Patent Application
20230250410
The present application relates to the prevention or treatment of the diseases, symptoms or conditions involving Angiotensin-Converting Enzyme 2 (ACE2) such as coronavirus disease 2019 (COVID-19) and related conditions.
Unless otherwise indicated herein, the materials described in this section are not prior art to the claims in this application and are not admitted to be prior art by inclusion in this section.
COVID-19 is an infectious disease caused by severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2). Complications of COVID-19 may include long-term lung damage, pneumonia, acute respiratory distress syndrome (ARDS), peripheral and olfactory nerve damage, multi-organ failure, septic shock, and death. A study of the first 41 cases of confirmed COVID-19, published in January 2020 in The Lancet, reported the earliest date of onset of symptoms as Dec. 1, 2019. By Mar. 11, 2020, the World Health Organization (WHO) declared the COVID-19 outbreak a pandemic. As of Sep. 26, 2020, more than 32.6 million cases have been reported across 188 countries and territories with more than 990,000 deaths, of which more than 7.5 million cases and 205,000 deaths were reported by the United States.
No medication or vaccine other than Remdesivir is approved with the specific indication to treat COVID-19. The US National Institute Health guidelines do not recommend any medication for prevention of COVID-19 outside the setting of a clinical trial, either before or after exposure to the SARS-CoV-2 virus. Nine vaccines have been authorized by at least one national regulatory authority for public use: two RNA vaccines from Pfizer-BioNTech and Moderna; three conventional inactivated vaccines from Sinopharm, Bharat Biotech, and Sinovac; three viral vector vaccines from Sputnik V, Oxford-AstraZeneca, and Janssen; and one peptide vaccine (EpiVacCorona).
Angiotensin-converting enzyme 2 (ACE2) is a zinc-containing metalloenzyme located on the cell membrane of mainly alveolar cells of the lung, enterocytes of the small intestine, endothelial cells of arterial and venous, smooth muscle cells of arteries, and other lineages of cells in the lungs, arteries, heart, kidney, intestines, and other tissues. ACE2 regulates the renin angiotensin system by counterbalancing angiotensin-converting enzyme activity in the cardiovascular, renal and respiratory systems, indicating its important role in the control of blood pressure. ACE2 plays a protective role in the physiology of hypertension, cardiac function, heart function, and diabetes. In the acute respiratory distress syndrome (ARDS), ACE, AngII, and AT1R promote the disease pathogenesis, whereas ACE2 and AT2R protect from ARDS. In addition, ACE2 has been identified as a receptor of severe acute respiratory syndrome (SARS) coronavirus and plays a key role in severe acute respiratory syndrome (SARS) pathogenesis. Of a family of coronaviruses, at least three viruses, SARS-CoV, MERS CoV, and SARS-CoV-2, use one of their viral proteins, also known as Spike, to bind to the ACE2 protein on the surface of human host cells for the viral entry into human body.
SARS-CoV-2 is one of seven known coronaviruses to infect humans, including SARS-CoV-1 and MERS CoV viruses that caused the outbreak of SARS in Asia in 2003 and in Middle East in 2012. The immune response to SARS-CoV-2 virus involves a combination of the cell-mediated immunity and antibody production. Although more than 100 million people have recovered from COVID-19, it remains unknown if the natural immunity to SARS-CoV-2 virus will be long-lasting in individuals. One of the concerns relates to the virus's continual accumulation of mutations, which may alter the spectrum of viral antigenicity and cause reinfection by mutant strains of the virus. As of January 2021, variant strains of SARS-CoV-2 virus identified in Europe and South Africa seem to be spreading so quickly. These variant strains may harbor mutations that ultimately enhance viral recognition and infection into host cells. Whether these or other foreseeable variants might diminish the potency of vaccines or overcome natural immunity and lead to a spate of reinfections remains unknown.
The other concern relates the phenomenon of antibody-dependent enhancement (ADE). ADE occurs when the binding of suboptimal antibodies enhances viral entry into host cells. In coronaviruses, antibodies targeting the viral spike (S) glycoprotein promote ADE. In cases of SARS-CoV-1 viruses, the antibodies that neutralized most variants were found to be able to enhance immune cell entry of the mutant virus, which, in turn, worsen the disease the vaccine was designed to protect against. Therefore, ADE can hamper vaccine development, as a vaccine may cause the production of suboptimal antibodies. In this context, any preventive strategy other than vaccines shall be considered as a viable alternative circumventing ADE, either before or after exposure to SARS-CoV-2 virus.
Therefore, there remains a significant need for effective treatments or preventions of the diseases or conditions involving Angiotensin-Converting Enzyme 2 (ACE2).
The following summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features will become apparent by reference to the drawings and the following detailed description.
The application provides, among others, fusion proteins, fusion protein complexes, protein complexes, immunoconjugates containing fusion protein complexes and pharmaceutical compositions containing fusion protein complexes. The application also provides methods of making the fusion proteins and fusion protein complexed and methods for using fusion proteins or fusion protein complexes to treat or prevent diseases.
In one aspect, the application provides fusion proteins that have ACE2 activity. In one embodiment, the fusion protein includes a variant angiotensin converting enzyme 2 (ACE2) domain covalently fused to a Fc domain. In one embodiment, the variant ACE2 domain comprises a N-terminal deletion, a C-terminal deletion, or both, relative to a full-length wildtype ACE2. In one embodiment, the full-length wildtype ACE2 domain has an amino acid sequence with at least 95%, 97%, or 98% sequence identity to SEQ ID NO: 1. In one embodiment, the variant ACE2 domain has ACE2 activity.
In one embodiment, the variant ACE2 domain comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to a segment of amino acid sequence from a full-length wildtype ACE2. In one embodiment, the segment may start with an amino acid residual selected from the residual 1-17 of a full-length wildtype ACE2. In one embodiment, the segment may end with an amino acid residual selected from the residual 615-740 of the full-length wildtype ACE2. For example, the variant ACE2 domain may have an amino acid sequence having at least 98% or 99% sequence identity to a segment of amino acid sequence from residual 1 to residual 615, from residual 2 to residual 618, from residual 2 to residual 740, from residual 4 to residual 615, from residual 17 to residual 615, from residual 17 to residual 740, or any other combination of the starting residual and ending residual, from a full-length wildtype ACE2.
In one embodiment, the variant ACE2 domain comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 3.
In one embodiment, the variant ACE2 domain may have a higher binding affinity to SARS-CoV, or SARS Spike protein than the full-length wildtype ACE2. For example, the variant ACE2 domain may have a binding affinity to SARS-CoV, or SARS spike protein with a KD from 0.1 nM to 100 nM.
In one embodiment, the variant ACE2 domain may have a higher binding avidity to SARS-CoV, or SARS Spike protein than the full-length wildtype ACE2. For example, the variant ACE2 domain may have a binding avidity to SARS-CoV, or SARS spike protein with a KD from 0.01 nM to 10 nM.
In one embodiment, the Fc domain is derived from a Fc domain of an immunoglobulin. The immunoglobulin may be IgG1, IgG2, IgG3, IgG4, IgA1 (d-IgA1, 5-IgA1), IgA2, IgD, IgE, or IgM. In one embodiment, the Fc domain may have a Fc hinge region. In one embodiment, the Fc hinge region may be engineered to C220S. In one embodiment, the Fc domain may include a null mutation selected from K322A, L234A, and L235A when compared to a wildtype Fc domain. In one embodiment, the wildtype Fc domain has an amino acid sequence having at least 98%, or 99% sequence identity to SEQ ID NO: 5.
In one embodiment, the Fc domain may lack effector function. In one embodiment, the Fc domain may lack antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). In one embodiment, the Fc domain comprises an IgG1 Fc domain.
In one embodiment, the Fc domain comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 6.
In one embodiment, the fusion protein may have an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% of sequence identity to SEQ ID NOs: 7, 9, 11, 13, 15, 16, 17, 18, 19, and 21.
In one embodiment, the fusion protein may have a molecular weight from about 50 kDa to 250 kDa. In one embodiment, the fusion protein may have a molecule weight of 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 120 kDa, 150 kDa, 180 kDa, 200 kDa, 250 kDa or any number in between.
In a second aspect, the application provides fusion protein complexes. In one embodiment, the fusion protein complex is a homodimer of the fusion protein as disclosed herein. In one embodiment, the fusion protein complex includes two variant ACE2 domains.
In one embodiment, the fusion protein complex comprises at least two fusion proteins. In one embodiment, the two fusion protein are paired through one or two disulfide bonds. In one embodiment, the disulfide bond is located on the hinge of the Fc domain.
In one embodiment, the fusion protein or fusion protein complex has a binding affinity to SARS-CoV-2, SARS-CoV, or SARS spike protein or a fragment thereof. In one embodiment, the binding affinity has an equilibrium dissociation constant not greater than 0.1 nM, 0.5 nM, 1 nM, 2 nM, 3 nM, 5 nM, 10 nM, 20 nM, 25 nM, 30 nM, 40 nM, 50 nM, 60 nM, 80 nM, or any number in between.
In one embodiment, the fusion protein or fusion protein complex has a binding avidity to SARS-CoV-2, SARS-CoV, or SARS spike protein or a fragment thereof. In one embodiment, the binding avidity has an equilibrium dissociation constant not greater than 0.01 nM, 0.05 nM, 1 nM, 2 nM, 3 nM, 5 nM, 10 nM, or any number in between.
In one embodiment, the fusion protein or fusion protein complex has a specific enzymatic activity from about from 50 pmol/min/μg to about 5000 pmol/min/μg. In one embodiment, the fusion protein has a specific enzymatic activity of about 568 pmol/min/μg.
In a third aspect, the application provides protein complexes. In one embodiment, the protein complex includes the fusion protein or fusion protein complex as disclosed thereof bound to a viral protein. In one embodiment, the viral protein comprises SARS-CoV-2, SARS-CoV, SARS spike protein, coronavirus, SARS virus, or a fragment or a combination thereof.
In a further aspect, the application provides isolated nucleic acid encoding the fusion protein as disclosed thereof.
In a further aspect, the application provides an expression vector comprising the isolated nucleic acid encoding the fusion protein as disclosed thereof.
In a further aspect, the application provides host cell comprising the nucleic acid that encodes the fusion protein as disclosed thereof. In one embodiment, the host cell is a prokaryotic cell. In one embodiment, the host cell is an eukaryotic cell.
In a further aspect, the application provides methods for producing the fusion protein and fusion protein complex as disclosed thereof. In one embodiment, the method comprises culturing the host cell with nucleic acid encoding the fusion protein or fusion protein complex so that the fused protein or fusion complex is produced.
In a further aspect, the application provides protein-conjugate. In one embodiment, the protein-conjugate includes the fusion protein or fusion protein complex as disclosed thereof and a drug moiety. The drug moiety may be linked to the fusion protein or fusion protein complex through a linker. In one embodiment, the linker may be a covalent bond selected from an ester bond, an ether bond, an amine bond, an amide bond, a disulphide bond, an imide bond, a sulfone bond, a phosphate bond, a phosphorus ester bond, a peptide bond, a hydrazone bond or a combination thereof.
In one embodiment, the drug moiety may be an antiviral agent, an immune regulatory reagent, an imaging agent or a combination thereof. In one embodiment, the antiviral agent may be favipiravir, ribavirin, galidesivir, remdesvir, or a combination thereof. In one embodiment, the imaging agent may be radionuclide, a florescent agent, a quantum dots, or a combination thereof.
In a further aspect, the application provides pharmaceutical compositions for treating disease or condition involving Angiotensin-Converting Enzyme 2 (ACE2). In one embodiment, the pharmaceutical composition includes the fusion protein or fusion complex as disclosed herein and a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutical composition further includes an antiviral agent. In one embodiment, the pharmaceutical composition includes the protein-conjugate as disclosed thereof and a pharmaceutically acceptable carrier.
In a further aspect, the application provides method of treating or preventing a viral infection, acute respiratory distress syndrome, pulmonary arterial hypertension, or acute lung injury in a subject. In one embodiment, the method includes the step of administering to the subject an effective amount of the fusion protein or fusion complex as disclosed herein. In one embodiment, the method further includes co-administering an effective amount of a therapeutic agent. In one embodiment, the therapeutic agent includes an antiviral agent. In one embodiment, the subject is a mammal.
In one embodiment, the viral infection may be the infection of SARS-CoV-2, SARS-CoV, SARS Spike protein, coronavirus, SARS virus, or a fragment or a combination thereof.
In one embodiment, the method may include administering the fusion protein or fusion protein complex intravenously, subcutaneously, through nasal passage (such as nasal spray), or through pulmonary passageway.
In a further aspect, the application provides a solution. In one embodiment, the solution includes an effective concentration of the fusion protein or fusion protein complex as disclosed herein. In one embodiment, the solution is blood plasma in a subject. In one embodiment, the solution includes the fusion protein, the fusion protein complex or the protein complex as disclosed herein, and the solution is blood plasma in a subject.
The foregoing and other features of this disclosure will become more fully apparent from the following description and appended claims, taken in conjunction with the accompanying drawings. Understanding that these drawings depict only several embodiments arranged in accordance with the disclosure and are, therefore, not to be considered limiting of its scope, the disclosure will be described with additional specificity and detail through use of the accompanying drawings, in which:
In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein, and illustrated in the Figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein.
The present application relates to, among others, the generation and characterization of fusion proteins such as recombinant human ACE2-Fc fusion proteins. In some embodiments, these fusion proteins are capable of protecting the membranous ACE2 of human host cells from the viral particles or virus. In one embodiment, the viral particles or virus may utilize viral spike proteins for viral entry into host cells after infection. In one embodiment, the viral particles include, but not limited to, SARS-CoV-2 virus, COVID-19 virus, variants of SARS-CoV-2, and other coronaviruses. In one embodiment, the virus may cause severe acute respiratory syndrome (SARS). In one embodiment, the SARS may include coronavirus disease 2019 or COVID-19.
In one embodiment, the recombinant human ACE2-Fc fusion proteins may be a fusion protein of ACE2 zinc metallopeptidase domain (also known as ACE2 extracellular domain, ACE2-ECD) and IgG1 Fc fragment. In one embodiment, the fusion protein is SI-F019, a fusion protein of ACE2-ECD and IgG1 Fc fragment with mutations of C220S, L234A, L235A, and K322A according to EU numbering system (Table 1 and
The terms “a”, “an” and “the” as used herein are defined to mean “one or more” and include the plural unless the context is inappropriate.
The term “recombinant fusion protein” refers to a protein that is created through genetic engineering of a fusion gene encoding two or more genes that originally coded for separate proteins.
The term “ACE2-Fc” refers to a recombinant fusion protein of a human ACE2 protein fragment and an engineered fragment of the fragment crystallizable region (Fc region) of a human immunoglobulin, where the human Immunoglobulin including, but not limited to, IgG1, IgG2, IgG3, IgG4, IgA1 (d-IgA1, S-IgA1), IgA2, IgD, IgE, and IgM.
The term “spike”, “Spikes”, “S protein”, or variants refers to the protein responsible for allowing the virus to attach (“S1 subunit” or “S1 protein”) to and fuse (“S2 subunit” or “S2 protein”) with the membrane of a host cell. In the case of COVID-19, SARS-CoV-2 has sufficient affinity to the ACE2 receptor on human cells to use them as a mechanism of cell entry, and SARS-CoV-2 has a higher affinity to human ACE2 than the original SARS virus.
The term “Fc domain”, “Fc fragment”, and “Fc region” refer to the identical domain or fragment of the Fc region (“Fc domain” and “Fc fragment”, respectively) in IgG, IgA, and IgD antibody isotypes, which is derived from the hinge, and the second and third constant domains (CH2-CH3) of the antibody's two heavy chains.
The term “affinity” refers to a measure of the attraction between two polypeptides, such as receptor/ligand, ACE2/spike protein or it's variants, for example. The intrinsic attractiveness between two polypeptides can be expressed as the binding affinity equilibrium constant (KD) of a particular interaction. A KD binding affinity constant can be measured, e.g., by Bio-Layer Interferometry.
The term “avidity” refers to the accumulated strength of multiple affinities of individual non-covalent binding interactions, such as between a protein receptor and its ligand, and is commonly referred to as functional affinity. As such, avidity is distinct from affinity, which describes the strength of a single interaction.
The term “antigenic drift” refers to random genetic mutation of an infectious virus resulting in a new strain of virus with minor changes in antigenicity, to which the antibodies that prevented infection by previous strains may not be effective.
The term “cytokine release syndrome” (CRS) refers to CRS in severe cases of COVID-19 associated with an increased level of inflammatory mediators including cytokines and chemokines, such as interleukin (IL)-2, IL-6, IL-7, IL-10, tumor necrosis factor (TNF), granulocyte colony-stimulating factor (G-CSF), monocyte chemoattractant protein-1 (MCP1; also known as CCL2), macrophage inflammatory protein 1 alpha (MIP1α; also known as CCL3), CXC-chemokine ligand 10 (CXCL10), C-reactive protein, ferritin, and D-dimers in blood upon SARS-CoV-2 infection.
Human membranous ACE-2 is the receptor critical for mediating SARS-CoV viral entry into host cells in human. The human ACE2 protein has at least three functional domains: a signal peptide (residues 1-17), zinc metallopeptidase domain (residues 18-615), and a TMPRSS2 protease cutting site (residues 697-716) (SEQ ID NO: 1 is the full length human ACE2 protein sequence from Genbank number: NP_001358344.1), of which the SARS-CoV viral protein, Spike, interacts with the zinc metallopeptidase domain (SEQ ID NO: 3 is the protein sequence of truncated ACE2 from residue 1 to 615). On the other hand, the Fc region of a human antibody (SEQ ID NO: 5) is capable of interacting with Fc receptors (FcRs) on many immune cells and some proteins of the complement system. Each Fc fragment of IgG1 Fc region contains a cysteine at C220 (according to EU numbering system), which may intrinsically form disulfide bond with either kappa or lambda light chain. To reduce the risk of having a free cysteine that may destabilize and/or inactivate the protein, C220 may be substituted for serine (C220S) or other amino acids. To reduce the Fc binding to FcγR and C1q, other point mutations, such as K322A, L234A, and L235A, may be engineered into wild type IgG1 Fc fragment. Collectively, the IgG1 Fc fragment harboring the four mutations is called IgG1 Fc null (SEQ ID NO: 6).
The recombinant human ACE2-Fc fusion proteins (as listed in Table 1) were engineered to produce soluble fusion proteins, of which SI-69R2 (SEQ ID NO: 7) is a recombinant fusion protein of a truncated ACE2 fragment without the TMPRSS2 protease cutting site and the IgG1 Fc null fragment. Other recombinant fusion proteins were created to provide a Fc fragment of Ig isotype, such as SI-69R2-G4 (IgG4 Fc, SEQ ID NO: 9), SI-69R2-A1 (IgA1 Fc, SEQ ID NO: 11), SI-69R2-A2(IgA2 Fc, SEQ ID NO: 13), or wild type IgG1 Fc fragment (IgG1 Fc, SEQ ID NO: 19). The recombinant fusion protein of a truncated ACE2 with all three domains and a wild type IgG1 Fc fragment was also created (SI-69R4, 1-740, SEQ ID NO: 21). Of all recombinant ACE2-Fc fusion proteins, the signal peptide (ACE2 residues 1-17) may be replaced with other signal peptides at different lengths, without affecting the function of other domains in either human ACE protein or ACE2-Fc fusion proteins.
The recombinant fusion genes encoding the fusion proteins in Table 1 were cloned into either pCGS3.0 (such as SI-69R2) or pTT5 expression vector (such as SI-69R4 and SI-69R10) and expressed in ExpiCHO cells. All the fusion proteins were purified following standard protein expression protocols, sterilized using a 0.22 um filter, and stored in a cryopreservation buffer at 4° C. During the expression and purification, each recombinant fusion protein may undergo post-translational modification, including N-glycosylation and the cleavage of N-terminal signal peptide (17 amino acids). In case of SI-69R2, the purified fusion protein was given a new name, SI-F019.
As shown in
The SI-F019 fusion protein likely undergoes post-translation modification, such as N-glycosylation, and homodimerization linked by the two disulfide bonds of Fc region. To assess the actual molecule weight of the SI-F019 dimer, the analytical size exclusion chromatography (SEC) was used, in a combination of multi-angle light scattering (MALS), absorbance (UV), and/or refractive index (RI) concentration detectors techniques, as shown in
SI-F019 was designed to block SARS-CoV viral entry into human by preventing the spike proteins from binding to the membranous ACE2 protein on human host cells. Spikes are the most distinguishing feature of coronaviruses, which are the knoblike structures responsible for the corona- or halo-like surface. The spike proteins are generally composed of glycoproteins, and each spike is composed of a trimer of the S protein, and the S protein is in turn composed of an S1 and S2 subunit. The homotrimeric S protein mediates the receptor binding and membrane fusion between the virus and host cell. The S1 subunit forms the head of the spike and has the receptor-binding domain (RBD). The S2 subunit forms the stem which anchors the spike in the viral envelope and on protease activation enables fusion. In a functionally active state, the subunit complex of S1 and S2 is split into individual subunits when the virus binds and fuses with the host cell under the action of proteases, such as cathepsin family and transmembrance protease serine 2 (TMPRSS2) of the host cell. Spikes play important roles in the viral entry of infection process by coronavirus. In case of COVID-19, SARS-CoV-2 virus docks onto the membrane bound ACE2 receptor on the host cell surface, and the interaction between spikes and the functional domain of ACE2 brings about the release of viral nucleocapsid into the host cell cytoplasm by triggering fusion between the viral envelope and host cell membranes.
SI-F019 was evaluated for the binding affinity and avidity of ACE-Fc fusion proteins to the viral spike proteins. In a Bio-Layer Interferometry analysis, the samples of spike proteins include SARS-CoV-2 spike trimer, SARS-CoV-2 S1 protein, SARS-CoV-2 S1 protein RBD domain, and SARS-CoV-1 RBD domain (Table 2). The binding affinity assay measured the binding of SI-F019 immobilized on the anti-human IgG Fc Capture Biosensors tip surface to the spike protein in solution. The avidity assay measured the binding of a biotinylated spike protein immobilized on the Streptavidin Biosensors tip surface to SI-F019 in solution. The data analysis utilized a 1:1 fitting model to calculate both the binding affinity and avidity. The result indicates that the binding affinity and avidity of SI-F019 to these spike proteins, fragments, or domains seem to be within their respective scales of KD in nanomolar (nM) (Table 2). This characteristic and informative data may be useful references for measuring the SI-F019 protein complex with variants of viral spike proteins indicative of potential antigenic drift among SARS-CoV-2 variants. The phenomenon is close to be reality referring to the viral mutations in certain strains of SARS-CoV-2 virus, such as D614G in the spike protein (Zhang et al., 2020), which has likely altered the viral affinity to membranous ACE2 and viral entry into the host cells.
In parallel to its binding to spikes, SI-F019 was evaluated for its binding to human FcγRs, C1q, and FcRn by using Bio-Layer Interferometry. As shown in Table 3, the binding to FcγRs, including FcγRI, FcγRIIa, FcγRIIb, and FcγRIIIa, was not detected, nor the binding to C1q. However, SI-F019 did bind to FcRn and the binding affinity was determined at a KD of 37.6 nM, which is comparable to that of human IgG1 Fc region.
Human ACE2 is subject to membranous protease hydrolysis by TMPRSS2, and monomeric extracellular ACE2 is shed from cells, which can be readily detected in serum. In the recombinant ACE2-Fc fusion proteins, the truncated ACE2 domain is fused to Fc fragment but still retains the binding affinity to the viral spike proteins.
SI-F019 was engineered without the TMPRSS2 cutting site in the truncated ACE2 domain. As shown in
SI-F019 is a fusion protein of a truncated ACE2 (residue 18-615) and IgG1 Fc null fragment. The truncated ACE2 encodes a zinc metallopeptidase, whose enzymatic activity may be re-evaluated by using an established assay. A peptide substrate of ACE2 with an MCA (7-Methoxycoumarin-4-acetic acid) fluorescent tag [MCA-YVADAPK (Dnp)-OH Fluorogenic Peptide Substrate] was used to measure ACE2 enzymatic activity of SI-F019. MCA molecule was prepared as standard curve calibration for free fluorophore quantification, and the substrate was diluted in DMSO to 0.97 mg/ml. SI-F019 was diluted to 100, 200, and 300 ng/ml and used to cleave fluorogenic peptide in-vitro to release free MCA. The assay was incubated at room temperature for 20 minutes, and data were collected for fluorescent signals at timepoints with 2 minute intervals.
The cleaved MCA was quantified in molar using MCA standard curve. The enzymatic activity was determined according to the slope of linear curve as shown in
SI-F019 was tested for the ability to inhibit live SARS-CoV-2 infection and lysis of VeroE6 (ATCC: CRL-1586) cells in vitro. SI-F019 test concentrations, ranging from 1.5 nM to 1200 nM, were preincubated with 3 concentrations of live SARS-CoV-2 virus (Strain USA-WA1/2020, representing a 100-fold range of Multiplicity Of Infection, MOI) for 1 hour and then added to 90% confluent monolayer of VeroE6 cells. After 1 hour, the medium containing the virus was removed and replaced with the medium containing SI-F019 at matching test concentrations, and the tests were conducted in triplicate. The cell viability was measured by neutral red dye uptake after 72 hours and the percentage of inhibition of lytic viral infection was determined by comparison to wells in which virus was added at each MOI without SI-F019. The 50% inhibitory concentrations (IC50) for each virus concentration (1 MOI=40,000 virus particles) were calculated using GraphPad Prism software and are shown on each graph. The preincubation of SI-F019 with live SARS-CoV-2 resulted in a dose-dependent blockade of infection that reached 100% at all three MOI of virus that were tested. As shown in
SI-F019 was tested for its ability to inhibit replication and reinfection, i.e. further transfer of infection to VeroE6 cells from the cells previously infected with a low MOI of SARS-CoV-2 or SARS-CoV-1 viruses. VeroE6 cells in a 90% confluent monolayer (˜20,000 cells) were exposed to either SARS-CoV-2 (Strain USA-WA1/2020) or SARS-CoV-1 (Strain Urbani 2003000592) for 1 hour at an MOI of 0.01 (calculated as 400 virus infective particles). After washing out free virus particles, SI-F019 was added to the cells in a range from 10 fM to 100 nM in triplicates and the cell culture was maintained for 72 hours. Cell viability was determined by neutral red dye uptake and % inhibition of viral cytotoxicity was calculated. Absorbance values were normalized on each plate using the maximum absorbance of the conditions with no virus or no drug (NVND) representing 100% cell viability, and the average absorbance value of the virus/no drug (VND) establishing the maximum cell death using the formula:
% Cell Survival=[(Well OD540−VND OD540)/(NVND OD540−VND OD540)]*100
As shown in
HEK293T (ATCC: CRL-3216)-3D4 clone cell line was generated by lentiviral transduction of human ACE2 protein. The function of expressed human ACE2 was confirmed by enzymatic substrate conversion assay and binding by specific antibody by FACS. SARS-CoV-2 S protein packaged pseudo-virus which containing a luciferase reporter gene was obtained from National Institute for the Control of Pharmaceutical & Biological Products. Testing was conducted according to the manufacturer's instructions. The S-pseudo virus stock solution was diluted in culture medium with MRD of 20 in order to yield 300 TCID50/well of virus load. SI-F019 at concentrations ranging from 0.07 nM to 1500 nM were preincubated with the diluted virus solution for 1 hour. HEK293T-3D4 cells were dispersed into a 96-well plate. After 1 hour, mixtures were added into cell plate. Infected cells were measured by testing luciferase activity after 24 hours of incubation. 50% inhibitory concentrations (IC50) for defined virus load were calculated using GraphPad Prism software.
Antibody-dependent enhancement (ADE) is a phenomenon in which binding of a virus to suboptimal antibodies enhances its entry into host cells. In case of COVID-19, the secondary infection of SARS-CoV-2 virus to the patient who has anti-SARS-CoV-2 antibodies developed from a primary infection or to an individual who has been vaccinated may lead to enhanced uptake of virus by monocytes and B cells. The anti-virus antibodies in contact with the virus may bind to Fc receptors expressed on certain immune cells or some of the complement proteins. The latter binding depends on the Fc region of the antibody. Typically, the virus undergoes degradation in a process called phagocytosis, by which viral particles are engulfed by host cells through plasma membrane. However, the antibody binding might result in virus escape if the virus is not neutralized by an antibody, either due to low affinity binding or targeting a non-neutralizing epitope. Then, the outcome is an antibody enhanced infection.
The antibodies developed through either natural immunity or vaccination possess a wild type Fc region. While SI-F019 is capable of competing with anti-spike antibodies for binding to SARS-CoV2 virus, the IgG1 Fc null fragment is incapable of binding to either Fc receptors or C1q (see Table 3). To demonstrate its comparative advantage in reducing the effect of ADE, SI-F019 was evaluated for its role in internalization, replication, and reinfection.
In an assay for measuring Fc mediated internalization, the SARS-CoV-2 S protein was packaged into GFP-expressing pseudo-virus (PsV), and two cell lines, THP1 (monocyte) and Daudi (B cell) that express Fc receptors and complement receptor 2 (CR2), were used for testing FcRγ and CR2-mediated ADE mechanisms. SI-69R3 was used as a control for SI-F019, having a wild type Fc in contrast to SI-F019 that has an IgG1 Fc null modification (see Table 1). After being exposed to PsV for 48 hours, the green fluorescent signal from the cells was quantified as an indicator of PsV infection. In the conditions treated with PsV and SI-69C1, anti-S1 antibody, or SI-69R3 low levels of green fluorescence were measured at 48h in THP1 (pH 7.2) (6A), THP1 (pH 6.0)(6B), and Daudi (6C) cells. This result indicated that some transfer of PsV could occur via the Fc receptor. In contrast, the condition with SI-F019 at the indicated concentrations resulted in no uptake of PsV by THP1 or Daudi cells, comparable with the green fluorescent signal measured in the negative control conditions including, assay media, formulation buffer, and SI-69C1(
SI-F019 may not mediate the internalization of S protein packaged GFP-expressing pseudo-virus (PsV) due to lack of a functional Fc fragment. To determine if SI-F019 can inhibit the uptake of the pseudovirus, SI-F019 was used as a co-treatment with either SI-69R3 or natural anti-SARS-CoV-2 antibody in a competition mode. The PsV was incubated for 1 hour with SI-F019 at a dose range from 1 pM to 100 nM, together with either 10 pM of anti-SARS-CoV-2 (51) antibody or 10 pM of SI-69R3 prior to infecting the same set of target cells. PsV derived GFP signals were detected as the virus load of infection. SI-F019 was able to inhibit the virus load of PsV in the target cells starting at 10 fM (
While both the antibody, such as anti-SARS-CoV-2 (51) antibody, and the fusion protein of truncated ACE2-wild type Fc fragment in SI-69R3, were shown to be able to mediate internalization of SARS-CoV-2 Spike pseudotyped lentivirus, SI-F019 failed to do so due to lack of a functional Fc fragment. Herein, SI-F019 helped reduce virus load of PsV in the presence of either 10 pM of anti-SARS-CoV-2 (51) antibody or 10 pM of SI-69R3, even at a low concentration of 10 fM. Together, these results indicate that SI-F019 may reduce the incidence of ADE induced by FcRγ and CR2 dependent mechanisms in THP1 monocytes and Daudi B cells, respectively.
HEK293-T cells (ATCC: CRL-3216) that stably express SARS-CoV-2 spike protein were established by transducing the lentivirus packaged with SARS-CoV-2 spike protein encoding cDNA (Accession: YP_009724390.1) and IRES expression and selection based on puromycin resistance driven by same expression construct (LPP-CoV219-Lv105-050, GeneCopoeia). The expression of SARS-CoV-2 spike protein was confirmed by binding of a human IgG clone AM001414, specific for SARS-CoV-2 Spike protein “Anti-Spike”, (SKU938701, Biolegend) and the Human IgG Isotype matched clone QA16A12 was used as control “Isotype”, (SKU403502, Biolegend). Bound protein was quantified by secondary incubation with polyclonal anti-human Fc AF647 Fab (SKU109-607-008, Jackson ImmunoResearch) and FACS evaluation as shown in
HEK293-T cells expressing either SARS-CoV-2 spike protein and the parental HEK293 cells were stained with the indicated materials for 30 minutes at 37° C. in the presence of internalization inhibitor sodium azide. After the removal of free SI-F019, SI-F109 was detected and quantified by using anti-human Fc AF647 fab (SKU109-607-008, Jackson ImmunoResearch) and flow cytometry analysis. Geometric mean signal intensity was used to quantify the binding of SI-F019 and target cells line as shown in
Antibody-dependent cellular cytotoxicity (ADCC) is one of important immune responses to viral infection, such as the infection of SARS-CoV-2 virus in the case of COVID-19. Following the initial viral infection, anti-virus antibodies directly bind to the viral particles for neutralization and agglutination. Binding of a virus-antibody complex to an Fc receptor on a phagocyte can trigger phagocytosis, resulting in destruction of the virus; binding to the Fc receptors on immune effector cells, such as monocytes, neutrophils, eosinophils and NK cells, can trigger the release of cytotoxic factors (e.g., antiviral interferons), creating a microenvironment that is hostile to virus replication.
To distinguish the effect of SI-F019 from anti-spike antibodies, HEK293-T cells expressing SARS-CoV-2 spike protein were loaded with Calcein-AM and co-cultured with purified human NK cells at a 5:1 effector to target ratio. Treatments tested included SI-F019 and S1-specific human IgG clone SI-69C3. SI-69C3 is the human antibody clone CC12.3, isolated from a hospitalized COVID-19 patient (10.1126/science.abc7520). After 12 hours in co-culture, cells were stained with propidium iodide and evaluated for viability. As shown in
ADCC mediated by NK cells can be directed toward HEK293-T cells expressing SARS-CoV-2 protein when exposed to SI-specific human IgG clone SI-69C3 (Clone CC12.3). SI-F019 did not mediate ADCC compared to SI-69C3 within the treatment range of 100 nM to 100 fM. Under these assay conditions, the SI-F019 drug variant with wt Fc (SI-69R3) was able to mediate ADCC in a dose-dependent fashion, but the level of activity was lower compared to the S1-specific human IgG clone CC12.3 as shown in
The role of the complement cascade in mediation of antibody-based cell and tissue injury in COVID-19 patients is evident in both the natural immune responses and neutralizing antibody-based therapy (Perico et al., 2021). Immune complexes formed of virus and specific IgG mediate complement-induced blood clotting, thromboembolism and systemic microangiopathy. These widespread complications in COVID-19 patients can be life-threatening and are dependent on the complement proteins binding to IgG. Virus immune complexes bridging red blood cells through C1q and platelets with FcγRIIA are mediators of the thromboembolism in COVID-19 patients (Nazy et al., 2020). The fixation of immune complexes to endothelial vessel walls and complement-mediated coagulation are a primary concern in patients with COVID-19 where the activation of endothelial cells is part of the thromboembolism cascade.
Unlike a natural IgG antibody, SI-F019 is unable to binding C1q as shown in Table 3. This feature eliminates the risk of the induction of cell death of infected epithelia and endothelium that may transiently express the SARS-CoV-2 spike protein on their surface. This protective effect of SI-F019 is demonstrated in comparison to anti-spike human IgG antibody.
To demonstrate the protective effect of SI-F019, HEK293-T cells expressing SARS-CoV-2 Spike protein were cultured in serum-free media (Optimem) with treatments for 30 minutes, followed by addition of human serum complement at 1:10 serum-to-media ratio. Treatments tested included SI-F019 and S1-specific human IgG clone AM001414. Cell were cultured at 37° C. for 3 hours prior to addition of Propidium Iodide staining and positive staining cells counted in each well. Red cells counted by Incucyte Zoom Software at 3 hours are evaluated as a measure of CDC as shown in
The protection of tissue cells from complement damage is further confirmed by the ability of these cells to further proliferate after human serum complement challenge. CDC mediated by human serum complement at a 1:10 volume to volume ratio with serum free media is evaluable toward HEK293T cells expressing SARS-Cov-2 S protein when exposed to S1-specific human IgG clone (Clone AM4141). The result indicated that both human soluble monomeric ACE2 and SI-F019 did not mediate CDC, whereas SI-69R3 had limited, dose dependent increase CDC activity compared to human IgG antibody. CDC cytolysis was reflected in reduced cell growth, based on well confluence at 96 hours post treatment.
SARS-CoV-2 has a tropism for ACE2-expressing epithelia of respiratory tract and small intestine. Clinical laboratory findings of elevated IL-2, IL-6, IL-7, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-γ inducible protein 10 (IP-10), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1-α (MIP-1α), and tumor necrosis factor-α (TNF-α) indicative of cytokine release syndrome (CRS) suggest an underlying immunopathology. CRS is a major adverse side effect that can limit the utility of treatment with biologics and is tested for using in vitro cytokine release assays.
SI-F019 is a fusion protein consisting of human ACE2 and a mutated form of human IgG1 Fc that is incapable of binding to Fcγ receptors. As such, SI-F019 is not expected to bind any target cells in peripheral blood or to elicit cytokine release. White blood cells (WBC) including neutrophils, isolated from 5 healthy donors were put in culture wells containing either plate-bound or soluble SI-F019 at 2000 nM and 200 nM concentrations.
As a positive control, the TGN1412 antibody was used at the same concentrations and in the same formats due to its well-documented ability to induce cytokine release in the plate-bound format of this assay. The potential contribution of the IgG1 Fc null fragment to reduce cytokine release was evaluated by comparison with SI-69R3 having a wild type Fc fragment that is capable of binding Fcγ receptors expressed by several cell types in peripheral blood. WBC cultures containing only the formulation buffer for SI-F019 at similar dilutions were used as a negative control. Culture supernatants were collected at 24 and 48-hour time points and the presence of 9 cytokines was detected using the Meso Scale Discovery (MSD) platform.
Included in the cytokine panel were the T cell-associated cytokines IFNγ, TNFα, GM-CSF, IL-2 and IL-10 as shown in
The results indicate that SI-F019 does not induce any of the tested cytokines from exposed to WBC in either plate-bound or soluble formats at 200 nM and 2000 nM concentrations. Cytokine levels in SI-F019 treated samples showed concentrations similar to buffer controls in all conditions. The positive control, TGN1412 strongly induced most of the cytokines in the plate-bound but not the soluble format, which is in an agreement with previously published results. Some intermediate production of IFNγ, GM-CSF, and TNFα were detectable when plate-bound ACE2-Fc wild type was used to stimulate the WBC indicating the increased safety of the Fc null fragment of SI-F019.
The pathogenic role of the humoral response against SARS-CoV-2 virus has recently been suggested in patients receiving interventional IgG therapy (Weinreich et al., 2021; Chen et al., 2021). The small vessel hyperinflammatory response underlies adverse events, including thrombocytosis, pruitus, pyrexia, and hypertension. The present application demonstrates that SI-F019 could provide the benefit of virus neutralization comparable to that of IgG therapy while protecting tissues and organs from multiple pathways of dysfunctions. Therefore, SI-F019 may be used for treating, preventing, or moderating a viral infection, specifically for preventing and managing the progression of COVID-19 with reduced clinical complications, and additionally for acute respiratory distress syndrome, pulmonary arterial hypertension, or acute lung injury.
RMSRSRINDAFRLNDNSLEFLGIQPTLGPPNQPPVSIWLIVFGVVMGVIVVGIVILIFTGIRDR
cutting site) -Fc-w2 (EU numbering 216-447) -protein sequence
RMSRSRINDAFRLNDNSLEFLGIQPTLGPPNQPPVSEPKSSDKTHTCPPCPAPELLGGPSVFLF
This application claims the benefit of the filing date of U.S. Provisional Application Ser. No. 62/976,344 filed Feb. 13, 2020, and U.S. Provisional Application Ser. No. 63/086,593 filed Oct. 1, 2020 under 35 U.S.C. 119(e), the entire disclosures of which are incorporated by reference herein.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/017305 | 2/10/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63086593 | Oct 2020 | US | |
| 62976344 | Feb 2020 | US |
