CA3179142A1 - Production of ace2-fc fusion protein in plants and uses thereof - Google Patents

Abstract

A method of making a fusion protein in plants is described. The method comprises (a) introducing a nucleic acid molecule encoding a fusion protein, wherein the fusion protein comprises ACE2, or a variant or fragment thereof, and the Fc domain of lgA or a variant or fragment thereof, into a plant or plant cell; and (b) growing the plant or plant cell to obtain a plant that expresses the fusion protein and/or fusion protein dimer. The method optionally further comprises expressing a second nucleic acid molecule the encodes J-Chain or a variant or fragment thereof in the plant or plant cell. The disclosure also relates to using the fusion protein to treat a coronavirus infection in a subject.

Description

TITLE: PRODUCTION OF ACE2-FC FUSION PROTEIN IN PLANTS AND USES
THEREOF
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63/255,256 filed October 13, 2021, the contents of which are incorporated herein by reference in their entirety.
FIELD

[0002] The present disclosure relates to methods of making an ACE2-Fc fusion protein plants as well as methods of using the same to prevent the spike protein of a coronavirus from attaching to a cell or in therapy to treat coronavirus infection.
INTRODUCTION

[0003] The Coronaviridae as a family are characterized by expression of a 'Spike' protein on the viral surface which is used for primary attachment to susceptible cells. The 2019 pandemic coronavirus (SARS-CoV-2) is somewhat similar to the previous SARS virus in that the Spike protein recognizes the ACE2 receptor on human cells (conversely, the related MERS virus binds to cells via the DPP4 receptor). A class of therapeutics is in development to interfere specifically in this interaction ¨ by in situ development of antibodies following vaccine administration; by use of ready made antibodies (convalescent patient plasma, monoclonal antibodies, or Llama-derived nanobodies); or by use of decoy molecules based on the ACE2 protein ectodomain (synthetic peptides or recombinant hACE2). This latter approach has the added benefit of being effective against the so-called 'escape mutants' which evade the normal immune response to infection or vaccination through mutations in the Spike protein but which retain the ability to gain cell entry via binding to the ACE2 receptor.
SUMMARY

[0004] The present disclosure describes the successful expression of plant-produced ACE2-IgA Fc fusion protein in N. benthamiana using an Agrobacterium tumefaciens-based transient expression system. The fusion protein was shown to exert a protective effect against SARS-CoV-2 infection.

Date Recue/Date Received 2022-10-13

[0005] Accordingly, the disclosure provides a method of making a fusion protein comprising:
(a) introducing a nucleic acid molecule encoding a fusion protein, wherein the fusion protein comprises ACE2, or a variant or fragment thereof, and the Fc domain of IgA or a variant or fragment thereof, into a plant or plant cell; and (b) growing the plant or plant cell to obtain a plant that expresses the fusion protein.

[0006] In one embodiment, the ACE2 or variant or fragment thereof comprises the ACE2 ectodomain.

[0007] In another embodiment, the IgA is IgA2.

[0008] In one embodiment, the nucleic acid molecule further comprises a flexible linker between the ACE2 or variant or fragment thereof and the Fc domain.

[0009] In one embodiment, the nucleic acid molecule is codon-optimized for expression in plants.

[0010] In another embodiment, the nucleic acid molecule encodes a sequence as shown in SEQ ID NO: 1, or a sequence at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99% identical to SEQ ID NO: 1.

[0011] In one embodiment, the method further comprises introducing a second nucleic acid molecule encoding a J-Chain or a variant or fragment thereof into the plant or plant cell.

[0012] In one embodiment, the second nucleic acid molecule is codon-optimized for expression in plants.

[0013] In another embodiment, the second nucleic acid molecule encodes a sequence as shown in SEQ ID NO: 3, or a sequence at least 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99% identical to SEQ ID NO: 3.

[0014] In one embodiment, the wherein the nucleic acid molecule encoding the fusion protein and the second nucleic acid molecule encoding J-Chain or a variant or fragment thereof are introduced on separate vectors.

Date Recue/Date Received 2022-10-13

[0015] In another embodiment, the nucleic acid molecule encoding the fusion protein and the second nucleic acid molecule encoding J-Chain or a variant or fragment thereof are introduced on the same vector.

[0016] In one embodiment, the method further comprises manipulating the ratio of the nucleic acid molecules encoding the fusion protein and the second nucleic acid molecule encoding J-Chain or a variant or fragment thereof to optimize expression of fusion protein tetramers or higher multimers.

[0017] In another embodiment, the plant is N. benthamiana and the plant cell is a N. benthamiana cell.

[0018] The present disclosure further provides a composition comprising a fusion protein as described herein and a pharmaceutically acceptable diluent, excipient, or carrier.

[0019] The present disclosure in addition provides a method of treating or preventing a coronavirus infection in a subject, comprising administering a fusion protein or a composition as described herein to a subject in need thereof.

[0020] In one embodiment, the coronavirus is SARS-CoV-2.

[0021] In another embodiment, the fusion protein or composition is administered within 1,6, 12, 18, 24 hours or 1, 2, 3,4, 5,6 or 7 days of exposure to the coronavirus.

[0022] In another embodiment, the fusion protein or composition is formulated for intranasal administration.

[0023] The present disclosure additionally provides a transgenic plant or plant cell that expresses encoding a fusion protein, wherein the fusion protein comprises ACE2, or a variant or fragment thereof, and the Fc domain of IgA or a variant or fragment thereof.

[0024] In one embodiment, the ACE2 or variant or fragment thereof comprises the ACE2 ectodomain.

[0025] In another embodiment, the IgA is IgA2.

[0026] In another embodiment, wherein the transgenic plant or plant cell further expresses a J-Chain.

Date Recue/Date Received 2022-10-13 Other features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific Example while indicating preferred embodiments of the disclosure are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The disclosure will now be described in relation to the drawings in which:

[0028] Figure 1 shows schematics for the two plant expression vectors as set out in Example 1. (A) shows pPFC4128_ -ABChACE2IgAFc, a vivoXPRESSO vector directing expression of a chimeric fusion protein consisting, N-terminus to C-terminus, of a version of the Arabidopsis thaliana basic chitinase signal peptide (21 amino acids), human ACE2 fragment (721 amino acids), and human IgA hinge plus Fc fragment (239 amino acids). Genetic control elements are a double-enhancer version version of the Cauliflower Mosaic Virus 35S promoter (EE35S) and a truncated version of the termination sequence from the Chrysanthemum moriflorium Rubisco gene. Plasmid backbone and Agrobacterium tumefaciens Ti-plasmid left- and right-border sequences (LB, RB) are indicated. (B) shows pPFC4123_ -hJChain, a vivoXPRESSO vector directing expression of a chimeric fusion protein consisting, N-terminus to C-terminus, of a version of the Arabidopsis thaliana basic chitinase signal peptide (21 amino acids), and the human IgA hinge plus Fc fragment (137 amino acids). Genetic control elements are a double-enhancer version version of the Cauliflower Mosaic Virus promoter (EE35S) and a truncated version of the termination sequence from the Chrysanthemum moriflorium Rubisco gene. Plasmid backbone and Agrobacterium tumefaciens Ti-plasmid left- and right-border sequences (LB, RB) are indicated.

[0029] Figure 2 shows neutralizing activity of ACE2 against SARS-CoV-2. (A) shows the virus (SARS-CoV-2 WT) and sample (ACE2-IgA2), (B) SARS-CoV-2 WT
and Tris-Glycine (negative control) and (C) shows SARS-CoV-2 WT and 1212C2 (positive control).

[0030] Figure 3 shows a Commassie blue gel of Peptide M purified 1gA2 fusion protein in the presence of J-chain.

Date Recue/Date Received 2022-10-13

[0031]
Figure 4 shows the interaction of ACE2-IgA-Fc with a fragment of the coronavirus spike protein (RBD or Receptor Binding Domain).
DETAILED DESCRIPTION
METHODS

[0032] An ACE2-IgA Fc fusion protein was transiently expressed in N.
benthamiana plants. A. tumefaciens clones carrying vectors designed to express an ACE2-IgA Fc fusion protein were introduced into N. benthamiana plants by vacuum infiltration. The fusion protein was shown to exert a protective effect against SARS-CoV-2 infection.

[0033] Accordingly, the present disclosure provides a method of making a fusion protein comprising:
(a) introducing a nucleic acid molecule encoding a fusion protein, wherein the fusion protein comprises ACE2, or a variant or fragment thereof, and the Fc domain of IgA or a variant or fragment thereof, into a plant or plant cell; and (b) growing the plant or plant cell to obtain a plant that expresses the fusion protein.

[0034] As used herein, the term "nucleic acid molecule" means a sequence of nucleoside or nucleotide monomers consisting of naturally occurring bases, sugars and intersugar (phosphodiester) linkages. The term also includes modified or substituted sequences comprising non-naturally occurring monomers or portions thereof. The nucleic acid sequences of the present disclosure may be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA) and may include naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil.
The sequences may also contain modified bases. Examples of such modified bases include pseudouridine, 5-methylcytosine, 6-methyladenosine, N1-methylpseudouridine, aza and deaza adenine, guanine, cytosine, thymidine and uracil; and xanthine and hypoxanthine.

[0035] As used herein, the term "variant", when referring to a peptide sequence, means a modified version of the sequence that includes, but not limited to, conservative substitutions, insertions and/or deletions. For example, variants of ACE2 useful in the present methods and compositions include the natural human S19P
and Date Recue/Date Received 2022-10-13 K26R ACE2 variants described in MacGowan et al 2021. It is believed that these two natural variants result in higher spike protein binding. Other variants include those described in Linsky et al 2020, Karoyan et al 2020, Higuchi et al 2020, Jing and Procko 2020, Glasgow et al 2020, Ferrari et al 2021, Curreli et al 2020, and Cogen-Dvashi et al, 2020.

[0036] As used herein, the term "fragment", when referring to a peptide sequence, means a part of portion of the sequence comprising fewer amino acid residues.

[0037] As used herein, the term "fusion protein" means a protein wherein two or more protein domains are fused together, wherein the domains are optionally separated by a linker, wherein the domains confer distinct functions.

[0038] "ACE2" refers to the protein angiotensin-converting enzyme 2, which is encoded by the ACE2 gene. The term "ACE2" includes ACE2 from any species, optionally from a mammal, including but not limited to humans, bats, pangolins, ducks, geese, chickens, bushmeat animals. The term "ACE2" also includes ACE2 from a recombinant source. ACE2 or the ACE2 gene may have any of the known published sequences for ACE2 which can be obtained from public sources such as GenBank.
In one embodiment, ACE2 is human ACE2. Examples of the human amino acid sequences for ACE2 include GenBank accession no. NP_001358344.1. Variants of ACE2 include amino acid sequences having at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99% sequence identity to the sequence set out in GenBank accession no.
NP_001358344.1. The aforementioned sequences are incorporated herein by reference.

[0039] The term "IgA Fc" refers to the Fc domain of IgA. "Fc"
refers to the heavy chain constant region of an antibody. The term "IgA" refers to a polypeptide belonging to a class of antibodies found in serum and in secretions. IgA in serum is mainly as a single covalently linked dimer, whereas IgA in secretions is found mainly as a tetramer (two non-covalently linked dimers). In human, two subtypes of IgA antibodies exist:
IgA1 and IgA2. In one embodiment, IgA Fc is human IgA Fc. In another embodiment, IgA Fc is IgA1 Fc or IgA2 Fc. Examples of human amino acid sequences for IgA
Fc include GenBank accession no. AAT74071.1. Examples of human nucleic acid Date Recue/Date Received 2022-10-13 sequences the encode IgA Fc include GenBank accession no. AY647979.1. The aforementioned sequences are incorporated herein by reference.

[0040] In an embodiment, the ACE2 or variant or fragment thereof comprises or consists of the ACE2 ectodomain. In one embodiment, the ACE2 ectodomain corresponds to amino acid residues 20-740 of the ACE2 protein sequence, optionally where the ACE2 ectodomain retains or enhances binding to the spike protein RBD
and prevents attachment of the virus to the target cell.

[0041] In an embodiment, the IgA is the IgA2 subtype.

[0042] In another embodiment, the fusion protein comprises a linker between the ACE2 or variant or fragment thereof and the Fc domain of IgA or variant or fragment thereof, optionally the linker is a flexible linker. In one embodiment, the linker is (GGGGS),, where n is 1-4 In another embodiment, the linker is (GGGS),, where n is 1-4.

[0043] As used herein, the term "linker" means a peptide that connects two protein domains in a fusion protein. The term "flexible linker" means a linker that allows for mobility of the connecting domains.

[0044] Linkers may be designed using methods known in the art (e.g. Chen et al., 2013).

[0045] In a specific embodiment, the nucleic acids in the nucleic acid molecule are optimized for plant codon usage. In a specific embodiment, coding sequences are optimized for expression in Nicotiana species with the goal of making the coding sequences more similar to those of Nicotiana species. Codon optimizations may be performed as known in the art, for example by utilizing online freeware, i.e., the Protein Back Translation program (Entelechon), and Nicotiana coding sequence preferences.
Codons with the highest frequencies for each amino acid in Nicotiana species (Nakamura, 2005) are thereby incorporated. Furthermore, potential intervening sequence splice-site acceptor and donor motifs can be identified (Shapiro et al., 1987;
CNR National Research Council) and subsequently removed by replacement with nucleotides that resulted in codons encoding the same amino acids. Inverted repeat sequences can be analyzed using the Genebee RNA Secondary Structure software package (Brodsky et al., 1995; GeneBee Molecular Biology Server); nucleotides can be changed to reduce the free energy (kilocalories per mole) of potential secondary Date Recue/Date Received 2022-10-13 structure while maintaining the polypeptide sequence. Likewise, repeated elements can be analyzed (CNR National Research Council) and replaced where present.
Potential methylation sites (i.e., CXG and CpG; Gardiner-Garden et al., 1987) can be replaced where possible and always without changing the encoded amino acid sequence. A Kozak (Kozak, 1984) optimized translation start site can be engineered.
Plant polyadenylation sites (i.e., AATAAA, AATGAA, AAATGGAAA, and AATGGAAATG (Li et al., 1995; Rothnie, 1996) and ATTTA RNA instability elements (Ohme-Takagi et al., 1993) can be likewise avoided.

[0046] In an embodiment, the nucleic acid molecule encodes a sequence as shown in SEQ ID NO: 1, or a sequence at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99% identical to SEQ ID NO: 1.

[0047] Nucleic acid and amino acid sequences described herein are set out in Table 1.

[0048] In an embodiment, the method further comprising introducing a second nucleic acid molecule encoding a J-Chain or a variant or fragment thereof into the plant or plant cell.

[0049] As used herein, "J-Chain" refers to a polypeptide that regulates the dimer formation of IgA. The term "J-Chain" includes J-Chain from any species, optionally from mammal and optionally from human. The term "J-Chain" also includes J-Chain from a recombinant source. In one embodiment, J-Chain is human J-Chain.
Examples of the human amino acid sequences for J-Chain include SEQ ID NO: 3.
Examples of the human nucleic acid sequences for J-chain include SEQ ID NO: 4.

[0050] In an embodiment, the second nucleic acid molecule encodes a sequence as shown in SEQ ID NO: 3, or a sequence at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99% identical to SEQ ID NO: 3.

[0051] Optionally, the nucleic acid molecule encoding the fusion protein and the nucleic acid molecule encoding a J-Chain or a variant or fragment thereof are introduced to the plant or plant cell on the separate vectors. In another embodiment, the nucleic acid molecule encoding the fusion protein and the nucleic acid molecule encoding J-Chain or a variant or fragment thereof are introduced to the plant or plant cell on the same vector Date Recue/Date Received 2022-10-13

[0052] In one embodiment, the fusion protein aggregates into a dimer. In addition, co-introduction of a nucleic acid molecule encoding a J-Chain or a variant or fragment thereof can promote multimer formation, in particular, tetramers.
Therefore, in one embodiment, the ratio of ACE2-IgA Fc to J-Chain expressed in the plant is manipulated to optimize expression of the fusion protein tetramer. In one embodiment, the ratio of fusion protein nucleic acid molecules to J-chain nucleic acid molecules introduced to the plant or plant cell is between 1:1 and 1:2 (fusion protein:J-chain).

[0053] As used here, the term "sequence identity" refers to the percentage of sequence identity between two polypeptide sequences or two nucleic acid sequences.
To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=number of identical overlapping positions/total number of positions multiplied by 100%). In one embodiment, the two sequences are the same length. The determination of percent identity between two sequences can also be accomplished using a mathematical algorithm. One non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990), modified as in Karlin and Altschul (1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al.
(1990). BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g., for score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the present disclosure.
BLAST
protein searches can be performed with the XBLAST program parameters set, e.g., to score=50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule of the present disclosure. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997).

Alternatively, PSI-BLAST can be used to perform an iterated search which detects Date Recue/Date Received 2022-10-13 distant relationships between molecules (Altschul et al., 1997). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g., the NCBI
website).
Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller (1988). Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG
sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

[0054] The coding sequences for the fusion protein and the J-Chain are optionally synthesized using standard procedures (for example, Almquist et al., 2004;
Almquist et al., 2006; McLean et al., 2007; Olea-Popelka et aL, 2005).

[0055] The nucleic acid vectors encoding the fusion protein and/or the J-Chain can also contain other elements suitable for the proper expression of the protein in the plant or plant cell. In particular, each vector can also contain a promoter that promotes transcription in plants or plant cells. Suitable promoters include, but are not limited to, cauliflower mosaic virus promoters (such as CaMV35S and 19S), nopaline synthase promoters, alfalfa mosaic virus promoter, and other plant virus promoters.
Constitutive promoters, such as plant actin gene promoters, and histone gene promoters can also be used.

[0056] Inducible promoters, such as light-inducible promoters:
ribulose-1,5-bisphosphate carboxylase oxidase (a.k.a. RUBISCO) small subunit gene promoter;
chlorophyll a/b binding (CAB) protein gene promoter; and other light inducible promoters may also be used. Other inducible promoters include chemically-inducible promoters, alcohol inducible promoters, and estrogen inducible promoters.

[0057] Synthetic promoters, such as the so-called superpromoter comprised of 3 mannopine synthase gene upstream activation sequences and the octopine synthase basal promoter sequence (Lee et al., 2007) can also be used.

[0058] Predicted promoters, such as those that can be found from genome database mining (Shahmuradov et al., 2003) may also be used.

Date Recue/Date Received 2022-10-13

[0059] The nucleic acid vectors can also contain suitable terminators useful for terminating transcription in the plant or plant cell. Examples of terminators include the nopaline synthase poly A addition sequence (nos poly A), cauliflower mosaic virus 19S
terminator, actin gene terminator, alcohol dehydrogenase gene terminator, or any other terminator from the GenBank database.

[0060] Seletectable marker genes can also be linked on the T-DNA, such as the kanamycin resistance gene (also known as neomycin phosphotransferase gene II, or nptI1), Basta resistance gene, hygromycin resistance gene, or others.

[0061] As used herein, the term "vector" means a nucleic acid molecule, such as a plasmid, comprising regulatory elements and a site for introducing transgenic DNA, which is used to introduce said transgenic DNA into a plant or plant cell. The transgenic DNA can encode a heterologous protein, which can be expressed in and isolated from a plant or plant cells. Vectors useful in the present methods are well known in the art. In one embodiment, the vector is a commercially-available vector.

[0062] As used herein, the term "expression cassette" means a single, operably linked set of regulatory elements that includes a promoter, a 5' untranslated region (5' UTR), an insertion site for transgenic DNA, a 3' untranslated region (3' UTR) and a terminator sequence.

[0063] In an embodiment, the plant is N. benthamiana and the plant cell is a N. benthamiana cell.

[0064] The phrases "introducing a nucleic acid molecule into a plant or plant cell" and "introducing a second nucleic acid molecule into a plant or plant cell" include both the stable integration of the nucleic acid molecule(s) into the genome of a plant cell to prepare a transgenic plant or plant cell as well as the transient integration of the nucleic acid(s) into a plant or part thereof.

[0065] The nucleic acid vectors may be introduced into the plant or plant cell using techniques known in the art including, without limitation, vacuum infiltration, electroporation, an accelerated particle delivery method, a cell fusion method or by any other method to deliver the nucleic acid vectors to a plant or plant cell, including Agrobacterium mediated delivery, or other bacterial delivery such as Rhizobium sp.
NGR234, Sinorhizobium meliloti and Mesorhizobium lot! (Chung et al., 2006).

Date Recue/Date Received 2022-10-13

[0066] In one embodiment, the method further comprises introducing nucleic acid molecule encoding a suppressor of post translational gene silencing into the plant of plant cell. In one embodiment, the suppressor of post translational gene silencing is P19. The nucleic acid molecule encoding the suppressor of post translational gene silencing may be introduced into the plant or plant cell on the same vector as the nucleic acid molecule encoding the fusion protein or a separate vector.

[0067] P19 is a viral protein that suppresses gene silencing. For example, P19 from Tomato bushy stunt virus (TBSV) is an example of a protein known to function as a potent suppressor of gene silencing in plants as well as in animals (for example, Accession No. NP_062901.1; Garabagi et al., 2012b). A nucleic acid sequence and an amino acid sequence of P19 from TBSV are provided herein as SEQ ID NOs: 5 and 6, respectively.

[0068] In another embodiment, the nucleic acid molecule encoding the fusion protein is introduced into a plant or a plant cell with reduced expression of one or more genes involved in gene silencing compared to a wild-type plant or plant cell.
For example, in one embodiment, the nucleic acid molecule encoding the fusion protein is introduced into a plant or a plant cell with reduced expression of one or more Argonaute proteins, for example AGO1 and AG04, compared to a wild-type plant or plant cell. ARGONAUTE (AGO) proteins are a conserved family of proteins which play a key role in the RNA-induced silencing complex (RISC). RISC is responsible for gene expression silencing through RNA interference (RNAi).

[0069] The plant or plant cell may be any plant or plant cell, including, without limitation, tobacco plants or plant cells, tomato plants or plant cells, maize plants or plant cells, alfalfa plants or plant cells, Nicotiana benthamiana, rice plants or plant cells, Lemna major or Lemna minor (duckweeds), safflower plants or plant cells or any other plants or plant cells that are both agriculturally propagated and amenable to genetic modification for the expression of recombinant or foreign proteins.

[0070] The phrase "growing a plant or plant cell to obtain a plant that expresses the antibody or antibody fragment" includes both growing transgenic plant cells into a mature plant as well as growing or culturing a mature plant that has received the nucleic acid molecules encoding the antibody. One of skill in the art can readily determine the appropriate growth conditions in each case.

Date Recue/Date Received 2022-10-13

[0071] In a specific embodiment, plasmids containing the nucleic acid molecules are introduced into A. tumefaciens strain by electroporation procedures.
The N. benthamiana plants can be vacuum infiltrated according to the protocol described by Marillonnet etal. (2005), Giritch etal. (2006) and Garabagi et aL
(2012) with several modifications. Briefly, all cultures can be grown at 28 C and 220 rpm to a final optical density at 600 nm (0D600) of 1.8 or 2Ø In one embodiment, equal volumes are combined and pelleted by centrifugation at 8,000 rpm for 4 minutes, resuspended and diluted by 103 in infiltration buffer (10 mM 1-(N-morpholino)ethanesulphonic acid (MES), pH 5.5, 10 mM MgSO4). Alternatively, each of the Agrobacterium cultures could be grown to lower OD values and Beer's Law could be applied to determine the volumes of each culture required to make a bacterial suspension cocktail whereby the concentrations of each bacterial strain were equivalent. Alternatively, higher or lower dilutions with infiltration buffer could be used.

[0072] In another specific embodiment, the aerial parts of five-week-old N.
benthamiana plants are submerged in a chamber containing the A. tumefaciens resuspension solution, after which a vacuum (0.5 to 0.9 bar) is applied for 90 seconds followed by a slow release of the vacuum, after which plants were returned to the greenhouse for 7 days before being harvested. In another embodiment, older or younger plants are used. In further embodiments, longer or shorter periods under vacuum, and/or vacuum release and/or longer or shorter periods of growth in greenhouse are used. Standard horticultural improvements of growth, maximized for recombinant protein production can also be used (see Colgan etal., 2010).

[0073] In another embodiment, instead of transient introduction of vectors (for example, pPFC4123 or pPFC4128-based vectors) containing the fusion protein or the J-Chain coding sequences, stable transgenic plants or plant cells are made.

[0074] In another embodiment, plant expression vector(s) containing the fusion protein and/or the J-Chain coding sequences are introduced into Agrobacterium tumefaciens At542 or other suitable Agrobacterium isolates such as At564, or other suitable bacterial species capable of introducing DNA to plants for transformation such as Rhizobium sp., Sinorhizobium meliloti, Mesorhizobium loti and other species (Broothaerts et al. 2005; Chung et al., 2006), by electroporation or other bacterial transformation procedures. Agrobacterium clones containing vectors can be propagated on Luria-Bertani (LB) plates containing rifampicin (30 mg/L) and Date Recue/Date Received 2022-10-13 kanamycin (50 mg/L), or other selectable media, depending on the nature of the selectable marker genes on the vector. Agrobacterium-mediated leaf disk transformation (Gelvin, 2003; Horsch et al. 1985), or similar protocols involving wounded tobacco (N. tabacum, variety 81V9 or tissue of other tobacco varieties such as those listed in Conley et aL, 2009) or N. benthamiana or other plant species such as those of the Solanaceae, maize, safflower, Lemna spp., etc. can be infected with the Agrobacterium culture (0D600 = 0.6) and plated on Murashige and Skoog plus vitamins medium (MS; Sigma), supplemented with agar (5.8%; Sigma) and containing kanamycin (100 mg/L) or 500 cefotaxime (mg/L) or other selectable media, depending on the nature of the selectable marker genes on the vector, for selection of transformed plant cells. Production of shoots can be induced with naphthalene acetic acid (NAA;
0.1 mg/L; Sigma) and benzyl adenine (BA; 1 mg/L; Sigma) in the medium. For induction of roots, the newly formed shoots were moved to Magenta boxes (Sigma-Aldrich, Oakville, ON) on MS medium (as above) that was lacking NAA and BA.
After roots are formed, plants can be transplanted to soil and could be raised in greenhouse culture. For plant transformation, as many as possible or at least 25 primary transgenic plants can be produced. ELISA and quantitative immunoblots can be performed on each plant to characterize levels of total and active antibody produced by the plants, respectively (Almquist etal., 2004; Almquist etal., 2006; Makvandi-Nejad et aL, 2005;
McLean et aL, 2007; Olea-Popelka et aL, 2005).

[0075] In another embodiment, after selection of the fusion protein- expressing primary transgenic plants with or without the expression of J-Chain, or concurrent with their selection, derivation of homozygous stable transgenic plant lines is performed.
Primary transgenic plants can be grown to maturity, allowed to self-pollinate, and produce seed. Homozygosity can be verified by the observation of 100%
resistance of seedlings on kanamycin plates (50 mg/L), or other selectable drug as indicated above.
A homozygous line with single T-DNA insertions, that are shown by molecular analysis to produce most amounts of the fusion protein with or without J-Chain, can be chosen for breeding to homozygosity and seed production, ensuring subsequent sources of seed for homogeneous production of the fusion protein with or without J-Chain by the stable transgenic or genetically modified crop (McLean et aL, 2007; Olea-Popelka et al., 2005; Yu et aL, 2008).

Date Recue/Date Received 2022-10-13

[0076] The fusion protein may be purified or isolated from the plants using techniques known in the art, including homogenization, clarification of homogenate and affinity purification. Homogenization is any process that crushes or breaks up plant tissues and cells and produces homogeneous liquids from plant tissues, such as using a blender, or juicer, or grinder, or pulverizer such as mortar and pestle, etc. Clarification involves either/and/or centrifugation, filtration, etc. Affinity purification uses Peptide M
and/or antibodies that bind IgA.

[0077] As used herein, the term "polishing" refers to post-purification removal of aggregates, endotoxin, DNA, viruses and any other impurities and contaminants in the preparation of an antibody or antibody fragment.

[0078] In another embodiment, the fusion protein and/or fusion protein dimer are purified and polished by contacting Butyl HP resin. Homogenate is clarified through several stages to remove gradually smaller particulate matter. Clarified extract is applied to a Peptide M column. The Peptide M eluate is applied to a Capto-Q
column in flow-through mode. The Capto-Q flow-through is then applied to a Butyl HP
resin column. The butyl eluate is then fill-finished.
Compositions

[0079] The disclosure also provides a composition comprising one or more of the fusion proteins described herein and optionally a carrier.

[0080] In one embodiment, the carrier is a carrier acceptable for administration to humans.

[0081] As used herein, the term "acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Optional examples of such carriers or diluents include, but are not limited to, water, saline, ringer's solutions and dextrose solution.

[0082] In one embodiment, a composition or combination described herein is formulated to be compatible with its intended route of administration.
Examples of Date Recue/Date Received 2022-10-13 routes of administration include oral and parenteral, e.g. intravenous, intradermal, subcutaneous.

[0083] For example, in one embodiment, the active ingredient such as a fusion protein described herein is prepared with a carrier that will protect it against rapid elimination from the body, such as a sustained/controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art, see for example Li et al, 2015.

[0084] In one embodiment, oral or parenteral compositions or combinations are formulated in dosage unit form for ease of administration and uniformity of dosage.
Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required carrier. The specification for the dosage unit forms are dictated by and directly dependent on the unique characteristics of the active ingredient and the particular therapeutic effect to be achieved, and the limitations inherent in the art of preparing such an active ingredient for the treatment of individuals.

[0085] In one embodiment, the compositions described herein comprise an agent that enhances the function or bioavailability of the fusion protein. The composition can also contain other active ingredients as necessary or beneficial for the particular indication being treated, optionally those with complementary activities that do not adversely affect each other. The other active ingredient is optionally an antiviral such as iota-karageenan, which has been shown to be active against respiratory viruses in vitro. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended.

[0086] The present disclosure further includes a transgenic plant or plant cell that expresses a fusion protein as described herein.

[0087] In an embodiment, the transgenic plant or plant cell further expresses a J-Chain.

Date Recue/Date Received 2022-10-13

[0088] The present disclosure includes all uses of a fusion protein or composition as described herein, including, without limitation, the use of a fusion protein or composition as described herein to prevent the spike protein of a coronavirus from attaching to a cell in a subject in need thereof. The disclosure further provides a use of a fusion protein or composition to treat or prevent a coronavirus infection.

[0089] Accordingly, the present disclosure provides a method of preventing the spike protein of a coronavirus from attaching to a cell comprising using or administering an effective amount of a fusion protein or composition as described herein.
The disclosure also provides a use of an effective amount of a fusion protein or a composition as described herein for preventing the spike protein of a coronavirus from attaching to a cell in a subject in need thereof. The disclosure also provides a use of a fusion protein or composition as described herein for the manufacture of a medicament for preventing the spike protein of a coronavirus from attaching to a cell.

[0090] The present disclosure also provides a method of treating or preventing a coronavirus infection comprising using or administering an effective amount of a fusion protein or composition as described herein. The disclosure also provides a use of an effective amount of a fusion protein or a composition as described herein for treating or preventing a coronavirus infection. The disclosure also provides a use of a fusion protein or composition as described herein for the manufacture of a medicament for treating or preventing a coronavirus infection.

[0091] In one embodiment, the coronavirus is SARS-CoV-2. As used herein, the term "SARS-CoV-2" includes variants of SARS-CoV-2 including, but not limited to the B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 (Delta), P.1 (Gamma), B.1.526 (Iota), B.1.427 (Epsilon), B.1.429 (Epsilon), B.1.617 (Kappa, Delta), B.1.525 (Eta) and P.2 (Zeta) variants.

[0092] The use or administration of the fusion protein or the composition as described herein, to a subject optionally comprises intranasal administration, ingestion, inhalation, or injection. The route of injection includes but not limited to intradermal, subcutaneous, intramuscular, intravenous, intraosseous, intraperitoneal, intrathecal, epidural, intracardiac, intraarticular, intracavernous, intravitreal, intracerebral, intracerebroventricular, or intraportal.

Date Recue/Date Received 2022-10-13

[0093] In an embodiment, the fusion protein or the composition as described herein is used as a prophylactic or an immediate post-exposure therapy. In another embodiment, the fusion protein or the composition as described herein is used as a post-exposure therapy within 1, 6, 12, 18, 24 hours or 1, 2, 3, 4, 5, 6 or 7 days of exposure.

[0094] The following non-limiting Examples are illustrative of the present disclosure:

Experimental Procedures A. Generation of Plant Expression Vectors

[0095] Synthetic DNA sequence with Sad l and BglIl restriction sites (5' and 3') was cloned into T-DNA region of binary vector pPFC3609 downstream of CaMV 35S
promoter with tandem upstream enhancer elements. Below are details of the cloning experiment.

[0096] Two plasmids were ordered from GeneArt: pMA-RQ contained a 2955bp insert that encoded the human ACE2-IgA2Fc pMA-T contained a 505bp insert that encoded the human J-Chain.

[0097] The ACE2-IgA2Fc and J-Chain inserts were cloned into the PF
expression vector at Sacl-BamHI sites: pMA-RQ- abcSP-hACE2IgA2-Fc was digested by Sad and BglIl and pMA-T-abcSP-hJChain were digested by Sad l and BamHI in 20pL of lx NEB CutSmart buffer at 37 C overnight. Reaction was inactivated at 80 C for 20 minutes. Ligation by T4 ligase of digested pMA-RQ and pMA-T
with digested pPFC1434 was carried out at room temperature for 2 hours in 10pL of lx T4 ligase buffer. 2pL of ligation mixture was transformed into 20pL of NEB 5a cells by heat shock using standard procedures. Colonies were screen by plasmid restriction digest using BamHI and Xhol. The resulting plasmids were transformed using standard electroporation procedures.

[0098] A flexible linker may be added between the hACE2 ectodomain and hIgA2 Fc such as (GGGGS)3.
B. Generation of transgenic plants Date Recue/Date Received 2022-10-13

[0099] Vector was transformed into Agrobacterium tumefaciens EHAl 05 by standard electroporation method.

[00100] Transformed strain was grown in LB (Miller) and diluted to an OD of 0.2 at 600 nm using 10 mM MES, 10 mM MgCL2 buffer pH 6.0 and vacuum infiltrated into 4-5 week old N.benthamiana KDFX plants. The plants were harvested after 7 days and proteins were extracted in PBS. Debris was removed by centrifugation and IgA
Fc proteins were recovered using Peptide M resin.
C. Analysis of Peptide M Purified ACE2-IgA2 Fc Fusion Protein

[00101] Peptide M purified ACE2-IgA2 Fc fusion protein (4 pg per lane) were analysed by 8% SDS-PAGE under reduced (FIG. 2, Lanes 2-5 from Left) & non-reduced (FIG. 2, lanes 7-10 from Left) conditions. The effect of J-chain expression was to shift protein from the dimer (-300 kDa) to a higher molecular mass band. In this experiment, the plants were infiltrated using the ACE2-IgA Fc vector at a constant OD
of 0.2 in the infiltration cocktail. The J-Chain vector was used at a range of OD values (0.05-0.2) to manipulate the ratio of ACE2-IgA Fc to J-Chain expressed in the plant.

Neutralizing activity of ACE2-IgA Fc fusion against SARS-CoV-2

[00102] A constant concentration of SARS-CoV-2 (-250 PFU) was incubated with decreasing 1:2 serial dilutions of the ACE2-IgA Fc fusion (starting concentration of 5ug) for 1 h at room temperature. Then, Vero E6 cells were infected with the virus-ACE2-IgA Fc fusion mixture for 1 h at room temperature and, after viral infection, we remove the virus-ACE2-IgA Fc fusion mixture was removed and the cells were incubated for ¨24h in post-infection media. Then, cells were fixed and stained with an anti-N SARS-CoV-2 antibody. As internal controls cells not treated with hsACE2 (FIG.
2B) and cells treated with a neutralizing hMAb, 1212C2 (FIG. 2C) were used.
Results demonstrate increasing neutralization of viral infection with increasing amounts of ACE2-IgA Fc fusion protein sample (FIG. 2A).

Effect of the ratio of ACE2-IgA Fc to J-Chain on dimer formation

[00103] Plants were infiltrated using the ACE2-IgA Fc vector at a constant OD
of 0.2 in the infiltration cocktail. The J-Chain vector was used at a range of OD values Date Recue/Date Received 2022-10-13 (0.05-0.2) to manipulate the ratio of ACE2-IgA Fc to J-Chain expressed in the plant (FIG. 3). Increasing intensity of the ACE2-Fc dimer band with decreasing J-Chain demonstrated a lower content of aggregate with lower proportion of J-Chain relative to the ACE2-IGA Fc molecule (FIG. 3). The optimal ratio of J-chain:ACE2-IgA Fc would result in a product with no detectable dimer, in this example by using the J-chain vector at an OD of 0.2 or higher. Alternative ways to manipulate the ratio of J-Chain to ACEs-IgA Fc include using different promoters of varying potency to increase or decrease the expression of either protein.

Binding of coronavirus spike protein (receptor binding domain; RBD) to ACE2-IgA2 protein

[00104] Figure 4 shows an in vitro interaction of the ACE2-IgA Fc with a fragment of the spike protein (receptor binding domain; RBD).

[00105] ACE2-IgA2 Fc fusion protein was produced as described in Example 1.
A construct comprising the RBD was fused to a human IgA1 Fc fragment.

[00106] In Figure 4, ACE2-IgA Fc is coated on the plate and probed under conditions where there is no RBD and secondly where there is RBD-IgG Fc;
detection on both cases using anti-hIgG Fc antibodies (HRP conjugated).

[00107] Each well was coated with hACE2-IgA2 (0.6, 0.3, 0.15, 0.075, 0.038 &
0.019 pg/well-100 pL), applied as dilutions from a starting solution of ACE2-IgA2 at a concentration of 6 pg/mL. RBD-TEV-hFc was added at 0.4 pg/well (prepared 4 pg/mL).
HRP-goat anti-human Fc (Jackson ImmunoResearch Inc, code # 109-035-098, lot #
126815) at 1:10000) was used for detection. TM B (Sigma T8665 Lot 5LBC7444V) was added for 25 min. Reaction was stopped with 0.2 N H2504 and read at OD 450 nm.

Date Recue/Date Received 2022-10-13 TABLE 1. Sequences SEQ ID NO:1 ATGGCGAAGACTAATTTGTTTCTGTTCTTGATATTTTCGT
Nucleic acid TGCTACTGTCACTTTCTTCTGCTACCATTGAAGAACAGG
sequence encoding CTAAAACCTTCCTTGACAAATTTAATCATGAGGCTGAGG
a ACE2-Fc IgA2 ATCTCTTTTATCAATCCAGCCTGGCTTCTTGGAACTACA
fusion protein ATACAAATATAACAGAAGAGAACGTGCAGAATATGAACA
ATGCTGGTGATAAATGGAGTGCTTTCTTAAAGGAACAGT
CTACTCTAGCTCAAATGTACCCTCTTCAAGAAATCCAAA
ATCTGACTGTGAAACTTCAACTGCAAGCATTGCAGCAAA
ATGGTAGCTCCGTTCTCTCAGAAGATAAATCGAAACGG
CTGAACACTATACTTAATACTATGTCTACTATCTACTCGA
CGGGAAAAGTGTGCAACCCTGACAATCCACAGGAGTGC
CTGCTACTTGAACCTGGTCTGAATGAAATCATGGCTAAT
TCTTTGGATTACAATGAGAGGCTTTGGGCTTGGGAGTC
CTGGAGATCGGAGGTGGGCAAGCAACTTCGGCCTCTCT
ATGAGGAATACGTGGTGTTGAAAAATGAGATGGCGCGT
GCCAACCATTATGAAGATTATGGTGACTATTGGCGAGG
GGATTATGAAGTCAATGGGGTCGATGGCTATGACTATA
GTCGAGGACAACTCATTGAGGATGTGGAACATACTTTC
GAGGAGATTAAACCTCTTTACGAGCATTTGCATGCTTAT
GTTCGAGCTAAGTTGATGAATGCATACCCAAGCTATATT
AGTCCCATTGGTTGTCTTCCTGCCCACTTGTTGGGCGA
CATGTGGGGTAGGTTTTGGACGAACCTTTATTCTCTAAC
GGTTCCTTTTGGTCAAAAGCCTAATATTGACGTCACTGA
TGCTATGGTCGATCAGGCTTGGGATGCCCAAAGAATTT
TCAAAGAAGCTGAAAAATTCTTCGTTAGTGTTGGATTAC
CTAACATGACTCAAGGTTTCTGGGAAAATTCCATGTTAA
CAGATCCAGGAAATGTTCAAAAAGCTGTGTGTCATCCTA
CAGCATGGGATCTTGGCAAGGGGGACTTCAGAATACTG
ATGTGTACTAAAGTGACTATGGATGATTTTTTAACTGCA
CATCATGAAATGGGTCATATCCAGTATGATATGGCTTAT
GCCGCCCAGCCCTTCCTTCTTCGTAATGGAGCAAACGA
AGGATTCCATGAAGCCGTCGGAGAAATTATGTCGCTCT
CTGCAGCGACTCCCAAACATTTAAAATCAATAGGGCTCT
TATCTCCTGATTTCCAAGAAGACAATGAAACCGAGATTA
ATTTTCTCCTTAAACAAGCGCTCACAATAGTAGGAACTC
TCCCATTTACATACATGTTGGAAAAATGGCGGTGGATG
GTATTTAAGGGAGAGATTCCAAAAGATCAATGGATGAAG
AAATGGTGGGAGATGAAAAGAGAAATAGTGGGTGTTGT
TGAACCAGTTCCACATGATGAAACTTATTGTGATCCTGC
ATCATTATTTCACGTATCAAATGATTATAGCTTTATCCGG
TATTATACAAGAACACTATACCAATTCCAGTTTCAAGAA
GCACTGTGTCAAGCTGCTAAGCATGAAGGACCTTTGCA
TAAATGTGACATTAGCAACTCTACCGAGGCTGGGCAAA
AGCTCTTCAACATGCTTCGTCTTGGAAAGTCGGAGCCG
TGGACCCTTGCCTTAGAAAACGTTGTCGGGGCAAAAAA
TATGAACACGCGCCCCCTTTTGAATTATTTTGAACCACT
GTTTACTTGGTTGAAAGACCAGAACAAAAATTCTTTTGTT
GGCTGGTCAACGGACTGGAGCCCTTATGCTGATCAATC

Date Recue/Date Received 2022-10-13 TATAAAAGTTAGGATCTCCTTAAAGTCAGCCTTGGGTGA
TAAGGCTTACGAATGGAATGATAACGAAATGTATCTATT
CAGGTCGTCCGTAGCATATGCAATGAGGCAATACTTTCT
AAAGGTCAAAAACCAGATGATTCTTTTTGGTGAGGAAGA
CGTTAGAGTAGCAAATCTTAAGCCACGTATCAGCTTCAA
TTTTTTTGTGACAGCTCCTAAGAACGTATCTGATATTATT
CCAAGGACCGAAGTTGAGAAGGCAATTAGAATGAGCCG
CAGTCGGATCAATGACGCCTTTCGTCTGAATGATAATTC
TTTGGAATTTCTTGGGATCCAGCCAACCCTTGGACCTCC
GAACCAACCACCTGTATCTCCAGTACCGCCGCCGCCGC
CTTGTTGCCATCCAAGGCTTTCCTTGCACAGGCCAGCA
CTTGAAGATTTGCTTCTAGGATCAGAGGCAAATCTCACA
TGCACGCTAACCGGACTGAGGGATGCATCTGGTGCTAC
CTTTACATGGACACCTTCATCAGGGAAGTCTGCTGTTCA
AGGGCCTCCCGAAAGAGACTTATGTGGTTGCTATTCAG
TTTCAAGTGTACTACCTGGATGTGCTCAGCCTTGGAATC
ACGGAGAGACATTCACTTGTACCGCCGCGCACCCAGAG
CTAAAAACTCCATTAACTGCCAATATAACAAAATCTGGC
AACACGTTTCGCCCTGAGGTTCACCTATTGCCTCCACC
CTCAGAAGAATTGGCCTTGAATGAGCTTGTAACTTTGAC
ATGTCTGGCAAGAGGATTTAGTCCAAAGGATGTTCTAGT
CAGATGGCTACAAGGGTCCCAGGAGTTACCTAGAGAAA
AGTATTTGACTTGGGCAAGTCGGCAAGAACCTTCACAG
GGTACAACTACTTTTGCTGTTACATCAATTCTTCGGGTC
GCGGCAGAAGATTGGAAAAAGGGTGATACCTTTAGTTG
CATGGTGGGACACGAGGCACTTCCTCTTGCATTTACAC
AGAAAACAATTGATAGGTTAGCTGGCAAGCCTACACAC
GTTAACGTGTCGGTTGTTATGGCAGAAGTTGATGGGAC
TTGCTACTGA
SEQ ID NO:2 Amino MAKTNLFLFLIFSLLLSLSSATIEEQAKTFLDKFNHEAEDLF
acid sequence of a YQSSLASWNYNTNITEENVQNMNNAGDKWSAFLKEQSTL
ACE2-Fc IgA fusion AQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTIL
protein NTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNE
RLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYED
YG DYWRGDYEVNGVDGYDYSRGQ LI EDVEHTFEEI KPLY
EHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWT
NLYSLTVPFGQ KPN I DVTDAMVDQAWDAQRI FKEAEKFFV
SVGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKG
DF RI LM CTKVTM DDFLTAHHEMGHIQYDMAYAAQPFLLRN
GANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNET
El NFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWM
KKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFI
RYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNSTEAGQK
LFNMLRLGKSEPWTLALENVVGAKNMNTRPLLNYFEPLFT
WLKDQNKNSFVGWSTDWSPYADQSIKVRISLKSALGDKA
YEWNDNEMYLFRSSVAYAMRQYFLKVKNQMILFGEEDVR
VANLKPRI SFNFFVTAPKNVSDI I PRTEVEKAI RMSRSRI ND
AFRLNDNSLEFLGIQPTLGPPNQPPVSPVPPPPPCCHPRL
SLHRPALEDLLLGSEANLTCTLTGLRDASGATFTWTPSSG
KSAVQGPPERDLCGCYSVSSVLPGCAQPWNHGETFTCT

Date Recue/Date Received 2022-10-13 AAHPELKTPLTANITKSGNTFRPEVHLLPPPSEELALNELV
TLTCLARGFSPKDVLVRWLQGSQ ELPREKYLTWASRQ EP
SQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFT
QKTIDRLAGKPTHVNVSVVMAEVDGTCY
SEQ ID NO: 3 ATGGCAAAAACAAATCTGTTCCTTTTCTTGATCTTTTCAC
Nucleic acid TCCTACTATCTCTTTCCTCTGCTCAAGAAGATGAAAGGA
sequence encoding TTGTCCTGGTGGATAACAAGTGCAAATGTGCTAGGATC
a human J-Chain ACG
AGCAGAATAATCCGATCAAGTGAAGATCCTAATGAAGAC
ATTGTCGAAAGAAACATTCGCATAATTGTTCCTTTGAATA
ATCGGGAAAATATTTCTGATCCTACTTCTCCCTTACGTA
CA
AGGTTTGTTTATCATTTGAGTGACCTTTGCAAGAAGTGT
GACCCAACTGAGGTAGAGCTTGATAATCAGATTGTGAC
AGCAACCCAATCAAACATATGTGATGAGGACTCGGCCA
CAG
AAACTTGTTACACTTATGATAGAAACAAATGCTACACCG
CTGTTGTACCGTTAGTGTATGGAGGTGAAACAAAAATG
GTTGAGACTGCATTGACTCCAGATGCTTGTTATCCTGAT
TGA
SEQ ID NO: 4 Amino MAKTNLFLFLIFSLLLSLSSAQEDERIVLVDNKCKCARITSRI
acid sequence of a IRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHL
human J-Chain SDLCKKCDPTEVELDNQIVTATQSNICDEDSATETCYTYD
RNKCY
TAVVPLVYGGETKMVETALTPDACYPD
SEQ ID NO: 5 ATGGAAAGGGCTATTCAGGGAAATGATGCTAGAGA
nucleic acid GCAGGCTAATTCTGAAAGATGGGATGGTGGATCTG
sequence of p19 GTGGAACTACTTCTCCATTCAAGCTTCCAGATGAGT
suppressor of CTCCATCTTG GACTGAGTG GAG GCTTCATAACGAT
gene-silencing GAGACTAACTCCAATCAGGATAACCCACTCGGATT
protein CAAAGAATCTTGGGGATTCGGAAAGGTTGTGTTCA
AG CGTTACCTTAG GTATGATAG GACTGAGG CTTCA
CTTCATAGGGTTCTCGGATCTTGGACTGGTGATTC
TGTTAACTACGCTGCTTCTCGTTTTTTTGGATTCGA
TCAGATCGGATGCACTTACTCTATTAGGTTCAGGG
GAGTGTCTATTACTGTTTCTGGTGGATCTAGGACTC
TTCAACACCTTTGCGAGATGGCTATTAGGTCTAAG
CAAGAGCTTCTTCAGCTTGCTCCAATTGAGGTTGA
GTCTAACGTTTCAAGAGGATGTCCAGAAGGTACTG
AGACTTTCGAGAAAGAATCCGAG
SEQ ID NO: 6 MERAIQGNDAREQAN SERWDGGSGGTTSP FKLP DE
amino acid SPSVVTEWRLHNDETNSNQDNPLGFKESWGFGKVVF
sequence of p19 KRYLRYDRTEASLHRVLGSVVTGDSVNYAASRFFGFD
suppressor of QI
GCTYSI RFRGVSITVSGGSRTLQHLCEMAIRSKQEL
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MacGowan et al 2021 BioRxiv 2021.05.21.4458v1 Date Recue/Date Received 2022-10-13

Claims (23)

1. A method of making a fusion protein comprising:
(a) introducing a nucleic acid molecule encoding a fusion protein, wherein the fusion protein comprises ACE2, or a variant or fragment thereof, and the Fc domain of IgA or a variant or fragment thereof, into a plant or plant cell; and (b) growing the plant or plant cell to obtain a plant that expresses the fusion protein.

2. The method according to claim 1, wherein the ACE2 or variant or fragment thereof comprises the ACE2 ectodomain.

3. The method according to claim 1 or 2, wherein the IgA is IgA2.

4. The method according to any one of claims 1 to 3, wherein the nucleic acid molecule further comprises a flexible linker between the ACE2 or variant or fragment thereof and the Fc domain.

5. The method according to any one of claims 1 to 4, wherein the nucleic acid molecule is codon-optimized for expression in plants.

6. The method according to any one of claims 1 to 4, wherein the nucleic acid molecule encodes a sequence as shown in SEQ ID NO: 1, or a sequence at least 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99% identical to SEQ ID NO: 1.

7. The method according to any one of claims 1 to 6, further comprising introducing a second nucleic acid molecule encoding a J-Chain or a variant or fragment thereof into the plant or plant cell.

8. The method according to claim 7, wherein the second nucleic acid molecule is codon-optimized for expression in plants.

9. The method according to claim 7, wherein the second nucleic acid molecule encodes a sequence as shown in SEQ ID NO: 3, or a sequence at least 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99% identical to SEQ ID NO: 3.

Date Recue/Date Received 2022-10-13

10. The method according to any one of claims 7 to 9, wherein the nucleic acid molecule encoding the fusion protein and the second nucleic acid molecule encoding J-Chain or a variant or fragment thereof are introduced on separate vectors.

11. The method of claim 10, wherein the ratio of the nucleic acid molecule encoding the fusion protein and the second nucleic acid molecule encoding J-Chain or a variant or fragment thereof is between 1:1 and 1:2.

12. The method according to any one of claims 7 to 9, wherein the nucleic acid molecule encoding the fusion protein and the second nucleic acid molecule encoding J-Chain or a variant or fragment thereof are introduced on the same vector.

13. The method according to any one of claims 1 to 12, wherein the plant is N.
benthamiana and the plant cell is a N. benthamiana cell.

14. A fusion protein obtained by the method of any one of claims 1 to 13.

15. A composition comprising a fusion protein obtained by the method of any one of claims 1 to 13 and a pharmaceutically acceptable diluent, excipient, or carrier.

16. A use of the fusion protein of claim 14 or the composition of claim 15 for treating or preventing a coronavirus infection.

17. The use of claim 16, wherein the fusion protein or composition is for intranasal administration.

18. The use of claim 16 or 17, wherein the coronavirus is SARS-CoV-2.

19. The use of any one of claims 16 to 18, wherein the fusion protein or the composition is for administration within 1, 6, 12, 18, 24 hours or 1, 2, 3, 4, 5, 6 or 7 days of exposure to the coronavirus.

20. A transgenic plant or plant cell that expresses a fusion protein comprising ACE2, or a variant or fragment thereof, and the Fc domain of lgA, or a variant or fragment thereof.

21. The transgenic plant or plant cell of claim 20, wherein the ACE2 or variant or fragment thereof comprises the ACE2 ectodomain.

22. The transgenic plant or plant cell of claim 20 or 21, wherein the lgA
is lgA2.

Date Recue/Date Received 2022-10-13

23. The transgenic plant or plant cell of any one of claims 20 to 22, wherein the transgenic plant or plant cell further expresses a J-Chain.

Date Recue/Date Received 2022-1 0-1 3