CN110894241A - Fusion protein for inducing differentiation of target antigen specific T cells to memory stem cells - Google Patents
Fusion protein for inducing differentiation of target antigen specific T cells to memory stem cells Download PDFInfo
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
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- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
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Abstract
The invention relates to an anti-PD-1 antibody and IL-21 fusion protein, which can block the combination of PD-L1 and PD-1 on the surface of a T cell, play a role in tumor treatment of the anti-PD-1 antibody, target IL-21 to a tumor specific T cell, induce the differentiation of the T cell, and regulate the function of the T cell.
Description
Technical Field
The invention relates to the field of bioengineering, in particular to a fusion protein for inducing differentiation of a target antigen specific T cell and enhancing an effect function of the target antigen specific T cell.
Background
The immune system functions to achieve an effective response of the body to the pathogenic agent. T cells play a key role in the process, normal cells are malignant to become cancer cells, and the cancer cells can be recognized by an immune system of an organism to kill and eliminate the cancer cells. The immune cells which can kill and eliminate growing tumors in the body are mainly T lymphocytes. After recognizing tumor antigens, T lymphocytes are activated, amplified in large numbers, differentiated into effector T cells, and reach tumor sites to attack tumor cells. However, during the long-term evolution of tumor cells, various mechanisms are generated to prevent immune cells from attacking, for example, the tumor cells highly express immunosuppressive molecule PD-L1, and the T lymphocytes can be inhibited from killing the T lymphocytes by binding with immunosuppressive receptor PD-1 on the surfaces of the T lymphocytes.
PD-1 (also known as programmed cell death-1), a member of the CD28 family, is expressed on the surface of activated T cells and functions to inhibit T cell responses by binding to the ligand PD-L1, preventing T cell hyperresponses from causing tissue damage, and is a negative regulatory mechanism of normal T cell responses. Tumor cells are combined with PD-1 on the surface of T cells through high expression PD-L1, so that the tumor cells can inhibit the killing of the anti-tumor T cells, and the tumor cells are an important mechanism for tumor immune escape. Scientists have invented an anti-PD-1 antibody which can block the binding of PD-L1 on the surface of tumor cells and PD-1 receptors on the surface of T lymphocytes, relieve the inhibition of the tumor cells on the T lymphocytes, and the T lymphocytes with recovered functions can kill the tumor cells and eliminate tumors in vivo, which is a widely concerned tumor therapeutic drug, namely the anti-PD-1 antibody in the world at present. Currently, many organizations in various countries around the world are investing in the development of PD-1, and hundreds of clinical trials are underway each year in the United states alone. And 1600 clinical trials are ongoing worldwide. PD-1/PD-L1 drug, developed by international well-known pharmaceutical manufacturers to date, has been approved by the U.S. FDA for marketing in 5 sets, including: PD-1 inhibitors nivolumab (opsivo), pembrolizumab (keytruda); the PD-L1 inhibitors atezolizumab (tecentriq), durvalumab (infinzi) and avelumab (bavencio); can be used for treating melanoma, squamous cell carcinoma of head and neck, non-small cell lung cancer, Hodgkin lymphoma, renal carcinoma, bladder cancer, etc. At least 100 biological companies including Xinda, Henrui, Baiji Shenzhou, Junshi and the like exist in China, and PD-1 blocking drugs are researched and developed and clinical tumor treatment experiments are carried out. Although the anti-PD-1 antibody drug achieves great success in tumor treatment, the treatment efficiency of most advanced tumors is only about 20%, and therefore, how to improve the anti-PD-1 antibody tumor treatment effect is an important direction for tumor drug development.
Only one kind of epitope can be recognized by one lymphocyte, thousands of T lymphocytes exist in a body, and only the tumor specific T lymphocyte can recognize and kill the specific tumor cell. T lymphocyte division into CD4+T cells and CD8+T cells. The main factor for recognizing and killing tumor cells in vivo is CD8+T cell, CD4+The function of T cells is primarily to assist the CD8+ T cell response. The resting T lymphocytes are inactive and can exert different biological functions only if activated, proliferated and differentiated into T cells of different functional states under antigen stimulation. CD8+T cells can be classified into primitive T cells (negative Tcells), memory stem T cells (memory stem cells), memory T cells (memory cells), and effector T cells (effector T cells) according to their differentiation state. Effect CD8+T cells are terminally differentiated cells with the strongest killing activity, but short survival time in vivo, and therefore, the weakest antitumor effect in vivo. Memory CD8+T cells have long survival time in vivo and can be differentiated into effector T cells to play a role after being stimulated by antigens, so that the antitumor effect in vivo is obviously enhanced. Memory dry CD8+The T cell has self-replication and renewal capacity, can survive in vivo for a long time, can be differentiated into memory and effector T cells by antigen stimulation, and has the strongest anti-tumor effect in vivo. Activation, proliferation and differentiation of T cells is a complex and precisely regulated reaction process in which various cytokines play an important role.
Cytokines (cytokines) are low molecular weight soluble proteins that are produced by various cells induced by immunogens, mitogens or other stimulants and have a variety of functions such as modulating immunity, hematopoiesis, cell growth and damaged tissue repair. Wherein, the interleukin can activate and regulate immune cells, mediate T, B cell activation and increaseColonisation and differentiation and play an important role in inflammatory responses. It is first produced by leukocytes and acts among leukocytes, so that it is named. The in vivo action mode of the interleukins is mainly that autocrine and paracrine of cells play important regulation and control roles and biological effects locally, and systemic application can cause serious side effects such as inflammatory reaction and the like. Interleukin 21 (IL-21 for short) is a major CD4+T cell production, on CD8+T cells, B lymphocytes, NK cells, mononuclear macrophages and other cytokines. IL-21 can induce activated CD8+Differentiation of T lymphocytes into a young, viable cell population, i.e., similar to memory-dried CD8+T lymphocytes. Since IL-21 can also act on other various immune cells and histiocytes, the anti-tumor effect of the systemic application clinical test is not obvious, and the long-term application can cause side effects such as inflammation and the like. Therefore, the key to the use of IL-21 for 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 for antigen-activated T cells. Recent studies have shown that: tumor-specific CD8 in tumor patients+All T cells express PD-1 and pass through PD-1 in vitro+The cell sorting can enrich tumor-specific T cells in tumor tissues or peripheral blood of tumor patients and is used for adoptive therapy of the T cells. Based on the above research findings, the present invention provides a fusion protein and a method for treating cancer using the same. The anti-PD-1 antibody can be combined with tumor specific T lymphocytes in a targeted manner, and IL-21 is targeted to the tumor specific T lymphocytes while the anti-tumor effect of the anti-PD-1 antibody is exerted, so that the tumor specific T cells are induced to be differentiated to memory (dry) T cells in vivo, and the tumor treatment effect of the anti-PD-1 antibody is improved. The fusion protein provided by the invention has the effect exceeding that of a single monoclonal antibody or cytokine, and can induce CD8+Furthermore, the inventor surprisingly found that the fusion protein prepared by the invention is obviously superior to the combined drug of a PD-1 inhibitor and IL-21 in the treatment effect of cancer.
Disclosure of Invention
Summary of The Invention
The object of the present invention is to overcome these disadvantages of the prior art. Specifically, the invention aims to provide a fusion protein which targets IL-21 to tumor-specific T lymphocytes and has the tumor treatment effect of an anti-PD-1 antibody and related application thereof.
The anti-PD-1 antibody is an anti-tumor drug which is currently most attractive internationally, and a plurality of pharmaceutical companies compete for research and development. The tumor treatment mechanism of the anti-PD-1 antibody is that the immunosuppression molecule PD-L1 on the surface of the tumor cell is blocked to be combined with the immunosuppression receptor PD-1 on the surface of the T lymphocyte, the inhibition of the tumor cell on the T lymphocyte is relieved, the function of the tumor specific T lymphocyte is recovered, the tumor cell is killed, and the tumor in vivo is eliminated. Activated tumor-specific T lymphocytes in patients are at different stages of differentiation, such as: terminally differentiated effector T cells effector memory T cells (effect memory T cells), central memory T cells (central memory T cells), and memory-drying T cells. Among them, the memory-stem T cells have the functions of self-renewal and reproduction, and can differentiate into different downstream various effector T cells, thus having the strongest antitumor effect in vivo. The invention discovers that IL-21 can induce activated CD8+T lymphocytes differentiate into memory-dry T lymphocytes.
The research and development motivation of the invention is to fuse the anti-PD-1 antibody and the IL-21 link together to form a novel fusion protein, and the anti-PD-L1 on the surface of the tumor cell is blocked from being combined with the PD-1 on the surface of the T lymphocyte to restore the function of the T lymphocyte, and simultaneously, the IL-21 is targeted to the T lymphocyte to enhance the anti-tumor function of the T lymphocyte.
The foregoing is merely a summary of aspects of the invention and is not, and should not be taken as, limiting the invention in any way.
All patents and publications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication was specifically and individually indicated to be incorporated herein by reference.
Detailed description of the invention
Unless otherwise indicated or defined, all terms used have the meanings commonly used in the art as is clear to the skilled person. For example, reference is made to standard manuals, such as Sambrook et al, "Molecular Cloning: a Laboratory Manual (molecular cloning: A Laboratory Manual) (2 nd edition), Vol.1-3, Green Publishing and Wiley Interscience, New York (1987); lewis, "Genes II (Gene II)", John Wiley & Sons, New York, N.Y. (1985); old et al, "Principles of Gene management: an Introduction to Genetic Engineering, 2 nd edition, University of California Press, Berkeley, Calif. (1981); roitt et al, "Immunology" (6 th edition), Mosby/Elsevier, Edinburgh (2001); roitt et al, Roitt's Essential Immunology, 10 th edition Blackwell Publishing, UK (2001); and Janeway et al, "immunology" (6 th edition), Garland Science Publishing/Churchill Livingstone, New York (2005), and the well-known background art cited therein;
the terms "amino-terminal" and "carboxy-terminal" as used herein refer to positions within a polypeptide. Where the context permits, these terms are used to indicate proximity or relative position with respect to a particular sequence or portion of a polypeptide. For example, a sequence that is at the carboxy-terminus of a reference sequence within a polypeptide is located near the carboxy-terminus of the reference sequence, but is not necessarily located at the carboxy-terminus of the entire polypeptide.
The term "cancer" or "cancer cell" as used herein refers to a tissue or cell found in an organism that has characteristics that distinguish it from normal tissue or tissue cells. Among these features include, but are not limited to: the extent of degenerative changes, irregularities in shape, indistinguishability of cell contours, changes in nuclear size, structural changes in the nucleus or cytoplasm, other phenotypic changes, the presence of cellular proteins predictive of a cancerous or pre-cancerous state, increased mitotic frequency, and the ability to metastasize. The term "cancer" includes carcinomas, sarcomas, tumors, epithelial cancers, leukemias, lymphomas, polyps, as well as hard cancers, transformations, and the like.
The term "combination therapy" as used herein refers to the administration of at least one therapeutically effective dose of a fusion protein component (including, but not limited to, "anti-PD-1 antibody and IL-21 fusion protein (PD-1Ab 21)") to a subject. The fusion protein can be a mature polypeptide, fragment, fusion or conjugate thereof having the biological activity of "targeting IL-21 to tumor-specific T lymphocytes, increasing the tumor therapeutic effect of an anti-PD-1 antibody".
' memory dry CD8+T cell "refers to activated CD8+T cells are in the early stage of differentiation, have the ability to self-replicate and renew, can survive in vivo for a long period, and can differentiate into memory and effector T cells upon antigen stimulation. Its cell phenotype is similar to that of the original
CD8+T cells, expressed as CD44 in micelowCD62LhighIn humans, it is expressed as CD45RA+CCR7+CD45RO-Meanwhile, IL-2R, Bcl-2, Scal-1 or CD95 is expressed.
"sequence" as used herein (e.g., in terms such as "immunoglobulin sequence", "antibody sequence", "domain sequence" or "protein sequence") should generally be understood to include both the relevant immunoglobulin sequence as well as the nucleic acid sequence or nucleotide sequence encoding it, unless the context requires a more limited interpretation.
Reference to "nucleic acid molecules" may relate to nucleic acids used for gene vaccination, or to nucleic acids encoding the immunoglobulin sequences of the invention, or to both, as will become clear from the specific context.
The present invention relates to immunoglobulin sequences that can bind to and/or have affinity for an antigen as defined herein. In the context of the present invention, "binds to and/or has affinity for an antigen" has the usual meaning in the art, e.g. as understood in the context of antibodies and their respective antigens.
In a particular embodiment of the invention, the term "binding" means that the immunoglobulin sequence specifically interacts with an antigen.
The nucleic acids of the invention may be prepared or obtained in a manner known per se, which is based on information about the cell-associated antigen or immunoglobulin sequence of the invention, and/or may be isolated from a suitable natural source.
The nucleic acid of the invention may also be in a form present in and/or part of a genetic construct, as should be clear to a person skilled in the art. Such genetic constructs typically comprise at least one nucleic acid of the invention, optionally linked to one or more genetic construct elements known per se, such as, for example, one or more suitable regulatory elements (such as suitable promoters, enhancers, terminators, etc.) and other construct elements mentioned herein.
Reference is made to the general manual, such as Sambrook et al and Ausubel et al, regarding some (other) non-limiting examples of promoters, selection markers, leader sequences, expression markers and other elements that may be present in/used in the genetic constructs of the invention, such as terminators, transcription and/or translation enhancers and/or integration factors.
In general, the genetic constructs of the invention are obtained by inserting the nucleotide sequences of the invention into suitable (expression) vectors known per se. Some preferred but non-limiting examples of suitable expression vectors are those used in the examples described below, as well as those mentioned herein.
The nucleic acids of the invention and/or the genetic constructs of the invention may be used for transforming a host cell or a host organism, i.e. for expressing and/or producing the fusion proteins of the invention. Suitable hosts or host cells will be clear to the skilled person and may be, for example, any suitable fungal, prokaryotic or eukaryotic cell or cell line, or any suitable fungal, prokaryotic or eukaryotic organism.
In particular, the invention provides in some particular aspects the following:
the 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 one selected from the group consisting of IL-21, IL-2, IL-15, IL-7 and IL-12; the T cell surface receptor molecule binding antagonist or agonist is selected from one of a PD-1 binding antagonist, a LAG-3 binding antagonist, a Tim-3 binding antagonist or a CD137(4-1BB) binding agonist, and preferably, the fusion protein is capable of inducing activation of T cells, simultaneously targeting cytokines to T cells, inducing T cell differentiation, and enhancing T cell function.
In a particular embodiment, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1; and/or the PD-1 binding antagonist inhibits the binding of PD-1 to both PD-L1 and PD-L2; preferably, 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 dimeric single chain antibody.
In another aspect of the present invention, a fusion protein is disclosed, the domain of which comprises, from amino terminus to carboxy terminus, the structure: -X-Linker 1-Y-wherein X is a PD-1 binding antagonist, Y is a 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 amino terminus to carboxy terminus: vH-Linker2-VLWherein, VHIs the variable region of the heavy chain of a PD-1 single-chain antibody, VLIs the variable region of the antibody light chain, Linker2 is the Linker; further preferably, the fusion protein may further include a tag sequence at the 3' end.
The invention also discloses a fusion protein, wherein, the nucleotide sequence of the PD-1 binding antagonist is shown in SEQ ID NO.5 or SEQ ID NO. 17; and/or the cytokine is IL-21, and the nucleotide sequence of the cytokine is shown as 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 to tumor-specific T cells, inducing differentiation of the T cells.
In one embodiment of the invention, the fusion protein is related, and the nucleotide sequence of the coding fusion protein is shown as SEQ ID NO.8 or SEQ ID NO. 20; further preferably, the fusion protein can also comprise a tag sequence at the 3' end, and the nucleotide sequence of the fusion protein containing the tag sequence is shown as SEQ ID NO.10 or SEQ ID NO. 21; preferably, the fusion protein is predominantly in the form of a homodimer.
Furthermore, the present invention relates to a polynucleotide encoding the fusion protein according to the present invention. And an expression vector comprising the polynucleotide operably linked to an expression control sequence. Meanwhile, the invention also discloses a host cell which comprises the expression vector related to the invention.
In one embodiment of the present invention, a method for preparing a fusion protein is disclosed, comprising the steps of: a) linking the cytokine coding gene to the C terminal of the PD-1 binding antagonist coding gene by a genetic engineering method to form a coding gene of a fusion protein; b) constructing an expression vector; c) transfecting cells, and performing protein expression and purification; d) obtaining the fusion protein.
In addition, the 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 comprises other anti-cancer drugs or tumor vaccines, and the fusion protein can be used in combination with other anti-cancer drugs or tumor vaccines; preferably, the other anti-cancer drug is Her2 antibody; more preferably, the other anticancer drug is herceptin;
in addition, the present invention relates to a use for the preparation of a medicament or kit for the prevention or treatment of cancer, comprising administering to the individual an effective amount of the fusion protein; preferably, the cancer is selected from the group consisting of 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, intestinal 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 according to the invention is administered continuously or intermittently; further preferred, the fusion protein is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally.
In particular embodiments of the present invention, it also relates to a method for preventing or treating cancer in an individual comprising administering to the individual an effective amount of a fusion protein; preferably, the cancer is selected from the group consisting of 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, intestinal 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; further preferred, the fusion protein is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally.
Drawings
FIG. 1. construction of anti-mouse PD-1 single-chain antibody and mouse IL-21 fusion protein (mPD-1Ab21) schematic A. DNA sequence of anti-mouse PD-1 single-chain antibody and mouse IL-21 fusion protein (mPD-1Ab 21); B. schematic representation of anti-mouse PD-1 single-chain antibody and mouse IL-21 fusion protein (mPD-1Ab21), wherein, VHAnd VLVariable regions of heavy and light chains, V, respectively, of an anti-murine PD-1 single-chain antibodyHAnd VLAnd IL-21 are respectively connected by two linkers with different lengths, and 3xFlag is positioned at the 3' end; C. a schematic of the conformation of the fusion protein (mPD-1Ab21) formed by the anti-mouse PD-1 single-chain antibody and mouse IL-21.
FIG. 2 expression purification and identification of mouse mPD-1Ab21 fusion protein A.SDS-PAGE protein gel the purity of purified mouse PD-1 single-chain antibody (mPD-1Ab) and mouse anti-PD-1 single-chain antibody and IL-21 fusion protein (mPD-1Ab21) was identified; B. the purified mPD-1Ab21 fusion protein was passed through a molecular sieve column, and the results showed that: the mPD-1Ab21 fusion protein exists mainly in a dimer form.
FIG. 3, anti-human PD-1 monoclonal antibody identification A. screening monoclonal anti-human PD-1 antibody by hybridoma technology to obtain 1G1 monoclonal antibody of high expression PD-1 antibody; incubating the supernatant of the cultured 1G1 cells and CHO cells expressing PD-1 for 30min, and carrying out flow detection on the binding capacity of the 1G1 supernatant and the PD-1 on the CHO cells; B. and (3) incubating the CHO cells and the PD-L1-IgFc for 30min, or adding 1G1 supernatant and incubating the CHO cells for 30min, then adding the PD-L1-IgFc, and carrying out flow detection on the capability of the 1G1 supernatant for blocking the combination of the PD-1 and the PD-L1-IgFc.
FIG. 4 is a schematic diagram of the construction of anti-human PD-1 antibody and human IL-21 fusion protein (PD-1Ab21) and functional characterization A. anti-human PD-1 antibody and human IL-21 fusion protein (PD-1Ab 21). VHIs the variable region of the heavy chain of murine PD-1 antibody, VLThe variable region of the light chain of the murine PD-1 antibody, and the constant region and Fc segment of the human antibody. The 3' end is human IL-21; B. v of anti-human PD-1 antibodyHAnd VLThe sequence of (a); C. stably expressing PD-1 CHO cells and PD-1Ab21 protein at 4 ℃ for 30min, and then flow detecting the binding capacity of PD-1Ab21 and PD-1; D. CHO cells were incubated with PD-L1-IgFc for 30min (black solid line), or PD-1Ab21 was incubated with CHO cells for 30min before PD-L1-IgFc (dotted line) was added and the ability of 1G1 supernatant to block the binding of PD-1 to PD-L1-IgFc was flow-tested.
FIG. 5 in vitro binding and blocking experiment of mPD-1Ab21 fusion protein A. flow assay for the ability of mPD-1Ab21 to bind PD-1 and mPD-1Ab21 to block the binding of PD-1 to its ligand PD-L1IgFc, left panel A: EG7 cells (PD-1)+) Co-incubation with mPD-1Ab21 protein at 4 ℃ for 30min (solid black line), or EG7 cells were incubated with anti-PD-1 antibody at 4 ℃ for 30min, followed by mPD-1Ab21 protein for 30min (dashed black line), negative control (shaded gray) without mPD-1Ab21, APC-anti Flag flow antibody detected the ability of mPD-1Ab21 to bind to PD-1, right panel a: EG7 cells were incubated with mPD-L1-IgFc protein at 4 ℃ for 30min (solid black line), or EG7 cells were incubated with mPD-1Ab21 protein for 30min and then with mPD-L1-IgFc protein for 30min (dashed black line). Without mPD-1Ab21 as a negative control (shaded in grey), the PE-anti-hIg flow antibody detects the ability of mPD-1Ab21 to block the binding of PD-1 to its ligand mPD-L1 IgFc; B. taking OT-1 transgenic mice (OVA antigen peptide specific T cell receptor transgenic mice) lymph node T cells, stimulating and activating for 48 hours in vitro with OVA peptide (0.1ng/ml), and highly expressing PD-1 molecules by the T cells. Repeating the above combination andblocking experiments.
FIG. 6 IL-21 biological function of mPD-1Ab21 fusion protein identifies Baf3 cells as pre-B cell line. Baf3 cells were plated in 96-well plates at 10000 cells per well. mIL-21 and mPD-1Ab21 proteins were added to the corresponding cells according to different concentration gradients. On the third day of cell culture, the Baf3 cells were counted. The cell proliferation rate (number of cells in experimental group/number of cells in control group-1) x 100%
FIG. 7 in vitro induction of CD44 by mPD-1Ab21lowCD62LhighA.OT-1 transgenic mouse T cell differentiated by a memory dry T cell is activated by OVA peptide (0.02ng/ml) in vitro, cell differentiation is carried out by adding cell factors or proteins with equimolar amount after 34h of activation, and the differentiation phenotype of the T cell is detected by flow on the third day; B. statistics of the proportion of cell subsets in the four quadrants marked by CD44 and CD62L in Panel A, CD44lowCD62LhiMemory-dry T cell phenotype.
FIG. 8 mPDIL21 induced CD44lowCD62LhighProliferation of memory-Dry T cells from OT-1 transgenic mice were labeled in vitro with CFSE dye. After activation for 47h with 0.02ng/ml OVA polypeptide, differentiation was performed with IL-2(10ng/ml) and mPD-1Ab21(25ng/ml), and the day after differentiation, flow-based assay: A.T cell proliferation; expression of CD44, CD62L and analysis of CD44lowCD62Lhigh，CD44highCD62LhighAnd CD44highCD62LlowCFSE-labeled cell proliferation levels of three cell subsets.
FIG. 9 mPD-1Ab 21-induced CD44lowCD62LhighMemory-drying T cells are composed of activated CD44highCD62LhighLymph node T cells from a.ot-1 mice were differentiated and labeled with CFSE in vitro and activated with OVA polypeptide. After 42h of activation, the activated T cells were flow sorted to give CD44highCD62LhighA subpopulation of cells; B. the T cells obtained after sorting are added with IL-2 and mPD-1Ab21 respectively for in vitro differentiation, the third day of differentiation, the expression of CD44 and CD62L is detected by flow and CD44 is analyzedlowCD62Lhigh，CD44highCD62LhighAnd CD44highCD62LlowThree cell subsets proliferate.
FIG. 10 shows cytokine secretion levels of memory-dried T cells induced by mPD-1Ab21 after antigen restimulation in murine naive OT-1T cells and OT-1 transgenic murine T cells differentiated for three days with mPD-1Ab21 and IL-2, respectively, after resting for two days with IL-7 cytokine, added to a pre-crimped (0.5 μ g/ml anti-CD 3 antibody) cell culture plate. After 12 hours of stimulation with anti-CD 3 antibody, BFA was added to block for 4 hours, and IFN-. gamma.and IL-2 expression levels in the cells were then flow-assayed.
FIG. 11 survival of Dry memory cells induced by mPD-1Ab 21A lymph node T cells from CD90.1+ OT-1 mice were activated in vitro with OVA peptide at a concentration of 0.02 ng/ml. After 40h of activation, IL-2(10ng/ml) and mPD-1Ab21(25ng/ml) were added for differentiation. Three-day differentiated cells were injected via tail vein into Co 60-irradiated (4Gy) C57 mice. On the day of injection, the mice were immunized subcutaneously with OVA peptide (50. mu.g) in complete adjuvant combination, and were injected intraperitoneally with IL-2 (5. mu.g/time) for seven consecutive days. At various time points, blood was drawn from the orbit and the proportion of CD90.1+ T cells to CD45+ cells in the peripheral blood was flow-assayed. B. In the mice, after 35 days of tail vein injection of T cells, the mice are killed, spleens of the mice are taken and counted, then the proportion of CD90.1+ T cells in the spleens is detected in a flow mode, and the absolute number of the CD90.1+ T cells in the spleens is calculated.
FIG. 12 mPD-1Ab21 targeting PD-1+T cells, promote differentiation of memory-drying T cells A. OT-1 mouse lymph node T cells were activated with OVA peptide (0.02ng/ml) in vitro. After 40h of activation, mIL-21 and mPD-1Ab21 at different concentrations were added for differentiation. Day three of differentiation, CD8 was detected by flow assay+Expression of CD44 and CD62L by T cells. For CD44lowCD62LhighThe proportion of the cell subsets was counted. B. T cells were prepared from wild-type C57 mice and PD-1 knockout mice, respectively, and activated in vitro with anti-CD 3 and CD28 antibodies. After 39 hours of activation, T cells were differentiated by adding mIL-21 and mPD-1Ab21 at different concentrations. On day four of differentiation, T cells were flow tested for expression of CD44 and CD62L vs CD44lowCD62LhighCounting the proportion of cell subsets。
FIG. 13 Rapid targeting of mPD-1Ab21 to activated tumor-specific T cells in peripheral blood and tumor tissue C57 mice subcutaneously inoculated with 1x106Melanoma cells B16-OVA, injected tail vein at 1X10 the day before inoculation6Primitive OT-1T cells. Six days after inoculation, mice were immunized subcutaneously with poly I C (50. mu.g) + OVA peptide (50. mu.g); injecting protein (antibody 200 mug, protein 100 mug) into the abdominal cavity of the mouse on the ninth day; blood and tumors were taken at different time points and analyzed by flow-based techniques for the expression of OT-1T cell surface PD-1 molecules.
FIG. 14. growing Balb/c mice with mPD-1Ab21 inhibiting colorectal cancer tumor cell CT26 were inoculated subcutaneously with 2x106Intestinal cancer cell CT26, and the third day after inoculation, antibody is injected intraperitoneally for treatment. The anti-PD-1 antibody group was injected three times a week at 200. mu.g each. The mPD-1Ab + mIL-21 group and mPD-1Ab21 group were injected once every other day for four times, each at 150. mu.g. The tumor size of CT26 was measured at different time points, and tumor volume (length × width)/2.
FIG. 15 combination therapy of mPD-1Ab21 and anti-Her 2/neu antibody significantly inhibits Her2+Growth of Tubo tumor Balb/c mice were inoculated subcutaneously with 1x10e6 breast cancer cells Tubo. The mPD-1Ab + mIL-21 group and the mPD-1Ab21 group were intraperitoneally injected from the fifteenth day, once every other day, four times, each at 150. mu.g. anti-Her 2/neu antibody was injected on days 16 and 19 at 200. mu.g each. Tubo tumor size was measured at different time points, tumor volume ═ length × width)/2.
FIG. 16 growth of C57 mice significantly inhibited melanoma B16-OVA by combination therapy of mPD-1Ab21 with tumor vaccine subcutaneous inoculation of 1x106Melanoma cells B16-OVA, 2X10 by tail vein injection one day before inoculation6Primitive OT-1T cells. Six days after inoculation, mice were immunized subcutaneously with poly I C (50. mu.g) + OVA peptide (50. mu.g). On the ninth day, the intraperitoneal protein injection treatment is started, and anti-PD-1 is injected once every two days for three times, wherein each time is 200 mu g. mPD-1Ab + mIL-21 and mPD-1Ab were injected once every other day for a total of five times, each time 150. mu.g. B16-OVA tumor sizes were measured at different time points, tumor volume ═ (length × width)/2.
FIG. 17 significant increase in mPD-1Ab21 treatmentMemory dry CD8+T cell ratio and tumor-specific T cell response A.Balb/c mice were inoculated subcutaneously with 2x106Colorectal cancer cells CT26, anti-PD-1 group were intraperitoneally injected with 200. mu.g of antibody on the fourth and seventh days. The mPD-1Ab21+ mIL-21 group and mPD-1Ab21 group were intraperitoneally injected with protein seven times a day, 100. mu.g each time, starting on the fourth day. Mice were killed on day 11, and single cell suspensions were prepared from draining lymph nodes and spleens of the mice, respectively. Flow detection of CD62L in different tissueshighCD44lowCD122+ScaI+Total CD8 from T cells+Proportion of T cells. B is&c.C57 mice were inoculated subcutaneously with 1x106Melanoma cells B16-OVA. The day before inoculation, tail vein injection 2X106Primitive OT-1T cells. Six days after inoculation, mice were immunized subcutaneously with poly I C (50. mu.g) + OVA peptide (50. mu.g). Intraperitoneal protein injection treatment was started on the ninth day, and mPD-1Ab21 was given once a day for seven times, each time 100 μ g. anti-PD-1 was injected on day 10 and day 14 at 200. mu.g each time. And killing mice on day 17, and preparing single cell suspension by respectively taking draining lymph nodes, spleens and tumors. Flow detection of OT-1T cell occupancy of CD45 in different tissues+Proportion of cells (panel B), CD127 in tumors was calculatedlowKLRG1highCD8+T cells and CD127highKLRG1lowCD8+T (FIG. C).
Detailed Description
The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
Main experimental materials
IL-2 cytokines: recombiant human IL-2 company Peprotech Cat No.: 200-02
IL-7 cytokine: recombiant human IL-7 company, peprotech cat #: 200-07
Recombinant human interleukin-2 for injection: shenyang Sansheng pharmaceutical Limited liability company
Complete Freund's adjuvant: company sigma; the cargo number is F5881-10ml
PolyI: C company sigma; cat number I3036-20mg
pTT3 expression vector: professor given in Liu Ying Fang
293T cells: professor given to Zhang uncle
CHO cells: professor presented in exhibition
EG7 cells: professor presented in exhibition
Baf3 cells: purchased from Beinana Chuanglian Biotech Ltd
PD-1KO mouse: professor presented in exhibition
Her2/neu + breast cancer Tubo cells: new professor given as present in paying yang
OT-1 transgenic mice: ovalbumin (OVA) antigen peptide (SIINFEKL) specific T Cell Receptor (TCR) transgenic mice were given to professor wang red yang.
C57 mice: c57BL/6 from Witonglihua
Balb/c mice: balb/c from Viton Hua
OVA antigen peptide: SIINFEKL (synthesized by Biotech Co., Ltd.)
anti-Flag beads:
M2 Affinity Gel Sigma A2220
CSFE dye: company: sigma cat #: 21888
Anti-murine PD-1 antibody (G4): the culture supernatant was purified by a Protein G column from hybridoma cells (G4) which were an anti-mouse PD-1 antibody.
Protein G Protein purification column: company GE Cat No. 17-0404-01
anti-CD 3 antibody: leaf purified anti-mouse CD3 ε: biolegend cargo number: 100331
anti-CD 28 antibody: leaf purified anti-mouse CD28 Co: biolegend cargo number: 102111
anti-Her 2/neu antibody: the culture supernatant was purified by using Protein G column from hybridoma cell line (7.16.4) with anti-mouse her2 antibody
APC-anti-Flag company Biolegend Cat No. 637307
PE-anti-hIg company ebioscience Cat No. 14-4998-82 BFA: ebioscience cargo number 00-4506-51
Example 1 construction of anti-murine PD-1 Single-chain antibody and mouse IL-21 fusion protein (mPD-1Ab21)
Anti-mouse PD-1 antibody (G4) hybridoma cells were subjected to exon sequencing, and the following primers were designed based on the antibody heavy and light chain gene sequences, and the antibody heavy and light chain variable region encoding genes were cloned by RT-PCR. The primer sequences are as follows:
anti-murine PD-1 antibody heavy chain (H) upstream primer (SEQ ID NO. 1):
GGC CAC TTG CCA TGA ACT
anti-murine PD-1 antibody heavy chain (H) downstream primer (SEQ ID NO. 2):
TGT TGT TGT GGT TGA GGA GAC
anti-murine PD-1 antibody light chain (L) upstream primer (SEQ ID NO. 3):
CAG TTT GTG CTA ACT CAG CC
anti-murine PD-1 antibody light chain (L) downstream primer (SEQ ID NO. 4):
TGTGACTTTGGGAGAAGACTT
after cloning to obtain genes encoding the heavy (H) and light (L) chain variable regions of the antibody, the genes are linked by a linker: GGGTCCAGTGGCGGT, connecting the encoding genes of the antibody heavy chain (H) and light chain (L) variable regions to form an encoding gene sequence of the anti-PD-1 single-chain antibody mPD-1Ab, wherein the sequence is shown in SEQ ID NO. 5.
Further, the other is composed of 36 amino acids, and the coding nucleotide sequence is a linker of a linking sequence shown in SEQ ID NO. 6. The obtained PD-1 single-chain antibody (mPD-1Ab) with the sequence shown in SEQ ID NO.5 is linked with the mouse IL-21 coding gene sequence with the sequence shown in SEQ ID NO. 7. The fusion protein (mPD-1Ab21) of the anti-PD-1 single-chain antibody and IL-21 is obtained, and the sequence is shown in SEQ ID NO. 8.
For subsequent protein purification and detection, optionally, a coding gene sequence of tag protein 3xFlag consisting of 22 amino acids is added at the C end of the obtained fusion protein, and the nucleotide sequence of 3xFlag is shown as SEQ ID NO. 9.
Finally, the coding gene encoding the mouse fusion protein (mPD-1Ab21) was constructed, the nucleotide sequence is shown in SEQ ID NO.10, see FIG. 1.
Example 2 expression and purification of mouse PDSIL21 fusion protein
The genes encoding the mouse mPD-1Ab single-chain antibody and mPD-1Ab21 fusion protein constructed in example 1 were inserted into pTT3 expression vector, transfected into 293T cells, and cell culture supernatant was collected and purified using anti-Flag beads to obtain mouse mPD-1Ab and mPD-1Ab21 proteins.
SDS-PAGF electrophoresis demonstrated that the mouse mPD-1Ab and mPD-1Ab21 proteins were single protein bands, as shown in FIG. 2. Molecular volume exclusion chromatography analysis showed that the mPD-1Ab21 fusion protein was predominantly in the dimeric form.
EXAMPLE 3 anti-human PD-1 antibody preparation
A hybridoma cell 1G1 producing a high-affinity anti-human PD-1 antibody was obtained by screening a mouse immunized with a human PD-1-IgFc fusion protein (PD-1: NCBI Reference Sequence: NM-005018.2, IgFc: Gene ID: 3500). Referring to FIG. 3, the results show that the anti-PD-1 antibody specifically binds to a CHO cell strain overexpressing PD-1 and is able to completely block the binding of PD-L1-IgFc to cell surface PD-1.
Example 4 construction, expression and characterization of anti-human PD-1 antibody and human IL-21 fusion protein (PD-1Ab21)
By RT-PCR technique, respectively using:
anti-human PD-1 antibody heavy chain (H) upstream primer, M is degenerate primer (SEQ ID NO. 11):
GAA GTG MAG CTG GTG GAG TCT GG
anti-human PD-1 antibody heavy chain (H) downstream primer (SEQ ID NO. 12):
ATA GAC AGA TGG GGG TGT CGT TTT GGC
anti-human PD-1 antibody light chain (L) upstream primer, M/R is degenerate primer (SEQ ID NO. 13):
GAC ATT GTG CTG ACM CAR TCT CCA
anti-human PD-1 antibody light chain (L) downstream primer (SEQ ID NO. 14):
GGA TAC AGT TGG TGC AGC ATC
the genes encoding the variable regions of the heavy and light chains of the antibody (see FIG. 4B) were cloned from anti-PD-1 antibody hybridoma cells (1G1), the sequences being shown in SEQ ID NO.15 and SEQ ID NO. 16. The heavy chain and light chain variable regions of the cloned anti-PD-1 antibody are used for replacing corresponding segments of heavy chains and light chains of human immunoglobulin to synthesize complete genes for encoding the heavy chains and the light chains of the human immunoglobulin, and the sequence of the constructed chimeric heavy chains is shown as SEQ ID NO.17, and the sequence of the light chains is shown as SEQ ID NO. 18. Meanwhile, the end C of the heavy chain is linked with human IL-21 shown in SEQ ID NO.19 to construct a humanized anti-PD-1 antibody heavy chain and IL-21 fusion protein, and 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 gene are respectively constructed into pTT3 expression vector, and are co-infected with CHO cells, and the anti-PD-1 antibody and IL-21 fusion protein (also called PD-1Ab21) is purified by a proteinG column. See fig. 4A.
The ability of PD-1Ab21 to bind to PD-1 was tested by incubating stably PD-1 expressing CHO cells with PD-1Ab21 for 30min and then detecting the bound PD-1Ab21 on the cells with anti-Flag antibody, showing that PD-1Ab21 binds well to cell surface expressed PD-1, see FIG. 4C. The ability of PD-1Ab21 to block the binding of PD-1 to PD-L1 was tested by incubating CHO cells with PD-L1-IgFc for 30min (solid black line), or by incubating PD-1Ab21 with CHO cells for 30min followed by addition of PD-L1-IgFc and detection of bound PD-L1-IgFc on the cells with anti-IgFc antibody (dotted line). The results show that PD-1Ab21 completely blocked the binding of PD-1 to PD-L1, see FIG. 4D.
Example 5 functional characterization of mPD-1Ab21 fusion protein
Flow cytometry analysis demonstrated that mPD-1Ab21 can bind to cell surface-expressed PD-1 molecules and this binding can be completely blocked by anti-PD-1 antibodies, see FIG. 5A, which illustrates that mPD-1Ab21 binds to cell surface-expressed PD-1 as well as PD-L1 binds to cell surface PD-1. Similarly, referring to FIG. 5B, the results show that mPD-1Ab21 is capable of binding to activated T lymphocytes and blocking the binding of PD-L1 to PD-1 on the surface of activated T lymphocytes.
Example 6 characterization of IL-21 biological function of mPD-1Ab21 fusion protein
Baf3 cells (Baf3 cells are pre-B cell line) were plated in 96-well plates at 1X10 cells per well4And (4) cells. Mouse IL-21 and mPD-1Ab21 proteins were present according to different concentration gradients: 0.1ng/ml, 1ng/ml, 10ng/ml, were added to the corresponding cell wells, three duplicate wells per concentration, and wells without protein added served as control wells. On the third day of cell culture, the Baf3 cells were counted. Calculating the average value of the number of the cells in each protein concentration in the three multiple wells, and calculating the cell proliferation rate, wherein the calculation formula is as follows: the cell proliferation rate was 100% (number of cells in experimental group/number of cells in control group-1). As can be seen from the results in FIG. 6, mPD-1Ab21 has the same biological activity of promoting B cell proliferation as recombinant mIL-21.
As can be seen from examples 5 and 6, mPD-1Ab21 fusion protein prepared by the invention has the function of blocking the binding of PD-L1 and PD-1 by an anti-PD-1 antibody and also has the biological activity of IL-21.
Example 7mPD-1Ab21 Induction of memory dryness CD8+T cell differentiation
The invention further researches the activation of CD8 by mPD-1Ab21+T cell differentiation was performed by activating OT-1 transgenic mouse T cells with OVA antigen peptide stimulation in vitro for 48 hours, then culturing and differentiating in a medium containing IL-2, mIL-21, mPD-1Ab or mPD-1Ab21, respectively, and analyzing the T cell differentiation by flow cytometry after 2 days.
The results are shown in FIG. 7, which shows: IL-2 promotes activated CD8+T cell to CD44highCD62LlowDifferentiation of effector or memory effector T cells, mIL-21 Induction of activated CD8+T cell to CD44lowCD62LhighMemory Stem T cell differentiation, mPD-1Ab alone vs activated CD8+T cell differentiation was not affected, mPD-1Ab in combination with mIL-21 had the same effect of inducing CD44 as mIL-21 alonelowCD62LhighThe activity of memory stem T cell differentiation is improved, and mPD-1Ab21 has stronger induction of D44lowCD62LhighThe biological role of memory-drying T cell differentiation.
Examples8 mPD-1Ab21 is a CD44 inducer by promoting cell proliferationlowCD62LhighMemory-drying T cell differentiation
To distinguish D44 induced differentiation of mPD-1Ab21lowCD62LhighMemory-drying T cells are derived from primitive cells that have not been activated for proliferation
CD44lowCD62LhighT cells, also activated T cells, were induced to differentiate into CD44 by the fusion protein mPD-1Ab21lowCD62LhighMemory of T cells in the Dry form, we labeled T cells of OT-1 transgenic mice with CSFE, followed by activation and differentiation under different conditions, and the results are shown in FIG. 8, CD44 differentiated in medium alonelowCD62LhighCell population proliferation was significantly lower than CD44highCD62LhighA population of T cells. mPD-1Ab21 induced differentiated CD44lowCD62LhighCell populations also had significant proliferation, with CD44highCD62LhighThere was no difference in the proliferation of the T cell population. CD44 indicating that mPD-1Ab21 induces differentiationlowCD62LhighThe cells are differentiated by proliferation from activated cells.
Example 9 mPD-1Ab21 Induction of activated CD44highCD62LhighCell differentiation back to CD44lowCD62LhighPrimitive cell phenotype
To further demonstrate that mPD-1Ab21 induces differentiated CD44lowCD62LhighThe cells are activated by CD44highCD62LhighT cell differentiation back to CD44lowCD62LhighCell population, CFSE-labeled OT-1 transgenic mouse T cells activated with OVA antigen peptide, activated CD44 purified by flow sortinghighCD62LhighT cell populations are then differentiated by the addition of cytokines or fusion proteins. Referring to fig. 9, the results show that: IL-2-induced activation of CD44highCD62LhighT cell to CD44highCD62LlowEffector T cell differentiation, while mPD-1Ab21 induces activated CD44highCD62LhighT cell to CD44lowCD62LhighMemory-drying T cell differentiation. And, with CD44highCD62LhighProliferation of T cell population compared to mPD-1Ab21 induced differentiated CD44lowCD62LhighMemory algebraic ratio of proliferation and division of dry T cells CD44highCD62LhighThe number of divisions of the T cell population is greater. Further, mPD-1Ab21 induced differentiated CD44lowCD62LhighThe cells are activated cells, and the cells are returned to CD44 through proliferation and differentiationlowCD62LhighMemory dry T cells.
Example 10 memory dryness induced by mPD-1Ab21 CD8+Functional characterization of T cells
To verify that mPD-1Ab21 induced differentiation of CD44lowCD62LhighBiological functional properties of cells, we cultured OT-1 transgenic mouse T cells induced to differentiate for 2-3 days by mPD-1Ab21 or IL-2 for 2 days in a medium containing IL-7, and then stimulated with anti-CD 3 to detect cytokine production, see FIG. 10, which shows: IL-2 induces differentiated cells to produce a large amount of IFN-gamma, and hardly expresses IL-2; mPD-1Ab21 induced the differentiated cells to produce a large amount of IL-2, and IFN-gamma expression was significantly lower than that of IL-2 induced differentiated cells. It shows that IL-2 induced differentiated cells have strong effector functions, while mPD-1Ab21 induced differentiated cells have stronger proliferation and differentiation capacities.
Example 11 memory-Dry T cells induced by mPD-1Ab21 have a stronger memory response (call response) and memory cell-forming ability in mice
To further verify the in vivo biological functional properties of mPD-1Ab 21-induced differentiated cells, we intravenously transfused mPD-1Ab21 or IL-2-induced differentiated OT-1 transgenic mouse T cells into C57 mice, injected OVA antigen peptide and Freund's complete adjuvant the next day for antigen stimulation, and then monitored the in vivo OT-1 transgenic mouse T cell responses, showing: following antigen stimulation, mPD-1Ab21 induced a 4-fold higher differentiated T cell response than IL-2 induced. After one month, the number of T cells in OT-1 transgenic mice in mice fed with mPD-1Ab21 differentiated cells was 2 times that in mice fed with IL-2 differentiated cells, as shown in FIG. 11, which illustrates that mPD-1Ab21 induces differentiated cells to have stronger memory response capability and capability of forming memory T cells in vivo.
Example 12
mPD-1Ab21 targeting PD-1+T cell promotion of CD44lowCD62LhighMemory-drying T cell differentiation
Example 7 shows that: mPD-1Ab21 induces CD44lowCD62LhighThe capacity of memory-drying T cells to differentiate is significantly higher than that of recombinant mIL-21 or mPD-1Ab and mIL-21. To verify whether mPD-1Ab21 could be activated by targeting IL-21 to activated PD-1+T cells, thereby promoting T cell differentiation, the present assay compares mPD-1Ab21 and mIL-21 induction of CD44lowCD62LhighThe dose-effect relationship of dry T cell differentiation was memorized. T cells of OT-1 transgenic mice are activated in vitro, then are induced to differentiate by recombinant mIL-21 or mPD-1Ab21 fusion proteins with different concentrations, and CD44 is analyzed by flow cytometrylowCD62LhighThe proportion of cells. Referring to fig. 12A, the results show that: mPD-1Ab21 fusion protein induces CD44lowCD62LhighThe activity of cell differentiation was more than 3 times higher than mIL-21. To further analyze the expression of PD-1 by T cells, CD44 was induced at mPD-1Ab21lowCD62LhighThe effect in cell differentiation is that normal mouse T cells or PD-1 gene knockout mouse T cells are stimulated and activated in vitro by anti-CD 3 and CD8 antibodies, then the differentiation is induced by recombinant IL-21 or mPD-1Ab21 fusion proteins with different concentrations, and CD44 is analyzed by flow cytometrylowCD62LhighThe proportion of cells. Referring to fig. 12B, the results show that: for normal mouse T cells, mPD-1Ab21 induced CD44lowCD62LhighThe capacity of memory stem T cell differentiation is obviously higher than that of recombinant IL-21; while for PD-1 knock-out mouse T cells, mPD-1Ab21 induced CD44lowCD62LhighThe capacity of memory-drying T cells to differentiate was slightly better than recombinant IL-21. This result illustrates that: mPD-1Ab21 targets IL-21 to activated T cells by binding to PD-1 on the surface of activated T cells, thereby promoting CD44lowCD62LhighMemory-drying T cell differentiation.
Example 13 mPD-1Ab21 Rapid targeting of activated tumor-specific T cells in peripheral blood and tumor tissue
Mice were inoculated with OVA antigen-expressing tumor cells B16-OVA, OVA-specific OT-1 primary T cells were infused into the mice one day prior to inoculation, mice were immunized subcutaneously with the adjuvant poly I C and OVA antigen peptides six days after inoculation, and mice were given intraperitoneal injections of mPD-1Ab21 or anti-PD-1 antibody the ninth day. The binding capacity of tumor-specific T cells to anti-PD-1 antibodies in peripheral blood and intratumoral infiltration was flow analyzed at different time points (30min, 6h, 12h, 24h and 48h) after protein administration to reflect the binding of mPD-1Ab21 protein or anti-PD-1 antibody to tumor-specific OT-1T cells in vivo. Referring to FIG. 13, the results show that the surface of activated OT-1T cells in peripheral blood and tumors of immunized mice highly express PD-1 molecules. After intraperitoneal injection of anti-PD-1 antibody or mPD-1Ab21 protein for 30 minutes, OT-1T cell surface PD-1 flow antibody staining in peripheral blood was blocked, indicating that both mPD-1Ab21 and anti-PD-1 antibody injected into the body can rapidly bind to tumor-specific T cell surface. The binding of the fusion protein or antibody to the tumor specific T cells in the tumor tissue is slower than that of peripheral blood, partial blocking starts at 6 hours, and PD-1 flow antibody staining is completely blocked at 12 hours, which indicates that the fusion protein or antibody is bound to the surface of the tumor specific T cells. In peripheral blood and tumor tissue, the binding of the fusion protein to OT-1T cells lasted for at least 24 hours, and only partial binding of the anti-PD-1 antibody was observed in tumor tissue after 48 hours. The results demonstrate that mPD-1Ab21 fusion protein can rapidly bind to activated tumor-specific T cells in peripheral blood and tumor tissue after injection into vivo.
Example 14 antitumor Effect of mPD-1Ab21 fusion protein
The mice are inoculated with mouse intestinal cancer tumor cells CT26 subcutaneously, after 3 days, the mice are injected with mPD-1Ab21 fusion protein, mPD-1Ab combined mIL-21 recombinant protein or anti-PD-1 antibody to treat, the tumor size is detected at regular time, and a tumor growth curve is drawn. Referring to fig. 14, the results show that: the mPD-1Ab and mIL-21 combination treatment only slightly slowed tumor growth in mice compared to untreated control mice, whereas anti-PD-1 antibody and mPD-1Ab21 fusion protein treatment resulted in complete tumor regression in 20% and 40% of mice, respectively. The results show that mPD-1Ab21 has more obvious tumor treatment effect compared with the combination treatment of mPD-1Ab and mIL-21, and the treatment effect is better than that of an anti-PD-1 antibody.
Example 15 combination therapy of mPD-1Ab21 significantly increases the anti-Her 2/neu (herceptin) vs. Her2+Therapeutic action of Breast cancer
Subcutaneous inoculation of mice with Her2/neu+And (3) injecting an anti-Her 2/neu antibody, or an anti-Her 2/neu antibody combined mPD-1Ab21 fusion protein, an anti-Her 2/neu antibody combined mPD-1Ab and mIL-21 into the abdominal cavity of the mouse after 16 days of breast cancer Tubo for treatment, detecting the size of the tumor at regular time, and drawing a tumor growth curve. Referring to fig. 15, the results show that: anti-Her 2/neu antibody treatment alone caused 40% tumor regression in mice, anti-Her 2/neu antibody treatment with mPD-1Ab and mIL-21 caused complete tumor regression in 60% of mice, and anti-Her 2/neu antibody treatment with mPD-1Ab21 caused complete tumor regression in all mice. The results show that the mPD-1Ab21 combination treatment can remarkably increase the tumor treatment effect of Her2/neu (herceptin), and is remarkably superior to the combination treatment with mPD-1Ab and mIL-21.
Example 16 combination treatment of mPD-1Ab21 significantly increases the therapeutic effect of the tumor vaccine on B16-OVA tumors
Tumor vaccines are an important future approach for tumor therapy, especially in combination with PD-1 inhibitors. The mPD-1Ab21 fusion protein targets IL-21 to tumor-specific T cells activated by the tumor vaccine through PD-1, promotes the formation of memory T cells, and can remarkably promote the tumor treatment effect of the tumor vaccine compared with a PD-1 inhibitor. For this purpose, mice were inoculated with OVA antigen-expressing tumor cells B16-OVA, OVA-specific OT-1 primary T cells were introduced into the mice one day before inoculation, subcutaneous immunization with the adjuvant poly I, C and OVA antigen peptides was administered to the mice six days after inoculation, intraperitoneal injection of mPD-1Ab21 fusion protein, mPD-1Ab in combination with mIL-21 recombinant protein, or anti-PD-1 antibody into the mice was started the ninth day for treatment, tumor size was periodically examined, and a tumor growth curve was plotted. Referring to fig. 16, the results show that: tumor antigen immunization is carried out on tumor-bearing mice, tumor growth can be obviously inhibited, the combination of mPD-1Ab and mIL-21 or anti-PD-1 antibody does not obviously increase the treatment effect of the tumor vaccine, but the combined application of mPD-1Ab21 can greatly improve the anti-tumor effect of the tumor vaccine.
Example 17mPD-1Ab21 treatment significantly increases memory dryness CD8+T cell proportion and tumor specific T cell response
The ratio of each T cell subset in the treated CT26 tumor-bearing mice was analyzed, and referring to FIG. 17A, it was found that mPD-1Ab21 treated mice had memory dryness in spleen and tumor draining lymph node (CD 62L)highCD44lowCD122+Scal-1+)CD8+The T cell ratio was significantly higher than that of mice treated with anti-PD-1 antibody or mPD-1Ab in combination with mIL-21.
Analysis of B16-OVA tumor-bearing mice treated with the above vaccine, tumor vaccine treatment stimulated in vivo tumor-specific T cell activation and proliferation, resulting in spleen, especially tumor-specific CD8 in tumor-bearing mice+The proportion of T cells increases significantly. The anti-PD-1 antibody combination therapy does not further increase the proportion of tumor-specific CD8+ T cells, but mPD-1Ab21 greatly improves the tumor-specific T cell response in a vaccine-treated mouse, so that the tumor-specific CD8+The proportion of T cells reaching intratumoral CD8 +80% of T cells. More importantly, anti-PD-1 antibody combination therapy prompted tumor-specific CD8 compared to vaccine-alone treated tumors+T cell effector (CD 127)lowKLRG1high) Differentiation, whereas combination therapy with mPD-1Ab21 significantly promoted tumor-specific CD8+T cell memory effect (CD 127)highKLRG1low) And (4) differentiation. See fig. 17B and 17C.
These findings demonstrate that mPD-1Ab21 treatment, in addition to blocking PD-1 immunosuppressive signaling, also promotes memory anti-tumor T cell responses, thereby greatly enhancing tumor therapeutic efficacy.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.
In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.
Sequence listing
<110> Wang Sheng Dian
Plum glume
Ni Suan Yan cluster
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<400>10
atgaactcag gactcaaatt ggttttcttt gtccttattc taaaaggtgt ccagtgtgag 60
gtgcagctgg tggagtctgg gggaggctta gcacaacctg gaaagtccct gaaactctcc 120
tgtgaggcct ctggattcac cttcagtgac tatggcatga actggttccg tcaggctcca 180
gggaaggggc tggagtgggt cgcatatatt agtagtggta gtaataagat cacatatgcc 240
gacactgtga agggccggtt caccgtctcc agagacaacg gcaagaacca actgttcctg 300
caaatgaaca atctcaagtc tgaggacaca gccatttatt actgtgtgga tagcggattt 360
aattcttact ctgatgtctg gggccaggga atccaggtca ccgtctcctc aaccacaaca 420
acagggtcca gtggcggtca gtttgtgcta actcagccaa agactgtgtc agagtctctg 480
gggagaacag tcaccatctc ctgcaaacgc agcagtggca gcgttgggga ctactatgtc 540
agctggcacc agcagcgctt tggaagctct cccaaaactg tgatctatct tgatgatgag 600
agaccatctg gggttcctaa aaggttctct ggctccattg acagctcatc caactcagcc 660
tcactgacca tcactgatct gcagactgac gatgaggctg actacttttg tctctcttat 720
gatagtaaca atcactttgt ttttggcagc ggaacccacc tcaccgtcct aggtggaccc 780
aagtcttctc ccaaagtcac aggtggttct ggcggcggtt ctgaaggtgg cggctccgaa 840
ggcggcggca gcgagggcgg tggtagcgaa ggtggtggct ccgagggtgg cggttccggc 900
ggcggtagcc aagggccaga tcgcctcctg attagacttc gtcaccttat tgacattgtt 960
gaacagctga aaatctatga aaatgacttg gatcctgaac ttctatcagc tccacaagat 1020
gtaaaggggc actgtgagca tgcagctttt gcctgttttc agaaggccaa actcaagcca 1080
tcaaaccctg gaaacaataa gacattcatc attgacctcg tggcccagct caggaggagg 1140
ctgcctgcca ggaggggagg aaagaaacag aagcacatag ctaaatgccc ttcctgtgat 1200
tcgtatgaga aaaggacacc caaagaattc ctagaaagac taaaatggct ccttcaaaag 1260
atgattcatc agcatctctc cgactacaaa gaccatgacg gtgattataa agatcatgac 1320
atcgattaca aggatgacga tgacaagtag 1350
<210>11
<211>23
<212>DNA
<213>artificially?synthesized
<400>11
gaagtgmagc tggtggagtc tgg 23
<210>12
<211>27
<212>DNA
<213>artificially?synthesized
<400>12
atagacagat gggggtgtcg ttttggc 27
<210>13
<211>24
<212>DNA
<213>artificially?synthesized
<400>13
gacattgtgc tgacmcartc tcca 24
<210>14
<211>21
<212>DNA
<213>artificially?synthesized
<400>14
ggatacagtt ggtgcagcat c 21
<210>15
<211>384
<212>DNA
<213>HUM
<400>15
gaagtgcagc tggtggagtc tgggggaggt ttagtgcagc ctggagggtc cctgaaactc 60
tcctgtgcag cctctggatt cactttcact agctatacca tgtcttgggt tcgccagact 120
cccgagaaga ggctggaatg ggtcgcattc attagtggtg gtggtggtga cacctactat 180
ccagacactg taaagggccg attcaccatc tccagagaca atgccaagaa caccctgtat 240
ttgcaaatga gcagtctgaa gtctgaggac acggccatgt attactgtgc aaggcatggt 300
tacgacggga cctggtttgc ttactggggc caagggactc tggtcactgt ctctgcagcc 360
aaaacgacac ccccatctgt ctat 384
<210>16
<211>342
<212>DNA
<213>HUM
<400>16
gtgctgaccc agtctccagc ctccctatct gcatctgtgg gagaaactgt caccatcaca 60
tgtcgagcaa gtgagaatat ttacagttat ttagcatggt atcagcagaa acagggaaaa 120
tctcctcaac tcctggtctc taatgcaaaa accttagcag agggtgtgcc atcaaggttc 180
agtggcagtg gatcaggcac acagttttct ctgaagatca acagcctgca gcctgaagat 240
tttgggagtt attactgtca acatcattat gctactccgt acacgttcgg gggggggacc 300
aagctggaaa taaaacgggc tgatgctgca ccaactgtat cc 342
<210>17
<211>1400
<212>DNA
<213>HUM
<400>17
atggcatgcc ctggcttcct gtgggcactt gtgatctcca cctgtcttga attttccatg 60
gccgaagtgc agctggtgga gtctggggga ggtttagtgc agcctggagg gtccctgaaa 120
ctctcctgtg cagcctctgg attcactttc actagctata ccatgtcttg ggttcgccag 180
actcccgaga agaggctgga atgggtcgca ttcattagtg gtggtggtgg tgacacctac 240
tatccagaca ctgtaaaggg ccgattcacc atctccagag acaatgccaa gaacaccctg 300
tatttgcaaa tgagcagtct gaagtctgag gacacggcca tgtattactg tgcaaggcat 360
ggttacgacg ggacctggtt tgcttactgg ggccaaggga ctctggtcac tgtctctgca 420
gcaagcttca agggcccatc ggtcttcccc ctggtgccct gctccaggag cacctccgag 480
agcacagccg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 540
tggaactcat gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 600
ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacgaagacc 660
tacacctgca acgtagatca caagcccagc aacaccaagg tggacaagag agttgagtcc 720
aaatatggtc ccccatgccc atcatgccca gcacctgagt tcctgggggg accatcagtc 780
ttcctgttcc ccccaaaacc caaggacact ctcatgatct cccggacccc tgaggtcacg 840
tgcgtggtgg tggacgtgag ccaggaagac cccgaggtcc agttcaactg gtacgtggat 900
ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagttcaa cagcacgtac 960
cgtgtggtca gggtcctcac cgtcctgcac caggactggc tgaacggcaa ggagtacaag 1020
tgcaaggtct ccaacaaagg cctcccgtcc tccatcgaga aaaccatctc caaagccaaa 1080
gggcagcccc gagagccaca ggtgtacacc ctgcccccat cccaggagga gatgaccaag 1140
aaccaggtca gcctgacctg cctggtcaaa ggcttctacc ccagcgacat cgccgtggag 1200
tgggagagca atgggcagcc ggaggacaac tacaagacca cgcctcccgt gctggactcc 1260
gacggctcct tcttcctcta cagcaggcta accgtggaca agagcaggtg gcaggagggg 1320
aatgtcttct catgctccgt gatgcatgag gctctgcaca accactacac acagaagagc 1380
ctctccctgt ctccgggtaa 1400
<210>18
<211>811
<212>DNA
<213>HUM
<400>18
atgaaatacc tattgcctac ggcagccgct ggattgttat tactcgcggc ccagccggcc 60
atggccgaaa ttgtgttgac gcagcttcca gccaccctgt ctttgtctgt gggagaaact 120
gtcaccatca catgtcgagc aagtgagaat atttacagtt atttagcatg gtatcagcag 180
aaacagggaa aatctcctca actcctggtc tctaatgcaa aaaccttagc agagggtgtg 240
ccatcaaggt tcagtggcag tggatcaggc acacagtttt ctctgaagat caacagcctg 300
cagcctgaag attttgggag ttattactgt caacatcatt atgctactcc gtacacgttc 360
ggcggaggga ccaaggtgga gatcaaacga actgtggctg caccatctgt cttcatcttc 420
ccgccatctg atgagcagtt gaaatctgga actgcctctg ttgtgtgcct gctgaataac 480
ttctatccca gagaggccaa agtacagtgg aaggtggata acgccctcca atcgggtaac 540
tcccaggaga gtgtcacaga gcaggacagc aaggacagca cctacagcct cagcagcacc 600
ctgacgctga gcaaagcaga ctacgagaaa cacaaagtct acgcctgcga agtcacccat 660
cagggcctga gctcgcccgt cacaaagagc ttcaacaggg gagagtgttc ggcgcgccag 720
tcgactccat tcgtttgtga atatcaaggc caatcgtctg acctgcctca acctcctgtc 780
aatgctggcg gcggctctgg tggtggttct g 811
<210>19
<211>399
<212>DNA
<213>HUM
<400>19
caaggtcaag atcgccacat gattagaatg cgtcaactta tagatattgt tgatcagctg 60
aaaaattatg tgaatgactt ggtccctgaa tttctgccag ctccagaaga tgtagagaca 120
aactgtgagt ggtcagcttt ttcctgcttt cagaaggccc aactaaagtc agcaaataca 180
ggaaacaatg aaaggataat caatgtatca attaaaaagc tgaagaggaa accaccttcc 240
acaaatgcag ggagaagaca gaaacacaga ctaacatgcc cttcatgtga ttcttatgag 300
aaaaaaccac ccaaagaatt cctagaaaga ttcaaatcac ttctccaaaa gatgattcat 360
cagcatctgt cctctagaac acacggaagt gaagattcc 399
<210>20
<211>1845
<212>DNA
<213>artificially?synthesized
<400>20
atggcatgcc ctggcttcct gtgggcactt gtgatctcca cctgtcttga attttccatg 60
gccgaagtgc agctggtgga gtctggggga ggtttagtgc agcctggagg gtccctgaaa 120
ctctcctgtg cagcctctgg attcactttc actagctata ccatgtcttg ggttcgccag 180
actcccgaga agaggctgga atgggtcgca ttcattagtg gtggtggtgg tgacacctac 240
tatccagaca ctgtaaaggg ccgattcacc atctccagag acaatgccaa gaacaccctg 300
tatttgcaaa tgagcagtct gaagtctgag gacacggcca tgtattactg tgcaaggcat 360
ggttacgacg ggacctggtt tgcttactgg ggccaaggga ctctggtcac tgtctctgca 420
gcaagcttca agggcccatc ggtcttcccc ctggtgccct gctccaggag cacctccgag 480
agcacagccg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 540
tggaactcat gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 600
ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacgaagacc 660
tacacctgca acgtagatca caagcccagc aacaccaagg tggacaagag agttgagtcc 720
aaatatggtc ccccatgccc atcatgccca gcacctgagt tcctgggggg accatcagtc 780
ttcctgttcc ccccaaaacc caaggacact ctcatgatct cccggacccc tgaggtcacg 840
tgcgtggtgg tggacgtgag ccaggaagac cccgaggtcc agttcaactg gtacgtggat 900
ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagttcaa cagcacgtac 960
cgtgtggtca gggtcctcac cgtcctgcac caggactggc tgaacggcaa ggagtacaag 1020
tgcaaggtct ccaacaaagg cctcccgtcc tccatcgaga aaaccatctc caaagccaaa 1080
gggcagcccc gagagccaca ggtgtacacc ctgcccccat cccaggagga gatgaccaag 1140
aaccaggtca gcctgacctg cctggtcaaa ggcttctacc ccagcgacat cgccgtggag 1200
tgggagagca atgggcagcc ggaggacaac tacaagacca cgcctcccgt gctggactcc 1260
gacggctcct tcttcctcta cagcaggcta accgtggaca agagcaggtg gcaggagggg 1320
aatgtcttct catgctccgt gatgcatgag gctctgcaca accactacac acagaagagc 1380
ctctccctgt ctccgggtaa aggtggtggt ggttctggtg gtggtggttc tggcggcggc 1440
ggctcccaag gtcaagatcg ccacatgatt agaatgcgtc aacttataga tattgttgat 1500
cagctgaaaa attatgtgaa tgacttggtc cctgaatttc tgccagctcc agaagatgta 1560
gagacaaact gtgagtggtc agctttttcc tgctttcaga aggcccaact aaagtcagca 1620
aatacaggaa acaatgaaag gataatcaat gtatcaatta aaaagctgaa gaggaaacca 1680
ccttccacaa atgcagggag aagacagaaa cacagactaa catgcccttc atgtgattct 1740
tatgagaaaa aaccacccaa agaattccta gaaagattca aatcacttct ccaaaagatg 1800
attcatcagc atctgtcctc tagaacacac ggaagtgaag attcc 1845
<210>21
<211>1914
<212>DNA
<213>artificially?synthesized
<400>21
atggcatgcc ctggcttcct gtgggcactt gtgatctcca cctgtcttga attttccatg 60
gccgaagtgc agctggtgga gtctggggga ggtttagtgc agcctggagg gtccctgaaa 120
ctctcctgtg cagcctctgg attcactttc actagctata ccatgtcttg ggttcgccag 180
actcccgaga agaggctgga atgggtcgca ttcattagtg gtggtggtgg tgacacctac 240
tatccagaca ctgtaaaggg ccgattcacc atctccagag acaatgccaa gaacaccctg 300
tatttgcaaa tgagcagtct gaagtctgag gacacggcca tgtattactg tgcaaggcat 360
ggttacgacg ggacctggtt tgcttactgg ggccaaggga ctctggtcac tgtctctgca 420
gcaagcttca agggcccatc ggtcttcccc ctggtgccct gctccaggag cacctccgag 480
agcacagccg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 540
tggaactcat gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 600
ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacgaagacc 660
tacacctgca acgtagatca caagcccagc aacaccaagg tggacaagag agttgagtcc 720
aaatatggtc ccccatgccc atcatgccca gcacctgagt tcctgggggg accatcagtc 780
ttcctgttcc ccccaaaacc caaggacact ctcatgatct cccggacccc tgaggtcacg 840
tgcgtggtgg tggacgtgag ccaggaagac cccgaggtcc agttcaactg gtacgtggat 900
ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagttcaa cagcacgtac 960
cgtgtggtca gggtcctcac cgtcctgcac caggactggc tgaacggcaa ggagtacaag 1020
tgcaaggtct ccaacaaagg cctcccgtcc tccatcgaga aaaccatctc caaagccaaa 1080
gggcagcccc gagagccaca ggtgtacacc ctgcccccat cccaggagga gatgaccaag 1140
aaccaggtca gcctgacctg cctggtcaaa ggcttctacc ccagcgacat cgccgtggag 1200
tgggagagca atgggcagcc ggaggacaac tacaagacca cgcctcccgt gctggactcc 1260
gacggctcct tcttcctcta cagcaggcta accgtggaca agagcaggtg gcaggagggg 1320
aatgtcttct catgctccgt gatgcatgag gctctgcaca accactacac acagaagagc 1380
ctctccctgt ctccgggtaa aggtggtggt ggttctggtg gtggtggttc tggcggcggc 1440
ggctcccaag gtcaagatcg ccacatgatt agaatgcgtc aacttataga tattgttgat 1500
cagctgaaaa attatgtgaa tgacttggtc cctgaatttc tgccagctcc agaagatgta 1560
gagacaaact gtgagtggtc agctttttcc tgctttcaga aggcccaact aaagtcagca 1620
aatacaggaa acaatgaaag gataatcaat gtatcaatta aaaagctgaa gaggaaacca 1680
ccttccacaa atgcagggag aagacagaaa cacagactaa catgcccttc atgtgattct 1740
tatgagaaaa aaccacccaa agaattcctagaaagattca aatcacttct ccaaaagatg 1800
attcatcagc atctgtcctc tagaacacac ggaagtgaag attccgacta caaagaccat 1860
gacggtgatt ataaagatca tgacatcgat tacaaggatg acgatgacaa gtag 1914
Claims (12)
1. A fusion protein comprising a cytokine and a T cell surface receptor molecule binding antagonist or agonist linked by a linker, wherein,
the cytokine is one selected from IL-21, IL-2, IL-15, IL-7 or IL-12;
the T cell surface receptor molecule binding antagonist or agonist is selected from one of a PD-1 binding antagonist, a LAG-3 binding antagonist, a Tim-3 binding antagonist or a CD137(4-1BB) binding agonist;
preferably, the fusion protein can induce and activate T cells, and simultaneously target cytokines to the T cells, induce the differentiation of the T cells and enhance the functions of the T cells.
2. The fusion protein of claim 1, wherein,
the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1; and/or
The PD-1 binding antagonist inhibits the binding of PD-1 to both PD-L1 and PD-L2;
preferably, 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 dimeric single chain antibody.
3. The fusion protein of claim 1 or 2, wherein the domain comprises the following structure from amino-terminus to carboxy-terminus:
-X-Linker1-Y-
wherein:
x is a PD-1 binding antagonist, Y is a 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 amino terminus to carboxy terminus:
VH-Linker2-VL
wherein, VHIs the variable region of the heavy chain of a PD-1 single-chain antibody, VLIs the variable region of the antibody light chain, Linker2 is the Linker;
further preferably, the fusion protein may further include a tag sequence at the 3' end.
4. The fusion protein of any of the preceding claims,
the PD-1 binding antagonist has a nucleotide sequence shown as SEQ ID NO.5 or SEQ ID NO. 17; and/or
The cell factor is IL-21, and the 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 to tumor-specific T cells, inducing differentiation of the T cells.
5. The fusion protein of any of the preceding claims, wherein the nucleotide sequence encoding the fusion protein is as set forth in SEQ ID No.8 or SEQ ID No. 20;
further preferably, the fusion protein can also comprise a tag sequence at the 3' end, and the nucleotide sequence which contains the tag sequence and encodes the fusion protein is shown as SEQ ID NO.10 or SEQ ID NO. 21;
preferably, the fusion protein is predominantly in the form of a homodimer.
6. A polynucleotide encoding the fusion protein of any one of claims 1-5.
7. An expression vector comprising the polynucleotide of claim 6 operably linked to an expression control sequence.
8. A host cell comprising the expression vector of claim 7.
9. A method of producing a fusion protein according to any one of claims 1 to 5, comprising the steps of:
a) linking the cytokine coding gene to the C terminal of the PD-1 binding antagonist coding gene by a genetic engineering method to form a coding gene of a fusion protein;
b) constructing an expression vector;
c) transfecting cells, and performing protein expression and purification;
d) obtaining the fusion protein.
10. A pharmaceutical composition comprising one or more fusion proteins of any one of claims 1-5, and a pharmaceutically acceptable carrier, diluent, or excipient;
and/or, the pharmaceutical composition also comprises other anti-cancer drugs or tumor vaccines, and the fusion protein can be used in combination with other anti-cancer drugs or tumor vaccines;
preferably, the other anti-cancer drug is Her2 antibody;
more preferably, the other anticancer drug is herceptin.
11. Use for the manufacture of a medicament or kit for the prevention or treatment of cancer comprising administering to the individual an effective amount of the fusion protein of any one of claims 1-5;
preferably, the cancer is selected from the group consisting of 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, intestinal 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 of any one of claims 1-5 is administered continuously or intermittently;
further preferred, the fusion protein is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally.
12. A method for preventing or treating cancer in an individual comprising administering to the individual an effective amount of the fusion protein of any one of claims 1-5;
preferably, the cancer is selected from the group consisting of 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, intestinal 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 of any one of claims 1-5 is administered continuously or intermittently;
further preferred, the fusion protein is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally.
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| CN113913379B (en) * | 2020-07-07 | 2024-02-06 | 深圳市菲鹏生物治疗股份有限公司 | T lymphocyte and application thereof |
| WO2023048516A1 (en) * | 2021-09-24 | 2023-03-30 | 바이오엔시스템스 주식회사 | Fusion protein dimer including pd-1 and il-21, and use thereof |
| CN114106198A (en) * | 2021-11-11 | 2022-03-01 | 武汉大学 | PD 1antibody and CCL7 fusion protein and preparation method and application thereof |
| WO2023104134A1 (en) * | 2021-12-09 | 2023-06-15 | Genequantum Healthcare (Suzhou) Co., Ltd. | Antibody-cytokine fusion proteins and applications thereof |
| WO2023116637A1 (en) * | 2021-12-21 | 2023-06-29 | 深圳市菲鹏生物治疗股份有限公司 | Transgenic immune cell, construction method therefor, and use thereof |
| CN115806627A (en) * | 2022-08-03 | 2023-03-17 | 深圳市先康达生命科学有限公司 | Autocrine IL-15 and anti-LAG3 combined fusion protein and application thereof |
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| CN110894241B (en) | 2022-01-18 |
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