CN109824780A - It is a kind of for researching and developing the bifunctional fusion proteins platform of drug - Google Patents
It is a kind of for researching and developing the bifunctional fusion proteins platform of drug Download PDFInfo
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- CN109824780A CN109824780A CN201811223401.8A CN201811223401A CN109824780A CN 109824780 A CN109824780 A CN 109824780A CN 201811223401 A CN201811223401 A CN 201811223401A CN 109824780 A CN109824780 A CN 109824780A
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- Peptides Or Proteins (AREA)
Abstract
The invention belongs to field of biotechnology, more particularly to a kind of bifunctional fusion proteins platform and its preparation method and application for researching and developing drug.The present invention is a kind of for researching and developing the bifunctional fusion proteins platform of drug, it includes bifunctional fusion proteins, the bifunctional fusion proteins are dimer, each bifunctional fusion proteins respectively include three structure function regions, and three structure function regions are the first receptor fragments, IgG1Fc segment and Co receptor segment.Bispecific fusion protein platform provided by the present invention includes the first acceptor moieties and Co receptor segment, can effectively be directed to the first acceptor moieties and Co receptor segment, has good bioactivity, specificity and stability.The structure of bispecific fusion protein can be effectively reduced the research and development cost of research and development drug, have high industrialization value.
Description
Technical field
The invention belongs to field of biotechnology, more particularly to a kind of for researching and developing the bifunctional fusion proteins platform of drug
And its bifunctional fusion proteins preparation method and purposes.
Background technique
Fc fusion protein, which refers to, has the Fc section of immunoglobulin (IgG, IgA etc.) with certain using technique for gene engineering
The functional protein molecule of biological activity merges and the novel protein of generation.Functional protein with biological activity can be
Cell factor, toxin, receptor, enzyme, Antigenic Peptide etc..Fc fusion protein can not only play the biological activity of institute's fusion protein, also
Property with some antibody can such as cause the cell of Antibody -dependent cell cytotoxicity effect (ADCC), Complement Dependent
The phagocytosis (ADCP) etc. that toxic action (CDC) and antibody-dependant cell mediate.
Protein medicaments half-life period in blood plasma is shorter, needs large dosage administration to reach therapeutic effect, can make in this way
At serious side effect, huge burden is caused to patient.Functional protein is merged with the Fc of immunoglobulin section, Ke Yiyan
Half-life period of the long drug in blood plasma, the immunogenicity of drug is reduced, there is important meaning in terms of the Clinics and Practices of disease
Justice.
Fc section of the Fc fusion protein by immunoglobulin is formed with the protein molecular with biological activity.Fc sections and function
Albumen have the function of relatively independent structural domain and, the physicochemical property and biology of fusion protein can be influenced from different perspectives
Activity.
The most important feature of Fc fusion protein is, the fusion protein similar with Fc sections of functions in monoclonal antibody comprising Fc sections
Fc section can play that half-life period of the extension function albumen in blood plasma, to propose high molecular stability, specific binding intracorporal
Fc receptor, and the effects of play corresponding biological function.In addition to this, protein A can be specifically bound for Fc sections, letter
The purification step for having changed Fc fusion protein is of great significance in the research and development preparation of associated biomolecule product.
The intracorporal immunoglobulin of people is dependent on Fc sections and the combination of neonatal Fc receptor (FcRn), under the protection of FcRn,
Half-life period can be up to 19~21d.Similar, Fc fusion protein can also rely on the half-life period of this principle extension in vivo: Fc
The combination of section and FcRn are in pH dependence, and under the physiological condition of pH7.4, FcRn is not combined with Fc；Contain body pH in the cell
Under 6.0~6.5 acid condition, the two is combined, so as to avoid fusion protein in the cell by fast degradations such as lysosomes.It removes
Except this, Fc sections are capable of increasing molecular volume, reduce renal clearance, also extend half-life period to a certain extent.
Fc fusion protein can link to form stable dimer by the disulfide bond of Fc hinge area.By to disulfide bond into
The further genetic engineering transformation of row, can also make Fc fusion protein form six more stable dimeric complexes.In addition, Fc
Region can independently fold, and ensure that the stability of molecule.
Fc sections are merged with functional protein, the expression secretion of albumen in mammals can be improved, while Fc sections can be with
Protein A is specifically bound, and is conducive to the purifying of fusion protein.
The FC structural domain of Fc fusion protein can play multiple biological function in conjunction with the Fc receptor on immunocyte surface,
As mediated across placenta and mucosal barrier, inflammatory reaction, the phagocytosis (ADCP) of antibody-dependant cell mediation, antibody-dependant
The cytotoxicity (CDC) of cell mediated cytotoxicity (ADCC), Complement Dependent promotes Dendritic Cells (DC) mature,
Cytokine secretion is adjusted, B cell proliferation differentiation etc. is adjusted.
The Fc structural domain of Fc fusion protein affects the physicochemical property and biological activity of fusion protein molecule, and function egg
White part then determines the pharmacological activity of Fc fusion protein.There are many type of functional protein, can be cell factor, toxin, by
Body, enzyme etc. act on also different.The effect of functional protein mainly has: anti-inflammatory infection, viral infection resisting, anti-tumor are exempted from
Epidemic disease prevents bone resorption, resisting transplant rejection, treatment hyperalgia, the treatment of autoimmune disease etc..
Interaction of the mechanism of action of most of Fc fusion protein drug between receptor and ligand, while being aided with Fc piece
The multiple biological function of section.End in September, 2014, has 9 kinds of human IgG-Fc fusion protein drugs through U.S.'s food and drug
Surveillance Authority (FDA) ratifies clinical use, including treatment hemophilia A drug antihemophilic factor Fc fusion protein
(Eloctate, 2014), graft rejection drug Belatacept (Nulojix, 2011), hemophilia B drug coagulation because
Sub- IXFc fusion protein (Alprolix, 2014), age-related macular degeneration drug Aflibercept (Eylea, 2011),
CAPS drug Rilonacept (Arcalyst, 2008), chronic immunity thrombocytopenic purpura drug Romiplostim
(Nplate, 2008), rheumatoid arthritis agents Abatacept (Orencia, 2005) and Etanercept (Enbrel,
And psoriasis and graft rejection drug Alefacept (Amevive, 2003) 1998).In addition to facing for above-mentioned successful conversion
Outside the Fc fusion protein of bed application, there are more Fc fusion protein drugs to be in clinical research.
In addition, Fc fusion protein can also be used as a kind of new generation vaccine form, by the incomplete antigen peptide and IgG- of pathogen
Fc segment is merged, and induction body generates antigen-specific immune response.Studies have shown that HIV-1Gag p24, gp120V3
And the fusion bacterin of influenza virus HA extracellular domain and mouse IgG 2a-Fc, it is anti-that murine antigen specific humoral immunity can be improved
It answers.
Fc fusion protein has a variety of new features compared with traditional protein class drug, receives significant attention in worldwide,
But Fc fusion protein itself is there is also many limitations, such as expensive, not Orally-administrable.With the relevant technologies and theory
Be constantly progressive, Fc fusion protein will play bigger effect in fields such as clinical treatment, medical biotechnology researchs.
Summary of the invention
In view of the foregoing deficiencies of prior art, one of the objects of the present invention is to provide a kind of for researching and developing drug
Bifunctional fusion proteins platform.
The second purpose of the present invention is to provide the preparation methods of the bifunctional fusion proteins platform for being used to research and develop drug.
The three of goal of the invention are to provide the purposes of the bifunctional fusion proteins platform for being used to research and develop drug.
In order to achieve the above objects and other related objects, first aspect present invention provides a kind of for researching and developing double function of drug
Energy fusion protein platform, it includes a bifunctional fusion proteins, the bispecific fusion protein is dimer, and every chain is equal
Including three structure function regions, three structure function regions are the first receptor fragments, IgG1Fc segment and Co receptor
Segment.
The IgG1Fc segment are as follows:
A) amino acid sequence polypeptide as shown in SEQ ID No.5；Or
B) amino acid sequence and SEQ ID No.5 have the function of 80% or more homology and have polypeptide c) limited
Polypeptide；
Specifically, it is described c) in polypeptide refer specifically to: amino acid sequence polypeptide as shown in SEQ ID No.2 is by taking
Generation, missing or addition it is one or more (specifically can be 1-50, be also possible to 1-30, be also possible to 1-20,
Can be 1-10, be also possible to 1-5, be also possible to 1-3) obtained from amino acid, and there is amino acid sequence such as
The polypeptide of polypeptide function shown in SEQ ID No.2.
It is described b) in polypeptide amino acid sequence can with SEQ ID No.1 have 80% or more homology, more specifically may be used
With 85% or more homology, can more specifically have 90% or more homology, can more specifically have 93% or more homology, more
Can specifically have 95% or more homology, can more specifically have 97% or more homology, further can specifically have 99% or more
Homology.
Specifically, the bifunctional fusion proteins from N-terminal successively include to C-terminal the first receptor fragments, IgG1Fc segment and
Co receptor segment is dimeric structure.
Specifically, dimer passes through two between the IgG1Fc segment in bifunctional fusion proteins provided by the present invention
Sulfide linkage combines, and so as to form Fc segment, and further can form the first receptor piece of two molecules in the N-terminal of Fc segment
Section, IgG1Fc segment form the Co receptor segment of two molecules in the C-terminal of Fc segment.
Second aspect of the present invention provides a kind of isolated polynucleotides, and coding is described for researching and developing the bifunctional fusion of drug
Albumen platform.
Specifically, the isolated polynucleotides include the first receptor fragments coded sequence, IgG1Fc fragment encoding se
With Co receptor fragment encoding sequence.
More specifically, the IgG1Fc fragment encoding se is as shown in SEQ ID No.10.
Third aspect present invention provides a kind of recombinant expression carrier, comprising encoding the bifunctional fusion egg for being used for drug
The polynucleotides of Bai Pingtai.
Specifically, the recombinant expression carrier is inserted into the multiple cloning sites of expression vector by the isolated polynucleotides
It is built-up.The expression vector specifically can be existing common expression vector well known to those skilled in the art, tool
The adoptable expression vector of body includes but is not limited to: pET series expression vector, pGEX series expression vector, pcDNA series of tables
Up to carrier etc..Those skilled in the art can choose suitable carrier, further can also carry out modification to existing carrier and change
It makes, obtains the recombinant expression carrier that can reach desired expression to construct.The desired expression can be more
High protein expression level is also possible to a relatively reasonable protein expression level, to give reasonably for Different Individual
Dosage.
Fourth aspect present invention provides a kind of expressing fusion protein system, and the expressing fusion protein system contains described heavy
The polynucleotides of external source are integrated in group expression vector or genome.
Specifically, the expressing fusion protein system is transfected into constructing host cell by the recombinant expression carrier and forms.
Any cell expressed suitable for expression vector all can serve as host cell.For example, yeast, insect, plant etc. is thin
Born of the same parents.Preferably, the host cell is eukaryocyte, the mammalian host cell line that will not generate antibody can be used, specifically
Adoptable cell line includes but is not limited to: kidney cell (BHK, the ATCC of the gonad cell (CHO) of Chinese hamster, young hamster
CCL 10), the Sertoli cell (sertoli cells) of young rat, monkey kidney cell (COS cell), pass through SV40
(the kidney CVI cell of 1) monkey that COS-7, ATCC CRL 165 is converted, people embryonic kidney cells (HEK-293), monkey kidney cell
The kidney cells (VERO-76, ATCC CRL-1587) of (CVI ATCC CCL 70), cercopithecus aethiops, people cervical cancer cell
(HELA, ATCC CCL 2) etc..
Fifth aspect present invention provides the preparation method of the bifunctional fusion proteins platform for drug, including as follows
Step:
1) the expressing fusion protein system is cultivated, the expression bifunctional fusion proteins for being used for drug are allowed to；
2) it collects containing described for researching and developing the culture of the bifunctional fusion proteins of drug；
3) bifunctional fusion proteins for researching and developing drug and thus shape are isolated from culture obtained by step 2)
At the bifunctional fusion proteins platform for drug.
Specifically, after obtaining the nucleic acid sequence for encoding the bifunctional fusion proteins for researching and developing drug of the invention, it can
The bifunctional fusion proteins that production purpose is used to research and develop drug are prepared in accordance with the following methods.Such as it will be used for containing encoding target
The recombinant expression carrier for researching and developing the polynucleotides of the bifunctional fusion proteins of drug is introduced directly into host cell, obtains for grinding
The bifunctional fusion proteins platform expression system of dispensing object, and cultivated under suitable condition, to induce encoded
For researching and developing the expression of the bifunctional fusion proteins of drug.
Recombinant expression carrier used in the present invention and host cell are the prior art, can directly be obtained by commercial sources
It takes, culture medium used in culture is also various conventional mediums, and those skilled in the art can rule of thumb select applicable training
Base is supported, is cultivated under conditions of being suitable for host cell growth.After host cell growth is to cell density appropriate, use
Cell is further cultured for a period of time by the promoter that suitable method (such as temperature transition or chemical induction) induces selection.Above
Method in, recombinant polypeptide can express in the cell or on cell membrane, and can interact to form dimer fusion protein
It structure and/or is secreted extracellularly.Once obtaining of the present invention for researching and developing the bifunctional fusion proteins of drug, so that it may
It is separated by various separation methods and purifies using its physics, chemical and other characteristics and is described for researching and developing double function of drug
Energy fusion protein, these methods are well-known to those skilled in the art, and the example of these methods includes but is not limited to: often
The renaturation process of rule breaks bacterium, super processing, ultracentrifugation, molecular sieve layer with protein precipitant processing (salting-out method), centrifugation, infiltration
Analyse (gel filtration), adsorption chromatography, ion-exchange chromatography, high performance liquid chroma- tography (HPLC) and various other liquid chromatography technologies
Deng.
It, can also be by the method for the insertion marker in expression vector, to for grinding in an embodiment of the present invention
The bifunctional fusion proteins for researching and developing drug that the bifunctional fusion proteins expression system culture of hair therapeutic agent obtains carry out
Label, it is specific to can be used in order to be separated, purified to the bifunctional fusion proteins for researching and developing drug in culture
Marker can be the marks that the various conventional bifunctional fusion proteins being suitable for for researching and developing drug in various this fields purify
Remember object.
Sixth aspect present invention provides a kind of composition, including being used to research and develop the difunctional of drug described in therapeutically effective amount
Fusion protein platform is described for researching and developing the bifunctional fusion proteins platform expression system (for example, host cell) of drug
Culture.
Herein, if can be reduced one or more symptom or clinical indices to represent the treatment is " effective ".
Specifically, the composition further includes pharmaceutically acceptable carrier.Pharmaceutically acceptable carrier refers to for controlling
The carrier of agent administration, including various excipient and diluent are treated, refer specifically to medicament carriers some in this way: themselves is not
Necessary active constituent, and there is no excessive toxicity after applying.Suitable carrier is well known to those of ordinary skill in the art
's.Can be found in Remington's Pharmaceutical Sciences (Mack Pub.Co., N.J.1991) about
Pharmaceutically acceptable excipient discusses fully.Pharmaceutically acceptable carrier may include liquid in the composition, as water,
Salt water, glycerol and ethyl alcohol.In addition, there is likely to be complementary substances in these carriers, such as disintegrating agent, wetting agent, emulsification
Agent, pH buffer substance, seaweed glue, pectin, sodium carboxymethylcellulose (CMC), xanthan gum, gellan gum, guar gum, carragheen,
Sucrose, maltitol, steviol glycoside etc..
Seventh aspect present invention provide it is described for research and develop the bifunctional fusion proteins platform of drug prepare or screen with
First acceptor moieties and/or Co receptor segment are the application in the drug of action target.
Described using the first acceptor moieties and/or Co receptor segment as action target drug includes but is not limited to tumour immunity
Therapeutic agent or rheumatoid arthritis immunotherapy medicaments or silver-colored clouds disease immunotherapy medicaments or ankylosing spondylitis immunization therapy
Drug.
The purposes refers specifically to: using the first acceptor moieties and/or Co receptor segment as action target, being used to grind by described
The bifunctional fusion proteins platform of dispensing object is used to prepare therapeutic agent as effective component, and the therapeutic agent can pass through drop
Low first acceptor moieties and/or the expression quantity of Co receptor segment inhibit the first acceptor moieties and/or Co receptor segment
The modes such as activity.Specifically, the expression quantity for reducing the first acceptor moieties refers specifically to, and before administration, the table of the first acceptor moieties
At least 10% can be reduced up to amount, at least 30% can be more specifically reduced, can more specifically reduce at least 50%, more specifically may be used
At least 70% is reduced, further can specifically reduce at least 90%.Specifically, the expression quantity for reducing Co receptor segment is specific
Refer to, before administration, the expression quantity of Co receptor segment can reduce at least 10%, can more specifically reduce at least 30%, more
At least 50% can be reduced to body, can more specifically reduce at least 70%, further can specifically reduce at least 90%.Specifically,
The first acceptor moieties activity is inhibited to refer specifically to, before administration, the first acceptor moieties activity can reduce at least 10%, more specifically
Ground can reduce at least 30%, can more specifically reduce at least 50%, can more specifically reduce at least 70%, further specifically
At least 90% can be reduced.Specifically, inhibiting Co receptor segment activity to refer specifically to, compared to Co receptor segment activity before being administered
At least 10% can be reduced, can more specifically reduce at least 30%, can more specifically reduce at least 50%, can more specifically be reduced
At least 70%, it further can specifically reduce at least 90%.
Eighth aspect present invention provides a kind for the treatment of method, and described pharmaceutical composition is applied to individual.
The individual refers to the animal (including mankind) of acceptable described pharmaceutical composition and/or treatment method, contains herein
Male and female two kinds of genders are covered, unless otherwise expressly specified.Therefore, the individual includes at least any mammal,
Including but not limited to: the mankind, inhuman primate, such as mammal, dog, cat, horse, sheep, pig, ox, it can be because utilizing
Described pharmaceutical composition is treated and is benefited.
Specifically, the treatment method is by reducing the expression quantity of the first acceptor moieties and/or Co receptor segment, inhibiting
The modes such as the activity of the first acceptor moieties and/or Co receptor segment.
As described above, the bifunctional fusion proteins platform provided by the present invention for researching and developing drug includes the first receptor piece
Disconnected and Co receptor segment, can effectively be directed to the first acceptor moieties and Co receptor segment, with good bioactivity,
Specificity and stability.The structure of bispecific fusion protein platform can be effectively reduced the research and development cost of research and development therapeutic agent,
With high industrialization value.
The action principle of bifunctional fusion proteins platform provided by the present invention for researching and developing drug is, using in receptor
In EDC and ligand, the interaction between EDC and ligand generate inhibition after useful effect.
Detailed description of the invention
Fig. 1 is shown as exempting from the bifunctional fusion proteins platform provided by the present invention for researching and developing drug for tumour
The schematic diagram of the SIRP α-IgG1Fc-PD-1 bifunctional fusion proteins of epidemic disease treatment.
Fig. 2 is shown as exempting from the bifunctional fusion proteins platform provided by the present invention for researching and developing drug for tumour
The schematic diagram of the LAG-3-IgG1Fc-PD-1 bifunctional fusion proteins of epidemic disease treatment.
Specific embodiment
Illustrate embodiments of the present invention below by way of specific specific example, those skilled in the art can be by this specification
Other advantages and efficacy of the present invention can be easily understood for disclosed content.The present invention can also be by addition different specific
Embodiment is embodied or practiced, and the various details in this specification can also not carried on the back based on different viewpoints and application
From carrying out various modifications or alterations under spirit of the invention.
Before further describing the specific embodiments of the present invention, it should be appreciated that protection scope of the present invention is not limited to down
State specific specific embodiment；It is also understood that term used in the embodiment of the present invention is specific specific in order to describe
Embodiment, rather than limiting the scope of protection of the present invention；In description of the invention and claims, unless literary
In in addition explicitly point out, singular "one", " one " and " this " include plural form.
When embodiment provides numberical range, it should be appreciated that except non-present invention is otherwise noted, two ends of each numberical range
Any one numerical value can be selected between point and two endpoints.Unless otherwise defined, the present invention used in all technologies and
Scientific term is identical as the normally understood meaning of those skilled in the art of the present technique.Except specific method used in embodiment, set
Outside standby, material, grasp and record of the invention according to those skilled in the art to the prior art can also be used
Any method, equipment and the material of the similar or equivalent prior art with method described in the embodiment of the present invention, equipment, material
Material is to realize the present invention.
Unless otherwise stated, disclosed in this invention experimental method, detection method, preparation method be all made of this technology neck
Molecular biology, biochemistry, chromatin Structure and analysis, analytical chemistry, the cell culture, recombinant DNA technology of domain routine
And the routine techniques of related fields.These technologies have perfect explanation in the prior art, and for details, reference can be made to Sambrook etc.
MOLECULAR CLONING:A LABORATORY MANUAL, Second edition, Cold Spring Harbor
Laboratory Press, 1989and Third edition, 2001；Ausubel etc., 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；With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin
Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..
Illustrate that the bifunctional fusion proteins platform for researching and developing drug of the invention is being ground below by several embodiments
The application of tumor of swelling immunotherapy medicaments, to illustrate the work of the bifunctional fusion proteins platform for researching and developing drug of the invention
With.But the following examples are not intended to limit the bifunctional fusion proteins platform for researching and developing drug of the invention and are only used for
Immunotherapy of tumors drug is researched and developed, can be also used for research and development with the first acceptor moieties and/or Co receptor segment as effect target
Target drug, such as rheumatoid arthritis immunotherapy medicaments, silver-colored clouds disease immunotherapy medicaments, ankylosing spondylitis are immunized and control
Treat drug etc..
Embodiment 1
1. the amino acid sequence as SIRP α receptor fragments is as shown in SEQ ID No.1, coded sequence such as SEQ ID
Shown in No.5；Amino acid sequence as LAG-3 receptor fragments is as shown in SEQ ID No.2, coded sequence such as SEQ ID
Shown in No.6, the amino acid sequence as PD-1 receptor fragments is as shown in SEQ ID No.4, coded sequence such as SEQ ID No.7
Shown, the amino acid sequence of IgG1Fc segment is as shown in SEQ ID No.5, and coded sequence is as shown in SEQ ID No.8；
The amino acid sequence (SEQ ID No.1) of SIRP α receptor fragments:
EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVS DLT
KRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSAPVVSGPAA RATPQHTVSFTCES
HGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDV HSQVICEVAHVTLQGDPLRGTANLS
ETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLT WLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSA
HRDDVKLTCQVEHDGQPAVSKSHDLKVS AHPKEQGSNTAAENTGSNERN
The coded sequence (SEQ ID No.5) of SIRP α receptor fragments:
GAGGAGGAGCTGCAGGTGATTCAGCCTGACAAGTCCGTGTTGGTTGCAGCTGGAGAGACAG CCACTC
TGCGCTGCACTGCGACCTCTCTGATCCCTGTGGGGCCCATCCAGTGGTTCAGAGGAGC TGGACCAGGCCGGGAAT
TAATCTACAATCAAAAAGAAGGCCACTTCCCCCGGGTAACAACTGTT TCAGACCTCACAAAGAGAAACAACATGG
ACTTTTCCATCCGCATCGGTAACATCACCCCAGCAG ATGCCGGCACCTACTACTGTGTGAAGTTCCGGAAAGGGA
GCCCCGATGACGTGGAGTTTAAGTC TGGAGCAGGCACTGAGCTGTCTGTGCGCGCCAAACCCTCTGCCCCCGTGG
TATCGGGCCCTGCG GCGAGGGCCACACCTCAGCACACAGTGAGCTTCACCTGCGAGTCCCACGGCTTCTCACCCA
GAG ACATCACCCTGAAATGGTTCAAAAATGGGAATGAGCTCTCAGACTTCCAGACCAACGTGGACCC CGTAGGA
GAGAGCGTGTCCTACAGCATCCACAGCACAGCCAAGGTGGTGCTGACCCGCGAGGAC GTTCACTCTCAAGTCATC
TGCGAGGTGGCCCACGTCACCTTGCAGGGGGACCCTCTTCGTGGGA CTGCCAACTTGTCTGAGACCATCCGAGTT
CCACCCACCTTGGAGGTTACTCAACAGCCCGTGAG GGCAGAGAACCAGGTGAATGTCACCTGCCAGGTGAGGAAG
TTCTACCCCCAGAGACTACAGCTG ACCTGGTTGGAGAATGGAAACGTGTCCCGGACAGAAACGGCCTCAACCGTT
ACAGAGAACAAGG ATGGTACCTACAACTGGATGAGCTGGCTCCTGGTGAATGTATCTGCCCACAGGGATGATGTG
AA GCTCACCTGCCAGGTGGAGCATGACGGGCAGCCAGCGGTCAGCAAAAGCCATGACCTGAAGGTC TCAGCCCA
CCCGAAGGAGCAGGGCTCAAATACCGCCGCTGAGAACACTGGATCTAATGAACGGA AC
The amino acid sequence (SEQ ID No.2) of LAG-3 receptor fragments:
VPVVWAQEGAPAQLPCSPTIPLQDLSLLRRAGVTWQHQPDSGPPAAAPGHPLAPGPHPAAPSSW GPR
PRRYTVLSVGPGGLRSGRLPLQPRVQLDERGRQRGDFSLWLRPARRADAGEYRAAVHLRDR ALSCRLRLRLGQAS
MTASPPGSLRASDWVILNCSFSRPDRPASVHWFRNRGQGRVPVRESPHHH LAESFLFLPQVSPMDSGPWGCILTY
RDGFNVSIMYNLTVLGLEPPTPLTVYAGAGSRVGLPCRL PAGVGTRSFLTAKWTPPGGGPDLLVTGDNGDFTLRL
EDVSQAQAGTYTCHIHLQEQQLNATVTL AI ITVTPKSFGSPGSLGKLLCEVTPVSGQERFVWSSLDTPSQRSFS
GPWLEAQEAQLLSQPWQC QLYQGERLLGAAVYFTELSSP
The coded sequence (SEQ ID No.6) of LAG-3 receptor fragments:
GTCCCGGTGGTGTGGGCCCAGGAGGGGGCTCCTGCCCAGCTCCCCTGCAGCCCCACAATCCCCCTCCA
GGATCTCAGCCTTCTGC GAAGAGCAGGGGTCACTTGGCAGCATCAGCCAGACAGTGGCCCGCCCGCTGCCGCCCC
CGGCCATCCCCTGGCCCCCGGCCCTCA CCCGGCGGCGCCCTCCTCCTGGGGGCCCAGGCCCCGCCGCTACACGGT
GCTGAGCGTGGGTCCCGGAGGCCTGCGCAGCGGGAGG CTGCCCCTGCAGCCCCGCGTCCAGCTGGATGAGCGCGG
CCGGCAGCGCGGGGACTTCTCGCTATGGCTGCGCCCAGCCCGGCGCG CGGACGCCGGCGAGTACCGCGCCGCGGT
GCACCTCAGGGACCGCGCCCTCTCCTGCCGCCTCCGTCTGCGCCTGGGCCAGGCCTC GATGACTGCCAGCCCCCC
AGGATCTCTCAGAGCCTCCGACTGGGTCATTTTGAACTGCTCCTTCAGCCGCCCTGACCGCCCAGCC TCTGTGCA
TTGGTTCCGGAACCGGGGCCAGGGCCGAGTCCCTGTCCGGGAGTCCCCCCATCACCACTTAGCGGAAAGCTTCCTCT
TCCTGCCCCAAGTCAGCCCCATGGACTCTGGGCCCTGGGGCTGCATCCTCACCTACAGAGATGGCTTCAACGTCTC
CATCATGTA TAACCTCACTGTTCTGGGTCTGGAGCCCCCAACTCCCTTGACAGTGTACGCTGGAGCAGGTTCCAG
GGTGGGGCTGCCCTGCCGC CTGCCTGCTGGTGTGGGGACCCGGTCTTTCCTCACTGCCAAGTGGACTCCTCCTGG
GGGAGGCCCTGACCTCCTGGTGACTGGAG ACAATGGCGACTTTACCCTTCGACTAGAGGATGTGAGCCAGGCCCA
GGCTGGGACCTACACCTGCCATATCCATCTGCAGGAACA GCAGCTCAATGCCACTGTCACATTGGCAATCATCAC
AGTGACTCCCAAATCCTTTGGGTCACCTGGATCCCTGGGGAAGCTGCTT TGTGAGGTGACTCCAGTATCTGGACA
AGAACGCTTTGTGTGGAGCTCTCTGGACACCCCATCCCAGAGGAGTTTCTCAGGACCTT GGCTGGAGGCACAGGA
GGCCCAGCTCCTTTCCCAGCCTTGGCAATGCCAGCTGTACCAGGGGGAGAGGCTTCTTGGAGCAGCAGT
GTACTTCACAGAGCTGTCTAGCCCA
The amino acid sequence (SEQ ID No.3) of PD-1 receptor fragments:
PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPE DRS
QPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERR
AEVPTAHPSPSPRPAGQFQ
The coded sequence (SEQ ID No.7) of PD-1 receptor fragments:
ccaggatggttcttagactccccagacaggccctggaaccccccaccttctccccagccct gctcgt
ggtgaccgaaggggacaacgccaccttcacctgcagcttctccaacacatcggagagc ttcgtgctaaactggta
ccgcatgagccccagcaaccagacggacaagctggccgccttccccg aggaccgcagccagcccggccaggactg
ccgcttccgtgtcacacaactgcccaacgggcgtga cttccacatgagcgtggtcagggcccggcgcaatgacag
cggcacctacctctgtggggccatc tccctggcccccaaggcgcagatcaaagagagcctgcgggcagagctcag
ggtgacagagagaa gggcagaagtgcccacagcccaccccagcccctcacccaggccagccggccagttccaaa
The amino acid sequence (SEQ ID No.4) of IgG1Fc segment:
EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISATPEVTCVVVDVSHEDPEVKFM WYVDGV
EVHNAKTKPAEEQYNSTYAVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPAEPQVYTLPPSRDE
LTYNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEAL
HNHYTQKSLSLSPGK
The coded sequence (SEQ ID No.8) of IgG1Fc segment:
GAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGG GAC
CGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGA GGTCACATGCGTGG
TGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTG GACGGCGTGGAGGTGCATAATGCCA
AGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACC GTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACT
GGCTGAATGGCAAGGAGTACAAGTGCAA GGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCA
AAGCCAAAGGGCAGCCC CGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGG
TCAGCC TGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCA GCCG
GAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTAC AGCAAGCTCACCGTG
GACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGC ATGAGGCTCTGCACAACCACTACACG
CAGAAGAGCCTCTCCCTGTCTCCGGGTAAA
2. the SIRP α-IgG1Fc-PD-1 bifunctional fusion proteins for immunotherapy of tumors are successively wrapped from N-terminal to C-terminal
It includes, SIRP α receptor fragments, IgG1Fc segment, PD-1 receptor fragments；SIRP α-IgG1Fc-PD-1 for immunotherapy of tumors
Bifunctional fusion proteins coded sequence successively includes that SIRP α receptor fragments coded sequence, IgG1Fc segment are compiled from N-terminal to C-terminal
Code sequence, PD-1 receptor fragments coded sequence.
SIRP α-IgG1Fc-PD-1 bifunctional fusion proteins amino acid sequence for immunotherapy of tumors:
EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVS DLT
KRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSAPVVSGPAA RATPQHTVSFTCES
HGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDV HSQVICEVAHVTLQGDPLRGTANLS
ETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLT WLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSA
HRDDVKLTCQVEHDGQPAVSKSHDLKVS AHPKEQGSNTAAENTGSNERNEPKSCDKTHTCPPCPAPELLGGPSVF
LFPPKPKDTLMISATPE VTCVVVDVSHEDPEVKFMWYVDGVEVHNAKTKPAEEQYNSTYAVVSVLTVLHQDWLNG
KEYKCK VSNKALPAPIEKTISKAKGQPAEPQVYTLPPSRDELTYNQVSLTCLVKGFYPSDIAVEWESNGQ PENN
YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSS SSGSSSSGSSSSPGW
FLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSP SNQTDKLAAFPEDRSQPGQDCRFRVT
QLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKE SLRAELRVTERRAEVPTAHPSPSPRPAGQFQ
SIRP α-IgG1Fc-PD-1 bifunctional fusion proteins coded sequence for immunotherapy of tumors:
GAGGAGGAGCTGCAGGTGATTCAGCCTGACAAGTCCGTGTTGGTTGCAGCTGGAGAGACAGCCA CTC
TGCGCTGCACTGCGACCTCTCTGATCCCTGTGGGGCCCATCCAGTGGTTCAGAGGAGCTGG ACCAGGCCGGGAAT
TAATCTACAATCAAAAAGAAGGCCACTTCCCCCGGGTAACAACTGTTTCA GACCTCACAAAGAGAAACAACATGG
ACTTTTCCATCCGCATCGGTAACATCACCCCAGCAGATG CCGGCACCTACTACTGTGTGAAGTTCCGGAAAGGGA
GCCCCGATGACGTGGAGTTTAAGTCTGG AGCAGGCACTGAGCTGTCTGTGCGCGCCAAACCCTCTGCCCCCGTGG
TATCGGGCCCTGCGGCG AGGGCCACACCTCAGCACACAGTGAGCTTCACCTGCGAGTCCCACGGCTTCTCACCCA
GAGACA TCACCCTGAAATGGTTCAAAAATGGGAATGAGCTCTCAGACTTCCAGACCAACGTGGACCCCGT AGGA
GAGAGCGTGTCCTACAGCATCCACAGCACAGCCAAGGTGGTGCTGACCCGCGAGGACGTT CACTCTCAAGTCATC
TGCGAGGTGGCCCACGTCACCTTGCAGGGGGACCCTCTTCGTGGGACTG CCAACTTGTCTGAGACCATCCGAGTT
CCACCCACCTTGGAGGTTACTCAACAGCCCGTGAGGGC AGAGAACCAGGTGAATGTCACCTGCCAGGTGAGGAAG
TTCTACCCCCAGAGACTACAGCTGACC TGGTTGGAGAATGGAAACGTGTCCCGGACAGAAACGGCCTCAACCGTT
ACAGAGAACAAGGATG GTACCTACAACTGGATGAGCTGGCTCCTGGTGAATGTATCTGCCCACAGGGATGATGTG
AAGCT CACCTGCCAGGTGGAGCATGACGGGCAGCCAGCGGTCAGCAAAAGCCATGACCTGAAGGTCTCA GCCCA
CCCGAAGGAGCAGGGCTCAAATACCGCCGCTGAGAACACTGGATCTAATGAACGGAACG AGCCCAAATCTTGTGA
CAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGG ACCGTCAGTCTTCCTCTTCCCCCCAAA
ACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAG GTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCC
TGAGGTCAAGTTCAACTGGTACGTGG ACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTA
CAACAGCACGTACCG TGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG
CAAG GTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCC GAGAAC
CACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCT GACCTGCCTGGTCAAAG
GCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG CCGGAGAACAACTACAAGACCACGCCTC
CCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACA GCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGA
ACGTCTTCTCATGCTCCGTGATGCA TGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGG
GTAAAGGAGGCGGA GGATCTGGAGGCGGAGGATCTGGAGGCGGAGGATCTccaggatggttcttagactccccag
aca ggccctggaaccccccaccttctccccagccctgctcgtggtgaccgaaggggacaacgccacc ttcacct
gcagcttctccaacacatcggagagcttcgtgctaaactggtaccgcatgagcccca gcaaccagacggacaagc
tggccgccttccccgaggaccgcagccagcccggccaggactgccg cttccgtgtcacacaactgcccaacgggc
gtgacttccacatgagcgtggtcagggcccggcgc aatgacagcggcacctacctctgtggggccatctccctgg
cccccaaggcgcagatcaaagaga gcctgcgggcagagctcagggtgacagagagaagggcagaagtgcccacag
cccaccccagccc ctcacccaggccagccggccagttccaaa
3. the LAG-3-IgG1Fc-PD-1 bifunctional fusion proteins for immunotherapy of tumors are successively wrapped from N-terminal to C-terminal
It includes, LAG-3 receptor fragments, IgG1Fc segment, PD-1 receptor fragments；LAG-3-IgG1Fc-PD-1 for immunotherapy of tumors
Bifunctional fusion proteins coded sequence successively includes that LAG-3 receptor fragments coded sequence, IgG1Fc segment are compiled from N-terminal to C-terminal
Code sequence, PD-1 receptor fragments coded sequence.
LAG-3-IgG1Fc-PD-1 bifunctional fusion proteins amino acid sequence for immunotherapy of tumors:
VPVVWAQEGAPAQLPCSPTIPLQDLSLLRRAGVTWQHQPDSGPPAAAPGHPLAPGPHPAAPSSW GPR
PRRYTVLSVGPGGLRSGRLPLQPRVQLDERGRQRGDFSLWLRPARRADAGEYRAAVHLRDR ALSCRLRLRLGQAS
MTASPPGSLRASDWVILNCSFSRPDRPASVHWFRNRGQGRVPVRESPHHH LAESFLFLPQVSPMDSGPWGCILTY
RDGFNVSIMYNLTVLGLEPPTPLTVYAGAGSRVGLPCRL PAGVGTRSFLTAKWTPPGGGPDLLVTGDNGDFTLRL
EDVSQAQAGTYTCHIHLQEQQLNATVTL AI ITVTPKSFGSPGSLGKLLCEVTPVSGQERFVWSSLDTPSQRSFS
GPWLEAQEAQLLSQPWQC QLYQGERLLGAAVYFTELSSPEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL
MISATPE VTCVVVDVSHEDPEVKFMWYVDGVEVHNAKTKPAEEQYNSTYAVVSVLTVLHQDWLNGKEYKCK VSN
KALPAPIEKTISKAKGQPAEPQVYTLPPSRDELTYNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGSS SSGSSSSGSSSSPGWFLDSPDRPWN
PPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSP SNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHM
SVVRARRNDSGTYLCGAISLAPKAQIKE SLRAELRVTERRAEVPTAHPSPSPRPAGQFQ
LAG-3-IgG1Fc-PD-1 bifunctional fusion proteins coded sequence for immunotherapy of tumors:
GTCCCGGTGGTGTGGGCCCAGGAGGGGGCTCCTGCCCAGCTCCCCTGCAGCCCCACAATCCC CCTCC
AGGATCTCAGCCTTCTGCGAAGAGCAGGGGTCACTTGGCAGCATCAGCCAGACAGTGGC CCGCCCGCTGCCGCCC
CCGGCCATCCCCTGGCCCCCGGCCCTCACCCGGCGGCGCCCTCCTCCT GGGGGCCCAGGCCCCGCCGCTACACGG
TGCTGAGCGTGGGTCCCGGAGGCCTGCGCAGCGGGAG GCTGCCCCTGCAGCCCCGCGTCCAGCTGGATGAGCGCG
GCCGGCAGCGCGGGGACTTCTCGCTA TGGCTGCGCCCAGCCCGGCGCGCGGACGCCGGCGAGTACCGCGCCGCGG
TGCACCTCAGGGACC GCGCCCTCTCCTGCCGCCTCCGTCTGCGCCTGGGCCAGGCCTCGATGACTGCCAGCCCCC
CAGG ATCTCTCAGAGCCTCCGACTGGGTCATTTTGAACTGCTCCTTCAGCCGCCCTGACCGCCCAGCC TCTGTG
CATTGGTTCCGGAACCGGGGCCAGGGCCGAGTCCCTGTCCGGGAGTCCCCCCATCACC ACTTAGCGGAAAGCTTC
CTCTTCCTGCCCCAAGTCAGCCCCATGGACTCTGGGCCCTGGGGCTG CATCCTCACCTACAGAGATGGCTTCAAC
GTCTCCATCATGTATAACCTCACTGTTCTGGGTCTG GAGCCCCCAACTCCCTTGACAGTGTACGCTGGAGCAGGT
TCCAGGGTGGGGCTGCCCTGCCGCC TGCCTGCTGGTGTGGGGACCCGGTCTTTCCTCACTGCCAAGTGGACTCCT
CCTGGGGGAGGCCC TGACCTCCTGGTGACTGGAGACAATGGCGACTTTACCCTTCGACTAGAGGATGTGAGCCAG
GCC CAGGCTGGGACCTACACCTGCCATATCCATCTGCAGGAACAGCAGCTCAATGCCACTGTCACAT TGGCAAT
CATCACAGTGACTCCCAAATCCTTTGGGTCACCTGGATCCCTGGGGAAGCTGCTTTG TGAGGTGACTCCAGTATC
TGGACAAGAACGCTTTGTGTGGAGCTCTCTGGACACCCCATCCCAG AGGAGTTTCTCAGGACCTTGGCTGGAGGC
ACAGGAGGCCCAGCTCCTTTCCCAGCCTTGGCAAT GCCAGCTGTACCAGGGGGAGAGGCTTCTTGGAGCAGCAGT
GTACTTCACAGAGCTGTCTAGCCC AGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACC
TGAACTCCTGGGG GGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCC
TG AGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGT GGACGGCG
TGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTAC CGTGTGGTCAGCGTCCTCA
CCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCA AGGTCTCCAACAAAGCCCTCCCAGCCCCCA
TCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCC CCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATG
AGCTGACCAAGAACCAGGTCAGC CTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGG
AGAGCAATGGGC AGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTA
CAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATG CATGAGGCTCT
GCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAGGAGGCG GAGGATCTGGAGGCGGAGGATC
TGGAGGCGGAGGATCTccaggatggttcttagactccccaga caggccctggaaccccccaccttctccccagcc
ctgctcgtggtgaccgaaggggacaacgcca ccttcacctgcagcttctccaacacatcggagagcttcgtgcta
aactggtaccgcatgagccc cagcaaccagacggacaagctggccgccttccccgaggaccgcagccagcccggc
caggactgc cgcttccgtgtcacacaactgcccaacgggcgtgacttccacatgagcgtggtcagggcccggc g
caatgacagcggcacctacctctgtggggccatctccctggcccccaaggcgcagatcaaaga gagcctgcgggc
agagctcagggtgacagagagaagggcagaagtgcccacagcccaccccagc
ccctcacccaggccagccggccagttccaaa
Embodiment 2
Expression is used for the SIRP α-IgG1Fc-PD-1 bifunctional fusion proteins of immunotherapy of tumors
The coded sequence for the SIRP α-IgG1Fc-PD-1 bifunctional fusion proteins for being used for immunotherapy of tumors is cloned into table
Up in the multiple cloning sites of carrier, the connection of coded sequence and expression vector is realized, obtain Plasmid DNA.By plasmid DNA transfection
Host cell, transfection can be carried out in 6 orifice plates.Positive cell strain after transfection is passaged in 1L shaking flask, temperature 37C, 5%
CO2In environment, 120RPM shaking table culture supplements nutriment, the Cell viabilities such as glucose, amino acid daily and 80-85% is reduced to stop
Only cultivate.Cell liquid 2000RCF is centrifuged after taking out cell, then 5000RCF centrifuging and taking supernatant carries out protein purification, is used
In the SIRP α-IgG1Fc-PD-1 bifunctional fusion proteins of immunotherapy of tumors.
Embodiment 3
Expression is used for the LAG-3-IgG1Fc-PD-1 bifunctional fusion proteins of immunotherapy of tumors
The coded sequence for the sLAG-3-IgG1Fc-PD-1 bifunctional fusion proteins for being used for immunotherapy of tumors is cloned into
In the multiple cloning sites of expression vector, the connection of coded sequence and expression vector is realized, obtain Plasmid DNA.Plasmid DNA is turned
Host cell is contaminated, transfection can be carried out in 6 orifice plates.Positive cell strain after transfection is passaged in 1L shaking flask, temperature 37C, and 5%
CO2In environment, 120RPM shaking table culture supplements nutriment, the Cell viabilities such as glucose, amino acid daily and is reduced to 80-85%
Stop culture.Cell liquid 2000RCF is centrifuged after taking out cell, then 5000RCF centrifuging and taking supernatant carries out protein purification, obtains
SLAG-3-IgG1Fc-PD-1 bifunctional fusion proteins for immunotherapy of tumors.
Embodiment 4
SIRP α-IgG1Fc-PD-1 bifunctional fusion proteins activity experiment for immunotherapy of tumors:
CD47 is cell surface one important " self " label, is an important letter for adjusting macrophage phagocytosis
Number.CD47 can be in conjunction with Macrophage Surface SIRP α, its ITIM of phosphorylation, then recruits SHP-1 albumen, generates a system
The cascade reaction of column inhibits the phagocytosis of macrophage.Almost all of tumour cell and the high expression CD47 of tissue, are pair
3 times for answering normal cell and tissue.By this " self " signal of CD47, tumour cell has effectively hidden macrophage
Phagocytosis.Thus it can enhance macrophage for the SIRP α-IgG1Fc-PD-1 bifunctional fusion proteins of immunotherapy of tumors
Killing to tumour cell, so the SIRP α-IgG1Fc-PD-1 bifunctional fusion proteins for immunotherapy of tumors can be passed through
Antagonism SIRP α-CD47 inhibits access to the killing of target cell strain to detect the SIRP α-for immunotherapy of tumors
The biological activity of IgG1Fc-PD-1 bifunctional fusion proteins.It is demonstrated experimentally that being used for the SIRP α-of immunotherapy of tumors
IgG1Fc-PD-1 bifunctional fusion proteins have stronger protective effect to macrophage, it is seen that for immunotherapy of tumors
SIRP α-IgG1Fc-PD-1 bifunctional fusion proteins have good activity and specificity for SIRP α.
Embodiment 5
LAG-3-IgG1Fc-PD-1 bifunctional fusion proteins activity experiment for immunotherapy of tumors:
LAG3 (lymphocyte activation gene 3, LAG3, CD223) is that a kind of immunologic test selects receptor egg
White, main expression is in the T cell of activation, NK cell, B cell and thick liquid cell dendritic cells.LAG3 can by with MHC II molecule
Combination, lower the activity of T cell.Meanwhile LAG3 can also enhance the inhibitory activity of regulatory T cells (Treg).
It can thus release for the LAG-3-IgG1Fc-PD-1 bifunctional fusion proteins of immunotherapy of tumors to T cell
Inhibit, so LAG-3- can be blocked by the LAG-3-IgG1Fc-PD-1 bifunctional fusion proteins for immunotherapy of tumors
MHCII inhibits access, and enhancing T cell detects the LAG-3-IgG1Fc- for immunotherapy of tumors to the killing of tumour cell
The biological activity of PD-1 bifunctional fusion proteins.It is demonstrated experimentally that being used for the LAG-3-IgG1Fc-PD-1 of immunotherapy of tumors
Bifunctional fusion proteins can effectively enhance the activity of T cell, it is seen that the sLAG-3-IgG1Fc- for immunotherapy of tumors
PD-1 bifunctional fusion proteins have good activity and specificity for sLAG-3.
In conclusion the present invention effectively overcomes various shortcoming in the prior art and has high industrial utilization value.
The above-described embodiments merely illustrate the principles and effects of the present invention, and is not intended to limit the present invention.It is any ripe
The personage for knowing this technology all without departing from the spirit and scope of the present invention, carries out modifications and changes to above-described embodiment.Cause
This, those of ordinary skill in the art institute without departing from the spirit and technical ideas disclosed in the present invention such as
All equivalent modifications or change completed, should be covered by the claims of the present invention.
Claims (10)
- It is described difunctional to melt it includes bifunctional fusion proteins 1. a kind of for researching and developing the bifunctional fusion proteins platform of drug Hop protein is dimer, and each bifunctional fusion proteins respectively include three structure function regions, three structure functions Region is the first receptor fragments, IgG1Fc segment and Co receptor segment；The IgG1Fc segment are as follows:A) amino acid sequence polypeptide as shown in SEQ ID No.5；OrB) amino acid sequence and SEQ ID No.5 have the function of 80% or more homology and have the more of polypeptide c) limited Peptide.
- 2. as described in claim 1 a kind of for researching and developing the bifunctional fusion proteins platform of drug, which is characterized in that described double Function fusion protein successively includes the first receptor fragments, IgG1Fc segment and Co receptor segment to C-terminal from N-terminal, is dimer.
- 3. as described in claim 1 a kind of for researching and developing the bifunctional fusion proteins platform of drug, which is characterized in that described two Aggressiveness passes through the disulfide-bonded between the IgG1Fc segments-segment.
- 4. a kind of isolated polynucleotides, coding is described to be used for for one kind described in any one of claims 1 to 3 claim Research and develop the bifunctional fusion proteins of drug.
- 5. a kind of recombinant expression carrier, described for a kind of use described in any one of claims 1 to 3 claim comprising encoding In the polynucleotides of the bifunctional fusion proteins of research and development drug.
- 6. it is a kind of for researching and developing the bifunctional fusion proteins platform expression system of drug, contain recombination table described in claim 5 Up to the polynucleotides for being integrated with external source in carrier or genome.
- 7. a kind of for researching and developing the system of the bifunctional fusion proteins platform of drug as described in claim 1-3 any claim Preparation Method includes the following steps:1) culture is described a kind of for researching and developing the bifunctional fusion proteins expression system of drug, is allowed to express described a kind of for grinding The bifunctional fusion proteins of dispensing object；2) it collects containing described a kind of for researching and developing the culture of the bifunctional fusion proteins of drug；3) a kind of bifunctional fusion proteins for researching and developing drug are isolated from culture obtained by step 2) and thus obtained The bifunctional fusion proteins platform of drug must be used to research and develop.
- 8. a kind of composition, one kind as described in claim 1-3 any claim including therapeutically effective amount is for researching and developing The bifunctional fusion proteins platform of drug or a kind of bifunctional fusion proteins for researching and developing drug as claimed in claim 6 are flat The culture of platform expression system.
- 9. as described in claim 1-3 any claim it is a kind of for research and develop the bifunctional fusion proteins of drug in preparation or Screening is using the first acceptor moieties and/or Co receptor segment as the application in the drug of action target.
- 10. application as claimed in claim 9, wherein described using the first acceptor moieties and/or Co receptor segment as action target Drug includes but is not limited to immunotherapy of tumors drug or rheumatoid arthritis immunotherapy medicaments or silver-colored clouds disease immunization therapy medicine Object or ankylosing spondylitis immunotherapy medicaments.
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| PCT/CN2019/086880 WO2020077991A1 (en) | 2018-10-19 | 2019-05-14 | Bifunctional fusion protein platform used for drug research and development |
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|---|---|---|---|---|
| CN101712964A (en) * | 2008-10-08 | 2010-05-26 | 上海富莼科芯生物技术股份有限公司 | Fusion protein for inhibiting formation of osteoclast and preparation method as well as pharmaceutical composition thereof |
| CN104327187A (en) * | 2014-10-11 | 2015-02-04 | 上海兴迪金生物技术有限公司 | Recombinant human GLP-1-Fc fusion protein |
| CN107857818A (en) * | 2017-08-07 | 2018-03-30 | 上海科新生物技术股份有限公司 | A kind of bispecific fusion protein for IL 17 and TNF α |
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| CN101712964A (en) * | 2008-10-08 | 2010-05-26 | 上海富莼科芯生物技术股份有限公司 | Fusion protein for inhibiting formation of osteoclast and preparation method as well as pharmaceutical composition thereof |
| CN104327187A (en) * | 2014-10-11 | 2015-02-04 | 上海兴迪金生物技术有限公司 | Recombinant human GLP-1-Fc fusion protein |
| CN107857818A (en) * | 2017-08-07 | 2018-03-30 | 上海科新生物技术股份有限公司 | A kind of bispecific fusion protein for IL 17 and TNF α |
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