Fusion proteins of hyperstable intrabodies targeting pdia3 and uses thereof

Abstract

The application discloses a kind of fusion protein of ultra-stable intracellular antibody targeting PDIA3 and application thereof.The fusion protein includes antigen binding peptide and intracellular stabilizing peptide for targeting PDIA3;The antigen binding peptide includes heavy chain variable region and light chain variable region.The application enables antibody to exist stably in cell and be specifically located in endoplasmic reticulum by optimizing design intracellular stabilizing peptide and signal peptide sequence.The fusion protein can effectively target and combine PDIA3 protein, significantly inhibit the expression level of human coronavirus and various capsid virus S and shell protein in cell, can be regulated endoplasmic reticulum stress by protein-protein interaction on host cell ER and thus promote its degradation, thereby effectively reduce the infection ability of virus.

Description

[0001] This application claims priority to Chinese Patent Application No. 2025101845592, filed on February 19, 2025, and Chinese Patent Application No. 2025119578327, filed on December 23, 2025. The full text of the aforementioned Chinese patent applications is incorporated herein by reference. Technical Field

[0002] This invention relates to the field of biomedical technology, specifically to a fusion protein of an ultrastable intracellular antibody targeting PDIA3 and its applications. Background Technology

[0003] Viral infections pose a significant challenge to global health, with emerging and re-emerging viruses constantly threatening public health (1). Viruses are intracellular pathogens that rely entirely on host cell mechanisms for replication and transmission (2). Throughout the viral life cycle, host factors—including proteins, enzymes, and cellular pathways—play an indispensable role in facilitating viral entry, genome replication, protein synthesis, and viral particle assembly (3). In these stages, viral replication is particularly dependent on the hijacking of host factors, as viruses lack the mechanisms required to independently replicate their genome or synthesize proteins (4). This close dependence on host cell components makes these factors attractive targets for antiviral interventions.

[0004] Host factors involved in viral replication include RNA-binding proteins, transcription factors, molecular chaperones, and components of protein synthesis and folding mechanisms (5). These factors are often co-utilized by a variety of viruses, ranging from RNA viruses (such as influenza virus, HIV, and SARS-CoV-2) to DNA viruses (such as herpesvirus and adenovirus) (6). For example, host molecular chaperones and proteases assist in the proper folding and processing of viral proteins, ensuring the generation of functional viral components (7). Because these host factors are often shared across multiple viral families, targeting them has the potential for broad-spectrum antiviral effects, which is a key advantage in combating diverse and rapidly evolving viral pathogens (8).

[0005] The importance of targeting host factors lies in their conservation and stability compared to viral proteins. Viral genomes, especially those of RNA viruses, are highly variable, capable of rapid evolution and the emergence of drug-resistant variants (9). In contrast, host factors are genetically more stable and less likely to develop resistance, making them more reliable targets in long-term treatment strategies (10). Furthermore, broad-spectrum antiviral drugs that disrupt host factors essential for viral replication can provide a first line of defense against novel or emerging viruses for which specific antiviral drugs are not yet available (11).

[0006] ERp57, also known as PDIA3 or GRP58, is a multifunctional protein disulfide isomerase (PDI) that plays a crucial role in protein folding, quality control, and redox regulation within the endoplasmic reticulum (ER). As a member of the sulfoxide-reduction protein superfamily, ERp57 possesses a sulfoxide-reduction protein domain, enabling it to catalyze the formation, reduction, and isomerization of disulfide bonds in nascent proteins (12). ERp57 is involved in the life cycle of various viruses, including influenza virus (13), hepatitis C virus (HCV) (14), and human immunodeficiency virus (HIV) (15). In these viruses, ERp57 interacts with viral proteins or regulates host pathways essential for viral replication.

[0007] To date, approved antibody therapies primarily target extracellular antigens, which is related to the properties of antibodies. The cell membrane is composed of a phospholipid bilayer, whose structure forms an effective barrier against large molecules such as antibodies. Previous research by the inventors has reported a method for generating ultra-stable cytoplasmic antibodies (STAND) (16), making it possible to target intracellular targets with antibody molecules.

[0008] Human rhinoviruses are among the most common respiratory pathogens in humans, belonging to the Picornaviridae family, and are a major cause of the common cold. Human rhinovirus type 14 is particularly common clinically, causing upper respiratory tract inflammation and serious complications in certain populations, such as those with asthma or chronic obstructive pulmonary disease. Currently, no specific antiviral drugs for human rhinoviruses have been approved for marketing, and clinical treatment remains primarily symptomatic and supportive. Existing technologies have explored methods such as RNA interference, neutralizing antibodies, or small molecule inhibitors to inhibit human rhinovirus replication. For example, some small molecule compounds, such as DHM, have been reported to inhibit viral replication, but their effects are limited and they may exhibit cytotoxicity. Furthermore, protein-based antiviral strategies, such as using engineered proteins to interfere with the viral life cycle, are still in the exploratory stage, but still suffer from problems such as low target specificity, poor expression stability, or high cytotoxicity. Therefore, there is an urgent need in this field to develop novel, highly effective, and low-toxicity antiviral agents, especially molecules that can specifically inhibit viral replication and reduce cell damage caused by viral infection. Summary of the Invention

[0009] The technical problem this invention aims to solve is the lack of existing technologies that design and utilize intracellular antibodies to target and regulate the endoplasmic reticulum stress molecular chaperone PDIA3. This invention provides a fusion protein of an ultrastable intracellular antibody targeting PDIA3 and its applications. The fusion protein comprises, from N-terminus to C-terminus, an intracellular stabilizing peptide (ISP), an endoplasmic reticulum signal peptide (SP), an anti-PDIA3 scFv, an HA tag, and an endoplasmic reticulum retention signal (ER). Alternatively, the fusion protein comprises, from N-terminus to C-terminus, an intracellular stabilizing peptide (ISP), an anti-PDIA3 scFv, and an HA tag. The fusion protein of this invention can exist stably within cells and can specifically locate in the endoplasmic reticulum to bind to PDIA3, thereby inhibiting its function in assisting viral protein folding.

[0010] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0011] A first aspect of the present invention provides a fusion protein comprising an antigen-binding peptide targeting PDIA3 and an intracellular stabilizing peptide; The antigen-binding peptide comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region are selected from any one of the following: (1) The heavy chain variable region comprises HCDR1 as shown in SEQ ID NO: 21, HCDR2 as shown in SEQ ID NO: 22, and HCDR3 as shown in SEQ ID NO: 23; the light chain variable region comprises LCDR1 as shown in SEQ ID NO: 25, LCDR2 as shown in SEQ ID NO: 26, and LCDR3 as shown in SEQ ID NO: 27; (2) The heavy chain variable region comprises HCDR1 with an amino acid sequence as shown in SEQ ID NO: 29, HCDR2 with an amino acid sequence as shown in SEQ ID NO: 30, and HCDR3 with an amino acid sequence as shown in SEQ ID NO: 31; the light chain variable region comprises LCDR1 with an amino acid sequence as shown in SEQ ID NO: 33, LCDR2 with an amino acid sequence as shown in SEQ ID NO: 34, and LCDR3 with an amino acid sequence as shown in SEQ ID NO: 35; (3) The heavy chain variable region comprises HCDR1 as shown in SEQ ID NO: 37, HCDR2 as shown in SEQ ID NO: 38, and HCDR3 as shown in SEQ ID NO: 39; the light chain variable region comprises LCDR1 as shown in SEQ ID NO: 41, LCDR2 as shown in SEQ ID NO: 42, and LCDR3 as shown in SEQ ID NO: 43; (4) The heavy chain variable region comprises HCDR1 as shown in SEQ ID NO: 45, HCDR2 as shown in SEQ ID NO: 46, and HCDR3 as shown in SEQ ID NO: 47; the light chain variable region comprises LCDR1 as shown in SEQ ID NO: 49, LCDR2 as shown in SEQ ID NO: 34, and LCDR3 as shown in SEQ ID NO: 35; (5) The heavy chain variable region comprises HCDR1 as shown in SEQ ID NO: 21, HCDR2 as shown in SEQ ID NO: 22, and HCDR3 as shown in SEQ ID NO: 51; the light chain variable region comprises LCDR1 as shown in SEQ ID NO: 53, LCDR2 as shown in SEQ ID NO: 54, and LCDR3 as shown in SEQ ID NO: 55; (6) The heavy chain variable region comprises HCDR1 as shown in SEQ ID NO: 29, HCDR2 as shown in SEQ ID NO: 30, and HCDR3 as shown in SEQ ID NO: 57; the light chain variable region comprises LCDR1 as shown in SEQ ID NO: 59, LCDR2 as shown in SEQ ID NO: 60, and LCDR3 as shown in SEQ ID NO: 61; (7) The heavy chain variable region comprises HCDR1 with an amino acid sequence as shown in SEQ ID NO: 63, HCDR2 with an amino acid sequence as shown in SEQ ID NO: 64, and HCDR3 with an amino acid sequence as shown in SEQ ID NO: 65; the light chain variable region comprises LCDR1 with an amino acid sequence as shown in SEQ ID NO: 67, LCDR2 with an amino acid sequence as shown in SEQ ID NO: 68, and LCDR3 with an amino acid sequence as shown in SEQ ID NO: 69; (8) The heavy chain variable region comprises HCDR1 as shown in SEQ ID NO: 71, HCDR2 as shown in SEQ ID NO: 72, and HCDR3 as shown in SEQ ID NO: 73; the light chain variable region comprises LCDR1 as shown in SEQ ID NO: 75, LCDR2 as shown in SEQ ID NO: 34, and LCDR3 as shown in SEQ ID NO: 76; (9) The heavy chain variable region comprises HCDR1 with an amino acid sequence as shown in SEQ ID NO: 78, HCDR2 with an amino acid sequence as shown in SEQ ID NO: 79, and HCDR3 with an amino acid sequence as shown in SEQ ID NO: 80; the light chain variable region comprises LCDR1 with an amino acid sequence as shown in SEQ ID NO: 49, LCDR2 with an amino acid sequence as shown in SEQ ID NO: 82, and LCDR3 with an amino acid sequence as shown in SEQ ID NO: 35; The fusion protein specifically binds to PDIA3.

[0012] In this invention, the CDR is defined according to the Kabat system.

[0013] In some embodiments, the intracellular stabilizing peptide is located at the N-terminus or C-terminus of the antigen-binding peptide.

[0014] In some embodiments, the amino acid sequence of the intracellular stable peptide is as shown in any one of SEQ ID NO: 1-5 or SEQ ID NO: 89-90.

[0015] In this invention, the intracellular stabilizing peptides described, for example, in International Patent Application WO2019 / 004213, the entire contents of which are incorporated herein by reference.

[0016] In some embodiments, the heavy chain variable region and the light chain variable region of the antigen-binding peptide are selected from any one of the following: (1) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 24 or having at least 80% sequence identity with SEQ ID NO: 24; the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 28 or having at least 80% sequence identity with SEQ ID NO: 28; (2) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 32 or having at least 80% sequence identity with SEQ ID NO: 32; the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 36 or having at least 80% sequence identity with SEQ ID NO: 36; (3) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 40 or having at least 80% sequence identity with SEQ ID NO: 40; the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 44 or having at least 80% sequence identity with SEQ ID NO: 44; (4) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 48 or having at least 80% sequence identity with SEQ ID NO: 48; the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 50 or having at least 80% sequence identity with SEQ ID NO: 50. (5) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 52 or having at least 80% sequence identity with SEQ ID NO: 52; the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 56 or having at least 80% sequence identity with SEQ ID NO: 56; (6) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 58 or having at least 80% sequence identity with SEQ ID NO: 58; the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 62 or having at least 80% sequence identity with SEQ ID NO: 62; (7) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 66 or having at least 80% sequence identity with SEQ ID NO: 66; the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 70 or having at least 80% sequence identity with SEQ ID NO: 70. (8) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 74 or having at least 80% sequence identity with SEQ ID NO: 74; the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 77 or having at least 80% sequence identity with SEQ ID NO: 77; (9) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 81 or having at least 80% sequence identity with SEQ ID NO: 81; the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 83 or having at least 80% sequence identity with SEQ ID NO: 83.

[0017] In some embodiments, the heavy chain variable region and the light chain variable region of the antigen-binding peptide are linked by a linker; the structure of the antigen-binding peptide from the N-terminus to the C-terminus is: heavy chain variable region-linker-light chain variable region; or, light chain variable region-linker-heavy chain variable region.

[0018] In some embodiments, the linker comprises an amino acid sequence as shown in any of SEQ ID NO: 84-88.

[0019] In some embodiments, the antigen-binding peptide comprises an amino acid sequence as shown in any of SEQ ID NO: 9-17 or having at least 80% sequence identity with any of SEQ ID NO: 9-17.

[0020] In some embodiments, the fusion protein further comprises an endoplasmic reticulum signal peptide, wherein one end of the intracellular stabilizing peptide is linked to the antigen-binding peptide and the other end is linked to the endoplasmic reticulum signal peptide.

[0021] In some embodiments, the endoplasmic reticulum signal peptide has an amino acid sequence as shown in SEQ ID NO: 6.

[0022] In some embodiments, the fusion protein further includes an HA tag and an endoplasmic reticulum (ER) residency signal; the end of the antigen-binding peptide not connected to the intracellular stabilizing peptide is connected to a tandem HA tag and ER residency signal.

[0023] In some embodiments, the HA tag has an amino acid sequence as shown in SEQ ID NO: 7, and the endoplasmic reticulum residency signal has an amino acid sequence as shown in SEQ ID NO: 8.

[0024] In some embodiments, the fusion protein comprises an amino acid sequence as shown in any of SEQ ID NO: 18-20 or having at least 80% sequence identity with any of SEQ ID NO: 18-20.

[0025] A second aspect of the present invention provides an antibody or antigen-binding fragment targeting PDIA3, the antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region as described in the first aspect.

[0026] A third aspect of the invention provides a polynucleotide that encodes a fusion protein as described in the first aspect or an antibody or antigen-binding fragment as described in the second aspect.

[0027] A fourth aspect of the present invention provides a recombinant expression vector comprising the polynucleotides described in the third aspect.

[0028] A fifth aspect of the present invention provides a recombinant cell expressing a fusion protein as described in the first aspect or an antibody or antigen-binding fragment as described in the second aspect, or the recombinant cell comprising a recombinant expression vector as described in the fourth aspect.

[0029] A sixth aspect of the present invention provides a method for preparing a fusion protein or an antibody or antigen-binding fragment targeting PDIA3, the method comprising culturing recombinant cells as described in the fifth aspect.

[0030] A seventh aspect of the present invention provides a pharmaceutical composition comprising a fusion protein as described in the first aspect, an antibody or antigen-binding fragment as described in the second aspect, a recombinant expression vector as described in the fourth aspect, and / or a recombinant cell as described in the fifth aspect, as well as a pharmaceutically acceptable carrier and / or excipients.

[0031] The eighth aspect of the present invention provides a fusion protein as described in the first aspect, an antibody or antigen-binding fragment as described in the second aspect, a recombinant expression vector as described in the fourth aspect, a recombinant cell as described in the fifth aspect, or a pharmaceutical composition as described in the seventh aspect for treating and / or preventing various capsid virus infections, including human coronavirus infection, retrovirus infection, rhinovirus infection, or influenza virus infection.

[0032] In some embodiments, the genetic material of the capsid virus is DNA or RNA.

[0033] In some implementations, the human coronavirus infection is selected from SARS-CoV, MERS-CoV, SARS-CoV-2 and their variants.

[0034] In some implementations, the retroviral infection is HIV infection.

[0035] In some embodiments, the rhinovirus infection is selected from HRV-A, HRV-B, and HRV-C virus infections, such as HRV-1, HRV-2, HRV-3, HRV-14, and HRV-16 virus infections.

[0036] In some implementations, the influenza virus infection is an influenza A virus infection.

[0037] The ninth aspect of the present invention provides the use of a fusion protein as described in the first aspect, an antibody or antigen-binding fragment as described in the second aspect, a recombinant expression vector as described in the fourth aspect, a recombinant cell as described in the fifth aspect, or a pharmaceutical composition as described in the seventh aspect in the preparation of a medicament for treating and / or preventing various capsid virus infections, including coronavirus infection, retrovirus infection, rhinovirus infection, or influenza virus infection.

[0038] In some embodiments, the genetic material of the capsid virus is DNA or RNA.

[0039] In some implementations, the human coronavirus infection is selected from SARS-CoV, MERS-CoV, SARS-CoV-2 and their variants.

[0040] In some implementations, the retroviral infection is HIV infection.

[0041] In some embodiments, the rhinovirus infection is selected from HRV-A, HRV-B, and HRV-C virus infections, such as HRV-1, HRV-2, HRV-3, HRV-14, and HRV-16 virus infections.

[0042] In some implementations, the influenza virus infection is an influenza A virus infection.

[0043] The tenth aspect of the present invention provides a method for treating and / or preventing various capsid virus infections, including human coronavirus infection, retrovirus infection, rhinovirus infection, or influenza virus infection, the method comprising administering an effective amount of a PDIA3 binder to a subject in need; the PDIA3 binder inhibits the replication of viral capsid proteins, such as coronavirus S protein, in host cells by specifically binding to PDIA3 intracellularly.

[0044] In some embodiments, the PDIA3 binder is selected from fusion proteins as described in the first aspect of the invention, antibody or antigen-binding fragments as described in the second aspect of the invention, recombinant expression vectors as described in the fourth aspect of the invention, recombinant cells as described in the fifth aspect of the invention, and pharmaceutical compositions as described in the seventh aspect of the invention.

[0045] In some embodiments, the genetic material of the capsid virus is DNA or RNA.

[0046] In some implementations, the human coronavirus infection is selected from SARS-CoV, MERS-CoV, SARS-CoV-2 and their variants.

[0047] In some implementations, the retroviral infection is HIV infection.

[0048] In some embodiments, the rhinovirus infection is selected from HRV-A, HRV-B, and HRV-C virus infections, such as HRV-1, HRV-2, HRV-3, HRV-14, and HRV-16 virus infections.

[0049] In some implementations, the influenza virus infection is an influenza A virus infection.

[0050] The eleventh aspect of the present invention provides a method for inhibiting the protein folding or assembly of capsid viruses, the method comprising introducing a PDIA binder into the host cells of the capsid virus, the PDIA binder disrupting the cytoplasmic stress state of the host cells through protein-protein interactions, reducing the protein folding or assembly of capsid viruses, and decreasing the infectivity of folded or assembled capsid virus proteins.

[0051] In some implementations, the method is in vivo or in vitro.

[0052] In some embodiments, the PDIA3 binder is selected from the fusion protein as described in the first aspect, the antibody or antigen-binding fragment as described in the second aspect, the recombinant expression vector as described in the fourth aspect, the recombinant cell as described in the fifth aspect, and the pharmaceutical composition as described in the seventh aspect.

[0053] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0054] The reagents and raw materials used in this invention are all commercially available.

[0055] The positive and progressive effects of this invention are as follows: The fusion protein of this invention, through optimized intracellular stabilizing peptide and signal peptide sequences, enables antibodies to exist stably within cells and specifically locate in the endoplasmic reticulum, solving the technical problem that conventional antibodies cannot function within cells. It can effectively target and bind to the PDIA3 protein, significantly inhibiting the expression levels of S proteins such as SARS-CoV, MERS-CoV, and SARS-CoV-2 in cells, with experimental evidence showing a reduction of over 90%. Compared to treatment strategies directly targeting the virus, this invention, by targeting the host factor PDIA3, has broad-spectrum activity and a lower risk of drug resistance, providing a new technical route for antiviral therapy. Experiments regulating endoplasmic reticulum stress response and proteasome degradation pathways demonstrate that the fusion protein of this invention can effectively reduce viral infectivity by inhibiting the replication of S proteins within host cells.

[0056] Furthermore, the fusion protein of this invention significantly inhibits virus-induced apoptosis, including that induced by rhinovirus, efficiently suppresses viral replication, and effectively blocks viral amplification in host cells; its endoplasmic reticulum-independent localization broadens its potential application in different cellular compartments. It shows promising development prospects. Attached Figure Description

[0057] Figure 1 This is a graph showing the ELISA results for detecting the binding ability of the antibody to PDIA3.

[0058] Figure 2 This is a schematic diagram of the scFv structure incorporating various peptide tags.

[0059] Figure 3 A comparison of the net charge (bars) of the scFv antibody fused with the specified peptide tag at a cytoplasmic pH of 6.6 and the isoelectric point (circles) determined by physicochemical analysis.

[0060] Figure 4 The graph shows the effect of STAND expression on PDIA3 protein expression levels.

[0061] Figure 5 The effect of STAND expression on Spike protein expression levels.

[0062] Figure 6 The expression level of S protein in SARS-CoV-2 WTS pseudovirus packaging system after treatment with the proteasome inhibitor MG-132.

[0063] Figure 7 STAND transfection inhibits the infectivity of SARS-CoV-2 WTS pseudoviruses.

[0064] Figure 8 The expression levels of PDIA3, Spike protein, and GRP78 in 293T cells transfected with STD-4 were determined by further treatment with the proteasome inhibitor MG-132. The expression level of Spike protein in SARS-CoV-2 WT cells was also determined. In the figure: Mock is untreated HEK293T cells, NC is HEK293T cells transfected with Spike and NL4-3 plasmids, Mut is cells treated with inactivated STD-4 mutant (Y93A); STD-4 is cells treated with STAND4, STD-4-M is cells treated with STAND4 and then infused with MG-132, Mut-M is cells treated with STD-4 mutant and then infused with MG-132, NC-M is NC cells treated with MG-132, and Mock-M is Mock cells treated with MG-132.

[0065] Figure 9This is a schematic diagram of the results of the pseudovirus infection experiment for STD-4.

[0066] Figure 10 This is a schematic diagram illustrating the anti-infective effect of STD-4 on viruses.

[0067] Figure 11 The antiviral effect of STD-4 against in vivo HRV-14 in H1-HeLa cells; Figure: A: Representative To-Pro-1 staining images of H1-HeLa cells under different treatment conditions: Mock (untreated group), NC (HRV-14 infected group), 6C (HRV-14 infected + control lentivirus group), and DHM (HRV-14 infected + DHM treated group). Scale bar is 200 μm. B: HRV-14 replication level of H1-HeLa cells in Figure (A) was detected by RT-qPCR. C: TCID 50 Viral titer in supernatant determined by the method. dpi: day post-infection. Data are expressed as mean ± standard deviation (n=3). Compared with the NC group, p < 0.001; statistical significance was determined by ordinary one-way ANOVA and multiple comparisons. Detailed Implementation

[0068] The present invention will be described in detail below with reference to embodiments, but this does not imply any adverse limitation on the invention. The present invention has been described in detail, and specific embodiments thereof have been disclosed. It will be apparent to those skilled in the art that various changes and modifications can be made to the specific embodiments of the present invention without departing from the concept and scope of the invention.

[0069] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the operational steps used herein, such as molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics, and recombinant DNA, are all conventional steps widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below: In this invention, the letters in the amino acid sequence represent single-letter abbreviations of amino acids known in the art, such as those described in J. Biol. Chem, 243, p3558 (1968): alanine: Ala-A, arginine: Arg-R, aspartic acid: Asp-D, cysteine: Cys-C, glutamine: Gln-Q, glutamic acid: Glu-E, histidine: His-H, glycine: Gly-G, asparagine: Asn-N, tyrosine: Tyr-Y, proline: Pro-P, serine: Ser-S, methionine: Met-M, lysine: Lys-K, valine: Val-V, isoleucine: Ile-I, phenylalanine: Phe-F, leucine: Leu-L, tryptophan: Trp-W, threonine: Thr-T.

[0070] In this invention, the amino acid sequences of the listed complementarity-determining regions (CDRs) are all as defined according to the IMGT numbering rules. However, it is well known to those skilled in the art that antibody CDRs can be defined in various ways, such as Chothia (Chothia et al. (1989) Nature 342: 877-883, Al-Lazikani et al., “Standard conformations for the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273, 927-948 (1997)) based on antibody sequence variability, Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), Kabat, and North CDR definitions based on affinity propagation clustering using a large number of crystal structures. Those skilled in the art will understand that, unless otherwise specified, the terms “CDR” and “complementary determination region” for a given antibody or its region (e.g., variable region) should be understood to encompass the complementary determination region defined by any of the above-described known schemes as described in this invention.

[0071] Therefore, when referring to antibodies defined by a specific CDR sequence as defined in this invention, the scope of said antibody also includes antibodies whose variable region sequence contains the specific CDR sequence, but whose claimed CDR boundaries differ from those defined in this invention due to the application of different schemes (e.g., different assignment system rules or combinations). Although the scope of protection claimed by this invention is based on the sequence defined according to the IMGT numbering rules, amino acid sequences corresponding to other CDR definition rules should also fall within the scope of protection of this invention.

[0072] In this invention, the term "full-length antibody" is used interchangeably to refer to a glycoprotein comprising at least two heavy chains (HC) and two light chains (LC) linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated as VH in this invention) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated as VL in this invention) and a light chain constant region (abbreviated as CL in this invention). The light chain constant region consists of one domain: CL. Mammalian heavy chains are classified as α, δ, ε, γ, and μ. Mammalian light chains are classified as λ or κ. Immunoglobulins containing α, δ, ε, γ, and μ heavy chains are classified as immunoglobulins IgA, IgD, IgE, IgG, and IgM. A complete antibody forms a "Y" shape. The stem of the Y is formed by the second and third constant regions of the two heavy chains (and, for IgE and IgM, a fourth constant region) linked together, and disulfide bonds (interchain) are formed in the hinge. The heavy chains γ, α, and δ have constant regions consisting of three tandem (in a row) Ig domains and hinge regions for increased flexibility; the heavy chains μ and ε have constant regions consisting of four immunoglobulin domains. The second and third constant regions are referred to as the "CH2 domain" and the "CH3 domain," respectively. Each arm of the Y chain includes a variable region of a single heavy chain and a first constant region that binds to a variable and constant region of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding.

[0073] In this invention, a "Fab fragment" consists of a light chain and a heavy chain, comprising the CH1 domain and a variable region. The heavy chain of the Fab molecule cannot form disulfide bonds with another heavy chain molecule. The "Fc" region contains two heavy chain fragments containing the CH2 and CH3 domains of the antibody. The two heavy chain fragments are held together by two or more disulfide bonds and through the hydrophobic interaction of the CH3 domain. A "Fab' fragment" contains a portion of a light chain and a heavy chain containing the VH domain, the CH1 domain, and the region between the CH1 and CH2 domains, thereby allowing interchain disulfide bonds to form between the two heavy chains of the two Fab' fragments to form the F(ab')2 molecule. An "F(ab')2 fragment" contains two light chains and two heavy chains containing portions of the constant region between the CH1 and CH2 domains, thereby forming interchain disulfide bonds between the two heavy chains. Therefore, the F(ab')2 fragment consists of two Fab' fragments held together by disulfide bonds between the two heavy chains. The term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody, but lacking the constant region.

[0074] In this invention, scFv refers to a single-chain antibody fragment, which includes a heavy chain variable region, a light chain variable region, and a linker peptide of 15-20 amino acids. The VL and VH domains enable the linker peptides to pair and form monovalent molecules as single polypeptide chains [see, for example, Bird et al., Science 242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)]. Such scFv molecules may have a general structure: NH2-VL-linker peptide-VH-COOH or NH2-VH-linker peptide-VL-COOH.

[0075] In this invention, "nucleic acid" refers to a nucleotide chain of any length and includes DNA and RNA. A nucleotide can be a deoxyribonucleotide, ribonucleotide, modified nucleotide or base, and / or its analogues, or any substrate capable of being incorporated into the chain by DNA or RNA polymerase.

[0076] In this invention, the term "recombinant expression vector" refers to a genetically modified oligonucleotide or polynucleotide construct that, when the construct contains a nucleotide sequence encoding mRNA, protein, polypeptide, or peptide, and the vector is contacted with a cell under conditions sufficient to allow the mRNA, protein, polypeptide, or peptide to be expressed in the cell, permits the expression of the mRNA, protein, polypeptide, or peptide by the host cell. The vectors of this invention are generally not naturally occurring. However, portions of the vector may be naturally occurring. The recombinant expression vectors of this invention can contain any type of nucleotide, including but not limited to DNA and RNA that can be single-stranded or double-stranded, synthetic or partially obtained from natural sources, and may contain natural, non-natural, or modified nucleotides. Recombinant expression vectors can contain naturally occurring or non-naturally occurring nucleotide linkages, or both. In an exemplary aspect, modified nucleotides or non-naturally occurring nucleotide linkages do not impede transcription or replication of the vector.

[0077] The recombinant expression vector of the present invention can be any suitable recombinant expression vector capable of being used to transform or transfect one or more genes or sequences of interest into any suitable host cell and preferably to express the genes or sequences in the host cell. Suitable vectors include those designed for amplification and expansion or for expression or both of the above, and examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, granules or phage vectors, DNA or RNA expression vectors associated with cationic condensers, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as production cells.

[0078] In this invention, the term "host cell" refers to any type of cell that may contain the nucleic acids or vectors described herein. In exemplary aspects, the host cell is a eukaryotic cell, such as a plant, animal, fungus, or algae; or it may be a prokaryotic cell, such as a bacterium or protozoan.

[0079] In this invention, the pharmaceutical composition may comprise a suitable pharmaceutically acceptable carrier, such as pharmaceutical excipients, including buffers, as known in the art. "Pharmaceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, isotonic agents, and absorption delay agents. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions, aqueous dextran, and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Pharmaceutical compositions comprising the invention can be prepared by mixing antibodies of the invention having the desired purity with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)). Preferably, the composition is in the form of a lyophilized formulation or an aqueous solution.

[0080] The pharmaceutical compositions of the present invention may also comprise more than one active ingredient required for a specific indication to be treated, preferably those active ingredients having complementary activities that do not adversely affect each other. For example, it is desirable to also provide other active ingredients, such as other antibodies, antiviral agents, small molecule drugs, or immunomodulators. The active ingredients are suitably combined in amounts effective for the intended use. Sustained-release formulations can be prepared, suitable examples of which include a semi-permeable matrix of a solid hydrophobic polymer containing the antibody of the present invention, said matrix being a shaped article, such as a film or microcapsule.

[0081] In this invention, "RNA virus infection-related diseases" refers to a series of infectious diseases caused by RNA viruses, including but not limited to: novel coronavirus infection, influenza, AIDS, Middle East Respiratory Syndrome, Marburg virus disease, and yellow fever.

[0082] In this invention, "capsid viruses" or "enveloped viruses" refer to viruses whose core genetic material (DNA or RNA) is encased in a protein capsid and further surrounded by a lipid bilayer envelope derived from the host cell. This envelope contains viral-encoded glycoproteins, which play a crucial role in the specific recognition, adsorption, and entry of the virus into the host cell. The basic structure of a capsid virus, from the inside out, includes: a core genome, a protein capsid, and a lipid envelope. Typical capsid viruses include, but are not limited to, coronaviruses, influenza viruses, and human immunodeficiency virus (HIV). Due to the presence of the outer envelope, these viruses are relatively sensitive to external environmental factors (such as dryness, heat, and certain disinfectants), but the specificity of their surface glycoproteins enables them to efficiently infect specific host cells.

[0083] The present invention is further illustrated below by way of embodiments, but these embodiments are not intended to limit the invention to their scope. Experimental methods in the following embodiments, unless otherwise specified, were performed according to conventional methods and conditions, or as selected in the product instructions. All virus culture and infection operations involved in the following embodiments were performed in the Level 2 Biosafety Laboratory of ShanghaiTech University.

[0084] Example 1: Screening of phage display antibodies targeting PDIA3

[0085] First, the inventors used phage display antibody technology to screen phages that bind to the PDIA3 protein (UniProt ID: P30101, brand Novus, catalog number NBP1-37081), obtaining 9 scFv sequences (see Table 1, where the variable region (CDR) is defined according to the Kabat numbering system), and linker sequences (see Table 2). These sequences were then constructed into the pFuse-IL2-Fc vector to create the pFuse-IL2-scFv-Fc plasmid, and the corresponding protein was expressed. ELISA experiments demonstrated that all 9 selected antibodies could bind to the target protein with an affinity at the nM level, such as... Figure 1 As shown. The control antibody is ab13507 (Abcam, Erp57 antibody).

[0086] Table 1. Amino acid sequence of the variable region of anti-PDIA3 antibody

[0087]

[0088]

[0089]

[0090] Table 2 Connecting subsequences

[0091] Example 2: STAND modification based on scFv sequence

[0092] Since the PDIA3 protein primarily functions within the endoplasmic reticulum (ER), the prerequisite for validating the intracellular function of candidate antibodies is their stable localization within the ER. Therefore, the inventors designed the candidate antibody as a stand for intracellular application. Through literature review and relevant patents, seven intracellular stabilizing peptides were selected (see Table 3). Intracellular stabilizing peptides generally contain 14-25 amino acids, with at least half being acidic amino acids. The inventors designed the candidate antibody to have an intracellular stabilizing peptide fused to the N-terminus and an HA tag fused to the C-terminus to ensure the stability of the scFv fragment in the cytoplasm. Furthermore, a cleavable ER signal peptide was fused to the N-terminus of this sequence, and an ER residency signal peptide was fused to the C-terminus (see Table 4 for the specific sequences of other elements) to ensure the scFv fragment is localized to the ER. Figure 2 .

[0093] Table 3. Amino acid sequences of the seven intracellular stable peptides ISP1-7

[0094] Table 4. Amino acid sequences of other STAND components

[0095] Connection relationships of STAND components: From N-end to C-end: SP-ISP-scFv-HA-ER A strong net negative charge (net charge < -8) and a low pI (pI < 5) at pH 6.6 are key parameters for maintaining the intracellular stability of antibodies. Physicochemical analysis of these five intracellular antibodies showed that, compared to scFv1, both the net negative charge and pI value of the five intracellular antibodies were reduced at pH 6.6. Firstly, taking scFv-1 as an example, the pI value of ISP1-scFv1-HA was above 7, while the pI values ​​of ISP3-scFv1-HA, ISP4-scFv1-HA, and ISP5-scFv1-HA were slightly higher than 5. Only ISP2-scFv1-HA best met the pI value requirements. (See...) Figure 3 Secondly, the net negative charge of all five intracellular antibodies was below -8. Finally, considering all factors, ISP2-scFv1-HA met the requirements of low pI and strong net negative charge. Based on the same principle and method, ISP2-scFv2-HA and ISP2-scFv4-HA also best met the requirements. Therefore, ultrastable intracellular antibodies STAND1, STAND2, and STAND4 were constructed based on the structures of ISP2-scFv1-HA, ISP2-scFv2-HA, and ISP2-scFv4-HA, respectively.

[0096] The amino acid sequence of STAND1: MKTNLFLFLIFSLLLSSAEFEEDQDDEDDEDQDDAAQPAMAQVQLQQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDKSRGQWLVSA FDIWGQGTTVTVSSGGGGGSDVVMTQSPGTLSLTPGERATLSCRASQSVSSTYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISNLEPEDFAVYYCQQYGSSPRTFGQGTKLEIKRGLGGLVYPYDVPDYAHDEL (SEQ ID NO: 18) The amino acid sequence of STAND2: MKTNLFLFLIFSLLLSSAEFEEDQDDEDDEDQDDAAQPAMAQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAREGELLPFDYWG QGTTVTVSSGGGGSQAVLTQPSSLSASPGASVSLTCTLRSGINVGSSTIYWYQQKAGSPPQSLLRYKSDSDKQQGSGVPSRFSGSRDASANAGILLISGLRSEDEADYHCAIWHSSAWVFGGGTKVTVLGGLGGLVYPYDVPDYAHDEL (SEQ ID NO: 19) The amino acid sequence of STAND4: MKTNLFLFLIFSLLLSSAEFEEDQDDEDDEDQDDAAQPAMAQVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQPPGKGLEWIGEIYHSGSTNYNPSLKSRVTISVDKSKNQFSLKLSSVTAADTAVYYCARDRGYSSSQVLDYWGQ GTLVTVSSGGGGSGGGSGGGGSQSALTQPRSVSGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGNAPKLMIYDVNQRPSGVPHRFSGSKSDNTASLTISGLQAEDEADYFCSSYAAGYTLVFGRGTQLTVLGGLGGLVYPYDVPDYAHDEL (SEQ ID NO: 20)

[0097] Example 3: Functional Study (STAND1 and STAND2 as examples)

[0098] First, to verify the effect of STAND on PDIA3 expression levels in 293T cells, 293T cells were transfected with STAND1 and STAND2, respectively, and the effect of STAND on PDIA3 expression levels was assessed using Western blotting. The results are as follows: Figure 4 As shown, compared with the NC group, there were no significant changes in PDIA3 expression levels in the STAND1 and STAND2 groups. This indicates that STAND1 and STAND2 do not affect PDIA3 protein expression levels in 293T cells.

[0099] Previous experiments using ELISA, BLI, and WB have demonstrated that scFv-1 and scFv-2 have an affinity interaction with PDIA3. Notably, the entire VH and VL regions (the regions interacting with the antigen) remained sequence-unchanged before and after antibody modification, thus not affecting the antibody-antigen binding pattern. This result suggests that STAND1 and STAND2 may exert their effect by binding to PDIA3 protein and inhibiting its promotion of correct S protein folding. Secondly, to verify the effect of STAND1 on the expression level of WT SARS-CoV-2 S protein, a WT SARS-CoV-2 S pseudovirus packaging system was established using 293T cells transfected with STAND1. Western blotting was used to assess the S protein expression level, and the results are as follows: Figure 5 As shown, both STAND1 and STAND2 transfections significantly reduced the expression level of WT SARS-CoV-2 S protein, with grayscale analysis revealing a reduction exceeding 90%. These results highlight the crucial role of STAND1 in regulating WT SARS-CoV-2 S protein expression, a mechanism likely closely related to the targeted binding of PDIA3. PDIA3 plays a critical role in glycoprotein folding and quality control, participating in the life cycle of many viruses. Therefore, STAND1 may indirectly inhibit SARS-CoV-2 S protein expression by binding to and suppressing PDIA3 function.

[0100] Finally, the proteasome inhibitor MG-132 was added to the above system, and the expression level of the S protein was further evaluated by Western blotting. The results are as follows: Figure 6 As shown, the expression level of the S protein was restored after the addition of MG-132. This indicates that, similar to siPDIA3, the proteasome degradation pathway is also involved in the mechanism by which STAND reduces the expression level of the S protein, and supports the hypothesis that STAND prevents the S protein from folding correctly by targeting PDIA3.

[0101] Example 4: STAND inhibits pseudovirus infection of coronavirus

[0102] To verify the effect of STAND on the infectivity of WT SARS-CoV-2 S pseudovirus, viral particles from the cell supernatant of WT SARS-CoV-2 S pseudovirus packaging systems under different treatment conditions were transduced into 293T-hACE2 cells, and the activity of the reporter gene Luciferase was detected. The results are as follows: Figure 7As shown, compared with the NC group, the Luciferase activity in both the STAND1 and STAND2 groups was significantly reduced (p<0.0001). Both STAND1 and STAND2 transfections significantly reduced the infectivity of WT SARS-CoV-2 S pseudoviruses, with reductions exceeding 90%. Figure 10 As shown, STD-4 expression can suppress multiple variants of SARS-CoV-2, as well as SARS-CoV, MERS-CoV, and HIV-1, in pseudovirus systems. Figure 10 As shown in Figure A, all three STAND constructs (STD-1, STD-2, and STD-4) containing non-selective or selective anti-hPDIA3scFv inhibited entry of wild-type and variant (Delta, OmicronBA.2, or BA.4 / 5) SARS-CoV-2 pseudoviruses by more than 95%. Importantly, the STAND constructs also strongly blocked infection by pseudo-SARS-CoV, MERS-CoV, and HIV. Figure 10 The results (BD) demonstrate its broad-spectrum antiviral activity. These results indicate that the intracellular antibody obtained and modified by the inventors through screening human PDIA3 can effectively inhibit the expression level of the SARS-CoV-2 S protein in cells, thereby inhibiting the production of infective SARS-CoV-2 pseudovirus particles.

[0103] These results clearly demonstrate that STAND effectively inhibits the expression of the WT SARS-CoV-2 WTS protein by targeting PDIA3, thereby reducing the infectivity of pseudoviruses. This finding indicates that STAND can target key proteins within host cells and affect important proteins on the viral surface. The binding of STAND to PDIA3 may lead to misfolding or degradation of the S protein, thereby reducing S protein expression and thus decreasing the virus's ability to invade. Future in-depth research into the interaction mechanism between STAND and PDIA3 will help optimize antibody design and enhance its potential for clinical application.

[0104] Example 5: Functional Study (Taking the highly specific intracellular antibody STAND-4 as an example, hereinafter referred to as STD-4)

[0105] Referring to Example 3, to verify the intracellular specificity of STD-4 for its target PDIA3, the inventors transfected 293T cells with STD-4 and evaluated its effects on the expression levels of PDIA3, SARS-CoV-2 Spike protein, and GRP78 using Western blotting. The results are as follows: Figure 8As shown, compared with the negative control (NC) group, STD-4 exhibited similar biological functions to earlier versions: Spike protein expression levels were significantly reduced, and intracellular GRP78 expression levels also decreased, indicating that STD-4 alleviated endoplasmic reticulum stress induced by viral packaging to some extent. Furthermore, STD-4 treatment did not significantly alter intracellular PDIA3 protein expression levels, consistent with previous results. Combined with the results of the BIL experiment, this further confirmed that STD-4 not only did not affect PDIA3 protein expression in 293T cells, but also exhibited higher family specificity and lower off-target effects in binding to PDIA3 compared to STD and STD2.

[0106] To confirm whether STD-4 also degrades misfolded Spike proteins via the proteasome pathway, the proteasome inhibitor MG-132 was added to the above experimental system. Western blot results are as follows: Figure 8 As shown, the addition of MG-132 effectively reversed the low expression of Spike protein induced by STD-4, and the degree of recovery was similar to that of the STD1 / STD2 group. This result indicates that although STD-4 is more potent, its mechanism of action is consistent with that of STD1 / STD2, namely, by inhibiting PDIA3 function, leading to misfolding of Spike protein and eventual degradation via the proteasome pathway.

[0107] Finally, the ultimate antiviral effect of STD-4 was evaluated using a pseudovirus infection experiment. Pseudovirus particles from different treatment conditions were collected, used to infect 293T-hACE2 cells, and the activity of the reporter gene luciferase was measured. The results are as follows: Figure 9 As shown, compared with the NC group, the Luciferase activity in the STD-4 group was significantly reduced (p < 0.0001). These results indicate that STD-4, based on scFv4 modified to specifically target PDIA3, can more efficiently inhibit the expression of SARS-CoV-2 Spike protein, thereby almost completely blocking the generation of infective pseudovirus particles.

[0108] In summary, STD-4, as an intracellular antibody with high family specificity to PDIA3, inherits the mechanism of action of STD1 / STD2 and achieves potent inhibition of SARS-CoV-2 Spike protein expression and pseudovirus infection through more precise targeting and stronger binding affinity. The fact that STD-4 does not bind to other members of the PDI family suggests higher safety and lower off-target risk in clinical applications. The superior performance of STD-4 demonstrates the success of antibody specificity optimization strategies and provides a more promising candidate molecule for developing broad-spectrum antiviral therapies targeting host PDIA3.

[0109] To verify the inhibitory effect of STAND on human rhinovirus type 14 (HRV-14) infection, lentivirus was packaged and H1-HeLa cells were infected with 6C (the same control plasmid as the STAND4 vector, red fluorescence, MOI=2), STAND4-NO ER (STAND4 without ISP and ER localization sequences), and STAND4, respectively, for 24 hours. Then, H1-HeLa cells in the above groups were infected with HRV-14 (MOI=0.3) for 1 hour. After washing twice with PBS, fresh culture medium was added and the cells were cultured for another 24 hours. The effect of STAND on HRV14 infection was evaluated by TO-PRO-1 staining and RT-qPCR experiments. The DHM group served as a positive control (Admas, #013226871), and DHM was co-incubated with H1-HeLa cells infected with HRV-14 for 24 hours.

[0110] TO-PRO-1 is a non-cellular DNA-binding fluorescent dye (Ex / Em = 515 nm / 531 nm) that can intercalate into base pairs of double-stranded DNA, causing necrotic cells with damaged cell membranes or late-stage apoptotic cells to exhibit green fluorescence under a fluorescence microscope. Results are as follows... Figure 11 As shown, compared with the NC group, the number of apoptotic cells in the STAND4-NO ER group, STAND4 group, and DHM group was significantly reduced, while the proportion of apoptotic cells in the 6C group did not change significantly. This indicates that STAND4 can effectively protect H1-HeLa cells and prevent them from undergoing HRV-14-induced apoptosis.

[0111] Cells from the above groups were collected for RT-qPCR experiments. Total RNA was extracted, reverse transcribed, and the HRV-14 nucleocapsid (N) gene level was detected. Figure 11 As shown in Figure B, compared with the control plasmid 6C group, the HRV-14 progeny viral RNA in cells treated with STAND4-NO ER, STAND4, and DHM decreased by more than 70%, indicating that STAND4 can effectively reduce the amplification of HRV-14 in H1-HeLa cells. Furthermore, both TO-PRO-1 staining and RT-qPCR experiments demonstrated that STAND4 significantly inhibited HRV-14 infection of H1-HeLa cells; this result was independent of endoplasmic reticulum targeting signaling.

[0112] References

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[0120] 8. Karlas, A., et al. (2010) Genome-wide RNAi screen identifies humanhost factors crucial for influenza virus replication Nature 463(7282), 818-822.

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Claims

1. A fusion protein, characterized in that, The fusion protein contains an antigen-binding peptide that targets PDIA3 and an intracellular stabilizing peptide. The antigen-binding peptide comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region are selected from any one of the following: (1) The heavy chain variable region comprises HCDR1 as shown in SEQ ID NO: 21, HCDR2 as shown in SEQ ID NO: 22, and HCDR3 as shown in SEQ ID NO: 23; the light chain variable region comprises LCDR1 as shown in SEQ ID NO: 25, LCDR2 as shown in SEQ ID NO: 26, and LCDR3 as shown in SEQ ID NO: 27; (2) The heavy chain variable region comprises HCDR1 with an amino acid sequence as shown in SEQ ID NO: 29, HCDR2 with an amino acid sequence as shown in SEQ ID NO: 30, and HCDR3 with an amino acid sequence as shown in SEQ ID NO: 31; the light chain variable region comprises LCDR1 with an amino acid sequence as shown in SEQ ID NO: 33, LCDR2 with an amino acid sequence as shown in SEQ ID NO: 34, and LCDR3 with an amino acid sequence as shown in SEQ ID NO: 35; (3) The heavy chain variable region comprises HCDR1 as shown in SEQ ID NO: 37, HCDR2 as shown in SEQ ID NO: 38, and HCDR3 as shown in SEQ ID NO: 39; the light chain variable region comprises LCDR1 as shown in SEQ ID NO: 41, LCDR2 as shown in SEQ ID NO: 42, and LCDR3 as shown in SEQ ID NO: 43; (4) The heavy chain variable region comprises HCDR1 as shown in SEQ ID NO: 45, HCDR2 as shown in SEQ ID NO: 46, and HCDR3 as shown in SEQ ID NO: 47; the light chain variable region comprises LCDR1 as shown in SEQ ID NO: 49, LCDR2 as shown in SEQ ID NO: 34, and LCDR3 as shown in SEQ ID NO: 35; (5) The heavy chain variable region comprises HCDR1 as shown in SEQ ID NO: 21, HCDR2 as shown in SEQ ID NO: 22, and HCDR3 as shown in SEQ ID NO: 51; the light chain variable region comprises LCDR1 as shown in SEQ ID NO: 53, LCDR2 as shown in SEQ ID NO: 54, and LCDR3 as shown in SEQ ID NO: 55; (6) The heavy chain variable region comprises HCDR1 as shown in SEQ ID NO: 29, HCDR2 as shown in SEQ ID NO: 30, and HCDR3 as shown in SEQ ID NO: 57; the light chain variable region comprises LCDR1 as shown in SEQ ID NO: 59, LCDR2 as shown in SEQ ID NO: 60, and LCDR3 as shown in SEQ ID NO: 61; (7) The heavy chain variable region comprises HCDR1 as shown in SEQ ID NO: 63, HCDR2 as shown in SEQ ID NO: 64, and HCDR3 as shown in SEQ ID NO: 65; the light chain variable region comprises LCDR1 as shown in SEQ ID NO: 67, LCDR2 as shown in SEQ ID NO: 68, and LCDR3 as shown in SEQ ID NO: 69; (8) The heavy chain variable region comprises HCDR1 as shown in SEQ ID NO: 71, HCDR2 as shown in SEQ ID NO: 72, and HCDR3 as shown in SEQ ID NO: 73; the light chain variable region comprises LCDR1 as shown in SEQ ID NO: 75, LCDR2 as shown in SEQ ID NO: 34, and LCDR3 as shown in SEQ ID NO: 76; (9) The heavy chain variable region comprises HCDR1 with an amino acid sequence as shown in SEQ ID NO: 78, HCDR2 with an amino acid sequence as shown in SEQ ID NO: 79, and HCDR3 with an amino acid sequence as shown in SEQ ID NO: 80; the light chain variable region comprises LCDR1 with an amino acid sequence as shown in SEQ ID NO: 49, LCDR2 with an amino acid sequence as shown in SEQ ID NO: 82, and LCDR3 with an amino acid sequence as shown in SEQ ID NO: 35; The fusion protein specifically binds to PDIA3.

2. The fusion protein as described in claim 1, characterized in that, The intracellular stabilizing peptide is located at the N-terminus or C-terminus of the antigen-binding peptide. And / or, the intracellular stable peptide comprises an amino acid sequence as shown in any of SEQ ID NO: 1-5 or SEQ ID NO: 89-90.

3. The fusion protein as described in claim 1 or 2, characterized in that, In the antigen-binding peptide, the heavy chain variable region and the light chain variable region are selected from any one of the following: (1) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 24 or having at least 80% sequence identity with SEQ ID NO: 24; the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 28 or having at least 80% sequence identity with SEQ ID NO: 28; (2) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 32 or having at least 80% sequence identity with SEQ ID NO: 32; the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 36 or having at least 80% sequence identity with SEQ ID NO: 36; (3) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 40 or having at least 80% sequence identity with SEQ ID NO: 40; the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 44 or having at least 80% sequence identity with SEQ ID NO: 44; (4) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 48 or having at least 80% sequence identity with SEQ ID NO: 48; the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 50 or having at least 80% sequence identity with SEQ ID NO:

50. (5) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 52 or having at least 80% sequence identity with SEQ ID NO: 52; the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 56 or having at least 80% sequence identity with SEQ ID NO: 56; (6) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 58 or having at least 80% sequence identity with SEQ ID NO: 58; the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 62 or having at least 80% sequence identity with SEQ ID NO: 62; (7) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 66 or having at least 80% sequence identity with SEQ ID NO: 66; the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 70 or having at least 80% sequence identity with SEQ ID NO:

70. (8) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 74 or having at least 80% sequence identity with SEQ ID NO: 74; the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 77 or having at least 80% sequence identity with SEQ ID NO: 77; (9) The heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 81 or having at least 80% sequence identity with SEQ ID NO: 81; the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 83 or having at least 80% sequence identity with SEQ ID NO:

83.

4. The fusion protein according to any one of claims 1-3, characterized in that, In the antigen-binding peptide, the heavy chain variable region and the light chain variable region are connected by a linker; the structure of the antigen-binding peptide from the N-terminus to the C-terminus is: heavy chain variable region-linker-light chain variable region; or, light chain variable region-linker-heavy chain variable region. Preferably, the linker comprises an amino acid sequence as shown in any of SEQ ID NO: 84-88.

5. The fusion protein according to any one of claims 1-4, characterized in that, The antigen-binding peptide comprises an amino acid sequence as shown in any of SEQ ID NO: 9-17 or having at least 80% sequence identity with any of SEQ ID NO: 9-17.

6. The fusion protein according to any one of claims 1-5, characterized in that, The fusion protein further includes an endoplasmic reticulum signal peptide, one end of which is linked to the antigen-binding peptide and the other end of which is linked to the endoplasmic reticulum signal peptide. Preferably, the endoplasmic reticulum signal peptide has the amino acid sequence shown in SEQ ID NO:

6.

7. The fusion protein according to any one of claims 1-6, characterized in that, The fusion protein also includes an HA tag and an endoplasmic reticulum (ER) residency signal; the end of the antigen-binding peptide not connected to the intracellular stabilizing peptide is connected to a tandem HA tag and ER residency signal. Preferably, the HA tag has an amino acid sequence as shown in SEQ ID NO: 7; and / or, the endoplasmic reticulum resident signal has an amino acid sequence as shown in SEQ ID NO:

8.

8. The fusion protein according to any one of claims 1-7, characterized in that, The fusion protein comprises an amino acid sequence as shown in any of SEQ ID NO: 18-20 or having at least 80% sequence identity with any of SEQ ID NO: 18-20.

9. An antibody or antigen-binding fragment targeting PDIA3, characterized in that, The antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region as described in any one of claims 1-3.

10. A polynucleotide, characterized in that, The polynucleotide encodes the fusion protein as described in any one of claims 1-8 or the antibody or antigen-binding fragment as described in claim 9.

11. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the polynucleotide as described in claim 10.

12. A recombinant cell, characterized in that, The recombinant cells express the fusion protein as described in any one of claims 1-8 or the antibody or antigen-binding fragment as described in claim 9, or the recombinant cells contain the recombinant expression vector as described in claim 11.

13. A method for preparing a fusion protein or an antibody or antigen-binding fragment targeting PDIA3, characterized in that, The method includes culturing the recombinant cells as described in claim 12.

14. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises a fusion protein as described in any one of claims 1-8, an antibody or antigen-binding fragment as described in claim 9, a recombinant expression vector as described in claim 11, and / or a recombinant cell as described in claim 12, as well as a pharmaceutically acceptable carrier and / or excipient.

15. The use of a fusion protein as described in any one of claims 1-8, an antibody or antigen-binding fragment as described in claim 9, a recombinant expression vector as described in claim 11, a recombinant cell as described in claim 12, or a pharmaceutical composition as described in claim 14 in the preparation of a medicament for treating and / or preventing various capsid virus infections, including human coronavirus infection, retrovirus infection, rhinovirus infection, or influenza virus infection; Preferably, the human coronavirus infection is selected from SARS-CoV, MERS-CoV, SARS-CoV-2 and their variants; the retrovirus infection is HIV infection; the influenza virus infection is influenza A virus infection; and / or, the genetic material of the capsid virus is DNA or RNA.

16. A method for inhibiting protein folding or assembly of capsid viruses, characterized in that, The method includes introducing a PDIA binder into the host cells of the capsid virus, the PDIA binder disrupting the cytoplasmic stress state of the host cells through protein-protein interactions, reducing the folding or assembly of capsid virus proteins, and decreasing the infectivity of folded or assembled capsid virus proteins. Preferably, the method is in vivo or in vitro; and / or, the PDIA3 binder is selected from the fusion protein of any one of claims 1-8, the antibody or antigen-binding fragment of claim 9, the recombinant expression vector of claim 11, the recombinant cell of claim 12, and the pharmaceutical composition of claim 14.

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