Fusion protein compositions, specific antibodies and methods of making and using the same

Abstract

The present application relates to a kind of fusion protein composition and the single / dual / multi-specific immunological drug constructed by the fusion protein composition, the fusion protein composition includes the fusion protein A of SpyCatcher003-albumin, and at least one protein-SpyTag003 fusion protein B;The fusion protein A is fused by SpyCatcher003 protein and albumin;The fusion protein B is fused by protein and SpyTag003, and the protein is selected from at least one single-chain antibody.The immunological drug is obtained by covalent bond after mixing with at least one fusion protein B after nano-assembly, the nano-assembly is obtained by the fusion protein A and hydrophobic degradable polyester or its derivative after forming by hydrophobic interaction.This application also provides the preparation method of the immunological drug, and the application of immunological drug in tumor immunotherapy or autoimmune disease treatment, inflammation treatment, infection treatment.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a fusion protein composition, a specific antibody, its preparation method, and its application. Background Technology

[0002] Bispecific antibodies (BsAbs) are a type of artificial antibody that can simultaneously and specifically bind to two antigens or two different epitopes on the same antigen. Bispecific antibodies have broad application prospects in areas such as tumor immunotherapy and autoimmune diseases. Currently, at least 30 bispecific antibody drugs are in clinical research stages. Furthermore, the development of polyclonal antibody technology, which combines bispecific antibodies with other methods, is ongoing.

[0003] With the development of technology, more and more technologies are being used to construct bispecific antibody drugs, among which non-natural nanoparticles are also commonly used in the construction of bispecific antibody drugs.

[0004] Albumin-polymer composite nanoassemblies can be obtained by combining serum albumin with hydrophobic polymers through ultrasonic emulsification and high-pressure homogenization. Therefore, linking single-chain antibody fragments (ScFv) of different antibodies to the surface of nanoparticles via surface immobilization is a highly promising method for rapidly constructing bispecific and multispecific antibody drugs. For example, our previously constructed fusion protein and nanoassemblies based on single-chain antibody fragments (CN114516921A), serum albumin-based fusion protein and nanoassemblies (CN114478800A), and protein nanoparticles for multispecific antibody delivery (CN112336873A) are examples.

[0005] To enable these nanoparticles to serve as a universal platform for single / dual / multi-specific drugs, the components used to construct them need modification. Previous methods involved directly constructing dual / multi-specific antibodies using ScFv-SA fusion proteins and hydrophobic polymers, a simple technique, but preparing different antibodies required the production of different fusion proteins, making the process cumbersome. Nanoaptamers constructed using FcγR-SA can effectively adsorb antibodies, enabling rapid construction of single / dual / multi-specific antibodies, but their non-covalent binding mechanism carries the risk of detachment. Therefore, obtaining nanoparticles with excellent stability and maintaining good biological activity is essential.

[0006] The SpyTag / SpyCatcher system was developed by the Howarth laboratory based on the internal isopeptide bonds within the CnaB2 domain of the fibronectin FbaB from *Streptococcus pyogenes*. Within this domain, an internal isopeptide bond spontaneously forms between the ε-amino group of lysine K3 and the carboxyl group of the aspartic acid D117 side chain, conferring high stability to the CnaB2 domain. This binding domain can be divided into two parts: the larger part, named SpyCatcher, contains 138 amino acid residues; the smaller part, named SpyTag, consists of 13 amino acid residues. These two parts can rapidly bind together to form a high-affinity covalent bond without external assistance, thus forming a complex. The Howarth laboratory further optimized this system into SpyCatcher003 and SpyTag003, increasing the rate of isopeptide bond formation by more than 100-fold. Summary of the Invention

[0007] Based on this, one of the objectives of the present invention is to provide a fusion protein composition and its application in constructing a dual multispecific drug, as well as the constructed dual multispecific drug and its preparation method.

[0008] In a first aspect, the present invention provides a fusion protein composition comprising a fusion protein A of SpyCatcher003-albumin and a fusion protein B of at least one protein-SpyTag003.

[0009] The fusion protein A is formed by fusing SpyCatcher003 protein with albumin;

[0010] The fusion protein B is formed by fusing a protein with SpyTag003, wherein the protein is selected from at least one single-chain antibody.

[0011] In some of these embodiments, the albumin is any one of human serum albumin, bovine serum albumin, mouse serum albumin, rat serum albumin, rabbit serum albumin, and chicken oocyte albumin.

[0012] In some of these embodiments, the single-chain antibody targets at least one of the following antigens: single-chain antibodies derived from humans or other species that target HER2, CD137L, CD137, 4-1BBL, 4-1BB, OX40L, OX40, ICOS, CD86, CD80, CD28, LFA3, CD2, CTLA4, PDL1, PD1, CD70, CD27, GAL9, TIM3, CD111, CD96, CD112, CD226, CD115, CD113, TIGIT, CD39, CD73, CD47, SIRPα, TNFα, IL1β, IL6, TGFβ, IL-10, and IL-12. That is, the single-chain antibody is selected from antibody fragments of at least one of the above antigens. Preferably, the single-chain antibody targets one, two or three antigens. Alternatively, the fusion protein B is a fusion protein including one single-chain antibody, or includes two fusion proteins, each fused with two different single-chain antibodies, or includes three fusion proteins, each fused with three different single-chain antibodies.

[0013] In some of these embodiments, when multiple fusion proteins B comprising various single-chain antibodies are prepared, the most preferred amount of each fusion protein B is equal.

[0014] In some of these embodiments, in the fusion protein A, the albumin can be linked to the N-terminus or C-terminus of SpyCatcher003 via a flexible linker peptide.

[0015] In some of these embodiments, the fusion protein B may be linked to the N-terminus or C-terminus of SpyTag003 via a flexible linker peptide.

[0016] In some of these embodiments, the residues of the linker peptide are (GGGGS)n, where n is an integer from 1 to 4.

[0017] In a second aspect, the invention provides encoding genes for the above-described fusion protein composition, including encoding genes for fusion protein A and fusion protein B.

[0018] In some of these embodiments, the gene encoding fusion protein A is shown as either SEQ ID No: 1 or SEQ ID No: 2.

[0019] In some of these embodiments, the gene encoding fusion protein B is shown as any one of SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7, SEQ ID No: 8, SEQ ID No: 9, SEQ ID No: 10.

[0020] A third aspect of the invention provides a gene encoding the above-described recombinant fusion protein composition, or a plasmid vector containing a gene encoding any of the above-described recombinant fusion protein compositions.

[0021] A fourth aspect of the present invention provides engineered bacteria, protoplasts, or in vitro cloning systems containing any gene or vector encoding the above-described recombinant fusion protein composition.

[0022] A fifth aspect of the present invention provides a single / double / multi-specific immunomodulatory drug, which is a nano-assembly formed by covalently bonding fusion protein A in a fusion protein composition with a hydrophobic biodegradable polyester or its derivative therefrom through hydrophobic interaction, and then mixing it with fusion protein B in the fusion protein composition.

[0023] In some of these embodiments, the biodegradable polyester is an aliphatic polyester, or aliphatic polyester modified with polyethylene glycol.

[0024] In some of these embodiments, the aliphatic polyester includes, but is not limited to, at least one of polylactide, polyglycolic acid, poly(lactide-co-glycolic acid), and polycaprolactone.

[0025] In some embodiments, the block molecular weight of the polylactide is 1 kDa to 1,100 kDa; or the block molecular weight of the poly(lactide-co-glycolic acid) is 1 kDa to 1,100 kDa, wherein the ratio of lactide to glycolide is in the range of 95 / 5 to 50 / 50.

[0026] In some embodiments, the molar ratio of the nanoassembly to the fusion protein B is (1-10):1, preferably (1-6):1, and more preferably (2-3):1.

[0027] In some embodiments, the ratio of the amount of fusion protein A to fusion protein B is (3-1):1, and most preferably, the ratio of the amount of fusion protein A to fusion protein B is 1:1.

[0028] A sixth aspect of the present invention provides a method for preparing a single / dual / multi-specific immunomodulatory drug, comprising the following steps:

[0029] (1) The fusion protein A is mixed with water or physiological saline at a concentration of 0.5 to 20 mg / mL to obtain an aqueous phase; the hydrophobic biodegradable polyester and its derivatives are mixed with an organic solvent to obtain an oil phase at a concentration of 0.5 to 10 mg / mL.

[0030] (2) Prepare an oil-in-water emulsion from the aqueous phase solution and the oil phase solution described in step (1);

[0031] (3) The emulsion is separated and purified to obtain nano-assemblies;

[0032] (4) The nano-assembly is mixed with at least one of the fusion proteins B at room temperature to obtain the drug.

[0033] In some of the embodiments, the organic solvent is at least one of chloroform, dichloromethane, and ethyl acetate.

[0034] In some embodiments, the concentration of the recombinant fusion protein A in the aqueous phase is 1–10 mg / ml.

[0035] In some embodiments, the concentration of the hydrophobic biodegradable polyester and its derivatives in the oil phase is...

[0036] The concentration is 1–10 mg / ml.

[0037] In some embodiments, the weight ratio of the hydrophobic biodegradable polyester and its derivatives to the fusion protein A is 1:0.1 to 10, preferably 1:1 to 10, and more preferably 1:2 to 6.

[0038] In some of these embodiments, the volume ratio of the aqueous phase to the oil phase is 1-10:1, preferably 2-6:1.

[0039] In some embodiments, the molar ratio of the nanoassembly to all fusion protein B is (1-10):1, preferably (1-6):1, and more preferably (2-3):1.

[0040] A seventh aspect of the present invention provides the use of any of the above-mentioned immunotherapeutic drugs in the preparation of products for tumor immunotherapy, autoimmune disease treatment, inflammation treatment, and infection treatment.

[0041] This invention creatively constructs a fusion protein containing SpyCatcher003 and albumin, which is then assembled with a hydrophobic biodegradable polyester via affinity. This assembly is then combined with ScFv containing SpyTag003 to form nanoassemblies for various applications. It was found that the SpyCatcher003 on the surface of these assemblies can conveniently, quickly, and covalently bind to the SpyTag003-containing fusion protein, constructing stable single / double / multi-specific antibody drugs. These drugs not only maintain their corresponding biological activity and are stable, but can also simultaneously bind multiple specific antibodies. The binding of these multiple specific antibodies within the nanoassembly is more robust and exhibits greater stability. Therefore, these single / double / multi-specific immunotherapies can be well applied to the treatment of diseases such as tumors. Attached Figure Description

[0042] Figure 1 : Carrier construction map.

[0043] Figure 2 SDS-PAGE characterization of the SpyCatcher003-HSA fusion protein.

[0044] Figure 3 Western blot characterization of the SpyCatcher003-HSA fusion protein.

[0045] Figure 4 HPLC was used to characterize the purity of the purified SpyCatcher003-HSA fusion protein.

[0046] Figure 5 SDS-PAGE characterizes the αHER2 ScFv-SpyTag003 fusion protein.

[0047] Figure 6 Western blot characterization of the αHER2 ScFv-SpyTag003 fusion protein.

[0048] Figure 7 HPLC was used to characterize the purity of the purified αHER2 ScFv-SpyTag003 fusion protein.

[0049] Figure 8 ELISA characterizes the affinity of the αHER2 ScFv-SpyTag003 fusion protein for the HER2 antigen.

[0050] Figure 9 SDS-PAGE characterizes the binding of αHER2 ScFv-SpyTag003 and SpyCatcher003-HSA.

[0051] Figure 10 Western blotting characterizes the binding of αHER2 ScFv-SpyTag003 and SpyCatcher003-HSA. Figure 11 : Detection of NPs prepared from recombinant proteins using a Zetasizer device SCHSA The particle size.

[0052] Figure 12 Scanning electron microscopy characterization of NP SCHSA .

[0053] Figure 13 NP SCHSA Particle stability assessment.

[0054] Figure 14: Zetasizer device for detecting NP in recombinant protein preparation SCHSA The changes after binding αHER2 ScFv-SpyTag003.

[0055] Figure 15 Nanoflow cytometry characterization of NP SCHSA The combination of αHER2 ScFv-SpyTag003.

[0056] Figure 16 Flow cytometry characterization of NP-binding SpyTag003 SCHSA It can enhance the binding ability to target cells.

[0057] Figure 17 Confocal characterization of NP-covalently bound SpyTag003 SCHSA It can enhance the binding ability to target cells.

[0058] Figure 18 Confocal characterization of αHER2 / CD3 ScFv-SpyTag003-equipped nanoparticles to enhance the binding of immune cells to tumor cells.

[0059] Figure 19 HPLC characterization of NP SCHSA It can combine three different ScFv-SpyTag003s simultaneously.

[0060] Figure 20 HPLC characterization of NP SCHSA The ratio of αCD22 ScFv-SpyTag003 fusion protein was determined.

[0061] Figure 21 NP SCHSA The ratio of αCD22 ScFv-SpyTag003 fusion protein was determined. Figure 21 This is a schematic diagram illustrating the principles of the fusion protein, assembly, and application of the present invention. Detailed Implementation

[0062] Unless otherwise specified, the experimental methods described in the following embodiments of the present invention are generally performed under conventional conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the embodiments are commercially available products.

[0063] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0064] To facilitate understanding of this technology, some terms and phrases are defined below.

[0065] The terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product, or device that includes a series of steps is not limited to the steps or modules listed, but may optionally include steps not listed, or may optionally include other steps inherent to such process, method, product, or device.

[0066] In this invention, "multiple" refers to two or more. "And / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A alone, A and B simultaneously, or B alone. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0067] Homologous: In the phylogenetic theory of biology, if two or more structures have the same ancestor, they are said to be homologous.

[0068] Antibody affinity refers to the binding strength between the antigen-binding cluster of an antibody and the antigenic determinant cluster, or the binding force between an antibody and an antigenic epitope or antigenic determinant cluster. It is essentially a non-covalent interaction force, including the attraction between amino acids, hydrogen bonds, hydrophobic interactions, etc.

[0069] Some embodiments of the present invention relate to a protein having at least a serum albumin fragment that can bind to a hydrophobic degradable polymer through hydrophobic interactions. In the present invention, the protein is albumin, i.e., serum albumin, which may be at least one of human serum albumin, bovine serum albumin, mouse serum albumin, rat serum albumin, rabbit serum albumin, and chicken oocyte albumin.

[0070] In some preferred embodiments, the protein is a human serum albumin protein having at least three or four hydrophobic domains, the protein having hydrophobic domains is a human serum albumin protein having at least five hydrophobic domains, or the above-mentioned protein having its hydrophobic domains intact through substitution, deletion and / or addition of one or more amino acids, more preferably, a human serum albumin protein having six or seven hydrophobic domains.

[0071] In this invention, a single-chain variable fragment (scFv) refers to a simplified portion of an immunoglobulin Fab fragment that specifically binds to antigens, artificially expressed through gene recombination technology. It consists of a heavy chain and a light chain, connected by a short polypeptide hinge. scFv retains the antigen recognition and binding properties, and its small molecular size allows it to recombine with polypeptides or proteins to form fusion proteins.

[0072] The single-chain antibody is derived from a functional fragment of an antibody and includes at least one light chain variable region (VL) and / or heavy chain variable region (VH) of the antibody.

[0073] The single-chain antibody is derived from humans, mice, or other species, and its species is consistent with that of serum albumin.

[0074] The linker peptide can be a conventional peptide sequence used to link polypeptides, capable of connecting two polypeptides and naturally folding them into the desired structure. It is typically a short peptide with hydrophobicity and a certain degree of extensibility. The purpose in this invention is to separate the two fused proteins to mitigate their mutual interference. The linker peptide can be flexible or rigid. In some embodiments, a flexible linker peptide may be advantageous, as it can link two protein / peptide components while maintaining their respective activities and functions. In some embodiments, the hydrophobic regions of the fused protein, and the functional fragments of immunomodulatory antibodies, are linked by a linker peptide, which in some embodiments, for example, uses [GlyGlyGlyGlySer]n, where n is an integer from 0 to 4, more preferably 1, 2, or 3. When n is 0, it means that the relevant fragments are directly linked.

[0075] Some embodiments of this invention involve hydrophobic biodegradable polyesters and their derivatives, which are currently known biodegradable biomaterials, and also include new biodegradable biomaterials to be further developed in the future, which can bind to the hydrophobic regions of the serum albumin moiety described above. The polyester is an aliphatic polyester or a derivative thereof.

[0076] This invention constructs and expresses the SpyCatcher003-SA fusion protein, and prepares nanoparticles by combining it with a hydrophobic polymer; simultaneously, it constructs a fusion protein by combining SpyTag003 with exogenous ScFv, etc.; the above nanoparticles are incubated with the fusion protein containing SpyTag003, and the fusion protein can be uniformly bound to the surface of the nanoparticles through isopeptide bonds to form a bispecific antibody.

[0077] The relevant sequences used in the following examples.

[0078] SpyCatcher003:VTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDAHT(SEQ ID No.11)

[0079] SpyTag003:RGVPHIVMVDAYKRYK(SEQ ID No.12)

[0080] SEQ ID No.1

[0081] SpyCatcher003-HSA

[0082] VTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDAHT GGGGSGGGGSGGGGS DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPY FYAPELLFFAKRYKAAFTECCQAADKAACLLPCLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDE MPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLHHHHHHHHHH

[0083] SEQ ID No.2

[0084] HSA-SpyCatcher003DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIAR RHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPCLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSL

[0085] AADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKKCCAAA

[0086] DPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEV

[0087] SRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRP

[0088] CFSALEVDETYVPKEFNAETTFFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVM

[0089] DDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGGGSGGGGSGGGGSVTTLSGLS

[0090] GEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISTGHVKDFYLY

[0091] PGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDAHTHHHHHHHHHHSEQ ID No.3

[0092] αHER2 ScFv-SpyTag003

[0093] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGCAPKLLIYSASFLYSGVPSRFSGSRS

[0094] GTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGSGGSGGCPPCGGGGSEVQLVE

[0095] SGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISA

[0096] DTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGCGTLVTVSS GGGGSGGGGSGGGG

[0097] S RGVPHIVMVDAYKRYKEQKLISEEDLNSAVDHHHHHH

[0098] SEQ ID No.4

[0099] SpyTag003-αHER2 ScFv

[0100] RGVPHIVMVDAYKRYKGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDV

[0101] NTAVAWYQQKPGCAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHY

[0102] TTPPTFGQGTKVEIKGGGSGGSGGCPPCGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNI

[0103] KDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAE

[0104] DTAVYYCSRWGGDGFYAMDYWGCGTLVTVSSEQKLISEEDLNSAVDHHHHHHSEQ ID No.5

[0105] αCD3 ScFv-SpyTag003

[0106] QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTP

[0107] ARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVGGGGSGGGGSGG

[0108] GGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIRSKYNN

[0109] YATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQ

[0110] GTLVTVSSGGGGSGGGGSGGGGSRGVPHIVMVDAYKRYKEQKLISEEDLNSAVDHHHHHHSEQ IDNo.6

[0111] SpyTag003-αCD3 ScFv

[0112] RGVPHIVMVDAYKRYKGGGGSGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCGSSTGAV

[0113] TTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYC

[0114] ALWYSNLWVFGGGTKLTVGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASG

[0115] FTFSTYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMN

[0116] SLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSEQKLISEEDLNSAVDHHHHHHSEQ IDNo.7

[0117] α4-1BB ScFv-SpyTag003

[0118] EVQLVQSGAEVKKPGESLRISCKGSGYSFSTYWISWVRQMPGKGLEWMGKIYPGDSYTNY

[0119] SPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGYGIFDYWGQGTLVTVSSGGGGS

[0120] GGGGSGGGGSSYELTQPPSVSVSPGQTASITCSGDNIGDQYAHWYQQKPGQSPVLVIYQDK

[0121] NRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCATYTGFGSLAVFGGGTKLTVLGGGGS

[0122] GGGGSGGGGSRGVPHIVMVDAYKRYKEQKLISEEDLNSAVDHHHHHHSEQ ID No.8

[0123] SpyTag003-α4-1BB ScFv

[0124] RGVPHIVMVDAYKRYKGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGESLRISCKGSGYSF

[0125] STYWISWVRQMPGKGLEWMGKIYPGDSYTNYSPSFQGQVTISADKSISTAYLQWSSLKASD

[0126] TAMYYCARGYGIFDYWGQGTLVTVSSGGGGSGGGGSGGGGSSYELTQPPSVSVSPGQTASI

[0127] TCSGDNIGDQYAHWYQQKPGQSPVLVIYQDKNRPSGIPERFSGSNSGNTATLTISGTQAMDE

[0128] ADYYCATYTGFGSLAVFGGGTKLTVLEQKLISEEDLNSAVDHHHHHHSEQ ID No.9

[0129] αCD22 ScFv-SpyTag003

[0130] DVQVTQSPSSLSASVGDRVTITCRSSQSLANSYGNTFLSWYLHKPGKAPQLLIYGISNRFSG

[0131] VPDRFSGSGSGTDFTLTISSLQPEDFATYYCLQGTHQPYTFGQGTKVEIKGGGGSGGGGSGG

[0132] GGSEVQLVQSGAEVKKPGASVKVSCKASGYRFTNYWIHWVRQAPGQGLEWIGGINPGNN

[0133] YATYRRKFQGRVTMTADTSTSTVMELSSLRSEDTAVYYCTREGYGNYGAWFAYWGQGTLV

[0134] TVSSGGGGSGGGGSGGGGSRGVPHIVMVDAYKRYKEQKLISEEDLNSAVDHHHHHHSEQ ID No.10

[0135] SpyTag003-αCD22 ScFv

[0136] RGVPHIVMVDAYKRYKGGGGSGGGGSGGGGSDVQVTQSPSSLSASVGDRVTITCRSSQSL

[0137] ANSYGNTFLSWYLHKPGKAPQLLIYGISNRFSGVPDRFSGSGSGTDFTLTISSLQPEDFATYY

[0138] CLQGTHQPYTFGQGTKVEIKGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGASVKVSCKA

[0139] SGYRFTNYWIHWVRQAPGQGLEWIGGINPGNNYATYRRKFQGRVTMTADTSTSTVMELSS

[0140] LRSEDTAVYYCTREGYGNYGAWFAYWGQGTLVTVSSEQKLISEEDLNSAVDHHHHHH

[0141] Raw materials used in the examples and sources thereof:

[0142] SpyCatcher003-HSA, αHER2 ScFv-SpyTag003, αCD3 ScFv-SpyTag003, α4-1BBScFv-SpyTag003, and αCD22 ScFv-SpyTag003 were all purified from HEK-293F cells after transient transfection expression using an AKTA purification system. All fusion proteins contained (GGGGS)3 flexible links.

[0143] Polylactic acid PLA134K, with a molecular weight of 134,000 Da and ester-terminated L-lactic acid, was purchased from Jinan Daigang Biotechnology Co., Ltd.

[0144] Dichloromethane: Purchased from Guangzhou Chemical Reagent Factory.

[0145] pXC17.4αHER2 ScFv-SpyTag003 was synthesized by GenScript Biotech Ltd. pXC17.4SpyCatcher003-HSA was synthesized by GenScript Biotech Ltd.

[0146] PEI40000: Polysciences, purchased from Morningstar Biotechnology (Guangzhou) Co., Ltd.

[0147] HEK-293F cells: purchased from Shanghai Fuheng Biotechnology Co., Ltd.

[0148] BT-474 cells: purchased from Xiamen Yimo Biotechnology Co., Ltd.

[0149] HT-29 cells: purchased from Guangzhou Zhenzhi Biotechnology Co., Ltd.

[0150] HEK CD02 culture medium: purchased from Shanghai Youyi Biotechnology Co., Ltd.

[0151] HER2 antigen: ACRO, purchased from Guangzhou Zhijianlian Biotechnology Co., Ltd.

[0152] ELISA reagents: purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.

[0153] Mouse anti-Myc tag antibody: Guangzhou Juyan Biotechnology Co., Ltd.

[0154] Anti-HSA antibody; purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.

[0155] anti-HIS antibody; purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.

[0156] Protein L: Purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.

[0157] Cy5-NHS dye: purchased from Xi'an Dianhua Biotechnology Co., Ltd.

[0158] Cy3-NHS dye: purchased from Xi'an Dianhua Biotechnology Co., Ltd.

[0159] FITC-NHS dye: purchased from Xi'an Dianhua Biotechnology Co., Ltd.

[0160] CellTrace Far Red dye: purchased from ThermoFisher Technology Co., Ltd.

[0161] Example 1: Construction and expression of SpyCatcher003-HSA fusion protein

[0162] 1. Construction of recombinant plasmids

[0163] Synthesize the gene sequence corresponding to the target protein sequence in the following order:

[0164] The target protein sequence SpyCatcher003-Link-HSA-10*HIS is named SpyCatcher003-HSA, and the specific sequence can be found in SEQ ID No: 1.

[0165] A signal peptide sequence was added to the N-terminus of the gene sequence corresponding to the above protein sequence, a start codon was added to the N-terminus, and a stop codon was added to the C-terminus. The full-length gene sequence was then cloned into the pXC17.4 vector, with HindIII and EcoRI restriction sites added upstream and downstream, respectively, along with the signal peptide. The above plasmid was synthesized by Genscript Biotech Co., Ltd., and named pXC17.4 SpyCatcher003-HSA. A schematic diagram of the recombinant vector is shown below. Figure 1 As shown in Figure A, the 10*HIS sequence contained therein is used for purification.

[0166] 2. Expression and purification of fusion proteins

[0167] Following standard procedures, the synthesized plasmid was transformed into E. coli, and single colonies were picked and amplified.

[0168] Plasmids were extracted during culture, and then HEK-293F cells were transiently transduced.

[0169] First, HEK-293F cells were cultured to a scale of 4E6 cells / mL, with a total culture volume of 100mL. 200μg of pXC17.4 SpyCatcher003-HSA was diluted to 5mL with 0.15M sodium chloride solution, and 400μg of PEI 40000 was diluted to 5mL with 0.15M sodium chloride solution. The PEI 40000 dilution was then slowly added to the plasmid dilution and mixed well. The mixture was allowed to stand for 15min. Finally, the mixture was slowly poured into the cell culture medium and placed in a CO2 incubator for culture at 37℃, 8% CO2, and 130rpm. After 7 days of batch culture, the supernatant was collected after centrifugation at 5000g for 15 min. The expressed fusion protein has a 10*HIS tag at its C-terminus, which can be purified by affinity chromatography through specific binding to NI. The supernatant was then collected in a solution containing 200mM imidazole and subjected to multiple ultrafiltrations using a 30kDa ultrafiltration tube at 3000g and 4℃. The solution was completely replaced with an aqueous solution for subsequent particle preparation. The molecular weight of the purified target fusion protein was characterized by SDS-PAGE, as shown below. Figure 2 SDS-PAGE results showed that the molecular weight of the SpyCatcher003-HSA fusion protein was 80 kDa+, consistent with the expected 80.91 kDa; further preliminary characterization of its domains was performed using Western blotting, such as... Figure 3 The results showed that it contained HSA and HIS domains, consistent with expectations; finally, the purity of the target protein was preliminarily characterized by HPLC, such as... Figure 4 The results showed that its purity was higher than 95%, which met the requirements for subsequent experiments.

[0170] Example 2: Expression and purification of αHER2 ScFv-SpyTag003

[0171] 1. Construction of recombinant plasmids

[0172] Synthesize the gene sequence corresponding to the target protein sequence in the following order:

[0173] The target protein sequence is αHER2 ScFv-Link-SpyTag003-Myc-6*HIS, named αHER2 ScFv-SpyTag003.

[0174] The specific sequence can be found in SEQ ID No: 3.

[0175] A signal peptide sequence was added to the N-terminus of the gene sequence corresponding to the above protein sequence, a start codon was added to the N-terminus, and a stop codon was added to the C-terminus. The full-length gene sequence was then cloned into the pXC17.4 vector. HindIII and EcoRI restriction sites were added upstream and downstream, respectively. A 6*HIS tag was used for purification, and a Myc tag was used for subsequent characterization. The plasmid was synthesized by GenScript Biotech Ltd., and named pXC17.4αHER2 ScFv-SpyTag003. A schematic diagram of the recombinant vector is shown below. Figure 1 As shown in B.

[0176] 2. Expression and purification of fusion proteins

[0177] The synthesized plasmid was transformed into *E. coli*, single colonies were picked, and the culture was expanded for plasmid extraction. After extraction, HEK-293F cells were transiently transduced. First, HEK-293F cells were cultured to 4E6 cells / mL, with a total culture volume of 100mL. 100μg of pXC17.4αHER2 ScFv-SpyTag003 was diluted to 5mL with 0.15M sodium chloride solution, and 200μg of PEI40000 was diluted to 5mL with 0.15M sodium chloride solution. The PEI40000 dilution was then slowly added to the plasmid dilution and mixed thoroughly. The mixture was allowed to stand for 15 minutes. Finally, the mixture was slowly poured into the cell culture medium and incubated in a CO2 incubator at 37℃, 8% CO2, and 130rpm. After 7 days of batch culture, the supernatant was collected after centrifugation at 5000g for 15 min. The expressed fusion protein has a 6*HIS tag at its C-terminus, which can be purified by affinity chromatography through specific binding to NI. The supernatant was then collected in a solution containing 200mM imidazole and subjected to multiple ultrafiltrations using a 30kDa ultrafiltration tube at 3000g and 4℃. The solution was then completely replaced with PBS and aseptically stored. The molecular weight of the purified fusion protein was characterized by SDS-PAGE. Figure 5 The results showed that the molecular weight of the pXC17.4αHER2 ScFv-SpyTag003 fusion protein was 30 kDa+, consistent with the expected 31.44 kDa; further preliminary characterization of its domains was performed using Western blotting, such as... Figure 6 The results showed that it contained ScFv and HIS domains, consistent with expectations; subsequently, the purity of the target protein was preliminarily characterized by HPLC, such as... Figure 7 The results showed that its purity was higher than 90%, which met the requirements for subsequent experiments.

[0178] 3. Enzyme-linked immunosorbent assay (ELISA) characterization of the affinity between the fusion protein and the antigen.

[0179] The affinity of the αHER2 ScFv-SpyTag003 fusion protein for the target antigen HER2 was characterized by enzyme-linked immunosorbent assay (ELISA). Human HER2 antigen was diluted to 1 μg / mL with coating buffer, 100 μL / well, and incubated at 37°C for 2 h or 4°C overnight for coating. The plates were washed three times with PBST. Blocking was performed using 200 μL / well of 1% protein-free blocking buffer, followed by three washes with PBST. The αHER2 ScFv-SpyTag003 fusion protein was diluted with PBST to 20 μg / mL, 2 μg / mL, 200 ng / mL, 20 ng / mL, 2 ng / mL, 0.2 ng / mL, 0.02 ng / mL, and 0.002 ng / mL, 100 μL / well, and incubated at 37°C for 2 h. The plates were washed six times with PBST. Mouse anti-Myc tag antibody (HRP) was used as the secondary antibody, followed by six washes with PBST. Using ELISA chromogenic buffer, the reaction was carried out in the dark for 10-20 minutes, and terminated with 2M sulfuric acid. The absorbance at 450-630 nm was measured using a microplate reader. The affinity curve for αHER2ScFv-SpyTag003 is shown below. Figure 8 The Kd value is 0.29 nM. The Kd value reflects the affinity of the compound for the target. The smaller the value, the stronger the affinity. The results show that the expressed αHER2 ScFv-SpyTag003 has a strong affinity for the target antigen, indicating that the fusion of SpyTag003 does not affect the affinity of the antibody ScFv.

[0180] Example 3: Characterization of the binding of SpyCatcher003-HSA and αHER2 ScFv-SpyTag003

[0181] The two fusion proteins (SpyCatcher003-HSA and αHER2 ScFv-SpyTag003) were mixed in different ratios to investigate their binding. The molar ratios were 1:2, 1:1, and 2:1. The mixing system was PBS buffer at pH 7.4. After mixing for 30 min, SDS-PAGE characterization was performed. The SDS-PAGE results are shown below. Figure 9 As shown, when the molar ratio of the two is 1:1, they can completely react to generate a new protein at 112 kDa. When the ratio is not 1:1, but 2:1 or 1:2, there will be residual protein. The structure after binding was also characterized by Western blotting using anti-HSA, anti-HIS, and anti-ScFv, respectively. The results are as follows. Figure 10 This shows that the two can be fully combined in a 1:1 ratio.

[0182] Example 4 Nanoparticles (NP)SCHSA Assembly and characterization of )

[0183] 1. NP SCHSA Preparation

[0184] A PLLA solution with a concentration of 10 mg / mL was prepared using dichloromethane as a solvent. 134k The solution was used as the oil phase, and a 5 mg / mL SpyCatcher003-HSA fusion protein aqueous solution was used as the aqueous phase. 100 μL of the oil phase and 1 mL of the aqueous phase were placed in a 5 mL centrifuge tube and emulsified using an ultrasonic cell disruptor. The ultrasonic disruptor probe diameter was 3 mm, the amplitude was 40%, and the sonication time was 5 seconds followed by a 2-second pause, for a total of 2 minutes. After sonication, the emulsion was washed and transferred to a rotary evaporator flask. Dichloromethane was removed by applying vacuums at 400 / 350 / 300 / 250 / 200 / 150 / 100 / 75 / 50 / 25 mbar for 2 minutes each. After evaporation, the aliphatic polyester nanoassemblies of the fusion protein were collected. The hydration diameter of the nanoassemblies was measured using a nanoparticle size analyzer, and the corresponding NP... SCHSA Particle size distribution diagram as follows Figure 11 As shown, the particle size is about 100 nm and the PDI is about 0.09, indicating that it has excellent dispersibility.

[0185] 2. Scanning electron microscopy characterization of NP SCHSA

[0186] NP SCHSA When the concentration was diluted and the ATTN value measured by the nanoparticle size analyzer was 9, 2 μL was dropped onto a silicon wafer. After the moisture evaporated, gold sputtering was performed, and then the wafer was observed under a scanning electron microscope. The electron microscope results are as follows. Figure 12 As shown, the nanoparticles exhibit an independent, full spherical shape.

[0187] 3. NP SCHSA Stability assessment

[0188] Nanoparticles were placed in aqueous solution and PBS solution and stored at 37°C with shaking. Samples were taken at 0, 2, 4, 8, 24, 36, 60, 96, 144, 180, and 252 hours to detect particle size and PDI. The results are as follows: Figure 13 As shown, over a period of up to ten days, the particle size and PDI did not change significantly, indicating that the nanoparticles prepared in this invention have good stability.

[0189] Example 5: Rapid preparation and characterization of nanomedicines

[0190] 1. Nanomedicine preparation

[0191] NP SCHSAThe αHER2 ScFv-SpyTag003 fusion protein was mixed with αHER2 ScFv-SpyTag003 in PBS solution at a molar ratio of 3:1 at room temperature, and the mixture was then named NP. SCHSA&αHER2-ScFv The particle size, PDI, and potential were then measured using a nanoparticle size analyzer, and the results are as follows: Figure 14 As shown, the overall particle size increases by 30 nm after the fusion protein is incorporated, and the potential also increases to some extent due to the incorporation of the fusion protein, which is consistent with expectations.

[0192] 2. Nanoflow cytometry characterization of NP SCHSA Binding to αHER2 ScFv-SpyTag003 fusion protein

[0193] The fluorescent molecule labeled with Cy5 on the SpyCatcher003-HSA fusion protein was named SpyCatcher003-HSA. Cy5 Simultaneously, the αHER2 ScFv-SpyTag003 fusion protein was labeled with the AF488 fluorescent molecule and named αHER2ScFv-SpyTag003. AF488 Then use SpyCatcher003-HSA Cy5 Fluorescent nanoparticles NP were prepared from the fusion protein using the particle preparation method described in Example 4. SCHSA-Cy5 Then combine it with αHER2 ScFv-SpyTag003 AF488 The particles were incubated in PBS for 1 hour, then centrifuged at 15000 rpm for 45 minutes. The supernatant was removed, and the particles were resuspended in PBS. Finally, the particles were characterized by nanoflow cytometry. The experimental results are as follows: Figure 15 As shown, nanoparticles NP SCHSA-Cy5 Able to interact with αHER2ScFv-SpyTag003 AF488 Effective binding was achieved, and αHER2ScFv-SpyTag003 was detected on nearly 90% of the particle surface. AF488 .

[0194] 3. HPLC characterization of NP SCHSA Combined with the ratio of αCD22 ScFv-SpyTag003 fusion protein

[0195] NP SCHSA Co-incubated with αCD22ScFv-SpyTag003 at molar ratios of 6:1, 3:1, 2:1, and 1:1, and after incubation at room temperature for 1 hour, samples were taken to detect the amount of free αCD22ScFv-SpyTag003. The experimental results are as follows: Figure 20 As shown, nanoparticles can be obtained with molar ratios ranging from 1 to 6:1, but this is not a perfect NP (NP-complete) method. SCHSAThe more the better. Among them, when the ratio of (2-3):1, there is very little free αCD22 ScFv-SpyTag003. In particular, when the ratio of the two is 2:1, there is almost no free αCD22 ScFv-SpyTag003 in the system. Therefore, this ratio can be used for subsequent binding.

[0196] Example 6: αHER2 ScFv-SpyTag003 combined with enhanced nanoparticle targeting

[0197] 1. Flow cytometry characterization of αHER2 ScFv-SpyTag003 covalently bound NP SCHSA It can promote the binding of nanoparticles to HER2 ++ BT-474 cells

[0198] The αHER2 ScFv-SpyTag003 fusion protein was labeled with a FITC fluorescent molecule and named αHER2ScFv-SpyTag003. FITC NP as shown in Example 5 SCHSA-Cy5 Combined with αHER2 ScFv-SpyTag003 FITC The particle resulting from the protein fusion was named NP. SCHSA-Cy5&αHER2-FITC .

[0199] BT474 cells were cultured to 80% confluence, the supernatant was discarded, and the cells were digested with trypsin and counted. 50W cells were transferred to 1.5mL EP tubes, and pre-prepared NP was added according to the different experimental objectives. SCHSA-Cy5 or NP SCHSA-Cy5&αHER2-FITC After incubation on ice for 15 minutes, the cells were centrifuged at 300g at 4°C for 5 minutes. The fluorescence intensity of the cell surface binding granules was then analyzed by flow cytometry. The experimental results are as follows: Figure 16 As shown, add αHER2 ScFv-SpyTag003 FITC Subsequently, the fluorescence intensity of Cy5 bound to the cell surface was significantly enhanced, indicating that NP SCHSA-Cy5 It can bind to the cell surface via αHER2 ScFv-SpyTag003.

[0200] 2. Confocal characterization of αHER2 ScFv-SpyTag003 covalently bound to NP SCHSA It can improve the binding of nanoparticles to HER2++ in BT-474 cells

[0201] BT-474 cells were cultured to 80% confluence, the supernatant was discarded, and the cells were digested with trypsin and counted. 20W cells were transferred to 1.5mL EP tubes, and pre-prepared NP was added according to the different experimental objectives. SCHSA-Cy5 or NP SCHSA-Cy5&αHER2-FITCAfter incubation on ice for 15 minutes, the cells were centrifuged at 300g at 4°C for 5 minutes. The fluorescence on the cell surface was then characterized using confocal microscopy. Figure 17 As shown, without adding αHER2 ScFv-SpyTag003 FITC At that time, no NP was detected on the surface of the cell. SCHSA-Cy5 The signal, when αHER2 ScFv-SpyTag003 is added. FITC Subsequently, strong FITC and Cy5 signals were observed on the cell surface, indicating that NP SCHSA-Cy5 It can bind to the cell surface via αHER2 ScFv-SpyTag003.

[0202] Example 7: αHER2 ScFv-SpyTag003 and αCD3 ScFv-SpyTag003 combined nanoparticles enhance the binding of immune cells to tumor cells.

[0203] 1. Confocal characterization of bispecific antibodies NP SCHSA&αHER2 / CD3 Promotes binding to target cells

[0204] αHER2 ScFv-SpyTag003 and αCD3 ScFv-SpyTag003 were compared with NP SCHSA Co-incubation yields NP SCHSA&αHER2 / CD3 , for use in subsequent experiments.

[0205] HT-29 cells were cultured to 80% confluence, the supernatant was removed, and the cells were digested with trypsin and resuspended in 1640 medium. 1100w / mL HT-29 cells were transferred to a sterile centrifuge tube, 1μL of CSFE dye was added, and the cells were treated at 37℃ for 15 min. Then, 10 times the volume of DMEM + 10% FBS was added and incubated for 5 min to terminate staining. The cells were centrifuged at 800 rpm to remove the supernatant, and 10w of cells were seeded into a confocal dish and cultured for 24 h.

[0206] 500W of T cells were centrifuged at 400g for 10 min, resuspended in 1 mL of serum-free 1640 medium, and stained with 1 μL CellTrace Far Red dye. The cells were treated at 37℃ for 15 min, followed by incubation with 10 times the volume of DMEM + 10% FBS for 5 min to terminate staining. The cells were centrifuged at 400g for 10 min, the supernatant was removed, and 50W of cells were seeded into corresponding confocal microscopy dishes. Appropriate amounts of PBS and 30 μg / mL of free αHER2 ScFv-SpyTag003 and αCD3 ScFv-SpyTag003 were added respectively. The cells were co-cultured for 4 H, the supernatant was gently aspirated, and the cells were gently washed three times with PBS. Finally, the binding of the two cell types was characterized by confocal microscopy. Figure 18As shown, compared to the other two groups, NP was added. SCHSA&αHER2 / CD3 It significantly increased the proportion of binding to target cells.

[0207] Example 8: HPLC characterization of NP SCHSA It can combine three different ScFv-SpyTag003 simultaneously

[0208] Three different α4-1BB ScFv-SpyTag003 (SEQ ID No: 7), αHER2 ScFv-SpyTag003 (SEQ ID No: 5), and αCD22 ScFv-SpyTag003 (SEQ ID No: 9) were stained with Cy5, Cy3, and FITC fluorescent molecules, respectively, and then NP... SCHSA The three fluorescently labeled ScFv-SpyTag003 were incubated in PBS at a molar ratio of 6:1:1:1 and incubated overnight at 4°C. The elution times of the particles and fluorescent molecules were detected by HPLC using both UV and fluorescence channels. The experimental results are as follows: Figure 19 As shown, the UV peak of the nanoparticles coincides with the elution times of the three different fluorescent molecules, indicating that the nanoparticles can bind to three different ScFv-SpyTag003 molecules and pass through the detector simultaneously. This demonstrates that the nanoparticles can simultaneously bind to multiple fusion proteins containing SpyTag003, providing guidance for the preparation of multispecific antibodies.

[0209] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A fusion protein composition, characterized in that, The fusion protein composition includes a SpyCatcher003-albumin fusion protein A and at least one protein-SpyTag003 fusion protein B. The fusion protein A is formed by fusing SpyCatcher003 protein with albumin; The fusion protein B is formed by fusing a protein with SpyTag003, and the protein is a single-chain antibody. In fusion protein A, the albumin is linked to the C-terminus of SpyCatcher003 via a flexible linker peptide; and in fusion protein B, the protein is linked to the N-terminus of SpyTag003 via a flexible linker peptide.

2. The fusion protein composition according to claim 1, characterized in that, The albumin is any one of human serum albumin, bovine serum albumin, mouse serum albumin, rat serum albumin, rabbit serum albumin, and chicken oocyte albumin.

3. The fusion protein composition according to claim 1, characterized in that, The single-chain antibody is an antibody targeting at least one of the following antigens: antibodies derived from humans or other species targeting HER2, CD137L, CD137, OX40L, OX40, ICOSL, ICOS, CD86, CD80, CD28, LFA3, CD2, CTLA4, PDL1, PD1, CD70, CD27, GAL9, TIM3, CD111, CD96, CD112, CD226, CD115, CD113, TIGIT, CD39, CD73, CD47, SIRPα, TNFα, IL1β, IL6, TGFβ, IL-10, and IL-12.

4. The fusion protein composition according to claim 1, characterized in that, The fusion protein B may be one, two, or three or more; each different fusion protein B may have a different single-chain antibody.

5. The fusion protein composition according to claim 1, characterized in that, The linker peptide is [GGGGS]n or (EAAAK)n, where n is any integer.

6. The fusion protein composition according to claim 5, characterized in that, n is an integer between 1 and 4.

7. The gene encoding the fusion protein composition according to any one of claims 1-6, wherein, The coding genes include the coding gene for fusion protein A and the coding gene for fusion protein B.

8. The encoding gene according to claim 7, characterized in that, The amino acid sequence of the fusion protein A is shown in SEQ ID No: 1; and / or The amino acid sequence of the fusion protein B is shown in any one of SEQ ID No: 3, SEQ ID No: 5, SEQ ID No: 7, or SEQ ID No:

9.

9. A plasmid vector, characterized in that, The plasmid vector contains the encoding gene as described in any one of claims 7-8.

10. An engineered bacterium, protoplast, or in vitro cloning system containing the coding gene of claim 7 or 8 or the plasmid vector of claim 9.

11. A single / double / multi-specific immunomodulatory drug, characterized in that, The immunomodulatory drug is obtained by covalently linking a nano-assembly with at least one fusion protein B in the fusion protein composition according to any one of claims 1-6. The nano-assembly is formed by the fusion protein A in the fusion protein composition according to any one of claims 1-6 and a hydrophobic biodegradable polyester through hydrophobic interaction.

12. The single / dual / multi-specific immunomodulatory agent according to claim 11, characterized in that, The biodegradable polyester is an aliphatic polyester, or an aliphatic polyester modified with polyethylene glycol.

13. The single / dual / multi-specific immunomodulatory agent according to claim 12, characterized in that, The aliphatic polyester is at least one of polylactide, polyglycolic acid, poly(lactide-co-glycolic acid), and polycaprolactone.

14. The single / dual / multi-specific immunomodulatory agent according to claim 13, characterized in that, The block molecular weight of the polylactide is 1 kDa to 1,100 kDa; or the block molecular weight of the poly(lactide-co-glycolic acid) is 1 kDa to 1,100 kDa, wherein the ratio of lactide to glycolide is in the range of 95 / 5 to 50 / 50.

15. The single / dual / multi-specific immunomodulatory agent according to claim 11, characterized in that, The ratio of the amount of fusion protein A to fusion protein B is (3-1):

1.

16. The single / dual / multi-specific immunomodulatory agent according to claim 15, characterized in that, The ratio of fusion protein A to fusion protein B is 1:

1.

17. A method for preparing a single / dual / multi-specific immunomodulatory drug as described in claim 11, characterized in that, Includes the following steps: (1) The fusion protein A is mixed with water or physiological saline at a concentration of 0.5-20 mg / mL to obtain an aqueous phase; the hydrophobic biodegradable polyester is mixed with an organic solvent to obtain an oil phase at a concentration of 0.5-10 mg / mL; (2) Prepare an oil-in-water emulsion from the aqueous phase solution and the oil phase solution described in step (1); (3) The emulsion is separated and purified to obtain nano-assemblies; (4) The nano-assembly is mixed with at least one of the fusion proteins B at room temperature to obtain the drug.

18. The preparation method according to claim 17, characterized in that, The organic solvent is at least one of chloroform, dichloromethane, and ethyl acetate.

19. The preparation method according to claim 17, characterized in that, The concentration of the fusion protein A in the aqueous phase is 1-10 mg / ml; and / or, the concentration of the hydrophobic biodegradable polyester in the oil phase is 1-10 mg / ml; and / or, the weight ratio of the hydrophobic biodegradable polyester to the fusion protein A is 1:1-10; and / or, the volume ratio of the aqueous phase to the oil phase is 1-10:

1.

20. The preparation method according to claim 19, characterized in that, The weight ratio of the hydrophobic biodegradable polyester to the fusion protein A is 1:5~10.

21. The preparation method according to claim 19, characterized in that, The volume ratio of the aqueous phase to the oil phase is 5-10:

1.

22. The preparation method according to claim 17, characterized in that, The molar ratio of the nanoassembly to the fusion protein B is (1-10):

1.

23. The preparation method according to claim 22, characterized in that, The molar ratio of the nanoassembly to the fusion protein B is (1-6):

1.

24. The preparation method according to claim 23, characterized in that, The molar ratio of the nanoassembly to the fusion protein B is (2-3):1.

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