WO2023011575A1 - Multifunctional fusion protein drug targeting and killing pathogens and/or tumor cells - Google Patents

Abstract

Provided are a fusion protein containing a recognition function domain, a recruitment function domain, an activation function domain, and an auxiliary killing function domain, a construction method therefor and an application thereof in the field of biomedicines. The fusion protein can recognize pathogens and mobilize an immune system to kill the pathogens, and has important application value for new drug research and development for treating pathogen infection or tumor cells.

Description

A multifunctional fusion protein drug targeting and killing pathogens and/or tumor cells technical field

The invention relates to the technical field of drug development, in particular to a multifunctional fusion protein drug targeting and killing pathogens and/or tumor cells.

Background technique

Microbial infections including pathogenic viruses, bacteria, fungi and some protists cause a large number of deaths every year. Although there is an immune system in the body that can completely or partially resist the infection of these pathogenic microorganisms, when the human body’s immunity declines, it cannot effectively clear the infection of the pathogen, causing the pathogen to multiply in the body, destroying healthy tissues and organs in various parts of the body, and directly life threatening. At the same time, some pathogenic bacteria are covered with capsules, which can avoid the recognition and attack of immune cells.

Cryptococcosis (torulosis) is a subacute or chronic infectious disease, caused by Cryptococcus neoformans, mainly invading the central nervous system. In recent years, fungal meningitis, brain abscess and granuloma have become rare. Intracranial diseases are confused and delayed treatment, so the mortality rate is high. The disease can also involve the lungs, skin, subcutaneous tissue, epiphysis, joints, and other internal organs and tissues, and can occur at any age. The human body is often exposed to environments containing various cryptococci, and the human body's immunity to cryptococci includes cellular immunity and humoral immunity. Macrophages, neutrophils, lymphocytes, natural killer cells play an important role. Humoral immunity includes: anti-capsular polysaccharide antibodies and complement participate in opsonophagocytosis, assisting phagocytes to phagocytose Cryptococcus. Only when the body's resistance is reduced, pathogenic bacteria can easily invade the human body and cause disease.

Amoeba infectious disease is an infectious disease caused by amoeba protozoa that grows in water and can erode the human brain. Amoeba will enter the human body through the nose, drill into the skull, and erode brain tissue, resulting in fatality. It is also due to the decline of the body's immunity that it is easy to make people fall ill.

After pathogens enter the body, they will induce specific immunity including cellular immunity and humoral immunity. Some pathogenic bacteria are not easy to be recognized by the human body, so they cannot be attacked. The recognition and killing effect will be greatly reduced, so a drug is needed to enhance this recognition and killing effect. For example, cryptococcal infection, amoeba infection and other pathogen infection diseases currently have no effective drug treatment. Therefore, it is particularly important to invent a new drug to combat the infection of pathogens.

In the prior art, such as the non-patent literature "Single human B cell-derived monoclonal anti-Candida antibodies enhance phagocytosis and protect against disseminated candidiasis" mainly uses antibodies to identify Candida, and utilizes the Fc structure to recruit immune cells and complement components to attack Invasive pathogenic bacteria, but not applied in vivo for pathogen or tumor cell therapy.

Contents of the invention

In order to overcome the deficiencies in the prior art, in the present invention, by constructing a fusion protein, the pathogen and/or tumor cell recognition functional domain, the immune cell recruitment functional domain, the immune cell activation functional domain and the auxiliary killing functional domain are fused together to form a multi-functional domain. The functional fusion protein also has the function of recognizing pathogens, tumor cells and/or B cell receptors and mobilizing the immune system to kill pathogens and tumor cells.

Specifically, the first aspect of the present invention provides a fusion protein comprising the following functional domains:

(1) a recognition domain that can recognize pathogens, tumor cells and/or B cell receptors; and,

(2) a recruitment domain, which can recruit immune cells.

Preferably, the fusion protein further comprises:

(3) an activation domain that activates immune cells; and/or,

(4) A secondary killer domain that activates a complement response.

Preferably, the recognition functional domain includes proteins that recognize pathogens, tumor cells and/or B cell receptors, further preferably, the proteins include but are not limited to proteins that recognize pathogens, tumor cells and/or B cell receptors Antibody variable regions or cell membranes can recognize pathogens, tumor cells and/or natural receptors of B cells.

Preferably, the natural receptors include but are not limited to Dectin1, DC-205 (dendritic cell receptor), MR (mannose receptor), DNGR-1 (dendritic cell natural killer cell lectin family receptor body 1), DNGR-2 (dendritic cell natural killer cell lectin family receptor 2), Mincle (macrophage-inducible C-type lectin receptor), CLECSF8, CLEC5A (C-type lectin 5A), DCIR One or more of (dendritic cell immune receptor), DC-SIGN (dendritic cell-specific intercellular adhesion molecule 3-binding non-integrin).

Preferably, the recognition functional domain includes antibody scFv and/or antigens that specifically recognize pathogens, tumor cells and/or B cell receptors.

Preferably, the recognition domain also includes a tag protein, further preferably, the tag protein includes but not limited to streptavidin-binding peptide tag, polyarginine, polyhistidine, calmodulin Binding polypeptide (CBP), c-myc protein, glutathione S-transferase (GST), FLAG polypeptide, staphylococcal protein A, maltose binding protein, thioredox protein, small molecule ubiquitin-like modifying protein (SUMO), HA protein, AviTag protein, enhanced green fluorescent protein (eGFP), enhanced yellow-green fluorescent protein (eCFP), enhanced yellow-green fluorescent protein (eYFP), monomeric red fluorescent protein (mCherry).

In one embodiment of the present invention, the tagged protein is a small molecule tagged protein, preferably, the tagged protein is 8aa.

Preferably, the pathogen includes but not limited to parasite, fungi, bacteria or virus.

Preferably, the parasite includes but is not limited to one of egg antigen, amoeba, helminth, malaria or leishmania or Various.

Preferably, the fungi include, but are not limited to, Cryptococcus, Malaria furfur, Trichomonas, Microsporum, Epidermophyton flocculus, colored fungi, Sporotrichum, Candida, Aspergillus, Mucor or One or more species of Pneumocystis carinii.

Preferably, the bacteria include but are not limited to Streptococcus (Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus viridans, Streptococcus agalactiae, Streptococcus equi, Streptococcus bovis), staphylococcus (Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus), Mycoplasma (Mycoplasma pneumoniae, Mycoplasma hominis, Mycoplasma genitalium, Mycoplasma penetrating, Ureaplasma urealyticum), Actinomycetes, Nocardia, Mycobacterium, Escherichia, Salmonella, Shigella, Spirochetes (Leptospira, Borrelia burgdorferi, Borrelia relapsing fever, Treponema pallidum, Treponema fragilis), Chlamydia (Chlamydia trachomatis, Chlamydia psittaci, Chlamydia pneumoniae), Rickettsia Rickettsia, Rickettsia rickettsii, Rickettsia mosonii, Rickettsia tsutsugamushi, Ehrlichia), Clostridium tetani, Clostridium botulinum, Clostridium perfringens, difficile Clostridium, Bacillus anthracis, Corynebacterium diphtheriae, Listeria monocytogenes, Brucella malta, Pseudomonas aeruginosa, Legionella pneumophila, Vibrio cholerae, Yersinia pestis, Haemophilus influenzae, Helicobacter pylori One or more of Neisseria meningitidis or Neisseria gonorrhoeae.

Preferably, the viruses include but are not limited to hepatitis virus (type A, type B, type C, type D, type E), norovirus, rotavirus, coxsackie virus, echovirus, star Virus, measles virus, varicella-zoster virus, rubella virus, Epstein-Barr virus, mumps virus, herpes virus, influenza virus, avian influenza virus or common coronavirus.

In one embodiment of the present invention, the pathogen includes cryptococcus or amoeba.

Preferably, the recruitment domain includes a domain of a protein that has a recruitment function and/or regulates the function of endogenously recruiting immune cells. Further preferably, the domain of a protein includes but is not limited to chemokines, other cellular Domains of factors or antibodies.

Preferably, the chemokine is selected from CC chemokine subfamily, CXC chemokine subfamily, XC chemokine subfamily or CX3C chemokine subfamily.

Preferably, the chemokines include but are not limited to CXCL8, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CCL11, CCL7, CCL8, CCL13, CCL5, CCL3, CCL4, CCL2, CCL20, CCL19, CCL21, CXCL12, CXCL13 , XCL1, CX ₃ CL1, CCL17, CCL22, CXCL9, CXCL10, CXCL11, CCL1, CCL25, CCL27, CCL28, CXCL16.

Preferably, the antibody is selected from full-length antibodies, chimeric antibodies, Fab fragments, Fab' fragments, Fd fragments, Fd' fragments, Fv fragments, dAb fragments, isolated CDR regions, F(ab') ₂ fragments, Single-domain antibody (DAB), single-chain antibody molecule (such as single-chain Fv; scFv), diabody, triabody, linear antibody or a combination of two or more.

Preferably, the recruitment domain can also activate immune cells.

In one embodiment of the present invention, the recognition functional domain is a protein domain that can recognize pathogenic bacteria such as scFv, nanobody or cell surface receptors (such as Dectin1, etc.), and the chemokine binding domain behind the recognition functional domain (including strepTactin, antibody ScFv, leucine zipper, etc.), can bind chemokines with small tags such as strep, Flag, HA, etc., and then recruit immune cells.

Preferably, the activation domain includes a domain of a protein that activates immune cells or regulates a protein that activates immune cells. More preferably, the protein domain includes a cytokine or antibody domain.

Preferably, the cytokines include but not limited to interferon or interleukin, further preferably, the cytokines include INF-γ, IL-4 or IL-13.

Preferably, the antibody is selected from full-length antibodies, chimeric antibodies, Fab fragments, Fab' fragments, Fd fragments, Fd' fragments, Fv fragments, dAb fragments, isolated CDR regions, F(ab') ₂ fragments, Single-domain antibody (DAB), single-chain antibody molecule (such as single-chain Fv; scFv), diabody, triabody, linear antibody or a combination of two or more.

Preferably, the activation domain also includes a protein domain that regulates endogenous cytokines.

In one embodiment of the present invention, the activation domain is the domain of anti-trem2.

Preferably, the immune cells described in the present invention include but are not limited to neutrophil (neutrophils), basophil (basophils), eosinophil (eosinophils), monocyte (monocytes), immature DC (not Mature dendritic cells), mature DC (mature dendritic cells), B cell (B cells), Tfh (follicular helper T cells), NK cell (NK cells), Naive T (initial T cells), Tcm (central memory T cell), Treg (regulatory T cell), Th1 cell, Th2 cell, Th17 cell, α4β7 ⁺ T cell, CLA ⁺ T cell, colon T (colon T cell), CD8 ⁺ T cell, NKT cell (NKT cells).

Preferably, the helper killing domain includes, but is not limited to, antibody crystallizable fragments or antigen receptor fragments of immune cells.

Preferably, the crystallizable fragment is an antibody constant region, further preferably, the constant region includes a KIH structure.

Preferably, the crystallizable fragment includes interaction sites with FcγR and C1q.

Preferably, the crystallizable fragment includes interaction sites with FcRn, SpA, and SpG.

Preferably, the effect of the helper killing domain includes a domain that induces antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) or complement-dependent cytotoxicity (CDC) .

In one embodiment of the present invention, the antigen receptor fragment is TCRα or β chain.

Preferably, the recognition domain, recruitment domain, activation domain or auxiliary killing domain is derived from human.

The second aspect of the present invention provides a nucleic acid encoding the above fusion protein.

The third aspect of the present invention provides an expression vector, said expression vector comprising the above-mentioned nucleic acid.

Preferably, the expression vector can be expressed in vivo or in vitro or in vitro. Further preferably, the expression vector is continuously expressed at a high level in cells in vivo.

Preferably, the expression vector may be a prokaryotic expression vector or a retrovirus vector.

Further preferably, the prokaryotic expression vector is Escherichia coli series.

More preferably Rous sarcoma virus (RSV), lentivirus, human immunodeficiency virus (HIV), murine leukemia virus (MLV), equine infectious anemia virus (EIAV), mouse breast cancer virus (MMTV), Fujinami Sarcoma virus (FuSV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine leukemia virus (Mo-MLV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV ), avian myeloproliferative virus 29 (MC29) or avian erythroblastosis virus (AEV), etc.

The fourth aspect of the present invention provides a host cell, said host cell comprising the above-mentioned nucleic acid or expression vector.

Preferably, said host cell can be eukaryotic or prokaryotic. Further preferably, the host cells include but not limited to yeast cells, animal cells (such as 293 cells, CHO cells), insect cells, Escherichia coli, Bacillus subtilis, Streptomyces and the like.

The fifth aspect of the present invention provides a method for preparing the above-mentioned fusion protein, comprising the following steps:

(1) Linking the nucleic acid of the above-mentioned fusion protein to the carrier backbone to obtain an expression vector;

(2) transforming or transfecting the expression vector obtained in step (1) into a host cell, and then inducing its expression;

(3) The fusion protein is obtained after purification.

The sixth aspect of the present invention provides a pharmaceutical composition, which includes the above-mentioned fusion protein and pharmaceutically acceptable excipients.

Preferably, the pharmaceutical composition can also be used together with other therapeutic agents. Further preferably, the therapeutic agent may be an immunomodulator.

The seventh aspect of the present invention provides the application of the above-mentioned fusion protein, the above-mentioned expression vector, the above-mentioned host cell or the fusion protein prepared by the method for preparing the above-mentioned fusion protein in the preparation of a pharmaceutical composition for treating pathogenic infection or tumor.

Preferably, the pathogen includes but not limited to parasite, fungi, bacteria or virus.

Preferably, the parasite includes but is not limited to one of egg antigen, amoeba, helminth, malaria or leishmania or Various.

Preferably, the fungi include, but are not limited to, Cryptococcus, Malaria furfur, Trichomonas, Microsporum, Epidermophyton flocculus, colored fungi, Sporotrichum, Candida, Aspergillus, Mucor or One or more species of Pneumocystis carinii.

Preferably, the bacteria include but are not limited to Streptococcus (Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus viridans, Streptococcus agalactiae, Streptococcus equi, Streptococcus bovis), staphylococcus (Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus), Mycoplasma (Mycoplasma pneumoniae, Mycoplasma hominis, Mycoplasma genitalium, Mycoplasma penetrating, Ureaplasma urealyticum), Actinomycetes, Nocardia, Mycobacterium, Escherichia, Salmonella, Shigella, Spirochetes (Leptospira, Borrelia burgdorferi, Borrelia relapsing fever, Treponema pallidum, Treponema fragilis), Chlamydia (Chlamydia trachomatis, Chlamydia psittaci, Chlamydia pneumoniae), Rickettsia Rickettsia, Rickettsia rickettsii, Rickettsia mosonii, Rickettsia tsutsugamushi, Ehrlichia), Clostridium tetani, Clostridium botulinum, Clostridium perfringens, difficile Clostridium, Bacillus anthracis, Corynebacterium diphtheriae, Listeria monocytogenes, Brucella malta, Pseudomonas aeruginosa, Legionella pneumophila, Vibrio cholerae, Yersinia pestis, Haemophilus influenzae, Helicobacter pylori One or more of Neisseria meningitidis or Neisseria gonorrhoeae.

Preferably, the viruses include but are not limited to hepatitis virus (type A, type B, type C, type D, type E), norovirus, rotavirus, coxsackie virus, echovirus, star Virus, measles virus, varicella-zoster virus, rubella virus, Epstein-Barr virus, mumps virus, herpes virus, influenza virus, avian influenza virus or common coronavirus.

In one embodiment of the present invention, the pathogenic infection includes cryptococcal infection or amoeba infection.

The eighth aspect of the present invention provides a method for treating pathogen infection or tumor disease, the method comprising administering to an individual an effective amount of the fusion protein of the present invention, the expression vector, the host cell, the fusion protein The fusion protein or the pharmaceutical composition prepared by the preparation method.

Preferably, the pathogen includes but not limited to parasite, fungi, bacteria or virus.

Preferably, the parasite includes but is not limited to one of egg antigen, amoeba, helminth, malaria or leishmania or Various.

Preferably, the fungi include, but are not limited to, Cryptococcus, Malaria furfur, Trichomonas, Microsporum, Epidermophyton flocculus, colored fungi, Sporotrichum, Candida, Aspergillus, Mucor or One or more species of Pneumocystis carinii.

Preferably, the bacteria include but are not limited to Streptococcus (Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus viridans, Streptococcus agalactiae, Streptococcus equi, Streptococcus bovis), staphylococcus (Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus), Mycoplasma (Mycoplasma pneumoniae, Mycoplasma hominis, Mycoplasma genitalium, Mycoplasma penetrating, Ureaplasma urealyticum), Actinomycetes, Nocardia, Mycobacterium, Escherichia, Salmonella, Shigella, Spirochetes (Leptospira, Borrelia burgdorferi, Borrelia relapsing fever, Treponema pallidum, Treponema fragilis), Chlamydia (Chlamydia trachomatis, Chlamydia psittaci, Chlamydia pneumoniae), Rickettsia Rickettsia, Rickettsia rickettsii, Rickettsia mosonii, Rickettsia tsutsugamushi, Ehrlichia), Clostridium tetani, Clostridium botulinum, Clostridium perfringens, difficile Clostridium, Bacillus anthracis, Corynebacterium diphtheriae, Listeria monocytogenes, Brucella malta, Pseudomonas aeruginosa, Legionella pneumophila, Vibrio cholerae, Yersinia pestis, Haemophilus influenzae, Helicobacter pylori One or more of Neisseria meningitidis or Neisseria gonorrhoeae.

Preferably, the viruses include but are not limited to hepatitis virus (type A, type B, type C, type D, type E), norovirus, rotavirus, coxsackie virus, echovirus, star Virus, measles virus, varicella-zoster virus, rubella virus, Epstein-Barr virus, mumps virus, herpes virus, influenza virus, avian influenza virus or common coronavirus.

In one embodiment of the present invention, the pathogenic infection includes cryptococcal infection or amoeba infection.

Preferably, the method for treating diseases also includes other synergistic treatment means, preferably one or a combination of two or more of chemotherapy, surgery, radiotherapy, gene therapy, hormone therapy, and immunotherapy.

The "tumor" mentioned in the present invention includes but not limited to lymphoma, non-small cell lung cancer, cervical cancer, leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, gastric cancer, bladder cancer , glioma, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophagus cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma Cytoma, melanoma, myelodysplastic syndrome, and sarcoma. Wherein, the leukemia is selected from acute lymphocytic (lymphoblastic) leukemia, acute myelogenous leukemia, myelogenous leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myelogenous leukemia; The lymphoma is selected from Hodgkin's lymphoma and non-Hodgkin's lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma , T-cell lymphoma, and Waldenstrom macroglobulinemia; said sarcoma is selected from the group consisting of osteosarcoma, Ewing sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma , and chondrosarcoma. In a specific embodiment of the present invention, the tumor is breast cancer, ovarian cancer, endometrial cancer, melanoma, kidney cancer, lung cancer, liver cancer.

As used herein, "treating" means slowing, interrupting, arresting, controlling, stopping, alleviating, or reversing the progression or severity of a sign, symptom, disorder, condition, or disease after the disease has begun to develop, but not necessarily The complete elimination of all disease-related signs, symptoms, conditions, or disorders is contemplated.

The "expression vector" in the present invention can be any vector in the prior art that can carry nucleic acid and stably replicate and express in the host. It contains an origin of replication, a promoter, marker genes and translational control elements. Preferably, they may be bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian viruses, and the like.

The "antibody" of the present invention includes, but is not limited to: Fab fragments, which have VL, CL, VH and CH1 domains; Fab' fragments, which have one or more cysteine residues at the C-terminal of the CH1 domain Fab fragment; Fd fragment, which has VH and CH1 domains; Fd' fragment, which has VH and CH1 domains and one or more cysteine residues at the C-terminus of CH1 domain; Fv fragment, which has a single antibody The VL and VH domains of the arms; the dAb fragment, which consists of either the VH domain or the VL domain; the isolated CDR regions; the F(ab') ₂ fragment, which is a Fab' fragment comprising two connected by a disulfide bridge at the hinge region A bivalent fragment of a single chain antibody molecule (e.g. single chain Fv; scFv); a "diabody" with two antigen binding sites comprising a heavy chain linked to a light chain variable domain (VL) in the same polypeptide chain a variable domain (VH); a "linear antibody" comprising a pair of tandem Fd segments (VH-CH1-VH-CH1) which together with a complementary light chain polypeptide form a pair of antigen-binding regions; and any of the foregoing A modified form of a substance that retains antigen binding activity, wherein a "CDR" is a short fragment of an immunoglobulin (Ig) or T cell antigen receptor (TCR) that binds an antigenic epitope, alone or in combination with other CDRs. The immunoglobulin can be an antibody, and the CDRs correspond to the complementarity determining regions in the variable sequence of the antibody. The system described by Kabat et al (Kabat et al, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides unambiguous residue numbering applicable to antibody variable regions system, but also provides residue boundaries defining the three CDRs. These CDRs may be referred to as Kabat CDRs. Each CDR may contain amino acid residues from a "complementarity determining region" as defined by Kabat. Chothia et al. ( Chothia & Lesk, J. Mol. Biol, 196:901-917 (1987) and Chothia et al., Nature 342:877-883 (-1989)) found that some sub-parts within the Kabat CDR adopt almost the same peptide backbone conformation , despite large diversity at the amino acid sequence level. These sub-parts are called "L" and "H", denoting the light and heavy chain regions, respectively. These regions can be called Chothia CDRs, which have overlap with the Kabat CDRs There are other CDR boundary definitions that may not strictly follow one of the above systems, but will still overlap with Kabat CDRs, the method used herein can utilize CDRs defined according to any of these systems, although preferred implementations use Kabat or Chothia defined CDR. The residue boundary of the CDR in the TCR is the same as above. The "antibody variable region" refers to the light chain and the heavy chain of the antibody molecule comprising a complementarity determining region (CDR) and a framework region (FR) part of the amino acid sequence. VH refers to the variable domain of the heavy chain. VL refers to the variable domain of the light chain.

In this application, by constructing a fusion protein, the pathogen and/or tumor cell recognition functional domain, the immune cell recruitment functional domain, the immune cell activation functional domain and the auxiliary killing functional domain are fused together to form a multifunctional protein, which has the ability to detect pathogens, tumor cells and /or the recognition of B cell receptors and the function of inducing the immune system to kill pathogens and tumor cells.

The fusion protein in the present application can effectively eliminate pathogenic infection and/or tumor cells, and can effectively identify and attack even pathogenic bacteria covered with capsules on the surface. Compared with ordinary antibody drugs, the fusion protein drug has a more effective ability to kill pathogens and/or tumor cells.

The advantages of the present invention are:

1. Some pathogenic bacteria are not easily recognized by the human body, so they cannot be attacked. The recognition domain of the present invention can recognize pathogens and/or tumor cells that cannot be recognized by the human immune system. The combination of the recognition domain and the recruitment domain of the present invention can be used as a multiple Attractant for immune cells, its small molecular structure may increase permeability, while a small tag (8aa) at the C-terminus of chemokines maintains its activity and facilitates the combination of different scFvs and chemokines.

2. Although the pathogens and/or tumor cells can be recognized and killed when the human immunity is insufficient, the recognition and killing effects will be greatly reduced. This drug can enhance the killing performance of the immune system.

3. The fusion protein of the present invention also includes a recruitment domain, which can recruit and mobilize the innate and adaptive immune systems in the body, thereby further improving the killing effect on pathogens and/or tumor cells.

4. Preferably, the functional domain of the fusion protein of the present invention can use the domain of the protein in the human body to avoid the immunogenicity of other protein drugs and the immune rejection caused by the immunogenicity.

5. Preferably, the preparation method of the fusion protein of the present invention is simple, and various functional domains can be easily replaced to realize the recognition of various diseases and the recruitment and killing of various corresponding immune cells.

6. The fusion protein or pharmaceutical composition of the present invention, because the recognition functional domain can recognize multiple pathogens or tumor cells, can treat multiple diseases at the same time, especially tumor patients, who are more likely to be infected due to chemotherapy and other reasons , the pharmaceutical composition of the present invention can not only treat tumors but also treat pathogens, simplify the method of administration, and realize the all-in-one administration mode.

The above only summarizes some aspects of the present invention, and it is not and should not be considered as limiting the present invention in any respect.

All patents and publications mentioned in this specification are hereby incorporated by reference in their entirety. Those skilled in the art would recognize that certain changes can be made in the present invention without departing from the spirit or scope of the invention.

The following examples further illustrate the present invention and should not be considered as limiting the scope of the present invention or the specific methods described in the present invention.

Description of drawings

Hereinafter, embodiments of the present invention will be described in detail in conjunction with the accompanying drawings, wherein:

Figure 1: Schematic diagram of fusion protein drug;

Figure 2: Schematic diagram of the recruitment functional domain;

Figure 3: Schematic diagram of the interaction between the recognition domain and the recruitment domain;

Figure 4: Flow diagram of the recognition function detection of fusion protein drugs on pathogens (taking Candida albicans as an example).

Detailed ways

The present invention will be further described below in conjunction with specific embodiments, and the advantages and characteristics of the present invention will become clearer along with the description. However, these embodiments are only exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

Example 1 Preparation of fusion protein drug

The nucleic acid of the fusion protein is connected to the carrier backbone to obtain the expression vector; the obtained expression vector is transformed into a host cell, and then its expression is induced; after purification, the fusion protein is obtained, and the structure of the fusion protein and the schematic diagram of the recruitment functional domain are shown in Figure 1 and As shown in FIG. 2 , in the constructed fusion protein, the interaction mechanism between the recognition functional domain and the recruitment functional domain is shown in FIG. 3 .

The specific fusion protein construction steps are as follows:

1. Construct the gene expressing the fusion protein into the expression vector pCDNA3.1;

2. Transfect the plasmid containing the fusion protein expression gene into 293 cells: the constructed plasmid and PEI are divided into 1 microgram of plasmid: 3 microliters of PEI, diluted and mixed evenly, and placed at room temperature for 30 minutes before adding to the expression cells;

3. Harvest the medium supernatant after 48 hours of culture, and use protein A to capture the fusion protein in the supernatant;

4. Purification by ion exchange and molecular exclusion chromatography further improves the purity of the protein;

5. Store the purified protein in sterile PBS solution at -80°C.

Example 2 In vitro experimental verification of drug function

Use flow cytometry to test the recognition function of fusion protein drugs on pathogens (take Candida albicans as an example, other pathogens can use this method). Fluorescence was detected by the fluorescently labeled antibody of the killing domain (Fc), as shown in Figure 4, after adding the protein drug, fluorescence could be significantly detected on the surface of Candida albicans.

The specific detection steps are as follows:

1. Rinse the 1E6 amount of Candida albicans in 2 tubes (A, B) with flow detection buffer (1XPBS+2%FBS), then centrifuge to remove the supernatant;

2. Resuspend tube A with 100 microliters of ordinary cell culture supernatant; resuspend tube B with 100 microliters of cell supernatant expressing the target protein drug. Incubate for 30 minutes;

3. Centrifuge to remove the supernatant, add 100 microliters of anti-Fc fluorescently labeled antibody (diluted with flow buffer 1:200) and mix well, incubate for 30 minutes.

4. Centrifuge to remove the supernatant, resuspend with flow buffer, pass through a 40-micron cell sieve, and test the leukocytes after filtration. The results shown in Figure 4 are obtained, which proves that the drug can identify Candida albicans.

Example 3 Functional verification of fusion protein drugs

The effectiveness of the drug is tested by constructing a mouse pathogen (Candida albicans, Cryptococcus or amoeba) infection model or a tumor model.

1. Take 4 groups of 10 male and female C57BL/6 mice aged 6-8 weeks, named group A/B/C/D, and inject 5×10 ⁵ CFU white rosary beads into group A/B/C at the same time Bacteria, group D was not treated;

2. After 24 hours, group A was injected with 100 microliters of PBS; group B was injected with 1 microgram of the fusion protein prepared in Example 1; group C/D was injected with 10 micrograms of the fusion protein prepared in example 1;

3. Observe the survival rate of the mice.

The experimental results showed that the survival period of mice injected with protein drugs was significantly longer than that of mice injected with PBS.

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention. These simple modifications All belong to the protection scope of the present invention.

In addition, it should be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable way if there is no contradiction. The combination method will not be described separately.

In addition, various combinations of different embodiments of the present invention can also be combined arbitrarily, as long as they do not violate the idea of the present invention, they should also be regarded as the disclosed content of the present invention.

Claims (27)

A fusion protein, characterized in that the fusion protein comprises the following functional domains: (1) a recognition domain that can recognize pathogens, tumor cells and/or B cell receptors; and, (2) a recruitment domain, which can recruit immune cells. The fusion protein according to claim 1, wherein the fusion protein also comprises: (3) an activation domain that activates immune cells; and/or, (4) A secondary killer domain that activates a complement response. According to the fusion protein according to any one of claims 1-2, it is characterized in that the recognition functional domain includes proteins that recognize pathogens, tumor cells and/or B cell receptor functions, preferably, the proteins include proteins that recognize pathogens , the antibody variable region of the tumor cell and/or B cell receptor or the natural receptor on the cell membrane that can recognize pathogens, tumor cells and/or B cells. The fusion protein according to claim 3, wherein the natural receptor is selected from Dectin1, DC-205, MR, DNGR-1, DNGR-2, Mincle, CLECSF8, CLEC5A, DCIR or DC-SIGN one or more of two. The fusion protein according to claim 3, wherein the recognition functional domain includes antibody scFv or antigen that specifically recognizes pathogens, tumor cells and/or B cell receptors. The fusion protein according to any one of claims 3-5, wherein the recognition domain also includes a tag protein, preferably, the tag protein is selected from the group consisting of streptavidin-binding peptide tag, polysperm amino acid, polyhistidine, calmodulin-binding polypeptide, c-myc protein, glutathione S-transferase, FLAG polypeptide, staphylococcal protein A, maltose-binding protein, thioredox protein, small molecule ubiquitin-like Modified protein, HA protein, AviTag protein, enhanced green fluorescent protein, enhanced yellow-green fluorescent protein, enhanced yellow-green fluorescent protein, monomeric red fluorescent protein. According to the fusion protein described in any one of claims 1-6, it is characterized in that the pathogen includes parasites, fungi, bacteria or viruses, preferably, the parasites include egg antigens, amoebas, One or more of worms, Plasmodium or Leishmania, and the fungus includes Cryptococcus. The fusion protein according to any one of claims 1-7, characterized in that, the recruitment domain includes a protein domain with the function of recruiting immune cells and/or regulating the function of endogenous recruitment, preferably, the protein Domains include those of chemokines, other cytokines, or antibodies. The fusion protein according to claim 8, wherein the chemokine is selected from the CC chemokine subfamily, the CXC chemokine subfamily, the XC chemokine subfamily or the CX3C chemokine subfamily. The fusion protein according to any one of claims 8-9, wherein the chemokine is selected from the group consisting of CXCL8, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CCL11, CCL7, CCL8, CCL13, CCL5, CCL3, One or a combination of two or more of CCL4, CCL2, CCL20, CCL19, CCL21, CXCL12, CXCL13, XCL1, _CX3 CL1, CCL17, CCL22, CXCL9, CXCL10, CXCL11, CCL1, CCL25, CCL27, CCL28 or CXCL16. The fusion protein according to any one of claims 8-10, wherein the antibody is selected from full-length antibodies, chimeric antibodies, Fab, Fab', F(ab') ₂ , single domain antibodies, Fv , scFv, diabody, triabody or a combination of two or more. The fusion protein according to any one of claims 1-11, characterized in that, the recruitment domain can also activate immune cells at the same time. The fusion protein according to any one of claims 2-12, wherein the activation domain includes a protein domain that activates immune cells, preferably, the protein domain includes cytokines or antibodies domain. The fusion protein according to claim 13, wherein the cytokines include interferon or interleukin, preferably, the cytokines include INF-γ, IL-4 or IL-13. The fusion protein according to any one of claims 13-14, wherein the antibody is selected from full-length antibodies, chimeric antibodies, Fab fragments, Fab' fragments, Fd fragments, Fd' fragments, Fv fragments, dAb One or a combination of two or more of fragments, isolated CDR regions, F(ab') ₂ fragments, single domain antibodies, single chain antibody molecules, diabodies, triabodies, and linear antibodies. The fusion protein according to any one of claims 1-15, wherein the immune cells are selected from the group consisting of neutrophils, basophils, eosinophils, monocytes, immature dendritic cells, mature dendritic cells, B cells, follicular helper T cells, NK cells, naive T cells, central memory T cells, regulatory T cells, Th1 cells, Th2 cells, Th17 cells, α4β7 ⁺ T cells, One or more of CLA ⁺ T cells, colon T cells, CD8 ⁺ T cells, and NKT cells. The fusion protein according to any one of claims 2-16, wherein the auxiliary killing domain includes antibody crystallizable fragments or antigen receptors of immune cells. The fusion protein according to any one of claim 17, wherein the crystallizable fragment is an antibody constant region, preferably, the constant region comprises a KIH structure. The fusion protein according to any one of claims 2-18, wherein the auxiliary killing domain includes a domain that induces ADCC, ADCP or CDC effects. The fusion protein according to any one of claims 1-19, wherein the recognition domain, recruitment domain, activation domain or auxiliary killing domain is derived from human. A nucleic acid encoding the fusion protein of any one of claims 1-20. An expression vector, characterized in that the expression vector comprises the nucleic acid according to claim 21. A host cell comprising the nucleic acid of claim 21 or the expression vector of claim 22. A method for preparing a fusion protein, characterized in that the method for preparing comprises the following steps: (1) connecting the nucleic acid of the fusion protein according to claim 21 to the carrier backbone to obtain an expression vector; (2) transforming or transfecting the expression vector obtained in step (1) into a host cell, and then inducing its expression; (3) The fusion protein is obtained after purification. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises the fusion protein according to any one of claims 1-20, and pharmaceutically acceptable auxiliary materials. The fusion protein according to any one of claims 1-20, the expression vector according to claim 22, the host cell according to claim 23 or the fusion protein prepared by the preparation method according to claim 24 is used in the preparation and treatment of pathogen infection or tumor application in pharmaceutical compositions. The pharmaceutical composition according to claim 25 or the application according to claim 26, wherein said pathogens include parasites, fungi, bacteria or viruses, preferably, said parasites include egg antigens, One or more of worms, amoebas, Plasmodium or Leishmania, and the fungi include Cryptococcus.

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