CN117957257A

CN117957257A - Multifunctional fusion protein medicine for targeting and killing pathogen and/or tumor cells

Info

Publication number: CN117957257A
Application number: CN202280062222.9A
Authority: CN
Inventors: 林欣; 袁建龙
Original assignee: Tsinghua University
Current assignee: Tsinghua University
Priority date: 2021-08-04
Filing date: 2022-08-04
Publication date: 2024-04-30
Also published as: WO2023011575A1

Abstract

The fusion protein can identify pathogens and mobilize an immune system to kill the pathogens, and has important application value for developing new drugs for treating pathogen infection or tumor cells.

Description

Multifunctional fusion protein medicine for targeting and killing pathogen and/or tumor cells

Technical Field

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

Background

Microbial infections, including pathogenic viruses, bacteria, fungi and some protozoa, cause a number of death events annually. Although the immune system in the body can completely or partially resist the infection of pathogenic microorganisms, when the immunity of the human body is reduced, the infection of the pathogens cannot be effectively cleared, so that the pathogens are greatly propagated in the body, the healthy tissues and organs of various parts of the body are damaged, and the life is directly threatened. Meanwhile, some pathogenic bacteria are covered with capsules on the surfaces, so that immune cell recognition and attack can be avoided.

Cryptococcosis (cryptococcosis, torulosis) is a subacute or chronic infectious disease, is caused by novel cryptococcosis, mainly invades the central nervous system, has been rare in recent years, is easy to confuse with other intracranial diseases and delays treatment, and has high death rate. The disease may also involve lung, skin, subcutaneous tissue, epiphysis, joints and other viscera, tissues, etc., and may occur at any age. The human body is often exposed to an environment containing various cryptococcus species, wherein immunity of the human body to cryptococcus species 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 are involved in opsonophagocytosis, assisting phagocytes in phagocytosis of cryptococcus. Pathogenic bacteria are liable to invade the human body to cause diseases only when the body resistance is lowered.

Amoeba infectious diseases, infectious diseases caused by amoeba growing in water and corroding human brain, amoeba enter human body from nose, penetrate into cranium and erode brain tissue, thus being fatal. It is also easy for people to develop diseases due to the decrease of body immunity.

The pathogen can induce the organism to generate specific immunity including cell immunity and humoral immunity after entering the organism, wherein some pathogenic bacteria are not easily identified by human body and cannot be attacked, and meanwhile, when the human body is insufficient in immunity, the identification and killing effects of the pathogen which can be identified and killed are greatly reduced, so that a medicament is needed to enhance the identification and killing effects. Pathogen infection diseases such as cryptococcus infection, amoeba infection and the like are not effectively treated by drugs at present. Therefore, it is particularly important to invent a new drug against pathogen infection.

In the prior art, as in non-patent literature "Single human B cell-derived monoclonal anti-Candida antibodies enhance phagocytosis and protect against disseminated candidiasis", candida is mainly identified by an antibody mode, immune cells and complement components are recruited by utilizing an Fc structure to attack invading pathogenic bacteria, but the method is not applied to in vivo pathogen or tumor cell treatment.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention fuses 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 together by constructing a fusion protein to form a multifunctional fusion protein, and simultaneously has the functions of recognizing pathogen, tumor cell and/or B cell receptor and mobilizing an immune system to kill the pathogen and the tumor cell.

In particular, according to a first aspect of the present invention there is provided a fusion protein comprising the following functional domains:

(1) A recognition domain that can recognize a pathogen, tumor cell, and/or B cell receptor; and, a step of, in the first embodiment,

(2) A recruitment domain that recruits immune cells.

Preferably, the fusion protein further comprises:

(3) An activation domain that activates an immune cell; and/or the number of the groups of groups,

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

Preferably, the recognition domain includes a protein that recognizes the function of a pathogen, tumor cell and/or B cell receptor, and more preferably, the protein includes, but is not limited to, an antibody variable region that recognizes the pathogen, tumor cell and/or B cell receptor or a natural receptor on the cell membrane that recognizes the pathogen, tumor cell and/or B cell receptor.

Preferably, the natural receptor includes, but is not limited to, one or more of Dectin1, DC-205 (dendritic cell receptor), MR (mannose receptor), DNGR-1 (dendritic cell natural killer lectin family receptor 1), DNGR-2 (dendritic cell natural killer lectin family receptor 2), mincle (macrophage-induced C-type lectin receptor), CLECSF, CLEC5A (C-type lectin 5A), DCIR (dendritic cell immunoreceptor), DC-SIGN (dendritic cell-specific intercellular adhesion molecule 3 binds to non-integrin).

Preferably, the recognition domain comprises an antibody scFv and/or antigen that specifically recognizes a pathogen, tumor cell and/or B cell receptor.

Preferably, the recognition domain further comprises a tag protein, further preferably, the tag protein includes, but is not limited to, a 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 modification 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 invention, the tag protein is a small molecule tag protein, preferably 8aa.

Preferably, the pathogen includes, but is not limited to, parasites (parapsite), fungi (furgi), bacteria (bacteria) or viruses (viruses).

Preferably, the parasites include, but are not limited to, one or more of an egg antigen (EGG ANTIGEN), amoeba (amoeba), helminths (helminth), plasmodium (malaria) or leishmania (leishmania).

Preferably, the fungus includes, but is not limited to, one or more of cryptococcus, blighted bran Ma Laxian, mao Xian, microsporobacteria, epidermophyton floccosum, chromomycosis, sporomyces, candida, aspergillus, mucor or pneumospora cassii.

Preferably, the method comprises the steps of, the bacteria include, but are not limited to, streptococcus pyogenes, streptococcus pneumoniae, streptococcus viridis, streptococcus agalactiae, streptococcus equi, streptococcus bovis), staphylococcus aureus, staphylococcus epidermidis, staphylococcus saprophyticus, mycoplasma (Mycoplasma pneumoniae, mycoplasma hominus, mycoplasma genitalium, mycoplasma penetrations, ureaplasma urealyticum), actinomycetes, nocardia, mycobacterium, escherichia, salmonella, shigella, spirochete (Leptospira, leptospira burgdorferi, leptospira thermosiphon, treponema pallidum, leptospira tenuifolia), mycoplasma genitalium, mycoplasma hyopneumoniae, mycoplasma, leptospira, mycobacterium, and Shigella chlamydia (chlamydia trachomatis, chlamydia psittaci, chlamydia pneumoniae), rickettsia (rickettsia praecox, rickettsia morse, rickettsia tsiae), clostridium tetani, clostridium botulinum, clostridium perfringens, clostridium difficile, bacillus anthracis, corynebacterium diphtheriae, listeria monocytogenes, maltesa Lu Sijun, pseudomonas aeruginosa, legionella pneumophila, vibrio cholerae, yersinia pestis, haemophilus influenzae, helicobacter pylori, neisseria meningitidis, or neisseria gonorrhoeae.

Preferably, the virus includes, but is not limited to, one or more of hepatitis virus (hepatitis A, B, C, D, E), norovirus, rotavirus, coxsackie virus, epstein-Barr virus, astrovirus, measles virus, varicella-zoster virus, rubella virus, EB virus, mumps virus, herpes virus, influenza virus, avian influenza virus or common coronavirus.

In one embodiment of the invention, the pathogen comprises cryptococcus or amoebae.

Preferably, the recruitment domain comprises a domain of a protein having recruitment function and/or modulating endogenous recruitment immune cell function, and more preferably, the domain of a protein comprises, but is not limited to, a domain of a chemokine, other cytokine, or antibody.

Preferably, the chemokine is selected from the CC chemokine subfamily, the CXC chemokine subfamily, the XC chemokine subfamily or the 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 ₃CL1、CCL17、CCL22、CXCL9、CXCL10、CXCL11、CCL1、CCL25、CCL27、CCL28、CXCL16.

Preferably, the antibody is selected from one or a combination of two or more of a full-length antibody, a chimeric antibody, a Fab fragment, a Fab ' fragment, a Fd ' fragment, a Fv fragment, a dAb fragment, an isolated CDR region, a F (ab ') ₂ fragment, a single Domain Antibody (DAB), a single chain antibody molecule (e.g., single chain Fv; scFv), a diabody, a triabody, a linear antibody.

Preferably, the recruitment domain also activates immune cells at the same time.

In one embodiment of the invention, the recognition domain is a protein domain that recognizes pathogenic bacteria, such as scFv, nanobody, or cell surface receptor (e.g., dectin1, etc.), and the chemokine binding domain (including STREPTACTIN, antibody ScFv, leucone zipper, etc.) following the recognition domain can bind to chemokines bearing small tags such as strep, flag, HA, etc., thereby recruiting immune cells.

Preferably, the activating domain comprises a domain having a protein that activates immune cell function or modulates a function of activating immune cells, and more preferably, the domain of the protein comprises a domain of a cytokine or an antibody.

Preferably, the cytokines include, but are not limited to, interferons or interleukins, and more preferably, the cytokines include INF-gamma, IL-4 or IL-13.

Preferably, the activation domain further comprises a protein domain that modulates an endogenous cytokine.

In one embodiment of the invention, the activating domain is the domain of anti-trem.

Preferably, the immune cells described in the present invention include, but are not limited to, neutrophil (neutrophil), basophil (basophil), eosinophil (eosinophil), monocyte (monocyte), immature DC (immature dendritic cell), texture DC (mature dendritic cell), B cell (B cell), tfh (follicular helper T cell), NK cell (NK cell), naive T (primitive T cell), 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 cell).

Preferably, the auxiliary killing domain includes, but is not limited to, an antibody crystallizable fragment or an antigen receptor fragment of an immune cell.

Preferably, the crystallizable fragment is an antibody constant region, and more preferably, the constant region comprises a KIH structure.

Preferably, the crystallizable fragment comprises a site of interaction with fcγ R, C1 q.

Preferably, the crystallizable fragment comprises a site of interaction with FcRn, spA, spG.

Preferably, the effect of the ancillary killing domain comprises 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 invention, the antigen receptor fragment is a TCR alpha or beta chain.

Preferably, the identification domain, the recruitment domain, the activation domain or the auxiliary killing domain is of human origin.

In a second aspect of the invention, there is provided a nucleic acid encoding the fusion protein described above.

In a third aspect of the invention there is provided an expression vector comprising a nucleic acid as described above.

Preferably, the expression vector is capable of expression in vivo or in vitro or ex vivo. Further preferred, 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 retroviral vector.

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

Further preferably, it may be 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 osteosarcoma virus (Mo-MSV), abelson murine leukemia virus (A-MLV), avian myeloproliferative virus 29 (MC 29) or avian myeloblastosis virus (AEV), etc.

In a fourth aspect of the invention, there is provided a host cell comprising a nucleic acid or expression vector as described above.

Preferably, the host cell may be eukaryotic or prokaryotic. Further preferred, the host cells include, but are not limited to, yeast cells, animal cells (e.g., 293 cells, CHO cells), insect cells, escherichia coli, bacillus subtilis, streptomycete, and the like.

In a fifth aspect of the present invention, there is provided a method for preparing the above fusion protein, comprising the steps of:

(1) Connecting the nucleic acid of the fusion protein to a vector skeleton to obtain an expression vector;

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

(3) And purifying to obtain the fusion protein.

In a sixth aspect of the present invention, a pharmaceutical composition is provided, which comprises the fusion protein and pharmaceutically acceptable excipients.

Preferably, the pharmaceutical composition may also be used in combination with other therapeutic agents. Further preferably, the therapeutic agent may be an immunomodulatory agent.

In a seventh aspect, the present invention provides the use of the fusion protein, the expression vector, the host cell or the fusion protein prepared by the method for preparing the fusion protein in the preparation of a pharmaceutical composition for treating pathogen infection or tumor.

In one embodiment of the invention, the pathogen infection comprises a cryptococcus infection or an amoeba infection.

In an eighth aspect of the invention, there is provided a method of treating a pathogen infection or a neoplastic disease, said method comprising administering to an individual an effective amount of a fusion protein of the invention, said expression vector, said host cell, fusion protein prepared by a method of preparing a fusion protein as described above, or said pharmaceutical composition.

Preferably, the method of treating a disease further comprises other synergistic therapeutic means, preferably one or a combination of two or more of chemotherapy, surgery, radiotherapy, gene therapy, hormonal therapy, immunotherapy.

The "tumor" as described herein includes, but is 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, esophageal cancer, renal cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, and sarcomas. Wherein the leukemia is selected from acute lymphoblastic (lymphoblastic) leukemia, acute 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's macroglobulinemia; the sarcoma is selected from osteosarcoma, ewing sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma. In one embodiment of the invention, the tumor is breast cancer, ovarian cancer, endometrial cancer, melanoma, kidney cancer, lung cancer, liver cancer.

The term "treatment" as used herein means slowing, interrupting, arresting, controlling, stopping, alleviating, or reversing the progression or severity of one sign, symptom, disorder, condition, or disease after the disease has begun to develop, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions, or disorders.

The "expression vector" according to the present invention may be any vector in the art that can carry a nucleic acid and stably replicate and express in a host. Which comprises an origin of replication, a promoter, a marker gene and translational control elements. Preferably, it may be a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian virus, etc.

The "antibody" of the present invention includes, but is not limited to: a Fab fragment having VL, CL, VH and CH1 domains; a Fab' fragment, which is a Fab fragment having one or more cysteine residues at the C-terminus of the CH1 domain; fd fragment with VH and CH1 domains; fd' fragments having VH and CH1 domains and one or more cysteine residues at the C-terminus of the CH1 domain; fv fragments having the VL and VH domains of a single arm of an antibody; a dAb fragment consisting of a VH domain or a VL domain; an isolated CDR region; a F (ab ') ₂ fragment, which is a bivalent fragment comprising two Fab' fragments linked by a disulfide bridge at the hinge region; single chain antibody molecules (e.g., single chain Fv; scFv); a "diabody" having two antigen binding sites comprising a heavy chain variable domain (VH) linked to a light chain variable domain (VL) in the same polypeptide chain; a "linear antibody" comprising a pair of tandem Fd segments (VH-CH 1-VH-CH 1) that form a pair of antigen-binding regions with complementary light chain polypeptides; and modified versions of any of the foregoing that retain antigen binding activity, wherein a "CDR" is a short fragment of an immunoglobulin (Ig) or T cell antigen receptor (TCR) that binds an epitope alone or in combination with other CDRs. The immunoglobulin may be an antibody, and the CDRs correspond to complementarity determining regions within the variable sequences 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)) provides not only a well-defined residue numbering system for the variable regions of antibodies, but also residue boundaries defining the three CDRs. These CDRs may be referred to as Kabat CDRs. Each complementarity determining region may comprise 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 of the sub-portions within the Kabat CDRs employed nearly identical peptide backbone conformations, albeit with great diversity at the amino acid sequence level. These subfractions are referred to as "L" and "H", respectively, representing the light and heavy chain regions, respectively. These regions may be referred to as Chothia CDRs, which have boundaries that overlap with Kabat CDRs. Still other CDR boundary definitions may not strictly follow one of the above systems, but will still overlap with Kabat CDRs, and the methods used herein may utilize CDRs defined according to any of these systems, although preferred embodiments use Kabat or Chothia defined CDRs. The residue boundaries of the CDRs in the TCR are as described above. The term "antibody variable region" refers to a portion of the amino acid sequence of the light and heavy chains of an antibody molecule that includes Complementarity Determining Regions (CDRs) and Framework Regions (FRs). VH refers to the variable domain of the heavy chain. VL refers to the variable domain of the light chain.

The application combines 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 together by constructing the fusion protein to form the multifunctional protein, and simultaneously has the functions of recognizing pathogen, tumor cell and/or B cell receptor and inducing the immune system to kill pathogen and tumor cell.

The fusion protein can effectively remove infection and/or tumor cells of pathogens, and can effectively identify and attack the pathogens covered with the capsules on the surfaces. Compared with the common antibody medicine, the fusion protein medicine has more effective pathogen and/or tumor cell killing ability.

The invention has the advantages that:

1. some pathogens are not easily recognized by human bodies and cannot be attacked, the recognition domain of the invention can recognize pathogens and/or tumor cells which cannot be recognized by the human immune system, the combination of the recognition domain and the recruitment domain of the invention can be used as an attractant for multiple immune cells, the small molecular structure of the small molecular structure possibly increases permeability, and the small tag (8 aa) at the C-terminal end of a chemokine keeps its activity and is easy to combine different scFvs and chemokines.

2. Although the human body has insufficient immunity, the pathogen and/or tumor cells can be identified and killed, the identification and killing effect can be greatly reduced, and the medicament can enhance the killing performance of an immune system.

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

4. Preferably, the functional domains of the fusion proteins of the invention may use domains of proteins in the human body, avoiding the immunogenicity of other protein drugs, as well as immune rejection due to such immunogenicity.

5. Preferably, the preparation method of the fusion protein is simple, and each functional domain can be conveniently replaced, so that the effects of identifying various diseases, recruiting and killing various corresponding immune cells and the like are realized.

6. The fusion protein or the pharmaceutical composition can identify a plurality of pathogens or tumor cells, can treat a plurality of diseases at the same time, especially can treat a tumor patient, is easier to be infected due to chemotherapy and other reasons, can treat the tumor and can treat the pathogens, simplifies the administration method and realizes the multi-in-one administration mode.

The foregoing is merely illustrative of some aspects of the present invention and is not, nor should it be construed as limiting the invention in any respect.

All patents and publications mentioned in this specification are incorporated herein by reference in their entirety. It will be appreciated by those skilled in the art that certain changes may be made thereto without departing from the spirit or scope of the invention.

The following examples further illustrate the invention in detail and are not to be construed as limiting the scope of the invention or the particular methods described herein.

Drawings

Embodiments of the present invention are described in detail below with reference to the attached drawing figures, wherein:

fig. 1: schematic representation of fusion protein drug;

fig. 2: recruitment domain schematics;

fig. 3: schematic of identifying functional domain interactions with recruitment functional domains;

fig. 4: the recognition function of the fusion protein drug against pathogens (e.g., candida albicans) is examined in a flow chart.

Detailed Description

The invention will be further described with reference to specific embodiments, and advantages and features of the invention will become apparent from the description. These examples are merely exemplary and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions of details and forms of the technical solution of the present invention may be made without departing from the spirit and scope of the present invention, but these changes and substitutions fall within the scope of the present invention.

EXAMPLE 1 preparation of fusion protein drug

Connecting nucleic acid of the fusion protein to a vector framework to obtain an expression vector; transforming the obtained expression vector into a host cell, and then inducing the expression thereof; after purification, a fusion protein is obtained, the structure of which and the schematic diagrams of the recruitment domains are shown in FIG. 1 and FIG. 2, respectively, and the interaction mechanism of the recognition domain and the recruitment domain in the constructed fusion protein is shown in FIG. 3.

The specific fusion protein is constructed as follows:

1. Constructing a gene for expressing the fusion protein into an expression vector pCDNA3.1;

2. transfection of plasmids containing fusion protein expressed genes into 293 cells: the constructed plasmid and PEI are diluted and uniformly mixed according to the proportion of 1 microgram of plasmid to 3 microliter of PEI, and the mixture is placed for 30 minutes at room temperature and then added into an expression cell;

3. Harvesting the culture supernatant after 48 hours of culture, and capturing fusion proteins in the supernatant by using protein A;

4. purifying by ion exchange and size exclusion chromatography to further increase protein purity;

5. The purified protein was stored in sterile PBS solution, -80 degrees.

Example 2 in vitro experimental verification of drug function

The identification function of the fusion protein drug on pathogens (candida albicans is taken as an example, other pathogens can use the method) is checked by adopting a flow cytometry mode, after the fusion protein drug is incubated with candida albicans, fluorescence is detected by using a fluorescence labeled antibody which can specifically bind an auxiliary killing structural domain (Fc) in the drug, as shown in figure 4, and after the protein drug is added, the surface of candida albicans can obviously detect the fluorescence.

The specific detection steps are as follows:

1, rinsing 2 tubes (A, B) 1E6 of candida albicans with a flow detection buffer (1XPBS+2% FBS), and centrifuging to remove the supernatant;

2, resuspension of tube A with 100 microliters of ordinary cell culture supernatant; b was resuspended with 100. Mu.l of the cell supernatant expressing the protein drug of interest. Incubating for 30 minutes;

3, centrifuging to remove supernatant, adding 100 microliters of anti-Fc fluorescence labeled antibody (diluted with flow buffer 1:200), mixing, and incubating for 30 minutes.

4, Centrifuging to remove supernatant, re-suspending by using a flow buffer, sieving with a 40-micrometer cell sieve, and performing on-machine detection on the filtered white blood cells to obtain a result shown in figure 4, wherein the result shows that the medicine can identify candida albicans.

Example 3 functional verification of fusion protein drugs

The effectiveness of the drug was tested by constructing a pathogen (candida albicans, cryptococcus or amoeba) infection model or tumor model of mice.

1. Taking 4 groups of 10C 57BL/6 mice with half ages of 6-8 weeks, naming A/B/C/D groups, injecting 5X 10 ⁵ CFU candida albicans into the A/B/C groups simultaneously, and not treating the D groups;

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. Mu.g of the fusion protein prepared in example 1;

3. The survival rate of the mice was observed.

Experimental results show that mice injected with protein drugs have significantly longer survival times than mice injected with PBS groups.

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

In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.

Moreover, any combination of the various embodiments of the invention can be made without departing from the spirit of the invention, which should also be considered as disclosed herein.

Claims

A fusion protein comprising the following domains:

(1) A recognition domain that can recognize a pathogen, tumor cell, and/or B cell receptor; and, a step of, in the first embodiment,

(2) A recruitment domain that recruits immune cells.
The fusion protein of claim 1, further comprising:

(3) An activation domain that activates an immune cell; and/or the number of the groups of groups,

(4) A secondary killing domain that activates a complement response.
The fusion protein of any one of claims 1-2, wherein the recognition domain comprises a protein that recognizes a pathogen, tumor cell and/or B cell receptor function, preferably wherein the protein comprises an antibody variable region that recognizes a pathogen, tumor cell and/or B cell receptor or a natural receptor on a cell membrane that recognizes a pathogen, tumor cell and/or B cell.
A fusion protein according to claim 3 wherein the natural receptor is selected from one or more of Dectin1, DC-205, MR, DNGR-1, DNGR-2, mincle, CLECSF8, CLEC5A, DCIR or DC-SIGN.
A fusion protein according to claim 3 wherein the recognition domain comprises an antibody scFv or antigen that specifically recognizes a pathogen, a tumor cell and/or a B cell receptor.
The fusion protein of any one of claims 3-5, wherein the recognition domain further comprises a tag protein, preferably wherein the tag protein is selected from the group consisting of streptavidin-binding peptide tag, polyarginine, polyhistidine, calmodulin-binding polypeptide, c-myc protein, glutathione S transferase, FLAG polypeptide, staphylococcal protein a, maltose-binding protein, thioredoxin, small molecule ubiquitin-like modified protein, HA protein, aviTag protein, enhanced green fluorescent protein, enhanced yellow-green fluorescent protein, monomeric red fluorescent protein.
The fusion protein of any one of claims 1-6, wherein the pathogen comprises a parasite, a fungus, a bacterium or a virus, preferably wherein the parasite comprises one or more of an egg antigen, an amoeba, a worm, a plasmodium or a leishmania, and wherein the fungus comprises cryptococcus.
A fusion protein according to any one of claims 1 to 7 wherein the recruitment domain comprises a domain of a protein having recruiting immune cell function and/or modulating endogenous recruitment function, preferably the domain of a protein comprises a domain of a chemokine, other cytokine or antibody.
The fusion protein of claim 8, wherein the chemokine is selected from the group consisting of the CC chemokine subfamily, the CXC chemokine subfamily, the XC chemokine subfamily, and the CX3C chemokine subfamily.
The fusion protein of any one of claims 8-9, wherein the chemokine is selected from one or a combination of two or more of CXCL8、CXCL1、CXCL2、CXCL3、CXCL5、CXCL6、CCL11、CCL7、CCL8、CCL13、CCL5、CCL3、CCL4、CCL2、CCL20、CCL19、CCL21、CXCL12、CXCL13、XCL1、CX ₃CL1、CCL17、CCL22、CXCL9、CXCL10、CXCL11、CCL1、CCL25、CCL27、CCL28 or CXCL 16.
The fusion protein of any one of claims 8-10, wherein the antibody is selected from the group consisting of a full length antibody, a chimeric antibody, fab ', F (ab') ₂, a single domain antibody, fv, scFv, diabody, and triabody.
The fusion protein of any one of claims 1-11, wherein the recruitment domain is also capable of activating immune cells.
Fusion protein according to any of claims 2-12, wherein the activating domain comprises a domain of a protein having the function of activating immune cells, preferably wherein the domain of the protein comprises a domain of a cytokine or an antibody.
The fusion protein of claim 13, wherein the cytokine comprises an interferon or an interleukin, preferably wherein the cytokine comprises INF- γ, IL-4 or IL-13.
The fusion protein of any one of claims 13-14, wherein the antibody is selected from one or a combination of two or more of a full length antibody, a chimeric antibody, a Fab fragment, a Fab ' fragment, a Fd ' fragment, a Fv fragment, a dAb fragment, an isolated CDR region, a F (ab ') ₂ fragment, a single domain antibody, a single chain antibody molecule, a diabody, a triabody, a linear antibody.
The fusion protein of any one of claims 1-15, wherein the immune cell is selected from one or more 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, α4β ⁺ T cells, CLA ⁺ T cells, colon T cells, CD8 ⁺ T cells, NKT cells.
The fusion protein of any one of claims 2-16, wherein the auxiliary killing domain comprises an antibody crystallizable fragment or an antigen receptor of an immune cell.
The fusion protein of any one of claims 17, wherein the crystallizable fragment is an antibody constant region, preferably wherein the constant region comprises a KIH structure.
The fusion protein of any one of claims 2-18, wherein the ancillary killing domain comprises a domain that induces ADCC, ADCP or CDC effect.
The fusion protein of any one of claims 1-19, wherein the recognition domain, recruitment domain, activation domain, or auxiliary killing domain is of human origin.
A nucleic acid encoding the fusion protein of any one of claims 1-20.
An expression vector comprising the nucleic acid of 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, which is characterized by comprising the following steps:

(1) Ligating the nucleic acid of the fusion protein of claim 21 to a vector backbone to obtain an expression vector;

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

(3) And purifying to obtain the fusion protein.
A pharmaceutical composition comprising the fusion protein of any one of claims 1-20, and a pharmaceutically acceptable excipient.
Use of a fusion protein according to any one of claims 1-20, an expression vector according to claim 22, a host cell according to claim 23 or a fusion protein prepared by a method of preparation according to claim 24 for the preparation of a pharmaceutical composition for the treatment of a pathogen infection or tumor.
The pharmaceutical composition of claim 25 or the use of claim 26, wherein the pathogen comprises a parasite, a fungus, a bacterium or a virus, preferably the parasite comprises one or more of an egg antigen, a helminth, an amoeba, a plasmodium or a leishmania, and the fungus comprises cryptococcus.