Deubiquitinase fc fusion proteins, products thereof, methods of making, and use in lead compound screening

Abstract

The application discloses a deubiquitinating enzyme Fc fusion protein and application thereof in lead compound screening, and belongs to the technical field of fusion proteins. The technical problem to be solved is that OTUD3 protein is unstable and easy to degrade in the prior art. The technical solution is characterized in that an OTUD3 and Fc fusion protein is provided, the fusion protein is easy to purify while maintaining the activity of OTUD3, and can be applied to lead compound screening.

Description

Technical Field

[0001] This invention belongs to the field of fusion protein technology, and relates to deubiquitinizing enzymes, specifically to a deubiquitinizing enzyme Fc fusion protein, its product, preparation method, and application in seed compound screening. Background Technology

[0002] The balance between protein production and degradation is crucial for biological activities. Organisms regulate this balance primarily through two pathways. Ubiquitination is a key post-translational modification of proteins, playing a vital role in intracellular protein degradation. Deubiquitination, on the other hand, involves deubiquitinating enzymes (DUBs) specifically hydrolyzing ubiquitin from ubiquitin-linked proteins or precursor proteins, preventing target protein degradation, and participating in ubiquitin molecule recycling, processing ubiquitin precursors, and editing ubiquitin chains, thereby affecting protein function. Active protein ubiquitination and degradation are root causes of many diseases, including the ubiquitination and degradation of many tumor suppressor factors that have inhibitory effects on cancer. Therefore, in the field of drug discovery, developing pharmacological strategies that selectively restore or enhance protein levels (such as tumor suppressor factors) to achieve therapeutic benefits holds broad application prospects. Targeted protein stabilization (TPS) therapy is one approach that involves discovering a small molecule recruiter of a deubiquitinating enzyme (DUB) to link with a target protein ligand to form a chimeric molecule, thereby achieving deubiquitination and stabilization of the target protein by the deubiquitinating enzyme. OTU domain-containing protein 3 (OTUD3), a member of the OTU family, deubiquitinates various proteins by specifically cleaving the K11-ubiquitin and K6-ubiquitin chains, playing an important role in neurodevelopment, innate immunity, inflammatory responses, and the occurrence and development of tumors, diabetes, and neurodegenerative diseases. OTUD3 is an ideal deubiquitinating enzyme for this therapeutic strategy, but small molecule recruiters targeting its binding have not yet been developed or investigated. Surface plasmon resonance (SPR) technology, with its real-time kinetic analysis, high-throughput potential, and label-free advantages, has become the gold standard for small molecule-protein interaction research. However, traditional protein immobilization techniques used in SPR, such as the biotin-avidin method, cannot balance low cost and effectiveness. Biotinylated reagents are expensive to synthesize, and the biotin-avidin binding dissociation constant is extremely low, making it difficult to break the binding with conventional regeneration buffers (such as hydrochloric acid and NaOH), leading to difficulties in chip regeneration. Secondly, due to the instability and easy degradation of the full-length OTUD3 protein, it is not suitable for traditional methods of continuous screening of small molecules after immobilization. Therefore, there is an urgent need to develop a screening method that is easy to dissociate from the chip and easily repeats the immobilization of fresh proteins.

[0003] Relevant patent documents retrieved: The document, published in China (CN116802198A) on September 22, 2023, discloses a fusion protein comprising: a. an effector domain containing a catalytic domain of a deubiquitinating enzyme or a functional fragment or variant thereof; and b. a targeting domain containing a targeting portion of a membrane protein that specifically binds to a non-ion channel; wherein the targeting domain portion specifically binding to the membrane protein comprises a full-length antibody, a single-chain variable fragment (scFv), scFv2, scFv-Fc, Fab, Fab', F(ab')2, F(v), VHH, or (VHH)2.

[0004] This document, published in China (CN116712433A) on September 8, 2023, discloses the use of a compound that inhibits the activity of the deubiquitinating enzyme OTUD3 in the treatment and / or prevention of tumors. The compound described in this invention can inhibit the binding of OTUD3 to ubiquitin, promoting the degradation of GRP78 protein, and can be used to treat cancer and / or viral infections.

[0005] The core of Fc fusion protein technology lies in leveraging the inherent properties of the immunoglobulin constant region (Fc segment) to optimize the pharmacokinetics and pharmacodynamics of therapeutic proteins. Structurally, a typical Fc fusion protein consists of three parts: an N-terminal functional protein domain, a connecting peptide sequence in the middle, and a C-terminal IgG immunoglobulin Fc domain. This chimeric design is not a simple molecular assembly but is based on a deep understanding of antibody structure and function. The IgG immunoglobulin Fc domain contains two constant region domains, CH2 and CH3, which can bind to the neonatal Fc receptor (FcRn), a key mechanism for prolonging serum half-life. FcRn binds to IgG in an acidic endosomal environment, protecting it from lysosomal degradation, and is subsequently released back into the bloodstream under neutral pH conditions. This recovery process can extend the half-life of Fc fusion proteins to 10-30 times that of traditional therapeutic proteins.

[0006] The design of linkers is equally crucial. They must be flexible enough to allow functional domains and Fc segments to maintain their correct spatial conformation, while also possessing appropriate rigidity to prevent protein hydrolysis. Commonly used linkers include flexible sequences rich in glycine and serine (such as GGGGS repeat sequences) or cleavable linkers containing specific protease cleavage sites. In some applications, linkers also play a role in regulating protein dimerization, for example, by introducing cysteine ​​residues to promote intermolecular disulfide bond formation, thereby creating recombinant proteins with specific oligomeric states.

[0007] By introducing intramolecular disulfide bonds, replacing easily degradable amino acid residues, and optimizing surface charge distribution, the thermal stability and resistance to protease degradation of fusion proteins can be significantly improved. Computational biology tools such as Rosetta and FoldX can predict the impact of mutations on protein stability, guiding rational design. Glycosylation modification is another key consideration; N-glycosylation on the Fc domain of immunoglobulins affects their binding to the Fcγ receptor and serum half-life. Through host cell engineering or optimization of culture conditions, glycan structure can be regulated, for example, reducing the core fucose content to enhance ADCC effects or increasing sialic acid content to reduce immunogenicity.

[0008] In addition to improving the binding affinity to the target, the binding characteristics of Fc and FcRn also need to be considered. Introducing mutations such as T250Q / M428L into the immunoglobulin Fc domain can enhance the affinity for FcRn and further prolong the half-life. Meanwhile, YTE mutations (M252Y / S254T / T256E) extend the half-life by up to four times by altering pH-dependent binding characteristics. These optimizations are not isolated; a systematic evaluation of their impact on protein expression, stability, immunogenicity, and other aspects is needed to find the optimal balance through high-throughput screening and in-depth characterization.

[0009] Upstream process development focuses on optimizing cell culture conditions. Serum-free culture media with defined chemical compositions have become industry standards. Precise control of amino acids, vitamins, and trace elements can increase yield while reducing batch-to-batch variability. The application of perfusion culture technology enables cell densities of tens of millions per milliliter, significantly increasing production capacity. Fine-grained control of process parameters such as pH, dissolved oxygen, and temperature, combined with real-time metabolite monitoring and feedback regulation, simultaneously improves the expression level and quality of fusion proteins. Modern bioreactor systems also integrate advanced sensors such as Raman spectroscopy to achieve online monitoring of key quality attributes.

[0010] Downstream purification processes are crucial for ensuring product safety and efficacy. Protein A affinity chromatography is the core step in capturing Fc fusion proteins; its specific binding ability to the Fc domain of IgG immunoglobulins enables purification efficiencies exceeding 95%. Subsequent multi-step chromatography, including ion exchange, hydrophobic interactions, and molecular sieving, further removes impurities such as host cell proteins, DNA, aggregates, and charge isoforms. Virus removal steps (such as nanofiltration and low-pH incubation) are essential for ensuring the safety of the biopharmaceutical. Process-related impurities such as Protein A leakage and endotoxins need to be strictly controlled at extremely low levels (typically <1 ppm). Summary of the Invention

[0011] The purpose of this invention is to provide: A fusion protein of OTUD3 and Fc, and related technologies, to solve the technical problems of instability and easy degradation of OTUD3 protein in the prior art, or a combination thereof.

[0012] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0013] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0014] The definition of the standard chemical term can be found in the reference "Antibody Technology (Second Edition)," Science Press, 2nd edition, 2018.

[0015] Unless otherwise stated, conventional methods within the scope of the art, such as plasmid construction, protein expression, and purification, shall be used.

[0016] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0017] As used herein, the term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to that of naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as modified amino acids such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids—that is, carbon bound to hydrogen, carboxyl, amino, and R groups—such as homoserine, ortholeucine, methionine sulfoxide, and methionine methylsulfonium. These analogs have modified R groups (e.g., ortholeucine) or modified peptide backbones but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimics are compounds whose structure differs from the general chemical structure of amino acids but function in a manner similar to that of naturally occurring amino acids.

[0018] The terms “activity,” “functional activity,” or “biological activity,” or “biological property,” or “biological characteristic,” as used herein, are used interchangeably and include, but are not limited to, epitope / antigen affinity and specificity, the ability to neutralize or antagonize antigen activity in vivo or in vitro, IC50, in vivo stability of the antibody, and immunogenic properties of the antibody. Other identifiable biological properties or characteristics of antibodies known in the art include, for example, cross-reactivity (i.e., cross-reactivity with non-human homologs of the target peptide, or with other proteins or tissues), and the ability to maintain high levels of protein expression in mammalian cells. The aforementioned properties or characteristics may be observed, measured, or evaluated using techniques known in the art, including, but not limited to, ELISA, FACS, or BIACORE plasma resonance analysis, unrestricted in vitro or in vivo neutralization assays, receptor binding, production and / or secretion of cytokines or growth factors, signal transduction, and immunohistochemistry of tissue sections from various sources, including human, primate, or any other source.

[0019] As used herein, the terms “nucleic acid” or “polynucleotide” refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and their polymers in single-stranded or double-stranded form. Unless explicitly limited, the term includes nucleic acids containing analogs of known natural nucleotides that have similar binding properties to a reference nucleic acid and are metabolized in a manner similar to that of naturally occurring nucleotides (see U.S. Patent No. 8,278,036, belonging to Kariko et al., which discloses mRNA molecules with uridine replaced by pseudouridine, methods for synthesizing said mRNA molecules, and methods for delivering therapeutic proteins in vivo). Unless otherwise indicated, a particular nucleic acid sequence also implicitly includes variants of its conserved modifications (e.g., degenerate codon substitutions), alleles, orthologs, SNPs and complementary sequences, and explicitly stated sequences. Specifically, degenerate codon substitutions can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is substituted with a mixture of bases and / or deoxyinosine residues (Batzer, Nucleic Acid Res . 19: 5081 (1991); Ohtsuka, J.Biol.Chem . 260:2605-2608 (1985); Rossolini, Mol.Cell.Probes 8: 91-98 (1994)).

[0020] As used herein, the term "vector" refers to any recombinant polynucleotide construct that can be used for transformation purposes (i.e., introducing heterologous DNA into host cells). One type of vector is the "plasmid," which is a circular double-stranded DNA loop into which an additional DNA segment can be ligated. Another type of vector is the viral vector, into which an additional DNA segment can be ligated into the viral genome. Some vectors are capable of autonomous replication in the host cells they are introduced into (e.g., bacterial vectors with bacterial origins of replication and free-living mammalian vectors). After introduction into the host cell, other vectors (e.g., non-free-living mammalian vectors) integrate into the host cell's genome and thus replicate along with the host genome. Furthermore, some vectors can guide the expression of operatively linked genes. Such vectors are referred to herein as "expression vectors."

[0021] As used in this article, the term "expression vector" refers to a nucleic acid molecule capable of replicating and expressing a target gene when transformed, transfected, or transduced into host cells. Expression vectors contain one or more phenotypic selection markers and origins of replication to ensure the maintenance of the vector and to provide amplification within the host when needed.

[0022] The terms “cell” and “cell line” used herein are used interchangeably, and all such names include their descendants. The term “host cell” refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli, fungal cells such as yeast cells, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0023] As used herein, the term "pharmaceutical composition" generally refers to a formulation which is present in a form that allows for the effective biological activity of the active ingredient and does not contain any additional ingredients that would have unacceptable toxicity to the subject to which the composition will be administered. The composition is sterile.

[0024] The term "antibody fragment" refers to a molecule that, in addition to the complete antibody, contains a portion of the complete antibody and binds to the antigen bound by the complete antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab"-, Fab"-SH, F(ab")2; bisomatic antibodies; linear antibodies; single-chain antibody molecules (e.g., scFv and scFab); single-domain antibodies; and multispecific antibodies formed from antibody fragments.

[0025] As used in this article, the terms "Fc" or "Fc region," "Fc domain," or "immunoglobulin Fc domain" refer to polypeptides composed of the CH2 and CH3 domains of IgA, IgD, and IgG, or the CH2, CH3, and CH4 domains of IgE and IgM, linked by a hinge region. Although the breakdown of immunoglobulin Fc domains is variable, the Fc domain of the heavy chain of human IgG typically refers to the polypeptide segment from A231 to its carboxyl terminus. Antibody Fc is an important component of antibody molecules, belonging to the constant region (C region) of the antibody. Specifically, the antibody Fc segment is located on the two heavy chains of the antibody molecule and mainly includes the CH2 and CH3 domains (in some cases, other domains may also be included, but these two are the most important). The name Fc segment comes from its ability to crystallize. The Fc segment is primarily responsible for mediating the interaction between antibodies and immune cells, such as binding to Fc receptors (FcRs), thereby triggering a series of immune responses. These responses include, but are not limited to, phagocytosis, extracellular killing, antibody-dependent cell-mediated cytotoxicity (ADCC), and antibody-dependent cell-mediated phagocytosis (ADCP). The Fc fragment of IgG antibodies can bind to Fcγ receptors (such as FcγRI, FcγRIIa, FcγRIIc, and FcγRIIIa) on the surface of immune cells, thereby mediating immune responses such as phagocytosis and extracellular killing. Human IgG antibodies have four Fc subtypes: IgG1, IgG2, IgG3, and IgG4.

[0026] Expression vectors in genetic engineering are constructed by adding expression elements (such as promoters and terminators) to cloning vectors, enabling the target gene to be expressed in recipient cells. Expression vectors typically consist of the target gene, promoter, terminator, and marker gene. Commonly used expression vectors include plasmids, bacteriophages, and artificial chromosomes, each with its own characteristics. For example, plasmids are suitable for rapid expression and screening, bacteriophage vectors have high capacity and high stability, and artificial chromosomes can carry larger DNA fragments. When constructing expression vectors, strategies such as codon optimization must be considered to improve expression efficiency. Plasmid expression vectors are further divided into prokaryotic and eukaryotic expression vectors. Prokaryotic expression vectors, such as those from *E. coli*, have a basic backbone containing a plasmid origin of replication and an antibiotic resistance gene, along with regulatory sequences that enable gene expression, such as promoters and terminators. Eukaryotic expression vectors include yeast expression vectors and mammalian cell expression vectors. They also contain the target gene, promoter, terminator, and marker gene, but their regulatory sequences and replication mechanisms are more complex to adapt to the transcription and translation processes in eukaryotic cells.

[0027] "Affinity" refers to the strength of the binding between the antibody-binding site and the antigen epitope. This binding strength determines the antibody's ability to recognize and bind to antigens, and is one of the important parameters of antibody function. Affinity describes the tightness of the interaction between the antibody and the antigen, reflecting the strength of the binding force between them. When an antibody binds to an antigen, if the interaction force between them is strong, then the affinity of this binding is high; conversely, if the interaction force is weak, the affinity is low. The affinity of an antibody can be measured by various methods, such as biomembrane interference (BLI) technology and surface plasmon resonance (SPR) technology. These methods can quantitatively assess the binding strength between antibodies and antigens, providing important evidence for the research and development and application of antibody drugs.

[0028] To achieve the above objectives, the present invention provides the following technical solution: On one hand, the present invention provides a fusion protein of deubiquitinating enzyme and Fc, wherein the fusion protein is formed by fusing OTUD3 protein and immunoglobulin Fc domain, and the CH3 domain of the heavy chain of the immunoglobulin Fc domain is introduced with a knob mutation.

[0029] Preferably, the OTUD3 protein has the amino acid sequence shown in SEQ ID NO.1, and the immunoglobulin Fc domain has the amino acid sequence shown in SEQ ID NO.5.

[0030] Preferably, the fusion protein further comprises a tag.

[0031] Preferably, the tag is selected from one or more of the following tags: His tag, GST tag, MBP tag, FLAG tag, SUMO tag, Strep-tag II tag, Twin-Strep-tag tag, MyC tag, HA tag, AviTag tag, HaloTag tag, EGFP tag, SNAP tag, V5 tag, thioredoxin tag, GFP tag, mCherry tag, protein C tag, BCCP tag, Spot-tag tag, Isopeptag, and SpyTag.

[0032] In some specific implementations, the label is a His label and a GST label.

[0033] In some specific embodiments, the fusion protein further includes an enzyme cleavage site.

[0034] On the other hand, the present invention provides a method for preparing the above-mentioned fusion protein, wherein the fusion protein is composed of OTUD3 protein and immunoglobulin Fc domain covalently linked by a linker.

[0035] Preferably, the connector comprises one or more linker peptides.

[0036] In some specific embodiments, the linker peptide comprises the amino acid sequence shown in SEQ ID NO.7.

[0037] On the other hand, the present invention provides a recombinant protein to which at least one of the following is attached: an immunoglobulin Fc domain, serum albumin, albumin-binding polypeptide, prealbumin, carboxyl-terminal peptide, elastin-like polypeptide, cytokine, single-chain antibody, virus-like particle, ligase, kinase, prodrug activating enzyme, chemotherapeutic agent, gold nanoparticle or liposome.

[0038] On the other hand, the present invention provides a nucleic acid that encodes the above-mentioned fusion protein or the above-mentioned recombinant protein.

[0039] On the other hand, the present invention provides an expression vector containing the above-mentioned nucleic acid, or the expression vector is used to express the above-mentioned fusion protein, or the expression vector is used to express the above-mentioned recombinant protein.

[0040] Preferably, the expression vector is a eukaryotic expression vector or a prokaryotic expression vector; the eukaryotic expression vector includes any one or more of pFastBac, pEGFP-N1, pcDNA3.1, and pcDNA3.4, and the prokaryotic expression vector includes any one or more of pGEX6P1, pET28a, pET32a, pGEX-4T, pMAL-p2x, and pMAL-c2X.

[0041] In some specific embodiments, the expression vector is pGEX6P1.

[0042] On the other hand, the present invention provides a cell that includes, expresses, or secretes the above-described fusion protein; or, the cell includes the above-described recombinant protein; or, the cell includes the above-described nucleic acid; or, the cell includes the above-described expression vector.

[0043] Preferably, the cells are eukaryotic cells or prokaryotic cells; the eukaryotic cells include any one or more of yeast, filamentous fungi, insect cells, mammalian cells, or plant cells; the prokaryotic cells include any one or more of Escherichia coli, Bacillus subtilis, lactic acid bacteria, Streptomyces, Proteus mirabilis, Corynebacterium glutamicum, thermophilic bacteria, cyanobacteria, and halophilic bacteria.

[0044] In some specific embodiments, the cells are Escherichia coli.

[0045] On the other hand, the present invention provides a pharmaceutical composition comprising the above-described fusion protein, the above-described recombinant protein, the above-described nucleic acid, the above-described expression vector, and / or the above-described cells.

[0046] Preferably, the pharmaceutical composition further includes pharmaceutical excipients.

[0047] Preferably, the pharmaceutical excipients include any one or more of the following: binders, fillers, disintegrants, lubricants, preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, solubilizers, or osmotic pressure regulators.

[0048] On the other hand, the present invention provides the application of the above-described fusion protein or the above-described recombinant protein in the screening of lead compounds.

[0049] On the other hand, the present invention provides the application of the above-mentioned nucleic acid or expression vector in the screening of seed compounds.

[0050] On the other hand, the present invention provides the application of the above-mentioned cells in the screening of seed compounds.

[0051] Related sequences: Human OTUD3 full-length sequence SEQ ID NO.1: MSRKQAAKSRPGSGSRKAEAERKRDERAARRALAKERRNRPESGGGGGCEEEFVSFANQLQALGLKLREVPGDGNCLFRALGDQLEGHSRNHLKHRQETVDYMIKQREDFEPFVEDDIPFEKHVASLAKPGTFAGNDAIVAFARNHQLNVVIHQLNAPLWQIRGTEKSVRELHIAYRYGEHYDSVRRINDNSEAPAHL QTDFQMLHQDESNKREKIKTKGMDSEDDLRDEVEDAVQKVCNATGCSDFNLIVQNLEAENYNIESAIIAVLRMNQGKRNNAEENLEPSGRVLKQCGPLWEEGGSGARIFGNQGLNEGRTENNKAQASPSEENKANKNQLAKVTNKQRREQQWMEKKKRQEERHRHKALESRGSHRDNNRSEAEANTQVTLVKTFAALNI, The corresponding nucleotide sequence is SEQ ID NO.2: The immunoglobulin Fc domain sequence of human IgG1, SEQ ID NO.3: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, The corresponding nucleotide sequence is SEQ ID NO.4: ATGGACAAAACCCATACCTGCCCACCTTGCCCAGCACCAGAATTACTTGGAGGTCCCTCGGTGTTCCTGTTCCCGCCAAAGCCCAAAGATACCCTGATGATCAGCCGAACTCCAGAGGTCACGTGCGTAGTTGTGGACGTGTCACACGAAGACCCGGAAGTAAAATTCAACTGGTATGTTGACGGCGTTGAAGTGCACAATGCTAAGACAAAGCCTCGGGAGGAACAGTATAACAGTACTTATCGTGTGGTCTCCGTCCTGACAGTACTTCATCAAGATTGGCTGAACGGAAAAGAATACAAATGTAAGGTGTCCAACAAGGCCCTCCCAGCACCGATTGAGAAGACGATTAGCAAAGCCAAGGGTCAGCCGAGAGAGCCCCAGGTGTATACACTCCCACCTTCCCGCGATGAACTGACCAAGAACCAGGTCAGCCTGACGTGCTTAGTAAAGGGCTTCTACCCCAGCGATATTGCAGTAGAGTGGGAGAGCAATGGCCAGCCAGAAAATAACTACAAGACCACTCCTCCTGTGCTGGACTCGGACGGTTCTTTCTTTTTGTACTCAAAGCTCACCGTCGATAAAAGCCGCTGGCAACAGGGCAACGTGTTCTCTTGCAGTGTGATGCACGAAGCCCTGCATAATCACTATACCCAAAAGAGCTTAAGCCTCAGTCCTGGAAAA; Fc mutant knob chain sequence, SEQ ID NO.5: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, The corresponding nucleotide sequence is SEQ ID NO.6: ATGGACAAAACCCATACCTGCCCACCTTGCCCAGCACCAGAATTACTTGGAGGTCCCTCGGTGTTCCTGTTCCCGCCAAAGCCCAAAGATACCCTGATGATCAGCCGAACTCCAGAGGTCACGTGCGTAGTTGTGGACGTGTCACACGAAGACCCGGAAGTAAAATTCAACTGGTATGTTGACGGCGTTGAAGTGCACAATGCTAAGACAAAGCCTCGGGAGGAACAGTATAACAGTACTTATCGTGTGGTCTCCGTCCTGACAGTACTTCATCAAGATTGGCTGAACGGAAAAGAATACAAATGTAAGGTGTCCAACAAGGCCCTCCCAGCACCGATTGAGAAGACGATTAGCAAAGCCAAGGGTCAGCCGAGAGAGCCCCAGGTGTATACACTCCCACCTTGTCGCGATGAACTGACCAAGAACCAGGTCAGCCTGTGGTGCTTAGTAAAGGGCTTCTACCCCAGCGATATTGCAGTAGAGTGGGAGAGCAATGGCCAGCCAGAAAATAACTACAAGACCACTCCTCCTGTGCTGGACTCGGACGGTTCTTTCTTTTTGTACTCAAAGCTCACCGTCGATAAAAGCCGCTGGCAACAGGGCAACGTGTTCTCTTGCAGTGTGATGCACGAAGCCCTGCATAATCACTATACCCAAAAGAGCTTAAGCCTCAGTCCTGGAAAA; The Fc mutant knob chain and the OTUD3 linker peptide sequence, SEQ ID NO.7: GGGGSGGGGS, The corresponding nucleotide sequence is SEQ ID NO.8: GGCGGTGGGGGCAGTGGCGGCGGCGGGTCC.

[0052] The present invention has at least the following beneficial effects: 1. This invention effectively inhibits the formation of homodimers (such as OTUD3-knob-knob-OTUD3) by introducing a "knob" mutation into the CH3 domain of the fused immunoglobulin Fc domain heavy chain. The recombinant protein formed by fusing OTUD3 with the immunoglobulin Fc domain containing the "knob" not only retains the activity of the functional protein but also endows it with antibody properties, such as prolonged half-life, mediating immune effector function, and enhanced stability. After the fusion protein binds to a Protein A microarray via the immunoglobulin Fc domain, small molecule recruiters of OTUD3 can be screened using SPR high-throughput microarray analysis. Since the binding of the immunoglobulin Fc domain to Protein A can be dissociated by conventional acid washing, microarray regeneration is simple, greatly reducing costs while maintaining effectiveness. Furthermore, it allows for repeated loading of fresh OTUD3 protein, avoiding the instability and degradation problems that occur during long-term screening of full-length proteins.

[0053] 2. The purified knot chain-OTUD3 can be directly bound to the Protein A chip for SPR screening. It can be dissociated by conventional acid washing, and the chip regeneration is simple and stable. Fresh OTUD3 protein can be repeatedly loaded, avoiding the instability and easy degradation problems that occur during long-term screening of full-length OTUD3 protein. Using this method, four small molecule compounds with nM affinity for OTUD3 were successfully screened. Attached Figure Description

[0054] Figure 1 The plasmid map in Example 1 is (knob chain-OTUD3-3C site-GST-His-pGEX6P1).

[0055] Figure 2 The results of SDS-PAGE and molecular sieve analysis for the knob chain-OTUD3 purification process in Example 1 are shown; the arrows indicate the target protein.

[0056] Figure 3 The results are the knock chain-OTUD3 enzyme activity assay results from the Ub-AMC experiment in Example 2.

[0057] Figure 4The results of protein-protein conjugation on the Protein A chip in each channel were screened by Knob-OTUD3 SPR in Example 3. The repeatability and stability of conjugation and detection were verified through parallel experiments in 8 channels to ensure data reliability. For the result image of each channel, the horizontal axis is time (min), which records the entire time sequence from baseline, conjugation, binding to dissociation and regeneration, and accurately marks key nodes such as sample injection and washing. The vertical axis is the response value (RU), which reflects the change in the amount of molecules bound to the chip surface.

[0058] Figure 5 The response results of the SPR high-throughput screening of 8-mix 1 compound groups in Example 3 are divided into 4 different groups (AD). The horizontal axis represents time (min) and the vertical axis represents the response value (RU).

[0059] Figure 6 The results of the kinetic fitting of the 8-mix 1 compound groups selected in Example 3 as having good affinity are shown. The 8 groups correspond to time (min) on the x-axis and response value (RU) on the y-axis.

[0060] Figure 7 The results of SPR high-throughput screening of single small molecule compounds in Example 3 include 32 groups of compounds with high response values ​​and good kinetic fit, corresponding to two different detection batches (A and B). The horizontal axis represents time (min) and the vertical axis represents response value (RU).

[0061] Figure 8 The results are kinetic fitting results of the responses of the eight small molecule compounds with good affinity in Example 3. The x-axis represents time (min) and the y-axis represents the response value (RU).

[0062] Figure 9 The results show the kinetic fitting of the response to different concentration gradients for the affinity detection of small molecule compounds in Example 3, corresponding to 4 different small molecule compounds. The horizontal axis represents time (min) and the vertical axis represents the response value (RU). Detailed Implementation

[0063] Unless otherwise specified, all raw materials and reagents used in this invention were purchased from commercial suppliers, and experiments were conducted in accordance with the operating instructions. Unless otherwise specified, all instruments, equipment, and apparatus used in this invention are conventional instruments, equipment, and apparatus, and experiments were conducted in accordance with the operating instructions and the accompanying reagents.

[0064] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conditions are performed under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments without specified manufacturers are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention.

[0065] Data analysis and statistical analysis were performed using professional data processing software. One-way ANOVA was used for significance analysis, and P<0.05 was considered to indicate a significant difference.

[0066] Material descriptions related to embodiments of the present invention: HRV3C enzyme was prepared and purified in-house; OTUD3(FL) was prepared and purified in-house; UB-AMC was purchased from R&D Company; the small molecule compound library was purchased from the Small Molecule Platform of the Big Health Research Institute of the Hefei Comprehensive National Science Center; and the positive compound (Rolapltant) was purchased from Aladdin.

[0067] LB liquid medium (1L): Add 10g tryptone, 5g yeast extract, and 10g sodium chloride to 950mL of water. Make up to 1L; autoclave at 121℃ for 20min.

[0068] Ampicillin-resistant LB medium: Add ampicillin to LB liquid medium to a final concentration of 50 μg / mL.

[0069] Ampicillin resistance plate: Add ampicillin to LB liquid medium at a final concentration of 50 μg / mL and agar at a final concentration of 1.2%.

[0070] Example 1: Preparation of the knob chain-OTUD3 fusion protein (1) Vector construction: Based on the optimized nucleic acid sequence of Fc (human) in the prokaryotic expression system (SEQ ID NO.4), mutant primers were synthesized using chemical synthesis methods. The nucleic acid sequence of the Fc mutant knob chain (SEQ ID NO.6) was amplified in vitro. Then, using YEASEN's homologous recombinase (Hieff Clone® Universal One Step Cloning Kit), the OTUD3 nucleic acid fragment, GST fragment, His fragment, and 3C restriction site were recombined in vitro and inserted downstream of the transcription promoter in the prokaryotic expression system pGEX6P1 (existing in the laboratory), constructing the knob chain-OTUD3-3C site-GST-His (e.g., Figure 1(As shown). The OTUD3 nucleic acid sequence in the cDNA is (SEQ ID NO.2), and the knot chain region contains the hinge, CH2, and CH3 regions of the human IgG heavy chain. The expressed protein knot chain-OTUD3-3C site-GST-His is present in the cytoplasm of *E. coli*. Because the OTUD3 protein loses its enzymatic activity in a low pH environment, one-step purification using Protein A agarose gel is not suitable. Instead, preliminary purification is performed using Ni packing material, followed by removal of nucleic acids via a heparin column. After HRV3C digestion, the protein is then purified using Ni packing material and gel filtration chromatography to obtain a high-purity and homogeneous knot chain-OTUD3 protein.

[0071] (2) Amplification of plasmid vector: The DNA sequence containing knob chain-OTUD3-3C site-GST-His was cloned into pGEX6P1 plasmid, and then heat-shocked transformation was performed in DH5α competent cells. Single colonies were formed by culturing on plates containing ampicillin. Single colonies were picked and gradually expanded to 100 mL. After overnight culture at 37℃ and 220 rpm, the cells were centrifuged and plasmids were extracted to obtain high-concentration plasmids.

[0072] (3) DNA sequence identification and verification: Sanger sequencing, a first-generation sequencing technology, was used to sequence the recombinant plasmid containing the target gene. The sequencing results were compared with the knob chain-OTUD3-3C site-GST-His sequence and were accurate.

[0073] (4) Expression of the knob chain-OTUD3-3C site-GST-His sequence fusion protein: To establish Bl21(DE3) stably expressing the knob chain-OTUD3-3C site-GST-His sequence, heat shock transformation was used to transform the recombinant target plasmid into Bl21(DE3). The cells were cultured overnight on plates containing ampicillin. Single colonies were picked and cultured overnight in 100 mL of LB broth containing ampicillin. The next day, large-volume fermentation was performed. The seed culture from the overnight culture was inoculated into a 5 L culture flask at a ratio of 1:100. The flask was incubated at 37°C with shaking for approximately 3 hours. When the OD600 value of the cells reached approximately 0.8, the cells were pre-cooled to 18°C, and IPTG at a final concentration of 0.3 mM was inoculated. Induction was performed overnight for approximately 20 hours, and the cells were collected by centrifugation.

[0074] (5) Purification and preparation of the knob chain-OTUD3-3C site-GST-His fusion protein: 5.1 HisTrap HP (5 mL) affinity: Take approximately 6 L of fermented knob chain-OTUD3-3C site-GST-His Bl21(DE3) bacterial block and resuspend it in 300 mL of lysis buffer (40 mM Tris-HCl, 500 mM NaCl, pH 7.4, 1 mM PMSF, 10 mM Imidazole, 2 mM DTT, 100 mM Arginine, 5% Glycerol). Sonicate at 240 W for 15 min. Centrifuge the cell lysate at 16000 g for 30 min, collect the supernatant, and filter through a 0.45 μm filter. The supernatant was collected and incubated with equilibrated Ni-NTA packing material for 2 hours. The sample was washed with buffer (40 mM Tris-HCl, 500 mM NaCl, pH 7.4, 10 mM Imidazole, 2 mM DTT, 100 mM Arginine, 5% Glycerol), and finally eluted with 500 mM Imidazole to remove the target protein. The sample fractions were analyzed by SDS-PAGE and Coomassie Brilliant Blue staining to assess yield and purification rate. The results are shown below. Figure 2 Lane A, lane 5 shows that the purity of the target protein is around 75%.

[0075] 5.2 Heparin Column (5 mL) Affinity: The NaCl concentration was diluted to 200 mM by adding an appropriate amount of dilution buffer (40 mM Tris-HCl, pH 7.4) to the eluted sample from the previous step. The sample was then purified using a heparin column. After equilibration with 5 column volumes of equilibration buffer (40 mM Tris-HCl, 150 mM NaCl, 5% Glycerol, pH 7.4), the sample was placed on ice and the column was loaded with a flow rate of 1 mL / min. Protein elution was performed using an AKTA protein purifier. The tubing was first flushed with equilibration buffer, and then 20 column volumes were set. Elution was performed using a linear gradient of 0-100% elution buffer (40 mM Tris-HCl, 2 M NaCl, 5% Glycerol, pH 7.4). The sample fractions were analyzed by SDS-PAGE and Coomassie Brilliant Blue staining to evaluate yield and purification rate. Results are as follows: Figure 2 In lane B, lanes 3-5 show that the purity of the target protein is around 80%.

[0076] 5.3 HRV3C protease digestion: After removing the nucleic acids bound to the target protein using Heparin affinity, the collected target protein was added to the knob chain-OTUD3-3C site-GST-His protein at a protein content ratio of 10:1. GST-HRV3C protease at a concentration of 1 mg / mL was added, and DTT was added to a final concentration of 1 mM. The reaction solution was incubated overnight at 4°C for digestion. The results are as follows... Figure 2 In the C, lane 1 is before enzyme digestion, and lane 2 is after enzyme digestion.

[0077] 5.4. HisTrap HP (2 mL) affinity after enzyme digestion: Protein samples after enzyme digestion were purified using an AKTA Pure protein purifier. Knob-OTUD3 digested with enzyme was purified using HisTrap (2 mL). The column volume was equilibrated with 3 times the column volume of equilibration buffer (40 mM Tris-HCl, 500 mM NaCl, pH 7.4, 2 mM DTT, 100 mM Arginine, 5% Glycerol). Then, a 20 mL column volume was set, and elution was performed using a linear gradient of 0-100% elution buffer (40 mM Tris-HCl, 500 mM NaCl, pH 7.4, 2 mM DTT, 100 mM Arginine, 500 mM Imidazole, 5% Glycerol). The sample fractions were analyzed by SDS-PAGE and Coomassie Brilliant Blue staining to evaluate yield and purification rate. Results are as follows: Figure 2 C, the target protein corresponding to lanes 8-10 was collected, showing a purity of about 85%.

[0078] 5.5 Gel Filtration Chromatography: The target protein was collected from the Histrap column using an ultrafiltration tube with a molecular cutoff of 50 kDa, concentrating to approximately 500 μL. Separation and purification were then performed using a Superdex 200 gel filtration chromatography column. Before treatment: A syringe was taken from the column inlet wall, connected to AKTA, and washed with buffer (40 mM HEPES, 150 mM NaCl, pH 7.4) for 1.5 column volumes. After column equilibration, 500 μL of Knob-OTUD3 was added to a 1 mL sample loop and passed through the Superdex 200 column at a flow rate of 0.5 mL / min. The sample flowed through the column elution buffer until the target protein was detected, and the protein was collected. The sample fractions were analyzed by SDS-PAGE and Coomassie Brilliant Blue staining to evaluate yield and purification rate. The results are as follows: Figure 2The target protein was collected from lanes D and E, corresponding to the molecular sieve peaks in lanes 2-7, with a purity of over 85%. Finally, the protein concentration was concentrated to 3 mg / mL using an ultrafiltration tube with a molecular cutoff of 50 kDa. Approximately 4 mg of Knob-OTUD3 protein was obtained from the collected cells in 6 L of culture medium, aliquoted, and stored at -80°C for later use.

[0079] Example 2: Assay of purified Knob chain-OTUD3 protease activity Ub-AMC assay for protein activity: Two batches of positive control protein (OTUD3(FL)) and target protein (Knob-OTUD3) were diluted in reaction buffer (40 mM Tris-HCl, 150 mM NaCl, 0.1 mg / mL BSA, 1% glycerol, pH 7.4) to achieve a final concentration of 500 nM for OTUD3(FL) and a final concentration range of 500 nM-3.8 μM for Knob-OTUD3. The mixture was incubated on ice for 20 min. The above mixture was then added to 10 μL per well of a 384-well plate. Ub-AMC was diluted in reaction buffer to a final concentration of 1 μM and 10 μL was added to each sample. Readings were taken using a microplate reader, with excitation wavelength set to 345 nm and emission wavelength to 445 nm, every 30 s for a total of 30 min. Results are as follows: Figure 3 As shown, at the same concentration, the activity of Knob-OTUD3 is about 1 / 16 of that of full-length untagged OTUD3 (FL). Although the activity is weaker, it still indicates that this fusion protein can be used for SPR screening of non-inhibitory small molecule compounds.

[0080] Example 3: SPR screening of small molecule compounds that can bind to Knob-OTUD3 (1) Screening of small molecule compounds binding to Knob-OTUD3: Take the stock of Knob-OTUD3 protein solution (4.04 mg / ml) and dilute it with running buffer to make the final concentration of protein 7 μg / mL; mix 8 small molecule compounds with a concentration of 10 mM each from the purchased compound library, and dilute each group of small molecule compounds with running buffer to a final concentration of 20 uM; then put the ProteinA chip into Biacore TM Insert the 8K chip chamber and connect the tubing labeled Inlet A into the prepared buffer solution (20 mM HEPES pH 7.4, 150 mM NaCl, 5% DMSO, 0.1% Twwen 20). Before starting the procedure, run the Change solutions option to flush the machine flow path system and the chip. Configure Biacore. TMThe sample chamber temperature of the 8K instrument is 10 ℃, and the temperature of the Protein A chip, i.e., the screening temperature, is 25 ℃. Open Biacore. TM The 8K program control interface was used to select the system's low molecular weight protein (LMW) multi-cycle coupling screening program and edit the method. The experimental workflow was set as follows: startup, buffer washing, solvent correction, positive control analysis, negative control analysis, and LMW screening. Each workflow included protein coupling, analyte flow, analyte elution (under 50% DMSO), and chip regeneration (under 10 mM Glycine-HCl pH 1.5). The LMW screening was set to run several cycles. Eight detection channels were selected, each including a protein coupling channel and a reference channel (i.e., dual channels). The detection parameters for solvent correction were set as follows: injection time 60 s, flow rate 30 μL / min. The detection parameters for LMW screening were set as follows: protein coupling time 60 s, small molecule compound binding time 60 s, small molecule compound dissociation time 60 s, chip regeneration time 60 s, and flow rate 30 μL / min. During the screening process, fresh Knob-OTUD3 protein was added to the sample container every 2 hours. The program was run to obtain the response signal spectrum, which was then analyzed using Biacore. TM 8K analysis software is used to analyze and process the data. Figure 4 The results of screening each channel of Knob-OTUD3 SPR for protein-coupled proteinA chip levels are shown in the figure. The results show that after running multiple rounds of screening, the protein-coupled chip levels remain stable, indicating that the chip regeneration is good. Figure 5 The graph shows the response results of SPR high-throughput screening of small molecule compounds. Figure 6 The kinetic fitting results are for the selected 8-to-1 compound groups considered to have good affinity. The 32 compound groups with high response values ​​and good kinetic fits were selected. Figure 7 Following the same method described above, individual compounds were screened, ultimately yielding eight small molecule compounds with high response values ​​and good kinetic fit. Figure 8 Then, affinity tests were conducted on them.

[0081] (2) Affinity detection of Knob-OTUD3 with small molecule compounds: The small molecule compounds were diluted with buffer (20 mM HEPES pH 7.4, 150 mM NaCl, 5% DMSO, 0.1% Twwen 20) to prepare concentrations ranging from 0.19 to 25 μM. 200 μL of each concentration was transferred to a round-bottom 96-well plate, and the 96-well plate was sealed with a sealing membrane for later use. Biacore was selected. TMThe kinetics / affinity program for the 8K system uses experimental buffer as the mobile phase to flush the machine flow path system, and sets up Biacore. TM The sample chamber temperature of the 8K instrument was 10 ℃, and the temperature of the ProteinA chip, i.e., the screening temperature, was 25 ℃. The experimental procedure was set as startup, solvent calibration, and kinetics / affinity detection, with dual-channel detection using channel 2 (Knob-OTUD3 protein coupling channel) and channel 1 (reference channel). Startup was set to 8 cycles; the detection parameters for solvent calibration were: injection time 60 s, flow rate 30 μL / min; the detection parameters for kinetics / affinity were: protein coupling time 60 s, small molecule compound binding time 60 s, small molecule compound dissociation time 60 s, chip regeneration time 60 s, and flow rate 30 μL / min. During the screening process, fresh Knob-OTUD3 protein was added to the sample chamber every 2 hours. The program was run to obtain the interaction mode between the small molecule compound and Knob-OTUD3. The affinity constant (KD) was obtained by fitting the affinity using a steady-state affinity model (1:1). Finally, four small molecule compounds with nM affinity for Knob-OTUD3 were obtained through detection. Figure 9 ).

[0082] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A fusion protein of a deubiquitinating enzyme and Fc, characterized in that, The fusion protein is formed by fusing OTUD3 protein and immunoglobulin Fc domain, and the CH3 domain of the heavy chain of the immunoglobulin Fc domain is mutated with knob; the OTUD3 protein has the amino acid sequence shown in SEQ ID NO.1, the immunoglobulin Fc domain has the amino acid sequence shown in SEQ ID NO.5, and the immunoglobulin Fc domain is located at the N-terminus of the OTUD3 protein.

2. A fusion protein, characterized in that, The fusion protein consists of the fusion protein as described in claim 1 and a tag.

3. The fusion protein according to claim 2, characterized in that, The tag is selected from one or more of the following tags: His tag, GST tag, MBP tag, FLAG tag, SUMO tag, Strep-tag II tag, Twin-Strep-tag tag, MyC tag, HA tag, AviTag tag, HaloTag tag, EGFP tag, SNAP tag, V5 tag, thioredoxin tag, GFP tag, mCherry tag, protein C tag, BCCP tag, Spot-tag tag, Isopeptag, and SpyTag.

4. The fusion protein according to claim 3, characterized in that, The tags are His and GST.

5. The fusion protein according to any one of claims 1-4, characterized in that, The OTUD3 protein and the immunoglobulin Fc domain are covalently linked by a linker.

6. The fusion protein according to claim 5, characterized in that, The connector contains one or more connecting peptides.

7. The fusion protein according to claim 6, characterized in that, The linker peptide has the amino acid sequence shown in SEQ ID NO.

7.

8. A nucleic acid, characterized in that, The nucleic acid encodes the fusion protein according to any one of claims 1-7.

9. An expression carrier, characterized in that, The expression vector contains the nucleic acid of claim 8, or the expression vector is used to express the fusion protein of any one of claims 1-7.

10. The expression vector according to claim 9, characterized in that, The expression vector is a eukaryotic expression vector or a prokaryotic expression vector; the eukaryotic expression vector is any one or more of pFastBac, pEGFP-N1, pcDNA3.1, and pcDNA3.4, and the prokaryotic expression vector is any one or more of pGEX6P1, pET28a, pET32a, pGEX-4T, pMAL-p2x, and pMAL-c2X.

11. The expression vector according to claim 10, characterized in that, The expression vector is pGEX6P1.

12. A cell, characterized in that, The cell comprises, expresses, or secretes the fusion protein according to any one of claims 1-7; or, the cell comprises the nucleic acid according to claim 8; or, the cell comprises the expression vector according to any one of claims 9-11.

13. The cell according to claim 12, characterized in that, The cells are eukaryotic or prokaryotic cells; the eukaryotic cells are any one or more of yeast, filamentous fungi, insect cells, mammalian cells, or plant cells; the prokaryotic cells are any one or more of Escherichia coli, Bacillus subtilis, lactic acid bacteria, Streptomyces, Proteus mirabilis, Corynebacterium glutamicum, thermophilic bacteria, cyanobacteria, and halophilic bacteria.

14. The cell according to claim 13, characterized in that, The cells were Escherichia coli.

15. The use of the fusion protein according to any one of claims 1-7 in screening compounds that bind to OTUD3.

16. The use of the nucleic acid of claim 8 or the expression vector of any one of claims 9-11 in screening compounds that bind to OTUD3.

17. Use of the cells according to any one of claims 12-14 in screening compounds that bind to OTUD3.

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