, such as PD-L1 or PD-L2CTL, which effectively kills the tumor cells specifically. Further, the inventors have further experimentally verified that the recombinant protein proposed in the examples of the present invention has a use in preparing a vaccine effective for preventing or treating a tumor.
在本发明的第九方面，本发明提出了前面所述的重组蛋白在制备疫苗中的用途，所述疫苗用于治疗病毒感染。根据本发明的实施例，发明人发现，HBV、HCV、HIV、EBV病毒感染的细胞表达PD-L1，本发明实施例所提出的重组蛋白所制备的疫苗可在病人体内刺激产生抗PD－L1抗体，调动病人体内已经存在的自发诱导产生的免疫细胞CTL，并且刺激产生抗免疫检查点，如PD-L1CTL，进而有效地特异性杀伤被上述病毒感染的细胞。In a ninth aspect of the invention, the invention provides the use of a recombinant protein as described above for the preparation of a vaccine for the treatment of a viral infection. According to an embodiment of the present invention, the inventors have found that HBV, HCV, HIV, EBV virus-infected cells express PD-L1, and the vaccine prepared by the recombinant protein of the present invention can stimulate anti-PD-L1 in a patient. The antibody mobilizes spontaneously induced immune cell CTLs already present in the patient and stimulates the production of anti-immunological checkpoints, such as PD-L1 CTL, to effectively kill cells infected with the above virus.
在本发明的第十方面，本发明提出了一种药物组合物。根据本发明的实施例，所述药物组合物包括：前面所述的重组蛋白；以及药学上可接受的佐剂。本发明实施例所提出的药物组合物中的重组蛋白可引起显著的特异性抗原免疫反应，加之佐剂的增强免疫应答的功能。根据本发明的实施例，本发明实施例所提出的药物组合物在病人体内有效刺激产生抗免疫检查点，如PD－L1或PD-L2抗体，调动病人体内已经存在的自发诱导产生的免疫细胞CTL，并且刺激产生抗免疫检查点，如PD-L1或PD-L2CTL，进而有效地特异性杀伤肿瘤细胞或被病毒(HBV、HCV、HIV、EBV)感染的细胞。In a tenth aspect of the invention, the invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises: the recombinant protein described above; and a pharmaceutically acceptable adjuvant. The recombinant protein in the pharmaceutical composition proposed in the examples of the present invention can cause a significant specific antigen immune response, and the function of the adjuvant to enhance the immune response. According to an embodiment of the present invention, the pharmaceutical composition provided by the embodiment of the present invention effectively stimulates an anti-immunization checkpoint, such as a PD-L1 or PD-L2 antibody, to mobilize an already existing spontaneously induced immune cell in a patient. CTL, and stimulate the production of anti-immunological checkpoints, such as PD-L1 or PD-L2CTL, to effectively kill tumor cells or cells infected with viruses (HBV, HCV, HIV, EBV).
在本发明的第十一方面，本发明提出了一种DC细胞。根据本发明的实施例，所述DC细胞负载前面所述的重组蛋白。根据本发明的实施例，本发明实施例所提出的DC细胞可将重组蛋白中的抗原(如前面所述的免疫检查点分子片段)、辅助T细胞抗原决定基片段以及免疫刺激分子片段分别呈递到细胞表面，进而有效刺激产生抗免疫检查点，如PD－L1或PD-L2抗体，调动病人体内已经存在的自发诱导产生的免疫细胞CTL，并且刺激产生抗免疫检查点，如PD-L1或PD-L2CTL，进而有效地特异性杀伤肿瘤细胞或被病毒(HBV、HCV、HIV、EBV)感染的细胞。In an eleventh aspect of the invention, the invention proposes a DC cell. According to an embodiment of the invention, the DC cells are loaded with the recombinant protein described above. According to an embodiment of the present invention, the DC cells proposed in the embodiments of the present invention can present antigens in the recombinant protein (such as the immunological checkpoint molecular fragment described above), helper T cell epitope fragments, and immunostimulatory molecule fragments, respectively. On the cell surface, it effectively stimulates the production of anti-immunological checkpoints, such as PD-L1 or PD-L2 antibodies, mobilizes spontaneously induced immune cell CTLs already present in patients, and stimulates the production of anti-immune checkpoints such as PD-L1 or PD-L2CTL, in turn, effectively kills tumor cells or cells infected with viruses (HBV, HCV, HIV, EBV).
在本发明的第十二方面，本发明提出了一种靶向性免疫细胞群。根据本发明的实施例，所述靶向性免疫细胞群是通过前面所述的DC细胞与淋巴细胞进行共培养获得的。根据本发明的实施例，本发明实施例所提出的靶向性免疫细胞群可特异性杀伤肿瘤细胞，分泌特异性结合肿瘤抗原的抗体，实现对肿瘤细胞的特异性清除。In a twelfth aspect of the invention, the invention provides a targeted immune cell population. According to an embodiment of the invention, the targeted immune cell population is obtained by co-culture of DC cells with lymphocytes as described above. According to an embodiment of the present invention, the targeted immune cell population proposed in the embodiments of the present invention can specifically kill tumor cells, secrete antibodies that specifically bind tumor antigens, and achieve specific removal of tumor cells.
在本发明的第十三方面，本发明提出了一种疫苗。根据本发明的实施例，所述疫苗包含前面所述的重组蛋白、前面所述的DC细胞或前面所述的靶向性免疫细胞群。如前所述，
本发明实施例所提出的重组蛋白、DC细胞以及靶向性免疫细胞群在患者体内可引起显著的特异性抗原免疫反应。根据本发明的实施例，本发明实施例所提出的疫苗可有效刺激产生抗免疫检查点，如PD－L1或PD-L2抗体，调动病人体内已经存在的自发诱导产生的免疫细胞CTL，并且刺激产生抗免疫检查点，如PD-L1或PD-L2CTL，进而有效地特异性杀伤肿瘤细胞或被病毒(HBV、HCV、HIV、EBV)感染的细胞。In a thirteenth aspect of the invention, the invention proposes a vaccine. According to an embodiment of the invention, the vaccine comprises a recombinant protein as described above, a DC cell as described above or a targeted immune cell population as described above. As mentioned earlier,
The recombinant protein, DC cell and targeted immune cell population proposed by the embodiments of the present invention can cause a significant specific antigen immune response in a patient. According to an embodiment of the present invention, the vaccine according to the embodiment of the present invention can effectively stimulate the production of an anti-immunization checkpoint, such as a PD-L1 or PD-L2 antibody, mobilize the spontaneously induced immune cell CTL already existing in the patient, and stimulate An anti-immunization checkpoint, such as PD-L1 or PD-L2CTL, is generated to effectively kill tumor cells or cells infected with viruses (HBV, HCV, HIV, EBV).
在本发明的第十四方面，本发明提出了一种抗体。根据本发明的实施例，所述抗体特异性识别前面所述的重组蛋白，进而本发明实施例所提出的抗体可特异性识别肿瘤抗原。根据本发明的实施例，发明发现，所述抗体可特异性识别抗原，与肿瘤细胞或被病毒(H BV、HCV、HIV、EBV)感染的细胞特异性结合，进而使得肿瘤细胞或被病毒(HBV、HCV、HIV、EBV)感染的细胞被吞噬细胞吞噬，实现对肿瘤细胞或被病毒(HBV、HCV、HIV、EBV)感染的细胞的特异性清除。In a fourteenth aspect of the invention, the invention provides an antibody. According to an embodiment of the present invention, the antibody specifically recognizes the recombinant protein described above, and the antibody proposed in the embodiment of the present invention specifically recognizes a tumor antigen. According to an embodiment of the present invention, the invention finds that the antibody specifically recognizes an antigen and specifically binds to a tumor cell or a cell infected by a virus (H BV, HCV, HIV, EBV), thereby causing the tumor cell or the virus to be infected ( Cells infected with HBV, HCV, HIV, EBV) are engulfed by phagocytic cells to achieve specific clearance of tumor cells or cells infected with viruses (HBV, HCV, HIV, EBV).
在本发明的第十五方面，本发明提出了一种制备抗体的方法。根据本发明的实施例，所述方法包括：利用前面所述的重组蛋白对动物进行免疫接种；采集经过免疫接种的动物的血清；以及从所述血清中纯化出目的抗体。本发明实施例所提出的制备抗体的方法，操作简便，抗体可特异性识别所述重组蛋白。In a fifteenth aspect of the invention, the invention provides a method of preparing an antibody. According to an embodiment of the invention, the method comprises: immunizing an animal with the recombinant protein described above; collecting serum of the immunized animal; and purifying the antibody of interest from the serum. The method for preparing an antibody proposed in the embodiments of the present invention is simple and convenient, and the antibody can specifically recognize the recombinant protein.
在本发明的第十六方面，本发明提出了一种治疗组合物。根据本发明的实施例，所述治疗组合物包括：前面所述的重组蛋白、前面所述的核酸、前面所述的构建体、前面所述的转基因细胞、前面所述的药物组合物、前面所述的DC细胞、前面所述的靶向性免疫细胞群、前面所述的疫苗或者前面所述的抗体。根据本发明的实施例，本发明实施例所提出的治疗组合物可直接或间接引起特异性抗原免疫反应，实现对肿瘤细胞或被病毒(HBV、HCV、HIV、EBV)感染的细胞的特异性杀伤和清除。In a sixteenth aspect of the invention, the invention provides a therapeutic composition. According to an embodiment of the invention, the therapeutic composition comprises: a recombinant protein as described above, a nucleic acid as described above, a construct as described above, a transgenic cell as described above, a pharmaceutical composition as described above, a front The DC cells, the aforementioned targeted immune cell population, the vaccine described above or the antibodies described above. According to an embodiment of the present invention, the therapeutic composition proposed by the embodiments of the present invention can directly or indirectly cause a specific antigen immune response, and achieve specificity to tumor cells or cells infected by viruses (HBV, HCV, HIV, EBV). Kill and clear.
在本发明的第十七方面，本发明提出了一种在患者体内刺激抗PD-L1抗体生成或细胞毒性T淋巴细胞反应的方法。根据本发明的实施例，所述方法是通过下列方式的至少之一实现的：1)前面所述的重组蛋白与取自患者的DC细胞共培养，将负载了前面所述的重组蛋白的DC细胞回输到患者体内；2)给患者给予前面所述的药物组合物；3)将前面所述的构建体导入取自患者的DC细胞，将导入所述构建体的DC细胞回输到患者体内；以及4)给患者给予前面所述的构建体。本发明实施例所提出的方式可在患者体内显著刺激抗PD-L1抗体生成或细胞毒性T淋巴细胞反应。In a seventeenth aspect of the invention, the invention provides a method of stimulating anti-PD-L1 antibody production or cytotoxic T lymphocyte response in a patient. According to an embodiment of the invention, the method is achieved by at least one of the following: 1) the recombinant protein described above is co-cultured with DC cells taken from a patient, and the DC of the recombinant protein described above is loaded The cells are returned to the patient; 2) the patient is administered the pharmaceutical composition described above; 3) the previously described construct is introduced into the DC cells taken from the patient, and the DC cells introduced into the construct are returned to the patient. In vivo; and 4) administering to the patient a construct as described above. The manner proposed in the examples of the present invention can significantly stimulate anti-PD-L1 antibody production or cytotoxic T lymphocyte reaction in a patient.
在本发明的第十八方面，本发明提出了一种治疗癌症的方法。根据本发明的实施例，是通过下列方式的至少之一实现的：1)前面所述的重组蛋白与取自癌症患者的DC细胞共培养，将负载了前面所述的重组蛋白的DC细胞回输到癌症患者体内；2)给癌症患者给予前面所述的药物组合物；3)将前面所述的构建体导入取自癌症患者的DC细胞，将导入所
述构建体的DC细胞回输到癌症患者体内；以及4)给癌症患者给予前面所述的构建体。本发明实施例所提出的方法可直接或间接引起特异性抗原免疫反应，实现对肿瘤细胞的特异性杀伤和清除。In an eighteenth aspect of the invention, the invention provides a method of treating cancer. According to an embodiment of the present invention, it is achieved by at least one of the following methods: 1) the recombinant protein described above is co-cultured with DC cells taken from a cancer patient, and the DC cells loaded with the recombinant protein described above are returned. Loss to a cancer patient; 2) administration of the aforementioned pharmaceutical composition to a cancer patient; 3) introduction of the aforementioned construct into DC cells taken from a cancer patient, and introduction into the body
The DC cells of the construct are returned to the cancer patient; and 4) the cancer patient is administered the construct described above. The method proposed by the embodiments of the present invention can directly or indirectly cause a specific antigen immune response, and achieve specific killing and elimination of tumor cells.
在本发明的第十九方面，本发明提出了一种治疗被病毒感染患者的方法。根据本发明的实施例，是通过下列方式的至少之一实现的：1)前面所述的重组蛋白与取自患者的DC细胞共培养，将负载了前面所述的重组蛋白的DC细胞回输到患者体内；2)给患者给予前面所述的药物组合物；3)将前面所述的构建体导入取自患者的DC细胞，将导入所述构建体的DC细胞回输到患者体内；以及4)给患者给予前面所述的构建体。本发明实施例所提出的方法可直接或间接引起特异性抗原免疫反应，实现对被病毒(HBV、HCV、HIV、EBV)感染的细胞的特异性杀伤和清除，进而有效治疗被病毒感染患者。In a nineteenth aspect of the invention, the invention provides a method of treating a patient infected with a virus. According to an embodiment of the present invention, it is achieved by at least one of the following methods: 1) the recombinant protein described above is co-cultured with DC cells taken from a patient, and the DC cells loaded with the recombinant protein described above are returned. To the patient; 2) administering to the patient a pharmaceutical composition as described above; 3) introducing the aforementioned construct into a DC cell taken from the patient, and returning the DC cells introduced into the construct to the patient; 4) The patient is administered the construct described above. The method proposed by the embodiments of the present invention can directly or indirectly cause a specific antigen immune reaction, and achieve specific killing and elimination of cells infected by viruses (HBV, HCV, HIV, EBV), thereby effectively treating patients infected with the virus.
根据本发明的实施例，上述方法还可以进一步包括如下附加技术特征至少之一：According to an embodiment of the present invention, the above method may further include at least one of the following additional technical features:
根据本发明的实施例，所述病毒包括选自HBV、HCV、HIV和EBV的至少之一。利用根据本发明实施例的上述方法，可实现对HBV、HCV、HIV或EBV感染患者的进一步有效治疗。According to an embodiment of the invention, the virus comprises at least one selected from the group consisting of HBV, HCV, HIV and EBV. Further effective treatment of patients infected with HBV, HCV, HIV or EBV can be achieved using the above method according to an embodiment of the invention.
图1是根据本发明实施例的融合蛋白结构示意图；1 is a schematic view showing the structure of a fusion protein according to an embodiment of the present invention;
图2是根据本发明实施例的负载了融合蛋白(PD-L1Δ-PADRE Th-GM-CSF)的DC疫苗能够显著诱导抗-PD-L1抗体反应；2 is a DC vaccine loaded with a fusion protein (PD-L1Δ-PADRE Th-GM-CSF) capable of significantly inducing an anti-PD-L1 antibody response according to an embodiment of the present invention;
图3是根据本发明实施例的负载了融合蛋白(PD-L1Δ-PADRE Th-GM-CSF)的DC疫苗能够显著诱导抗-PD-L1CTL反应；以及3 is a DC vaccine loaded with a fusion protein (PD-L1Δ-PADRE Th-GM-CSF) capable of significantly inducing an anti-PD-L1 CTL reaction according to an embodiment of the present invention;
图4是根据本发明实施例的负载了融合蛋白(PD-L1Δ-PADRE Th-GM-CSF)的DC疫苗能够显著控制PD-L1+肺癌的生长。4 is a DC vaccine loaded with a fusion protein (PD-L1Δ-PADRE Th-GM-CSF) capable of significantly controlling the growth of PD-L1 + lung cancer according to an embodiment of the present invention.
下面详细描述本发明的实施例，所述实施例的示例在附图中示出，其中自始至终相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附图描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。The embodiments of the present invention are described in detail below, and the examples of the embodiments are illustrated in the drawings, wherein the same or similar reference numerals are used to refer to the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are intended to be illustrative of the invention and are not to be construed as limiting.
需要说明的是，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括一个或者更多个该特征。进一步地，在本发明的描述中，除非另有说明，“多个”的含义是两个或两个以上。It should be noted that the terms "first" and "second" are used for descriptive purposes only, and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, features defining "first" and "second" may include one or more of the features either explicitly or implicitly. Further, in the description of the present invention, the meaning of "a plurality" is two or more unless otherwise specified.
重组蛋白及其用途
Recombinant protein and its use
一方面，本发明提出了一种重组蛋白。根据本发明的实施例，该重组蛋白包括：免疫检查点分子片段；辅助T细胞抗原决定基片段；以及免疫刺激分子片段。本发明实施例所提出的重组蛋白在患者体内刺激产生抗免疫检查点抗体，调动体内已经存在的自发诱导产生的免疫细胞CTL，并且刺激产生抗免疫检查点的CTL，进而特异性杀伤肿瘤细胞。本发明实施例所提出的重组蛋白引起的对肿瘤细胞的主动免疫杀伤效果显著。In one aspect, the invention proposes a recombinant protein. According to an embodiment of the invention, the recombinant protein comprises: an immunological checkpoint molecule fragment; a helper T cell epitope fragment; and an immunostimulatory molecule fragment. The recombinant protein proposed in the embodiments of the present invention stimulates the production of anti-immunological checkpoint antibodies in the patient, mobilizes the spontaneously induced immune cell CTLs already existing in the body, and stimulates the production of CTLs against the immune checkpoints, thereby specifically killing the tumor cells. The active immune killing effect on tumor cells caused by the recombinant protein proposed by the embodiments of the present invention is remarkable.
具体地，根据本发明的实施例，免疫检查点分子可选自但不限于PD-L1和PD-L2的至少之一。PD-L1或PD-L2在肿瘤细胞中特异性表达，进而本发明实施例所提出的重组蛋白可引起的肿瘤抗原免疫反应的特异性更强。Specifically, according to an embodiment of the present invention, the immune checkpoint molecule may be selected from, but not limited to, at least one of PD-L1 and PD-L2. PD-L1 or PD-L2 is specifically expressed in tumor cells, and the specificity of the tumor antigen immune response caused by the recombinant protein proposed in the examples of the present invention is stronger.
更具体地，根据本发明的实施例，免疫检查点分子片段为所述PD-L1或PD-L2的去除跨膜区的细胞外分子片段(PD-L1Δ/PD-L2Δ)。PD-L1或PD-L2的去除跨膜区的细胞外分子片段仅具有肿瘤抗原性，不具有肿瘤免疫抑制功能，进而PD-L1或PD-L2的去除跨膜区的细胞外分子片段被抗原呈递细胞如DC细胞呈递在细胞的表面，可有效地引起肿瘤抗原免疫反应，且特异性进一步提高。More specifically, according to an embodiment of the present invention, the immunological checkpoint molecule fragment is an extracellular molecular fragment (PD-L1Δ/PD-L2Δ) of the transmembrane region of the PD-L1 or PD-L2. The extracellular molecular fragment of the PD-L1 or PD-L2 removal transmembrane region has only tumor antigenicity and does not have tumor immunosuppressive function, and then the extracellular molecular fragment of the transmembrane region of PD-L1 or PD-L2 is antigen-removed. Presenting cells such as DC cells are presented on the surface of the cells, which can effectively elicit a tumor antigen immune response, and the specificity is further improved.
另外，根据本发明的实施例，辅助T细胞抗原决定基可为广谱PADRE辅助T细胞抗原决定基(PADRE Th)。广谱PADRE辅助T细胞抗原决定基是一种与广谱人类白细胞抗原DR(HLA-DR)结合的抗原决定基肽段，这种肽段以高亲和力或中间亲和力与16种最普遍的HLA-DR类型中的15种相结合。由于它有普遍的结合力，进而PADRE需要克服HLA-DR分子在人群中的多样性带来的问题。PADRE作为辅助T细胞抗原决定基与抗原结合，结合后高效、长时间激活Ag-特异性抗原反应。PADRE肽段被特别加工成在人体中可以免疫性激活辅助T淋巴细胞1(Th1)以协助杀伤性免疫T细胞的激活以及激活辅助T淋巴细胞2(Th2)以协助B淋巴细胞分泌抗体，进而进一步增强重组蛋白所引起的抗原免疫反应。Additionally, according to an embodiment of the invention, the helper T cell epitope can be a broad spectrum PADRE helper T cell epitope (PADRE Th). The broad-spectrum PADRE helper T cell epitope is an epitope peptide that binds to a broad spectrum of human leukocyte antigen DR (HLA-DR) with high affinity or intermediate affinity and 16 of the most prevalent HLA- 15 of the DR types are combined. Because of its universal binding, PADRE needs to overcome the problems caused by the diversity of HLA-DR molecules in the population. PADRE acts as a helper T cell epitope and binds to the antigen. After binding, it activates the Ag-specific antigen reaction efficiently and for a long time. The PADRE peptide is specifically processed to immunologically activate helper T lymphocyte 1 (Th1) in humans to assist in the activation of killer immune T cells and to activate helper T lymphocyte 2 (Th2) to assist B lymphocytes to secrete antibodies, thereby Further enhancing the antigenic immune response caused by the recombinant protein.
根据本发明的具体实施例，免疫刺激分子可选自粒细胞集落刺激生物因子(GM-CSF)、白细胞介素-12(IL-12)或趋化因子(RANTES)。上述免疫刺激分子具有生物活性。According to a particular embodiment of the invention, the immunostimulatory molecule may be selected from the group consisting of granulocyte colony stimulating biological factor (GM-CSF), interleukin-12 (IL-12) or chemokine (RANTES). The above immunostimulatory molecules are biologically active.
其中，GM-CSF在动物模型和临床试验中用来增强免疫反应。在免疫治疗中，GM-CSF也被广泛的用作佐剂以增强免疫反应。在多种啮齿类动物肿瘤模型中，均被免疫接种经辐射造成的肿瘤细胞，这种肿瘤细胞分泌GM-CSF，以此来刺激强有力的、特异性的和长时间的抗肿瘤免疫反应。这种免疫接种在晚期黑色素瘤的转移性病变中会诱导CD4+和CD8+T淋巴细胞和浆细胞的渗入，进而引起大量肿瘤细胞的坏死。除了黑色素瘤，使用GM-CSF-分泌肿瘤细胞的临床试验已经被报道用在非小细胞肺癌、胰腺癌、前列腺癌、肾癌的治疗中。GM-CSF–分泌肿瘤细胞的免疫增强作用与GM-CSF能够征集DC细胞并使DC细胞成熟和活化，进而激活免疫杀伤T淋巴细胞和B淋巴细胞的作用相关。根据本发明的实施例，本发明实施例所提出的重组蛋白中的GM-CSF可显著增强树突状细胞(DC细胞)的抗原呈
递功能和增强细胞毒性T淋巴细胞(CTL)和B淋巴细胞的活性，进而本发明实施例的重组蛋白可更为有效地引起肿瘤抗原免疫反应。Among them, GM-CSF is used to enhance the immune response in animal models and clinical trials. In immunotherapy, GM-CSF is also widely used as an adjuvant to enhance the immune response. In a variety of rodent tumor models, tumor cells are irradiated by radiation, which secretes GM-CSF to stimulate a potent, specific, and prolonged anti-tumor immune response. This immunization induces infiltration of CD4+ and CD8+ T lymphocytes and plasma cells in metastatic lesions of advanced melanoma, which in turn causes necrosis of a large number of tumor cells. In addition to melanoma, clinical trials using GM-CSF-secreting tumor cells have been reported for use in the treatment of non-small cell lung cancer, pancreatic cancer, prostate cancer, and renal cancer. GM-CSF - The immunopotentiating effect of secreting tumor cells and GM-CSF are capable of recruiting DC cells and maturation and activation of DC cells, thereby activating the role of immune killing of T lymphocytes and B lymphocytes. According to an embodiment of the present invention, the GM-CSF in the recombinant protein proposed by the embodiment of the present invention can significantly enhance the antigen presentation of dendritic cells (DC cells).
The function and the activity of cytotoxic T lymphocytes (CTL) and B lymphocytes are enhanced, and the recombinant protein of the embodiment of the present invention can more effectively induce a tumor antigen immune response.
趋化因子在免疫反应、造血过程和常规免疫监视中控制着特定白细胞种群的迁移。RANTES(regulated upon activation normal T-cell expressed，CCL5)是一种免疫趋化因子。它与表达在T淋巴细胞、单核细胞、成熟免疫杀伤细胞和DC细胞上的CCR1和CCR5有亲和力。趋化因子，如RANTES在不同的疫苗中，通过大量征集相关免疫细胞，以此来进行肿瘤细胞识别、免疫致敏和杀伤肿瘤细胞。Chemokines control the migration of specific leukocyte populations in immune responses, hematopoiesis, and routine immune surveillance. RANTES (regulated upon activation normal T-cell expressed, CCL5) is an immunochemokine. It has affinity for CCR1 and CCR5 expressed on T lymphocytes, monocytes, mature immune killer cells and DC cells. Chemokines, such as RANTES, in a variety of vaccines, through the large collection of relevant immune cells, in order to carry out tumor cell recognition, immunosensitization and kill tumor cells.
IL-12是一种多效细胞因子，IL-12激活能够引起自身免疫的和适应性免疫的相互关联。基于它的反应，IL-12被认为是“细胞毒性淋巴细胞成熟因子”和“自然杀伤细胞刺激因子”。由于IL-12可建立起自身免疫和适应性免疫的相互关联，进而它能够强有力地刺激IFN-γ—a的生成，进而协调人体自身的抗癌机制。IL-12已经在人体中被用于肿瘤的免疫治疗。IL-12在多种免疫细胞中发挥作用，包括T淋巴细胞和B淋巴细胞。IL-12在促进Th1细胞的抗肿瘤免疫反应中发挥着关键作用。IL-12 is a pleiotropic cytokine that activates the association between autoimmune and adaptive immunity. Based on its response, IL-12 is considered to be "cytotoxic lymphocyte maturation factor" and "natural killer cell stimulating factor". Since IL-12 can establish the correlation between autoimmune and adaptive immunity, it can strongly stimulate the production of IFN-γ-a, thereby coordinating the body's own anti-cancer mechanism. IL-12 has been used in the human body for immunotherapy of tumors. IL-12 plays a role in a variety of immune cells, including T lymphocytes and B lymphocytes. IL-12 plays a key role in promoting the anti-tumor immune response of Th1 cells.
辅助T细胞抗原决定基片段以及免疫刺激分子片段的连接顺序如下所述：辅助T细胞抗原决定基片段N端与免疫检查点分子片段的C端相连，辅助T细胞抗原决定基片段C端与所述免疫刺激分子片段的N端相连。本发明实施例的重组蛋白中的相关分子片段在上述连接状态下，重组蛋白中相应分子片段可分别呈递在DC细胞的表面。DC细胞，主要的抗原呈递细胞(APCs)，通过产生前免疫细胞因子和呈递抗原给T淋巴细胞来调节自身和适应性免疫反应以此来抵抗病毒感染。本发明实施例所提出的重组蛋白允许DC细胞呈递细胞内抗原到细胞表面上，其中通过内吞途径呈递到MHC class II，通过交叉启动途径呈递到MHC class I，进而导致产生抗原特异性Th细胞和CTL细胞反应。本发明实施例所提出的重组蛋白呈递在DC细胞的表面引起强有力的抗体反应。一直以来认为，DC细胞激活体液反应，是通过CD4+Th细胞引发T淋巴细胞和B淋巴细胞的相互作用而实现的。然而，现有的体外和体内实验证实，DC细胞激活体液反应是一种直接的作用方式。特别地，DC细胞被证实可强有力地促进细胞分化和CD40-激活的B淋巴细胞的抗体的产生。接种负载有抗原的DC细胞能够诱导保护性体液免疫反应。本发明实施例的重组蛋白可更为有效地引起肿瘤抗原免疫反应。The ligation sequence of the helper T cell epitope fragment and the immunostimulatory molecule fragment is as follows: the N-terminus of the helper T cell epitope is linked to the C-terminus of the immunological checkpoint molecule fragment, and the helper T cell epitope fragment C-end and The N-terminus of the immunostimulatory molecule fragment is linked. In the above-described ligation state, the corresponding molecular fragment in the recombinant protein of the present invention can be presented on the surface of the DC cell, respectively. DC cells, the main antigen-presenting cells (APCs), regulate their own and adaptive immune responses by producing pre-immune cytokines and presenting antigens to T lymphocytes to counteract viral infections. The recombinant protein proposed by the embodiments of the present invention allows DC cells to present intracellular antigens to the cell surface, which is presented to the MHC class II by endocytic pathway, and is presented to the MHC class I by a cross-priming pathway, thereby leading to the production of antigen-specific Th cells. Reacts with CTL cells. The recombinant protein presented by the embodiments of the present invention causes a strong antibody response on the surface of DC cells. It has long been believed that the activation of humoral responses by DC cells is achieved by the interaction of T lymphocytes and B lymphocytes by CD4+ Th cells. However, existing in vitro and in vivo experiments have demonstrated that DC cells activate a humoral response as a direct mode of action. In particular, DC cells have been shown to strongly promote cell differentiation and production of antibodies to CD40-activated B lymphocytes. Inoculation of antigen-loaded DC cells is capable of inducing a protective humoral immune response. The recombinant protein of the embodiments of the present invention can more effectively cause a tumor antigen immune response.
根据本发明的实施例，上述重组蛋白具有SEQ ID NO：1～9所示的氨基酸序列。其中SEQ ID NO：1是人类PD-L1Δ-PADRE Th-人类GM-CSF重组蛋白的氨基酸序列，SEQ ID NO：2是人类PD-L1Δ-PADRE Th-人类RANTES重组蛋白的氨基酸序列，SEQ ID NO：3是人类PD-L1Δ-PADRE Th-IL-12重组蛋白的氨基酸序列，SEQ ID NO：4是人类PD-L2Δ-PADRE Th-人类GM-CSF重组蛋白的氨基酸序列，SEQ ID NO：5是人类PD-L2Δ-PADRE
Th-人类RANTES重组蛋白的氨基酸序列，SEQ ID NO：6是人类PD-L2Δ-PADRE Th-人类IL-12重组蛋白的氨基酸序列，SEQ ID NO：7是人类PD-L1-PD-L2Δ-PADRE Th-人类GM-CSF重组蛋白的氨基酸序列，SEQ ID NO：8是人类PD-L1-PD-L2Δ-PADRE Th-人类RANTES重组蛋白的氨基酸序列，SEQ ID NO：9是人类PD-L1-PD-L2Δ-PADRE Th-人类IL-12重组蛋白的氨基酸序列。本发明实施例所述提出的重组蛋白可引起肿瘤特异性抗原免疫反应，可引起细胞毒性T淋巴细胞(CTL)的特异性杀伤和B细胞分泌特异性抗体，实现对肿瘤细胞的特异性杀伤。According to an embodiment of the present invention, the recombinant protein has the amino acid sequences shown in SEQ ID NOS: 1 to 9. Wherein SEQ ID NO: 1 is the amino acid sequence of human PD-L1Δ-PADRE Th-human GM-CSF recombinant protein, and SEQ ID NO: 2 is the amino acid sequence of human PD-L1Δ-PADRE Th-human RANTES recombinant protein, SEQ ID NO :3 is the amino acid sequence of human PD-L1Δ-PADRE Th-IL-12 recombinant protein, SEQ ID NO: 4 is the amino acid sequence of human PD-L2Δ-PADRE Th-human GM-CSF recombinant protein, SEQ ID NO: 5 is Human PD-L2Δ-PADRE
The amino acid sequence of the Th-human RANTES recombinant protein, SEQ ID NO: 6 is the amino acid sequence of the human PD-L2Δ-PADRE Th-human IL-12 recombinant protein, and SEQ ID NO: 7 is the human PD-L1-PD-L2Δ-PADRE The amino acid sequence of the Th-human GM-CSF recombinant protein, SEQ ID NO: 8 is the amino acid sequence of the human PD-L1-PD-L2Δ-PADRE Th-human RANTES recombinant protein, and SEQ ID NO: 9 is the human PD-L1-PD -L2Δ-PADRE Th-amino acid sequence of human IL-12 recombinant protein. The recombinant protein proposed in the embodiment of the invention can cause tumor-specific antigen immune response, can cause specific killing of cytotoxic T lymphocytes (CTL) and secrete specific antibodies of B cells, and achieve specific killing of tumor cells.
同时，本发明提出了一种编码前面所述的重组蛋白的核酸，根据本发明的实施例，编码前面所述的重组蛋白的核酸具有SEQ ID NO：10～18所示的核苷酸序列。其中SEQ ID NO：10是编码人类PD-L1Δ-PADRE Th-人类GM-CSF重组蛋白的核酸的核苷酸序列，SEQ ID NO：11是编码人类PD-L1Δ-PADRE Th-人类RANTES重组蛋白的核酸的核苷酸序列，SEQ ID NO：12是编码人类PD-L1Δ-PADRE Th-IL-12重组蛋白的核酸的核苷酸序列，SEQ ID NO：13是编码人类PD-L2Δ-PADRE Th-人类GM-CSF重组蛋白的核酸的核苷酸序列，SEQ ID NO：14是编码人类PD-L2Δ-PADRE Th-人类RANTES重组蛋白的核酸的核苷酸序列，SEQ ID NO：15是编码人类PD-L2Δ-PADRE Th-人类IL-12重组蛋白的核酸的核苷酸序列，SEQ ID NO：16是编码人类PD-L1-PD-L2Δ-PADRE Th-人类GM-CSF重组蛋白的核酸的核苷酸序列，SEQ ID NO：17是编码人类PD-L1-PD-L2Δ-PADRE Th-人类RANTES重组蛋白的核酸的核苷酸序列，SEQ ID NO：18是编码人类PD-L1-PD-L2Δ-PADRE Th-人类IL-12重组蛋白的核酸的核苷酸序列。本发明实施例所述提出的核酸编码的重组蛋白可引起肿瘤特异性抗原免疫反应，引起细胞毒性T淋巴细胞(CTL)的特异性杀伤和B细胞分泌特异性抗体，实现对肿瘤细胞的特异性杀伤。Meanwhile, the present invention proposes a nucleic acid encoding the recombinant protein described above, and according to an embodiment of the present invention, the nucleic acid encoding the recombinant protein described above has the nucleotide sequences shown in SEQ ID NOS: 10 to 18. Wherein SEQ ID NO: 10 is the nucleotide sequence of a nucleic acid encoding a human PD-L1Δ-PADRE Th-human GM-CSF recombinant protein, and SEQ ID NO: 11 is a recombinant protein encoding human PD-L1Δ-PADRE Th-human RANTES The nucleotide sequence of the nucleic acid, SEQ ID NO: 12 is the nucleotide sequence of the nucleic acid encoding the human PD-L1Δ-PADRE Th-IL-12 recombinant protein, and SEQ ID NO: 13 is the encoding human PD-L2Δ-PADRE Th- The nucleotide sequence of the nucleic acid of the human GM-CSF recombinant protein, SEQ ID NO: 14 is the nucleotide sequence of the nucleic acid encoding the human PD-L2Δ-PADRE Th-human RANTES recombinant protein, and SEQ ID NO: 15 is the coding human PD -L2Δ-PADRE Th-nucleotide sequence of a nucleic acid of human IL-12 recombinant protein, SEQ ID NO: 16 is a nucleoside encoding a nucleic acid of human PD-L1-PD-L2Δ-PADRE Th-human GM-CSF recombinant protein The acid sequence, SEQ ID NO: 17 is the nucleotide sequence of the nucleic acid encoding the human PD-L1-PD-L2Δ-PADRE Th-human RANTES recombinant protein, and SEQ ID NO: 18 encodes the human PD-L1-PD-L2Δ- The nucleotide sequence of the nucleic acid of the PADRE Th-human IL-12 recombinant protein. The recombinant protein encoded by the nucleic acid proposed in the embodiments of the present invention can cause tumor-specific antigen immune response, cause specific killing of cytotoxic T lymphocytes (CTL) and secrete specific antibodies of B cells, and achieve specificity to tumor cells. Killing.
同时，本发明提出了一种携带前面所述的核酸的构建体。根据本发明的实施例，本发明实施例所提出的构建体导入受体细胞，可实现前面所述的核酸的高效表达，进而在受体细胞中高效表达前面所述的重组蛋白。根据本发明的具体实施例，该构建体的载体为pET系列载体、pGEX系列载体、pPIC系列载体、BacPAK、pSV系列载体或pCMV系列载体，其中，pET系列载体在E.coli中在T7启动子下调控表达重组蛋白，pGEX系列载体用于在E.coli中在tac启动子下调控表达重组蛋白，pPIC系列载体用于在酵母菌中在AOX1启动子下调控表达重组蛋白，BacPAK载体用于在杆状病毒中在pPolh启动子的调控下表达重组蛋白，pSV系列载体或pCMV系列载体用于在哺乳动物细胞中在CMV、SV40和(EF)-1启动子的调控下表达重组蛋白。本发明实施例的上述载体可实现在原核细胞或真核细胞中进一步高效表达上述重组蛋白。Meanwhile, the present invention proposes a construct which carries the nucleic acid described above. According to an embodiment of the present invention, the construct introduced into the recipient cell of the embodiment of the present invention can achieve high-efficiency expression of the nucleic acid described above, thereby efficiently expressing the recombinant protein described above in the recipient cell. According to a particular embodiment of the invention, the vector of the construct is a pET series vector, a pGEX series vector, a pPIC series vector, a BacPAK, a pSV series vector or a pCMV series vector, wherein the pET series vector is in the T7 promoter in E. coli Down-regulated expression of recombinant protein, pGEX series vectors were used to regulate the expression of recombinant proteins in E. coli under the tac promoter, pPIC series vectors were used to regulate the expression of recombinant proteins in yeast under the AOX1 promoter, and BacPAK vectors were used in Recombinant proteins are expressed in baculovirus under the control of the pPolh promoter, and the pSV series vector or pCMV series vectors are used to express recombinant proteins in mammalian cells under the control of the CMV, SV40 and (EF)-1 promoters. The above vector of the embodiment of the present invention can achieve further efficient expression of the above recombinant protein in prokaryotic cells or eukaryotic cells.
具体地，根据本发明的实施例，上述构建体携带下列核酸分子:(1)编码免疫检查点分子
片段的核酸分子，该免疫检查点分子片段具有SEQ ID NO：19～21所示的氨基酸序列，其中SEQ ID NO：19是人类PD-L1Δ的氨基酸序列，SEQ ID NO：20是人类PD-L2Δ的氨基酸序列，SEQ ID NO：21是人类PD-L1-PD-L2Δ的氨基酸序列，所述编码免疫检查点分子片段的核酸分子具有SEQ ID NO：22～24所示的核苷酸序列，其中SEQ ID NO：22是编码人类PD-L1Δ的核酸分子的核苷酸序列，SEQ ID NO：23是编码人类PD-L2Δ的核酸分子的核苷酸序列，SEQ ID NO：24是编码人类PD-L1-PD-L2Δ的核酸分子的核苷酸序列；(2)编码辅助T细胞抗原决定基片段的核酸分子，辅助T细胞抗原决定基片段具有SEQ ID NO：25所示的氨基酸序列，编码辅助T细胞抗原决定基片段的核酸分子具有SEQ ID NO：26所示的核苷酸序列；以及(3)编码免疫刺激分子片段的核酸分子，免疫刺激分子片段具有SEQ ID NO：27～29所示的氨基酸序列，其中，SEQ ID NO：27是人类GM-CSF片段的氨基酸序列，SEQ ID NO：28是人类RANTES片段的氨基酸序列，SEQ ID NO：29是人类IL-12片段的氨基酸序列，所述编码免疫刺激分子片段的核酸分子具有SEQ ID NO：30～32所示的核苷酸序列，其中，SEQ ID NO：30是编码人类GM-CSF片段的核酸分子的核苷酸序列，SEQ ID NO：31是编码人类RANTES片段的核酸分子的核苷酸序列，SEQ ID NO：32是编码人类IL-12片段的核酸分子的核苷酸序列。Specifically, according to an embodiment of the present invention, the above construct carries the following nucleic acid molecules: (1) encoding an immunological checkpoint molecule
A nucleic acid molecule of the fragment having the amino acid sequence of SEQ ID NOS: 19 to 21, wherein SEQ ID NO: 19 is the amino acid sequence of human PD-L1Δ, and SEQ ID NO: 20 is human PD-L2Δ The amino acid sequence of SEQ ID NO: 21 is the amino acid sequence of human PD-L1-PD-L2Δ, and the nucleic acid molecule encoding the immunological checkpoint molecule fragment has the nucleotide sequence shown by SEQ ID NOs: 22-24, wherein SEQ ID NO: 22 is the nucleotide sequence of a nucleic acid molecule encoding human PD-L1Δ, SEQ ID NO: 23 is the nucleotide sequence of a nucleic acid molecule encoding human PD-L2Δ, and SEQ ID NO: 24 is encoding a human PD- a nucleotide sequence of a nucleic acid molecule of L1-PD-L2Δ; (2) a nucleic acid molecule encoding a helper T cell epitope fragment having the amino acid sequence of SEQ ID NO: 25, encoding assistance The nucleic acid molecule of the T cell epitope fragment has the nucleotide sequence of SEQ ID NO: 26; and (3) the nucleic acid molecule encoding the immunostimulatory molecule fragment having the SEQ ID NOS: 27-29 Amino acid sequence Wherein SEQ ID NO: 27 is the amino acid sequence of the human GM-CSF fragment, SEQ ID NO: 28 is the amino acid sequence of the human RANTES fragment, and SEQ ID NO: 29 is the amino acid sequence of the human IL-12 fragment, the coding immunization The nucleic acid molecule stimulating the molecular fragment has the nucleotide sequence shown in SEQ ID NOs: 30 to 32, wherein SEQ ID NO: 30 is the nucleotide sequence of the nucleic acid molecule encoding the human GM-CSF fragment, SEQ ID NO: 31 Is the nucleotide sequence of a nucleic acid molecule encoding a human RANTES fragment, and SEQ ID NO: 32 is the nucleotide sequence of a nucleic acid molecule encoding a human IL-12 fragment.
任选地，所述构建体的载体为原核或真核细胞蛋白表达载体。本发明实施例所述提出的构建体在受体细胞中高效表达含有免疫检查点分子片段、辅助T细胞抗原决定基片段和免疫刺激分子片段的重组蛋白，该重组蛋白在肿瘤患者体内可显著引起肿瘤特异性抗原免疫反应，进而引起细胞毒性T淋巴细胞(CTL)的特异性杀伤和B淋巴细胞分泌特异性抗体，实现对肿瘤细胞的特异性杀伤。Optionally, the vector of the construct is a prokaryotic or eukaryotic protein expression vector. The construct according to the embodiment of the present invention efficiently expresses a recombinant protein containing an immunological checkpoint molecular fragment, a helper T cell epitope fragment and an immunostimulatory molecule fragment in a recipient cell, and the recombinant protein can be significantly caused in a tumor patient. The tumor-specific antigen immunoreaction, which in turn causes specific killing of cytotoxic T lymphocytes (CTL) and specific antibodies against B lymphocyte secretion, achieves specific killing of tumor cells.
另外，本发明还提出了一种转基因细胞。根据本发明的实施例，所述转基因细胞携带前面所述的构建体，进而本发明实施例所提出的转基因细胞可高表达前面所述的重组蛋白通过主动免疫，在病人体内刺激产生抗免疫检查点，如PD－L1或PD-L2的抗体，调动病人体内已经存在的自发诱导产生的免疫细胞CTL，并且刺激产生抗免疫检查点，如PD-L1或PD-L2的CTL，进而特异性杀伤肿瘤细胞。In addition, the present invention also proposes a transgenic cell. According to an embodiment of the present invention, the transgenic cell carries the construct described above, and the transgenic cell proposed in the embodiment of the present invention can express the recombinant protein as described above to actively stimulate the anti-immunoassay in the patient body by active immunization. Point, such as antibodies to PD-L1 or PD-L2, mobilize spontaneously induced immune cell CTLs already present in the patient, and stimulate the production of anti-immunological checkpoints, such as PD-L1 or PD-L2 CTL, thereby specifically killing Tumor cells.
根据本发明的具体实施例，所述转基因细胞为BL21，BL21(DE3)，BL21(DE3)pLysS，DH10B，XL1-Blue，Pichia pastors，Kluyveromyces lactis，Sf9，Sf21，High-Five T，CHO细胞系，HEK细胞系，Hela细胞系或COS细胞系。其中，BL21，BL21(DE3)，BL21(DE3)pLysS，DH10B和XL1-Blue是E.coli细胞，Pichia pastors和Kluyveromyces lactis是酵母细胞，Sf9，Sf21，和High-Five T用于杆状病毒的表达，CHO细胞系，HEK细胞系，Hela细胞系或COS细胞系是哺乳动物细胞系。根据本发明的实施例，上述的转基因细胞可高效表达前面所述的重组蛋白，进而通过蛋白纯化，获得的重组蛋白给予患者，可在
患者体内通过主动免疫，在病人体内进一步有效刺激产生抗PD－L1或PD-L2抗体，调动病人体内已经存在的自发诱导产生的免疫细胞CTL，并且刺激产生抗PD-L1或PD-L2CTL，进而特异性杀伤肿瘤细胞。According to a specific embodiment of the present invention, the transgenic cells are BL21, BL21 (DE3), BL21 (DE3) pLysS, DH10B, XL1-Blue, Pichia pastors, Kluyveromyces lactis, Sf9, Sf21, High-Five T, CHO cell line , HEK cell line, Hela cell line or COS cell line. Among them, BL21, BL21 (DE3), BL21 (DE3) pLysS, DH10B and XL1-Blue are E. coli cells, Pichia pastors and Kluyveromyces lactis are yeast cells, Sf9, Sf21, and High-Five T are used for baculovirus. Expression, CHO cell line, HEK cell line, Hela cell line or COS cell line is a mammalian cell line. According to an embodiment of the present invention, the transgenic cell described above can efficiently express the recombinant protein described above, and then the recombinant protein obtained by protein purification can be administered to a patient.
The patient actively stimulates the production of anti-PD-L1 or PD-L2 antibodies in the patient's body, mobilizes the spontaneously induced immune cell CTLs already present in the patient, and stimulates the production of anti-PD-L1 or PD-L2CTL. Specific killing of tumor cells.
根据本发明的具体实施例，所述转基因细胞可为抗原呈递细胞，所述转基因细胞为DC细胞。根据本发明的实施例，所述抗原呈递细胞来源于病人，进而携带前面所述构建体的抗原呈递细胞可进一步回输入病人体内，进而在病人体内实现前面所述重组蛋白的持续表达，进而可在患者体内通过主动免疫，在体内进一步有效刺激产生抗PD－L1或PD-L2抗体，调动病人体内已经存在的自发诱导产生的免疫细胞CTL，并且刺激产生抗PD-L1或PD-L2CTL，进而特异性杀伤肿瘤细胞。According to a particular embodiment of the invention, the transgenic cell can be an antigen presenting cell and the transgenic cell is a DC cell. According to an embodiment of the present invention, the antigen presenting cell is derived from a patient, and the antigen presenting cell carrying the aforementioned construct can be further input into the patient, thereby realizing the continuous expression of the recombinant protein described above in the patient, and further Actively immunizing in the body to produce anti-PD-L1 or PD-L2 antibodies in vivo, mobilizing the spontaneously induced immune cell CTLs already present in the patient, and stimulating the production of anti-PD-L1 or PD-L2CTL, Specific killing of tumor cells.
另一方面，在应用方面，发明人提出了前面所述的重组蛋白在制备药物、疫苗中的用途，所述药物或疫苗用于预防或治疗肿瘤。本发明实施例所提出的重组蛋白在肿瘤患者体内可引起显著的肿瘤特异性抗原免疫反应，有效刺激产生抗免疫检查点，如PD－L1或PD-L2抗体，调动病人体内已经存在的自发诱导产生的免疫细胞CTL，并且刺激产生抗免疫检查点，如PD-L1或PD-L2CTL，进而有效地特异性杀伤肿瘤细胞。进而发明人进一步经过实验验证发现，本发明实施例所提出的重组蛋白具有在制备有效用于预防或治疗肿瘤的药物或疫苗中的用途。On the other hand, in terms of application, the inventors have proposed the use of the aforementioned recombinant protein for the preparation of a medicament or a vaccine for preventing or treating a tumor. The recombinant protein proposed by the embodiments of the present invention can cause a significant tumor-specific antigen immune response in a tumor patient, and effectively stimulates the production of an anti-immunological checkpoint, such as PD-L1 or PD-L2 antibody, to mobilize the spontaneous induction already existing in the patient. The resulting immune cell CTL is stimulated to produce an anti-immunological checkpoint, such as PD-L1 or PD-L2CTL, which effectively kills the tumor cells specifically. Further, the inventors have further experimentally verified that the recombinant protein proposed in the examples of the present invention has a use in the preparation of a medicament or vaccine effective for preventing or treating a tumor.
另外，本发明还提出了前面所述的重组蛋白在制备疫苗中的用途，所述疫苗用于治疗病毒感染。根据本发明的实施例，发明人发现，HBV、HCV、HIV、EBV病毒感染的细胞表达PD-L1，本发明实施例所提出的重组蛋白所制备的疫苗可在病人体内刺激产生抗PD－L1抗体，调动病人体内已经存在的自发诱导产生的免疫细胞CTL，并且刺激产生抗免疫检查点，如PD-L1CTL，进而有效地特异性杀伤被上述病毒感染的细胞。In addition, the present invention also proposes the use of the aforementioned recombinant protein for the preparation of a vaccine for the treatment of a viral infection. According to an embodiment of the present invention, the inventors have found that HBV, HCV, HIV, EBV virus-infected cells express PD-L1, and the vaccine prepared by the recombinant protein of the present invention can stimulate anti-PD-L1 in a patient. The antibody mobilizes spontaneously induced immune cell CTLs already present in the patient and stimulates the production of anti-immunological checkpoints, such as PD-L1 CTL, to effectively kill cells infected with the above virus.
治疗组合物Therapeutic composition
一方面，本发明提出了一种药物组合物。根据本发明的实施例，所述药物组合物包括：前面所述的重组蛋白；以及药学上可接受的佐剂。本发明实施例所提出的药物组合物中的重组蛋白可引起显著的特异性抗原免疫反应，加之佐剂的增强免疫应答的功能。根据本发明的实施例，根据本发明的实施例，本发明实施例所提出的药物组合物在肿瘤病人体内有效刺激产生抗免疫检查点，如PD－L1或PD-L2抗体，调动病人体内已经存在的自发诱导产生的免疫细胞CTL，并且刺激产生抗免疫检查点，如PD-L1或PD-L2CTL，进而有效地特异性杀伤肿瘤细胞或被病毒(HBV、HCV、HIV、EBV)感染的细胞。In one aspect, the invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises: the recombinant protein described above; and a pharmaceutically acceptable adjuvant. The recombinant protein in the pharmaceutical composition proposed in the examples of the present invention can cause a significant specific antigen immune response, and the function of the adjuvant to enhance the immune response. According to an embodiment of the present invention, according to an embodiment of the present invention, the pharmaceutical composition provided by the embodiment of the present invention effectively stimulates an anti-immunological checkpoint, such as a PD-L1 or PD-L2 antibody, in a tumor patient, and mobilizes the patient already. The presence of spontaneously induced immune cell CTLs and stimulation of anti-immunological checkpoints, such as PD-L1 or PD-L2CTL, to effectively kill tumor cells or cells infected with viruses (HBV, HCV, HIV, EBV) .
另一方面，本发明提出了一种DC细胞。根据本发明的实施例，所述DC细胞负载前面所述的重组蛋白。根据本发明的实施例，本发明实施例所提出的DC细胞可将重组蛋白中的肿瘤抗原(如前面所述的免疫检查点分子片段)、辅助T细胞抗原决定基片段以及免疫刺
激分子片段分别呈递到细胞表面，进而有效刺激产生抗免疫检查点，如PD－L1或PD-L2抗体，调动病人体内已经存在的自发诱导产生的免疫细胞CTL，并且刺激产生抗免疫检查点，如PD-L1或PD-L2CTL，进而有效地特异性杀伤肿瘤细胞或被病毒(HBV、HCV、HIV、EBV)感染的细胞。。In another aspect, the invention provides a DC cell. According to an embodiment of the invention, the DC cells are loaded with the recombinant protein described above. According to an embodiment of the present invention, the DC cells proposed in the embodiments of the present invention can treat tumor antigens in the recombinant protein (such as the immunological checkpoint molecular fragment described above), helper T cell epitope fragments, and immunospins.
The excimer fragments are presented to the cell surface, respectively, thereby effectively stimulating the production of anti-immunological checkpoints, such as PD-L1 or PD-L2 antibodies, mobilizing the spontaneously induced immune cell CTLs already present in the patient, and stimulating the production of anti-immune checkpoints, For example, PD-L1 or PD-L2CTL, thereby effectively killing tumor cells or cells infected with viruses (HBV, HCV, HIV, EBV). .
再一方面，本发明提出了一种靶向性免疫细胞群。根据本发明的实施例，所述靶向性免疫细胞群是通过前面所述的DC细胞与淋巴细胞进行共培养获得的。根据本发明的实施例，本发明实施例所提出的靶向性免疫细胞群可特异性杀伤肿瘤细胞，分泌特异性结合肿瘤抗原的抗体，实现对肿瘤细胞的特异性清除。In still another aspect, the invention provides a targeted immune cell population. According to an embodiment of the invention, the targeted immune cell population is obtained by co-culture of DC cells with lymphocytes as described above. According to an embodiment of the present invention, the targeted immune cell population proposed in the embodiments of the present invention can specifically kill tumor cells, secrete antibodies that specifically bind tumor antigens, and achieve specific removal of tumor cells.
再一方面，本发明提出了一种疫苗。根据本发明的实施例，所述疫苗包含前面所述的重组蛋白、前面所述的DC细胞或前面所述的靶向性免疫细胞群。如前所述，本发明实施例所提出的重组蛋白、DC细胞以及靶向性免疫细胞群在患者体内可引起显著的特异性抗原免疫反应。根据本发明的实施例，本发明实施例所提出的疫苗可有效刺激产生抗免疫检查点，如PD－L1或PD-L2抗体，调动病人体内已经存在的自发诱导产生的免疫细胞CTL，并且刺激产生抗免疫检查点，如PD-L1或PD-L2CTL，进而有效地特异性杀伤肿瘤细胞或被病毒(HBV、HCV、HIV、EBV)感染的细胞。In still another aspect, the invention provides a vaccine. According to an embodiment of the invention, the vaccine comprises a recombinant protein as described above, a DC cell as described above or a targeted immune cell population as described above. As described above, the recombinant protein, DC cell, and targeted immune cell population proposed in the examples of the present invention can cause a significant specific antigen immune response in a patient. According to an embodiment of the present invention, the vaccine according to the embodiment of the present invention can effectively stimulate the production of an anti-immunization checkpoint, such as a PD-L1 or PD-L2 antibody, mobilize the spontaneously induced immune cell CTL already existing in the patient, and stimulate An anti-immunization checkpoint, such as PD-L1 or PD-L2CTL, is generated to effectively kill tumor cells or cells infected with viruses (HBV, HCV, HIV, EBV).
再一方面，本发明提出了一种抗体。根据本发明的实施例，所述抗体特异性识别前面所述的重组蛋白，进而本发明实施例所提出的抗体可特异性识别肿瘤抗原。根据本发明的实施例，发明发现，所述抗体可特异性识别抗原，与肿瘤细胞或被病毒(HBV、HCV、HIV、EBV)感染的细胞特异性结合，进而使得肿瘤细胞或被病毒(HBV、HCV、HIV、EBV)感染的细胞被吞噬细胞吞噬，实现对肿瘤细胞或被病毒(HBV、HCV、HIV、EBV)感染的细胞的特异性清除。同时，本发明提出了一种制备抗体的方法。根据本发明的实施例，所述方法包括：利用前面所述的重组蛋白对动物进行免疫接种；采集经过免疫接种的动物的血清；以及从所述血清中纯化出目的抗体。本发明实施例所提出的制备抗体的方法，操作简便，抗体可特异性识别所述重组蛋白。In still another aspect, the invention provides an antibody. According to an embodiment of the present invention, the antibody specifically recognizes the recombinant protein described above, and the antibody proposed in the embodiment of the present invention specifically recognizes a tumor antigen. According to an embodiment of the present invention, the present invention finds that the antibody specifically recognizes an antigen, specifically binds to a tumor cell or a cell infected by a virus (HBV, HCV, HIV, EBV), thereby causing a tumor cell or a virus (HBV) Cells infected with HCV, HIV, EBV) are engulfed by phagocytic cells to achieve specific clearance of tumor cells or cells infected with viruses (HBV, HCV, HIV, EBV). Meanwhile, the present invention proposes a method of preparing an antibody. According to an embodiment of the invention, the method comprises: immunizing an animal with the recombinant protein described above; collecting serum of the immunized animal; and purifying the antibody of interest from the serum. The method for preparing an antibody proposed in the embodiments of the present invention is simple and convenient, and the antibody can specifically recognize the recombinant protein.
根据本发明的实施例，本发明提出的治疗组合物可包括前面所述的重组蛋白、前面所述的核酸、前面所述的构建体、前面所述的转基因细胞、前面所述的药物组合物、前面所述的DC细胞、前面所述的靶向性免疫细胞群、前面所述的疫苗或者前面所述的抗体的任意一种。根据本发明的实施例，本发明实施例所提出的治疗组合物可直接或间接引起特异性抗原免疫反应，实现对肿瘤细胞或被病毒(HBV、HCV、HIV、EBV)感染的细胞的特异性杀伤和清除。According to an embodiment of the present invention, the therapeutic composition proposed by the present invention may comprise the recombinant protein described above, the nucleic acid described above, the aforementioned construct, the above-described transgenic cell, the aforementioned pharmaceutical composition The DC cells described above, the targeted immune cell population described above, the vaccine described above, or any of the antibodies described above. According to an embodiment of the present invention, the therapeutic composition proposed by the embodiments of the present invention can directly or indirectly cause a specific antigen immune response, and achieve specificity to tumor cells or cells infected by viruses (HBV, HCV, HIV, EBV). Kill and clear.
相应地，对患者给予治疗有效量的前面所述的重组蛋白、前面所述的药物组合物、前面所述的DC细胞、前面所述的靶向性免疫细胞群、前面所述的疫苗或者前面所述的抗体均可
有效治疗或预防表达PD-L1或PD-L2的肿瘤。Accordingly, the patient is administered a therapeutically effective amount of the recombinant protein described above, a pharmaceutical composition as described above, a DC cell as described above, a previously described targeted immune cell population, a vaccine as described above, or a front The antibodies can be
Effectively treat or prevent tumors that express PD-L1 or PD-L2.
在本文中所使用的术语“给予”指将预定量的物质通过某种适合的方式引入病人。本发明实施例中的重组蛋白、药物组合物、DC细胞、靶向性免疫细胞群、疫苗或抗体可以通过任何常见的途径给药，只要它可以到达预期的组织。给药的各种方式是可以预期的，包括腹膜，静脉，肌肉，皮下，皮层，口服，局部，鼻腔，肺部和直肠，但是本发明不限于这些已举例的给药方式。然而，由于口服给药时，口服给药的组合物的活性成分应该被包被或被配制以防止其在胃部被降解。优选地，本发明的组合物可以注射制剂被给药。此外，本发明的药物组合物可以使用将活性成分传送到靶细胞的特定器械来给药。The term "administering" as used herein refers to introducing a predetermined amount of a substance into a patient in some suitable manner. The recombinant protein, pharmaceutical composition, DC cell, targeted immune cell population, vaccine or antibody in the embodiments of the present invention can be administered by any common route as long as it can reach the intended tissue. Various modes of administration are contemplated, including peritoneal, venous, muscular, subcutaneous, cortical, oral, topical, nasal, pulmonary, and rectal, but the invention is not limited to these exemplary modes of administration. However, due to oral administration, the active ingredient of the orally administered composition should be coated or formulated to prevent its degradation in the stomach. Preferably, the compositions of the invention may be administered as an injectable preparation. Furthermore, the pharmaceutical compositions of the invention may be administered using a particular device that delivers the active ingredient to the target cells.
本发明实施例中的重组蛋白、药物组合物、DC细胞、靶向性免疫细胞群、疫苗或抗体的给药频率和剂量可以通过多个相关因素被确定，该因素包括要被治疗的疾病类型，给药途径，病人年龄，性别，体重和疾病的严重程度以及作为活性成分的药物类型。根据本发明的一些实施例，日剂量可分为适宜形式的1剂、2剂或多剂，以在整个时间段内以1次、2次或多次给药，只要达到治疗有效量即可。The frequency and dose of administration of the recombinant protein, pharmaceutical composition, DC cell, targeted immune cell population, vaccine or antibody in the examples of the present invention can be determined by a plurality of related factors including the type of disease to be treated. , route of administration, patient age, sex, weight and severity of the disease, and the type of drug as the active ingredient. According to some embodiments of the invention, the daily dose may be divided into 1 dose, 2 doses or multiple doses in a suitable form for administration once, twice or more times throughout the time period, as long as a therapeutically effective amount is achieved. .
术语“治疗有效量”是指足以显著改善某些与疾病或病症相关的症状的量，也即为给定病症和给药方案提供治疗效果的量。术语“治疗”用于指获得期望的药理学和/或生理学效果。本文使用的“治疗”涵盖将发明实施例中的重组蛋白、药物组合物、DC细胞、靶向性免疫细胞群、疫苗或抗体给予个体以治疗，包括但不限于将含本文所述的给予有需要的个体。The term "therapeutically effective amount" refers to an amount sufficient to significantly ameliorate certain symptoms associated with a disease or condition, that is, an amount that provides a therapeutic effect for a given condition and dosage regimen. The term "treatment" is used to mean obtaining the desired pharmacological and/or physiological effect. As used herein, "treatment" encompasses administration of a recombinant protein, pharmaceutical composition, DC cell, targeted immune cell population, vaccine or antibody in an embodiment of the invention to a subject, including, but not limited to, administration comprising the agents described herein. The individual in need.
需要说明的是，根据本发明实施例的重组蛋白及其用途、药物组合物、DC细胞、靶向性免疫细胞群、疫苗、抗体、治疗和诊断癌症的方法和系统是本申请的发明人经过艰苦的创造性劳动和优化工作才发现和完成的。It should be noted that the recombinant protein and the use thereof, the pharmaceutical composition, the DC cell, the targeted immune cell population, the vaccine, the antibody, the method and system for treating and diagnosing cancer according to the embodiment of the present invention are the inventors of the present application. Hard creative labor and optimization work were discovered and completed.
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社)或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。The solution of the present invention will be explained below in conjunction with the embodiments. Those skilled in the art will appreciate that the following examples are merely illustrative of the invention and are not to be considered as limiting the scope of the invention. In the examples, the specific techniques or conditions are not mentioned, according to the techniques or conditions described in the literature in the field (for example, refer to J. Sambrook et al., Huang Peitang et al., Molecular Cloning Experimental Guide, Third Edition, Science Press) or in accordance with the product manual. The reagents or instruments used are not indicated by the manufacturer, and are conventional products that can be obtained commercially.
实施例1Example 1
在以下实施例中，所用的材料和方法如下所述：In the following examples, the materials and methods used are as follows:
制备树突状(DC)细胞Preparation of dendritic (DC) cells
从鼠骨髓分离DC细胞的方法如下所述：从小鼠的四肢中冲出骨髓，并将骨髓过尼龙网，并用氯化铵去除红细胞。然后用RPMI-1640培养基充分润洗细胞，之后培养在2.5ml RPMI-1640培养基中，培养基中含有10％FBS，20ng/ml重组小鼠GM-CSF(rmGM-CSF)
和20ng/ml重组小鼠IL-4(rmIL-4)(购自PeproTech，Inc.，Rocky Hill，NJ)。在培养过程的第2天和第4天，去除细胞上清并更换新鲜培养基，新鲜培养基中含有20ng/ml的rmGM-CSF和20ng/ml的rmIL-4。细胞培养在37℃、5％CO2的培养箱中。在培养过程的第48小时，去除非粘连粒细胞并更换新鲜培养基。细胞培养7天后，通过FACS分析，约80％以上的细胞表达DC细胞特有标记物。The method of isolating DC cells from mouse bone marrow is as follows: bone marrow is punched out from the limbs of the mouse, and the bone marrow is passed through a nylon mesh, and red blood cells are removed with ammonium chloride. The cells were then thoroughly washed with RPMI-1640 medium, and then cultured in 2.5 ml of RPMI-1640 medium containing 10% FBS and 20 ng/ml recombinant mouse GM-CSF (rmGM-CSF).
And 20 ng/ml recombinant mouse IL-4 (rmIL-4) (available from PeproTech, Inc., Rocky Hill, NJ). On days 2 and 4 of the culture process, the cell supernatant was removed and fresh medium was replaced with 20 ng/ml of rmGM-CSF and 20 ng/ml of rmIL-4. The cells were cultured in an incubator at 37 ° C in a 5% CO 2 atmosphere. At 48 hours of the culture process, non-adherent granulocytes were removed and fresh medium was replaced. After 7 days of cell culture, about 80% or more of the cells expressed DC cell-specific markers by FACS analysis.
DC细胞免疫和肿瘤模型DC cell immunity and tumor model
向骨髓来源的DC细胞(骨髓细胞培养5-7天后获得)中加入重组蛋白，用来激活DC细胞，8小时后用PBS润洗细胞3次，并继续培养136小时后，将DC细胞用作免疫模型。在一些实验中，抗原激活的DC细胞用100ng/ml LPS(Sigma，St.Louis，MO)刺激24小时后PBS润洗细胞并将DC细胞通过小鼠的脚掌注入小鼠(C57BL/6，Jackson Laboratory)体内。在肿瘤模型中，啮齿类动物肺癌细胞CMT167(C57BL)(购自European Collection of Authenticated Cell Cultures(ECAC))通过皮下注射被到导入同系C57BL/6小鼠的右侧腹腔。肿瘤接种后，小鼠被随机分组并且在不同天数，不同的组分别注射抗原激活的DC细胞或PBS。利用测径器每周2或3次测量肿瘤体积。Recombinant protein was added to bone marrow-derived DC cells (obtained after 5-7 days of bone marrow cell culture) to activate DC cells, and after 8 hours, the cells were washed with PBS three times, and after further culture for 136 hours, DC cells were used as Immunization model. In some experiments, antigen-activated DC cells were stimulated with 100 ng/ml LPS (Sigma, St. Louis, MO) for 24 hours, and the cells were washed with PBS and injected into mice via the soles of mice (C57BL/6, Jackson). Laboratory) In vivo. In the tumor model, rodent lung cancer cells CMT167 (C57BL) (purchased from the European Collection of Authenticated Cell Cultures (ECAC)) were introduced into the right abdominal cavity of syngeneic C57BL/6 mice by subcutaneous injection. After tumor inoculation, mice were randomized and injected into antigen-activated DC cells or PBS in different groups on different days. Tumor volume was measured 2 or 3 times a week using a caliper.
免疫杀伤细胞(CTL)分析Immunological killer cell (CTL) analysis
CD8+CTL反应是通过标准铬释放实验评估的。标准铬释放评估实验是通过测量体外再激活脾细胞溶解靶细胞的能力实现的。取自免疫小鼠的脾细胞体外用包含多肽的RPMI再刺激4～6天。靶细胞和对照组细胞用51Cr铬酸钠盐溶液标记90min。不同数目的效应细胞与一定数目(1X 104/孔)的靶细胞在37℃下在96-孔板v-底板(每孔加有200微升培养基)中共培养3小时。从每3个孔中收集100微升上清。溶解率通过以下公式计算：(实验铬释放量-自发铬释放量)/(最大铬释放量–自发铬释放量)X 100。其中，铬释放量是通过如下操作实现的：共培养后，将孔板放到离心机上离心，通过γ计数器(购自Beckman Coulter，Inc.，Fullerton，CA)计算上清液中的辐射活性(铬释放量)。The CD8+ CTL response was assessed by standard chromium release assays. Standard chromium release assessment experiments were performed by measuring the ability of spleen cells to lyse target cells in vitro. Splenocytes from immunized mice were restimulated in vitro with RPMI containing the polypeptide for 4 to 6 days. Target cells and control cells were labeled with 51Cr sodium chromate solution for 90 min. Different numbers of effector cells were co-cultured with a certain number (1×104/well) of target cells for 3 hours at 37° C. in a 96-well plate v-bottom plate (200 microliters of medium per well). 100 microliters of supernatant was collected from every 3 wells. The dissolution rate is calculated by the following formula: (experimental chromium release amount - spontaneous chromium release amount) / (maximum chromium release amount - spontaneous chromium release amount) X 100. Among them, the amount of chromium released was achieved by co-cultivation, placing the wells on a centrifuge and centrifuging, and calculating the radiation activity in the supernatant by a gamma counter (purchased from Beckman Coulter, Inc., Fullerton, CA) ( Chromium release).
实施例2构建融合蛋白表达载体Example 2 Construction of Fusion Protein Expression Vector
合成融合基因一：包含部分人PD-L1序列或PD-L2序列(Accession number GenBank:AF177937.1)，完整的PADRE辅助T细胞抗原决定基序列和完整的GM-CSF、IL-12或RANTES序列(Accession number GenBank:M11734.1)以及侧翼克隆位点序列(融合基因一结构如图1所示)(由GENEWIZ，South Plainfield，NJ，USA合成)。合成融合基因二：包含部分人PD-L1序列或PD-L2序列(GenBank:AF177937.1)和完整的辅助T细胞PADRE抗原决定基序列。这些合成基因被NdeI和XhoI(购自Boehringer Mannheim)限制性内切酶消化后克隆到pET21a(+)表达载体上(购自Novagen)，通过酶切和测序鉴定，连接正确且无突变
的目标重组质粒。Synthesis of fusion gene 1: contains part of human PD-L1 sequence or PD-L2 sequence (Accession number GenBank: AF177937.1), intact PADRE helper T cell epitope sequence and complete GM-CSF, IL-12 or RANTES sequence (Accession number GenBank: M11734.1) and flanking cloning site sequences (fusion gene-structure shown in Figure 1) (synthesized by GENEWIZ, South Plainfield, NJ, USA). Synthetic Fusion Gene 2: Contains a partial human PD-L1 sequence or a PD-L2 sequence (GenBank: AF177937.1) and an intact helper T cell PADRE epitope sequence. These synthetic genes were digested with restriction endonucleases of NdeI and XhoI (purchased from Boehringer Mannheim) and cloned into the pET21a(+) expression vector (purchased from Novagen), identified by restriction enzyme digestion and sequencing, and ligated correctly and without mutation.
Target recombinant plasmid.
实施例3制备和纯化包含人PD-L1的融合蛋白Example 3 Preparation and Purification of Fusion Proteins Containing Human PD-L1
为了制备和纯化重组蛋白，目标重组质粒电转入Escherichia coli BL21(DE3)(Novagen)感受态细胞，之后Escherichia coli BL21(DE3)接种在LB琼脂培养板(包含50微克/ml的氨苄西林)上进行扩增培养。以下所描述的重组蛋白表达的方法是一系列在不同实验条件下的实验中的一个。To prepare and purify the recombinant protein, the target recombinant plasmid was electroporated into Escherichia coli BL21(DE3) (Novagen) competent cells, and then Escherichia coli BL21 (DE3) was seeded on LB agar plates (containing 50 μg/ml of ampicillin). Amplification culture is carried out. The method of recombinant protein expression described below is one of a series of experiments under different experimental conditions.
为了表达重组蛋白，包含氨苄西林的4YT培养基(包含32g Bacto胰蛋白胨，20g酵母提取物和5g NaCl/L，pH 7.2)与一个克隆共同孵育，孵育条件是：在180rpm和37℃下震荡培养24h。之后加入IPTG(购自Sigma)至终浓度为1mM，再继续培养4～5小时以便表达重组蛋白。最后在4℃，15，900×g的条件下离心收集细胞。To express the recombinant protein, 4YT medium containing ampicillin (containing 32 g of Bacto tryptone, 20 g of yeast extract and 5 g of NaCl/L, pH 7.2) was incubated with a clone under shaking conditions at 180 rpm and 37 °C. 24h. IPTG (purchased from Sigma) was then added to a final concentration of 1 mM, and incubation was continued for a further 4 to 5 hours to express the recombinant protein. Finally, the cells were collected by centrifugation at 4 ° C, 15,900 × g.
为了从包涵体中纯化重组蛋白，冰冻的细胞小块用裂解液(50mM Tris，pH 8.0，1mM EDTA和1mM PMSF)重悬，细胞小块与裂解液的质量体积比是1：10。包含重组蛋白的包涵体在French压力(Constant Systems LTD)137.9MPa的条件下恢复活性。在离心之前，加入等体积的裂解液液稀释以减少粘滞度，从而更有利于获得包涵体。裂解后的溶液在48，000×g的条件下离心30min，以致使包涵体沉淀。弃上清，然后对沉淀清洗三次以去除宿主细胞中的内毒素、蛋白和DNA。第一遍清洗所用溶液包含50mM Tris，pH 8.0，5mM EDTA和2％Triton×-100。第二遍清洗所用溶液包含50mM Tris，pH 8.0，5mM EDTA，1％脱氧胆酸钠。第三遍清洗所用溶液包含50mM Tris，pH 8.0，5mM EDTA，and 1M NaCl.清洗之后，沉淀用专门的裂解液在室温下重悬(质量体积比为1：40)，搅拌30min并在离心条件下重沉淀。包涵体中重组蛋白的溶解和变性需要利用一种溶解剂(8M尿素)。室温下，沉淀溶解后，蛋白的浓度为2mg/ml，并继续搅拌30min。加入醋酸调pH到8.0。通过两步透析法(MWCO 6，000–8，000Da)在12–16小时内浓缩190mL的溶解蛋白。第一步透析在50mM Tris HCl pH 8.0的溶液中进行，第二步透析在pH 4.5的25mM醋酸钠溶液中进行。溶解的蛋白进一步通过Ni-NTA Fast Start Kit(Qiagen)进行纯化。洗脱出的蛋白通过12％SDS-PAGE凝胶电泳分析，并且蛋白浓度通过Bradford et al.(Bio-Rad Laboratories)测定。纯度大于90％的重组蛋白保存到-20℃，以备后续研究。To purify the recombinant protein from the inclusion bodies, the frozen cell pellet was resuspended in lysate (50 mM Tris, pH 8.0, 1 mM EDTA and 1 mM PMSF) with a mass to volume ratio of cell pellet to lysate of 1:10. The inclusion bodies containing the recombinant protein were restored to activity under the conditions of French Pressure (Constant Systems LTD) of 137.9 MPa. Prior to centrifugation, an equal volume of lysate is added to dilute to reduce viscosity, which is more advantageous for obtaining inclusion bodies. The lysed solution was centrifuged at 48,000 x g for 30 min to cause inclusion bodies to precipitate. The supernatant was discarded and the pellet was washed three times to remove endotoxin, protein and DNA from the host cells. The solution used for the first pass of cleaning contained 50 mM Tris, pH 8.0, 5 mM EDTA and 2% Triton x-100. The solution used for the second pass of cleaning contained 50 mM Tris, pH 8.0, 5 mM EDTA, 1% sodium deoxycholate. The solution used for the third cleaning consisted of 50 mM Tris, pH 8.0, 5 mM EDTA, and 1 M NaCl. After washing, the pellet was resuspended at room temperature with a special lysate (mass to volume ratio of 1:40), stirred for 30 min and centrifuged. Heavy sedimentation. The dissolution and denaturation of the recombinant protein in the inclusion body requires the use of a solvent (8 M urea). After the precipitate was dissolved at room temperature, the protein concentration was 2 mg/ml and stirring was continued for 30 min. Add acetic acid to adjust the pH to 8.0. 190 mL of solubilized protein was concentrated in 12–16 hours by a two-step dialysis process (MWCO 6,000–8,000 Da). The first step of dialysis was carried out in a solution of 50 mM Tris HCl pH 8.0, and the second step of dialysis was carried out in a 25 mM sodium acetate solution at pH 4.5. The solubilized protein was further purified by a Ni-NTA Fast Start Kit (Qiagen). The eluted proteins were analyzed by 12% SDS-PAGE gel electrophoresis, and the protein concentration was determined by Bradford et al. (Bio-Rad Laboratories). Recombinant proteins with a purity greater than 90% were stored at -20 °C for subsequent studies.
实施例4负载了包含人PD-L1的融合蛋白的DC细胞在小鼠体内可有效诱导抗PD-L1抗体的产生和CTL反应Example 4 DC cells loaded with a fusion protein comprising human PD-L1 were effective in inducing anti-PD-L1 antibody production and CTL responses in mice
在本实施例中，一系列的实验是为了验证负载了PD-L1融合蛋白的DC细胞是否能够在小鼠中诱导抗PD-L1抗体的产生和CTL反应。负载了PD-L1融合蛋白的DC细胞在小鼠
体内能够引起PD-L1-特异性反应的能力是通过利用DC细胞免疫小鼠验证的。In the present example, a series of experiments were conducted to verify whether DC cells loaded with the PD-L1 fusion protein were able to induce anti-PD-L1 antibody production and CTL responses in mice. DC cells loaded with PD-L1 fusion protein in mice
The ability of the body to elicit a PD-L1-specific response was verified by immunizing mice with DC cells.
将雌性B6小鼠(the Jackson Laboratory，Bar Harbor，ME，USA)(n＝4)用骨髓来源的DC细胞进行免疫，此DC细胞分别负载了重组蛋白(PD-L1Δ-PADRE Th-GM-CSF)(iPD-L1-Vax)，蛋白(PD-L1Δ)，免疫刺激因子(重组GM-CSF，Genzyme，Tarzana，CA)或PBS。每只小鼠接种1×106个DC细胞，通过每周间隔两次的脚板注射50μg/ml的细胞溶液实现。两周后，每组鼠取脾和血清。每组鼠血清中的PD-L1-特异性IgG的水平通过ELISA测定，ELISA板每3个孔中铺有重组PD-L1蛋白(Abcam，Cambridge，MA，USA)，ELISA数值是通过血清(1：100倍稀释)的OD450nm值的平均值±SD获得的。Female B6 mice (the Jackson Laboratory, Bar Harbor, ME, USA) (n=4) were immunized with bone marrow-derived DC cells loaded with recombinant protein (PD-L1Δ-PADRE Th-GM-CSF) (iPD-L1-Vax), protein (PD-L1Δ), immunostimulatory factor (recombinant GM-CSF, Genzyme, Tarzana, CA) or PBS. Each mouse was inoculated with 1 x 106 DC cells by injection of 50 μg/ml of cell solution twice a week. Two weeks later, spleens and serum were taken from each group of rats. The levels of PD-L1-specific IgG in the serum of each group of rats were determined by ELISA, and the recombinant PD-L1 protein (Abeam, Cambridge, MA, USA) was plated every 3 wells of the ELISA plate, and the ELISA value was passed through serum (1). : 100 times dilution) The mean value of OD450nm values ± SD was obtained.
结果如图2所示，图2显示了负载了iPD-L1-Vax DC细胞能够诱导显著的抗-PD-L1抗体反应，然而负载了PD-L1Δ蛋白DC细胞只能诱导微弱的抗-PD-L1抗体反应。The results are shown in Figure 2. Figure 2 shows that iPD-L1-Vax DC cells can induce significant anti-PD-L1 antibody responses, whereas PD-L1Δ protein-loaded DC cells can only induce weak anti-PD- L1 antibody reaction.
为了评估CTL反应，从接种免疫小鼠的肿瘤细胞悬液中分离脾细胞。分离出的T细胞利用PD-L1重组蛋白激活的DC细胞(10微克/ml)再刺激，进而进行体外51Cr释放试验，51Cr释放试验按照指定的T/E(靶细胞：效应细胞)比进行。靶细胞是PD-L1+鼠肺癌细胞系CMT167(C57BL)(购自European Collection of Authenticated Cell Cultures(ECACC))。To assess the CTL response, spleen cells were isolated from tumor cell suspensions vaccinated with immunized mice. The isolated T cells were re-stimulated by PD-L1 recombinant protein-activated DC cells (10 μg/ml), and then the in vitro 51Cr release test was performed, and the 51Cr release test was performed according to the specified T/E (target cell: effector cell) ratio. The target cell was the PD-L1+ murine lung cancer cell line CMT167 (C57BL) (available from the European Collection of Authenticated Cell Cultures (ECACC)).
结果如图3所示，图3显示了负载了iPD-L1-Vax DC细胞能够诱导显著的抗-PD-L1CTL反应，然而负载了PD-L1Δ蛋白的DC细胞仅诱导微弱的抗-PD-L1CTL反应。The results are shown in Figure 3. Figure 3 shows that iPD-L1-Vax DC cells were able to induce significant anti-PD-L1 CTL responses, whereas DC cells loaded with PD-L1Δ protein induced only weak anti-PD-L1 CTLs. reaction.
实施例5在同系小鼠中，负载了包含人PD-L1的融合蛋白的DC免疫接种控制PD-L1+肺癌的生长Example 5 DC Immunization Loaded with Fusion Protein Containing Human PD-L1 Controls Growth of PD-L1+ Lung Cancer in Syngeneic Mice
为了评估验证负载了PD-L1融合蛋白的DC细胞诱导抗肿瘤免疫反应，C57BL/6小鼠(n＝6只/组)皮下接种CMT167肿瘤细胞(1x105)，3d后，免疫接种1.5x106骨髓来源的DC细胞，DC细胞分别负载了重组蛋白(PD-L1Δ-PADRE Th-GM-CSF)(iPD-L1-Vax)，蛋白PD-L1Δ，免疫刺激因子(GMCSF)，或PBS，并且DC细胞预先体外利用LPS一周间隔两次刺激成熟。免疫接种后，每隔3～4天测量肿瘤生长。C57BL/6 mice (n=6/group) were subcutaneously inoculated with CMT167 tumor cells (1×105), and after 1.5 days, 1.5×106 bone marrow sources were immunized in order to evaluate the induction of anti-tumor immune responses by DC cells loaded with PD-L1 fusion protein. DC cells, DC cells were loaded with recombinant protein (PD-L1Δ-PADRE Th-GM-CSF) (iPD-L1-Vax), protein PD-L1Δ, immunostimulatory factor (GMCSF), or PBS, and DC cells were pre-treated Maturation was stimulated twice in vitro using LPS in vitro. Tumor growth was measured every 3 to 4 days after immunization.
结果如图4所示，图4显示了利用负载了iPD-L1-Vax DC免疫接种小鼠后，能够显著抑制PD-L1+肺癌的生长，然而利用负载了蛋白PD-L1Δ或PBS的DC免疫接种小鼠，却不能抑制PD-L1+肺癌的生长。The results are shown in Figure 4. Figure 4 shows that the immunization of mice with iPD-L1-Vax DC can significantly inhibit the growth of PD-L1+ lung cancer, but with DC immunization with protein PD-L1Δ or PBS. Mice did not inhibit the growth of PD-L1+ lung cancer.
实施例6Example 6
在本实施例中，发明人考察了负载了重组蛋白(PD-L1Δ-PADRE Th-IL-12或PD-L1Δ-PADRE Th-RANTES或PD-L2Δ-PADRE Th-GM-CSF或PD-L2Δ-PADRE Th-IL-12或
PD-L1Δ-PADRE Th-RANTES或PD-L1/L2Δ-PADRE Th-GM-CSF或PD-L1/L2Δ-PADRE Th-IL-12或PD-L1/L2Δ-PADRE Th-RANTES)的DC细胞在小鼠体内诱导抗PD-L1或抗PD-L2抗体的产生和CTL反应的情况以及免疫接种控制PD-L1+或PD-L2+肺癌的生长的情况，实验方法如实施例4和实施例5所描述的，结果显示，负载了重组蛋白(PD-L1Δ-PADRE Th-IL-12或PD-L1Δ-PADRE Th-RANTES或PD-L2Δ-PADRE Th-GM-CSF或PD-L2Δ-PADRE Th-IL-12或PD-L1Δ-PADRE Th-RANTES或PD-L1/L2Δ-PADRE Th-GM-CSF或PD-L1/L2Δ-PADRE Th-IL-12或PD-L1/L2Δ-PADRE Th-RANTES)DC细胞能够诱导显著的抗PD-L1或抗PD-L2抗体反应和诱导显著的抗PD-L1或抗PD-L2CTL反应，利用负载了重组蛋白(PD-L1Δ-PADRE Th-IL-12或PD-L1Δ-PADRE Th-RANTES或PD-L2Δ-PADRE Th-GM-CSF或PD-L2Δ-PADRE Th-IL-12或PD-L1Δ-PADRE Th-RANTES或PD-L1/L2Δ-PADRE Th-GM-CSF或PD-L1/L2Δ-PADRE Th-IL-12或PD-L1/L2Δ-PADRE Th-RANTES)DC细胞免疫接种小鼠后，能够显著抑制PD-L1+或PD-L2+肺癌的生长。In this example, the inventors examined the loading of recombinant protein (PD-L1Δ-PADRE Th-IL-12 or PD-L1Δ-PADRE Th-RANTES or PD-L2Δ-PADRE Th-GM-CSF or PD-L2Δ- PADRE Th-IL-12 or
DC cells of PD-L1Δ-PADRE Th-RANTES or PD-L1/L2Δ-PADRE Th-GM-CSF or PD-L1/L2Δ-PADRE Th-IL-12 or PD-L1/L2Δ-PADRE Th-RANTES) The condition of inducing anti-PD-L1 or anti-PD-L2 antibody production and CTL reaction in mice and the case of vaccination controlling the growth of PD-L1+ or PD-L2+ lung cancer, the experimental methods are as described in Example 4 and Example 5. The results showed that the recombinant protein was loaded (PD-L1Δ-PADRE Th-IL-12 or PD-L1Δ-PADRE Th-RANTES or PD-L2Δ-PADRE Th-GM-CSF or PD-L2Δ-PADRE Th-IL- 12 or PD-L1Δ-PADRE Th-RANTES or PD-L1/L2Δ-PADRE Th-GM-CSF or PD-L1/L2Δ-PADRE Th-IL-12 or PD-L1/L2Δ-PADRE Th-RANTES) DC cells Ability to induce significant anti-PD-L1 or anti-PD-L2 antibody responses and induce significant anti-PD-L1 or anti-PD-L2CTL responses using recombinant proteins (PD-L1Δ-PADRE Th-IL-12 or PD-L1Δ) -PADRE Th-RANTES or PD-L2Δ-PADRE Th-GM-CSF or PD-L2Δ-PADRE Th-IL-12 or PD-L1Δ-PADRE Th-RANTES or PD-L1/L2Δ-PADRE Th-GM-CSF or PD-L1/L2Δ-PADRE Th-IL-12 or PD-L1/L2Δ-PADRE Th-RANTES) DC cells can significantly inhibit PD-L1+ or PD-L2+ after immunization of mice The growth of cancer.
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。In the description of the present specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" and the like means a specific feature described in connection with the embodiment or example. A structure, material or feature is included in at least one embodiment or example of the invention. In the present specification, the schematic representation of the above terms is not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in a suitable manner in any one or more embodiments or examples. In addition, various embodiments or examples described in the specification, as well as features of various embodiments or examples, may be combined and combined.
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。
Although the embodiments of the present invention have been shown and described, it is understood that the above-described embodiments are illustrative and are not to be construed as limiting the scope of the invention. The embodiments are subject to variations, modifications, substitutions and variations.
Claims (29)
- 一种重组蛋白，其特征在于，包括：A recombinant protein characterized by comprising:免疫检查点分子片段；Immunological checkpoint molecular fragment;辅助T细胞抗原决定基片段；以及Helper T cell epitope fragment;免疫刺激分子片段。Immunostimulatory molecular fragments.
- 根据权利要求1所述的重组蛋白，其特征在于，所述免疫检查点分子包括选自PD-L1和PD-L2的至少之一。The recombinant protein according to claim 1, wherein the immunological checkpoint molecule comprises at least one selected from the group consisting of PD-L1 and PD-L2.
- 根据权利要求2所述的重组蛋白，其特征在于，所述免疫检查点分子片段为所述PD-L1和PD-L2的至少之一的去除跨膜区的细胞外分子片段。The recombinant protein according to claim 2, wherein the immunological checkpoint molecular fragment is an extracellular molecular fragment of the transmembrane region at least one of the PD-L1 and PD-L2.
- 根据权利要求1所述的重组蛋白，其特征在于，所述辅助T细胞抗原决定基为广谱PADRE辅助T细胞抗原决定基。The recombinant protein according to claim 1, wherein the helper T cell epitope is a broad-spectrum PADRE helper T cell epitope.
- 根据权利要求1所述的重组蛋白，其特征在于，所述免疫刺激分子为粒细胞集落刺激生物因子、白细胞介素-12或趋化因子。The recombinant protein according to claim 1, wherein the immunostimulatory molecule is a granulocyte colony-stimulating biological factor, interleukin-12 or a chemokine.
- 根据权利要求1所述的重组蛋白，其特征在于，所述辅助T细胞抗原决定基片段的N端与所述免疫检查点分子片段的C端相连，所述辅助T细胞抗原决定基片段的C端与所述免疫刺激分子片段的N端相连。The recombinant protein according to claim 1, wherein the N-terminus of the helper T cell epitope fragment is linked to the C-terminus of the immunological checkpoint molecule fragment, and the helper T cell epitope fragment C The end is linked to the N-terminus of the immunostimulatory molecule fragment.
- 一种重组蛋白，其特征在于，所述重组蛋白具有SEQ ID NO：1～9所示的氨基酸序列。A recombinant protein characterized by having the amino acid sequence of SEQ ID NOS: 1 to 9.
- 一种核酸，其特征在于，所述核酸编码权利要求1～7任一项所述的重组蛋白，并且所述核酸具有SEQ ID NO：10～18所示的核苷酸序列。A nucleic acid encoding the recombinant protein according to any one of claims 1 to 7, wherein the nucleic acid has the nucleotide sequence shown in SEQ ID NOS: 10 to 18.
- 一种构建体，其特征在于，所述构建体携带权利要求8所述的核酸。A construct characterized in that the construct carries the nucleic acid of claim 8.
- 根据权利要求9所述的构建体，其特征在于，所述构建体的载体为pET系列载体、pGEX系列载体、pPIC系列载体、BacPAK、pSV系列载体或pCMV系列载体。The construct according to claim 9, wherein the vector of the construct is a pET series vector, a pGEX series vector, a pPIC series vector, a BacPAK, a pSV series vector or a pCMV series vector.
- 一种构建体，其特征在于，所述构建体携带下列核酸分子:A construct characterized in that the construct carries the following nucleic acid molecules:(1)编码免疫检查点分子片段的核酸分子，所述免疫检查点分子片段具有SEQ ID NO：19～21所示的氨基酸序列，所述编码免疫检查点分子片段的核酸分子具有SEQ ID NO：22～24所示的核苷酸序列；(1) A nucleic acid molecule encoding an immunological checkpoint molecule fragment having the amino acid sequence of SEQ ID NOS: 19 to 21, the nucleic acid molecule encoding the immunological checkpoint molecule fragment having SEQ ID NO: Nucleotide sequences shown in 22-24;(2)编码辅助T细胞抗原决定基片段的核酸分子，所述辅助T细胞抗原决定基片段具有SEQ ID NO：25所示的氨基酸序列，所述编码辅助T细胞抗原决定基片段的核酸分子具有SEQ ID NO：26所示的核苷酸序列；以及(2) A nucleic acid molecule encoding a helper T cell epitope fragment having the amino acid sequence set forth in SEQ ID NO: 25, wherein the nucleic acid molecule encoding the helper T cell epitope fragment has a nucleotide sequence set forth in SEQ ID NO: 26;(3)编码免疫刺激分子片段的核酸分子，所述免疫刺激分子片段具有SEQ ID NO：27～29所示的氨基酸序列，所述编码免疫刺激分子片段的核酸分子具有SEQ ID NO：30～32所示的核苷酸序列， (3) A nucleic acid molecule encoding an immunostimulatory molecule fragment having the amino acid sequence of SEQ ID NOS: 27 to 29, wherein the nucleic acid molecule encoding the immunostimulatory molecule fragment has SEQ ID NOS: 30 to 32 The nucleotide sequence shown,任选地，所述构建体的载体为pET系列载体、pGEX系列载体、pPIC系列载体、BacPAK、pSV系列载体或pCMV系列载体。Optionally, the vector of the construct is a pET series vector, a pGEX series vector, a pPIC series vector, a BacPAK, a pSV series vector or a pCMV series vector.
- 一种转基因细胞，其特征在于，所述转基因细胞携带权利要求9～11任一项所述的构建体。A transgenic cell, characterized in that the transgenic cell carries the construct of any one of claims 9 to 11.
- 根据权利要求12所述的转基因细胞，其特征在于，所述转基因细胞为BL21，BL21(DE3)，BL21(DE3)pLysS，DH10B，XL1-Blue，Pichia pastors，Kluyveromyces lactis，Sf9，Sf21，High-Five T，CHO细胞系，HEK细胞系，Hela细胞系或COS细胞系。The transgenic cell according to claim 12, wherein the transgenic cell is BL21, BL21 (DE3), BL21 (DE3) pLysS, DH10B, XL1-Blue, Pichia pastors, Kluyveromyces lactis, Sf9, Sf21, High- Five T, CHO cell line, HEK cell line, Hela cell line or COS cell line.
- 根据权利要求12所述的转基因细胞，其特征在于，所述转基因细胞为抗原呈递细胞。The transgenic cell according to claim 12, wherein the transgenic cell is an antigen presenting cell.
- 根据权利要求14所述的转基因细胞，其特征在于，所述转基因细胞为树突状细胞。The transgenic cell according to claim 14, wherein the transgenic cell is a dendritic cell.
- 权利要求1～7任一项所述的重组蛋白在制备药物中的用途，所述药物用于预防或治疗肿瘤。Use of the recombinant protein according to any one of claims 1 to 7 for the preparation of a medicament for preventing or treating a tumor.
- 权利要求1～7任一项所述的重组蛋白在制备疫苗中的用途，所述疫苗用于预防或治疗肿瘤。Use of the recombinant protein according to any one of claims 1 to 7 for the preparation of a vaccine for preventing or treating a tumor.
- 权利要求1～7任一项所述的重组蛋白在制备疫苗中的用途，所述疫苗用于治疗病毒感染。Use of the recombinant protein of any one of claims 1 to 7 for the preparation of a vaccine for the treatment of a viral infection.
- 一种药物组合物，其特征在于，包括：A pharmaceutical composition comprising:权利要求1～7任一项所述的重组蛋白；以及The recombinant protein of any one of claims 1 to 7;药学上可接受的佐剂。A pharmaceutically acceptable adjuvant.
- 一种DC细胞，其特征在于，所述DC细胞负载权利要求1～7任一项所述的重组蛋白。A DC cell, wherein the DC cell is loaded with the recombinant protein according to any one of claims 1 to 7.
- 一种靶向性免疫细胞群，其特征在于，所述靶向性免疫细胞群是通过将权利要求20所述的DC细胞与淋巴细胞进行共培养获得的。A targeted immune cell population characterized in that the targeted immune cell population is obtained by co-culturing the DC cells of claim 20 with lymphocytes.
- 一种疫苗，其特征在于，包含权利要求1～7任一项所述的重组蛋白、权利要求20所述的DC细胞或权利要求21所述的靶向性免疫细胞群。A vaccine comprising the recombinant protein of any one of claims 1 to 7, the DC cell of claim 20, or the targeted immune cell population of claim 21.
- 一种抗体，其特征在于，所述抗体特异性识别权利要求1～7任一项所述的重组蛋白。An antibody which specifically recognizes the recombinant protein according to any one of claims 1 to 7.
- 一种制备抗体的方法，其特征在于，包括：A method of preparing an antibody, comprising:利用权利要求1～7任一项所述的重组蛋白对动物进行免疫接种；Immunizing an animal with the recombinant protein of any one of claims 1 to 7;采集经过免疫接种的动物的血清；以及Collecting serum from immunized animals;从所述血清中纯化出目的抗体。The antibody of interest is purified from the serum.
- 一种治疗组合物，其特征在于，包括：A therapeutic composition comprising:权利要求1～7任一项所述的重组蛋白、权利要求8所述的核酸、权利要求9～11任一项所述的构建体、权利要求12～15任一项所述的转基因细胞、权利要求19所述 的药物组合物、权利要求20所述的DC细胞、权利要求21所述的靶向性免疫细胞群、权利要求22所述的疫苗或者权利要求23所述的抗体。The recombinant protein according to any one of claims 1 to 7, the nucleic acid according to claim 8, the construct according to any one of claims 9 to 11, and the transgenic cell according to any one of claims 12 to 15, Claim 19 The pharmaceutical composition according to claim 20, the targeted immune cell population according to claim 21, the vaccine according to claim 22, or the antibody according to claim 23.
- 一种在患者体内刺激抗PD-L1抗体生成或细胞毒性T淋巴细胞反应的方法，其特征在于，是通过下列方式的至少之一实现的：A method of stimulating anti-PD-L1 antibody production or cytotoxic T lymphocyte response in a patient, which is achieved by at least one of the following means:1)权利要求1～7任一项所述的重组蛋白与取自患者的DC细胞共培养，将负载了权利1～7任一项所述的重组蛋白的DC细胞回输到患者体内；1) The recombinant protein according to any one of claims 1 to 7 is co-cultured with a DC cell obtained from a patient, and DC cells loaded with the recombinant protein according to any one of claims 1 to 7 are returned to a patient;2)给患者给予权利要求19所述的药物组合物；2) administering to the patient the pharmaceutical composition of claim 19;3)将权利要求9～11任一项所述的构建体导入取自患者的DC细胞，将导入所述构建体的DC细胞回输到患者体内；以及3) introducing the construct of any one of claims 9 to 11 into a DC cell taken from a patient, and returning the DC cell introduced into the construct to the patient;4)给患者给予权利要求9～11任一项所述的构建体。4) The patient according to any one of claims 9 to 11 is administered to the patient.
- 一种治疗癌症的方法，其特征在于，是通过下列方式的至少之一实现的：A method of treating cancer, which is achieved by at least one of the following:1)权利要求1～7任一项所述的重组蛋白与取自癌症患者的DC细胞共培养，将负载了权利1～7任一项所述的重组蛋白的DC细胞回输到癌症患者体内；1) The recombinant protein according to any one of claims 1 to 7 is co-cultured with DC cells obtained from a cancer patient, and DC cells loaded with the recombinant protein according to any one of claims 1 to 7 are returned to a cancer patient. ;2)给癌症患者给予权利要求19所述的药物组合物；2) administering to a cancer patient a pharmaceutical composition according to claim 19;3)将权利要求9～11任一项所述的构建体导入取自癌症患者的DC细胞，将导入所述构建体的DC细胞回输到癌症患者体内；以及3) introducing the construct according to any one of claims 9 to 11 into a DC cell obtained from a cancer patient, and returning the DC cell introduced into the construct to a cancer patient;4)给癌症患者给予权利要求9～11任一项所述的构建体。4) A construct according to any one of claims 9 to 11 is administered to a cancer patient.
- 一种治疗被病毒感染患者的方法，其特征在于，是通过下列方式的至少之一实现的：A method of treating a patient infected with a virus, characterized in that it is achieved by at least one of the following:1)权利要求1～7任一项所述的重组蛋白与取自患者的DC细胞共培养，将负载了权利1～7任一项所述的重组蛋白的DC细胞回输到患者体内；1) The recombinant protein according to any one of claims 1 to 7 is co-cultured with a DC cell obtained from a patient, and DC cells loaded with the recombinant protein according to any one of claims 1 to 7 are returned to a patient;2)给患者给予权利要求19所述的药物组合物；2) administering to the patient the pharmaceutical composition of claim 19;3)将权利要求9～11任一项所述的构建体导入取自患者的DC细胞，将导入所述构建体的DC细胞回输到患者体内；以及3) introducing the construct of any one of claims 9 to 11 into a DC cell taken from a patient, and returning the DC cell introduced into the construct to the patient;4)给患者给予权利要求9～11任一项所述的构建体。4) The patient according to any one of claims 9 to 11 is administered to the patient.
- 根据权利要求28所述的方法，其特征在于，所述病毒包括选自HBV、HCV、HIV和EBV的至少之一。 The method of claim 28, wherein the virus comprises at least one selected from the group consisting of HBV, HCV, HIV, and EBV.
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| CN101002937A (en) * | 2006-08-23 | 2007-07-25 | 中国人民解放军第四军医大学 | Recombinant His-IT-B7-H11gV tumor vaccine, and its preparing method |
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| US20230167164A1 (en) * | 2020-12-23 | 2023-06-01 | Immunowake Inc. | Immunocytokines and uses thereof |
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| CN107286245B (en) | 2021-10-08 |
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