WO2020077991A1 - 一种用于研发药物的双功能融合蛋白平台 - Google Patents

一种用于研发药物的双功能融合蛋白平台 Download PDF

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WO2020077991A1
WO2020077991A1 PCT/CN2019/086880 CN2019086880W WO2020077991A1 WO 2020077991 A1 WO2020077991 A1 WO 2020077991A1 CN 2019086880 W CN2019086880 W CN 2019086880W WO 2020077991 A1 WO2020077991 A1 WO 2020077991A1
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fusion protein
bifunctional fusion
fragment
drug development
bifunctional
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French (fr)
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祝道成
包骏
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包骏
上海科新生物技术股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the invention belongs to the field of biotechnology, and in particular relates to a dual-function fusion protein platform for drug development and a preparation method and use of the dual-function fusion protein.
  • Fc fusion protein refers to a novel protein produced by fusing the Fc segment of immunoglobulin (IgG, IgA, etc.) with a biologically active functional protein molecule using genetic engineering technology.
  • Functional proteins with biological activity may be cytokines, toxins, receptors, enzymes, antigen peptides and the like.
  • Fc fusion protein can not only exert the biological activity of the fusion protein, but also has some antibody properties, such as can cause antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and antibody-dependent cells Mediated phagocytosis (ADCP), etc.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement-dependent cytotoxicity
  • ADCP antibody-dependent cells Mediated phagocytosis
  • Protein drugs have a short half-life in the plasma, and large doses are required to achieve the therapeutic effect, which will cause serious side effects and cause a huge burden to patients.
  • the fusion of the functional protein and the Fc segment of immunoglobulin can prolong the half-life of the drug in the plasma and reduce the immunogenicity of the drug, which is of great significance in the diagnosis and treatment of diseases.
  • Fc fusion protein is composed of Fc segment of immunoglobulin and protein molecules with biological activity.
  • the Fc segment and the functional protein have relatively independent domains and functions, and can affect the physical and chemical properties and biological activity of the fusion protein from different angles.
  • the main feature of the Fc fusion protein is that it contains an Fc segment, which is similar to the function of the Fc segment in monoclonal antibodies.
  • the Fc segment of the fusion protein can extend the half-life of the functional protein in the plasma, improve the stability of the molecule, and specifically bind to the body. Fc receptor, and play the corresponding biological function and other functions.
  • the Fc segment can specifically bind protein A, which simplifies the purification step of Fc fusion protein, and is of great significance in the research and development of related biological products.
  • the immunoglobulin in the human body depends on the binding of the Fc segment and the nascent Fc receptor (FcRn). Under the protection of FcRn, the half-life can be as long as 19 to 21 days.
  • the Fc fusion protein can also rely on this principle to extend the half-life in the body: the binding of the Fc segment to FcRn is pH-dependent, and under physiological conditions of pH 7.4, FcRn does not bind to Fc; at the cell endosome pH 6.0 Under the acidic condition of ⁇ 6.5, the two combine to avoid rapid degradation of the fusion protein by lysosomes in the cell.
  • the Fc segment can increase the molecular volume, reduce renal clearance, and extend the half-life to some extent.
  • the Fc fusion protein can form a stable dimer through a disulfide bond in the Fc hinge region. Through further genetic engineering of the disulfide bond, the Fc fusion protein can also form a more stable hexamer complex. In addition, the Fc region can be folded independently, ensuring the stability of the molecule.
  • Fusion of Fc segment and functional protein can improve protein expression and secretion in mammals, and Fc segment can specifically bind to protein A, which is conducive to purification of fusion protein.
  • the FC domain of the Fc fusion protein can bind to Fc receptors on the surface of immune cells and perform a variety of biological functions, such as mediating crossing the placenta and mucosal barrier, inflammation, antibody-dependent cell-mediated phagocytosis (ADCP), Antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), promote the maturation of dendritic cells (DC), regulate cytokine secretion, regulate B cell proliferation and differentiation.
  • ADCP antibody-dependent cell-mediated phagocytosis
  • ADCC Antibody-dependent cell-mediated cytotoxicity
  • CDC complement-dependent cytotoxicity
  • DC dendritic cells
  • the Fc domain of the Fc fusion protein affects the physicochemical properties and biological activity of the fusion protein molecule, while the functional protein part determines the pharmacological activity of the Fc fusion protein.
  • functional proteins can be cytokines, toxins, receptors, enzymes, etc., and their functions are also different.
  • the main functions of functional proteins are: anti-inflammatory infection, anti-viral infection, anti-tumor immunity, prevention of osteolysis, anti-graft rejection, treatment of hyperalgesia, treatment of autoimmune diseases, etc.
  • Fc fusion protein drugs have been approved for clinical use by the US Food and Drug Administration (FDA), including anti-hemophilia factor Fc fusion protein (Eloctate) , 2014), transplant rejection drug Belatacept (Nulojix, 2011), hemophilia B drug coagulation factor IXFc fusion protein (Alprolix, 2014), age-related macular degeneration drug Aflibercept (Eylea, 2011), CAPS drug Rilonacept (Arcalyst , 2008), chronic immune thrombocytopenic purpura drug Romiplostim (Nplate, 2008), rheumatoid arthritis drugs Abatacept (Orencia, 2005) and Etanercept (Enbrel, 1998), and psoriasis and transplant rejection drugs Alefacept (Amevive, 2003 ).
  • FDA US Food and Drug Administration
  • the Fc fusion protein can also be used as a new form of vaccine, fusing part of the antigen peptide of the pathogen with the IgG-Fc fragment to induce the body to produce an antigen-specific immune response.
  • Fc fusion protein Compared with traditional protein drugs, Fc fusion protein has many new characteristics and is widely concerned all over the world. However, Fc fusion protein itself also has many limitations, such as high price and cannot be administered orally. With the continuous advancement of related technologies and concepts, Fc fusion proteins will play a greater role in clinical treatment, medical biological research and other fields.
  • one of the objectives of the present invention is to provide a bifunctional fusion protein platform for drug development.
  • the second object of the present invention is to provide a preparation method of the dual-functional fusion protein platform for drug development.
  • the third object of the invention is to provide the use of the bifunctional fusion protein platform for drug development.
  • the first aspect of the present invention provides a bifunctional fusion protein platform for drug development, which comprises a bifunctional fusion protein, the bispecific fusion protein is a dimer, each chain Each includes three structural functional regions, and the three structural functional regions are a first receptor fragment, an IgG1 Fc fragment, and a second receptor fragment.
  • the IgG1Fc fragment is:
  • polypeptide in c) specifically refers to: a polypeptide whose amino acid sequence is as shown in SEQ ID No. 2 has been substituted, deleted or added one or more (specifically, 1-50, or 1-30 , Or 1-20, 1-10, 1-5, or 1-3) amino acids, and has an amino acid sequence such as SEQ ID No. 2.
  • the peptides shown are functional polypeptides.
  • the amino acid sequence of the polypeptide in b) may have more than 80% homology with SEQ ID No. 1, more specifically more than 85% homology, more specifically more than 90% homology, and more specifically It has 93% or more homology, more specifically 95% or more homology, more specifically 97% or more homology, and more specifically 99% or more homology.
  • the bifunctional fusion protein includes a first receptor fragment, an IgG1 Fc fragment, and a second receptor fragment in sequence from the N-terminus to the C-terminus, and is a dimer structure.
  • the dimer is bound by the disulfide bond between the IgG1 Fc fragments, thereby forming an Fc fragment, and further forming a first of two molecules at the N-terminus of the Fc fragment
  • the receptor fragment, IgG1 Fc fragment forms two molecules of the second receptor fragment at the C-terminus of the Fc fragment.
  • the second aspect of the present invention provides an isolated polynucleotide encoding the dual-function fusion protein platform for drug development.
  • the isolated polynucleotide includes a first receptor fragment coding sequence, an IgG1 Fc fragment coding sequence, and a second receptor fragment coding sequence.
  • the coding sequence of the IgG1 Fc fragment is shown in SEQ ID No. 10.
  • the third aspect of the present invention provides a recombinant expression vector comprising a polynucleotide encoding the bifunctional fusion protein platform for drugs.
  • the recombinant expression vector is constructed by inserting the isolated polynucleotide into a multiple cloning site of the expression vector.
  • the expression vector may specifically be an existing commonly used expression vector well known to those skilled in the art.
  • Specific expression vectors that may be used include, but are not limited to: pET series expression vectors, pGEX series expression vectors, pcDNA series expression vectors, and the like.
  • a person skilled in the art can select a suitable vector, and can further modify the existing vector to construct a recombinant expression vector that can achieve a desired expression level.
  • the desired expression level may be a higher protein expression level, or a relatively reasonable protein expression level, so as to give a reasonable dosage for different individuals.
  • a fourth aspect of the present invention provides a fusion protein expression system, the fusion protein expression system containing the recombinant expression vector or genome integrated with the polynucleotide.
  • the fusion protein expression system is constructed by transfecting the recombinant expression vector into a host cell.
  • a host cell Any cell suitable for expression in an expression vector can be used as a host cell.
  • the host cell is a eukaryotic cell, and a mammalian host cell line that does not produce antibodies can be used.
  • Specific cell lines that can be used include, but are not limited to: Chinese hamster ovary cells (CHO), baby hamster kidney cells (BHK, ATCC CCL 10), Sertoli cells of young rats, monkey kidney cells (COS cells), monkey kidney CVI cells transformed by SV40 (COS-7, ATCC CRL 165), Human embryonic kidney cells (HEK-293), monkey kidney cells (CVI ATCC CCL 70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical cancer cells (HELA, ATCC CCL 2) )Wait.
  • CHO Chinese hamster ovary cells
  • BHK baby hamster kidney cells
  • COS cells monkey kidney cells
  • COS-7 monkey kidney CVI cells transformed by SV40
  • HEK-293 Human embryonic kidney cells
  • CVI ATCC CCL 70 monkey kidney cells
  • African green monkey kidney cells VERO-76, ATCC CRL-1587
  • human cervical cancer cells HELA, ATCC CCL 2
  • a fifth aspect of the present invention provides a method for preparing the dual-function fusion protein platform for medicine, which includes the following steps:
  • step 3 Separate the bifunctional fusion protein for drug development from the culture obtained in step 2) and thereby form a bifunctional fusion protein platform for drugs.
  • the bifunctional fusion protein for production drug development can be prepared according to the following method. For example, a recombinant expression vector containing a polynucleotide encoding a bifunctional fusion protein targeted for drug development is directly introduced into a host cell, a bifunctional fusion protein platform expression system for drug development is obtained, and cultured under appropriate conditions, Thus, the expression of the bifunctional fusion protein encoded for the development of drugs is induced.
  • the recombinant expression vectors and host cells used in the present invention are all existing technologies and can be obtained directly through commercial channels.
  • the culture medium used in the cultivation is also various conventional culture mediums, and those skilled in the art can select suitable culture mediums based on experience , Cultured under conditions suitable for the growth of host cells.
  • the selected promoter is induced by an appropriate method (such as temperature conversion or chemical induction), and the cell is cultured for a period of time.
  • the recombinant polypeptide can be expressed in the cell or on the cell membrane, and can interact to form a dimeric fusion protein structure and / or be secreted out of the cell.
  • the bifunctional fusion protein for drug development described in the present invention can be separated and purified by various separation methods using its physical, chemical and other characteristics.
  • These Methods are well known to those skilled in the art, and examples of these methods include but are not limited to: conventional renaturation treatment, treatment with protein precipitation agent (salting out method), centrifugation, osmotic disruption, ultra-treatment, ultra-centrifugation, molecular sieve Chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques.
  • the method of inserting a marker in an expression vector may also be used to label the bifunctional fusion protein for drug development obtained by cultivating a bifunctional fusion protein expression system for drug development and development, so as
  • the specific markers that can be used can be various conventional markers suitable for the purification of the bifunctional fusion protein used in the development of drugs in the art. Thing.
  • a sixth aspect of the present invention provides a composition comprising a therapeutically effective amount of the dual-function fusion protein platform for drug development or the dual-function fusion protein platform expression system (eg, host cell) for drug development Cultures.
  • the dual-function fusion protein platform expression system eg, host cell
  • the composition further includes a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier refers to a carrier used for the administration of a therapeutic agent, including various excipients and diluents, and specifically refers to such pharmaceutical carriers: they are not essential active ingredients themselves, and they are not excessively toxic after administration. Suitable carriers are well known to those of ordinary skill in the art. A full discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceuticals (Mack Pub. Co., N. J. 1991). Pharmaceutically acceptable carriers in the composition can include liquids such as water, saline, glycerin, and ethanol.
  • auxiliary substances may be present in these carriers, such as disintegrants, wetting agents, emulsifiers, pH buffer substances, algin gum, pectin, sodium carboxymethyl cellulose (CMC), xanthan gum, knot Cold gum, guar gum, carrageenan, sucrose, maltitol, stevioside, etc.
  • the seventh aspect of the present invention provides the application of the dual-functional fusion protein platform for drug development in the preparation or screening of drugs that target the first receptor fragment and / or the second receptor fragment.
  • the drugs targeted by the first receptor fragment and / or the second receptor fragment include but are not limited to tumor immunotherapy drugs or rheumatoid arthritis immunotherapy drugs or Yinxiao disease immunotherapy drugs or ankylosing spondylitis immunotherapy drug.
  • the use specifically refers to: using the first receptor fragment and / or the second receptor fragment as a target, using the dual-functional fusion protein platform for drug development as a medicinal effect component to prepare a therapeutic drug, the treatment
  • the drug can be obtained by reducing the expression level of the first receptor fragment and / or the second receptor fragment, and inhibiting the activity of the first receptor fragment and / or the second receptor fragment.
  • reducing the expression level of the first receptor fragment specifically means that the expression level of the first receptor fragment can be reduced by at least 10%, more specifically by at least 30%, more specifically by at least 30% compared to before administration 50%, more specifically can be reduced by at least 70%, further specifically can be reduced by at least 90%.
  • reducing the expression level of the second receptor fragment specifically means that the expression level of the second receptor fragment can be reduced by at least 10%, more specifically by at least 30%, more specifically by at least 30% compared to before administration 50%, more specifically can be reduced by at least 70%, further specifically can be reduced by at least 90%.
  • inhibiting the activity of the first receptor fragment specifically means that the activity of the first receptor fragment can be reduced by at least 10%, more specifically by at least 30%, more specifically by at least 50%, more than before administration Specifically, it can be reduced by at least 70%, further specifically by at least 90%.
  • inhibiting the activity of the second receptor fragment specifically means that the activity of the second receptor fragment can be reduced by at least 10%, more specifically by at least 30%, more specifically by at least 50%, more specifically than before administration Ground can be reduced by at least 70%, further specifically by at least 90%.
  • the eighth aspect of the present invention provides a method of treatment in which the pharmaceutical composition is administered to an individual.
  • the individual refers to an animal (including a human) that can receive the pharmaceutical composition and / or treatment method, and covers both male and female genders unless specifically stated otherwise. Therefore, the individual contains at least any mammals, including but not limited to: humans, non-human primates, such as mammals, dogs, cats, horses, sheep, pigs, cattle, etc., who can use the drug
  • the composition benefits from treatment.
  • the treatment method includes reducing the expression level of the first receptor fragment and / or the second receptor fragment, and inhibiting the activity of the first receptor fragment and / or the second receptor fragment.
  • the bifunctional fusion protein platform for drug development includes a first receptor fragment and a second receptor fragment, which can effectively target the first receptor fragment and the second receptor fragment, and has a good Biological activity, specificity and stability.
  • the structure of the bispecific fusion protein platform can effectively reduce the R & D cost of R & D therapeutic drugs, and has extremely high industrial value.
  • the function principle of the dual-functional fusion protein platform for drug development is to use the EDC in the receptor to neutralize the ligand, and the interaction between the EDC and the ligand can produce inhibition after effective action.
  • FIG. 1 shows a schematic diagram of the SIRP ⁇ -IgG1Fc-PD-1 bifunctional fusion protein for tumor immunotherapy in the bifunctional fusion protein platform for drug development provided by the present invention.
  • FIG. 2 shows a schematic diagram of the LAG-3-IgG1Fc-PD-1 bifunctional fusion protein for tumor immunotherapy in the bifunctional fusion protein platform for drug development provided by the present invention.
  • the experimental methods, detection methods, and preparation methods disclosed in the present invention adopt conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields in the technical field. Conventional technology. These technologies have been well described in the existing literature. For details, see Sambrook et al.
  • MOLECULAR CLONING A LABORATORY MANUAL, Second Edition, Cold Spring Harbor Laboratory Laboratory Press, 1989 and Third Edition, 2001; Ausubel etc., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; the series METHODS INZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE AND AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODSROMIN, ENZYMOLOGY, Wassarman and APWolffe, eds.), Academic, Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, Chromatin Protocols (PBBecker, ed.) Humana Press, Totowa, 1999, etc.
  • the following uses several examples to illustrate the application of the dual-function fusion protein platform of the present invention for drug development in the development of tumor immunotherapy drugs, and to illustrate the role of the dual-function fusion protein platform of the present invention for drug development.
  • the following examples do not limit the dual-functional fusion protein platform of the present invention for drug development can only be used to develop tumor immunotherapy drugs, which can also be used to develop first receptor fragments and / or second receptors
  • the fragments are drugs that act as targets, such as rheumatoid arthritis immunotherapy drugs, Yinxiao disease immunotherapy drugs, ankylosing spondylitis immunotherapy drugs, etc.
  • the amino acid sequence of the SIRP ⁇ receptor fragment is shown in SEQ ID No. 1, and the coding sequence is shown in SEQ ID No. 5; the amino acid sequence of the LAG-3 receptor fragment is shown in SEQ ID No. 2 The sequence is shown in SEQ ID No. 6, the amino acid sequence of the PD-1 receptor fragment is shown in SEQ ID No. 4, the coding sequence is shown in SEQ ID No. 7, and the amino acid sequence of the IgG1 Fc fragment is shown in SEQ ID No. As shown in 5, the coding sequence is shown in SEQ ID No. 8;
  • SIRP ⁇ receptor fragment SEQ ID No. 1
  • SIRP ⁇ receptor fragment coding sequence (SEQ ID No. 5):
  • amino acid sequence of the LAG-3 receptor fragment (SEQ ID No. 2):
  • the coding sequence of the LAG-3 receptor fragment (SEQ ID No. 6):
  • Amino acid sequence of PD-1 receptor fragment (SEQ ID No. 3):
  • Coding sequence of PD-1 receptor fragment (SEQ ID No. 7):
  • the SIRP ⁇ -IgG1Fc-PD-1 bifunctional fusion protein for tumor immunotherapy includes, from the N-terminus to the C-terminus, SIRP ⁇ receptor fragment, IgG1Fc fragment, PD-1 receptor fragment; SIRP ⁇ for tumor immunotherapy -
  • the IgG1Fc-PD-1 bifunctional fusion protein coding sequence includes, from the N-terminus to the C-terminus, the SIRP ⁇ receptor fragment coding sequence, the IgG1Fc fragment coding sequence, and the PD-1 receptor fragment coding sequence.
  • SIRP ⁇ -IgG1Fc-PD-1 bifunctional fusion protein coding sequence for tumor immunotherapy :
  • LAG-3-IgG1Fc-PD-1 bifunctional fusion protein used for tumor immunotherapy includes N-terminal to C-terminal in sequence, including LAG-3 receptor fragment, IgG1Fc fragment, PD-1 receptor fragment; for tumor
  • the LAG-3-IgG1 Fc-PD-1 bifunctional fusion protein coding sequence for immunotherapy includes, from the N-terminus to the C-terminus, the LAG-3 receptor fragment coding sequence, the IgG1Fc fragment coding sequence, and the PD-1 receptor fragment coding sequence.
  • SIRP ⁇ -IgG1Fc-PD-1 bifunctional fusion protein for tumor immunotherapy
  • the coding sequence of the SIRP ⁇ -IgG1Fc-PD-1 bifunctional fusion protein used for tumor immunotherapy was cloned into the multi-cloning site of the expression vector, and the coding sequence was connected to the expression vector to obtain plasmid DNA.
  • the plasmid DNA is transfected into the host cell, and the transfection can be performed in a 6-well plate. After transfection, the positive cell lines were passaged into 1L shake flasks, 37C, 5% CO 2 environment, 120RPM shaker culture, supplemented daily with glucose, amino acids and other nutrients, the cell viability was reduced to 80-85% to stop the culture. After the cell fluid was centrifuged at 2000RCF to remove the cells, the supernatant was centrifuged at 5000RCF for protein purification to obtain the SIRP ⁇ -IgG1Fc-PD-1 bifunctional fusion protein for tumor immunotherapy.
  • the coding sequence of the sLAG-3-IgG1Fc-PD-1 bifunctional fusion protein used for tumor immunotherapy was cloned into the multi-cloning site of the expression vector, and the coding sequence was connected to the expression vector to obtain plasmid DNA.
  • the plasmid DNA is transfected into the host cell, and the transfection can be performed in a 6-well plate. After transfection, the positive cell lines were passaged into 1L shake flasks, 37C, 5% CO 2 environment, 120RPM shaker culture, supplemented daily with glucose, amino acids and other nutrients, the cell viability was reduced to 80-85% to stop the culture.
  • the cell fluid was centrifuged at 2000RCF to remove the cells, the supernatant was centrifuged at 5000RCF for protein purification to obtain the sLAG-3-IgG1Fc-PD-1 bifunctional fusion protein for tumor immunotherapy.
  • CD47 is an important "self” marker on the cell surface and an important signal for regulating macrophage phagocytosis.
  • CD47 can bind to SIRP ⁇ on the surface of macrophages, phosphorylate its ITIM, and then recruit SHP-1 protein to produce a series of cascade reactions to inhibit the phagocytosis of macrophages.
  • Almost all tumor cells and tissues highly express CD47, which is 3 times higher than that of normal cells and tissues.
  • tumor cells effectively avoid the phagocytosis of macrophages.
  • the SIRP ⁇ -IgG1Fc-PD-1 bifunctional fusion protein used for tumor immunotherapy can enhance the killing of tumor cells by macrophages, so it can be antagonized by the SIRP ⁇ -IgG1Fc-PD-1 bifunctional fusion protein used for tumor immunotherapy.
  • SIRP ⁇ -CD47 inhibition pathway kills target cell lines to detect the biological activity of SIRP ⁇ -IgG1Fc-PD-1 bifunctional fusion protein for tumor immunotherapy.
  • the experiment proves that the SIRP ⁇ -IgG1Fc-PD-1 bifunctional fusion protein used for tumor immunotherapy has a strong protective effect on macrophages, and it can be seen that the SIRP ⁇ -IgG1Fc-PD-1 bifunctional fusion protein used for tumor immunotherapy targets SIRP ⁇ has good activity and specificity.
  • LAG3 lymphocyte activation 3, LAG3, CD2263 is an immune checkpoint receptor protein, mainly expressed in activated T cells, NK cells, B cells and plasma cells dendritic cells. LAG3 can downregulate the activity of T cells by binding to MHC II molecules. At the same time, LAG3 can also enhance the inhibitory activity of regulatory T cells (Treg).
  • the LAG-3-IgG1Fc-PD-1 bifunctional fusion protein used for tumor immunotherapy can release the inhibition of T cells, so the LAG-3-IgG1Fc-PD-1 bifunctional fusion protein used for tumor immunotherapy can be used Block the LAG-3-MHCII inhibitory pathway and enhance T cell killing of tumor cells to detect the biological activity of LAG-3-IgG1Fc-PD-1 bifunctional fusion protein for tumor immunotherapy.
  • the LAG-3 -IgG1Fc-PD-1 bifunctional fusion protein used for tumor immunotherapy can effectively enhance the activity of T cells. It can be seen that the sLAG-3-IgG1Fc-PD-1 bifunctional fusion used for tumor immunotherapy The protein has good activity and specificity against sLAG-3.
  • the present invention effectively overcomes various shortcomings in the prior art and has high industrial utilization value.

Abstract

本发明属于生物技术领域，特别是涉及一种用于研发药物的双功能融合蛋白平台及其制备方法和用途。本发明一种用于研发药物的双功能融合蛋白平台，其包含双功能融合蛋白，所述双功能融合蛋白为二聚体，每个双功能融合蛋白均分别包括三个结构功能区域，所述三个结构功能区域为第一受体片段、IgG1Fc片段和第二受体片段。本发明所提供的双特异性融合蛋白平台包括第一受体片断和第二受体片断，能够有效针对第一受体片断和第二受体片断，具有良好的生物活性、特异性和稳定性。双特异性融合蛋白的结构能够有效降低研发药物的研发成本，具有极高的产业化价值。

Description

一种用于研发药物的双功能融合蛋白平台 技术领域
本发明属于生物技术领域，特别是涉及一种用于研发药物的双功能融合蛋白平台及其该双功能融合蛋白制备方法和用途。
背景技术
Fc融合蛋白是指利用基因工程技术将免疫球蛋白(IgG、IgA等)的Fc段与某种具有生物学活性的功能蛋白分子融合而产生的新型蛋白。具有生物学活性的功能蛋白可以是细胞因子、毒素、受体、酶、抗原肽等。Fc融合蛋白不仅可发挥所融合蛋白的生物学活性，还具有一些抗体的性质，如可引起抗体依赖细胞介导的细胞毒作用(ADCC)、补体依赖的细胞毒作用(CDC)与抗体依赖细胞介导的吞噬作用(ADCP)等。
蛋白类药物在血浆内半衰期较短，为了达到治疗效果需要大剂量给药，这样会造成严重的副作用，给患者造成巨大的负担。将功能蛋白与免疫球蛋白的Fc段融合，可以延长药物在血浆内的半衰期，降低药物的免疫原性，在疾病的诊断与治疗方面有重要的意义。
Fc融合蛋白由免疫球蛋白的Fc段与具有生物学活性的蛋白分子组成。Fc段与功能蛋白具有相对独立的结构域与功能，能够从不同角度影响融合蛋白的理化性质与生物学活性。
Fc融合蛋白最主要的特点是包含Fc段，与单克隆抗体中Fc段功能类似，融合蛋白的Fc段可以起到延长功能蛋白在血浆内的半衰期、提高分子的稳定性、特异性结合体内的Fc受体,并发挥相应的生物学功能等作用。除此之外，Fc段可以特异性结合protein A，简化了Fc融合蛋白的纯化步骤，在相关生物制品的研发制备具有重要意义。
人体内的免疫球蛋白依赖于Fc段与新生Fc受体(FcRn)的结合，在FcRn的保护下，半衰期可以长达19～21d。与此类似，Fc融合蛋白也能依赖此原理延长在体内的半衰期：Fc段与FcRn的结合呈pH依赖性，在pH7.4的生理条件下，FcRn与Fc不结合；在细胞内涵体pH 6.0～6.5的酸性条件下，两者结合，从而避免了融合蛋白在细胞内被溶酶体等快速降解。除此之外，Fc段能够增大分子体积，降低肾清除率，也从一定程度上延长了半衰期。
Fc融合蛋白可以通过Fc铰链区的二硫键链接形成稳定的二聚体。通过对二硫键进行进一步的基因工程改造，还可以使Fc融合蛋白形成更加稳定的六聚体复合物。另外，Fc区域可以独立折叠，保证了分子的稳定性。
将Fc段与功能蛋白融合，可提高蛋白在哺乳动物中的表达分泌，同时Fc段可以与protein A发生特异性结合，有利于融合蛋白的纯化。
Fc融合蛋白的FC结构域可与免疫细胞表面的Fc受体结合，发挥多种生物学功能，如介导穿过胎盘和粘膜屏障、炎症反应、抗体依赖细胞介导的吞噬作用(ADCP)、抗体依赖细胞介导的细胞毒作用(ADCC)、补体依赖的细胞毒作用(CDC)，促进树突状细胞(DC)成熟，调节细胞因子分泌，调节B细胞增殖分化等。
Fc融合蛋白的Fc结构域影响了融合蛋白分子的理化性质与生物学活性，而功能蛋白部分则决定了Fc融合蛋白的药理活性。功能蛋白的种类很多，可以是细胞因子、毒素、受体、酶等，其作用也各不相同。功能蛋白的作用主要有：抗炎性感染、抗病毒感染、抗瘤免疫、防止溶骨、抗移植排斥、治疗痛觉过敏、自身免疫性疾病的治疗等。
大部分Fc融合蛋白药物的作用机制为受体与配体之间的相互作用，同时辅以Fc片段的多种生物学功能。截止2014年9月，已有9种人IgG-Fc融合蛋白药物经美国食品及药品监督管理局(FDA)批准临床使用，包括治疗甲型血友病药物抗血友病因子Fc融合蛋白(Eloctate，2014)、移植排斥药物Belatacept(Nulojix，2011)、乙型血友病的药物凝血因子IXFc融合蛋白(Alprolix，2014)、年龄相关性黄斑变性药物Aflibercept(Eylea，2011)、CAPS药物Rilonacept(Arcalyst，2008)、慢性免疫性血小板减少性紫癜药物Romiplostim(Nplate，2008)、风湿性关节炎药物Abatacept(Orencia，2005)和Etanercept(Enbrel，1998)以及银屑病和移植排斥药物Alefacept(Amevive，2003)。除了上述已成功转化的临床应用的Fc融合蛋白外，有更多的Fc融合蛋白药物处于临床研究中。
另外，Fc融合蛋白也可以作为一种新型疫苗形式，将病原体的部分抗原肽与IgG-Fc片段进行融合，诱导机体产生抗原特异性免疫应答。研究表明，HIV-1 Gag p24、gp120 V3以及流感病毒HA胞外域与小鼠IgG2a-Fc的融合疫苗，可提高小鼠抗原特异性体液免疫反应。
Fc融合蛋白较传统蛋白类药物具有多种新特性，在全世界范围内受到广泛关注，但Fc融合蛋白本身也存在许多局限，如价格昂贵、不可口服给药等。随着相关技术与理念的不断进步，Fc融合蛋白在临床治疗、医学生物研究等领域必将发挥更大的作用。
发明内容
鉴于以上所述现有技术的缺点，本发明的目的之一在于提供一种用于研发药物的双功能融合蛋白平台。
本发明目的之二在于提供该用于研发药物的双功能融合蛋白平台的制备方法。
发明目的之三在于提供该用于研发药物的双功能融合蛋白平台的用途。
为实现上述目的及其他相关目的，本发明第一方面提供一种用于研发药物的双功能融合蛋白平台，其包含一双功能融合蛋白，所述双特异性融合蛋白为二聚体，每条链均包括三个结构功能区域，所述三个结构功能区域为第一受体片段、IgG1Fc片段和第二受体片段。
所述IgG1Fc片段为：
a)氨基酸序列如SEQ ID No.5所示的多肽；或
b)氨基酸序列与SEQ ID No.5具有80％以上同源性、且具有c)限定的多肽的功能的多肽；
具体的，所述c)中的多肽具体指：氨基酸序列如SEQ ID No.2所示的多肽经过取代、缺失或者添加一个或多个(具体可以是1-50个，也可以是1-30个，也可以是1-20个，也可以是1-10个，也可以是1-5个，也可以是1-3个)氨基酸而得到的，且具有氨基酸序列如SEQ ID No.2所示的多肽功能的多肽。
所述b)中的多肽的氨基酸序列可与SEQ ID No.1具有80％以上同源性，更具体可具有85％以上同源性，更具体可具有90％以上同源性，更具体可具有93％以上同源性，更具体可具有95％以上同源性，更具体可具有97％以上同源性，进一步具体可具有99％以上同源性。
具体的，所述双功能融合蛋白自N端至C端依次包括第一受体片段、IgG1Fc片段和第二受体片段，为二聚体结构。
具体的，本发明所提供的双功能融合蛋白中，二聚体通过所述IgG1Fc片段间的二硫键结合，从而可以形成Fc片段，并可以进一步在Fc片段的N端形成两分子的第一受体片段、IgG1Fc片段，在Fc片段的C端形成两分子的第二受体片段。
本发明第二方面提供一种分离的多核苷酸，编码所述用于研发药物的双功能融合蛋白平台。
具体的，所述分离的多核苷酸包括第一受体片段编码序列、IgG1Fc片段编码序列和第二受体片断编码序列。
更具体的，所述IgG1Fc片段编码序列如SEQ ID No.10所示。
本发明第三方面提供一种重组表达载体，包含编码所述用于药物的双功能融合蛋白平台的多核苷酸。
具体的，所述重组表达载体由所述分离的多核苷酸插入到表达载体的多克隆位点构建而成。所述表达载体具体可以是本领域的技术人员所熟知的现有常用的表达载体，具体可采用的表达载体包括但不限于：pET系列表达载体、pGEX系列表达载体、pcDNA系列表达载体等。本领域技术人员可以选择合适的载体，还可以进一步对现有的载体进行修饰改造，以构建获得能够达到期望的表达水平的重组表达载体。所述期望的表达水平可以是更高的 蛋白表达水平，也可以是一个相对合理的蛋白表达水平，以针对不同个体给予合理的给药量。
本发明第四方面提供一种融合蛋白表达系统，所述融合蛋白表达系统含有所述重组表达载体或基因组中整合有外源的所述的多核苷酸。
具体的，所述融合蛋白表达系统由所述重组表达载体转染到宿主细胞构建而成。任何适用于表达载体进行表达的细胞都可以作为宿主细胞。例如，酵母、昆虫、植物等的细胞。优选的，所述宿主细胞为真核细胞，可采用不会产生抗体的哺乳动物宿主细胞系，具体可采用的细胞系包括但不限于：中国仓鼠的卵巢细胞(CHO)、幼仓鼠的肾脏细胞(BHK,ATCC CCL 10)、幼鼠的塞尔托利细胞(sertoli cells)、猴的肾脏细胞(COS细胞)、通过SV40(COS-7,ATCC CRL 165 1)转化的猴的肾脏CVI细胞、人的胚肾细胞(HEK-293)、猴肾脏细胞(CVI ATCC CCL 70)、非洲绿猴的肾脏细胞(VERO-76,ATCC CRL-1587)、人的子宫颈癌细胞(HELA,ATCC CCL 2)等。
本发明第五方面提供所述用于药物的双功能融合蛋白平台的制备方法，包括如下步骤：
1)培养所述融合蛋白表达系统，使之表达所述用于药物的双功能融合蛋白；
2)收集含有所述用于研发药物的双功能融合蛋白的培养物；
3)从步骤2)所得培养物中分离出所述用于研发药物的双功能融合蛋白并由此形成用于药物的双功能融合蛋白平台。
具体的，在获得编码本发明的用于研发药物的双功能融合蛋白的核酸序列后，可按照以下方法制备生产目的用于研发药物的双功能融合蛋白。例如将含有编码目标用于研发药物的双功能融合蛋白的多核苷酸的重组表达载体直接导入宿主细胞，获得用于研发药物的双功能融合蛋白平台表达系统，并在适当的条件下进行培养，从而诱导出被编码用于研发药物的双功能融合蛋白的表达。
本发明中所用的重组表达载体和宿主细胞均为现有技术，可通过商业途径直接获取，培养中所用的培养基亦为各种常规培养基，本领域技术人员可根据经验选择适用的培养基，在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。在上面的方法中，重组多肽可在细胞内或在细胞膜上表达，并可以互相作用形成二聚体融合蛋白结构和/或被分泌到细胞外。一旦获得本发明所述的用于研发药物的双功能融合蛋白，就可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化所述用于研发药物的双功能融合蛋白，这些方法是本领域技术人员所熟知的，这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层 析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术等。
在本发明一实施方式中，还可以通过在表达载体中插入标记物的方法，对用于研发治疗药物的双功能融合蛋白表达系统培养获得的用于研发药物的双功能融合蛋白进行标记，以便于对培养物中的用于研发药物的双功能融合蛋白进行分离、提纯，具体可使用的标记物可以是各种本领域各种常规的适用于用于研发药物的双功能融合蛋白纯化的标记物。
本发明第六方面提供一种组合物，包括治疗有效量的所述用于研发药物的双功能融合蛋白平台或所述用于研发药物的双功能融合蛋白平台表达系统(例如，宿主细胞)的培养物。
在本文中，若能减少一个或多个病征或临床指标即代表该治疗是“有效”的。
具体的，所述组合物还包括药学上可接受的载体。药学上可接受的载体指用于治疗剂给药的载体，包括各种赋形剂和稀释剂，具体指这样一些药剂载体：它们本身并不是必要的活性成分，且施用后没有过分的毒性。合适的载体是本领域普通技术人员所熟知的。在Remington's Pharmaceutical Sciences(Mack Pub.Co.，N.J.1991)中可找到关于药学上可接受的赋形剂的充分讨论。在组合物中药学上可接受的载体可包括液体，如水、盐水、甘油和乙醇。另外，这些载体中还可能存在辅助性的物质，如崩解剂、润湿剂、乳化剂、pH缓冲物质、海藻胶、果胶、羧甲基纤维素钠(CMC)、黄原胶、结冷胶、瓜尔胶、卡拉胶、蔗糖、麦芽糖醇、甜菊糖苷等。
本发明第七方面提供所述用于研发药物的双功能融合蛋白平台在制备或筛选以第一受体片断和/或第二受体片断为作用靶标的药物中的应用。
所述以第一受体片断和/或第二受体片断为作用靶标药物包括但不限于肿瘤免疫治疗药物或类风湿关节炎免疫治疗药物或银霄病免疫治疗药物或强直性脊柱炎免疫治疗药物。
所述用途具体指：以第一受体片断和/或第二受体片断为作用靶标，将所述用于研发药物的双功能融合蛋白平台作为药效成分用于制备治疗药物，所述治疗药物可通过降低第一受体片断和/或第二受体片断的表达量、抑制第一受体片断和/或第二受体片断的活性等方式。具体的，降低第一受体片断的表达量具体指，相比给药前，第一受体片断的表达量可降低至少10％，更具体地可降低至少30％，更具体地可降低至少50％，更具体地可降低至少70％，进一步具体地可降低至少90％。具体的，降低第二受体片断的表达量具体指，相比给药前，第二受体片断的表达量可降低至少10％，更具体地可降低至少30％，更具体地可降低至少50％，更具体地可降低至少70％，进一步具体地可降低至少90％。具体的，抑制第一受体片断活性具体指，相比给药前，第一受体片断活性可降低至少10％，更具体地可降低至 少30％，更具体地可降低至少50％，更具体地可降低至少70％，进一步具体地可降低至少90％。具体的，抑制第二受体片断活性具体指，相比给药前第二受体片断活性可降低至少10％，更具体地可降低至少30％，更具体地可降低至少50％，更具体地可降低至少70％，进一步具体地可降低至少90％。
本发明第八方面提供一种治疗方法，将所述药物组合物施用于个体。
所述个体是指可接受所述药物组合物和/或治疗方法的动物(包括人类)，在此涵盖了雄性与雌性两种性别，除非另有具体说明。因此，所述个体至少包含任何哺乳类动物，包括但不限于：人类、非人类的灵长类，如哺乳动物、狗、猫、马、羊、猪、牛等，其可因利用所述药物组合物进行治疗而获益。
具体的，所述治疗方法通过降低第一受体片断和/或第二受体片断的表达量、抑制第一受体片断和/或第二受体片断的活性等方式。
如上所述，本发明所提供的用于研发药物的双功能融合蛋白平台包括第一受体片断和第二受体片断，能够有效针对第一受体片断和第二受体片断，具有良好的生物活性、特异性和稳定性。双特异性融合蛋白平台的结构能够有效降低研发治疗药物的研发成本，具有极高的产业化价值。
本发明所提供的用于研发药物的双功能融合蛋白平台的作用原理是,利用受体中EDC中和配体，EDC与配体之间的相互作用，有效作用后产生抑制。
附图说明
图1显示为本发明所提供的用于研发药物的双功能融合蛋白平台中的用于肿瘤免疫治疗的SIRPα-IgG1Fc-PD-1双功能融合蛋白的示意图。
图2显示为本发明所提供的用于研发药物的双功能融合蛋白平台中的用于肿瘤免疫治疗的LAG-3-IgG1Fc-PD-1双功能融合蛋白的示意图。
具体实施方式
以下通过特定的具体实例说明本发明的实施方式，本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用，本说明书中的各项细节也可以基于不同观点与应用，在没有背离本发明的精神下进行各种修饰或改变。
在进一步描述本发明具体实施方式之前，应理解，本发明的保护范围不局限于下述特 定的具体实施方案；还应当理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围；在本发明说明书和权利要求书中，除非文中另外明确指出，单数形式“一个”、“一”和“这个”包括复数形式。
当实施例给出数值范围时，应理解，除非本发明另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义，本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外，根据本技术领域的技术人员对现有技术的掌握及本发明的记载，还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
除非另外说明，本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、染色质结构和分析、分析化学、细胞培养、重组DNA技术及相关领域的常规技术。这些技术在现有文献中已有完善说明，具体可参见Sambrook等MOLECULAR CLONING：A LABORATORY MANUAL，Second edition，Cold Spring Harbor Laboratory Press，1989 and Third edition，2001；Ausubel等，CURRENT PROTOCOLS IN MOLECULAR BIOLOGY，John Wiley&Sons，New York，1987and periodic updates；the series METHODS IN ENZYMOLOGY，Academic Press，San Diego；Wolffe，CHROMATIN STRUCTURE AND FUNCTION，Third edition，Academic Press，San Diego，1998；METHODS IN ENZYMOLOGY，Vol.304，Chromatin(P.M.Wassarman and A.P.Wolffe，eds.)，Academic Press，San Diego，1999；和METHODS IN MOLECULAR BIOLOGY，Vol.119，Chromatin Protocols(P.B.Becker，ed.)Humana Press，Totowa，1999等。
下面通过几个实施例来说明本发明的用于研发药物的双功能融合蛋白平台在研发肿瘤免疫治疗药物的应用，来说明本发明的用于研发药物的双功能融合蛋白平台的作用。但是下面的实施例并不限制本发明的用于研发药物的双功能融合蛋白平台只能用于研发肿瘤免疫治疗药物，其还可以用于研发以第一受体片断和/或第二受体片断为作用靶标的药物，例如类风湿关节炎免疫治疗药物、银霄病免疫治疗药物、强直性脊柱炎免疫治疗药物等。
实施例1
1.作为SIRPα受体片段的氨基酸序列如SEQ ID No.1所示，编码序列如SEQ ID No.5所示；作为LAG-3受体片段的氨基酸序列如SEQ ID No.2所示，编码序列如SEQ ID No.6所示，作为PD-1受体片段的氨基酸序列如SEQ ID No.4所示，编码序列如SEQ ID No.7 所示，IgG1Fc片段的氨基酸序列如SEQ ID No.5所示，编码序列如SEQ ID No.8所示；
SIRPα受体片段的氨基酸序列(SEQ ID No.1)：
Figure PCTCN2019086880-appb-000001
SIRPα受体片段的编码序列(SEQ ID No.5)：
Figure PCTCN2019086880-appb-000002
LAG-3受体片段的氨基酸序列(SEQ ID No.2)：
Figure PCTCN2019086880-appb-000003
Figure PCTCN2019086880-appb-000004
LAG-3受体片段的编码序列(SEQ ID No.6)：
Figure PCTCN2019086880-appb-000005
PD-1受体片段的氨基酸序列(SEQ ID No.3)：
Figure PCTCN2019086880-appb-000006
PD-1受体片段的编码序列(SEQ ID No.7)：
Figure PCTCN2019086880-appb-000007
IgG1Fc片段的氨基酸序列(SEQ ID No.4)：
Figure PCTCN2019086880-appb-000008
IgG1Fc片段的编码序列(SEQ ID No.8)：
Figure PCTCN2019086880-appb-000009
2.用于肿瘤免疫治疗的SIRPα-IgG1Fc-PD-1双功能融合蛋白自N端至C端依次包括，SIRPα受体片段、IgG1Fc片段、PD-1受体片段；用于肿瘤免疫治疗的SIRPα-IgG1Fc-PD-1双功能融合蛋白编码序列自N端至C端依次包括，SIRPα受体片段编码序列、IgG1Fc片段编码序列、PD-1受体片段编码序列。
用于肿瘤免疫治疗的SIRPα-IgG1Fc-PD-1双功能融合蛋白氨基酸序列:
Figure PCTCN2019086880-appb-000010
用于肿瘤免疫治疗的SIRPα-IgG1Fc-PD-1双功能融合蛋白编码序列:
Figure PCTCN2019086880-appb-000011
Figure PCTCN2019086880-appb-000012
3.用于肿瘤免疫治疗的LAG-3-IgG1Fc-PD-1双功能融合蛋白自N端至C端依次包括，LAG-3受体片段、IgG1Fc片段、PD-1受体片段；用于肿瘤免疫治疗的LAG-3-IgG1Fc-PD-1双功能融合蛋白编码序列自N端至C端依次包括，LAG-3受体片段编码序列、IgG1Fc片段编码序列、PD-1受体片段编码序列。
用于肿瘤免疫治疗的LAG-3-IgG1Fc-PD-1双功能融合蛋白氨基酸序列:
Figure PCTCN2019086880-appb-000013
Figure PCTCN2019086880-appb-000014
用于肿瘤免疫治疗的LAG-3-IgG1Fc-PD-1双功能融合蛋白编码序列:
Figure PCTCN2019086880-appb-000015
Figure PCTCN2019086880-appb-000016
实施例2
表达用于肿瘤免疫治疗的SIRPα-IgG1Fc-PD-1双功能融合蛋白
将用于肿瘤免疫治疗的SIRPα-IgG1Fc-PD-1双功能融合蛋白的编码序列克隆至表达载体的多克隆位点中，实现编码序列与表达载体的连结，获得质粒DNA。将质粒DNA转染宿主细胞，转染可以在6孔板进行。转染后的阳性细胞株传代至1L摇瓶中，温度37C，5％的CO 2环境中，120RPM摇床培养，每天补充葡萄糖、氨基酸等养料，细胞活率降低至80-85％停止培养。将细胞液2000RCF离心取出细胞后，再5000RCF离心取上清进行蛋白纯化，获得用于肿瘤免疫治疗的SIRPα-IgG1Fc-PD-1双功能融合蛋白。
实施例3
表达用于肿瘤免疫治疗的LAG-3-IgG1Fc-PD-1双功能融合蛋白
将用于肿瘤免疫治疗的sLAG-3-IgG1Fc-PD-1双功能融合蛋白的编码序列克隆至表达 载体的多克隆位点中，实现编码序列与表达载体的连结，获得质粒DNA。将质粒DNA转染宿主细胞，转染可以在6孔板进行。转染后的阳性细胞株传代至1L摇瓶中，温度37C，5％的CO 2环境中，120RPM摇床培养，每天补充葡萄糖、氨基酸等养料，细胞活率降低至80-85％停止培养。将细胞液2000RCF离心取出细胞后，再5000RCF离心取上清进行蛋白纯化，获得用于肿瘤免疫治疗的sLAG-3-IgG1Fc-PD-1双功能融合蛋白。
实施例4
用于肿瘤免疫治疗的SIRPα-IgG1Fc-PD-1双功能融合蛋白活性实验：
CD47是细胞表面一个重要的"self"标记，是调节巨噬细胞吞噬作用的一个重要信号。CD47可以与巨噬细胞表面SIRPα结合，磷酸化其ITIM，随后招募SHP-1蛋白，产生一系列的级联反应抑制巨噬细胞的吞噬作用。几乎所有的肿瘤细胞和组织都高表达CD47，是对应正常细胞和组织的3倍。通过CD47这个"self"信号，肿瘤细胞有效的躲避了巨噬细胞的吞噬作用。因而用于肿瘤免疫治疗的SIRPα-IgG1Fc-PD-1双功能融合蛋白可增强巨噬细胞对肿瘤细胞的杀伤，所以可通过用于肿瘤免疫治疗的SIRPα-IgG1Fc-PD-1双功能融合蛋白拮抗SIRPα-CD47抑制通路对靶细胞细胞株的杀伤来检测用于肿瘤免疫治疗的SIRPα-IgG1Fc-PD-1双功能融合蛋白的生物学活性。实验证明，用于肿瘤免疫治疗的SIRPα-IgG1Fc-PD-1双功能融合蛋白对巨噬细胞具有较强的保护作用，可见用于肿瘤免疫治疗的SIRPα-IgG1Fc-PD-1双功能融合蛋白针对SIRPα具有良好的活性和特异性。
实施例5
用于肿瘤免疫治疗的LAG-3-IgG1Fc-PD-1双功能融合蛋白活性实验：
LAG3(lymphocyte activation gene 3，LAG3，CD223)是一种免疫检查点受体蛋白，主要表达在活化的T细胞、NK细胞、B细胞和浆细胞树突细胞。LAG3可通过和MHC II分子的结合，下调T细胞的活性。同时，LAG3也可增强调节性T细胞(Treg)的抑制活性。
因而用于肿瘤免疫治疗的LAG-3-IgG1Fc-PD-1双功能融合蛋白可解除对T细胞的抑制，所以可通过用于肿瘤免疫治疗的LAG-3-IgG1Fc-PD-1双功能融合蛋白阻断LAG-3-MHCII抑制通路，增强T细胞对肿瘤细胞的杀伤来检测用于肿瘤免疫治疗的LAG-3-IgG1Fc-PD-1双功能融合蛋白的生物学活性。实验证明，用于肿瘤免疫治疗的LAG-3 -IgG1Fc-PD-1双功能融合蛋白可有效地增强T细胞的活性，可见用于肿瘤免疫治疗的sLAG-3-IgG1Fc-PD-1双功能融合蛋白针对sLAG-3具有良好的活性和特异性。
综上所述，本发明有效克服了现有技术中的种种缺点而具高度产业利用价值。
上述实施例仅例示性说明本发明的原理及其功效，而非用于限制本发明。任何熟悉此技术的人士皆可在不违背本发明的精神及范畴下，对上述实施例进行修饰或改变。因此，举凡所属技术领域中具有通常知识者在未脱离本发明所揭示的精神与技术思想下所完成的一切等效修饰或改变，仍应由本发明的权利要求所涵盖。

Claims (10)

  1. 一种用于研发药物的双功能融合蛋白平台，其包含双功能融合蛋白，所述双功能融合蛋白为二聚体，每个双功能融合蛋白均分别包括三个结构功能区域，所述三个结构功能区域为第一受体片段、IgG1Fc片段和第二受体片段；
    所述IgG1Fc片段为：
    a)氨基酸序列如SEQ ID No.5所示的多肽；或
    b)氨基酸序列与SEQ ID No.5具有80％以上同源性、且具有c)限定的多肽的功能的多肽。
  2. 如权利要求1所述的一种用于研发药物的双功能融合蛋白平台，其特征在于，所述双功能融合蛋白自N端至C端依次包括第一受体片段、IgG1Fc片段和第二受体片段，为二聚体。
  3. 如权利要求1所述的一种用于研发药物的双功能融合蛋白平台，其特征在于，所述二聚体通过所述IgG1Fc片段片段间的二硫键结合。
  4. 一种分离的多核苷酸，编码所述针对权利要求1至3任一项权利要求所述的一种用于研发药物的双功能融合蛋白。
  5. 一种重组表达载体，包含编码所述针对权利要求1至3任一项权利要求所述的一种用于研发药物的双功能融合蛋白的多核苷酸。
  6. 一种用于研发药物的双功能融合蛋白平台表达系统，其含有权利要求5所述重组表达载体或基因组中整合有外源的所述的多核苷酸。
  7. 如权利要求1-3任一权利要求所述的一种用于研发药物的双功能融合蛋白平台的制备方法，包括如下步骤：
    1)培养所述一种用于研发药物的双功能融合蛋白表达系统，使之表达所述一种用于研发药物的双功能融合蛋白；
    2)收集含有所述一种用于研发药物的双功能融合蛋白的培养物；
    3)从步骤2)所得培养物中分离出所述一种用于研发药物的双功能融合蛋白并由此获得用于研发药物的双功能融合蛋白平台。
  8. 一种组合物，包括治疗有效量的如权利要求1-3任一权利要求所述的一种用于研发药物的双功能融合蛋白平台或如权利要求6所述的一种用于研发药物的双功能融合蛋白平台表达系统的培养物。
  9. 如权利要求1-3任一权利要求所述的一种用于研发药物的双功能融合蛋白在制备或 筛选以第一受体片断和/或第二受体片断为作用靶标的药物中的应用。
  10. 权利要求9所述的应用，其中所述以第一受体片断和/或第二受体片断为作用靶标药物包括但不限于肿瘤免疫治疗药物或类风湿关节炎免疫治疗药物或银霄病免疫治疗药物或强直性脊柱炎免疫治疗药物。
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