WO2021092719A1 - 一种靶向抗原特异性t细胞诱导其向记忆干性细胞分化的融合蛋白 - Google Patents

一种靶向抗原特异性t细胞诱导其向记忆干性细胞分化的融合蛋白 Download PDF

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WO2021092719A1
WO2021092719A1 PCT/CN2019/117035 CN2019117035W WO2021092719A1 WO 2021092719 A1 WO2021092719 A1 WO 2021092719A1 CN 2019117035 W CN2019117035 W CN 2019117035W WO 2021092719 A1 WO2021092719 A1 WO 2021092719A1
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cells
fusion protein
cancer
mpd
tumor
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PCT/CN2019/117035
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French (fr)
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王盛典
李颖
丛艳妮
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王盛典
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins

Definitions

  • the present invention generally relates to the field of bioengineering, and particularly relates to a fusion protein that targets antigen-specific T cells to induce their differentiation and enhance their effector functions.
  • T cells play a key role in it.
  • Normal cells become cancerous cells, which can be recognized by the body's immune system to kill and eliminate them.
  • the main immune cells in the body that can kill and eliminate tumors are T lymphocytes. After T lymphocytes recognize tumor antigens, they are activated, undergo a large amount of expansion, and differentiate into effector T cells, reaching the tumor site and attacking tumor cells.
  • T lymphocytes recognize tumor antigens, they are activated, undergo a large amount of expansion, and differentiate into effector T cells, reaching the tumor site and attacking tumor cells.
  • a variety of mechanisms have been developed to prevent immune cells from attacking.
  • tumor cells highly express the immunosuppressive molecule PD-L1 and bind to the immunosuppressive receptor PD-1 on the surface of T lymphocytes. It can inhibit T lymphocytes from killing it.
  • PD-1 also known as programmed cell death-1
  • programmed cell death-1 is a member of the CD28 family and is expressed on the surface of activated T cells. Its function is to inhibit T cell response by binding to the ligand PD-L1 and prevent excessive T cells. The reaction causes tissue damage, which is a negative regulation mechanism of normal T cell response.
  • Tumor cells express PD-L1 and bind to PD-1 on the surface of T cells to inhibit anti-tumor T cells from killing them, which is an important mechanism for tumor immune escape.
  • scientists have invented an anti-PD-1 antibody that can block the binding of PD-L1 on the surface of tumor cells to the PD-1 receptor on the surface of T lymphocytes, release tumor cells from inhibiting T lymphocytes, and recover T lymphocytes.
  • PD-1/PD-L1 drugs developed by internationally renowned pharmaceutical manufacturers have been approved by the US FDA, including: PD-1 inhibitor Nivolumab (Opdivo), Pembrolizumab (Keytruda); PD-L1 inhibition Atezolizumab (Tecentriq), Durvalumab (Imfinzi) and Avelumab (Bavencio); used for the treatment of melanoma, head and neck squamous cell carcinoma, non-small cell lung cancer, Hodgkin lymphoma, kidney cancer, bladder cancer and other cancers.
  • Nivolumab Opdivo
  • Pembrolizumab Keytruda
  • Tecentriq PD-L1 inhibition Atezolizumab
  • Durvalumab Imfinzi
  • Avelumab Avelumab
  • At least more than 100 domestic biological companies including Cinda, Hengrui, BeiGene, and Junshi, are also conducting PD-1 blocking drug research and development and tumor treatment clinical trials.
  • anti-PD-1 antibody drugs have achieved great success in tumor treatment, the effective rate of treatment for most advanced tumors is only about 20%. Therefore, how to improve the effect of anti-PD-1 antibody tumor treatment is an important direction for tumor drug research and development. .
  • a lymphocyte can only recognize one antigen epitope.
  • T lymphocytes There are thousands of T lymphocytes in the body. Only tumor-specific T lymphocytes can recognize and kill specific tumor cells.
  • T lymphocytes are divided into CD4 + T cells and CD8 + T cells.
  • the main body that recognizes and kills tumor cells is CD8 + T cells, and the function of CD4 + T cells is mainly to assist CD8 + T cell response. Resting T lymphocytes are inactive. Only when activated, proliferated, and differentiated into T cells in different functional states under antigen stimulation can they perform different biological functions.
  • CD8 + T cells can be divided into naive T cells, memory stem T cells, memory T cells and effector T cells according to their differentiation status.
  • Effector CD8 + T cells are terminally differentiated cells with the strongest killing activity, but their survival time in the body is short. Therefore, the anti-tumor effect in the body is the weakest. Memory CD8 + T cells survive for a long time in vivo, and can be differentiated into effector T cells by antigen stimulation, so the anti-tumor effect in vivo is significantly enhanced. Memory stem CD8 + T cells have the ability to self-replicate and renew, can survive in the body for a long time, and can be differentiated into memory and effector T cells by antigen stimulation, and have the strongest anti-tumor effect in the body.
  • the activation, proliferation and differentiation of T cells is a complex and precisely regulated reaction process, in which various cytokines play an important role in this process.
  • Cytokine is a low molecular weight soluble protein produced by immunogens, mitogens or other stimulants induced by a variety of cells. It has various functions such as regulation of immunity, hematogenesis, cell growth, and repair of damaged tissues. Among them, interleukins play an important role in activating and regulating immune cells, mediating the activation, proliferation and differentiation of T and B cells, and in inflammatory reactions. Since it was originally produced by white blood cells and then played a role in white blood cells, it was named after it. The mode of action of interleukins in the body is mainly based on the autocrine and paracrine of cells to play an important regulatory role and biological effects in the local area, and systemic application can cause severe inflammatory reactions and other side effects.
  • Interleukin 21 is a cytokine that is mainly produced by CD4 + T cells and acts on CD8 + T cells, B lymphocytes, NK cells, monocytes and macrophages, etc. .
  • IL-21 can induce activated CD8 + T lymphocytes to differentiate into young and vigorous cell populations, which are similar to memory stem CD8 + T lymphocytes. Since IL-21 can also act on a variety of other immune cells and tissue cells, the anti-tumor effect of systemic clinical trials is not obvious, and long-term application can cause side effects such as inflammation. Therefore, the key to using IL-21 in tumor therapy is how to target it to tumor-specific T cells.
  • PD-1 is expressed on the surface of activated T cells and is considered to be a marker of antigen activated T cells.
  • the latest research shows that tumor-specific CD8 + T cells in tumor patients all express PD-1, and PD-1 + cell sorting in vitro can enrich tumor-specific T cells in tumor tissues or peripheral blood of tumor patients for T Cell adoptive therapy.
  • the present invention provides a fusion protein and a method of using the fusion protein to treat cancer.
  • the use of anti-PD-1 antibody can target tumor-specific T lymphocytes, and while exerting its own anti-tumor effect, IL-21 can be targeted to tumor-specific T lymphocytes to induce tumor-specific T cells to memory ( Stem) T cells differentiate to increase the tumor treatment effect of anti-PD-1 antibodies.
  • the fusion protein provided by the present invention exceeds the effect of using monoclonal antibodies or cytokines alone, and at the same time can induce the differentiation of CD8 + T cells into memory stem T cells.
  • the inventors surprisingly found that the use of this The fusion protein prepared by the invention is obviously better than the combination of PD-1 inhibitor and IL-21.
  • the purpose of the present invention is to overcome these shortcomings of the prior art. Specifically, the purpose of the present invention is to provide a fusion protein that targets IL-21 to tumor-specific T lymphocytes and has the tumor therapeutic effect of anti-PD-1 antibody and related applications.
  • Anti-PD-1 antibody is currently the most eye-catching anti-tumor drug in the world, and many drug companies are competing for research and development.
  • the anti-PD-1 antibody tumor therapy mechanism is to block the immunosuppressive molecule PD-L1 on the surface of tumor cells from binding to the immunosuppressive receptor PD-1 on the surface of T lymphocytes to relieve the suppression of T lymphocytes by tumor cells and restore the tumor.
  • the function of specific T lymphocytes can kill tumor cells and eliminate tumors in the body.
  • the tumor-specific T lymphocytes activated in the patient are in different stages of differentiation, such as terminally differentiated effector T cells, effect memory T cells, central memory T cells, and memory Stem T cells.
  • memory stem T cells have the functions of self-renewal and reproduction, and can differentiate into different downstream effector T cells, so they have the strongest anti-tumor effect in the body.
  • the present invention found that IL-21 can induce activated CD8 + T lymphocytes to differentiate into memory stem T lymphocytes.
  • the research engine of the present invention is to link the anti-PD-1 antibody and IL-21 together to become a new fusion protein, which can block the binding of PD-L1 on the surface of tumor cells and PD-1 on the surface of T lymphocytes.
  • IL-21 is targeted to T lymphocytes to enhance the anti-tumor function of T lymphocytes.
  • amino terminus and “carboxy terminus” as used herein refer to positions within a polypeptide. Where the context permits, these terms are used to denote proximity or relative positions for a specific sequence or part of a polypeptide. For example, a certain sequence located at the carboxy terminus of the reference sequence in the polypeptide is located close to the carboxy terminus of the reference sequence, but not necessarily at the carboxy terminus of the complete polypeptide.
  • cancer refers to a tissue or cell that is found in an organism and has characteristics that distinguish it from a normal tissue or tissue cell. These characteristics include, but are not limited to: the degree of degenerative changes, irregular shapes, indistinguishability of cell contours, nuclear size, changes in the structure of the nucleus or cytoplasm, other phenotypic changes, predicting cancer or precancerous The state of the presence of cellular proteins, increased number of mitosis, and ability to metastasize. Terms related to “cancer” include cancer, sarcoma, tumor, epithelial cancer, leukemia, lymphoma, polyps, as well as sclerocarcinoma, transformation, etc.
  • fusion therapy refers to the administration of at least one therapeutically effective dose of the fusion protein component (including but not limited to "anti-PD-1 antibody and IL-21 fusion protein (PD-1Ab21)" ).
  • the fusion protein may be a mature polypeptide, fragments, fusions, or conjugates that have the biological activity of "targeting IL-21 to tumor-specific T lymphocytes and increasing the tumor therapeutic effect of anti-PD-1 antibodies” .
  • “Memory stem CD8 + T cells” means that activated CD8 + T cells are in the early stage of differentiation, have self-replication and renewal capabilities, can survive in the body for a long time, and can be differentiated into memory and effector T cells by antigen stimulation. Its cell phenotype is similar to the original CD8 + T cells express CD44 low CD62L high in mice and CD45RA + CCR7 + CD45RO - in humans. At the same time, they express IL-2R, Bcl-2, Scal-1 or CD95.
  • sequence when used herein (for example, in terms such as “immunoglobulin sequence”, “antibody sequence”, “domain sequence” or “protein sequence”) should generally be understood to include related immunoglobulin sequences as well as Both the nucleic acid sequence or the nucleotide sequence encoding it, unless the context requires a more limited explanation.
  • nucleic acid molecule may refer to nucleic acid used for genetic vaccination, or nucleic acid encoding the immunoglobulin sequence of the present invention, or both, which will become clear from the specific context.
  • the present invention relates to immunoglobulin sequences that can bind to the antigens defined herein and/or have affinity for the antigens defined herein.
  • binding to a certain antigen and/or having affinity for a certain antigen has a meaning commonly used in the art, for example, as understood in the context of antibodies and their respective antigens .
  • binding means that the immunoglobulin sequence specifically interacts with an antigen.
  • the nucleic acid of the present invention can be prepared or obtained by a method known per se, based on information about the cell-associated antigen or immunoglobulin sequence of the present invention, and/or can be isolated from a suitable natural source.
  • the nucleic acid of the present invention may also be in such a form, exist in and/or be part of a genetic construct, which should be clear to those skilled in the art.
  • Such genetic constructs generally include at least one nucleic acid of the present invention, which is optionally linked to one or more genetic construct elements known per se, such as, for example, one or more suitable regulatory elements (such as a suitable promoter). , Enhancer, terminator, etc.) and other construct elements mentioned herein.
  • promoters such as terminator, transcription and/or translation enhancement Sub and/or integration factors-refer to general manuals such as Sambrook et al. and Ausubel et al.
  • the genetic construct of the present invention is obtained by inserting the nucleotide sequence of the present invention into an appropriate (expression) vector known per se.
  • suitable expression vectors are those used in the following examples, as well as those mentioned herein.
  • the nucleic acid of the present invention and/or the genetic construct of the present invention can be used to transform host cells or host organisms, that is, to express and/or produce the fusion protein of the present invention.
  • the appropriate host or host cell is clear to the skilled person, and for example, can be any suitable fungal, prokaryotic or eukaryotic cell or cell line, or any suitable fungal, prokaryotic or eukaryotic organism .
  • the present invention provides the following in some specific aspects:
  • the present invention relates to a fusion protein comprising a cytokine and a T cell surface receptor molecule binding antagonist or agonist connected by a linker, wherein the cytokine is selected from IL-21, IL-2, and IL- 15.
  • the cytokine is selected from IL-21, IL-2, and IL- 15.
  • One of IL-7 or IL-12; the T cell surface receptor molecule binding antagonist or agonist is selected from PD-1 binding antagonist, LAG-3 binding antagonist, Tim-3 binding antagonist Or one of CD137 (4-1BB) binding agonists.
  • the fusion protein can induce activation of T cells, and at the same time target cytokines to T cells, induce T cell differentiation, and enhance T cell function.
  • the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1; and/or the PD-1 binding antagonist inhibits PD-1 to both PD-L1 and PD-L2
  • the PD-1 binding antagonist is an antibody; more preferably, the PD-1 binding antagonist is a single-chain antibody; further preferably, the PD-1 binding antagonist is a dimer Single chain antibody.
  • a fusion protein in another technical solution of the present invention, is disclosed, the domain of which includes the following structure from the amino terminus to the carboxy terminus: -X-Linker1-Y- where X is a PD-1 binding antagonist, and Y is The cytokine, Linker1 is a linker; preferably, the PD-1 binding antagonist is an antibody, and the cytokine is IL-21; more preferably, the PD-1 binding antagonist comprises the following structure from the amino terminus to the carboxy terminus : V H -Linker2-V L where, V H variable region of PD-1 is a single chain antibody heavy chain, V L variable region of an antibody light chain, Linker2 is a linker; more preferably, the fusion protein A tag sequence can also be included at the 3'end.
  • the present invention also discloses a fusion protein, wherein the PD-1 binding antagonist has a nucleotide sequence as shown in SEQ ID NO: 5 or SEQ ID NO: 17; and/or the cytokine is IL-21 , Its nucleotide sequence is shown in SEQ ID NO: 7 or SEQ ID NO: 19; preferably, the fusion protein blocks the binding of PD-LI to PD-1 on the surface of T cells, and targets IL-21 Tumor-specific T cells induce T cell differentiation.
  • the PD-1 binding antagonist has a nucleotide sequence as shown in SEQ ID NO: 5 or SEQ ID NO: 17; and/or the cytokine is IL-21 , Its nucleotide sequence is shown in SEQ ID NO: 7 or SEQ ID NO: 19; preferably, the fusion protein blocks the binding of PD-LI to PD-1 on the surface of T cells, and targets IL-21 Tumor-specific T cells induce T cell differentiation.
  • the nucleotide sequence encoding the fusion protein is shown in SEQ ID NO: 8 or SEQ ID NO: 20; further preferably, the fusion protein is The 3'end may also include a tag sequence.
  • the nucleotide sequence of the fusion protein containing the tag sequence is shown in SEQ ID NO: 10 or SEQ ID NO: 21; preferably, the fusion protein is mainly homodimeric. Body form.
  • the present invention also relates to a polynucleotide, which encodes the fusion protein of the present invention.
  • an expression vector which comprises a polynucleotide operably linked to an expression control sequence.
  • the present invention also discloses a host cell, which contains the expression vector of the present invention.
  • a method for preparing a fusion protein includes the following steps: a) Link the cytokine encoding gene to the C of the PD-1 binding antagonist encoding gene by means of genetic engineering. The end becomes the coding gene of the fusion protein; b) constructing an expression vector; c) transfecting cells to perform protein expression and purification; d) obtaining the fusion protein.
  • the present invention also discloses a pharmaceutical composition, which comprises one or more fusion proteins, and a pharmaceutically acceptable carrier, diluent or excipient; and/or, the pharmaceutical composition also includes other Anti-cancer drugs or tumor vaccines, the fusion protein can be used in combination with other anti-cancer drugs or tumor vaccines; preferably, the other anti-cancer drugs are Her2 antibodies; more preferably, the other anti-cancer drugs are Herceptin ;
  • the present invention also relates to an application for preparing a drug or kit for preventing or treating cancer, which includes administering an effective amount of the fusion protein to the individual; preferably, the cancer is selected from renal cell carcinoma, bladder cancer, and non- Small cell lung cancer, head and neck squamous cell carcinoma, squamous non-small cell lung cancer, non-squamous non-small cell lung cancer, bowel cancer, Hodgkin's lymphoma, colorectal cancer, melanoma, ovarian cancer, breast cancer, hormone receptors
  • the fusion protein is administered transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecal, intravenously, or intranasally.
  • the present invention also relates to a method for preventing or treating cancer in an individual, which comprises administering an effective amount of the fusion protein to the individual; preferably, the cancer is selected from renal cell carcinoma, bladder Cancer, non-small cell lung cancer, head and neck squamous cell carcinoma, squamous non-small cell lung cancer, non-squamous non-small cell lung cancer, bowel cancer, Hodgkin’s lymphoma, colorectal cancer, melanoma, ovarian cancer, breast cancer, Hormone receptor-positive breast cancer, Her2-positive breast cancer, and triple-negative breast cancer; more preferably, the fusion protein is administered continuously or intermittently; more preferably, intravenously, intramuscularly, subcutaneously, superficially, orally, via The fusion protein is administered cutaneously, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecal, intravenously, or intranasally.
  • the cancer is selected from renal cell carcinoma, bladder Cancer, non-small cell lung cancer
  • FIG. 1 Schematic diagram of the construction of anti-mouse PD-1 single-chain antibody and mouse IL-21 fusion protein (mPD-1Ab21)
  • FIG. 1 Expression, purification and identification of mouse mPD-1Ab21 fusion protein
  • A SDS-PAGE protein gel pair purified mouse PD-1 single-chain antibody (mPD-1Ab) and mouse anti-PD-1 single-chain antibody and IL The purity of the -21 fusion protein (mPD-1Ab21) was identified;
  • B The purified mPD-1Ab21 fusion protein was passed through a molecular sieve chromatography column, and the results showed that the mPD-1Ab21 fusion protein mainly exists as a dimer.
  • FIG. 3 Identification of anti-human PD-1 monoclonal antibodies
  • A Screening monoclonal anti-human PD-1 antibodies by hybridoma technology to obtain 1G1 monoclonal antibodies with high expression of PD-1 antibodies; the supernatant of cultured 1G1 cells and expressing PD -1 CHO cells were incubated for 30 minutes, and the ability of 1G1 supernatant to bind to PD-1 on CHO cells was detected by flow cytometry;
  • B Screening monoclonal anti-human PD-1 antibodies by hybridoma technology to obtain 1G1 monoclonal antibodies with high expression of PD-1 antibodies; the supernatant of cultured 1G1 cells and expressing PD -1 CHO cells were incubated for 30 minutes, and the ability of 1G1 supernatant to bind to PD-1 on CHO cells was detected by flow cytometry; B.
  • CHO cells were incubated with PD-L1-IgFc for 30 minutes, or 1G1 supernatant and CHO were added first The cells were incubated for 30 minutes, and then PD-L1-IgFc was added, and the 1G1 supernatant was tested for its ability to block the binding of PD-1 to PD-L1-IgFc by flow cytometry.
  • FIG. 4 Schematic diagram of construction and functional identification of anti-human PD-1 antibody and human IL-21 fusion protein (PD-1Ab21)
  • A Schematic diagram of construction of anti-human PD-1 antibody and human IL-21 fusion protein (PD-1Ab21) .
  • V H variable region is murine PD-1 antibody heavy chain, V L variable region of the murine PD-1 antibody light chain, and the constant region of a human antibody and the Fc fragment. 3 'end of the human IL-21;
  • C stably expressing PD-1 CHO cells and PD-1Ab21 protein at 4 °C incubated 30min, then flow Detect the ability of PD-1Ab21 to bind PD-1; D.
  • FIG. 5 In vitro binding and blocking experiment of mPD-1Ab21 fusion protein A. Flow cytometry to detect the binding of mPD-1Ab21 to PD-1 and the ability of mPD-1Ab21 to block the binding of PD-1 to its ligand PD-L1IgFc, A left Figure: EG7 cells (PD-1 + ) and mPD-1Ab21 protein were incubated at 4°C for 30 minutes (solid black line), or EG7 cells were incubated with anti-PD-1 antibody at 4°C for 30 minutes, and then with mPD-1Ab21 Incubate the protein for 30 minutes (black dotted line), and the negative control without mPD-1Ab21 (gray shading).
  • APC-anti-Flag flow cytometry antibody detects the ability of mPD-1Ab21 to bind to PD-1.
  • Right picture EG7 cells and mPD- The L1-IgFc protein was incubated at 4°C for 30 minutes (black solid line), or EG7 cells were incubated with mPD-1Ab21 protein for 30 minutes, and then with mPD-L1-IgFc for 30 minutes (black dotted line).
  • PE-anti-hIg flow cytometry antibody detects the ability of mPD-1Ab21 to block the binding of PD-1 to its ligand mPD-L1IgFc; B.
  • OVA Antigen peptide-specific T cell receptor transgenic mice
  • lymph node T cells were stimulated and activated with OVA peptide (0.1ng/ml) in vitro for 48 hours, and the T cells highly expressed PD-1 molecules. Repeat the above binding and blocking experiments.
  • FIG. 6 IL-21 biological function identification of mPD-1Ab21 fusion protein
  • FIG. 7 mPD-1Ab21 induces the differentiation of CD44 low CD62L high memory stem T cells in vitro into A.OT-1 transgenic mouse T cells, which were activated in vitro with OVA peptide (0.02ng/ml). After activation for 34 hours, an equimolar amount was added Perform cell differentiation with cytokines or proteins, and on the third day of differentiation, flow cytometry to detect the differentiation phenotype of T cells; B. Count the proportions of cell subpopulations in the four quadrants marked by CD44 and CD62L in Figure A, CD44 low CD62L hi It is a memory stem T cell phenotype.
  • FIG. 8 The proliferation of CD44 low CD62L high memory stem T cells induced by mPDIL21.
  • T cells from OT-1 transgenic mice were labeled with CFSE dye in vitro.
  • flow cytometry AT cell proliferation; B.CD44 , CD62L expression and analysis of CD44 low CD62L high , CD44 high CD62L high and CD44 high CD62L low three cell subgroups of CFSE-labeled cell proliferation levels.
  • CD44 low CD62L high memory stem T cells induced by mPD-1Ab21 are differentiated from activated CD44 high CD62L high cells.
  • Lymph node T cells of A.OT-1 mice are labeled with CFSE in vitro and then OVA is used.
  • the polypeptide is activated. After 42 hours of activation, the activated T cells were sorted by flow cytometry to obtain a subset of CD44 high and CD62L high ; B.
  • the T cells obtained after sorting were differentiated in vitro by adding IL-2 and mPD-1Ab21 respectively, and the third Day, flow cytometry to detect the expression of CD44 and CD62L and analyze the proliferation of three cell subgroups : CD44 low CD62L high , CD44 high CD62L high and CD44 high CD62L low.
  • FIG. 10 Cytokine secretion levels of memory stem T cells induced by mPD-1Ab21 after antigen re-stimulation.
  • Original mouse OT-1 T cells and OT-1 transgenic mouse T differentiated with mPD-1Ab21 and IL-2 for three days.
  • the cells were first resting with IL-7 cytokine for two days, and then added to a cell culture plate that had been pre-platen (anti-CD3 antibody 0.5 ⁇ g/ml). After 12 hours of anti-CD3 antibody stimulation, BFA was added to block for 4 hours, and then the expression levels of IFN- ⁇ and IL-2 in the cells were detected by flow cytometry.
  • FIG. 11 Survival of stem memory cells induced by mPD-1Ab21 in vivo A. Take CD90.1+OT-1 mouse lymph node T cells and activate them in vitro with OVA peptide at a concentration of 0.02ng/ml. After 40 hours of activation, IL-2 (10ng/ml) and mPD-1Ab21 (25ng/ml) were added for differentiation. The three-day-differentiated cells were injected through the tail vein into C57 mice that had been irradiated with Co60 (4Gy). On the day of injection, complete adjuvant mixed with OVA peptide (50 ⁇ g) was used for subcutaneous immunization, and IL-2 (5 ⁇ g/time) was injected intraperitoneally for seven consecutive days.
  • mice 35 days after the injection of T cells into the tail vein, kill the mice, take their spleens, count their cells, and then flow cytometry to detect the ratio of CD90.1+T cells in the spleen to calculate the CD90.1+T in the spleen The absolute number of cells.
  • mPD-1Ab21 targets PD-1 + T cells to promote the differentiation of memory stem T cells.
  • A. Take OT-1 mouse lymph node T cells and activate them with OVA peptide (0.02ng/ml) in vitro. After 40 hours of activation, different concentrations of mIL-21 and mPD-1Ab21 were added for differentiation. On the third day of differentiation, the expression of CD44 and CD62L of CD8 + T cells was detected by flow cytometry. Calculate the proportion of CD44 low CD62L high cell subsets.
  • B Prepare T cells from wild-type C57 mice and PD-1 knockout mice, respectively, and activate them with anti-CD3 and CD28 antibodies in vitro.
  • FIG 13.mPD-1Ab21 rapid targeting tumor-specific T cells subcutaneously C57 mice tumor tissues and peripheral blood activated cells were seeded 1x10 6 melanoma B16-OVA, day before inoculation intravenous injection of 1x10 6 cells original OT-1T.
  • mice On the sixth day after inoculation, mice were subcutaneously immunized with polyI:C (50 ⁇ g) + OVA peptide (50 ⁇ g); on the ninth day, mice were injected with protein (antibody 200 ⁇ g, protein 100 ⁇ g); blood and tumor were taken at different time points, flow cytometry Technical analysis of the expression of PD-1 molecules on the surface of OT-1T cells.
  • mPD-1Ab21 inhibits the growth of colorectal cancer tumor cells CT26.
  • Balb/c mice were subcutaneously inoculated with 2x10 6 colorectal cancer cells CT26.
  • antibodies were injected intraperitoneally for treatment.
  • the anti-PD-1 antibody group was injected with 200 ⁇ g three times a week.
  • the mPD-1Ab+mIL-21 group and mPD-1Ab21 group were injected once every other day, a total of four times, each 150 ⁇ g.
  • FIG. 15 Combination therapy of mPD-1Ab21 and anti-Her2/neu antibody significantly inhibited the growth of Her2 + Tubo tumors.
  • Balb/c mice were subcutaneously inoculated with 1x10e6 breast cancer cells Tubo.
  • the mPD-1Ab+mIL-21 group and mPD-1Ab21 group were injected intraperitoneally from the fifteenth day, once every other day, a total of four times, each 150 ⁇ g.
  • Anti-Her2/neu antibody was injected on the 16th and 19th day, 200 ⁇ g each time.
  • FIG 16.mPD-1Ab21 combination therapy and tumor vaccine significantly inhibited the growth of B16-OVA melanoma C57 mice were inoculated subcutaneously with 1x10 6 melanoma B16-OVA, day before inoculation intravenous injection of 2x10 6 cells original OT-1T. On the sixth day after inoculation, mice were subcutaneously immunized with polyI:C (50 ⁇ g) + OVA peptide (50 ⁇ g). On the ninth day, intraperitoneal injection of protein was started, and anti-PD-1 was injected every two days, a total of three times, 200 ⁇ g each time. mPD-1Ab+mIL-21 and mPD-1Ab were injected every other day, a total of five times, each 150 ⁇ g. The B16-OVA tumor size was measured at different time points, tumor volume (length ⁇ width ⁇ width)/2.
  • mPD-1Ab21 treatment significantly increased the proportion of memory stem CD8 + T cells and tumor-specific T cell response A.
  • Balb/c mice were subcutaneously inoculated with 2x10 6 colorectal cancer cells CT26, the fourth day of anti-PD-1 group On the seventh day, the antibody was injected intraperitoneally, 200 ⁇ g each time.
  • the mPD-1Ab21+mIL-21 group and the mPD-1Ab21 group were injected intraperitoneally with protein from the fourth day, once a day for a total of seven times, each 100 ⁇ g each.
  • the mice were killed on the 11th day, and the draining lymph nodes and spleens of the mice were taken to prepare a single cell suspension.
  • mice were inoculated with melanoma B16-OVA.
  • 2 ⁇ 10 6 primitive OT-1 T cells were injected into the tail vein.
  • mice were subcutaneously immunized with polyI:C (50 ⁇ g) + OVA peptide (50 ⁇ g).
  • mPD-1Ab21 was once a day for seven times, 100 ⁇ g each time.
  • Anti-PD-1 was injected on the 10th and 14th day, 200 ⁇ g each time. On the 17th day, the mice were killed, and the draining lymph nodes, spleen and tumor were taken separately to prepare a single cell suspension. Flow cytometry detected the ratio of OT-1 T cells to CD45 + cells in different tissues ( Figure B), and calculated the ratio of CD127 low KLRG1 high CD8 + T cells to CD127 high KLRG1 low CD8 + T in tumors ( Figure C).
  • IL-2 Cytokine Recombinant human IL-2 Company: peprotech Item No.: 200-02
  • IL-7 Cytokine Recombinant human IL-7 Company: peprotech Item No.: 200-07
  • PolyI C: company sigma; article number I3036-20mg
  • pTT3 expression vector donated by Professor Liu Yingfang
  • Baf3 cells purchased from BeiNa Chuanglian Biotechnology Co., Ltd.
  • Her2/neu+ breast cancer Tubo cells donated by Professor Fu Yangxin
  • Ovalbumin (OVA) antigen peptide (SIINFEKL) specific T cell receptor (TCR) transgenic mice donated by Professor Wang Hongyang.
  • C57 mouse C57BL/6 purchased from Weitong Lihua
  • Balb/c mice Balb/c purchased from Weitong Lihua
  • OVA antigen peptide SIINFEKL (synthesized by Shenggong Biological Company)
  • Anti-Flag beads M2 Affinity Gel Sigma A2220
  • Anti-mouse PD-1 antibody Obtained from the culture supernatant of anti-mouse PD-1 antibody hybridoma cells (G4) and purified with a Protein G column.
  • Protein G protein purification column Company: GE Item No.: 17-0404-01
  • Anti-CD3 antibody Leaf purified anti-mouse CD3 Company: biolegend Item No.: 100331
  • Anti-CD28 antibody Leaf purified anti-mouse CD28 Company: biolegend Item No.: 102111
  • Anti-Her2/neu antibody Obtained from the culture supernatant of anti-mouse her2 antibody hybridoma cells (7.16.4) and purified by Protein G column
  • PE-anti-hIg Company ebioscience Article number: 14-4998-82BFA: Company: ebioscience Article number: 00-4506-51
  • Example 1 Construction of the fusion protein of anti-mouse PD-1 single chain antibody and mouse IL-21 (mPD-1Ab21)
  • the anti-mouse PD-1 antibody (G4) hybridoma cells were subjected to exon sequencing, and the following primers were designed according to the antibody heavy chain and light chain gene sequences, and the antibody heavy chain and light chain variable region coding genes were cloned by RT-PCR.
  • the primer sequence is as follows:
  • Anti-mouse PD-1 antibody heavy chain (H) upstream primer SEQ ID NO.1:
  • Anti-mouse PD-1 antibody heavy chain (H) downstream primer SEQ ID NO.2
  • Anti-mouse PD-1 antibody light chain (L) upstream primer SEQ ID NO.3:
  • Anti-mouse PD-1 antibody light chain (L) downstream primer SEQ ID NO.4:
  • the antibody heavy chain (H) and light chain (L) variable region coding genes are compared Connected to become an anti-PD-1 single-chain antibody mPD-1Ab coding gene sequence, the sequence is shown in SEQ ID NO.5.
  • linker composed of 36 amino acids, whose coding nucleotide sequence is as shown in SEQ ID NO.6.
  • mPD-1Ab21 anti-PD-1 single-chain antibody and IL-21 fusion protein (mPD-1Ab21), the sequence is shown in SEQ ID NO.8.
  • a 22 amino acid coding gene sequence of the tag protein 3xFlag is added to the C-terminus of the obtained fusion protein.
  • the nucleotide sequence of 3xFlag is shown in SEQ ID NO.9.
  • mice mPD-1Ab single-chain antibody and mPD-1Ab21 fusion protein constructed in Example 1 was loaded into pTT3 expression vector, 293T cells were transfected, cell culture supernatant was collected, and purified with anti-Flag beads to obtain small Mouse mPD-1Ab and mPD-1Ab21 proteins.
  • mice Immunize mice with human PD-1-IgFc fusion protein (PD-1: NCBI Reference Sequence: NM_005018.2, IgFc: Gene ID: 3500), and obtain a strain that produces high-affinity anti-human PD-1 antibody through hybridoma technology screening Hybridoma cell 1G1.
  • PD-1 NCBI Reference Sequence: NM_005018.2, IgFc: Gene ID: 3500
  • IgFc Gene ID: 3500
  • the results show that the anti-PD-1 antibody specifically binds to CHO cell lines overexpressing PD-1, and can completely block the binding of PD-L1-IgFc to PD-1 on the cell surface.
  • Anti-human PD-1 antibody heavy chain (H) upstream primer M is a degenerate primer (SEQ ID NO.11):
  • Anti-human PD-1 antibody heavy chain (H) downstream primer SEQ ID NO.12:
  • Anti-human PD-1 antibody light chain (L) upstream primer M/R is a degenerate primer (SEQ ID NO.13):
  • Anti-human PD-1 antibody light chain (L) downstream primer SEQ ID NO.14:
  • the genes encoding the variable regions of the antibody heavy and light chains were cloned from anti-PD-1 antibody hybridoma cells (1G1) (see Figure 4B), and the sequences are shown in SEQ ID NO.15 and SEQ ID NO.16.
  • the sequence is shown in SEQ ID NO.17, and the light chain sequence is shown in SEQ ID NO.18.
  • the C-terminus of the heavy chain is linked with the human IL-21 shown in SEQ ID NO.19 to construct a humanized anti-PD-1 antibody heavy chain and IL-21 fusion protein.
  • the nucleotide sequence of the fusion protein is shown in SEQ ID NO. .20.
  • the synthesized anti-PD-1 antibody heavy chain and IL-21 fusion protein and light chain genes were respectively constructed into pTT3 expression vector, and CHO cells were co-infected.
  • the anti-PD-1 antibody and IL-21 fusion protein were purified by proteinG column ( Also known as PD-1Ab21). See Figure 4A.
  • mPD-1Ab21 can bind to PD-1 molecules expressed on the cell surface, and this binding can be completely blocked by anti-PD-1 antibodies.
  • Figure 5A shows that mPD-1Ab21 is as capable as anti-PD-1 antibodies. It binds to PD-1 expressed on the cell surface and can block the binding of PD-L1 to PD-1 on the cell surface.
  • Figure 5B the results show that mPD-1Ab21 can bind to activated T lymphocytes and can block the binding of PD-L1 to PD-1 on the surface of activated T lymphocytes.
  • Binf3 cells are pre-B cell lines
  • Mouse IL-21 and mPD-1Ab21 protein are added to the corresponding cell wells according to different concentration gradients: 0.1ng/ml, 1ng/ml, and 10ng/ml. Each concentration has three multiple wells, and no protein is added. Wells serve as control wells.
  • Example 5 It can be seen from Example 5 and Example 6 that the mPD-1Ab21 fusion protein prepared by the present invention not only has the effect of anti-PD-1 antibody to block the binding of PD-L1 and PD-1, but also has the biological activity of IL-21 .
  • Example 7 mPD-1Ab21 induces the differentiation of memory stem CD8 + T cells
  • the present invention further studied the effect of mPD-1Ab21 on the differentiation of activated CD8 + T cells.
  • the OT-1 transgenic mouse T cells were used to stimulate the activation of OVA antigen peptides in vitro for 48 hours, and then the cells were activated in the presence of IL-2, mIL-21, and mIL-21, respectively.
  • the differentiation of mPD-1Ab or mPD-1Ab21 was cultured in the medium of mPD-1Ab21. After 2 days, the differentiation of T cells was analyzed by flow cytometry.
  • the results are shown in Figure 7.
  • the results show that IL-2 promotes the differentiation of activated CD8 + T cells into CD44 high CD62L low effector T cells or memory effector T cells, and mIL-21 induces activated CD8 + T cells to differentiate into CD44 low CD62L high memory stem T cell differentiation, mPD-1Ab alone has no effect on the differentiation of activated CD8 + T cells.
  • mPD-1Ab combined with mIL-21 has the same induction of CD44 low CD62L high memory stem T cell differentiation as mIL-21 alone MPD-1Ab21 has a stronger biological effect of inducing the differentiation of D44 low CD62L high memory stem T cells.
  • Example 8 mPD-1Ab21 induces the differentiation of CD44 low CD62L high memory stem T cells by promoting cell proliferation
  • CD44 low CD62L high memory stem T cells induced by mPD-1Ab21 are derived from the original without activated proliferation.
  • CD44 low CD62L high T cells, or activated T cells are induced to differentiate into CD44 low CD62L high memory stem T cells by the fusion protein mPD-1Ab21.
  • OT-1 transgenic mouse T cells We labeled OT-1 transgenic mouse T cells with CSFE, and then activated them. Differentiated under different conditions, the results are shown in Figure 8.
  • the proliferation of the CD44 low CD62L high cell population differentiated in the simple medium is significantly lower than that of the CD44 high CD62L high T cell population.
  • the CD44 low CD62L high cell population induced by mPD-1Ab21 also has significant proliferation, which is indistinguishable from the proliferation of CD44 high CD62L high T cell population. It shows that mPD-1Ab21 induced differentiation of CD44 low CD62L high cells is derived from activated cells through proliferation and differentiation.
  • Example 9 mPD-1Ab21 induces the differentiation of activated CD44 high CD62L high cells back to the CD44 low CD62L high primitive cell phenotype
  • CD44 low CD62L high cells induced by mPD-1Ab21 were differentiated from activated CD44 high CD62L high T cells and returned to the CD44 low CD62L high cell population
  • CFSE-labeled OT-1 transgenic mouse T cells with OVA antigen After the peptide is activated, the activated CD44 high CD62L high T cell population is purified by flow sorting, and then cytokine or fusion protein is added for differentiation.
  • mPD-1Ab21 or IL-2 to induce differentiation of OT-1 transgenic mouse T cells for 2-3 days in a medium containing IL-7 Cultured in medium for 2 days, then stimulated with anti-CD3 to detect cytokine production, see Figure 10, the results show: IL-2 induced differentiated cells produced a large amount of IFN- ⁇ , almost no IL-2; mPD-1Ab21 induced differentiated cells A large amount of IL-2 is produced, and the expression of IFN- ⁇ is significantly lower than that of IL-2 induced differentiation cells. It shows that IL-2 induced differentiated cells have strong effector function, while mPD-1Ab21 induced differentiated cells have stronger proliferation and differentiation ability.
  • Example 11 Memory stem T cells induced by mPD-1Ab21 have stronger memory response and ability to form memory cells in mice
  • mice were injected subcutaneously with OVA antigen peptide and Freund's complete adjuvant for antigen stimulation, and then monitored the response of OT-1 transgenic mouse T cells in mice.
  • OVA antigen peptide and Freund's complete adjuvant for antigen stimulation were injected subcutaneously with OVA antigen peptide and Freund's complete adjuvant for antigen stimulation, and then monitored the response of OT-1 transgenic mouse T cells in mice.
  • the results showed that mPD-1Ab21 induced differentiated T cell responses after being stimulated by antigen. It is 4 times that of IL-2 induced differentiated T cell response.
  • mPD-1Ab21 targets PD-1 + T cells to promote the differentiation of CD44 low CD62L high memory stem T cells
  • Example 7 shows that the ability of mPD-1Ab21 to induce the differentiation of CD44 low CD62L high memory stem T cells is significantly higher than that of recombinant mIL-21 or the combination of mPD-1Ab and mIL-21.
  • the present invention analyzes and compares mPD-1Ab21 and mIL-21 to induce CD44 low CD62L high memory stem T cells. The dose-effect relationship of cell differentiation.
  • OT-1 transgenic mouse T cells were activated in vitro, and then different concentrations of recombinant mIL-21 or mPD-1Ab21 fusion protein were used to induce differentiation, and the ratio of CD44 low CD62L high cells was analyzed by flow cytometry.
  • the results show that the activity of mPD-1Ab21 fusion protein to induce the differentiation of CD44 low CD62L high cells is more than 3 times higher than that of mIL-21.
  • mPD-1Ab21 has a significantly higher ability to induce CD44 low CD62L high memory stem T cells to differentiate than recombinant IL-21; for PD-1 knockout mouse T cells The ability of mPD-1Ab21 to induce the differentiation of CD44 low CD62L high memory stem T cells is slightly better than that of recombinant IL-21. This result indicates that mPD-1Ab21 targets IL-21 to activated T cells by binding to PD-1 on the surface of activated T cells, thereby promoting the differentiation of CD44 low CD62L high memory stem T cells.
  • Example 13 mPD-1Ab21 quickly targets activated tumor-specific T cells in peripheral blood and tumor tissues
  • mice Inoculate mice with tumor cells expressing OVA antigen B16-OVA, inoculate mice with OVA-specific OT-1 primitive T cells one day before inoculation, and adjuvant polyI:C and OVA antigen peptides to mice on the sixth day after inoculation
  • mice were given intraperitoneal injection of mPD-1Ab21 or anti-PD-1 antibody on the ninth day.
  • flow cytometry analysis of the binding ability of tumor-specific T cells and anti-PD-1 antibodies in the peripheral blood and tumor infiltration to reflect in vivo The binding of mPD-1Ab21 protein or anti-PD-1 antibody to tumor-specific OT-1T cells.
  • the results show that the surface of activated OT-1T cells in the peripheral blood and tumors of the immunized mice highly express PD-1 molecules.
  • 30 minutes after intraperitoneal injection of anti-PD-1 antibody or mPD-1Ab21 protein the PD-1 flow cytometric antibody staining on the surface of OT-1T cells in the peripheral blood was blocked, indicating that both mPD-1Ab21 and anti-PD-1 antibodies injected into the body It can quickly bind to the surface of tumor-specific T cells.
  • the binding of fusion protein or antibody to tumor-specific T cells in tumor tissue is slower than that in peripheral blood.
  • the binding effect of the fusion protein and OT-1 T cells can last at least 24 hours, and only partial binding of anti-PD-1 antibodies can be observed in tumor tissues after 48 hours.
  • the results show that the mPD-1Ab21 fusion protein can quickly bind to the activated tumor-specific T cells in peripheral blood and tumor tissues after injection into the body.
  • mice Subcutaneously inoculate mice with mouse colorectal cancer tumor cells CT26. Three days later, mice were injected with mPD-1Ab21 fusion protein, mPD-1Ab combined with mIL-21 recombinant protein, or anti-PD-1 antibody for treatment, and the tumor size was measured regularly , Draw the tumor growth curve.
  • the results show that: compared with untreated control mice, the combined treatment of mPD-1Ab and mIL-21 only slightly slowed down the tumor growth in mice, while anti-PD-1 antibody and mPD-1Ab21 fusion protein The treatment can be 20% and 40% of the mouse tumors have completely resolved.
  • the results show that compared with the combination therapy of mPD-1Ab and mIL-21, mPD-1Ab21 has a more significant tumor treatment effect, and is superior to the therapeutic effect of anti-PD-1 antibody.
  • Example 15 Combination therapy of mPD-1Ab21 significantly increases the therapeutic effect of anti-Her2/neu (Herceptin) on Her2 + breast cancer
  • mice Inoculate mice subcutaneously with Her2/neu + breast cancer Tubo. After 16 days, mice were injected with anti-Her2/neu antibody, or anti-Her2/neu antibody combined with mPD-1Ab21 fusion protein, anti-Her2/neu antibody combined with mPD-1Ab and mIL -21 for treatment, regularly check the size of the tumor, and draw the tumor growth curve. See Figure 15, the results show that: anti-Her2/neu antibody treatment alone can make 40% of mouse tumors regression, anti-Her2/neu antibody combined with mPD-1Ab and mIL-21 treatment can make 60% of mouse tumors completely regression, anti-Her2/ The combination of neu antibody and mPD-1Ab21 treatment can completely regression of tumors in all mice. The results show that mPD-1Ab21 combination therapy can significantly increase the anti-Her2/neu (Herceptin) tumor treatment effect, which is significantly better than the combination therapy with mPD-1Ab and mIL-21.
  • Example 16 Combination therapy of mPD-1Ab21 significantly increases the therapeutic effect of tumor vaccine on B16-OVA tumor
  • Tumor vaccines are an important direction for cancer therapy in the future, especially in combination with PD-1 inhibitors.
  • the mPD-1Ab21 fusion protein uses PD-1 to target IL-21 to tumor-specific T cells activated by tumor vaccines, and promotes the formation of memory T cells, which will promote the tumor therapeutic effect of tumor vaccines more significantly than PD-1 inhibitors .
  • mice were inoculated with tumor cells expressing OVA antigen B16-OVA, the mice were inoculated with OVA-specific OT-1 primitive T cells one day before the inoculation, and the mice were given adjuvant polyI:C and polyI:C and Subcutaneous immunization of OVA antigen peptides.
  • mice were given intraperitoneal injection of mPD-1Ab21 fusion protein, mPD-1Ab combined with mIL-21 recombinant protein, or anti-PD-1 antibody for treatment, and tumor size was measured regularly and tumor growth was plotted. curve.
  • the results show that tumor antigen immunization of tumor-bearing mice can significantly inhibit tumor growth.
  • Combining mPD-1Ab and mIL-21, or anti-PD-1 antibody does not significantly increase the therapeutic effect of tumor vaccines.
  • Combined application with mPD-1Ab21 can greatly improve the anti-tumor effect of tumor vaccines.
  • the tumor vaccine treatment stimulated the activation and proliferation of tumor-specific T cells in the body, resulting in a significant increase in the proportion of tumor-specific CD8 + T cells in the spleen of tumor-bearing mice, especially the tumor.
  • Anti-PD-1 antibody combination therapy did not further increase the proportion of tumor-specific CD8+ T cells, but mPD-1Ab21 greatly improved the tumor-specific T cell response in vaccine-treated mice, resulting in the proportion of tumor-specific CD8+ T cells Reach 80% of the CD8 + T cells in the tumor.
  • anti-PD-1 antibody combination therapy promotes tumor-specific CD8 + T cells to differentiate into effector (CD127 low KLRG1 high ), while mPD-1Ab21 combination therapy significantly promotes tumors Specific CD8 + T cells differentiate to memory effect (CD127 high KLRG1 low ). See Figures 17B and 17C.
  • mPD-1Ab21 therapy can also promote memory anti-tumor T cell responses, thereby greatly improving tumor treatment effects.

Abstract

一种抗PD-1抗体与IL-21融合蛋白，所述融合蛋白既能阻断PD-L1与T细胞表面PD-1的结合，发挥抗PD-1抗体的肿瘤治疗作用，又能将IL-21靶向肿瘤特异性T细胞，诱导T细胞的分化，调控T细胞功能，所述融合蛋白具有显著增加抗PD-1抗体的肿瘤治疗效应的功能，具有广阔的医用治疗前景。

Description

一种靶向抗原特异性T细胞诱导其向记忆干性细胞分化的融合蛋白 技术领域
本发明一般涉及生物工程领域，尤其涉及一种靶向抗原特异性T细胞诱导其分化，增强其效应功能的融合蛋白。
背景技术
免疫系统的作用在于实现机体对于致病体的有效应答。T细胞在其中起到了关键的作用，正常细胞发生恶变成为癌细胞，能被机体免疫系统识别将其杀伤清除。机体内能杀伤和消除生长肿瘤的免疫细胞主要是T淋巴细胞。T淋巴细胞识别肿瘤抗原后，被激活，进行大量扩增，并分化为效应性T细胞，到达肿瘤部位攻击肿瘤细胞。但是，肿瘤细胞在长期发生进化过程中，产生多种机制来防止免疫细胞的攻击，例如肿瘤细胞高表达免疫抑制分子PD-L1，通过与T淋巴细胞表面的免疫抑制受体PD-1结合，就可以抑制T淋巴细胞对它的杀伤。
PD-1(也称为程序化细胞死亡-1)是CD28家族的一个成员，表达于活化的T细胞表面，其功能在于通过与配体PD-L1结合，抑制T细胞反应，防止T细胞过度反应造成组织损伤，是正常T细胞反应的一种负调控机制。肿瘤细胞通过高表达PD-L1,与T细胞表面的PD-1结合，抑制抗肿瘤T细胞对其杀伤，是肿瘤免疫逃逸的一种重要机制。科学家发明了一种抗PD-1抗体，可以阻断肿瘤细胞表面的PD-L1与T淋巴细胞表面的PD-1受体结合，解除肿瘤细胞对T淋巴细胞的抑制，功能恢复的T淋巴细胞就可以把肿瘤细胞杀死，清除体内肿瘤，这就是目前国际上广受关注的肿瘤治疗药——抗PD-1抗体。目前世界各国多家机构都在投入PD-1的研发当中，仅在美国每年就有数百个临床试验的项目在进行当中。而在全球范围内已有1600多个临床试验正在进行。截至目前由国际知名制药厂商所研发的PD-1/PD-L1药物已有5款已被美国FDA批准上市，包括：PD-1抑制剂Nivolumab(Opdivo)、Pembrolizumab(Keytruda)；PD-L1抑制剂Atezolizumab(Tecentriq)、Durvalumab(Imfinzi)和Avelumab(Bavencio)；用于黑色素瘤、头颈鳞状细胞癌、非小细胞肺癌、霍奇金淋巴瘤、肾癌、膀胱癌等癌症的治疗。国内有包括信达、恒瑞、百济神州、君实等至少100多家生物公司，也在进行PD-1阻断药物的研发和肿瘤治疗临床实验。尽管抗PD-1抗体药的肿瘤治疗取得巨大成功，但是对大多数晚期肿瘤的治疗有效率也只有20％左右，因此，如何提高抗PD-1抗体肿瘤治疗效果是肿瘤药物研发的一个重要方向。
一个淋巴细胞只能识别一种抗原表位，机体内有成千上万种T淋巴细胞，只有肿瘤特异性T淋巴细胞才能识别和杀伤特定的肿瘤细胞。T淋巴细胞分为CD4 +T细胞和CD8 +T细胞。体内识别和杀伤肿瘤细胞的主要是CD8 +T细胞，CD4 +T细胞的功能主要是辅助CD8+T细胞反应。静息的T淋巴细胞是没有活性的， 只有在抗原刺激下发生活化、增殖、分化成不同功能状态的T细胞，才能发挥不同的生物学功能。CD8 +T细胞根据分化状态可以分为原始T细胞(naive Tcells)、记忆干性T细胞(memory stemT cells)、记忆性T细胞(memoryTcells)和效应T细胞(effector T cells)。效应CD8 +T细胞是终末分化细胞，具有最强杀伤活性，但是体内存活时间短，因此，体内抗肿瘤作用最弱。记忆性CD8 +T细胞体内存活时间长，受到抗原刺激可以分化为效应性T细胞发挥作用，因而体内抗肿瘤作用显著增强。记忆干性CD8 +T细胞具有自我复制更新能力，可以在体内长期存活，受到抗原刺激可以分化为记忆性和效应性T细胞，体内抗肿瘤作用最强。T细胞的活化、增殖和分化这是一个复杂而精准调控的反应过程，其中，各种细胞因子在这一过程中发挥重要作用。
细胞因子(cytokine)是免疫原、丝裂原或其他刺激剂诱导多种细胞产生的低分子量可溶性蛋白质，具有调节免疫、血细胞生成、细胞生长以及损伤组织修复等多种功能。其中，白细胞介素在激活与调节免疫细胞，介导T、B细胞活化、增殖与分化及在炎症反应中起重要作用。由于最初是由白细胞产生又在白细胞间发挥作用，所以由此得名。白细胞介素体内的作用方式，主要是以细胞的自分泌和旁分泌在局部发挥重要调控作用和生物学效应，全身应用会导致严重的炎症反应等副作用。白细胞介素21(interleukin 21，简称IL-21)是一种主要有CD4 +T细胞产生，作用于CD8 +T细胞、B淋巴细胞、NK细胞、单核巨噬细胞等多种细胞的细胞因子。IL-21可以诱导活化的CD8 +T淋巴细胞分化成年轻、富有活力的细胞群体，即类似于记忆干性CD8 +T淋巴细胞。由于IL-21还可以作用其它多种免疫细胞和组织细胞，全身应用临床试验抗肿瘤效果不明显，而且长期应用会导致炎症等副作用。因此，IL-21用于肿瘤治疗的关键是如何将其靶向作用于肿瘤特异性T细胞。
PD-1表达于活化T细胞表面，被认为是抗原活化T细胞的标志。最新研究显示：肿瘤患者体内肿瘤特异性CD8 +T细胞均表达PD-1，体外通过PD-1 +细胞分选，可以富集肿瘤患者肿瘤组织或外周血中肿瘤特异性T细胞，用于T细胞过继治疗。基于以上研究发现，本发明提供了一种融合蛋白和利用该融合蛋白治疗癌症的方法。利用抗PD-1抗体可以靶向结合肿瘤特异性T淋巴细胞，在发挥其自身抗肿瘤作用的同时，将IL-21靶向肿瘤特异性T淋巴细胞，体内诱导肿瘤特异性T细胞向记忆(干性)T细胞分化，增加抗PD-1抗体的肿瘤治疗效果。本发明提供的融合蛋白超过单独使用单克隆抗体或细胞因子的效果，同时可以诱导CD8 +T细胞向记忆干性T细胞分化，此外，发明人惊奇的发现，在癌症的治疗效果上，使用本发明所制备的融合蛋白明显优于PD-1抑制剂与IL-21的联合用药。
发明内容
发明概述
本发明的目的是克服现有技术的这些缺点。具体地，本发明的目的是提供一种将IL-21靶向肿瘤特异性T淋巴细胞，并具有抗PD-1抗体的肿瘤治疗效果的融合蛋白及其相关的应用。
抗PD-1抗体是目前国际上最受瞩目的抗肿瘤药，众多药物公司在竞相研发。抗PD-1抗体的肿瘤治疗机制是通过阻断肿瘤细胞表面的免疫抑制分子PD-L1与T淋巴细胞表面的免疫抑制受体PD-1结合，解 除肿瘤细胞对T淋巴细胞的抑制，恢复肿瘤特异性T淋巴细胞的功能，杀死肿瘤细胞，清除体内肿瘤。患者体内活化的肿瘤特异性T淋巴细胞处于不同的分化阶段，如：终末分化效应性T细胞效应记忆性T细胞(effect memory T cells)、中枢记忆性T细胞(central memory T cells)和记忆干性T细胞。其中，记忆干性T细胞具有自我更新和繁殖的功能，同时又能分化成不同的下游各种效应性T细胞，因而在体内具有最强抗肿瘤作用。本发明发现，IL-21可以诱导活化的CD8 +T淋巴细胞分化为记忆干性T淋巴细胞。
本发明的研发动机，是将抗PD-1抗体与IL-21链接融合在一起，成为一个新的融合蛋白，在发挥其阻断肿瘤细胞表面的PD-L1与T淋巴细胞表面PD-1结合，恢复T淋巴细胞功能的同时，将IL-21靶向T淋巴细胞，增强T淋巴细胞抗肿瘤的功能。
以上只是概括了本发明的一些方面，不是也不应该认为是在任何方面限制本发明。
本说明书提到的所有专利和出版物都是通过参考文献作为整体而引入本发明的。
发明的详细说明
除非另外指明或定义，所用的所有术语具有本领域常用的意思，这是技术人员所清楚的。例如，参考标准手册，如Sambrook等，″Molecular Cloning：A Laboratory Manual(分子克隆：实验室手册)″(第2版)，卷1-3，Green Publishing and Wiley Interscience，纽约(1987)；Lewin，“Genes II(基因II)”，John Wiley&Sons，纽约，N.Y.，(1985)；Old等.，“Principles of Gene Manipulation：An Introduction to Genetic Engineering(基因操作原理：基因工程入门)”，第2版，University of California Press(加利福尼亚大学出版社)，Berkeley，CA(1981)；Roitt等.，“Immunology(免疫学)”(第6版)，Mosby/Elsevier，Edinburgh(2001)；Roitt等.，Roitt’s Essential Immunology(Roitt’s基础免疫学)，第10版.Blackwell Publishing，英国(2001)；和Janeway等.，“Immunobiology(免疫学)”(第6版)，Garland Science Publishing/Churchill Livingstone，纽约(2005)，以及其中所引用的公知背景技术；
此处所用的术语“氨基末端”和“羧基末端”是指多肽内的位置。在上下文允许的情况下，针对多肽的特定序列或部分，这些术语用于表示临近或相对位置。例如，位于多肽内参照序列的羧基末端的某一序列位于靠近该参照序列的羧基端，但不一定位于完整多肽的羧基端。
此处所用的术语“癌症”或“癌细胞”是指在生物中发现的、具有使其和正常组织或组织细胞区分开的特征的组织或细胞。在这些特征中包括但不限于：退行性变化的程度、形状上的不规则、细胞轮廓的不可分辨性、细胞核大小、细胞核或细胞质的结构上的改变、其他表型改变、预示癌症或前癌状态的细胞蛋白的存在、增加的有丝分裂次数和转移的能力。和“癌症”有关的术语包括癌、肉瘤、肿瘤、上皮癌、白血病、淋巴癌、息肉，以及硬癌、转化等。
此处所用的术语“组合治疗”是指给受治疗者施用至少一种治疗有效剂量的融合蛋白组分(包括但不限于“抗PD-1抗体与IL-21融合蛋白(PD-1Ab21)”)。所述融合蛋白可以是具有“将IL-21靶向肿瘤特异性T淋巴细胞，增加抗PD-1抗体的肿瘤治疗效果”的生物学活性的成熟的多肽、其片段、融合物或缀合物。
“记忆干性CD8 +T细胞”是指活化的CD8 +T细胞处于分化的早期阶段，具有自我复制更新能力，可以在体内长期存活，受到抗原刺激可以分化为记忆性和效应性T细胞。其细胞表型类似于原始
Figure PCTCN2019117035-appb-000001
CD8 +T细胞，在小鼠表现为CD44 lowCD62L high，在人表现为CD45RA +CCR7 +CD45RO -，同时，又表达IL-2R、Bcl-2、Scal-1或CD95。
“序列”在用于本文时(例如，在术语如“免疫球蛋白序列”，“抗体序列”，“结构域序列”或“蛋白序列”中)通常应该理解为包括相关的免疫球蛋白序列以及编码其的核酸序列或核苷酸序列二者，除非上下文需要更局限性的解释。
“核酸分子”的引用可以涉及用于基因疫苗接种的核酸，或编码本发明的免疫球蛋白序列的核酸，或二者，这将通过具体的上下文变得清楚。
本发明涉及可以结合本文定义的抗原和/或具有针对本文定义的抗原的亲和性的免疫球蛋白序列。在本发明的情形中，“结合某种抗原和/或具有针对某种抗原的亲和性”具有本领域中常用的意思，例如，如在抗体及其各自的抗原的情形中所理解的意思。
在本发明的具体实施方案中，术语“结合”意指所述免疫球蛋白序列特异性与抗原相互作用。
本发明的核酸可以以本身已知的方法制备或获得，其基于关于本发明的细胞相关抗原或免疫球蛋白序列的信息，和/或可以从适当的天然来源分离。
本发明的核酸还可以是这样的形式，存在于和/或是遗传构建体的一部分，这对于本领域的技术人员应该是清楚的。这样的遗传构建体通常包括至少一种本发明的核酸，其任选地与本身已知的一个或多个遗传构建体元件连接，诸如例如一个或多个适宜的调节元件(诸如适当的启动子、增强子、终止子、等等)和本文提到的其它构建体元件。
关于可以存在于/用于本发明的遗传构建体的启动子、选择标记、前导序列、表达标记和其它元件的一些(其它)非限制性的实例——诸如终止子、转录和/或翻译增强子和/或整合因子——参考通用手册，诸如Sambrook等和Ausubel等。
通常，本发明的遗传构建体通过将本发明的核苷酸序列插入到本身已知的适当的(表达)载体中而获得。适当的表达载体的一些优选的但非限制性的实例是下述实施例中所用的那些，以及本文提及的那些。
本发明的核酸和/或本发明的遗传构建体可以用于转化宿主细胞或宿主生物体，即，用于表达和/或生产本发明的融合蛋白。适当的宿主或宿主细胞是专业技术人员所清楚的，并且例如可以是任何适当的真菌的、原核的或真核的细胞或细胞系，或者任何适当的真菌的、原核的或真核的生物体。
特别地，本发明在一些具体的方面中提供下述：
本发明涉及一种融合蛋白，包含通过连接子相连接的细胞因子和T细胞表面受体分子结合拮抗剂或激动剂，其中，所述细胞因子是选自IL-21、IL-2、IL-15、IL-7或IL-12中的一种；所述T细胞表面受体分子结合拮抗剂或激动剂选自PD-1结合拮抗剂，LAG-3结合拮抗剂，Tim-3结合拮抗剂或CD137(4-1BB)结合激动剂中的一种，优选的，所述融合蛋白能够诱导激活T细胞，同时将细胞因子靶向T细胞，诱导T细胞分化，增强T细胞功能。
在一个具体的实施例中，PD-1结合拮抗剂抑制PD-1对PD-L1的结合；和/或所述PD-1结合拮抗剂抑制PD-1对PD-L1和PD-L2二者的结合；优选的，所述PD-1结合拮抗剂是抗体；更优选的，所述PD-1结合拮抗剂是单链抗体；进一步优选的，所述PD-1结合拮抗剂为二聚体单链抗体。
在本发明的另一个技术方案中，公开了一种融合蛋白，其结构域从氨基末端到羧基末端包含如下结构：-X-Linker1-Y-其中，X是PD-1结合拮抗剂，Y是细胞因子，Linker1是连接子；优选的，所述PD-1结合拮抗剂是抗体，细胞因子是IL-21；更优选的，所述PD-1结合拮抗剂从氨基末端到羧基末端包含如下结构：V H-Linker2-V L其中，V H为PD-1单链抗体重链的可变区，V L为抗体轻链的可变区，Linker2为连接子；进一步优选的，所述融合蛋白在3’端还可以包括标签序列。
本发明还公开了一种融合蛋白，其中，PD-1结合拮抗剂，其核苷酸序列如SEQ ID NO:5或SEQ ID NO:17所示；和/或所述细胞因子为IL-21，其核苷酸序列如SEQ ID NO:7或SEQ ID NO:19所示；优选的，所述融合蛋白阻断PD-LI与T细胞表面PD-1的结合，并将IL-21靶向肿瘤特异性T细胞，诱导T细胞的分化。
在本发明的一个具体实施例中，涉及一种融合蛋白，所述编码融合蛋白的核苷酸序列如SEQ ID NO:8或SEQ ID NO:20所示；进一步优选的，所述融合蛋白在3’端还可以包括标签序列，所述含有标签序列的融合蛋白的核苷酸序列如SEQ ID NO:10或SEQ ID NO:21所示；优选的，所述融合蛋白主要是同源二聚体形式。
此外，本发明还涉及一种多核苷酸，其编码本发明所述的融合蛋白。以及一种表达载体，其包含与表达控制序列操作性连接的上的多核苷酸。同时，本发明还公开了一种宿主细胞，其包含本发明涉及的表达载体。
在本发明的一个具体实施例中个，公开了一种融合蛋白的制备方法，包括如下步骤：a)通过基因工程手段方法，将细胞因子编码基因链接到PD-1结合拮抗剂编码基因的C末端，成为融合蛋白的编码基因；b)构建表达载体；c)转染细胞，进行蛋白表达和纯化；d)获得所述融合蛋白。
此外，本发明还公开了一种药物组合物，其包含一种或多种融合蛋白，以及药学上可接受的载体、稀释剂或赋形剂；和/或，所述药物组合物还包括其他抗癌药物或肿瘤疫苗，所述融合蛋白可以和其他抗癌药物或肿瘤疫苗联合使用；优选的，所述其他抗癌药物为Her2抗体；更优选的，所述其他抗癌药物为赫赛汀；
另外，本发明还涉及一种用于制备预防或治疗癌症药物或试剂盒的应用，其包括施用对该个体有效量的融合蛋白；优选的，所述癌选自由肾细胞癌，膀胱癌，非小细胞肺癌，头颈鳞状细胞癌，鳞状非小细胞肺癌，非鳞状非小细胞肺癌，肠癌，霍奇金淋巴瘤，结直肠癌，黑色素瘤，卵巢癌，乳腺癌，激素受体阳性乳腺癌，Her2阳性乳腺癌，和三重阴性乳腺癌组成的组；更优选的，连续或间歇施用本发明涉及的融合 蛋白；进一步优选的，通过静脉内，肌肉内，皮下，表面，口服，经皮，腹膜内，眶内，通过植入，通过吸入，鞘内，室内，或鼻内施用该融合蛋白。
在本发明的具体实施例中，还涉及一种用于在个体中预防或治疗癌症的方法，其包括施用对该个体有效量的融合蛋白；优选的，所述癌选自由肾细胞癌，膀胱癌，非小细胞肺癌，头颈鳞状细胞癌，鳞状非小细胞肺癌，非鳞状非小细胞肺癌，肠癌，霍奇金淋巴瘤、结直肠癌，黑色素瘤，卵巢癌，乳腺癌，激素受体阳性乳腺癌，Her2阳性乳腺癌，和三重阴性乳腺癌组成的组；更优选的，连续或间歇施用融合蛋白；进一步优选的，通过静脉内，肌肉内，皮下，表面，口服，经皮，腹膜内，眶内，通过植入，通过吸入，鞘内，室内，或鼻内施用该融合蛋白。
附图说明
图1.抗鼠PD-1单链抗体与小鼠IL-21融合蛋白(mPD-1Ab21)的构建示意图 A.抗鼠PD-1单链抗体与小鼠IL-21融合蛋白(mPD-1Ab21)的DNA序列；B.抗鼠PD-1单链抗体与小鼠IL-21融合蛋白(mPD-1Ab21)的示意图，其中，V H和V L分别为抗鼠PD-1单链抗体的重链和轻链的可变区，V H和V L以及IL-21之间分别由两个不同长度的linker连接，3xFlag位于3’端；C.抗鼠PD-1单链抗体与小鼠IL-21形成的融合蛋白(mPD-1Ab21)的构象示意图。
图2.小鼠mPD-1Ab21融合蛋白的表达纯化和鉴定 A.SDS-PAGE蛋白胶对纯化的小鼠PD-1单链抗体(mPD-1Ab)和小鼠抗PD-1单链抗体与IL-21融合蛋白(mPD-1Ab21)的纯度进行鉴定；B.将纯化的mPD-1Ab21融合蛋白过分子筛层析柱，结果显示：mPD-1Ab21融合蛋白主要以二聚体形式存在。
图3.抗人PD-1单克隆抗体鉴定 A.通过杂交瘤技术筛选单克隆抗人PD-1抗体，得到高表达PD-1抗体的1G1单克隆；将培养1G1细胞的上清与表达PD-1的CHO细胞共孵育30min，流式检测1G1上清与CHO细胞上的PD-1结合的能力；B.将CHO细胞与PD-L1-IgFc共孵育30min，或者先加入1G1上清与CHO细胞孵育30min，然后再加入PD-L1-IgFc，流式检测1G1上清阻断PD-1与PD-L1-IgFc结合的能力。
图4.抗人PD-1抗体和人IL-21融合蛋白(PD-1Ab21)的构建示意图和功能鉴定 A.抗人PD-1抗体和人IL-21融合蛋白(PD-1Ab21)的构建示意图。V H为鼠源PD-1抗体重链的可变区，V L为鼠源PD-1抗体轻链的可变区，以及人源抗体的恒定区和Fc段。3’端为人IL-21；B.抗人PD-1抗体的V H和V L的序列；C.稳定表达PD-1的CHO细胞与PD-1Ab21蛋白于4℃共孵育30min，然后流式检测PD-1Ab21与PD-1结合的能力；D.将CHO细胞与PD-L1-IgFc共孵育30min(黑色实线)，或者先将PD-1Ab21与CHO细胞孵育30min，然后再加入PD-L1-IgFc(虚线)，流式检测1G1上清阻断PD-1与PD-L1-IgFc结合的能力。
图5.mPD-1Ab21融合蛋白的体外结合和阻断实验 A.流式技术检测mPD-1Ab21与PD-1结合以及mPD-1Ab21阻断PD-1与其配体PD-L1IgFc结合的能力，A左图：EG7细胞(PD-1 +)与mPD-1Ab21蛋白于4℃条件下共孵育30min(黑色实线)，或EG7细胞先与抗PD-1抗体于4℃孵育30min，再与mPD-1Ab21蛋白孵育30min(黑色虚线)，不加mPD-1Ab21的为阴性对照(灰色阴影)，APC-抗Flag流式抗体检测mPD-1Ab21与PD-1结合的能力，A右图：EG7细胞与mPD-L1-IgFc蛋白于4℃条件下共孵育30min(黑色实线)，或者EG7细胞先与mPD-1Ab21蛋白孵育30min，再和mPD-L1-IgFc共孵育30min(黑色虚线)。 不加mPD-1Ab21为阴性对照(灰色阴影)，PE-抗hIg流式抗体检测mPD-1Ab21阻断PD-1与其配体mPD-L1IgFc结合的能力；B.取OT-1转基因小鼠(OVA抗原肽特异性T细胞受体转基因小鼠)淋巴结T细胞，体外用OVA肽(0.1ng/ml)刺激活化48小时，T细胞高表达PD-1分子。重复上述结合和阻断实验。
图6.mPD-1Ab21融合蛋白的IL-21生物学功能鉴定 Baf3细胞为pre-B细胞系。Baf3细胞铺于96孔板中，每孔10000个细胞。mIL-21和mPD-1Ab21蛋白按照不同的浓度梯度加入到相应的细胞中。细胞培养第三天，对Baf3细胞进行计数。细胞增殖率＝(实验组细胞数/对照组细胞数-1)x 100％
图7.mPD-1Ab21体外诱导CD44 lowCD62L high记忆干性T细胞分化 A.OT-1转基因小鼠T细胞，体外用OVA肽(0.02ng/ml)进行活化，活化34h后，加入等摩尔量的细胞因子或蛋白进行细胞分化，分化第三天，流式检测T细胞的分化表型；B.对图A中CD44和CD62L标记的四个象限的细胞亚群比例进行统计，CD44 lowCD62L hi为记忆干性T细胞表型。
图8.mPDIL21诱导的CD44 lowCD62L high记忆干性T细胞的增殖情况 使用OT-1转基因小鼠的T细胞，体外用CFSE染料进行标记。加入0.02ng/ml的OVA多肽活化47h后，再加IL-2(10ng/ml)和mPD-1Ab21(25ng/ml)进行分化，分化第二天，流式检测：A.T细胞增殖；B.CD44、CD62L的表达以及分析CD44 lowCD62L high，CD44 highCD62L high和CD44 highCD62L low三个细胞亚群的CFSE标记的细胞增殖水平。
图9.mPD-1Ab21诱导的CD44 lowCD62L high记忆干性T细胞是由活化的CD44 highCD62L high细胞分化而来 A.OT-1小鼠的淋巴结T细胞，体外用CFSE标记后，再用OVA多肽进行活化。活化42h后，流式分选活化的T细胞，得到CD44 highCD62L high的亚群细胞；B.分选后得到的T细胞，分别加入IL-2和mPD-1Ab21进行体外分化，分化的第三天，流式检测CD44、CD62L的表达以及分析CD44 lowCD62L high，CD44 highCD62L high和CD44 highCD62L low三个细胞亚群细胞增殖。
图10.mPD-1Ab21诱导的记忆干性T细胞受到抗原再刺激后细胞因子分泌水平 小鼠原始OT-1T细胞和用mPD-1Ab21及IL-2分别分化三天的OT-1转基因小鼠T细胞，先用IL-7细胞因子静息两天后，加入事先扣板(抗CD3抗体0.5μg/ml)的细胞培养板中。抗CD3抗体刺激12小时后，加入BFA阻断4小时，然后流式检测细胞中IFN-γ和IL-2的表达水平。
图11.mPD-1Ab21诱导的干性记忆性细胞在体内的存活情况 A.取CD90.1+OT-1小鼠淋巴结T细胞，体外用0.02ng/ml浓度的OVA肽进行活化。活化40h后，加入IL-2(10ng/ml)和mPD-1Ab21(25ng/ml)进行分化。将分化三天的细胞，通过尾静脉注射到经过Co60照射(4Gy)过的C57小鼠体内。注射当天用完全佐剂混合OVA肽(50μg)进行皮下免疫，同时连续七天腹腔注射IL-2(5μg/次)。在不同的时间点，眼眶取血，流式检测外周血中CD90.1+T细胞占CD45+细胞的比例。B.上述小鼠，T细胞尾静脉注射35天后，杀老鼠，取其脾脏，对其进行细胞计数，然后流式检测脾脏中CD90.1+T细胞的比例，计算脾脏中CD90.1+T细胞的绝对数。
图12.mPD-1Ab21靶向PD-1 +T细胞，促进记忆干性T细胞的分化 A.取OT-1小鼠淋巴结T细胞，体外 用OVA肽(0.02ng/ml)进行活化。活化40h后，再加入不同浓度的mIL-21和mPD-1Ab21进行分化。分化第三天，流式检测CD8 +T细胞的CD44和CD62L的表达。对CD44 lowCD62L high细胞亚群所占的比例进行统计。B.分别制备野生型C57小鼠和PD-1敲除小鼠的T细胞，体外用抗CD3和CD28抗体进行活化。活化39小时后，再加入不同浓度的mIL-21和mPD-1Ab21对T细胞进行分化。分化第四天，流式检测T细胞的CD44和CD62L的表达，对CD44 lowCD62L high细胞亚群所占的比例进行统计。
图13.mPD-1Ab21快速靶向外周血及肿瘤组织中活化的肿瘤特异性T细胞 C57小鼠皮下接种1x10 6黑色素瘤细胞B16-OVA，接种前一天尾静脉注射1x10 6原始OT-1T细胞。接种后第六天，polyI:C(50μg)+OVA肽(50μg)皮下免疫小鼠；第九天给小鼠腹腔注射蛋白(抗体200μg，蛋白100μg)；不同时间点取血和肿瘤，流式技术分析其中OT-1T细胞表面PD-1分子的表达。
图14.mPD-1Ab21抑制结直肠癌肿瘤细胞CT26的生长 Balb/c小鼠皮下接种2x10 6肠癌细胞CT26，接种后第三天，腹腔注射抗体进行治疗。抗PD-1抗体组每周注射三次，每次200μg。mPD-1Ab+mIL-21组和mPD-1Ab21组每隔一天注射一次，共四次，每次各150μg。在不同的时间点对CT26肿瘤大小进行测量，肿瘤体积＝(长×宽×宽)/2。
图15.mPD-1Ab21和抗Her2/neu抗体的联合治疗显著抑制Her2 +Tubo肿瘤的生长 Balb/c小鼠皮下接种1x10e6乳腺癌细胞Tubo。mPD-1Ab+mIL-21组和mPD-1Ab21组从第十五天开始腹腔注射，每隔一天注射一次，共四次，每次各150μg。抗Her2/neu抗体在第16天和第19天注射，每次200μg。在不同的时间点对Tubo肿瘤大小进行测量，肿瘤体积＝(长×宽×宽)/2。
图16.mPD-1Ab21和肿瘤疫苗联合治疗显著抑制黑色素瘤B16-OVA的生长 C57小鼠皮下接种1x10 6黑色素瘤细胞B16-OVA，接种前一天尾静脉注射2x10 6原始OT-1T细胞。接种后第六天，polyI:C(50μg)+OVA肽(50μg)皮下免疫小鼠。第九天开始腹腔注射蛋白治疗，anti-PD-1每隔两天注射一次，共三次，每次200μg。mPD-1Ab+mIL-21和mPD-1Ab每隔一天注射一次，共五次，每次各150μg。在不同的时间点测量B16-OVA肿瘤大小，肿瘤体积＝(长×宽×宽)/2。
图17.mPD-1Ab21治疗显著增加记忆干性CD8 +T细胞比例和肿瘤特异性T细胞反应 A.Balb/c小鼠皮下接种2x10 6结直肠癌细胞CT26,anti-PD-1组第四天和第七天腹腔注射抗体，每次200μg。mPD-1Ab21+mIL-21组及mPD-1Ab21组从第四天开始腹腔注射蛋白，每天一次共七次，每次各100μg。第11天杀老鼠，分别取小鼠的引流淋巴结和脾脏，制备单细胞悬液。流式检测不同组织中CD62L highCD44 lowCD122 +ScaI +T细胞占总CD8 +T细胞的比例。B&C.C57小鼠皮下接种1x10 6黑色素瘤细胞B16-OVA。接种前一天，尾静脉注射2x10 6原始OT-1T细胞。接种后第六天，polyI:C(50μg)+OVA肽(50μg)皮下免疫小鼠。第九天开始腹腔注射蛋白治疗，mPD-1Ab21每天一次共七次，每次100μg。anti-PD-1第10天和第14天注射，每次200μg。第17天杀老鼠，分别取引流淋巴结、脾脏和肿瘤，制备单细胞悬液。流式检测不同组织中OT-1T细胞占CD45 +细胞的比例(图B)，计算肿瘤中CD127 lowKLRG1 highCD8 +T细胞与CD127 highKLRG1 lowCD8 +T的比值(图C)。
具体实施方式
下面结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。但这些实 施例仅是范例性的，并不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本发明的保护范围内。
主要实验材料
IL-2细胞因子：recombinant human IL-2 公司:peprotech 货号：200-02
IL-7细胞因子：recombinant human IL-7 公司:peprotech 货号：200-07
注射用重组人白介素-2：沈阳三生制药有限责任公司
完全弗氏佐剂：公司sigma；货号F5881-10ml
PolyI:C:公司sigma；货号I3036-20mg
pTT3表达载体：受赠于刘迎芳教授
293T细胞：受赠予张叔人教授
CHO细胞：受赠于陈列平教授
EG7细胞：受赠于陈列平教授
Baf3细胞：购自北纳创联生物技术有限公司
PD-1KO小鼠：受赠于陈列平教授
Her2/neu+乳腺癌Tubo细胞：受赠于付阳新教授
OT-1转基因小鼠：卵清白蛋白(OVA)抗原肽(SIINFEKL)特异性T细胞受体(TCR)转基因小鼠，受赠于王红阳教授。
C57小鼠：C57BL/6    购自维通利华
Balb/c小鼠：Balb/c    购自维通利华
OVA抗原肽：SIINFEKL(由生工生物公司合成)
抗Flag beads：
Figure PCTCN2019117035-appb-000002
M2 Affinity Gel Sigma A2220
CSFE染料：公司：Sigma 货号：21888
抗鼠PD-1抗体(G4)：由抗鼠PD-1抗体杂交瘤细胞(G4)培养上清，用Protein G柱纯化获得。
Protein G蛋白纯化柱：公司:GE 货号:17-0404-01
抗CD3抗体：Leaf purified anti-mouse CD3ε 公司：biolegend 货号：100331
抗CD28抗体：Leaf purified anti-mouse CD28 公司：biolegend 货号：102111
抗Her2/neu抗体：由抗鼠her2抗体杂交瘤细胞(7.16.4)培养上清，用Protein G柱纯化获得
APC-抗Flag   公司:Biolegend   货号:637307
PE-抗hIg   公司:ebioscience   货号:14-4998-82BFA：公司:ebioscience   货号:00-4506-51
实施例1 抗鼠PD-1单链抗体与小鼠IL-21融合蛋白(mPD-1Ab21)的构建
将抗鼠PD-1抗体(G4)杂交瘤细胞进行外显子测序，根据抗体重链和轻链基因序列设计以下引物，通过RT-PCR克隆抗体重链和轻链可变区编码基因。引物序列如下：
抗鼠PD-1抗体重链(H)上游引物(SEQ ID NO.1)：
Figure PCTCN2019117035-appb-000003
抗鼠PD-1抗体重链(H)下游引物(SEQ ID NO.2)：
Figure PCTCN2019117035-appb-000004
抗鼠PD-1抗体轻链(L)上游引物(SEQ ID NO.3)：
Figure PCTCN2019117035-appb-000005
抗鼠PD-1抗体轻链(L)下游引物(SEQ ID NO.4)：
Figure PCTCN2019117035-appb-000006
克隆获得抗体重链(H)和轻链(L)可变区编码基因后，通过一段链接序列(linker)：GGGTCCAGTGGCGGT，将抗体重链(H)和轻链(L)可变区编码基因相连接，成为一个抗PD-1单链抗体mPD-1Ab的编码基因序列，序列如SEQ ID NO.5所示。
进一步，通过另一个由36个氨基酸组成的，其编码核苷酸序列如SEQ ID NO.6所示的链接序列linker。将获得的序列如SEQ ID NO.5所示PD-1单链抗体(mPD-1Ab)与序列如SEQ ID NO.7所示的小鼠IL-21编码基因序列链接。获得抗PD-1单链抗体与IL-21融合蛋白(mPD-1Ab21)，序列如SEQ ID NO.8所示。
为了后续蛋白纯化和检测，可选择的，在获得的融合蛋白C端加上一个由22个氨基酸组成标签蛋白3xFlag的编码基因序列，3xFlag的核苷酸序列如SEQ ID NO.9所示。
最终，构建获得编码小鼠融合蛋白(mPD-1Ab21)的编码基因，核苷酸序列如SEQ ID NO.10所示，参见图1。
实施例2 小鼠PDSIL21融合蛋白的表达和纯化
将实施例1中构建的编码小鼠mPD-1Ab单链抗体和mPD-1Ab21融合蛋白的编码基因装入pTT3表达载体，转染293T细胞，收集细胞培养上清，用抗Flag beads纯化，获得小鼠mPD-1Ab和mPD-1Ab21蛋白。
SDS-PAGF电泳证明，小鼠mPD-1Ab和mPD-1Ab21蛋白均为单一蛋白条带，如图2所示。分子体积排除色谱分析显示，mPD-1Ab21融合蛋白主要是二聚体形式。
实施例3 抗人PD-1抗体制备
用人PD-1-IgFc融合蛋白(PD-1:NCBI Reference Sequence:NM_005018.2,IgFc:Gene ID:3500)免疫小鼠，通过杂交瘤技术筛选获得一株产生高亲和力抗人PD-1抗体的杂交瘤细胞1G1。参见图3，结果显示该抗PD-1抗体特异性结合过表达PD-1的CHO细胞株，而且能完全阻断PD-L1-IgFc与细胞表面PD-1的结 合。
实施例4 抗人PD-1抗体与人IL-21融合蛋白(PD-1Ab21)的构建、表达和鉴定
通过RT-PCR技术，分别使用：
抗人PD-1抗体重链(H)上游引物，M为简并引物(SEQ ID NO.11)：
Figure PCTCN2019117035-appb-000007
抗人PD-1抗体重链(H)下游引物(SEQ ID NO.12)：
Figure PCTCN2019117035-appb-000008
抗人PD-1抗体轻链(L)上游引物，M/R为简并引物(SEQ ID NO.13)：
Figure PCTCN2019117035-appb-000009
抗人PD-1抗体轻链(L)下游引物(SEQ ID NO.14)：
Figure PCTCN2019117035-appb-000010
从抗PD-1抗体杂交瘤细胞(1G1)中克隆编码抗体重链和轻链可变区的基因(参见图4B)，序列如SEQ ID NO.15和SEQ ID NO.16所示。用克隆的抗PD-1抗体重链和轻链可变区替代人免疫球蛋白重链和轻链的相应片段，合成完整的编码人免疫球蛋白重链和轻链基因，构建嵌合重链序列如SEQ ID NO.17，轻链序列如SEQ ID NO.18所示。同时在重链C末端与SEQ ID NO.19所示的人IL-21链接，构建成人源化抗PD-1抗体重链与IL-21融合蛋白，融合蛋白的核苷酸序列如SEQ ID NO.20。
将合成的抗PD-1抗体重链与IL-21融合蛋白和轻链基因，分别构建到pTT3表达载体中，共传染CHO细胞，用proteinG柱纯化抗PD-1抗体与IL-21融合蛋白(也称为PD-1Ab21)。参见图4A。
检测PD-1Ab21与PD-1结合的能力，将稳定表达PD-1的CHO细胞与PD-1Ab21孵育30min，然后用抗Flag抗体检测细胞上结合的PD-1Ab21，结果显示PD-1Ab21能够很好结合细胞表面表达的PD-1，参见图4C。检测PD-1Ab21阻断PD-1与PD-L1的结合的能力，将CHO细胞与PD-L1-IgFc共孵育30min(黑色实线)，或者先将PD-1Ab21与CHO细胞孵育30min，然后再加入PD-L1-IgFc，用抗IgFc抗体检测细胞上结合的PD-L1-IgFc(虚线)。结果显示PD-1Ab21能完全阻断PD-1与PD-L1的结合，参见图4D。
实施例5 mPD-1Ab21融合蛋白功能鉴定
流式细胞分析证明，mPD-1Ab21可以结合细胞表面表达的PD-1分子，这种结合能被抗PD-1抗体完全阻断，参见图5A，说明mPD-1Ab21和抗PD-1抗体一样能结合细胞表面表达的PD-1，而且能阻断PD-L1与细胞表面PD-1结合。同样，参见图5B，结果显示，mPD-1Ab21能够结合活化的T淋巴细胞，能阻断PD-L1与活化T淋巴细胞表面PD-1的结合。
实施例6 mPD-1Ab21融合蛋白的IL-21生物学功能鉴定
将Baf3细胞(Baf3细胞为pre-B细胞系)铺于96孔板中，每孔1×10 4个细胞。小鼠IL-21和mPD-1Ab21蛋白按照不同的浓度梯度：0.1ng/ml，1ng/ml，10ng/ml，加入到相应的细胞孔中，每个浓度设三个复孔，不加蛋白的孔作为对照孔。细胞培养第三天，对Baf3细胞进行计数。计算每个蛋白浓度三个复孔细胞数的平均值，计算细胞增殖率，计算公式为：细胞增殖率＝(实验组细胞数/对照组细胞数-1)x 100％。从图6结果可以看出，mPD-1Ab21具有和重组mIL-21同样促进B细胞增殖的生物学活性。
从实施例5和实施例6可以看出，本发明制备的mPD-1Ab21融合蛋白既具有抗PD-1抗体阻断PD-L1与PD-1结合的作用，又具有IL-21的生物学活性。
实施例7 mPD-1Ab21诱导记忆干性CD8 +T细胞分化
本发明进一步研究了mPD-1Ab21对活化CD8 +T细胞分化的作用，利用OT-1转基因小鼠T细胞，体外用OVA抗原肽刺激活化48小时，然后分别在有IL-2、mIL-21、mPD-1Ab或mPD-1Ab21的培养基中培养分化，2天后用流式细胞技术分析T细胞的分化。
结果如图7所示，结果显示：IL-2促进活化的CD8 +T细胞向CD44 highCD62L low效应性T细胞或记忆效应性T细胞分化，mIL-21诱导活化的CD8 +T细胞向CD44 lowCD62L high记忆干性T细胞分化，单独mPD-1Ab对活化的CD8 +T细胞的分化没有影响，mPD-1Ab联合mIL-21具有和单独mIL-21同样诱导CD44 lowCD62L high记忆干性T细胞分化的活性，而mPD-1Ab21具有更强诱导D44 lowCD62L high记忆干性T细胞分化的生物学作用。
实施例8 mPD-1Ab21是通过促进细胞增殖诱导CD44 lowCD62L high记忆干性T细胞分化
为了区分mPD-1Ab21诱导分化的D44 lowCD62L high记忆干性T细胞是来源于没有活化增殖的原始
Figure PCTCN2019117035-appb-000011
CD44 lowCD62L high T细胞，还是活化的T细胞被融合蛋白mPD-1Ab21诱导分化为CD44 lowCD62L high记忆干性T细胞，我们对OT-1转基因小鼠T细胞进行CSFE标记，然后进行活化和在不同条件下分化，结果参见图8所示，单纯培养基中分化的CD44 lowCD62L high细胞群增殖显著低于CD44 highCD62L highT细胞群。mPD-1Ab21诱导分化的CD44 lowCD62L high细胞群也具有明显增殖，与CD44 highCD62L highT细胞群的增殖没有区别。说明mPD-1Ab21诱导分化的CD44 lowCD62L high细胞是由活化的细胞通过增殖分化来的。
实施例9 mPD-1Ab21诱导活化的CD44 highCD62L high细胞分化返回到CD44 lowCD62L high原始细胞表型
为了进一步证明mPD-1Ab21诱导分化的CD44 lowCD62L high细胞是由活化的CD44 highCD62L highT细胞分化返回到CD44 lowCD62L high细胞群，我们将CFSE标记的OT-1转基因小鼠T细胞用OVA抗原肽活化后，用流式分选纯化活化的CD44 highCD62L highT细胞群，然后在加入细胞因子或融合蛋白进行分化。参见图9，结果显示：IL-2诱导活化的CD44 highCD62L highT细胞向CD44 highCD62L low效应性T细胞分化，而mPD-1Ab21诱导活化的CD44 highCD62L highT细胞向CD44 lowCD62L high记忆干性T细胞分化。而且，与CD44 highCD62L highT细胞群的增殖相比较，mPD-1Ab21诱导分化的CD44 lowCD62L high记忆干性T细胞增殖分裂的代数比 CD44 highCD62L highT细胞群的分裂代数更多。进一步说明，mPD-1Ab21诱导分化的CD44 lowCD62L high细胞是由活化的细胞通过增殖、分化返回到CD44 lowCD62L high记忆干性T细胞。
实施例10 mPD-1Ab21诱导的记忆干性CD8 +T细胞功能鉴定
为了验证mPD-1Ab21诱导分化的CD44 lowCD62L high细胞的生物学功能特性，我们将mPD-1Ab21或IL-2诱导分化2-3天的OT-1转基因小鼠T细胞在含有IL-7培养基中培养2天，然后用抗CD3刺激，检测细胞因子产生，参见图10，结果显示：IL-2诱导分化的细胞产生大量IFN-γ，几乎不表达IL-2；mPD-1Ab21诱导分化的细胞产生大量IL-2，IFN-γ表达显著低于IL-2诱导分化的细胞。说明IL-2诱导分化的细胞具有很强的效应功能，而mPD-1Ab21诱导分化的细胞具有更强的增殖分化能力。
实施例11 mPD-1Ab21诱导的记忆干性T细胞在小鼠体内具有更强的记忆应答反应(recall response)及形成记忆细胞的能力
为了进一步验证mPD-1Ab21诱导分化的细胞在体内的生物学功能特性，我们将mPD-1Ab21或IL-2诱导分化的OT-1转基因小鼠T细胞静脉输入到C57小鼠体内，第二天给小鼠皮下注射OVA抗原肽和弗氏完全佐剂进行抗原刺激，然后监控小鼠体内OT-1转基因小鼠T细胞的反应，结果显示：受到抗原刺激后，mPD-1Ab21诱导分化的T细胞反应是IL-2诱导分化的T细胞反应的4倍。一个月后检测发现，输入mPD-1Ab21分化细胞的小鼠体内OT-1转基因小鼠T细胞数量是输入IL-2分化细胞小鼠的2倍，参见图11，说明mPD-1Ab21诱导分化的细胞在体内具有更强的记忆反应能力和形成记忆性T细胞的能力。
实施例12
mPD-1Ab21靶向PD-1 +T细胞促进CD44 lowCD62L high记忆干性T细胞分化
实施例7显示：mPD-1Ab21诱导CD44 lowCD62L high记忆干性T细胞分化的能力显著高于重组mIL-21、或者mPD-1Ab与mIL-21联合。为了验证mPD-1Ab21是否能通过将IL-21靶向活化的PD-1 +T细胞，从而促进T细胞分化，本发明分析比较了mPD-1Ab21和mIL-21诱导CD44 lowCD62L high记忆干性T细胞分化的剂量效应关系。将OT-1转基因小鼠T细胞体外活化，然后用不同浓度的重组mIL-21、或mPD-1Ab21融合蛋白诱导分化，流式细胞技术分析CD44 lowCD62L high细胞的比例。参见图12A，结果显示：mPD-1Ab21融合蛋白诱导CD44 lowCD62L high细胞分化的活性比mIL-21高3倍以上。为进一步分析T细胞表达的PD-1在mPD-1Ab21诱导CD44 lowCD62L high细胞分化中的作用，将正常小鼠T细胞、或PD-1基因敲除小鼠T细胞在体外用抗-CD3和CD8抗体刺激活化，然后用不同浓度的重组IL-21、或mPD-1Ab21融合蛋白诱导分化，流式细胞技术分析CD44 lowCD62L high细胞的比例。参见图12B，结果显示：对于正常小鼠T细胞，mPD-1Ab21诱导CD44 lowCD62L high记忆干性T细胞分化的能力显著高于重组IL-21；而对于PD-1基因敲除小鼠T细胞，mPD-1Ab21诱导CD44 lowCD62L high记忆干性T细胞分化的能力略好于重组IL-21。这一结果说明： mPD-1Ab21通过结合活化T细胞表面的PD-1将IL-21靶向活化的T细胞，从而促进CD44 lowCD62L high记忆干性T细胞分化。
实施例13 mPD-1Ab21快速靶向外周血及肿瘤组织中活化的肿瘤特异性T细胞
给小鼠接种表达OVA抗原的肿瘤细胞B16-OVA,在接种前一天给小鼠输入OVA特异的OT-1原始T细胞，接种后第六天给小鼠进行佐剂polyI:C和OVA抗原肽的皮下免疫，第九天给予小鼠腹腔注射mPD-1Ab21或者抗-PD-1抗体。在给蛋白后的不同时间点(30min，6h，12h，24h和48h)分别流式分析外周血中和肿瘤内浸润的肿瘤特异性的T细胞与抗PD-1抗体的结合能力，来反映体内mPD-1Ab21蛋白或抗-PD-1抗体与肿瘤特异性OT-1T细胞的结合情况。参见图13，结果显示，免疫后的小鼠外周血和肿瘤中活化的OT-1T细胞表面均高表达PD-1分子。腹腔注射抗PD-1抗体或者mPD-1Ab21蛋白30分钟后，外周血中OT-1T细胞表面PD-1流式抗体染色被阻断，说明注射到体内的mPD-1Ab21和抗PD-1抗体都能快速结合到肿瘤特异性T细胞表面。肿瘤组织中融合蛋白或抗体与肿瘤特异性T细胞的结合比外周血慢，6小时开始部分阻断，12小时则完全阻断了PD-1流式抗体染色，说明融合蛋白或抗体结合到肿瘤特异性T细胞表面。在外周血和肿瘤组织中，融合蛋白和OT-1T细胞的结合作用能持续至少24小时，48小时后仅在肿瘤组织能观察到抗PD-1抗体的部分结合。结果说明，mPD-1Ab21融合蛋白注射到体内后能够快速结合外周血及肿瘤组织中活化的肿瘤特异性T细胞。
实施例14 mPD-1Ab21融合蛋白的抗肿瘤作用
给小鼠皮下接种小鼠肠癌肿瘤细胞CT26，3天后给小鼠腹腔注射mPD-1Ab21融合蛋白、mPD-1Ab联合mIL-21重组蛋白、或者抗-PD-1抗体进行治疗，定时检测肿瘤大小，绘制肿瘤生长曲线。参见图14，结果显示：与不治疗的对照小鼠相比，mPD-1Ab和mIL-21联合治疗仅仅使小鼠的肿瘤生长稍有减慢，而抗PD-1抗体和mPD-1Ab21融合蛋白治疗可分别是20％和40％小鼠肿瘤完全消退。结果说明，与mPD-1Ab和mIL-21联合治疗相比，mPD-1Ab21具有更显著的肿瘤治疗效果，而且优于抗PD-1抗体的治疗作用。
实施例15 mPD-1Ab21的联合治疗显著增加抗Her2/neu(赫赛汀)对Her2 +乳腺癌的治疗作用
给小鼠皮下接种Her2/neu +乳腺癌Tubo，16天后给小鼠腹腔注射抗Her2/neu抗体、或者抗Her2/neu抗体联合mPD-1Ab21融合蛋白、抗Her2/neu抗体联合mPD-1Ab和mIL-21进行治疗，定时检测肿瘤大小，绘制肿瘤生长曲线。参见图15，结果显示：单独抗Her2/neu抗体治疗可使40％小鼠肿瘤消退，抗Her2/neu抗体联合mPD-1Ab和mIL-21治疗可使60％小鼠肿瘤完全消退，抗Her2/neu抗体联合mPD-1Ab21治疗可使所有小鼠肿瘤完全消退。结果说明，mPD-1Ab21联合治疗能够显著增加抗Her2/neu(赫赛汀)的肿瘤治疗效应，显著优于与mPD-1Ab和mIL-21的联合治疗。
实施例16 mPD-1Ab21的联合治疗显著增加肿瘤疫苗对B16-OVA肿瘤的治疗作用
肿瘤疫苗是肿瘤治疗未来的一个重要方向，尤其是与PD-1抑制剂联合应用。mPD-1Ab21融合蛋白通过PD-1将IL-21靶向肿瘤疫苗激活的肿瘤特异性T细胞，促进记忆性T细胞形成，将会比PD-1抑制剂更显著地促进肿瘤疫苗的肿瘤治疗效果。为此，给小鼠接种表达OVA抗原的肿瘤细胞B16-OVA,在接种前一天给小鼠输入OVA特异的OT-1原始T细胞，接种后第六天给小鼠进行佐剂polyI:C和OVA抗原肽的皮下免疫，第九天开始给予小鼠腹腔注射mPD-1Ab21融合蛋白、mPD-1Ab联合mIL-21重组蛋白、或者抗-PD-1抗体进行治疗，定时检测肿瘤大小，绘制肿瘤生长曲线。参见图16，结果显示：给荷瘤小鼠进行肿瘤抗原免疫，可以明显抑制肿瘤生长，联合mPD-1Ab和mIL-21、或者抗-PD-1抗体没有显著增加肿瘤疫苗的治疗效果，但是，与mPD-1Ab21联合应用可以极大提高肿瘤疫苗的抗肿瘤作用。
实施例17 mPD-1Ab21治疗显著增加记忆干性CD8 +T细胞比例和肿瘤特异性T细胞反应
分析上述治疗的CT26荷瘤小鼠体内各T细胞亚群比例，参见图17A,发现mPD-1Ab21治疗小鼠脾脏和肿瘤引流淋巴结中记忆干性(CD62L highCD44 lowCD122 +Scal-1 +)CD8 +T细胞比例显著高于抗-PD-1抗体或mPD-1Ab联合mIL-21治疗的小鼠。
分析上述疫苗治疗的B16-OVA荷瘤小鼠，肿瘤疫苗治疗刺激体内肿瘤特异性T细胞活化和增殖，导致荷瘤小鼠脾脏、尤其是肿瘤内肿瘤特异性CD8 +T细胞比例显著增加。抗PD-1抗体联合治疗没有进一步增加肿瘤特异性CD8+T细胞比例，但是，mPD-1Ab21极大提高了疫苗治疗小鼠体内肿瘤特异性T细胞反应，使肿瘤特异性CD8 +T细胞的比例到达肿瘤内CD8 +T细胞的80％。更重要的是，与单独疫苗治疗的肿瘤相比，抗PD-1抗体联合治疗促使肿瘤特异性CD8 +T细胞向效应性(CD127 lowKLRG1 high)分化，而mPD-1Ab21的联合治疗显著促进肿瘤特异性CD8 +T细胞向记忆性效应(CD127 highKLRG1 low)分化。参加图17B和17C。
这些研究结果说明，mPD-1Ab21治疗除了阻断PD-1免疫抑制信号，还能促进记忆性抗肿瘤T细胞反应，从而极大提高肿瘤治疗效果。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于上述实施方式中的具体细节，在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，这些简单变型均属于本发明的保护范围。
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本发明对各种可能的组合方式不再另行说明。
此外，本发明的各种不同的实施方式之间也可以进行任意组合，只要其不违背本发明的思想，其同样应当视为本发明所公开的内容。

Claims (12)

  1. 一种融合蛋白，包含通过连接子相连接的细胞因子和T细胞表面受体分子结合拮抗剂或激动剂，其中，
    所述细胞因子是选自IL-21、IL-2、IL-15、IL-7或IL-12中的一种；
    所述T细胞表面受体分子结合拮抗剂或激动剂选自PD-1结合拮抗剂，LAG-3结合拮抗剂，Tim-3结合拮抗剂或CD137(4-1BB)结合激动剂中的一种；
    优选的，所述融合蛋白能够诱导激活T细胞，同时将细胞因子靶向T细胞，诱导T细胞分化，增强T细胞功能。
  2. 如权利要求1所述的融合蛋白，其中，
    所述PD-1结合拮抗剂抑制PD-1对PD-L1的结合；和/或
    所述PD-1结合拮抗剂抑制PD-1对PD-L1和PD-L2二者的结合；
    优选的，所述PD-1结合拮抗剂是抗体；
    更优选的，所述PD-1结合拮抗剂是单链抗体；
    进一步优选的，所述PD-1结合拮抗剂为二聚体单链抗体。
  3. 如权利要求1或2所述的融合蛋白，其特征在于，所述结构域从氨基末端到羧基末端包含如下结构：
    -X-Linker1-Y-
    其中：
    X是PD-1结合拮抗剂，Y是细胞因子，Linker1是连接子；
    优选的，所述PD-1结合拮抗剂是抗体，细胞因子是IL-21；
    更优选的，所述PD-1结合拮抗剂从氨基末端到羧基末端包含如下结构：
    V H-Linker2-V L
    其中，V H为PD-1单链抗体重链的可变区，V L为抗体轻链的可变区，Linker2为连接子；
    进一步优选的，所述融合蛋白在3’端还可以包括标签序列。
  4. 如上述权利要求任一项所述的融合蛋白，其特征在于，
    所述PD-1结合拮抗剂，核苷酸序列如SEQ ID NO:5或SEQ ID NO:17所示；和/或
    所述细胞因子为IL-21，核苷酸序列如SEQ ID NO:7或SEQ ID NO:19所示；
    优选的，所述融合蛋白阻断PD-LI与T细胞表面PD-1的结合，并将IL-21靶向肿瘤特异性T细胞，诱导T细胞的分化。
  5. 如上述权利要求任一项所述的融合蛋白，其特征在于编码所述融合蛋白的核苷酸序列如SEQ ID NO:8或SEQ ID NO:20所示；
    进一步优选的，所述融合蛋白在3’端还可以包括标签序列，所述含有标签序列的编码融合蛋白的核苷酸序列如SEQ ID NO:10或SEQ ID NO:21所示；
    优选的，所述融合蛋白主要是同源二聚体形式。
  6. 一种多核苷酸，其编码如权利要求1-5中任一项所述的融合蛋白。
  7. 一种表达载体，其包含与表达控制序列操作性连接的如权利要求6所述的多核苷酸。
  8. 一种宿主细胞，其包含权利要求7所述的表达载体。
  9. 如权利要求1-5任一项所述的融合蛋白的制备方法，其特征在于，包括如下步骤：
    a)通过基因工程手段方法，将细胞因子编码基因链接到PD-1结合拮抗剂编码基因的C末端，成为融合蛋白的编码基因；
    b)构建表达载体；
    c)转染细胞，进行蛋白表达和纯化；
    d)获得所述融合蛋白。
  10. 一种药物组合物，其包含权利要求1-5中任一项所述的一种或多种融合蛋白，以及药学上可接受的载体、稀释剂或赋形剂；
    和/或，所述药物组合物还包括其他抗癌药物或肿瘤疫苗，所述融合蛋白可以和其他抗癌药物或肿瘤疫苗联合使用；
    优选的，所述其他抗癌药物为Her2抗体；
    更优选的，所述其他抗癌药物为赫赛汀；
  11. 一种用于制备预防或治疗癌症药物或试剂盒的应用，其包括施用对该个体有效量的权利要求1-5任一项所述的融合蛋白；
    优选的，所述癌选自由肾细胞癌，膀胱癌，非小细胞肺癌，头颈鳞状细胞癌，鳞状非小细胞肺癌，非鳞状非小细胞肺癌，肠癌，霍奇金淋巴瘤，结直肠癌，黑色素瘤，卵巢癌，乳腺癌，激素受体阳性乳腺癌，Her2阳性乳腺癌，和三重阴性乳腺癌组成的组；
    更优选的，连续或间歇施用权利要求1-5任一项所述的融合蛋白；
    进一步优选的，通过静脉内，肌肉内，皮下，表面，口服，经皮，腹膜内，眶内，通过植入，通过吸入，鞘内，室内，或鼻内施用该融合蛋白。
  12. 一种用于在个体中预防或治疗癌症的方法，其包括施用对该个体有效量的权利要求1-5任一项所述的融合蛋白；
    优选的，所述癌选自由肾细胞癌，膀胱癌，非小细胞肺癌，头颈鳞状细胞癌，鳞状非小细胞肺癌，非鳞状非小细胞肺癌，肠癌，霍奇金淋巴瘤、结直肠癌，黑色素瘤，卵巢癌，乳腺癌，激素受体阳性乳腺癌，Her2阳性乳腺癌，和三重阴性乳腺癌组成的组；
    更优选的，连续或间歇施用权利要求1-5任一项所述的融合蛋白；
    进一步优选的，通过静脉内，肌肉内，皮下，表面，口服，经皮，腹膜内，眶内，通过植入，通过吸入，鞘内，室内，或鼻内施用该融合蛋白。
PCT/CN2019/117035 2019-11-11 2019-11-11 一种靶向抗原特异性t细胞诱导其向记忆干性细胞分化的融合蛋白 WO2021092719A1 (zh)

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