Nucleocapsid antigen immunotherapy of covid-19 fusion proteins and methods of use

Abstract

The present disclosure provides a recombinant prepared fusion protein comprising an SARS-CoV-2 nucleocapsid protein (N protein) fragment linked to a human Fc fragment, or an analog thereof, for use in association with a 2019 novel coronavirus (COVID-19). Embodiments include administering the fusion protein to a patient having recovered from COVID-19 as a booster vaccination, administering to an antibody-negative patient to produce an antibody against the SARS-CoV-2 virus to make the patient become a plasma donor for recovery, administering to a patient having infected with the SARS-CoV-2 virus and having contacted with COVID-19 to limit the range of infection and ameliorate disease, and administering to an antibody-negative patient to produce an antibody against the SARS-CoV-2 virus to make the patient become a plasma donor for recovery. And as a prophylactic COVID-19 vaccine. Exemplary Fc fusion proteins and pharmaceutical formulations of the exemplary Fc fusion proteins are provided in addition to methods of use and preparation.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 488,565, filed March 6, 2023, entitled “Nucleocapsid antigen immunotherapy for COVID-19 fusion protein and methods of use,” which is incorporated herein by reference in its entirety.

[0003] sequence list

[0004] The following application contains a sequence list of electronic submissions of an XML file conforming to the ST.26 standard, named “ABC-047PCT.xml”, created on February 15, 2024, and with a file size of 23,503 bytes, the entire contents of which are incorporated herein by reference. Technical Field

[0005] This technology relates to a fusion protein comprising a truncated form of the SARS-CoV-2 nucleocapsid N protein or an analogue thereof linked to a human Fc fragment, and its use in relation to the 2019 novel coronavirus (COVID-19). Background Technology

[0006] The following description of the background art is provided only to aid in understanding the art and does not acknowledge any prior art that describes or constitutes the art.

[0007] Fc fusion protein

[0008] Fc fusion proteins consist of a species-specific immunoglobulin Fc domain linked to another peptide, such as a protein or peptide with therapeutic potential. As used herein, the terms "fusion protein" and "Fc fusion protein" refer to proteins comprising more than one part, for example, from different sources (e.g., different proteins, peptides, cells, etc.), which are covalently linked by peptide bonds. Fc fusion proteins are preferably covalently linked by (i) linking the genes encoding each part into a single nucleic acid molecule, and (ii) expressing the protein encoded by the nucleic acid molecule in a host cell (e.g., HEK cells or CHO cells). Fully recombinant synthetic methods are superior to methods that separately synthesize the therapeutic protein and Fc fragment and then chemically conjugate them. The chemical conjugation step and subsequent purification process increase production complexity, reduce product yield, and increase costs.

[0009] The terms“Fc fragment,”“Fc region,”“Fc domain,” or“Fc polypeptide” are used herein to define a C-terminal region of an immunoglobulin heavy chain. The Fc fragment, region, domain, or polypeptide can be a native sequence Fc region or a variant / mutated Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, they typically include some or all of the hinge region of the heavy chain, some or all of the CH2 region of the heavy chain, and some or all of the CH3 region of the heavy chain. The hinge region of an Fc fragment (e.g., canine or human Fc fragment) includes the amino acid sequence that connects the CH1 domain of the heavy chain to the CH2 region of the heavy chain and contains one or more cysteines that form one or more inter-heavy chain disulfide bonds to form a homodimer of the Fc fusion protein from two identical but separate Fc fusion protein monomers. The hinge region can include all or part of a naturally occurring amino acid sequence or a non-naturally occurring amino acid sequence.

[0010] The presence of the Fc domain increases the plasma half-life due to its interaction with the neonatal Fc receptor (FcRn), and in addition, the Fc fusion protein is cleared more slowly from the kidney due to the large size of the molecule, resulting in prolonged in vivo circulation of the molecule to enable the activity of the connecting peptide and to increase the solubility and stability of the Fc fusion protein molecule. The Fc domain also enables the Fc fusion protein to interact with Fc receptors on immune cells. In some examples, the therapeutic protein or peptide is linked to the immunoglobulin Fc domain via a linker. The therapeutic protein or peptide and the linker effectively replace the variable region of an antibody while leaving the Fc region intact.

[0011] Fc receptor (FcR) refers to a receptor that binds to an Fc fragment of an antibody or to the Fc region of an antibody. In examples, the FcR is a native sequence canine or human FcR, and the FcR is a receptor that binds an Fc fragment of an IgG antibody (a gamma receptor) and includes, but is not limited to, Fc(gamma) receptor I, Fc(gamma) receptor IIa, Fc(gamma) receptor IIb, and Fc(gamma) receptor III subtypes, including allelic variants and alternatively spliced forms of these receptors. “FcR” also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus and also responsible for the prolonging the in vivo half-life of antibodies and Fc fusion proteins in the body. In examples, a human-derived FcR is used in vitro (e.g., in an assay) to measure the binding of Fc fusion proteins comprising an Fc fragment of any mammalian origin, to assess their FcR binding properties. Those skilled in the art will appreciate that a mammalian FcR from one species (e.g., a human-derived FcR) is sometimes capable of binding an Fc fragment from a second species (e.g., a canine-derived FcR) in vitro. SUMMARY

[0012] Described herein are fusion proteins each comprising a respective viral nucleocapsid protein fragment and an Fc fragment, wherein the viral nucleocapsid fragment and the Fc fragment are connected by a peptide linker. In one or more embodiments, the nucleocapsid fragment comprises a SARS-CoV-2 nucleocapsid protein fragment comprising a functional fragment, analog, or variant / mutant thereof. In one or more embodiments, the nucleocapsid protein fragment comprises the nucleocapsid fragment of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, or a functional fragment, analog, or variant / mutant thereof. In one or more embodiments, the Fc fragment comprises the sequence of SEQ ID NO: 1 or a functional fragment. In one or more embodiments, the linker comprises SEQ ID NO: 14. In one or more embodiments, the fusion protein comprises, consists essentially of, or even consists of the sequence of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 19, or SEQ ID NO: 20. In one or more embodiments, the fusion protein is a homodimer. In one or more embodiments, the Fc fragment is glycosylated.

[0013] Also described herein are immunogenic compositions comprising, or consisting essentially of, one or more fusion proteins according to any embodiment or combination of embodiments described herein and a pharmaceutically acceptable carrier. In one or more embodiments, the fusion proteins are dispersed in a carrier. In one or more embodiments, the composition further comprises an adjuvant. In one or more embodiments, the adjuvant is Montanide ISA-720. In one or more embodiments, the fusion proteins are emulsified with the adjuvant. In one or more embodiments, the emulsion is prepared extemporaneously prior to administration. In one or more embodiments, the prepared emulsion is stable at cold (4°C) or at room temperature for at least 8 hours, preferably up to 24 hours. In one or more embodiments, the composition is an injectable formulation. In one or more embodiments, the composition is suitable for subcutaneous administration. In one or more embodiments, the composition is suitable for prophylactic vaccination. In one or more embodiments, the composition is suitable for therapeutic vaccination. TM ISA-720. In one or more embodiments, the fusion proteins are emulsified with the adjuvant. In one or more embodiments, the emulsion is prepared extemporaneously prior to administration. In one or more embodiments, the prepared emulsion is stable at cold (4°C) or at room temperature for at least 8 hours, preferably up to 24 hours. In one or more embodiments, the composition is an injectable formulation. In one or more embodiments, the composition is suitable for subcutaneous administration. In one or more embodiments, the composition is suitable for prophylactic vaccination. In one or more embodiments, the composition is suitable for therapeutic vaccination.

[0014] This document also describes various methods for increasing antibody production against an antigenic agent in subjects. These methods typically involve administering to the subject a therapeutically effective amount of one or more fusion proteins or one or more immunogenic compositions according to any embodiment or combination thereof described herein. In one or more embodiments, the subject has a measurable antibody titer against the antigenic agent prior to administration of the fusion protein or immunogenic composition. In one or more embodiments, the subject is in a null state (unsensitized) before administration of the fusion protein or immunogenic composition. In one or more embodiments, the fusion protein or immunogenic composition is administered via injection. In one or more embodiments, the fusion protein or immunogenic composition is administered subcutaneously or intramuscularly. In one or more embodiments, the fusion protein or immunogenic composition is provided as a unit dosage form. In one or more embodiments, the fusion protein or immunogenic composition is co-administered with an adjuvant. In one or more embodiments, the method further includes preparing the fusion protein or immunogenic composition for administration, wherein the preparation includes premixing the fusion protein or immunogenic composition with an adjuvant prior to administration. In one or more embodiments, the premixing includes emulsifying the adjuvant and the fusion protein to produce an emulsion, and administering the emulsion to the subject. In one or more embodiments, the prepared emulsion is stable for at least 8 hours at refrigeration (4°C) or room temperature, preferably for up to 24 hours after preparation.

[0015] This document also describes a method for inducing an immune response against a viral infection, preferably SARS-CoV-2, and more preferably COVID-19, in subjects. The method generally includes administering to the subject a therapeutically effective amount of one or more fusion proteins or one or more immunogenic compositions according to any embodiment or combination thereof described herein. In one or more embodiments, the subject has a measurable antibody titer against the viral infection prior to administration of the fusion protein or immunogenic composition. In one or more embodiments, the subject is in a pre-antibody state prior to administration of the fusion protein or immunogenic composition. In one or more embodiments, the fusion protein or immunogenic composition is administered via injection. In one or more embodiments, the fusion protein or immunogenic composition is administered subcutaneously or intramuscularly. In one or more embodiments, the fusion protein or immunogenic composition is provided as a unit dosage form. In one or more embodiments, the fusion protein or immunogenic composition is co-administered with an adjuvant. In one or more embodiments, the method further includes preparing the fusion protein or immunogenic composition for administration, wherein the preparation includes premixing the fusion protein or immunogenic composition with an adjuvant prior to administration. In one or more embodiments, premixing includes emulsifying the adjuvant and fusion protein to produce an emulsion, and administering the emulsion to a subject. In one or more embodiments, the prepared emulsion is stable for at least 8 hours at refrigeration (4°C) or room temperature, preferably for up to 24 hours after preparation.

[0016] This document also describes methods for producing fusion proteins according to any embodiment or combination of embodiments described herein. The method typically involves transiently transfecting a nucleic acid encoding the fusion protein into HEK293 cells, wherein the transfected HEK293 cells express the fusion protein. In one or more embodiments, the fusion protein is secreted by the cells into a cell culture medium, further comprising purifying or isolating the fusion protein from the culture medium. Advantageously, the yield of the purified or isolated fusion protein in any of the aforementioned expression systems is greater than 100 mg / L.

[0017] This document also describes cells engineered to express fusion proteins, which are fusion proteins according to any embodiment or combination of embodiments described herein. In one or more embodiments, the cells are HEK293 cells.

[0018] As described herein, one or more fusion proteins or one or more immunogenic compositions according to any embodiment or combination of embodiments described herein may be used for treatment and / or as a medicine.

[0019] As described herein, one or more fusion proteins or one or more immunogenic compositions according to any embodiment or combination of embodiments described herein may be used to increase antibody production in subjects.

[0020] As described herein, fusion proteins (or multiple fusion proteins) or immunogenic compositions (or multiple immunogenic compositions) according to any embodiment or combination thereof described herein may be used to treat and / or prevent viral infections, preferably SARS-CoV-2 virus, more preferably COVID-19.

[0021] As described herein, one or more fusion proteins or one or more immunogenic compositions according to any embodiment or combination of embodiments described herein may be used as prophylactic, therapeutic and / or booster vaccines.

[0022] Specific embodiments relate to one or more fusion proteins selected from the group consisting of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:19, and SEQ ID NO:20, or pharmaceutical compositions thereof, for the treatment and / or prevention of viral infection, preferably SARS-CoV-2 virus, more preferably COVID-19.

[0023] As described herein, one or more fusion proteins or one or more immunogenic compositions according to any embodiment or combination of embodiments described herein can be used to produce medicaments for the treatment and / or prevention of viral infections. Attached Figure Description

[0024] Figure 1 A schematic diagram of the insulin-Fc fusion protein homodimer is shown.

[0025] Figure 2 A schematic diagram of an exemplary SARS-CoV-2 N-Fc fusion protein homodimer is shown.

[0026] Figure 3 The binding of Fc(γ) receptor I to the insulin-Fc fusion proteins of SEQ ID NO:5 and SEQ ID NO:7 is shown.

[0027] Figure 4 The study showed that in six beagle dogs with chemically induced diabetes, the human titer of the anti-insulin antibody (AIA) against RHI was more than 200-fold higher after eight consecutive doses of the insulin-Fc fusion protein of SEQ ID NO:5.

[0028] Figure 5The percentage change in anti-insulin antibody (AIA) titer against RHI was shown in six beagle dogs with chemically induced diabetes after eight consecutive weekly administrations of the insulin-Fc fusion protein of SEQ ID NO:5, starting from day 0 of the trial.

[0029] Figure 6 Normalized AIA titers of eight customer dogs treated with the insulin-Fc fusion protein of SEQ ID NO:5 according to scheme 1 as described in Example 18 or scheme 2 as described in Example 19 are shown.

[0030] Figure 7 Normalized AIA titer is shown in a single dog that was treated with the insulin-Fc fusion protein of SEQ ID NO:5 for diabetes and whose treatment was interrupted.

[0031] Figure 8 The illustration shows the number of dogs with AIA after treatment with the insulin-Fc fusion protein of SEQ ID NO:5 produced in the HEK transient cell pool or the CHO stable cell pool.

[0032] Figure 9 Normalized AIA titers of eight dogs treated with the insulin-Fc fusion protein of SEQ ID NO:7 are shown.

[0033] Figure 10 A diagram showing the number of dogs exhibiting AIA after treatment with the insulin-Fc fusion protein of SEQ ID NO:5 or SEQ ID NO:7 for diabetes.

[0034] Figure 11 APC processing of the Fc fusion protein via the Fc(γ) receptor is shown.

[0035] Figure 12 This demonstrates that when BALB / c mice were administered the insulin-Fc fusion protein of SEQ ID NO:15 on days 0, 21, and 46, the anti-SP / RBD mouse IgG titer was absent (not detected).

[0036] Figure 13 This study demonstrates the presence of anti-nucleocapsid protein mouse IgG titer when BALB / c mice were administered the insulin-Fc fusion protein of SEQ ID NO:15 on days 0, 21, and 46.

[0037] Figure 14 The parallel sequence comparison of the SARS-CoV-2 nucleocapsid fragment of SEQ ID NO:10 and the truncated nucleocapsid fragment of SEQ ID NO:11 is shown.

[0038] Figure 15 The parallel sequence comparison of the SARS-CoV-2 nucleocapsid fragment of SEQ ID NO:10 with another truncated nucleocapsid fragment of SEQ ID NO:12 is shown.

[0039] Figure 16 The parallel sequence comparison of the SARS-CoV-2 nucleocapsid fragments of SEQ ID NO:10 and SEQ ID NO:12 with the nucleocapsid fragment of SEQ ID NO:13 is shown.

[0040] Figure 17 The parallel sequence comparison of the SARS-CoV-2 nucleocapsid fragment of SEQ ID NO:10 and the nucleocapsid fragment of SEQ ID NO:8 is shown.

[0041] Figure 18 The parallel sequence comparison of the SARS-CoV-2 nucleocapsid fragment of SEQ ID NO:9 and the nucleocapsid fragment of SEQ ID NO:8 is shown.

[0042] Figure 19 The parallel sequence comparison of the SARS-CoV-2 nucleocapsid fragments of SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:12 with the nucleocapsid fragment of SEQ ID NO:13 is shown.

[0043] Figure 20 The parallel sequence comparison of the SARS-CoV-2 nucleocapsid fragment of SEQ ID NO:12 and the nucleocapsid fragment of SEQ ID NO:13 is shown.

[0044] Figure 21 A 96-well microplate was demonstrated, which can be used as a universal serological assay to evaluate the titer of existing SARS-CoV-2 antibodies in serum. Detailed Implementation

[0045] Novel Coronavirus Disease 2019

[0046] Novel coronavirus disease 2019 (COVID-19) is a severe acute respiratory illness caused by the SARS-CoV-2 virus. SARS-CoV-2 has spread globally and had infected more than 400 million people by the end of 2021. The consensus among experts is that society cannot return to normal unless, and until, a sufficient level of immunity is achieved in the population. It is estimated that to achieve natural herd immunity, at least 70% of the population would need to be infected, which would result in millions of deaths globally—a morally unacceptable outcome.

[0047] ACE2 receptor

[0048] Angiotensin-converting enzyme 2 (ACE2) is the host cell receptor responsible for mediating SARS-CoV-2 infection (i.e., binding to the receptor-binding domain of the SARS-CoV-2 spike protein to infect cells). ACE2 is a type 1 transmembrane metallocarboxypeptidase. Polymerase chain reaction (PCR) analysis has shown that ACE2 is expressed on lung epithelium, vascular endothelium, and specific neuronal cells, which appears to explain the major clinical manifestations of COVID-19, including pulmonary, cardiovascular, and neurological complications. Based on the sequence similarity of the receptor-binding domain between SARS-CoV-2 and SARS-CoV, researchers have shown that SARS-CoV-2 can utilize ACE2 expressed on the surface of human cells to enter ACE2-expressing HeLa cells.

[0049] Convalescent serum used to treat viral patients

[0050] Human convalescent serum has been used as an option for the prevention and treatment of COVID-19, especially in immunocompromised patients.

[0051] Convalescent serum is a form of passive antibody therapy. This therapy involves infusing serum containing antiviral antibodies from infected or recovered individuals into susceptible or infected individuals, providing them with a level of immunity to prevent or reduce the severity of the disease. This treatment differs from vaccines, which induce an immune response, allowing the individual to produce its own antiviral antibodies. Experience from the 2002 SARS-CoV outbreak and the 2009-2010 H1N1 influenza outbreak has shown that serum from patients who have contracted and recovered from the virus (human convalescent serum) contains antibodies capable of neutralizing the virus and can be used as an intervention for individuals with severe symptoms of illness or as a preventative vaccine.

[0052] Using human convalescent serum carries risks and limitations. First, the transfer of blood material from one person to another carries the risk of accidental infection with another infectious disease and the risk of reactions to other serum components. Another challenge with convalescent serum is that some patients recovering from viral illness do not have high-titer neutralizing antibodies. In one case involving another human coronavirus, Middle East Respiratory Syndrome (MERS-CoV), three patients in South Korea received convalescent serum treatment, but only two recipients had neutralizing antibodies in their serum. Among those who do have neutralizing antibodies after recovering from viral illness, some may not have sufficiently high titers to be viable as living donors. Further investigations related to SARS-CoV found that 87 out of 99 convalescent serum samples from SARS patients had neutralizing antibodies, with a geometric mean titer of 1:61. These and other studies indicate that few patients produce high-titer responses, and that neutralizing antibody titers decline over time. Many companies are seeking to overcome this challenge by producing recombinant antibodies rather than relying solely on antibodies from recovered patients; however, the scale of production is insufficient, and the burden of requiring medical interventions every few weeks to months, likely administered intravenously or by infusion, is immense. A significant limitation of recombinant antibody therapy is that existing monoclonal antibody therapies may become ineffective as the SARS-CoV-2 virus mutates. In fact, as of November 2022, all six monoclonal antibody therapies previously approved by the FDA between 2020 and 2022 for the treatment of COVID-19 had their approval revoked due to their lack of ability to neutralize novel Omicron subtype variants of the SARS-CoV-2 virus. This has led to the re-application of convalescent serum to immunocompromised individuals infected with COVID-19, as this serum contains a large number of different antibodies with broad therapeutic potential not found in monoclonal antibodies.

[0053] However, a limitation is that treating COVID-19 patients with convalescent serum relies on preparations containing high-titer SARS-CoV-2 neutralizing antibodies. This requires a large donor pool of individuals who have recovered from the disease or developed antibodies through vaccination or booster vaccines and can donate convalescent serum. Identifying individuals who have already had the disease and developed some immunity presents challenges. COVID-19 exhibits symptoms of varying severity, and many individuals with mild cases may not be aware that they have the disease.

[0054] However, even if recovered patients with high-titer neutralizing antibodies can be identified, plasma from a single individual is unlikely to treat multiple patients. Therefore, while current convalescent serum therapy may be able to prevent or treat COVID-19 in a small number of patients, this solution does not meet the greater human need during and after the pandemic.

[0055] Current Vaccine Overview and Challenges

[0056] Clinicians and researchers around the world have developed various solutions to mitigate the pandemic caused by the SARS-CoV-2 virus. These solutions have included vaccines that can prevent or reduce the severity of COVID-19, and antiviral treatments that reduce the severity and symptoms of the disease when contracted. The foreseeable future anticipates that natural and vaccine-induced immunity is unlikely to be long-lasting; therefore, cost-effective and safe booster vaccines, administered frequently every six months if necessary, will be required to maintain robust immunity in the population. Therefore, the key design features of an effective preventive COVID-19 vaccine are: i) a potent ability to induce SARS-CoV-2 virus neutralizing IgG titers and a significant T helper type 1 (Th1) cell response in both antibody-naïve and antibody-titer subjects (preferably after a single dose); ii) acceptable safety and tolerability characteristics, particularly for inflammation caused by reactive (systemic effects) and injection site (local effects); a favorable cost of goods and services (COG) in terms of manufacturability and vaccine efficacy; dose-frequency and dose levels; and appropriate supply chain pathways, including sufficient shelf life and robust test sample preparation and administration procedures.

[0057] Live attenuated or inactivated whole-virus vaccines represent a classic strategy. The main advantage of whole-virus vaccines is their inherent immunogenicity and ability to stimulate Toll-like receptors (TLRs, including TLR 3, TLR 7 / 8, and TLR 9). However, live virus vaccines typically require extensive additional testing to confirm their safety. This is particularly problematic for coronavirus vaccines, given the findings of increased infectivity following immunization with live or inactivated whole-virus SARS coronavirus vaccines. Johnson & Johnson utilizes its Janssen's... Adenovirus vector platform, based on PER. Cell line technology was used to generate its lead vaccine, JNJ-78436735. This technology attempted to produce a viral vector to replace the whole virus with a supposedly benign adenovirus vector carrying a portion of the SARS-CoV-2 viral DNA. However, the use of JNJ-78436735 resulted in a severely serious adverse event (SAE) that led to the suspension of clinical trials.

[0058] Two other obstacles in the early development of SARS coronavirus vaccines were the discovery of 1) unintended immunomodulatory enhancement in the form of Th2-mediated eosinophilic infiltration and 2) ADE-driven increased viral infectivity, which occurred after challenge infection following immunization with whole-virus and complete SP vaccines. The risk of Th2-mediated eosinophilic infiltration and lung pathology in SARS-CoV-2 infection is still under investigation, but it has been observed in infants and animals challenged with respiratory syncytial virus (RSV) or immunized with a complete RSV vaccine.

[0059] ADE is an unfavorable characteristic of other viral vaccines, including those for original SARS-CoV, dengue virus, and Zika virus infections, in which the vaccine-induced Ab concentration or affinity is too low to neutralize viral infection, instead forming immune complexes with viruses that tend to interact with Fcγ receptors on the surface of bone marrow cells via the Fc domain of the Ab. These Abs do not neutralize viral infection or induce Fcγ-mediated viral clearance (Li), but instead contribute to viral infection by directly increasing viral uptake via Fcγ receptors or by activating downstream pathways to promote intracellular viral replication, thus antagonizing innate immunity (reviewed in Sun). In both ADE and Th2 immune enhancement, there is evidence that feline IgG2a mAb (likely a Th2 isotype) can mediate both unfavorable conditions, while IgG1 mAb (known for its strong effector function, i.e., a Th1 isotype) can avoid this effect.

[0060] In addition to the risk of ADE and / or Th2 immune enhancement, another challenge of viral vector vaccines is the relatively low manufacturable yield, resulting in higher commodity costs (COG) due to egg-based production or cell expression systems (Ewer).

[0061] As an alternative, nucleic acid expression vector vaccine platforms for COVID-19 encode the major coronavirus target antigen (Ag), namely the spike protein (SP), which mediates viral infection through its binding to the host receptor ACE2. BioNTech / Pfizer and Moderna have developed monovalent mRNA vaccines encoding the full-length SP. Recently, bivalent COVID-19 vaccines from Pfizer-BioNTech and Moderna have been approved for use in many countries worldwide as booster vaccines. These bivalent booster vaccines target the original strain as well as the Omeprone BA.4 and BA.5 variants, and their rollout is designed to generate a broader immune response and improve the strength and duration of protection against circulating variants. The concept of immunization using RNA or DNA began in 1993 with promising results showing protective immunity against influenza in mice, but these findings have not translated into similar results in humans for decades. Furthermore, while these RNA and DNA expression vector vaccines are non-replicating, many of them still endogenously produce target viral Ag well after inducing the expected immune response. This could ultimately lead to immune tolerance to the virus, a growing concern and a practical risk that may be the basis for what is now known as COVID-19 mRNA vaccines. Other challenges with these nucleic acid vaccines include low durability of response, potentially requiring overly frequent dosing, and unfavorable COG due to the cumbersome manufacturability of chemical synthesis. Additionally, the inherent instability of RNA necessitates the preservation and transport of the products under frozen conditions, making them difficult to access in most parts of the world. Monovalent vaccines have proven effective in preventing symptomatic SARS-CoV-2 infection of more than 90% of the original SARS-CoV-2 virus, while bivalent vaccines offer protection against some Omeprone variants. However, persistent mutations in the spike protein could lead to decreased vaccine efficacy.

[0062] As an alternative, recombinant subunit vaccines rely on inducing an immune response against SP to prevent it from docking with the host target protein ACE2. These vaccines comprise all or part of the SP, rather than the DNA or RNA encoding the protein, and are then mixed with an adjuvant to enhance the immune response. Because proteins have inherent stability relative to RNA and DNA, subunit vaccines have less stringent storage and transportation requirements. Companies developing recombinant subunit vaccines include Novavax, which has developed and produced immunogenic virus-like nanoparticles based on recombinant expression of SP, NVX-Cov2373, which utilizes the saponin-based adjuvant system Matrix-M. TM Formulated, and Clover Biopharmaceuticals, the company is using its patents. The technology involves developing subunit vaccines composed of trimeric SARS-CoV-2 SP. However, the full-length SP target Ag is known to be expressed at low yields in cellular expression systems, and when used in SARS vaccines, it is known to induce anti-SP IgG titers against non-neutralizing epitopes of SP, which in turn can mediate increased viral infectivity (i.e., ADE) and inflammation caused by pulmonary eosinophilia (i.e., Th2-mediated immune enhancement, discussed below).

[0063] A consortium led by the Texas Children's Hospital Center for Vaccine Development at Baylor College of Medicine has developed and tested a subunit vaccine consisting solely of the receptor-binding domain (RBD) of SARS SP. When formulated with alum, this RBD-based vaccine, in addition to avoiding ADE and immune enhancement, elicits high levels of protective immunity upon homologous viral challenge (Hotez.PJ et al. A novel SARS immunogenic composition (2014) US20160376321). Preliminary findings indicate that SARS and SARS-CoV-2 RBDs exhibit over 80% amino acid similarity and bind to the same ACE2 target, providing an opportunity to develop one of the proteins (Ag) as a subunit vaccine. In fact, proof-of-concept for this subunit vaccine has been successfully demonstrated against the SP / RBD Ag of MERS and SARS-infected coronaviruses.

[0064] Each vaccine strategy has its unique advantages and challenges in terms of production, safety, and efficacy, and all must be managed in the best possible way.

[0065] The SARS-CoV-2 virus has multiple spike protein variants and subvariants. Furthermore, numerous naturally occurring mutations have been found in the SP / RBD of the SARS-CoV-2 spike protein, which may enhance the ability of these variants to evade natural infection or immunity conferred by vaccination. As SARS-CoV-2 variants and mutations evolve, existing vaccines targeting all or part of the SARS-CoV-2 spike protein may become less effective, thus reducing their efficacy. Therefore, a new generation of SARS-CoV-2 vaccines capable of preventing infection by SARS-CoV-2 variants is needed.

[0066] This disclosure relates to a method for preparing and using a novel nucleocapsid-based Fc fusion protein (SARS-CoV-2 N-Fc fusion protein) that can cost-effectively produce large quantities of recombinant subunit vaccines that are potent against variants of the SARS-CoV-2 virus spike protein region and can be transported and stored at mild temperatures. Specifically, this disclosure relates to a method for preparing and using the nucleocapsid-based Fc fusion protein for prophylactic, therapeutic, or booster vaccines that effectively induce antiviral antibodies against the SARS-CoV-2 virus in patients, for example, by reducing the SARS-CoV-2 viral load in cells, thereby reducing viral replication capacity and alleviating symptom severity. Using a prophylactic vaccine based on the novel SARS-CoV-2 N-Fc fusion protein to induce endogenous antibodies targeting the SARS-CoV-2 virus in patients is expected to be significantly more cost-effective than recombinantly generated anti-SARS-CoV-2 therapeutic monoclonal antibodies (which are subsequently injected into patients and gradually become ineffective as mutations occur in the spike protein and RBD regions of the SARS-CoV-2 virus).

[0067] The main structural proteins of the SARS-CoV-2 virus include the membrane protein (M protein), spike protein (S protein), envelope protein (E protein), and nucleocapsid protein (N protein). The N protein is more conserved than the spike protein and has a lower mutation rate. The N protein is highly immunogenic and is expressed in large quantities during infection.(Marra MA, Jones SJ, AstellCR, et al. The Genome sequence of the SARS-associated coronavirus. Science. 2003; 300 (5624): 1399-1404; Drosten C, Günther S, Preiser W, et al. Identification of anovel coronavirus in patients with severe acute respiratory syndrome. N Holmes KV, Enjuanes L. Virology. The SARS coronavirus: a postgenomic era. Science. 2003; 300 (5624): 1377-1378; Rota PA, Oberste MS, Monroe SS, et al. Characterization of anovel coronavirus associated with severe acute respiratory syndrome. Science. 2003; 300 (5624): 1394-1399; Zhu Y, Liu M, Zhao W, et al. Isolation of virus from a SARS patient and genome-wide analysis of genetics Mutations related to pathogenesis and epidemiology from 47 SARS-CoV isolates. VirusGenes. 2005; 30(1):93-102. Engl J Med. 2003; 348(20):1967-1976.; Cong Y, Ulasli M, Schepers H, et al. Nucleocapsid protein recruitment to replication-transcription complexes plays a crucial role in coronaviral life cycle. JVirol. 2020; 94(4):e01925-e19.) Previous studies have shown that high levels of anti-N protein antibodies have been detected in COVID-19 patients, suggesting that the N protein can stimulate an immune response to the SARS-CoV-2 virus in humans.(Algaissi A,AlfalehMA,Hala S,et al.SARS-CoV-2S1 and N-based serological assays reveal rapidseroconversion and induction of specific antibody response in COVID-10patents.Sci Rep.2020;10(1):16561;Jiang HW,Li Y,Zhang HN,et al.SARS-CoV-2

[0001] proteome microarray for global profiling of COVID-19specific IgG andIgM responses. Nat Commun. 2020;11(1):3581.).

[0068] In one example, a pharmaceutical composition of a novel SARS-CoV-2 N-Fc fusion protein vaccine was administered to patients infected with the SARS-CoV-2 virus and suffering from COVID-19 to limit the extent of infection and improve disease progression. For instance, the novel SARS-CoV-2 N-Fc fusion protein is expected to stimulate both humoral and cell-mediated immunity in subjects, generating high levels of IgG and IgM antibodies. These antibodies bind to the N protein of the natural virus, inhibiting viral RNA replication and blocking viral transmission in the body. In another example, the novel SARS-CoV-2 N-Fc fusion protein is expected to produce high levels of interferon-γ (IFN-γ), which plays a crucial role in inducing immune responses by: promoting macrophage activation, mediating antiviral and antibacterial immunity, enhancing antigen presentation, coordinating activation of the innate immune system, coordinating lymphocyte-endothelial cell interactions, regulating Th1 / Th2 balance, and controlling cell proliferation and apoptosis.

[0069] In one instance, a pharmaceutical composition of the novel SARS-CoV-2 N-Fc fusion protein was administered as a prophylactic COVID-19 vaccine to individuals not infected with the SARS-CoV-2 virus, resulting in the individuals developing their own pool of anti-SARS-CoV-2 antibodies and immunity.

[0070] Equivalents and Definitions

[0071] As used herein, the article “a / an” refers to one or more of the grammatical objects of the article, such as at least one / an. When used in conjunction with the term “comprising” as used herein, the word “a / an” may mean “a / an,” but it also aligns with the meanings of “one or more / one or more kinds,” “at least one / at least one,” and “one or more / one or more kinds.” As used herein, the phrase “and / or” when used in a list of two or more items means that any one of the listed items may be used alone, or any combination of two or more listed items may be used. For example, if a composition is described as containing or excluding components A, B, and / or C, the composition may contain or exclude a single A; a single B; a single C; a combined A and B; a combined A and C; a combined B and C; or a combined A, B, and C.

[0072] As used in this article, “about” and “approximately” generally indicate the acceptable degree of error of a given measurement in terms of its properties or precision.

[0073] As used herein, the amount of a molecule, compound, conjugate, or substance that is effective in treating a condition (e.g., the condition described herein), the “therapeutic effective amount,” or the “effective amount” means the amount of that molecule, compound, conjugate, or substance that, after being administered to a subject in a single or multiple doses, results in treating, curing, reducing, alleviating, or improving a subject with a condition (e.g., the condition described herein) in a manner exceeding the outcome expected without such treatment.

[0074] As used herein, the term "analogue" refers to a compound or conjugate that has a chemical structure similar to another compound or conjugate but is different in at least one respect (e.g., the compound or conjugate described herein, such as a nucleocapsid).

[0075] As used in this article, the term "antigen" refers to any substance that causes a patient's immune system to produce antibodies against it. Antigens can be substances from the environment, such as chemicals, bacteria, viruses, or pollen, or they can be formed within the body. An example of an antigen is the SARS-CoV-2 virus.

[0076] As used herein, the term "antibody" or "antibody molecule" refers to an immunoglobulin molecule (Ig), or the immunologically active portion of an immunoglobulin (Ig) molecule, i.e., a molecule containing a binding site that specifically binds to an antigen (e.g., reacts with an antigen in an immune response). As used herein, the term "antibody domain" refers to the variable or constant region of an immunoglobulin. Human antibodies are described in the art as including several classes, such as IgA, IgM, or IgG in the case of mammals (e.g., humans and dogs). The classes of mammalian IgG immunoglobulins can be further subdivided into different subtypes, such as canine IgGA, IgGB, IgGC, and IgGD, and human IgG1, IgG2, IgG3, and IgG4. Those skilled in the art will recognize that immunoglobulin subtypes of a given immunoglobulin class will include amino acid sequences, structural and functional properties that differ from one another (e.g., different binding affinities to Fc(γ) receptors or ACE2 receptors). "Specific binding" or "responding with an immune response" means that the antibody reacts with one or more antigenic determinants of the desired antigen and has a lower affinity for other peptides, e.g., does not react with other peptides.

[0077] As used herein, the term "dimer" refers to a protein or fusion protein comprising two covalently linked polypeptides. In embodiments, two identical polypeptides covalently linked (e.g., via disulfide bonds) form a "homidimer" (in... Figure 1 and Figure 2 The text is presented in a chart format. Figure 1 This is a diagram of the insulin-Fc fusion protein for reference. Figure 2 (Illustration of the novel SARS-CoV-2 N-Fc fusion protein). See also: [link to more details] Figure 1 Insulin peptides (including insulin B-chain analogs linked to insulin A-chain analogs via C-chain peptides) may have one or more amino acid mutations from natural insulin. Insulin peptides are linked to Fc fragments via linkers. Disulfide bonds (the actual total number of disulfide bonds may be greater than or less than...) Figure 1 The quantities shown are homodimers formed from two identical Fc fusion proteins. See reference for more details. Figure 2 The novel SARS-CoV-2 N-Fc fusion protein may include a nucleocapsid protein fragment, which may comprise a portion of the complete SARS-CoV-2 nucleocapsid protein. This nucleocapsid protein fragment may contain one or more amino acid mutations compared to the native SARS-CoV-2 nucleocapsid protein. The nucleocapsid protein fragment is linked to the Fc fragment using a peptide linker. Disulfide bonds are used to construct a homodimer from two identical novel SARS-CoV-2 N-Fc fusion proteins (in fact, the total number of disulfide bonds can be greater than or less than...). Figure 2(The quantities shown). The novel SARS-CoV-2 N-Fc fusion protein homodimer can be encoded by a single nucleic acid molecule, wherein the homodimer is prepared by intracellular recombination by first forming a novel SARS-CoV-2 N-Fc fusion protein monomer, and then assembling two identical novel SARS-CoV-2 N-Fc fusion protein monomers into a homodimer after further intracellular processing.

[0078] As used herein, the terms "multimer," "multimeric," or "multimeric state" refer to a non-covalently associated form of the Fc fusion protein dimer that can be in equilibrium with the Fc fusion protein dimer or can exist as a permanently aggregated form of the Fc fusion protein dimer (e.g., a dimer of the Fc fusion protein homodimer, a trimer of the Fc fusion protein homodimer, a tetramer of the Fc fusion protein homodimer, or a higher-order aggregate containing five or more Fc fusion protein homodimers). It is anticipated that the multimeric form of the Fc fusion protein may possess different physical, stable, or pharmacological activities than the homodimer of the fusion protein.

[0079] As used herein, SARS-CoV-2 nucleocapsid-Fc fusion protein and novel SARS-CoV-2 N-Fc fusion protein (these terms are used interchangeably) refer to the human immunoglobulin Fc domain linked to a SARS-CoV-2 nucleocapsid fragment or its analogues, which can be used to generate antibodies that specifically bind to the N protein of SARS-CoV-2. For ease of reference, unless the context otherwise requires, the term “nucleocapsid” encompasses protein residues that retain the nucleocapsid protein activity and consist of a SARS-CoV-2 nucleocapsid protein fragment. As used herein, the general terms “fusion protein” and “Fc fusion protein” refer to proteins comprising more than one part, for example, from different sources (e.g., different proteins, peptides, cells, etc.), which are covalently linked by peptide bonds. Fc fusion proteins are covalently linked by (i) linking the genes encoding each part into a single nucleic acid molecule, and (ii) expressing the protein encoded by the nucleic acid molecule in a host cell (e.g., HEK cells or CHO cells). Fully recombinant synthesis is superior to methods that separately synthesize therapeutic proteins and Fc fragments and then chemically conjugate them. Chemical conjugation steps and subsequent purification processes increase production complexity, reduce product yield, and increase costs.

[0080] As used herein, the terms “bioactivity,” “activity,” “biological activity,” “potency,” “bioactive potency,” or “biopotency” refer to the extent to which an Fc fusion protein binds to or activates a cellular receptor and / or produces or reduces a native or foreign substance. As used herein, “in vitro activity” or “receptor activity” refers to the affinity of an Fc fusion protein for binding to a cellular receptor and is typically measured by the concentration of Fc fusion protein that achieves half of its maximum binding (i.e., the EC50 value). For example, the “bioactivity” of a novel SARS-CoV-2 N-Fc fusion protein refers to the extent to which the novel SARS-CoV-2 N-Fc fusion protein induces the production of anti-SARS-CoV-2 antibodies in cellular assays or target subjects.

[0081] As used herein, the terms “biosynthesis,” “recombinant synthesis,” or “recombinant preparation” refer to the process of expressing an Fc fusion protein within a host cell by transfecting the host cell with a nucleic acid molecule (e.g., a vector) encoding an Fc fusion protein (e.g., where the entire Fc fusion protein is encoded by a single nucleic acid molecule). Exemplary host cells include mammalian cells, such as HEK293 cells or CHO cells. Cells can be cultured using standard methods in the art, and the expressed Fc fusion protein can be harvested and purified from cell cultures using standard methods in the art.

[0082] As used herein, the term "cell surface receptor" refers to a molecule (such as a protein) that is typically found on the outer surface of a cell membrane and interacts with soluble molecules (e.g., molecules circulating in the blood supply). In some embodiments, cell surface receptors may include host cell receptors (e.g., ACE2 receptors) or Fc receptors that bind to Fc fragments or Fc regions of antibodies (e.g., Fc(γ) receptors, such as Fc(γ) receptor I, or Fc neonatal receptors, such as FcRn). As used herein, “in vitro activity” or “Fc(γ) receptor activity” or “Fc(γ) receptor binding” or “FcRn receptor activity” or “FcRn binding” refers to the affinity of an Fc fusion protein for binding to an Fc receptor (e.g., an Fc(γ) receptor or an FcRn receptor), and is typically measured by the concentration of the Fc fusion protein that causes it to reach half of its maximum binding capacity (i.e., the EC50 value), as measured in an assay (e.g., an enzyme-linked immunosorbent assay (ELISA)) using an OD 450 nm value measured on a microplate reader.

[0083] As used herein, the term "immunogenic" or "immunogenicity" refers to the ability of a given molecule (e.g., the Fc fusion protein of the present invention) to stimulate the immune system of a target subject such that, upon administration of the molecule, the subject produces antibodies (i.e., antidrug antibodies or ADAs) capable of binding all or a specific portion of the molecule. As used herein, the terms "neutralization," "neutralizing antibody," or "neutralizing antidrug antibody" refer to the ability of an antibody to interfere with all or part of the biological activity of the Fc fusion protein in a target subject. For example, in the case of administration of a novel SARS-CoV-2 N-Fc fusion protein molecule (or a pharmaceutical composition thereof) to a human, immunogenicity refers to antibodies binding to the SARS-CoV-2 nucleocapsid portion of the molecule, because the hIgG-Fc portion of the molecule is endogenous to humans and is therefore unlikely to elicit anti-hIgG-Fc antibodies. Similarly, antibodies generated by administration of novel SARS-CoV-2 N-Fc fusion protein molecules (or pharmaceutical compositions thereof) have a neutralizing effect, while those anti-SARS-CoV-2 antibodies inhibit the binding between SARS-CoV-2 N protein and host cells, the binding process of which is directly related to the bioactivity of SARS-CoV-2 RBD in the subject.

[0084] As used herein, the term "monomer" refers to a protein or fusion protein comprising a single polypeptide. In embodiments, a "monomer" is a protein or fusion protein, such as a single polypeptide comprising a nucleocapsid fragment polypeptide and an Fc fragment polypeptide, wherein the nucleocapsid fragment and the Fc fragment polypeptide are linked by peptide bonds to form a single polypeptide. In embodiments, the monomer is encoded by a single nucleic acid molecule.

[0085] As used in this article and as Figure 1 and Figure 2 As shown, "N-terminus" refers to the starting point of a protein or polypeptide that is initiated by an amino acid containing a free amine group, which is the α-amino group of the amino acid (e.g., a free amino group covalently linked to a carbon atom located adjacent to a second carbon atom, wherein the second carbon atom is part of the carbonyl group of the amino acid). As used herein and as... Figure 1 and Figure 2 As shown, "C-terminus" refers to the end of a protein or polypeptide, which is terminated by an amino acid containing a carboxylic acid group, wherein the carbon atom of the carboxylic acid group is located adjacent to the α-amino group of the amino acid.

[0086] As used herein, the term "carrier" refers to a diluent, excipient, solvent, etc., in which one or more Fc fusion proteins can be dispersed, emulsified, or encapsulated for administration. A suitable carrier will be pharmaceutically acceptable. As used herein, the term "pharmaceutically acceptable" means that it is not biologically or otherwise undesirable because it can be administered to a subject without excessive toxicity, irritation, or anaphylactic reactions, and does not cause unacceptable biological effects or interact harmfully with any other component of the composition containing it. As is well known to those skilled in the art, pharmaceutically acceptable carriers will be naturally selected to minimize any degradation of the compound or other reagent and to minimize any adverse side effects in the subject. Pharmaceutically acceptable ingredients include those that can be used for veterinary purposes as well as for human pharmaceutical purposes, depending on the route of administration. Any carrier compatible with one or more excipients and one or more Fc fusion proteins can be used.

[0087] As used in this article, "pharmacodynamics" or "PD" generally refers to the biological effect of the Fc fusion protein in subjects. For example, in this article, the PD of the novel SARS-CoV-2 N-Fc fusion protein refers to the measurement of anti-SARS-CoV-2 antibody titer over time in subjects after administration of the novel SARS-CoV-2 N-Fc fusion protein.

[0088] As used herein, “pharmacokinetic” or “PK” generally refers to the characteristic interaction between an Fc fusion protein and the subject’s body in terms of its absorption, distribution, metabolism, and excretion. For example, in this article, PK refers to the concentration of the novel SARS-CoV-2N-Fc fusion protein in the blood or serum of a subject at a given time after administration of the novel SARS-CoV-2N-Fc fusion protein. As used herein, “half-life” refers to the time it takes for the concentration of the Fc fusion protein in the blood or serum of a subject to reach half of its original value, calculated from a first-order exponential decay model of drug elimination. Fc fusion proteins with longer “half-lives” exhibit a longer duration of action in target subjects.

[0089] As used herein, the terms “sequence identity,” “sequence homology,” “homology,” or “identical” in the context of amino acid or nucleotide sequences describe the presence of identical nucleotide or amino acid residues in the variant and the reference sequence when a specified contiguous segment of the variant’s nucleotide or amino acid sequence is aligned and compared with the nucleotide or amino acid sequence of the reference sequence. Methods for sequence alignment and determining identity between sequences are known in the art, including the use of ClustalOmega, which organizes, aligns, and compares the similarity of sequences, wherein the software highlights each sequence position and compares all sequences at that position, and assigns one of the following scores: "*" (asterisk) indicates a sequence position with a single completely conserved residue; ":" (colon) indicates conservation between groups with a strong similarity attribute score greater than 0.5 in the Gonnet PAM 250 matrix; "." (period) indicates conservation between groups with a weak similarity attribute score less than or equal to 0.5 in the Gonnet PAM 250 matrix; "-" (hyphen) indicates a sequence vacancy, indicating the absence of local homology in a specific comparison set within a certain range of sequences; and a space "" indicates little or no sequence homology at that specific position in the compared sequences.

[0090] For optimal alignment of two nucleotide sequences, consecutive segments of the variant nucleotide sequence may have additional or missing nucleotides relative to the reference nucleotide sequence. Similarly, for optimal alignment of two amino acid sequences, consecutive segments of the variant amino acid sequence may have additional or missing amino acid residues relative to the reference amino acid sequence. In some embodiments, the consecutive segments used for comparison with the reference nucleotide or amino acid sequence will comprise at least 6, 10, 15, or 20 consecutive nucleotide or amino acid residues, and may be 30, 40, 50, 100, or more. Increased sequence identity associated with the inclusion of vacancies in the variant nucleotide or amino acid sequence can be corrected by assigning a vacancy penalty. Methods for sequence alignment are known in the art.

[0091] In implementations, mathematical algorithms are used to determine the percentage of identity or "homology" between two sequences. For example, the Smith-Waterman homology search algorithm is used to determine the percentage of identity between amino acid sequences, employing an affine 6-vacancy search with a 12 vacancy opening penalty and a 2 vacancy extension penalty, and a BLOSUM matrix of 62. In another implementation, the Smith-Waterman homology search algorithm is used to determine the percentage of identity between nucleotide sequences, employing a 25 vacancy opening penalty and a 5 vacancy extension penalty. This determination of sequence identity can be performed using, for example, a DeCypher hardware accelerator from TimeLogic.

[0092] As used herein, the term "homology" is used to compare two or more proteins by locating common structural features and common spatial distributions, such as β-chains, helices, and folds. Therefore, homologous protein structures are defined by spatial analysis. Measuring structural homology involves computing the geometric topological features of space. One approach used to generate and analyze three-dimensional (3D) protein structures is homology modeling (also known as comparative modeling or knowledge-based modeling), which works by finding similar sequences based on the fact that 3D similarity reflects 2D similarity. Homologous structures do not imply that sequence similarity is a necessary condition.

[0093] As used herein, the terms “subject” and “patient” are intended to include mice, non-human primates (NHPs), rabbits, canines, and humans. Exemplary canine subjects include dogs with a disease or condition (e.g., diabetes or another disease or condition described herein), or healthy subjects. Exemplary human subjects include individuals with a disease, such as COVID-19, including variants of COVID-19 or SARS-CoV-2 infection, or another virus, who have a history of the disease or condition described herein, or healthy subjects.

[0094] As used herein, the terms “potency” or “yield” refer to the amount of fusion protein product (e.g., the Fc fusion protein described herein) produced per volume of cell culture by biosynthesis (e.g., in mammalian cells, such as HEK293 cells or CHO cells). The amount of product may be determined at any step of the production process (e.g., before or after purification), but yield or potency is always expressed per volume of raw cell culture. As used herein, the terms “product yield” or “total protein yield” refer to the total amount of Fc fusion protein expressed by cells and purified by at least one affinity chromatography step (e.g., protein A or protein G) and include Fc fusion protein monomers, homodimers of Fc fusion protein, and higher-order molecular aggregates of Fc fusion protein homodimers. As used herein, the terms “homodimer percentage” or “homodimer %” refer to the proportion of fusion protein product (e.g., the Fc fusion protein described herein) as a desired homodimer. As used herein, the term "homodimer potency" refers to the product of the homodimer percentage and total protein yield per volume of cell culture reported after the protein A purification step.

[0095] As used herein, the terms “treatment” or “treat” or “treating” for a subject suffering from a disease or condition refer to an intervention intended to prevent the development of an infection or to alter the pathology of an infection. Therefore, “treatment” refers to therapeutic treatment and preventative or preventative measures. Therapeutic agents can directly reduce the pathology of an infection or make the infection more susceptible to treatment by other therapeutic agents or, for example, by the host's immune system. Improvement following treatment may manifest as a reduction or elimination of such symptoms. Therefore, compositions can be used to treat an infection by preventing the development of observable clinical symptoms of the infection, and / or reducing the incidence or severity of clinical symptoms and / or the effects of the infection, and / or shortening the duration of the infection / symptom / effect. Treating a subject suffering from a disease or condition can refer to a subject suffering from a disease or condition receiving a treatment regimen, such as administration of a fusion protein (like the Fc fusion protein described herein), or a pharmaceutical composition of a fusion protein (like the Fc fusion protein described herein), such that at least one symptom of the disease or condition is cured, healed, alleviated, relieved, altered, remedied, improved, or ameliorated. Treatment includes administering an effective dose to reduce, alleviate, alter, remedy, improve, enhance, or influence a disease or condition, or its symptoms. Treatment includes administering an effective dose to induce antibodies against the disease or condition in normal subjects or subjects with a history of the disease or condition. Treatment may suppress the worsening or exacerbation of symptoms of the disease or condition.

[0096] As used herein, a "preventive vaccine" refers to the introduction of an antigen into a patient's body with the goal of causing the patient's immune system to produce antibodies against that antigen and increasing or improving the subject's immune response to the associated disease or virus. In other words, a vaccinated subject has a higher degree of resistance to the disease or symptoms caused by the associated virus compared to an unvaccinated subject. This resistance can be manifested by a reduction in the severity or duration of disease symptoms, a decrease or elimination of viral shedding, and, in some cases, the presence of symptoms of infection observable in the vaccinated subject. In this implementation, the patient treated with the preventive vaccine did not have antibodies against the antigen prior to treatment (also referred to as the patient being "antibody-naïve").

[0097] As used in this article, a "therapeutic vaccine" refers to a treatment that introduces an antigen into a patient who already has a related disease or virus. The goal is for the patient's immune system to produce antibodies against that antigen, enabling the patient's body to fight the disease or virus more effectively.

[0098] As used in this article, a "boost vaccine" refers to an additional dose of vaccine administered after a patient has previously received an initial dose of a vaccine, or after a patient has acquired antibodies through having and recovering from a related disease or virus. In some instances, additional doses of vaccine are required periodically to "enhance" a patient's immunity to the disease or the virus that causes the antigen by increasing the patient's antigen-antibody titer.

[0099] As used herein, when referring to the SARS-CoV-2 nucleocapsid, such as an amino acid in a portion of a SARS-CoV-2 nucleocapsid fragment, the referenced amino acid position is referred to as the position of the amino acid in the SARS-CoV-2 nucleocapsid of SEQ ID NO:10. For example, referring to a mutation at amino acid position 41 of a SARS-CoV-2 nucleocapsid fragment refers to amino acid position 41 in SEQ ID NO:10, even if the SARS-CoV-2 nucleocapsid fragment includes only a portion of SEQ ID NO:10, such as only the portion of SEQ ID NO:10 starting from amino acid position 41.

[0100] As used herein, a “nucleocapsid fragment” refers to a portion of the novel SARS-CoV-2 N-Fc fusion protein, which comprises portions of the nucleocapsid protein of the SARS-CoV-2 virus as shown in SEQ ID NO:10. In this example, the nucleocapsid fragment is linked to a human Fc fragment or an analogue thereof, such as… Figure 2 As shown.

[0101] Basic principles of Fc fusion protein vaccines

[0102] As mentioned above, compared to inactivated or attenuated live viruses and nucleic acid vector-based vaccine formats, recombinant protein-based subunit vaccine methods offer advantages in safety and multiple booster doses, and also allow for the selective use of the most dominant epitope to generate effective neutralizing antibody (Ab) titers. Furthermore, such protein-based vaccines are more cost-effective for large-scale production and are stable at mild temperatures, facilitating transportation and storage. However, considering the challenge of inducing strong protective immune responses in immunologically naive populations with recombinant SARS-CoV-2 spike protein or nucleocapsid subunit vaccines, the antigen must be modified and / or formulated to possess additional immunomodulatory features to overcome the activation thresholds of naive T and B cells.

[0103] Experimental experience with insulin-Fc fusion protein in canines

[0104] An example of a fusion protein formed by linking a therapeutic protein to the Fc domain of an immunoglobulin is an insulin-Fc fusion protein. This construct has been used to provide ultra-long-acting basal insulin therapy to diabetic subjects. The combination of an insulin analog as a therapeutic protein with the Fc domain via a peptide linker has been shown to achieve significantly longer activity in vivo, on a daily basis. An example of an ultra-long-acting insulin-Fc fusion protein for the treatment of diabetes in cats and dogs is described in WO2020006529A1. In the example from WO 2020006529A1, an exemplary insulin analog is:

[0105] FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCT STCSLDQLENYC(SEQ ID NO:4)

[0106] An exemplary connector for linking a therapeutic protein (i.e., an insulin analog) to the Fc domain is: GGGGGQGGGGGQGGGGGG (SEQ ID NO:3).

[0107] Insulin-Fc fusion protein molecules for ultra-long-acting treatment of diabetes in a given species (e.g., dogs, cats, or humans) should be capable of production in mammalian cells (e.g., human embryonic kidney (HEK, e.g., HEK293) cells) with an acceptable potency of the desired homodimer product (e.g., a homodimer potency greater than 50 mg / L from transiently transfected HEK cells, greater than 75 mg / L from transiently transfected HEK cells, greater than 100 mg / L from transiently transfected HEK cells, etc.). Experience suggests that homodimer potencies below 50 mg / L are unlikely to produce commercially viable homodimer potencies in Chinese hamster ovary (CHO) cells, thus failing to meet the stringent low production cost requirements for veterinary products.

[0108] The insulin-Fc fusion protein for dogs, WO 2020006529A1 (incorporated herein by reference) and described herein, was produced in HEK cells according to Example 10 or in CHO cells according to Example 11. The insulin-Fc fusion protein was purified according to Example 12. Using conventional purification methods, compounds comprising only canine IgGA and canine IgGB immunoglobulin Fc fragments showed any considerable protein yield. The structure of the insulin-Fc fusion protein was confirmed by non-reducing and reducing CE-SDS according to Example 13, and the sequence was confirmed by LC-MS with glycan removal according to Example 14. Purity was measured according to Example 15 (e.g., assessed by the percentage of homodimer in the fusion protein yield). The insulin-Fc fusion protein in the canine IgGA form was highly aggregated and exhibited low levels of biological activity, while the insulin-Fc fusion protein in the canine IgGB form showed low degree of aggregation (i.e., high homodimer %), high desired homodimer titer (i.e., homodimer titer greater than 50 mg / L), and considerable levels of long-term glucose-reducing biological activity in dogs. Therefore, the canine IgGB (SEQ ID NO:2) immunoglobulin Fc fragment is the preferred Fc fragment for canine insulin-Fc fusion protein.

[0109] DCPKCPAPEMLGGPSVFIFPPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISK ARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ ID NO:2)

[0110] Exemplary canine ultra-long-acting insulin-Fc fusion proteins include insulin analogs of SEQ ID NO:4 having a canine native IgGB fragment of SEQ ID NO:2 via a peptide linker of SEQ ID NO:3:

[0111] FVNQHLCGSHLVEALELVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFNGT YRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ ID NO:5)

[0112] The binding of the insulin-Fc fusion protein of SEQ ID NO:5 to Fc(γ) receptor I (RI) was evaluated according to Example 16. Since canine receptor I is not commercially available, human Fc(γ) receptor I (i.e., rhFc(γ) receptor I) was used as an alternative mammalian receptor. OD values ​​proportional to the binding of rhFc(γ) receptor I to SEQ ID NO:5 were plotted against the logarithmic concentration of rhFc(γ) receptor I added to each protein to generate binding curves using GraphPad Prism software. Results are as follows: Figure 3 As shown, the OD450 value increases with increasing dose of the insulin-Fc fusion protein of SEQ ID NO:5 for all Fc(γ) receptors.

[0113] In vivo pharmacodynamics (PD) following periodic administration of the insulin-Fc fusion protein of SEQ ID NO:5 produced in HEK cells according to Example 10 was evaluated according to Example 18 or Example 19. The test population consisted of six beagle dogs with diabetes induced by alloxan-streptozotocin, each weighing approximately 10 kg. Anti-insulin antibody (AIA) titers were measured weekly for eight weeks in six chemically induced diabetic beagle dogs serving as subjects for laboratory testing of SEQ ID NO:5, according to Example 18. Figure 4 The AIA titer of the insulin-Fc fusion protein of SEQ ID NO:5 in six beagle dogs with chemically induced diabetes was shown after eight consecutive weekly administrations. Figure 5The data show the percentage change in AIA titer of the insulin-Fc fusion protein of SEQ ID NO:5 in six beagle dogs with chemically induced diabetes after eight consecutive weekly administrations starting from day 0 of the trial. The data indicate that there was no significant increase in AIA titer in the beagle dogs compared to eight weekly administrations of the insulin-Fc fusion protein of SEQ ID NO:5.

[0114] Based on these positive laboratory test results in chemically induced diabetic beagle dogs, field trials were initiated in actual customer-owned, naturally occurring diabetic dogs of different ages, breeds, and degrees of diabetes, according to Example 18 or Example 19. The customer dogs in the field trials had been receiving insulin treatment with known veterinary or human insulin products prior to the start of the trials, and were administered SEQ ID NO:5 according to Protocol 1 described in Example 18 or Protocol 2 described in Example 19.

[0115] During the treatment regimen according to Example 19, AIA titer was measured weekly again. Unexpectedly, several (8 / 20) of the client dogs in this “wild” patient population showed a significant increase in anti-insulin antibodies compared to results obtained in chemically induced diabetic beagle dogs. A normalized AIA titer of 0.15 was considered the minimum measurement at which client dogs were considered immunogenic to SEQ ID NO:5. For client dogs with a non-zero AIA titer at the start of treatment, the insulin-Fc fusion protein of SEQ ID NO:5 was considered immunogenic to that particular client dog if AIA increased more than double after treatment with the insulin-Fc fusion protein of SEQ ID NO:5. Figure 6 This is a normalized AIA potency plot for each client dog, where the insulin-Fc fusion protein of SEQ ID NO:5 is considered immunogenic, as measured weekly during treatment (treatment was administered according to Protocol 1 as described in Example 18 or Protocol 2 as described in Example 19, with each dog's treatment indicated). Figure 6 In each dog shown, the therapeutic effect of AIA neutralization of the insulin-Fc fusion protein of SEQ ID NO:5 rendered it unable to control the glycemic levels of the diabetic dogs owned by the client. The observed immunogenicity was also unexpected, as the insulin analog portion of the insulin-Fc fusion protein is a nearly natural canine peptide. Furthermore, the IgGB Fc fragment portion of the insulin-Fc fusion protein is a natural canine Fc fragment. The results indicate that the specific activity of the canine IgGB Fc fragment can induce a significant and durable increase in antibody titer specific to the therapeutic protein region of the fusion protein (i.e., insulin). In addition to showing a significant increase in neutralizing AIA titer, these dogs remained healthy in other respects with repeated dosing and did not exhibit any signs of treatment-related anaphylactic reactions or cytokine storms.

[0116] In some cases, administration of the insulin-Fc fusion protein of SEQ ID NO:5 was interrupted when the customer dog began to exhibit high levels of AIA, at which point the AIA titer began to decline (see example...). Figure 4 Dog in 2). Figure 7 This is a normalized AIA potency plot for dog 2 during a 12-week period of once-weekly dosing. It can be seen that AIA potency begins to increase significantly after dose 4 (day 21), and begins to increase sharply after dose 6 (day 35). Dosing is discontinued after dose 8 (day 49), and no further medication is administered on day 56 or 63. Figure 7 The study showed that the increase in AIA titer slowed immediately, and then began to decline after day 56. The dosing regimen was resumed on day 70, with administration repeated on day 70 and again on day 77. The increase in AIA titer following resumption of dosing on day 70 matched or exceeded the maximum AIA titer increase over 7 weeks until discontinuation of dosing, indicating that the anti-drug antibody titer recovered unexpectedly in a significantly shorter time compared to the improvement from initial continuous dosing. This strong memory response may indicate a reactive memory immune cell population.

[0117] Another observation from a field trial in diabetic dogs owned by a customer was that there was a significant difference between dogs treated with the insulin-Fc fusion protein of SEQ ID NO:5 prepared in CHO cells according to Example 11 and the insulin-Fc fusion protein of SEQ ID NO:5 prepared in HEK cells according to Example 10. The CHO-prepared insulin-Fc fusion protein of SEQ ID NO:5 showed a significantly higher prevalence of antidrug antibodies, such as... Figure 8 As shown.

[0118] Each IgG fragment contains a conserved asparagine (N)-glycosylation site in the CH2 domain of each heavy chain in the Fc region. In this paper, the symbol used to refer to the conserved N-glycosylation site is "cNg" (e.g., Figure 1 and Figure 2 (As shown). In therapeutic monoclonal antibodies, glycosylation occurs at the conserved amino acid N297 in the CH2 region (e.g., Figure 1 and Figure 2(As shown). For insulin-Fc fusion proteins, the absolute position of the cNg site relative to the N-terminus of the B chain of the insulin-Fc fusion protein depends on the length of the insulin polypeptide, the length of the linker, and any amino acids deleted in the Fc fragment preceding the cNg site. In this paper, the symbol used to refer to the absolute position of the cNg site in a given insulin-Fc fusion protein sequence (as measured by counting from the N-terminus of the B chain of the insulin-Fc fusion protein) is “NB (number)”. For example, if the cNg site is located at the 151st amino acid position counting from the N-terminus of the B chain, the absolute position of that site is called cNg-NB151. As another example, if the cNg site is located at the 151st amino acid position counting from the N-terminus of the B chain, and the asparagine at that site is mutated to a serine, the mutation is labeled “cNg-NB151-S”.

[0119] One possible difference between the fusion protein recombinantly produced in HEK cells according to Example 10 and the fusion protein recombinantly produced in CHO cells according to Example 11 is the composition of the oligosaccharide attached at the cNg site. Given the interaction between the canine IgGB subtype and the Fc(γ) receptor, there is a risk of unintended immunogenicity following repeated injections. One method for reducing Fc(γ) interaction involves deglycosylation of the Fc fragment or preventing its glycosylation during host cell synthesis. Due to the therapeutic value of antibody-neutralizing drugs, constructing antibodies is clearly unsuitable for treating chronic diseases such as diabetes. Therefore, this has led to attempts to construct non-glycosylated canine insulin-Fc fusion proteins. One method for removing the attached glycan from the synthesized insulin-Fc fusion protein is to mutate the cNg site to completely prevent glycan attachment during host cell production. In this document, the symbol used to describe cNg mutations is cNg- (the substituted amino acid). For example, if the asparagine at the cNg site is mutated to serine, the mutation is labeled "cNg-S".

[0120] The canine insulin-Fc fusion protein was designed, comprising an IgGB Fc fragment of SEQ ID NO:2 having cNg-S (hereinafter bold residues) at position 73, a linker of SEQ ID NO:3, and the following insulin analogues:

[0121] FVNQHLCGSHLVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCT STCSLDQLENYC(SEQ ID NO:6)

[0122] The resulting insulin-Fc fusion protein is shown below:

[0123] FVNQHLCGSHLVEALALVCGERGFHYGGGGGGSGGGGGIVEQCCTSTCSLDQLENYCGGGGGQGGGGQGGGGQGGGGGDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVDLDPEDPEVQISWFVDGKQMQTAKTQPREEQFSGT YRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPG(SEQ ID NO:7)

[0124] The insulin-Fc fusion protein of SEQ ID NO:7 was produced in HEK cells according to Example 10 or in CHO cells according to Example 11. The insulin-Fc fusion protein was purified according to Example 12. The structure of the insulin-Fc fusion protein was confirmed by non-reducing and reducing CE-SDS according to Example 13, and the sequence was confirmed by LC-MS with glycan removal according to Example 14. Purity was measured according to Example 15 (e.g., assessed by the percentage of homodimers in the yield of the fusion protein).

[0125] This fusion protein exhibits similar expected in vitro and in vivo properties to SEQ ID NO:5. Figure 3 As shown, the only difference is that the insulin-Fc fusion protein of SEQ ID NO:7 has a significantly reduced Fc(γ)RI binding affinity compared to the insulin-Fc fusion protein of SEQ ID NO:5. A field trial was conducted on five diabetic customer dogs of different ages, breeds, and degrees of diabetes. All five customer dogs in the field trial were on insulin therapy with known veterinary or human insulin products prior to the start of the trial, and were given the insulin-Fc fusion protein of SEQ ID NO:7 once weekly.

[0126] According to Example 19, AIA titer was measured again weekly or as frequently as possible during the treatment course. None of the client dogs in this “wild-type” patient population exhibited insulin anti-drug antibodies when administered the non-glycosylated insulin Fc fusion protein of SEQ ID NO:7, compared to dogs receiving the insulin-Fc fusion protein of SEQ ID NO:5. A normalized AIA titer of 0.15 was considered the minimum measurement value at which client dogs were considered immunogenic to SEQ ID NO:7. For client dogs with a non-zero AIA titer at the start of treatment, immunogenicity was considered if AIA increased more than double after treatment with the insulin-Fc fusion protein of SEQ ID NO:7. Figure 9 The normalized AIA potency plots, measured for each client dog during the duration of treatment, showed that none of the five client dogs in this "wild" patient population exhibited insulin resistance antibodies when administered the non-glycosylated insulin-Fc fusion protein of SEQ ID NO:7. Figure 10 As shown, this contrasts with dogs that received the insulin-Fc fusion protein of SEQ ID NO:5, of which a total of twelve dogs exhibited insulin-resistant antibodies, compared to none of the dogs that received the non-glycosylated insulin-Fc fusion protein of SEQ ID NO:7 exhibiting insulin-resistant antibodies.

[0127] In summary, the results unexpectedly demonstrate that, outside of laboratory animal populations, certain Fc fusion proteins possess the potential to induce high-titer antibodies against therapeutic peptides or protein fractions, and that this response may subsequently present the therapeutic peptides or protein fractions. Furthermore, these preliminary data suggest that inducing antibodies against specific therapeutic proteins or peptides is more likely in individuals who have already developed an immune response to them. These results contrast with numerous published findings demonstrating the potential of Fc fusion proteins to induce immune tolerance to fused therapeutic peptides or proteins, and that haptens such as DNPs, nucleosides, or peniciloyl groups are highly tolerable hapten carrier conjugates when chemically coupled to IgG carriers.

[0128] For therapeutic proteins used to treat chronic diseases such as diabetes, such as insulin, anti-insulin antibodies render treatment ineffective. However, the aforementioned findings have led to unique insights into how to effectively engineer Fc fusion proteins to induce antibodies against, for example, viral antigens to neutralize their activity. Based on field experiments, the desired Fc fusion protein should be at least native to the target subject (e.g., human Fc or hFc for human subjects, canine Fc or dFc for canine subjects), appropriately glycosylated at the Fc-cNg site, and capable of binding to the Fc(γ)I receptor. These findings provide opportunities for developing novel therapeutic Fc fusion proteins against, for example, the novel coronavirus SARS-CoV-2.

[0129] Novel SARS-CoV-2 Fc fusion protein

[0130] The COVID-19 outbreak poses a serious threat to public health. There is an urgent need for safe and effective solutions to reduce or prevent infection by its pathogen, the SARS-CoV-2 virus. Surface glycoproteins, including the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein (SP), have been identified, and SARS-CoV-2SP / RBD has been found to bind strongly to the human and bat angiotensin-converting enzyme 2 (ACE2) receptor. SARS-CoV-2SP / RBD is a foreign antigen, and a fusion protein comprising this antigen and a glycosylated human immunoglobulin Fc fragment (referred to herein as the SARS-CoV-2-RBD-hIgG-Fc fusion protein or SP / RBD-Fc fusion protein) represents a promising approach to constructing fusion proteins that can amplify existing antibody titers in patients or induce new antibody titers in patients with no or low immune responses to SARS-CoV-2. However, the continuous evolution of spike protein variants over time presents a challenge, rendering existing vaccines targeting extinct variants less effective or ineffective in preventing COVID-19. The method for preparing and using Fc fusion proteins for prevention or booster vaccines can effectively induce patients to produce antiviral antibodies against the SARS-CoV-2 virus in response to novel mutations in the SARS-CoV-2 spike protein, thereby meeting this urgent need and having significant public health value.

[0131] Therefore, the goal is to construct an Fc fusion protein that includes a portion of the SARS-CoV-2 nucleocapsid protein (or an analogue thereof) and a human Fc fragment (e.g., human IgG1 or hIgG1) containing sites or residues that are prone to glycosylation to construct a producible conjugate, which presents the antigen (SARS-CoV-2 nucleocapsid) in a novel manner, enabling patients to rapidly produce high-titer anti-SARS-CoV-2 antibodies.

[0132] like Figure 2 As shown, the novel SARS-CoV-2 N-Fc fusion protein includes a bivalent analog of the SARS-CoV-2 nucleocapsid protein that is partially recombinantly fused with human IgG1 Fc. This fusion protein will help deliver the nucleocapsid antigen to local APCs, which can internalize the novel SARS-CoV-2 N-Fc nucleocapsid fusion protein via the Fc(γ) receptor.

[0133] The complete nucleocapsid protein of SARS-CoV-2 is shown below (GenBank:QHD43416.1):

[0134] MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASS RSSSRSRNSSRNSTPGSSRGTSPARMAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFP(SEQ IDNO:10)

[0135] Previous studies on insulin-Fc fusion proteins, such as those described in WO 2018107117A1 and WO 2020006529A1, have demonstrated that the choice of protein sequence, adaptor sequence, and Fc domain composition can potentially affect protein yield, purity, and biological activity.

[0136] When selecting viral proteins for the novel SARS-CoV-2 N-Fc fusion protein, it is conceivable that viral subtypes including portions of the nucleocapsid could be chosen. The viral protein used for the novel SARS-CoV-2 N-Fc fusion protein may include all or part of the SARS-CoV-2 nucleocapsid protein. The viral protein of the novel SARS-CoV-2 N-Fc fusion protein may include all or part of the non-nucleocapsid portion of the SARS-CoV-2 virus, such as the spike protein portion, M protein portion, or E protein portion of SARS-CoV-2. For example, the viral protein of the novel SARS-CoV-2 N-Fc fusion protein may include all or part of the SARS-CoV-2 nucleocapsid and all or part of the non-nucleocapsid portion of the SARS-CoV-2 virus. For example, one or more amino acids in the nucleocapsid fragment of the novel SARS-CoV-2 N-Fc fusion protein may be mutated from its native state.

[0137] Based on experience in producing insulin-Fc fusion proteins, different viral protein designs will result in varying protein yields of novel SARS-CoV-2N-Fc fusion proteins. For example, a larger or shorter portion of the nucleocapsid protein sequence of SEQ ID NO:10 can be selected, and optionally, certain amino acids can be mutated to produce the desired viral portion of the Fc fusion protein. The protein yield produced when the selected viral protein is attached to the Fc fragment can be determined experimentally. Furthermore, the length and composition of the linker connecting the selected viral protein to the Fc fragment will similarly affect protein yield, as will the selection of the Fc fragment and the portion of the Fc fragment hinge region attached to the viral protein.

[0138] Figure 2 An illustration of an exemplary novel SARS-CoV-2 N-Fc fusion protein according to this disclosure is shown. This novel SARS-CoV-2 N-Fc fusion protein may include a peptide linker. In an example, some amino acids in the nucleocapsid fragment of the novel SARS-CoV-2 N-Fc fusion protein may be mutated from their native state. In an example, a therapeutic protein comprising a SARS-CoV-2 nucleocapsid protein fragment is located on the N-terminal side of the Fc fragment. The novel SARS-CoV-2 N-Fc fusion protein includes a domain oriented as follows from the N-terminus to the C-terminus: (N-terminus)--therapeutic protein--peptide linker--Fc fragment--(C-terminus) (e.g., (N-terminus)--nucleocapsid protein--peptide linker--Fc fragment--(C-terminus)). Figure 2 The nucleocapsid fragment of the novel SARS-CoV-2 N-Fc fusion protein shown may be well expressed or poorly expressed as part of a fusion protein recombinantly produced in a host cell (i.e., produced in HEK cells according to Example 1).

[0139] In all the following descriptions, the amino acid positions referred to are those of the amino acids in the SARS-CoV-2 nucleocapsid of SEQ ID NO:10. The complete SARS-CoV-2 nucleocapsid protein consists of 419 amino acids. The sequence and domain architecture of the SARS-CoV-2 nucleocapsid protein were analyzed using IUPred2A. (Baker, NA, Sept, D., Joseph, S., Holst, MJ & McCammon, J. Electrostatics of nanosystems: application to microtubules and the ribosome. Proc. Natl Acad. Sci. USA 98, 10037-10041 (2001). The SARS-CoV-2 nucleocapsid protein is composed of an N-terminal domain (NTD), an RNA-binding domain (RNABD), an adaptor domain, a dimerization domain, and a C-terminal domain (CTD). (Cubuk, J., Alston, JJ, Incicco, JJ et al. The SARS-CoV-2 nucleocapsid protein is dynamic, disordered, and phase separates with RNA. Nat Commun 12, 1936 (2021)).

[0140] As the first attempt to create a novel SARS-CoV-2 N-Fc fusion protein, a viral nucleocapsid fragment (SEQ ID NO: 10) including an N-terminal domain (NTD), an RNA-binding domain (RNABD), an adapter domain, and a dimerization domain was selected, resulting in a nucleocapsid fragment of 364 amino acids.

[0141] The peptide linker SGGGSGGGS (SEQ ID NO:14) was used to link the nucleocapsid fragment (SEQ ID NO:10) to a human IgG1 Fc fragment including the following sequence:

[0142] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:1)

[0143] The human IgG1 fragment in SEQ ID NO:1 indicates that the N-terminal lysine residue on the natural human IgG1 fragment has been removed to improve production yield and purity. Furthermore, asparagine at the cNg site on the human IgG1 fragment was preserved to retain glycan attachment during fusion protein production in host cells.

[0144] The obtained SARS-CoV-2 N-Fc fusion protein is shown below:

[0145] (SEQ ID NO:17)

[0146] According to Example 1, the SARS-CoV-2 N-Fc fusion protein of SEQ ID NO:17 was prepared in HEK293 cells. Unexpectedly, this resulted in an extremely low protein yield of only 14 mg / L.

[0147] Studies have shown that the N-terminal domain of nucleocapsid proteins is disordered, flexible, and transiently interacts with the RNABD domain of nucleocapsid proteins. (Cubuk, J., Alston, JJ, Incicco, JJ et al.) It is speculated that the introduction of the entire NTD leads to low protein yield of SEQ ID NO:17. In the second attempt to create the SARS-CoV-2 N-Fc fusion protein, most of the N-terminal domain of the nucleocapsid protein fragment of SEQ ID NO:10 was removed. Specifically, the SARS-CoV-2 nucleocapsid protein of SEQ ID NO:10 was truncated, and the first 40 amino acids (amino acids 1-40 of SEQ ID NO:10) were eliminated. The resulting SARS-CoV-2 nucleocapsid protein fragment includes amino acids 41 to 364 of SEQ ID NO:10, and is as follows:

[0148] RPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSRG TSPARMAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFP(SEQ ID NO:11)

[0149] Clustal Omega was used to perform a side-by-side comparison of the SARS-CoV-2 nucleocapsid fragment of SEQ ID NO:10 and the truncated nucleocapsid fragment of SEQ ID NO:11, and as shown... Figure 14 As shown. "*" indicates complete homology among all sequences at a given sequence position. ":" (colon) indicates conservation among groups with a high similarity score greater than 0.5 in the Gonnet PAM 250 matrix. "-" (dash) indicates a sequence gap, meaning that there is no local homology within a specific comparison set within a certain range of sequences.

[0150] The nucleocapsid fragment of SEQ ID NO:11 was ligated to a human IgG1 Fc fragment including the following sequence via the peptide linker SGGGSGGGS (SEQ ID NO:14):

[0151] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:1)

[0152] In the case of the SARS-CoV-2 N-Fc fusion protein of SEQ ID NO:17, as shown in SEQ ID NO:1 above, the N-terminal lysine residue on the human IgG1 fragment was removed in an attempt to improve yield and purity. Furthermore, asparagine at the cNg site on the human IgG1 fragment was preserved to retain glycan attachment during fusion protein production in host cells. The resulting SARS-CoV-2 N-Fc fusion protein is shown below:

[0153] (SEQ ID NO:18)

[0154] According to Example 1, the SARS-CoV-2 N-Fc fusion protein of SEQ ID NO:18 was produced in HEK293 cells. Although the N-terminal domain was removed to prevent transient interaction between the N-terminal domain and the RNABD domain of the nucleocapsid protein fragment, this unexpectedly resulted in a protein yield titer as low as 18 mg / L.

[0155] It is speculated that the total length of the SARS-CoV-2 Fc fusion protein molecule is the reason for the low protein yield. The study also showed that the central linker of the SARS-CoV-2 nucleocapsid is highly dynamic, and the interaction between the nucleocapsid and the dimerization domain is minimal. However, in the case of complete absence of the dimerization and CTD domains, it is speculated that the central linker domain may engage in self-interaction or interact with the RNABD domain. (Cubuk, J., Alston, JJ, Incicco, JJ et al.) We did not remove the dimerization and CTD domains and retain the complete central linker domain, but rather retained a portion of the linker domain. Specifically, the complete linker domain comprises amino acids 174 to 243, where amino acid 1 is the first amino acid of SEQ ID NO:10. To further attempt to produce a novel SARS-CoV-2 N-Fc fusion protein with acceptable production yields, a portion of the linker domain from amino acids 174 to 208 was retained, and 38 amino acids at the C-terminus of the linker domain were removed, yielding the nucleocapsid fragment of SEQ ID NO:12, as shown below:

[0156] RPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSRGTSPA(SEQ ID NO:12)

[0157] Clustal Omega was used to perform a side-by-side comparison of the SARS-CoV-2 nucleocapsid fragment of SEQ ID NO:10 and another truncated nucleocapsid fragment of SEQ ID NO:12, and as... Figure 15 As shown. "*" indicates complete homology among all sequences at a given sequence position. ":" (colon) indicates conservation among groups with a high similarity score greater than 0.5 in the Gonnet PAM 250 matrix. "-" (dash) indicates a sequence gap, meaning that there is no local homology within a specific comparison set within a certain range of sequences.

[0158] The nucleocapsid fragment of SEQ ID NO:12 was ligated to a human IgG1 Fc fragment including the following sequence via the peptide linker SGGGSGGGS (SEQ ID NO:14):

[0159] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:1)

[0160] Similar to the SARS-CoV-2 N-Fc fusion proteins of SEQ ID NO:17 and SEQ ID NO:18, as shown in SEQ ID NO:1 above, the N-terminal lysine residue on the human IgG1 fragment was removed in an attempt to improve yield and purity. Furthermore, asparagine at the cNg site on the human IgG1 fragment was preserved to retain glycan attachment during fusion protein production in host cells. The resulting novel SARS-CoV-2 N-Fc fusion protein is shown below:

[0161] RPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSRGTSPASGGGSGGGSDKTHTCPPCPAPELLGGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:19)

[0162] According to Example 1, the novel SARS-CoV-2 N-Fc fusion protein of SEQ ID NO:19 was produced in HEK293 cells. Furthermore, the truncated novel SARS-CoV-2 N-Fc fusion protein significantly increased the production yield, achieving a protein titer of 163 mg / L.

[0163] Given that vaccines utilizing the spike protein region of SARS-CoV-2 are less effective in the presence of mutations, it is noteworthy that the nucleocapsid fragment of SEQ ID NO:12 (shown below) includes two amino acids, R203 and G204, which are mutated in the N protein present in the α, γ, and Omeprón SARS-CoV-2 variants, and whose native forms are highlighted in bold below, where the mutations occur.

[0164]

[0165] To address any potential decrease in efficacy in the α, γ, and omeprone SARS-CoV-2 variants, two amino acids in the nucleocapsid fragment of SEQ ID NO:12 were modified, with arginine (R) at position 203 being modified to lysine (K) and glycine (G) at position 203 being modified to arginine (R), as highlighted in bold below. This resulted in the nucleocapsid fragment of SEQ ID NO:13, as shown below.

[0166]

[0167] In this nucleocapsid fragment, as in SEQ ID NO:12, the linker domain from amino acid 174 to amino acid 208 is retained, and 38 amino acids at the C-terminus of the linker domain are removed, thus forming the nucleocapsid fragment.

[0168] Clustal Omega was used to perform side-by-side comparisons of SARS-CoV-2 nucleocapsid fragments from SEQ ID NO:10, SEQ ID NO:12, and SEQ ID NO:13 with the mutation, and as shown... Figure 16 As shown. "*" indicates complete homology among all sequences at a given sequence position. ":" (colon) indicates conservation among groups with a high similarity score greater than 0.5 in the Gonnet PAM 250 matrix. "-" (dash) indicates a sequence gap, meaning that there is no local homology within a specific comparison set within a certain range of sequences.

[0169] The nucleocapsid fragment of SEQ ID NO:13 was ligated to a human IgG1 Fc fragment including the following sequence via the peptide linker SGGGSGGGS (SEQ ID NO:14):

[0170] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:1)

[0171] Similar to the SARS-CoV-2 N-Fc fusion proteins of SEQ ID NO:17 and SEQ ID NO:18, as shown in SEQ ID NO:1 above, the N-terminal lysine residue on the human IgG1 fragment was removed in an attempt to improve yield and purity. Furthermore, asparagine at the cNg site on the human IgG1 fragment was preserved to retain glycan attachment during fusion protein production in host cells. The resulting novel SARS-CoV-2 N-Fc fusion protein is shown below:

[0172] RPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSKRTSPASGGGSGGGSDKTHTCPPCPAPELLGGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:20)

[0173] According to Example 1, a novel SARS-CoV-2 N-Fc fusion protein (SEQ ID NO: 20) was produced in HEK293 cells. The novel SARS-CoV-2 N-Fc fusion protein with a variant amino acid mutation at the central linker region of the nucleocapsid fragment achieved a protein titer of 117 mg / L, which is acceptable based on the design target (protein yield greater than 100 mg / L). However, this yield is lower than that of the novel SARS-CoV-2 N-Fc fusion protein without the mutation at the central linker region of the nucleocapsid fragment.

[0174] To maintain the high yield obtained by removing a portion of the dimer portion and central linker portion of the SARS-CoV-2 nucleocapsid protein from the novel SARS-CoV-2 N-Fc fusion protein construct of SEQ ID NO:19, we used an additional nucleocapsid fragment in which the entire central linker portion was eliminated, leaving the complete RNABD of the nucleocapsid protein and a small portion of the N-terminal domain (NTD). The resulting nucleocapsid fragment is shown below, namely SEQ ID NO:8.

[0175] RSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAE(SEQ ID NO:8)

[0176] The SARS-CoV-2 nucleocapsid fragments of SEQ ID NO:10 and SEQ ID NO:8 were compared side-by-side using Clustal Omega, and as shown... Figure 17 As shown. "*" indicates complete homology among all sequences at a given sequence position. ":" (colon) indicates conservation among groups with a high similarity score greater than 0.5 in the Gonnet PAM 250 matrix. "-" (dash) indicates a sequence gap, meaning that there is no local homology within a specific comparison set within a certain range of sequences.

[0177] The nucleocapsid fragment of SEQ ID NO:8 was ligated to a human IgG1 Fc fragment including the following sequence via the peptide linker SGGGSGGGS (SEQ ID NO:14):

[0178] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:1)

[0179] As shown in SEQ ID NO:1 above, the N-terminal lysine residue on the human IgG1 fragment was removed in an attempt to improve production yield and purity. Furthermore, asparagine at the cNg site on the human IgG1 fragment was preserved to retain glycan attachment during fusion protein production in host cells. The resulting novel SARS-CoV-2 N-Fc fusion protein is shown below:

[0180] RSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAESGGGSGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:15)

[0181] According to Example 1, the novel SARS-CoV-2 N-Fc fusion protein of SEQ ID NO:15 was produced in HEK293 cells. The novel SARS-CoV-2 N-Fc fusion protein with the nucleocapsid fragment removed from the central linker portion achieved a protein titer of 140 mg / L, which is an improvement compared to the novel SARS-CoV-2 N-Fc fusion protein (SEQ ID NO:20) containing a mutant central linker portion of the nucleocapsid fragment. According to Example 1, the novel SARS-CoV-2 N-Fc fusion protein of SEQ ID NO:15 was produced in HEK293 cells in a considerably large batch (0.5 L instead of 0.03 L). In this larger batch, the novel SARS-CoV-2 N-Fc fusion protein with the nucleocapsid fragment removed from the central linker portion achieved a protein titer of 198 mg / L, which is a further improvement compared to the novel SARS-CoV-2 N-Fc fusion protein (SEQ ID NO:20) containing a mutant central linker portion of the nucleocapsid fragment.

[0182] Additionally, an attempt was made to create a novel SARS-CoV-2 N-Fc fusion protein whose nucleocapsid fragment contains only the complete N-terminal domain (NTD) and RNABD domain, as preserving the NTD portion may be more advantageous. In this novel SARS-CoV-2 N-Fc fusion protein, the nucleocapsid fragment shown in SEQ ID NO:9 was used.

[0183] MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAE(SEQ ID NO:9)

[0184] Clustal Omega was used to perform a side-by-side comparison of the SARS-CoV-2 nucleocapsid fragments of SEQ ID NO:9 and SEQ ID NO:8, and as shown Figure 18 As shown. "*" indicates complete homology among all sequences at a given sequence position. ":" (colon) indicates conservation among groups with a high similarity score greater than 0.5 in the Gonnet PAM 250 matrix. "-" (dash) indicates a sequence gap, meaning that there is no local homology within a specific comparison set within a certain range of sequences.

[0185] The nucleocapsid fragment of SEQ ID NO:9 was ligated to a human IgG1 Fc fragment including the following sequence via the peptide linker SGGGSGGGS (SEQ ID NO:14):

[0186] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:1)

[0187] As shown in SEQ ID NO:1 above, the N-terminal lysine residue on the human IgG1 fragment was removed in an attempt to improve production yield and purity. Furthermore, asparagine at the cNg site on the human IgG1 fragment was preserved to retain glycan attachment during fusion protein production in host cells. The resulting novel SARS-CoV-2 N-Fc fusion protein is shown below:

[0188] MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAESGGGSGGGSDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:16)

[0189] According to Example 1, a novel SARS-CoV-2 N-Fc fusion protein of SEQ ID NO:16 was produced in HEK293 cells. The novel SARS-CoV-2 N-Fc fusion protein of SEQ ID NO:16 achieved an acceptable protein titer of 173 mg / L.

[0190] Therefore, the four candidate novel SARS-CoV-2 N-Fc fusion proteins met the design goals, with protein yields greater than 100 mg / L: SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:19, and SEQ ID NO:20. The novel SARS-CoV-2 N-Fc fusion proteins of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:19, and SEQ ID NO:20, recombinantly produced in HEK cells according to Example 1, were purified according to Example 2. The Fc fusion protein structures of the novel SARS-CoV-2 N-Fc fusion proteins of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:19, and SEQ ID NO:20 were confirmed according to Example 3, and sequence identification was performed according to Example 4. To obtain the homodimer titer of each of the produced novel SARS-CoV-2 N-Fc fusion proteins, the homodimer percentage was measured according to Example 5, and the homodimer titer was calculated by multiplying the homodimer percentage by the protein titer of the recombinantly produced fusion protein. The homodimer titers of the novel SARS-CoV-2 N-Fc fusion proteins of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:19 and SEQ ID NO:20, along with their protein yields, are shown in Table 1 below.

[0191]

[0192] According to Example 6, the binding of the SARS-CoV-2 N-Fc fusion proteins SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:19, and SEQ ID NO:20 produced in HEK293 cells according to Example 1 to Fc(γ) receptor I was measured. As described in Example 6, the OD450 measurements of binding to human Fc(γ) receptor I, Fc(γ) receptor IIA, Fc(γ) receptor IIB, Fc(γ) receptor III, FcRn, and ACE2 receptor are expected to increase with increasing SARS-CoV-2 N-Fc fusion protein concentration.

[0193] The nucleocapsid framework domains of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:12, and SEQ ID NO:13 were compared side-by-side using Clustal Omega, and as shown... Figure 19 As shown in the diagram. "*" indicates complete homology among all sequences at a given sequence position. ":" (colon) indicates conservation between groups with a high similarity score greater than 0.5 in the Gonnet PAM 250 matrix. "-" (dash) indicates a sequence vacancy, meaning there is no local homology within a specific comparison set within a certain range of the sequence, while ":", ".", or a space indicate conserved, moderately different, or very different amino acid mutations in the sequence at a given sequence position, respectively.

[0194] To highlight the amino acid mutations in the nucleocapsid region, the nucleocapsid framework domains of SEQ ID NO:12 and SEQ ID NO:13 were compared side-by-side using Clustal Omega, and as shown... Figure 20 As shown. "*" indicates complete homology of all sequences at a given sequence position. ":" (colon) indicates conservation between groups with a score greater than 0.5 in the Gonnet PAM 250 matrix that have a high similarity attribute. "-" (dash) indicates a sequence gap, meaning there is no local homology within a specific comparison set within a certain range of the sequence, while ":", ".", or a space indicate conserved, moderately different, or very different amino acid mutations in the sequence at a given sequence position, respectively.

[0195] SARS-CoV-2 N-Fc fusion protein used as a primary vaccine

[0196] The SARS-CoV-2 N-Fc fusion protein can be used as a primary vaccine. In one or more embodiments, the SARS-CoV-2 N-Fc fusion protein is provided in the form of a pharmaceutical composition. Injection of any protein can induce an immune response, the magnitude and type of which are highly dependent on the “state” of the corresponding immune system. For example, injection of a foreign antigen (Ag) relative to self-antigens will induce a stronger immune response in the immune system that maintains central and peripheral tolerance mechanisms. Furthermore, administration of a foreign Ag to an immune system that has been primordially immunized by prior exposure to the corresponding Ag (e.g., viral infection) will produce a faster and stronger immune response relative to the Ag-primordially immunized system. The immunological basis for this primordially immunized state has two aspects: 1) the Ag-primordially immunized immune system has naïve B and T lymphocytes with a much higher activation threshold than the Ag-primordially immunized “memory” cells of the Ag-primordially immunized immune system, such that the antigen-presenting cells (APCs) that present Ag require less Ag to activate the primordially immunized memory T cells, and 2) due to the expansion of memory T cells during Ag-primordially immunized exposure, the number of these cells is inherently greater upon re-exposure to the injected Ag. Please note that dominant APCs are dendritic cells (DCs) and macrophages, which present Ag in combination with the major histocompatibility complex (MHC) molecules on their surface to T cell Ag receptors. Figure 11 For illustrative purposes, this diagram depicts an example pattern of how antigens can interact with antigen-presenting cells (such as dendritic cells).

[0197] APCs can influence the “amplitude” and “type” of the response to antigens. B cells directly participate in the immune response through humoral immunity (antibody production) and also participate in T cell immune responses as specific APCs that selectively capture antigens and present them to T cells. Both of these B cell functions are achieved by activating surface B cell receptors (BCRs), which are essentially membrane-bound antibodies that specifically bind to specific antigens. Multivalent soluble antigens, such as Fc fusion homodimers containing specific antigens, can be recognized and activated by BCRs. Therefore, the SARS-CoV-2 N-Fc fusion protein homodimer can activate B cells through antigen-specific BCR activation, thereby increasing antibody production, and through B cell-mediated APC activity, it also leads to increased T cell specificity and responsiveness to RBD epitopes. Thus, these fusion proteins activate both humoral and cellular immunity after administration. More specifically, induced Th1 (cellular) immunity activates the body’s cell-killing mechanisms, such as cytotoxic T cells, NK cells, and macrophages, which target cells already infected with the virus. Simultaneously, in Th2-type immunity, T helper cells stimulate B cell proliferation and differentiation into plasma cells that secrete antigen-specific antibodies. These antibodies help control infection by: (i) neutralizing the virus by binding to the viral surface RBD portion required for cell entry, wherein the antibody also helps clear antibody-bound viruses via Fc-mediated phagocytosis; (ii) antibody-directed cytotoxicity (ADC), which occurs when the antibody binds to antigens presented on the surface of the infected cell and instructs NK cells to destroy them; or (iii) potential neutralization of intracellularly exposed viral internal antigens: wherein the antibody comes into contact with the exposed viral internal environment (e.g., within the cell).

[0198] adjuvant

[0199] In some instances, Th1 cell responses are essential for clearing most viral and bacterial infections, where virus-like or bacterial-like substances (not inherently Ag) cause APCs to express key cytokines and surface co-stimulatory molecules that drive T cells to become Th1-type during Ag presentation. In fact, this APC activation is the conceptual basis for many immune-enhancing substances called adjuvants. Dominant APCs are dendritic cells (DCs) and macrophages, which present Ag in complex form with the major histocompatibility complex (MHC) molecules on their surface to T cell Ag receptors. These APCs can influence the “amplitude” and “type” of the Ag response. Some adjuvants are designed to induce the immune system to respond to injected vaccine Ag as if it were part of a persistent infection (i.e., the infectious agent provides this natural viral or bacterial adjuvant substance). Thus, adjuvants activate APCs to achieve greater Ag presentation capacity, necessary to overcome the high activation threshold of naive T cells, and further to develop them into a Th1 response for effective clearance of the corresponding infection. Please note that these T cells provide crucial assistance to B cells, which specifically bind to the corresponding Ag to produce Ag-specific antibody (Ab) titers.

[0200] Fc fusion proteins used as primary vaccines can be co-administered with adjuvants to enhance or otherwise alter the immune response in target subjects. Adjuvants activate APCs to gain greater Ag-presenting capacity, necessary to overcome the high activation threshold of naive T cells, and can further develop them into a Th1 response for efficient clearance of the corresponding infection. In examples, adjuvants are known to be used in pharmaceutical compositions of SARS-CoV-2 N-Fc fusion proteins to enhance the induction of anti-SARS-CoV-2 antibodies. Known adjuvants include those for respiratory viral infections, including trivalent or monovalent influenza vaccines, pandemic H1N1, H5N1, and SARS-CoV vaccines, which have been used in human clinical trials over the past decade.

[0201] Examples of adjuvants that can be used in the pharmaceutical compositions disclosed herein include, but are not limited to, oil-in-water, amorphous aluminum hydroxyphosphate sulfate (AAHS), aluminum hydroxide, aluminum phosphate, potassium aluminum sulfate (Alum), Freund's adjuvant (complete and / or incomplete), squalene, AS02, AS03, AS04, MF59, AS01B, QS-21, CpG 1018, ISCOMS, Montanide TM ISA-51, Montanide TMISA-720, polylactic acid co-glycolic acid (PLG), monophospholipid A (MPL), Detox, AGP [RC-529], DC_Chol, OM-174 (lipid A derivative), CpG motifs (synthetic oligonucleotides containing immunostimulatory CpG motifs), modified LT and CT, hGM-CSF, hIL-12, Immudaptin, inert solvents such as gold particles, and various experimental adjuvants from sources such as Advax (Australia), such as AddaVax (Invivogen) or other Advax-based vaccine adjuvants.

[0202] In some instances, the adjuvants chosen may be MF59 (Novartis) and AS-03 (GlaxoSmithKline). Custom formulations or equivalents of MF59 (Novartis), such as AddaVax (Invivogen) or other Advax-based vaccine adjuvants from Vaxine Pvt Ltd. (Australia), may be used in pharmaceutical compositions of the SARS-CoV-2 N-Fc fusion protein. In a preferred embodiment, the SARS-CoV-2 N-Fc fusion protein is combined with Montanide... TM ISA-720 adjuvants are co-administered to enhance or otherwise alter the immune response in target subjects. Many different adjuvants for respiratory viral infections have been extensively tested in seasonal trivalent influenza vaccines and pandemic H1N1 and H5N1 vaccines in Australia (Protein Sciences), as well as in the recent NIH-supported human influenza vaccine trial at Sanofi Pasteur in the United States, and can be used in pharmaceutical compositions containing the SARS-CoV-2 N-Fc fusion protein.

[0203] In one or more embodiments, the SARS-CoV-2 N-Fc fusion protein formulation is prepared on-site for administration. In one aspect, the SARS-CoV-2 N-Fc fusion protein is mixed on-site with an adjuvant under aseptic mixing conditions. In another aspect, the SARS-CoV-2 N-Fc fusion protein and the adjuvant are thoroughly mixed and / or emulsified to prepare a homogeneous emulsion for administration to a subject. Adjuvant formulations of the SARS-CoV-2 N-Fc fusion protein, or pharmaceutical compositions thereof, administered to patients via subcutaneous (sc) or intramuscular (im) injection are more likely to induce a strong antibody response at the sc or im injection site due to the presence of more dendritic cells (DCs) in the subcutaneous and intramuscular spaces.

[0204] As described above, in some cases, the use of adjuvants in the pharmaceutical composition to increase the virus-neutralizing capacity of the anti-SARS-CoV-2 antibody titer as measured according to Example 7 and / or the anti-SARS-CoV-2 antibody titer as measured according to Example 9 may be advantageous. The use of adjuvants may be particularly advantageous for antibody-negative patients who do not have a potential immune response to the virus or RNABD. Furthermore, adjuvants may be beneficial for older subjects whose immune capacity changes with age, a condition known as immunosenescence, which results from changes in multiple levels of the immune system over time. Once a patient has measurable antibodies, upon re-attack with the SARS-CoV-2 virus, the patient will exhibit very rapid development of anti-SARS-CoV-2 antibodies to establish their own defense against COVID-19.

[0205] Primary SARS-CoV-2 N-Fc fusion vaccine evaluated in mice

[0206] The efficacy of the exemplary SARS-CoV-2 N-Fc fusion protein or pharmaceutical compositions thereof disclosed herein can be preliminarily evaluated in mouse immunization studies according to the procedures in Example 9, assessing its ability to induce high-titer anti-nucleocapsid IgG antibodies. BALB / c mice are a relevant animal model widely used for preclinical immunogenicity assessment of vaccines. This strain produces a strong Ab response when immunized with adjuvanted and unadjuvanted candidate vaccines. Furthermore, mouse-specific reagents are widely used to evaluate the kinetics and characteristics of various immune responses to vaccination, including associated Ab subtypes and T-cell responses (e.g., Th1 versus Th2 responses). Therefore, the BALB / c mouse model was chosen to evaluate the immunogenicity of the SARS-CoV-2 N-Fc fusion protein vaccine in terms of Ag dosage, adjuvant enhancement, route of administration, and dosing frequency required to achieve an optimal Ab response.

[0207] In short, at predetermined time intervals (e.g., day 0, day 21, and day 46), target mice (e.g., BALB / c mice) were injected with an exemplary SARS-CoV-2 N-Fc fusion protein (with or without Montanide). TM Serum was collected three times with ISA 720 adjuvant or its pharmaceutical composition at regular intervals (every 7 days starting from day 14).

[0208] Following treatment with the SARS-CoV-2 N-Fc fusion protein of SEQ ID NO:15 once or more in N=7 BALB / c mice according to Example 8, the anti-SP / RBD SARS-CoV-2 IgG antibody titer was measured according to Example 7. As expected, and as... Figure 12As shown, since the exemplary SARS-CoV-2 N-Fc fusion protein of SEQ ID NO:15 does not contain any part of the SP / RBD of SARS-CoV-2, no measurable anti-SARS-CoV-2 antibody titer was observed after treatment.

[0209] Serum anti-SARS-CoV-2 nucleocapsid protein antibody titer was measured according to the procedure in Example 9.

[0210] At a dose level of 10 μg of adjuvant-free SARS-CoV-2 N-Fc fusion protein, the SARS-CoV-2 N-Fc fusion protein of SEQ ID NO:15 began to induce measurable anti-SARS-CoV-2 nucleocapsid antibody titers approximately 40 days after the first injection on day 0. It is anticipated that at a dose level of 10 μg of adjuvant-free SARS-CoV-2 N-Fc fusion protein, the SARS-CoV-2 N-Fc fusion proteins of SEQ ID NO:16, SEQ ID NO:19, and SEQ ID NO:20 will begin to induce measurable anti-SARS-CoV-2 nucleocapsid antibody titers approximately 40 days after the first injection on day 0.

[0211] As previously mentioned, adjuvants activate APCs to gain greater Ag-presenting capacity, which is necessary to overcome the high activation threshold of naive T cells. Furthermore, it can develop them into a Th1 response for effective clearance of the corresponding infection. When the SARS-CoV-2 N-Fc fusion protein of SEQ ID NO:15 is combined with Montanide... TM When bound to ISA 720 adjuvant (30% / 70% v / v), starting approximately 40 days after the first injection on day 0, the antibody titer against the SARS-CoV-2 nucleocapsid protein was higher compared to that without adjuvant. Further progress is expected when the SARS-CoV-2 N-Fc fusion proteins of SEQ ID NO:16, SEQ ID NO:19, and SEQ ID NO:20 are combined with Montanide. TM When bound to ISA 720 adjuvant (30% / 70% v / v), starting approximately 40 days after the first injection on day 0, the antibody titer against the SARS-CoV-2 nucleocapsid protein was significantly higher compared to that without adjuvant. These data strongly support the selection of Montanide. TM ISA 720 is the lead adjuvant candidate for this vaccine development program.

[0212] Following 1, 2, and 3 doses, the kinetic response (i.e., duration of response) at dose levels ranging from 1 μg to 100 μg was projected to demonstrate increased antibody titers against SARS-CoV-2 nucleocapsid protein at all dose levels, with a maximum duration of at least 56 days post-vaccination.

[0213] SARS-CoV-2 N-Fc fusion protein used as a booster vaccine

[0214] In examples, exemplary SARS-CoV-2 N-Fc fusion proteins of this disclosure, such as those in SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:19, or SEQ ID NO:20, can be used as booster vaccines. Administration of the fusion protein to subjects who already have low but measurable antibody levels against the SARS-CoV-2 antigen amplifies their antibody titer, thereby increasing their antiviral protection. Synthetic N protein (nucleocapsid) fragment variants are used to maximize antigenicity while prolonging the antigen residence time in the Fc region. Not wishing to be bound by any particular mechanistic theory, it is believed that during the extended in vivo residence time, the naturally glycosylated human Fc fragment will help bind to the Fc(γ) receptor on antigen-presenting cells (APCs), which in turn will cause more SARS-CoV-2 nucleocapsid analog antigen to be presented to T cells and / or B cells (e.g., T cells and / or B cells). Figure 11 (As shown), this is expected to elicit a strong immune response to the SARS-CoV-2 nucleocapsid antigen. Specifically, APCs internalize the SARS-CoV-2 RBD nucleocapsid antigen via Fc(γ) receptors and then process and present nucleocapsid fragments to CD4+Th cells, which in turn promote (“help”) B cell activation and the production of anti-SARS-CoV-2 nucleocapsid IgG (i.e., Ab). Antigen-presenting cells can be, for example, dendritic cells (DCs), monocytes, or macrophages, which can perform phagocytosis via Fc receptor-mediated processes (e.g., as shown). Figure 11 As shown, the SARS-CoV-2 N-Fc fusion protein of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:19, or SEQ ID NO:20 is internalized via the binding of the Fc region of the SARS-CoV-2 N-Fc fusion protein to the Fc(γ) receptor in immune cells. The SARS-CoV-2 N-Fc fusion protein is internalized via Fc-mediated binding, for example, by DCs (e.g., cDCs). 2SThe uptake by the SARS-CoV-2 subpopulation promotes the differentiation of anti-SARS-CoV-2 T helper 2 (Th2) cells through the secretion of IL-10 and IL-33. Anti-SARS-CoV-2 Th2 cells activate anti-SARS-CoV-2 B cells, for example, by cross-linking their antigen receptors to attract Th2 cells. B cell antigen receptor (BCR)-mediated uptake binds to the SARS-CoV-2 RNABD of the SARS-CoV-2 N-Fc fusion protein molecule, then delivers the SARS-CoV-2 antigen to an intracellular site, where it is degraded and returned to the B cell surface as a peptide bound to MHC class II molecules. The peptide MHC class II complex can be recognized by SARS-CoV-2 specific helper T cells, mimicking them to produce proteins, leading to B cell proliferation, whose progeny differentiate into B cells that secrete anti-SARS-CoV-2 antibodies. Due to the presence of the Fc fragment, the SARS-CoV-2 N-Fc fusion proteins of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:19, or SEQ ID NO:20 can directly expose the SARS-CoV-2 RNA BD (nucleocapsid) fragment to antigen-producing cells for a prolonged period. Furthermore, and as previously described, the glycosylated Fc fragments in the SARS-CoV-2 N-Fc fusion proteins of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:19, or SEQ ID NO:20 are expected to induce a very strong immune response against the therapeutic or antigenic portion of the fusion protein. These combined properties significantly increase the amount of antiviral antibodies while simultaneously reducing the amount of antigen required to generate the desired immune response.

[0215] In examples, treatment of patients with the SARS-CoV-2 N-Fc fusion protein or a pharmaceutical composition thereof, as specified in SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:19, or SEQ ID NO:20, can serve as a booster vaccine for recovered COVID-19 patients who have tested positive for SARS-CoV-2 antibodies, as a means of amplifying their antibody titer and affinity so that when these treated patients subsequently encounter the virus, they will have sufficient immunity to prevent infection and / or severe symptoms associated with SARS-CoV-2 infection. Furthermore, treatment of patients with the SARS-CoV-2 N-Fc fusion protein or a pharmaceutical composition thereof, as specified in SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:19, or SEQ ID NO:20, can serve as a booster vaccine for subjects previously immunized with a vaccine against SARS-CoV-2, as a means of amplifying their antibody titer and affinity specifically against the nucleocapsid of the SARS-CoV-2 N-Fc fusion protein. This therapy is crucial when vaccines are not 100% effective and / or the induced antibody titers diminish over time. In this case, the SARS-CoV-2 N-Fc fusion protein or pharmaceutical compositions thereof, as specified in SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:19, or SEQ ID NO:20, can be administered to patients via subcutaneous (sc) or intramuscular (im) injection. Because the subcutaneous and intramuscular spaces contain more dendritic cells (DCs), sc or im injection sites are more likely to induce a strong antibody response.

[0216] Fc fusion protein production

[0217] In this implementation, the fusion protein may be expressed by cells, as described in more detail in the Examples section.

[0218] Expression and purification

[0219] The SARS-CoV-2 N-Fc fusion protein can be recombinantly expressed, for example in eukaryotic cells, such as mammalian cells or non-mammalian cells. Exemplary mammalian cells for expression include HEK cells (e.g., HEK293 cells) or CHO cells. CHO cells can be further subdivided into various strains or subclasses (e.g., CHO DG44, CHO-M, CHO-SE). TM(and CHO-K1), some of which can be genetically engineered for optimal use with specific types of nucleic acid molecules (e.g., vectors including DNA) or specific cell growth medium compositions described in the Examples section. Cells can be transfected with nucleic acid molecules (e.g., vectors) encoding the SARS-CoV-2 N-Fc fusion protein (e.g., where the entire SARS-CoV-2 N-Fc fusion protein is encoded by a single nucleic acid molecule). HEK293 cells can be transfected with vectors encoding the SARS-CoV-2 N-Fc fusion protein, but this process only results in transient expression of the SARS-CoV-2 N-Fc fusion protein for a period of time (e.g., 3 days, 4 days, 5 days, 7 days, 10 days, 12 days, 14 days, or longer), after which the host cells cease expressing detectable levels of the SARS-CoV-2 N-Fc fusion protein (i.e., transient transfection). Transient transfection of HEK293 cells encoding the nucleic acid sequence of the SARS-CoV-2 N-Fc fusion protein typically leads to more rapid production of recombinant proteins, facilitating the preparation and screening of multiple SARS-CoV-2 N-Fc fusion protein candidates. CHO cells can be transfected with vectors permanently integrated into the host cell DNA, and with proper cell culture, this results in sustained and permanent expression (i.e., stable transfection) of the SARS-CoV-2 N-Fc fusion protein. Stable transfection of CHO cells and cell lines encoding the nucleic acid of the SARS-CoV-2 N-Fc fusion protein typically requires longer development times, but they generally yield higher protein yields and are better suited for producing low-cost products (e.g., for the veterinary market). Cells and cell lines can be cultured using standard methods in the art.

[0220] For example, the SARS-CoV-2 N-Fc fusion protein can be purified or isolated from cells (e.g., by lysing cells). The SARS-CoV-2 N-Fc fusion protein is secreted by cells and can be purified or isolated from cell culture media in which cells are grown. Purification of the SARS-CoV-2 N-Fc fusion protein may include using column chromatography (e.g., affinity chromatography) or other separation methods based on differences in size, charge, and / or affinity for certain molecules. Purification of the SARS-CoV-2 N-Fc fusion protein involves screening or enriching proteins containing the Fc fragment, for example, by using protein A beads or a protein A column, allowing the protein containing the Fc fragment to bind with high affinity to protein A covalently conjugated to protein A beads at a neutral solution pH. The bound SARS-CoV-2 N-Fc fusion protein can then be eluted from the protein A beads by changing the solution variable (e.g., decreasing the solution pH). Other separation methods, such as ion exchange chromatography and / or gel filtration chromatography, may also be applied alternatively or additionally. Purification of the SARS-CoV-2 N-Fc fusion protein may also include filtration or centrifugation of the protein formulation, percolation, ultrafiltration, and filtration through porous membranes of various sizes, as well as final formulation with excipients.

[0221] Purified SARS-CoV-2 N-Fc fusion protein can be characterized using various methods, such as purity, protein yield, structure, and / or activity: for example, absorbance at 280 nm (e.g., to determine protein yield), size exclusion or capillary electrophoresis (e.g., to determine molecular weight, aggregation percentage, and / or purity), mass spectrometry (MS) and / or liquid chromatography (LC-MS) (e.g., to determine purity and / or glycosylation), and / or ELISA (e.g., to determine the degree of binding to SARS-CoV-2 antibodies or ACE2, such as affinity). Exemplary characterization methods are also described in the Examples section.

[0222] The SARS-CoV-2 N-Fc fusion protein, after being produced in transiently transfected HEK cells and purified by protein A, can yield greater than 5 mg / L, 10 mg / L, or 20 mg / L, or more preferably greater than 50 mg / L (e.g., greater than 60 mg / L, greater than 70 mg / L, greater than 80 mg / L, greater than 90 mg / L, greater than 100 mg / L). The homodimer percentage of the SARS-CoV-2 N-Fc fusion protein, after being produced in transiently transfected HEK cells and purified by protein A, is greater than 70% (e.g., greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%). After being produced in transiently transfected HEK cells and purified by protein A, the homodimer titer of the SARS-CoV-2 N-Fc fusion protein (calculated as the product of the SARS-CoV-2 N-Fc fusion protein yield and the homodimer percentage) may be greater than 50 mg / L (e.g., greater than 60 mg / L, greater than 70 mg / L, greater than 80 mg / L, greater than 90 mg / L, greater than 100 mg / L).

[0223] Pharmaceutical Compositions and Routes of Administration

[0224] The amount and concentration of the SARS-CoV-2 N-Fc fusion protein in the drug composition, as well as the amount of the drug composition administered to the subject, can be selected based on clinically relevant factors, such as the subject's medical characteristics (e.g., age, weight, sex, other medical conditions), the solubility of the compounds in the drug composition, the potency and activity of the compounds, and the route of administration of the drug composition.

[0225] The formulations disclosed herein include those suitable for parenteral administration. As used herein, the phrases “parenteral administration” and “external administration” refer to a route of administration other than enteral and topical administration, typically via intravenous, intramuscular, or subcutaneous injection.

[0226] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of this disclosure include water, saline, ethanol, salt, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate, buffers such as potassium phosphate and / or sodium phosphate, pH buffers such as hydrochloric acid and / or sodium hydroxide, etc. For example, appropriate flowability can be maintained by using coating or emulsifying materials (such as lecithin), by maintaining the desired particle size in the case of dispersions, and by using surfactants, such as Tween-like surfactants. In some instances, the pharmaceutical composition (e.g., as described herein) includes a Tween-like surfactant, such as polysorbate-20, Tween-20, or Tween-80. In some instances, the pharmaceutical composition (e.g., as described herein) includes a Tween-like surfactant, such as Tween-80, at a concentration of about 0.001% to about 2%, or about 0.005% to about 0.1%, or about 0.01% to about 0.5%.

[0227] SARS-CoV-2 N-Fc fusion protein can be administered via bolus injection, infusion, or intravenous bolus, or via syringe injection, pump, pen, needle, or indwelling catheter. SARS-CoV-2 N-Fc fusion protein can also be administered via subcutaneous bolus injection. In practice, SARS-CoV-2 N-Fc fusion protein or pharmaceutical compositions thereof are administered to patients via subcutaneous injection (sc) or intramuscular injection (im), where the subcutaneous and intramuscular spaces contain more dendritic cells (DCs), making sc or im injection sites more likely to induce a strong antibody response. The method of introduction can also be provided via rechargeable or biodegradable devices. In recent years, various sustained-release polymer devices have been developed and tested in vivo for the controlled delivery of drugs, including protein biopharmaceuticals. Various biocompatible polymers (including hydrogels), including both biodegradable and non-degradable polymers, can be used to form implants for sustained release of compounds at specific target sites. Other pharmaceutically acceptable components used in compositions include buffers, salts, stabilizers, diluents, preservatives, antibiotics, isotonic agents, etc.

[0228] dose

[0229] In use, a therapeutically effective amount of the SARS-CoV-2 N-Fc fusion protein is administered to a subject in need. Administration of the SARS-CoV-2 N-Fc fusion protein can elicit an immune response in the subject, and more specifically, an immune response against a coronavirus infection (more specifically, SARS-CoV-2 or a variant). The immune response will be demonstrated by the absence of observable clinical symptoms or a reduction in clinical symptoms typically exhibited by infected subjects, reduced viral shedding, faster recovery time, and / or a shorter duration of infection. In another embodiment, a method for activating immune cells at a site of infection or disease is provided, comprising administering a therapeutically effective amount of the SARS-CoV-2 N-Fc fusion protein to a mammal. In another aspect, a method for increasing antibody production in a subject is provided, comprising administering a therapeutically effective amount of the SARS-CoV-2 N-Fc fusion protein to a mammal.

[0230] It should be understood that the treatments and preventative measures described herein are applicable to humans and any suitable warm-blooded animals, including but not limited to dogs, cats and other companion animals, as well as rodents, primates, horses, cattle, sheep, pigs, etc. These methods may also be applied to clinical research and / or surveys.

[0231] As used herein, the phrase "effective amount" or "therapeutic effective amount" means a therapeutic or preventative amount of the SARS-CoV-2 N-Fc fusion protein applicable to embodiments of this disclosure, which, when administered according to the desired treatment regimen, will elicit the desired therapeutic or preventative effect or response, including relief of some or all symptoms of infection or reduction of susceptibility to infection. Those skilled in the art will recognize that even if the disease is not completely eradicated or prevented, but its symptoms and / or effects are partially improved or alleviated in a subject, the amount can be considered therapeutically "effective." Therapeutic effective doses of the SARS-CoV-2 N-Fc fusion peptide may vary depending on the subject's body size and species, as well as the route of administration.

[0232] The actual dose level of the SARS-CoV-2 N-Fc fusion protein can be varied to obtain the amount of active ingredient that effectively achieves the desired therapeutic response in a specific subject. The chosen dose level will depend on a variety of factors, including the activity of the specific fusion protein used or its esters, salts, or amides, the route of administration, the timing of administration, the excretion rate of the specific compound used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the specific fusion protein used, the age, sex, weight, condition, general health status, and medical history of the subject being treated, and similar factors well known in the medical field. Generally, an appropriate dose of the SARS-CoV-2 N-Fc fusion protein will be the lowest dose that effectively produces a therapeutic effect. This effective dose typically depends on the factors mentioned above.

[0233] In all respects, immunogenic formulations are provided in unit dosage forms to facilitate administration and uniformity of dosage. As used herein, a “unit dosage form” refers to a physically discrete unit suitable as a unit dose for a subject to be treated, calculated to contain a predetermined amount of SARS-CoV-2N-Fc fusion protein to produce the desired therapeutic effect associated with the desired drug delivery system. The quality criteria for the unit dosage form depend on and are directly dependent on the unique characteristics of one or more excipients and one or more therapeutic agents and the specific biological effect to be achieved. In one or more embodiments, the formulation is provided as a kit for administering the SARS-CoV-2N-Fc fusion protein to a subject. In one or more embodiments, a pharmaceutical composition comprising the SARS-CoV-2N-Fc fusion protein dispersed in a suitable delivery system is provided in unit dosage forms (e.g., vials). In one or more embodiments, the kit further includes discrete unit dosage forms (e.g., vials) containing adjuvants and / or other delivery system for on-site mixing of the SARS-CoV-2N-Fc fusion protein for administration. In one or more embodiments, the kit includes one or more emulsifying needles and syringes for on-site mixing of the immunogenic preparation for administration. In one or more embodiments, the kit includes one or more administration syringes for administering the prepared immunological composition to a subject. In one or more embodiments, the kit also includes instructions for preparing the immunogenic composition and / or administering the immunogenic composition.

[0234] In examples following the procedures described above and in the examples below, 42 days after the first injection on day 0 and the second injection on day 21, a 10 μg dose level of the SARS-CoV-2 N-Fc fusion protein induced significant anti-nucleocapsid Ab titers in mice. Efficacy at 10 μg and 30 μg dose levels is expected in rabbits and non-human primates.

[0235] This disclosure covers the formulation of the SARS-CoV-2 N-Fc fusion protein into any of the above-described pharmaceutical compositions and formulations. Furthermore, this disclosure covers administration via any of the above-described routes of administration. Those skilled in the art can select appropriate formulations, dosage levels, and routes of administration based on the symptoms being treated and the overall health, age, and body type of the patient being treated.

[0236] Example

[0237] The present technology is further illustrated by the following embodiments. However, it should be understood that these embodiments are provided by way of illustration, and nothing therein should be considered as a limitation on the overall scope of the present technology.

[0238] General Examples of Synthesis, Purification and Validation of SARS-CoV-2 N-Fc Fusion Protein

[0239] Example 1: A method for synthesizing and preparing SARS-CoV-2 N-Fc fusion protein in HEK293 cells.

[0240] The synthesis of the SARS-CoV-2 N-Fc fusion protein was as follows. The target gene sequence was constructed using proprietary software (LakePharma, Belmont, CA) and cloned into a high-expression mammalian vector. HEK293 cells were seeded in shake flasks 24 hours prior to transfection and grown in a serum-free, chemically defined medium. Using the standard transient transfection procedure (LakePharma, Belmont, CA), the DNA expression construct encoding the target SARS-CoV-2 N-Fc fusion protein was transiently transfected into the HEK293 cell suspension. After 20 hours, cell counts were performed to determine viability and live cell count, and the results were analyzed by… (Pall FortéBio LLC, Fremont, CA) Measure titer. Obtain additional readings throughout the transient transfection production run. Harvest cultures on or after day 5.

[0241] Example 2: Purification of SARS-CoV-2 N-Fc fusion protein produced in HEK293 cells.

[0242] Purification of the SARS-CoV-2 N-Fc fusion protein was performed as follows. Conditioned medium supernatant containing the secreted SARS-CoV-2 N-Fc fusion protein was harvested from the HEK293 production run and clarified by centrifugation. The supernatant containing the desired SARS-CoV-2 N-Fc fusion protein was passed through a Protein A column and washed and eluted using a low pH gradient. The elution fractions containing the desired protein were then combined and buffer-exchanged to a 200 mM HEPES, 100 mM NaCl, 50 mM NaOAc, pH 7.0 buffer. A final filtration step was performed using a 0.2 μm membrane filter. The final protein concentration was calculated based on the optical density of the solution at 280 nm. Additional optional purification was performed as needed by ion-exchange chromatography (e.g., using anion-exchange or cation-exchange beads), gel filtration chromatography, or other methods.

[0243] Example 3: Confirmation of the structure of SARS-CoV-2 N-Fc fusion protein by non-reducing and reduced CE-SDS.

[0244] Capillary electrophoresis of sodium dodecyl sulfate (CE-SDS) analysis in The purified SARS-CoV-2 N-Fc fusion protein solution dissolved in 200 mM HEPES, 100 mM NaCl, 50 mM NaOAc, pH 7.0 buffer was analyzed and electrophoresis plotted in GXII (Perkin Elmer, Waltham, MA). Samples were run against a protein standard of known molecular weight (MW) under non-reducing conditions, and the elution peaks represent the "apparent" MW of the fusion protein homodimer.

[0245] Under reducing conditions (e.g., by using β-mercaptoethanol to break the disulfide bonds of the SARS-CoV-2 N-Fc fusion protein homodimer), the apparent molecular weight (MW) of the resulting SARS-CoV-2 N-Fc fusion protein monomer is compared with half the molecular weight of the SARS-CoV-2 N-Fc fusion protein homodimer to determine whether the structural purity of the SARS-CoV-2 N-Fc fusion protein is likely correct.

[0246] Example 4: The sequence of the SARS-CoV-2 N-Fc fusion protein was identified by LC-MS with glycans removed.

[0247] To obtain an accurate estimate of the amount of SARS-CoV-2 N-Fc fusion protein via mass spectrometry (MS), the sample was first treated to remove naturally occurring glycans that could interfere with MS analysis. Using a Zeba desalting column (Pierce, Thermo Fisher Scientific, Waltham, MA), 100 μL of 2.5 mg / mL SARS-CoV-2 N-Fc fusion protein dissolved in a 200 mM HEPES, 100 mM NaCl, 50 mM NaOAc, pH 7.0 buffer was first buffer-exchanged to a 0.1 M Tris, pH 8.0 buffer containing 5 mM EDTA. 1.67 μL of PNGase F (Prozyme N-glycanase) was added to this solution to remove N-linked glycans (e.g., glycans linked to the asparagine side chain at the cNg-N site) present in the fusion protein, and the mixture was incubated overnight at 37°C. The sample was then analyzed by LC-MS (NovaBioassays, Woburn, MA) to obtain the molecular weight, which corresponds to the expected homodimer without the glycan. This mass was then further corrected because the enzymatic process used to cleave the glycan from cNg-asparagine also deaminates the asparagine side chain to form aspartic acid, and in doing so, the enzymatically treated homodimers as a whole gained 2 Da, corresponding to a mass of 1 Da per chain present in the homodimer. Therefore, the actual molecular weight is the measured mass minus 2 Da to correct for each enzymatic modification of the SARS-CoV-2 N-Fc fusion protein structure in the analyzed sample.

[0248] Example 5: Analysis of homodimers of SARS-CoV-2 N-Fc fusion protein by size exclusion chromatography.

[0249] Size exclusion chromatography (SEC-HPLC) of the SARS-CoV-2 N-Fc fusion protein was performed using a Waters 2795HT HPLC (Waters Corporation, Milford, MA) connected to a 2998 photodiode array at a wavelength of 280 nm. 100 μL or less of the sample containing the target SARS-CoV-2 N-Fc fusion protein was injected into a MAbPac SEC-1, 5 μm, 4 × 300 mm column (ThermoFisher Scientific, Waltham, MA) at a flow rate of 0.2 mL / min. The mobile phase consisted of 50 mM sodium phosphate, 300 mM NaCl, and 0.05% w / v sodium azide at pH 6.2. The MAbPac SEC-1 column operates based on molecular size separation principles. Therefore, larger soluble SARS-CoV-2 N-Fc aggregates (e.g., multimers of the SARS-CoV-2 N-Fc fusion protein homodimer) elute at earlier retention times, and non-aggregated homodimers elute at later retention times. The purity of the SARS-CoV-2 N-Fc fusion protein solution (as a percentage of non-aggregated homodimers) is determined by separating the homodimer mixture from the aggregated multi-homogeneous homodimers via analytical SEC-HPLC.

[0250] Example 6: In vitro binding affinity of SARS-CoV-2 N-Fc fusion protein to Fc(γ), FcRn and ACE2 receptors.

[0251] The binding of the SARS-CoV-2 N-Fc fusion protein to the Fc(γ) receptor at pH 7.4 was determined using the following ELISA assay. Human Fc(γ) receptors I, IIa, IIb, III, and FcRn were used as mammalian receptors. The SARS-CoV-2 N-Fc fusion protein was diluted to 10 μg / mL in sodium bicarbonate buffer at pH 9.6 and coated onto Maxisorp (Nunc) microtiter plates overnight at 4°C. The microplates were then washed five times with PBST (PBS / 0.05% Tween-20) buffer and blocked with Superblock blocking reagent (ThermoFisher). Biotinylated rhFc(γ) receptors (recombinant human Fc(γ)RI, Fc(γ)RIIa, Fc(γ)RIIb, Fc(γ)RIII, FcRn; R&D Systems) were prepared in PBST / 10% Superblock buffer at concentrations ranging from 6000 ng / mL to 8.2 ng / mL and loaded at 100 μL / well onto microplates coated with SARS-CoV-2 N-Fc fusion protein. Recombinant human ACE2 receptors were purchased from R&D Systems and biotinylated using a known conjugation procedure, then prepared in PBST / 10% Superblock buffer from concentrations ranging from 6000 ng / mL to 8.2 ng / mL and loaded at 100 μL / well onto microplates coated with SARS-CoV-2 N-Fc fusion protein. The microtiter plates were incubated at room temperature for 1 hour, then the microplate strips were washed 5 times with PBST, and then loaded with 100 μL / well of streptavidin-HRP diluted 1:10000 in PBST / 10% Superblock buffer. After incubation for 45 min, the microplate strips were washed again 5 times with PBST. TMB was added to visualize the binding of Fc(γ), FcRn, or ACE2 receptor proteins, and the ELISA was terminated with an ELISA termination reagent (Boston Bioproducts). The plates were read at 450 nm in an ELISA reader, and the OD values ​​(proportional to the binding of each rhFc(γ), FcRn, or ACE2 receptor to the SARS-CoV-2 N-Fc fusion protein) were plotted using GraphPad Prism software against the logarithmic concentration of each rhFc(γ) receptor, FcRn, or ACE2 receptor added to each well to generate binding curves.

[0252] General Examples of In Vivo Serological Assays for Evaluating the Function of SARS-CoV-2 N-Fc Fusion Protein in Serum

[0253] Example 7: In vivo quantitative ELISA for evaluating the titer of SARS-CoV-2RBD IgG antibodies in the serum of vaccinated mice.

[0254] The titer of anti-SP / RBD IgG Ab in serum and plasma samples (including heat-inactivated serum or plasma) from vaccinated mice was measured using a quantitative SARS-CoV-2 SP / RBD-specific ELISA. The ELISA method used recombinant SP / RBD (e.g., SARS-CoV-2 SP / RBD specific IgG) immobilized on the plastic wells of a 96-well microtiter ELISA plate. Figure 21 (As shown) serves as a capture antigen that binds to anti-SP / RBD specific antibodies (IgG) in serum samples during incubation in microplate wells.

[0255] Study serum samples diluted 1:100–1:500 in sample dilution buffer (SDB; a mixture containing 10% Superblock (Thermo) in PBS / 0.05% Tween 20) were added in duplicate or individually to SP / RBD-coated wells and incubated for one hour. After washing the plate with PBS / 0.05% Tween 20 (PBST) buffer to remove all unbound molecules, HRP-conjugated anti-mouse IgG secondary antibody (1:40,000 dilution) was added as the detection reagent and incubated for 45 minutes. After washing with PBST buffer, trimethylbenzidine (TMB) reagent was added to each well catalyzed by the HRP enzyme and incubated for 10–20 minutes. This resulted in a colorimetric change proportional to the amount of HRP-antibody conjugate bound. The enzyme substrate reaction was then terminated by adding a stop reagent (1% H2SO4), and the color intensity (optical density, OD) of each well was measured at a wavelength of 450 nm using a spectrophotometer microplate reader.

[0256] A standard curve was generated using serial dilutions of a known amount of purified mouse IgG sample (i.e., μg / mL) directly bound to the plastic wells (i.e., wells excluding bound SP / RBD or any serum sample), and was developed using the same colorimetric method as for the serum samples described above. The amount of SP / RBD-specific antibody (i.e., efficacy value) of each serum sample was expressed in μg / mL units, which were obtained from the standard curve using a 4-parameter curve fitting model with appropriate software (e.g., SoftMaxPro or Gen 5).

[0257] Example 8: Universal preclinical in vivo assessment of the effectiveness of SARS-CoV-2 N-Fc formulations in inducing anti-SP / RBD IgG Ab titers or anti-nucleocapsid IgG titers in mice.

[0258] The following in vivo studies were conducted in mice to evaluate the efficacy of the SARS-CoV-2 N-Fc fusion protein formulation.

[0259] BALB / c mice (n=7 / group) (Jackson Laboratories, Bar Harbor, ME) were acclimatized for at least 7 days, weighed, and then assigned to study groups for drug administration. Mice were randomly assigned to each study group. Mice were housed in ventilated cages with 5 animals per cage, and were provided with standard irradiated feed and filtered water. Following standard operating procedures (SOPs) and the specific study protocol, mouse ears were individually tagged for identification, and study cages were labeled with animal ID, study name, study group identifier, and study records, including individual and group dose tables, blood / serum collection sheets, weight measurements, and health observation records.

[0260] Mice were administered up to three subcutaneous doses of 10 μg / dose of the SARS-CoV-2 N-Fc fusion protein of SEQ ID NO:15, with or without Montanide. TM ISA 720 (30% / 70% v / v), the fusion protein, was synthesized in transiently transfected HEK293 cells according to Example 1. Administration was performed on days 0, 21, and 46. Mice were observed for one to three hours after dose administration to observe any immediate response, and then the general health status of the mice was observed daily. All mice underwent non-terminal blood collection via submandibular vein puncture on day 0 prior to the first immunization, and serum samples were then obtained on days 14, 21, 28, 35, 42, 55, 70, 83, and 105 for evaluating SARS-CoV-2 nucleocapsid IgG Ab titers. The collected blood was coagulated, serum was separated by centrifugation of micro-blood collection tubes, and aliquoted and frozen for antibody analysis by ELISA according to the method described in Example 7 or Example 9.

[0261] Example 9: Measure in vitro IgG antibodies against SARS-CoV-2 nucleocapsid protein (e.g., N protein) in human serum after treatment with the SARS-CoV-2 N-Fc fusion protein of SEQ ID NO:15 using ELISA.

[0262] The SARS-CoV-2 N-Fc fusion protein (SEQ ID NO: 15) was synthesized according to Example 1 and purified according to Example 2. The fusion protein structure was confirmed by non-reducing and reducing CE-SDS according to Example 3, and the fusion protein sequence was confirmed by LC-MS with glycan removal according to Example 4. Following the procedure of Example 8, mice in groups of N=7 BALB / c (Jackson Laboratories) were introduced into the group with or without Montanide on days 0, 21, and 46. TMThe SARS-CoV-2 N-Fc fusion protein of SEQ ID NO:15 was administered subcutaneously with ISA 720 adjuvant.

[0263] The COVID-19 N Protein Human IgG ELISA Kit (Abcam#ab274339) is an in vitro indirect ELISA method for the quantitative measurement of human IgG antibodies against SARS-CoV-2 nucleocapsid proteins (e.g., N protein) in human serum. Standard 96-well plates (e.g.) are used. Figure 21 As shown, 12 strips (8 wells / strip) were coated with the SARS-CoV-2N protein, which binds to the corresponding antibody present in human serum samples. A positive control was used as a calibration curve for interpretation purposes. The wells were washed, and mouse IgG secondary antibody (1:40,000 dilution) was added to the mouse samples. After washing to remove unbound biotinylated antibody, HRP-conjugated streptavidin was pipetted into the wells. The wells were washed again, and TMB substrate solution was added, with the color formation proportional to the amount of COVID-19 N protein-bound human / mouse IgG antibody. The stop solution changed the color from blue to yellow, and the color intensity was measured at 450 nm. The positive control was derived from an inactivated serum sample containing human IgG antibody against the SARS-CoV-2N protein.

[0264] Anti-nucleocapsid IgG titers were measured in mice administered the SARS-CoV-2 N-Fc fusion protein of SEQ ID NO:15 on days 14 and 21 (after dose 1), days 28 and 42 (after dose 2), and days 55, 70, 83, and 105 (after dose 3). Figure 13 As shown, the SARS-CoV-2 N-Fc fusion protein of SEQ ID NO:15 induced significant anti-nucleocapsid protein IgG titers when measured on days 42, 55, 70, and 83. This was achieved by combining the SARS-CoV-2 N-Fc fusion protein of SEQ ID NO:15 with Montanide. TM The anti-nucleocapsid IgG titer levels induced by ISA 720 and measured on days 42, 55, 70 and 83 were measurably increased, indicating that the adjuvant produced a strong synergistic effect when delivered together with the SARS-CoV-2 N-Fc fusion protein of SEQ ID NO:15, supporting its dose-saving characteristics and selection as a primary adjuvant in clinical settings.

[0265] General Examples of Synthesis, Purification, and Validation of Canine Insulin-Fc Fusion Protein

[0266] Example 10: Synthesis and preparation method of insulin-Fc fusion protein in HEK293 cells.

[0267] The insulin-Fc fusion protein was synthesized as follows. The target gene sequence was constructed using proprietary software (LakePharma, Belmont, CA) and cloned into a high-expression mammalian vector. HEK293 cells were seeded in shake flasks 24 hours prior to transfection and grown in a serum-free, chemically defined medium. The DNA expression construct encoding the target insulin-Fc fusion protein was transiently transfected into the HEK293 cell suspension using standard operating procedures for transient transfection (LakePharma, Belmont, CA). After 20 hours, cell counts were performed to determine viability and live cell count, and the results were analyzed by... (Pall FortéBio LLC, Fremont, CA) Measure titer. Obtain additional readings throughout the transient transfection production run. Harvest cultures on or after day 5.

[0268] Example 11: Synthesis and preparation method of insulin-Fc fusion protein in CHO cells.

[0269] The CHO cell line was originally derived from CHO-K1 (LakePharma, Belmont, CA), and the endogenous glutamine synthase (GS) gene was knocked out using recombinant technology with methods known in the art. Stable expression DNA vectors were designed and optimized for CHO expression and GS selection, and integrated into a high-expression mammalian vector (LakePharma, Belmont, CA). The sequence of each completed construct was confirmed before starting scale-up experiments. CHO cells adapted to suspension were cultured in chemically defined media (CDOptiCHO; Invitrogen, Carlsbad, CA) at 37°C in a humidified 5% CO2 incubator. No serum or other animal-derived products were used to culture the CHO cells.

[0270] use The system (MaxCyte, Inc., Gaithersburg, MD) was used to construct stable CHO cell lines for each insulin-Fc fusion protein by electroporation using 80 μg of DNA to transfect approximately 80 million suspension-adapted CHO cells grown in CD OptiCHO medium during the exponential growth phase. The DNA constructs contained the full-length sequence of the insulin-Fc fusion protein. Twenty-four hours later, transfected cells were counted and selected for stable integration of the insulin-Fc fusion gene. Transfected cells were seeded into CD OptiCHO selection medium containing 0–100 μM methionine sulfoxide (MSX) at a cell density of 0.5 × 10⁶ cells / mL in shake flasks and incubated at 37°C and 5% CO₂. During selection, cells were centrifuged and resuspended in fresh selection medium every 2–3 days until the CHO stable pool regained its growth rate and viability. Cell culture growth and titer were monitored.

[0271] Cells were grown to 2.5 × 10⁶ cells / mL. Viability exceeded 95% at cell bank harvest. The cells were then centrifuged, and the cell pellet was resuspended in CD OptiCHO medium containing 7.5% dimethyl sulfoxide (DMSO) to achieve a cell count of 15 × 10⁶ cells / mL / vial. The viables were then cryopreserved in liquid nitrogen.

[0272] The following describes small-scale production using CHO cells. Cells were scaled up for production at 37°C in CDOptiCHO growth medium containing 100 μM MSX, with replenishment every 2–4 days as needed. The CDOptiCHO growth medium was supplemented with glucose and additional amino acids as needed for approximately 14–21 days. The conditioned medium supernatant harvested from the stable tank production run was clarified by centrifugation. Protein was run on a Protein A (MabSelect, GE Healthcare, Little Chalfont, UK) column pre-equilibrated with binding buffer. Wash buffer was then passed through the column until the OD280 value (NanoDrop, Thermo Scientific) was measured to be at or near background levels. Insulin-Fc fusion protein was eluted with a low-pH buffer, and the elution fractions were collected, with the OD280 value of each fraction recorded. Fractions containing the target insulin-Fc fusion protein were combined and optionally further filtered using a 0.2 μM membrane filter.

[0273] The cell line is optionally further subcloned into a single clone, and optionally further selected for high-titer insulin-Fc fusion protein expression clones using limiting dilution (a method known to those skilled in the art). After obtaining a high-titer monoclonal insulin-Fc fusion protein expression cell line, insulin-Fc fusion protein production is completed as described above in growth medium without MSX, or optionally in growth medium containing MSX, to obtain a cell culture supernatant containing insulin-Fc fusion protein prepared from recombinant CHO. The MSX concentration is optionally increased over time to impose additional selectivity on clones capable of producing higher product titers.

[0274] Example 12: Purification of insulin-Fc fusion protein.

[0275] Purification of the insulin-Fc fusion protein was performed as follows. Conditioned medium supernatant containing secreted insulin-Fc fusion protein was collected from transiently or stably transfected HEK production runs and clarified by centrifugation. The supernatant containing the desired insulin-Fc fusion protein was passed through a Protein A column and eluted using a low pH gradient. The elution fractions containing the desired protein were then combined and exchanged with a buffer of 200 mM HEPES, 100 mM NaCl, 50 mM NaOAc, pH 7.0. A final filtration step was performed using a 0.2 μm membrane filter. The final protein concentration was calculated based on the optical density of the solution at 280 nm. Further optional purification was performed as needed by ion exchange chromatography (e.g., using anion exchange beads or cation exchange beads), gel filtration chromatography, or other methods.

[0276] Example 13: Confirmation of the structure of the insulin-Fc fusion protein by non-reduced and reduced CE-SDS.

[0277] Capillary electrophoresis of sodium dodecyl sulfate (CE-SDS) analysis in The purified insulin-Fc fusion protein solution dissolved in 200 mM HEPES, 100 mM NaCl, 50 mM NaOAc, pH 7.0 buffer was analyzed and electrophoresis was performed in GXII (Perkin Elmer, Waltham, MA). Samples were run according to known molecular weight (MW) protein standards under non-reducing conditions, and the elution peaks represent the "apparent" MW of the insulin-Fc fusion protein homodimer.

[0278] Under reducing conditions (e.g., by using β-mercaptoethanol to break the disulfide bonds of the insulin-Fc fusion protein homodimer), the apparent molecular weight (MW) of the resulting insulin-Fc fusion protein monomer is compared with half the molecular weight of the insulin-Fc fusion protein homodimer to determine whether the structural purity of the insulin-Fc fusion protein is likely correct.

[0279] Example 14: The sequence of the insulin-Fc fusion protein was identified by LC-MS with glycans removed.

[0280] To obtain an accurate estimate of the amount of insulin-Fc fusion protein by mass spectrometry (MS), the sample was first treated to remove native glycans that might interfere with MS analysis. Using a Zeba desalting column (Pierce, Thermo Fisher Scientific, Waltham, MA), 100 μL of 2.5 mg / mL insulin-Fc fusion protein buffer dissolved in 200 mM HEPES, 100 mM NaCl, 50 mM NaOAc, pH 7.0 buffer was first exchanged for a 0.1 M Tris, pH 8.0 buffer containing 5 mM EDTA. 1.67 μL of PNGase F enzyme (Prozyme N-glycanase) was added to this solution to remove N-linked glycans (e.g., glycans linked to the asparagine side chain at the cNg-N site) present in the insulin-Fc fusion protein, and the mixture was incubated overnight at 37°C. The sample was then analyzed by LC-MS (NovaBioassays, Woburn, MA) to obtain the molecular weight, which corresponds to the expected homodimer without the glycan. This mass was then further corrected because the enzymatic process used to cleave the glycan from cNg-asparagine also deaminates the asparagine side chain to form aspartic acid, and in doing so, the enzymatically treated homodimers as a whole gained 2 Da, corresponding to a mass of 1 Da per chain present in the homodimer. Therefore, the actual molecular weight is the measured mass minus 2 Da to correct for the enzymatic modification of the insulin-Fc fusion protein structure in the analyzed sample.

[0281] Example 15: Determination of homodimer of insulin-Fc fusion protein by size exclusion chromatography.

[0282] Insulin-Fc fusion protein was subjected to size exclusion chromatography (SEC-HPLC) using a Waters 2795HT HPLC (Waters Corporation, Milford, MA) connected to a 2998 photodiode array at a wavelength of 280 nm. 100 μL or less of the sample containing the target insulin-Fc fusion protein was injected into a MAbPac SEC-1, 5 μm, 4 × 300 mm column (ThermoFisher Scientific, Waltham, MA) at a flow rate of 0.2 mL / min. The mobile phase consisted of 50 mM sodium phosphate, 300 mM NaCl, and 0.05% w / v sodium azide at pH 6.2. The MAbPac SEC-1 column operates based on molecular size separation principles. Therefore, larger soluble insulin-Fc aggregates (e.g., multimers of the insulin-Fc fusion protein homodimer) eluted at earlier retention times, while non-aggregated homodimers eluted at later retention times. The purity of the insulin-Fc fusion protein solution (as a percentage of non-aggregated homodimers) was determined by separating the homodimer mixture from aggregated polydimers using analytical SEC-HPLC.

[0283] Example 16: In vitro Fc(γ) receptor I binding affinity assay of insulin-Fc fusion protein.

[0284] The binding of the insulin-Fc fusion protein to Fc(γ) receptor I at pH 7.4 was determined using the following ELISA assay. Since canine Fc(γ) receptor I was not commercially available, human Fc(γ) receptor I (i.e., rhFc(γ) receptor I) was used as an alternative mammalian receptor. The insulin-Fc compound was diluted to 10 μg / mL in sodium bicarbonate buffer at pH 9.6 and coated overnight at 4°C on Maxisorp (Nunc) microtiter plates. The microplate strips were then washed five times with PBST (PBS / 0.05% Tween-20) buffer and blocked with Superblock blocking reagent (ThermoFisher). Serial dilutions of biotinylated rhFc(γ) receptor I (recombinant human Fc(γ)RI; R&D Systems) ranging from 6000 ng / mL to 8.2 ng / mL were prepared in PBST / 10% Superblock buffer and loaded at 100 μL / well onto microplates coated with insulin-Fc fusion protein. The microtiter plates were incubated at room temperature for 1 hour, followed by washing the microplates five times with PBST, and then loading 100 μL / well of streptavidin-HRP, diluted 1:10000 in PBST / 10% Superblock buffer. After incubation for 45 min, the microplates were washed five times again with PBST. TMB was added to visualize the bound Fc(γ) receptor I protein, and the ELISA was terminated with an ELISA stop kit (Boston Bioproducts). The plates were read at 450 nm in an ELISA reader, and the OD values ​​(proportional to the binding of each rhFc(γ) receptor to the insulin-Fc fusion protein) were plotted using GraphPad Prism software against the logarithmic concentration of each rhFc(γ) receptor added to each well to generate binding curves.

[0285] Example 17: In vivo pharmacodynamics (PD) of regularly administered insulin Fc fusion protein in customer-owned dogs.

[0286] The bioactive insulin-Fc fusion homodimer construct was synthesized according to Example 10 or Example 11 and purified according to Example 12. Its effect on fasting blood glucose levels was evaluated as follows.

[0287] Option 1 was an open, self-controlled, single-arm, pilot-scale field efficacy study of insulin-Fc fusion protein in customer-owned dogs diagnosed with diabetes. Efficacy was assessed by comparing glycemic control (based on clinical signs, fructosamine levels, and interstitial glucose concentration using a continuous glucose monitoring device (CGMS)) with standard insulin therapy (for one week) versus 8 weeks of escalating insulin-Fc fusion protein therapy. The subcutaneous dose was initiated at 0.1 mg / kg and then increased weekly up to a maximum of 0.5 mg / kg based on CGMS results and clinical signs. Table 2 provides the study timeline for some dogs in the Option 1 study.

[0288]

[0289]

[0290] Option 2 was an open, self-controlled, single-arm, pilot-scale field efficacy study involving the treatment of customer-owned dogs diagnosed with diabetes with insulin-Fc fusion protein. Efficacy was assessed by comparing glycemic control (based on clinical signs, fructosamine levels, and interstitial glucose concentration using a continuous glucose monitoring device (CGMS)) with standard insulin therapy (for one week) versus 5 weeks of escalating insulin-Fc fusion protein therapy. A subcutaneous dose of 0.1 mg / kg was initiated and then increased weekly up to a maximum of 0.5 mg / kg based on CGMS results and clinical signs. Table 3 provides the study timeline for some dogs in the Option 2 study.

[0291]

[0292]

[0293] Upon completion of any of the above protocols, a "home use" evaluation for up to one year may be selected. During this phase, each dog will be treated by a veterinarian as is the case for any other patient using regular insulin. Throughout the study, clinical signs and interstitial glucose concentrations will be monitored using a continuous glucose monitoring device (CGMS). Additionally, blood samples will be collected as frequently as possible to assess fructosamine levels, blood chemistry, and blood cell counts, and to detect the presence of anti-drug and anti-insulin antibodies.

[0294] Example 18: Measurement of in vivo anti-insulin antibody (AIA) titer in dogs after periodic administration of insulin-Fc fusion protein – Protocol 1.

[0295] Maxisorp ELISA plates (Nunc) were coated with purified RHI diluted 30 μg / mL in coating buffer (pH 9.6 carbonate-biocarbonate buffer) and incubated overnight at 4°C. The plates were then washed five times with PBST (PBS + 0.05% Tween 20) and blocked with SuperBlock blocking solution (Thermo Fisher) for ≥1 hour (or overnight). To calculate the AIA of canine IgG units, strips were directly coated with serially diluted canine IgG (Jackson Immunoresearch) at a concentration of 300–4.69 ng / mL in pH 9.6 Carb-Biocarb coating buffer at a 1:2 ratio and incubated overnight at 4°C, then used to create a 7-point pseudo-standard curve. Standard strip plates were also washed and blocked with SuperBlock blocking solution for ≥1 hour (or overnight).

[0296] Serum samples were diluted to ≥1:100 (typically 1:200) in PBST / SB / 20% HS sample dilution buffer (PBS + 0.1% Tween 20 + 10% SuperBlock + 20% horse serum) and RHI-coated strips were added, 100 μL / well for replicates. Duplicate strips of canine IgG-coated standard strips were also added to each plate, and 100 μL / well was filled with PBST / SB (PBS + 0.1% Tween 20 + 10% SuperBlock) buffer. The plates were incubated at room temperature for 1 hour. After incubation, the plates were washed 5 times with PBST. To detect AIA, HRP-conjugated goat anti-canine IgG F(ab')2 (Jackson Immunoresearch), which cross-reacts with canine IgG, was diluted to 1:10,000 in PBST / SB, 100 μL / well was added to both sample and standard wells, and incubated at room temperature in the dark for 45 minutes. The plate was washed five times with PBST and developed by adding 100 μL / well of TMB substrate (Invitrogen) to the plate in the dark at room temperature for 15–20 minutes. Development was then stopped by adding 100 μL / well of ELISA stop solution (Boston Bioproducts), and absorbance was read at 450 nm using a SpectraMax microplate reader after 30 minutes. Anti-drug antibody concentration was determined by inserting OD values ​​into a 4-PL pseudo-standard curve using SoftMax Pro software.

[0297] Example 19: Measurement of in vivo anti-insulin antibody (AIA) titer in dogs after periodic administration of insulin-Fc fusion protein – Protocol 2.

[0298] The general procedure is as follows: Serum containing labeled antigen is incubated overnight with and without cold insulin. Antibody-bound labeled antigen is precipitated in 96-well plates using Protein A / G agarose (Sepharose), with each serum sample tested repeatedly. The 96-well plates are washed to remove unbound labeled antigen. Each well is counted using a 96-well plate beta counter. Results are expressed as an index, which corrects for the Δcpm of positive and negative control sera in a specific assay by the Δcpm of the test serum.

[0299] The specific procedure involves the preparation of the following two buffers: Buffer 1 (150 mM NaCl, 20 mM Tris-HCl, 1% BSA, 0.15% Tween-20, 0.1% sodium azide, pH 7.4) and Buffer 2 (same as Buffer 1, except that 0.1% BSA is used instead of 1% BSA). Each serum sample is centrifuged if necessary to remove fibrin clots. A stock solution of radiolabeled insulin is then prepared by dissolving 10 μCi of 125I-insulin powder in 1 mL of 5% BSA PBS. A “hot” insulin antigen solution is prepared using 3040 μL of Buffer 1 and 160 μL of radiolabeled insulin stock solution. A stock solution of 2784 μL of Buffer 1, 160 μL of radiolabeled insulin stock solution, and 256 μL of… "Cold-inhibitory" insulin antigen solutions were prepared using Eli Lilly (IN). All solutions were stored on ice before use. In PCR tubes, 6 μL of each serum sample was mixed with 30 μL of "warm" insulin antigen solution, and 6 μL of each serum sample was mixed with 30 μL of "cold-inhibitory" insulin antigen solution. The resulting mixtures were incubated overnight at 4°C. Then, by adding 150 μL of Buffer 1 to each well and incubating overnight at room temperature under aluminum foil caps, the plate was washed and the wash buffer was removed to allow the plate to be coated with BSA. The protein-A / G agarose mixture was prepared in two parts. The protein-A agarose solution was prepared at a volume concentration of 62.5% in Buffer 1. The protein-G agarose solution was prepared at a volume concentration of 40% in Buffer 1. Finally, the protein-A / G agarose mixture (final concentration: 50% protein-A / 8% protein-G agarose) was prepared by mixing the protein-A and protein-G agarose solutions in a 4:1 ratio. For assay, 50 μL of protein A / G-agarose mixture and 30 μL of overnight incubated serum solution were added to each well in duplicate. The plate was mixed on a plate shaker at 4°C for 45 minutes, then washed seven times using a Millipore plate washer with 200 μL of wash buffer added to each well, and then incubated at 37°C for 15 minutes to dry. 50 μL of scintillation mixture (Microscint-20) was added to each well, and the plate was counted using a 96-well plate counter to determine the cpm of each well.

[0300] equivalent

[0301] In the claims, unless indicated to the contrary or obvious from the context, articles such as “a,” “an,” and “the” may indicate one or more. Claims or descriptions containing “or” are considered to include “or” among one or more members of a group if one, more than one, or all members of that group are present in, used in, or otherwise associated with a given product or process, unless indicated to the contrary or obvious from the context. This disclosure includes embodiments in which one member of that group is present in, used in, or otherwise associated with a given product or process. This disclosure also includes embodiments in which more than one or all members of that group are present in, used in, or otherwise associated with a given product or process.

[0302] Furthermore, this disclosure includes all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims are introduced into another claim. For example, any claim dependent on another claim may be modified to include one or more limitations found in any other claim dependent on the same basic claim. Where elements are presented in list form, such as in Markush groups, each subgroup of elements is also disclosed, and any one or more elements may be removed from the group. It should be understood that, generally, where an aspect of this disclosure is referred to as including a particular element and / or feature, certain embodiments of this disclosure or aspects of this disclosure consist of or are substantially composed of such elements and / or features. For simplicity, these embodiments are not specifically described herein. It should also be noted that the terms “comprise(s)”, “comprising”, “contain(s)”, and “containing” are intended to be open-ended, and their use allows for the inclusion of additional elements or steps. Where a scope is given, endpoints are included. Furthermore, unless otherwise stated or otherwise apparent from the context and from the understanding of one of ordinary skill in the art, values ​​expressed as ranges may be assumed to be any particular value or subrange within the range described in different embodiments of this disclosure, up to one-tenth of the lower limit unit of the range, unless the context expressly specifies otherwise.

[0303] Other advantages of various embodiments of the present technology will become apparent to those skilled in the art after reading this disclosure and the following working examples. It should be understood that, unless otherwise stated herein, the various embodiments described herein are not necessarily mutually exclusive. For example, features described or depicted in one embodiment may also be included in other embodiments, but are not necessarily required to be included. Therefore, the present invention covers various combinations and / or arrangements of the specific embodiments described herein.

[0304] This specification also uses numerical ranges to quantify certain parameters relating to various embodiments of the invention. It should be understood that when numerical ranges are provided, these ranges are to be interpreted as providing textual support for claims that list only the lower limit of the range and claims that list only the upper limit of the range. For example, the disclosed numerical range of about 10 to about 100 provides literal support for claims listing “greater than about 10” (no upper limit) and claims listing “less than about 100” (no lower limit).

Claims

1. A fusion protein comprising a nucleocapsid domain and an Fc fragment, wherein, The nucleocapsid domain and the Fc fragment are connected via a peptide linker, wherein the nucleocapsid domain includes the following sequence: RSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAE (SEQ ID NO: 8).

2. The fusion protein according to claim 1, wherein, The Fc fragment includes the following sequence: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:1).

3. The fusion protein according to claim 1, wherein, The connector includes the following sequence: SGGGSGGGS (SEQ ID NO: 14).

4. The fusion protein according to claim 1, wherein, The fusion protein includes the following sequence: RSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAESGGGSGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:15).

5. The fusion protein according to claim 1, wherein, The fusion protein is a homodimer.

6. The fusion protein according to claim 1, wherein, The Fc fragment is glycosylated.

7. An immunogenic composition comprising the fusion protein according to claim 1 and a pharmaceutically acceptable carrier.

8. The immunogenic composition according to claim 7, further comprising an adjuvant.

9. The immunogenic composition according to claim 8, wherein, The adjuvant is Montanide. TM ISA-720.

10. The immunogenic composition according to claim 8, wherein, The fusion protein is emulsified with the adjuvant.

11. A method for increasing antibody production against an antigen in a subject, the method comprising administering to the subject a therapeutically effective amount of the fusion protein according to claim 1.

12. The method according to claim 11, wherein, Prior to administration of the fusion protein, the subject had a measurable antibody titer against the antigen.

13. The method according to claim 11, wherein, Prior to administration of the fusion protein, the subject was in the antibody null state.

14. The method according to claim 11, wherein, The fusion protein is administered via injection.

15. The method according to claim 11, wherein, The fusion protein is administered subcutaneously or intramuscularly.

16. The method according to claim 11, wherein, The fusion protein was administered co-administered with an adjuvant.

17. The method of claim 16, further comprising premixing the fusion protein with the adjuvant prior to administration.

18. The method according to claim 17, wherein, The premixing includes emulsifying the adjuvant and the fusion protein to produce an emulsion, and administering the emulsion to the subject.

19. A method for inducing an immune response against a viral infection in a subject, the method comprising administering to the subject a therapeutically effective amount of the fusion protein according to claim 1.

20. The method according to claim 19, wherein, The subjects had measurable antibody titers against the viral infection prior to administration of the fusion protein.

21. The method according to claim 19, wherein, Prior to administration of the fusion protein, the subject was in the antibody null state.

22. The method according to claim 19, wherein, The fusion protein is administered via injection.

23. The method according to claim 22, wherein, The fusion protein is administered subcutaneously or intramuscularly.

24. The method according to claim 19, wherein, The fusion protein was administered co-administered with an adjuvant.

25. The method of claim 24, further comprising premixing the fusion protein with the adjuvant prior to administration.

26. The method of claim 25, wherein, The premixing includes emulsifying the adjuvant and the fusion protein to produce an emulsion, and administering the emulsion to the subject.

27. A method for producing the fusion protein according to claim 1, the method comprising transiently transfecting a nucleic acid encoding the fusion protein into HEK293 cells, wherein, The transfected HEK293 cells express the fusion protein, and wherein, in any of the aforementioned expression systems, the yield of the purified or isolated fusion protein is greater than 100 mg / L.

28. A cell engineered to express the fusion protein according to claim 1.

29. A cDNA encoding the fusion protein according to claim 1.

30. The use of a fusion protein comprising the sequence of SEQ ID NO:15 or a pharmaceutical composition thereof for the treatment and / or prevention of viral infection with SARS-CoV-2 virus.

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