Modified antibodies and their uses

Abstract

This invention falls under the field of biomedicine. Specifically, it relates to a novel species of antibody fragments (AFs) fused to a stabilizing motif, their use in pharmaceuticals, and methods for producing them.

Description

[Technical Field]

[0001] This invention falls under the field of biomedicine. Specifically, this invention relates to novel species of antibodies fused to stabilizing motifs and their fragments, their use in pharmaceuticals, and methods for producing them. [Background technology]

[0002] Monoclonal antibodies, antibody fragments, and derived fusion proteins are widely used in the diagnosis and therapy of numerous diseases, including cancer, inflammatory diseases, autoimmune diseases, neurodegenerative diseases, immune diseases, or rare hematological disorders, as well as for the prevention and treatment of solid organ transplant rejection.

[0003] The transformation of antibodies in pharmaceuticals is evidenced by the increase in antibody-based products. Because antibodies generally exhibit high specificity for specific targets, they tend to have less off-target toxicity than small molecule therapies.

[0004] Despite the importance of biopharmaceuticals in medicine, including antibodies and their derivatives, the biopharmaceutical industry faces significant challenges in their production (high costs and long processing times associated with mammalian cell production) and routes of administration. For example, antibodies are unstable and expensive to produce, so they are delivered in intravenous or subcutaneous injection forms, but these are associated with adverse effects such as systemic inflammatory responses, fluid reactions, and low tolerance for pain. Due to these factors, the use of these drugs is currently limited to more severely ill patients.

[0005] To overcome these factors, several attempts have been made, including engineered antibody derivatives, alternative production hosts, or complex formulations, all aimed at increasing stability and efficiency in specific target tissues / organs / cells.

[0006] Novel formulations that significantly stabilize mAbs under unfavorable conditions such as low concentrations or body temperature have shown promising results with the anti-VEGF antibodies bevacizumab, ranibizumab, and aflibercept, all of which tend to lose function when removed from the manufacturer's vial and diluted (Giannos et al. Pharm Res. 2018; 35(4): 78).

[0007] Recombinant antibodies against tumor necrosis factor α (TNFα) and anti-IL23 are being developed for the oral treatment of inflammatory bowel disease. Manipulated anti-TNF-modified antibodies showed improved permeability to affected tissues in the gastrointestinal tract (GI) (Nurbhai, Suhail, et al., Scientific Reports, 2019, 9, Article number: 14042, Roberts et al., Sci Rep. 2021, 11: 19422).

[0008] Antibodies for combating infectious gastrointestinal diseases are of interest for oral administration in both animals and humans. Fusion of antibodies or antibody fragments with recombinant moieties has been shown to improve resistance to intestinal proteases and to degradation.

[0009] Oral administration of a fusion of an antibody derivative to the Fc region of mucosal IgA has been proven effective in protecting piglets from infection by enterohemorrhagic Escherichia coli (F4-ETEC) with F4 pili (Virdi V. et al., Nat Biotechnol. 2019 May; 37(5):527-530). Another example is patent document US20150252100A1, which describes a fusion protein containing anti-enterotoxitogenic Escherichia coli (anti-ETEC) VHH fused to the IgA Fc domain.

[0010] A modified antibody with a sequence similar to that of colostrum bovine antibodies demonstrated efficacy as an oral therapeutic agent (Kailash C. Bhol et al., Inflamm Bowel Dis. 2013 October; 19(11): 2273-2281).

[0011] Pegylation (Certolizumab PEGOL) (Pasut G. et al., BioDrugs. 2014 Apr; 28 Suppl 1:S15-23), pasylation (Somayeh Mazaheri et al., Scientific Reports volume 10, Article number: 18464, 2020), or anti-albumin antibodies (Ralph Adams et al., MAbs. 2016 Oct; 8(7): 1336-1346) are also options to improve the stability of antibodies in serum, thereby extending the half-life.

[0012] Topical, non-invasive routes of administration for biopharmaceuticals offer potential advantages over injections in terms of ease of administration, high patient tolerance, lower manufacturing costs, and potential for local efficacy.

[0013] To meet the growing demand for stable and affordable biologics via non-injectable delivery routes, there is a strong need for new biologic formulations that are easily and safely administered. This is especially true for conditions requiring local effects without systemic exposure (e.g., skin, intestinal, or respiratory diseases). Non-injectable biologics reduce systemic adverse effects, avoid drug metabolism and dilution, and therefore reduce the required dose. Novel antibody derivatives that interact with various therapeutic targets are being continuously developed. Cost-effective and efficient production of these and other antibody derivatives is crucial for their further success. [Overview of the Initiative] [Problems that the invention aims to solve]

[0014] Despite the fact that research on the oral delivery of biologics has been conducted for nearly a century, current therapeutic administrations remain unchanged and are limited to injections. The main barrier to oral delivery is the stability of these biologics under the harsh conditions within the gastrointestinal (GI) tract. The challenges to making oral delivery of biologics a reality that need to be overcome are to improve the stability of biologics in the digestive tract and achieve higher permeability and targeted delivery.

Means for Solving the Problems

[0015] Summary of the Invention The inventors of the present invention have found that a novel modified antibody fused to a glycomodule motif is superior to an unmodified antibody in terms of potency, stability, and aggregation resistance. The inventors have shown that by adding a glycomodule motif, high neutralizing activity is imparted compared to unmodified antibodies, and the antibody fused to the glycomodule motif is more stable against temperature and proteases.

[0016] Regarding the above, a first aspect of the present invention relates to a modified antibody (hereinafter, the "modified antibody" of the present invention) comprising a first antibody chain comprising a VH region and a CH1 region, and a second antibody chain comprising a VL region and a CL region, wherein at least one of the antibody chains is fused to at least one glycomodule motif (GM).

[0017] In a second aspect, the present invention relates to a polynucleotide composition (hereinafter, the "polynucleotide composition of the present invention") comprising a polynucleotide encoding the antibody chain of the modified antibody of the present invention (hereinafter, the "first polynucleotide of the present invention") or a first polynucleotide encoding the first antibody chain of the modified antibody of the present invention and a second polynucleotide encoding the second antibody chain of the modified antibody of the present invention.

[0018] In a third aspect, the present invention relates to a vector comprising the polynucleotide of the present invention (hereinafter, "the first vector of the present invention"), or a vector composition in which each vector comprises one of the polynucleotides of the polynucleotide composition of the present invention (hereinafter, "the vector composition of the present invention").

[0019] In another aspect, the present invention relates to a host cell comprising the first vector of the present invention or the vector composition of the present invention (hereinafter, "the host cell of the present invention").

[0020] In another aspect, the present invention relates to a pharmaceutical composition comprising the modified antibody of the present invention, the first polynucleotide or polynucleotide composition of the present invention, the first vector or vector composition of the present invention, or the host cell of the present invention and at least one pharmaceutically acceptable excipient (hereinafter, "the pharmaceutical composition of the present invention").

[0021] In another aspect, the present invention relates to the modified antibody of the present invention, the first polynucleotide or polynucleotide composition of the present invention, the first vector or vector composition of the present invention, the host cell of the present invention, or the pharmaceutical composition of the present invention for use in medicine.

[0022] In another aspect, the present invention relates to the modified antibody of the present invention, the first polynucleotide or polynucleotide composition of the present invention, the first vector or vector composition of the present invention, the host cell of the present invention, or the pharmaceutical composition of the present invention, wherein the modified antibody is a TNFα-neutralizing modified antibody, for use in the treatment of inflammatory diseases.

[0023] In another aspect, the present invention relates to the modified antibody of the present invention, the first polynucleotide or polynucleotide composition of the present invention, the first vector or vector composition of the present invention, the host cell of the present invention, or the pharmaceutical composition of the present invention for use in the treatment of digestive diseases.

[0024] In another embodiment, the present invention relates to a modified antibody of the present invention, wherein the modified antibody is against VEGF, a first polynucleotide or polynucleotide composition of the present invention, a first vector or vector composition of the present invention, a host cell of the present invention, or a pharmaceutical composition of the present invention, for use in the treatment of diseases associated with unwanted angiogenesis.

[0025] In another embodiment, the present invention relates to a modified antibody of the present invention, in which the modified antibody is specific to VEGF, a first polynucleotide or polynucleotide composition of the present invention, a first vector or vector composition of the present invention, a host cell of the present invention, or a pharmaceutical composition of the present invention, for use in the treatment of intravascular age-related macular degeneration, macular edema after retinal vein occlusion, diabetic macular edema, diabetic retinopathy, or myopic choroidal neovascularization.

[0026] In another embodiment, the present invention is: A method for producing the modified antibody of the present invention, "the first method of the present invention," (i) Proliferating cells comprising the first polynucleotide or polynucleotide composition of the present invention under conditions suitable for enabling the expression of modified antibodies from the polynucleotide or from the polynucleotide of the polynucleotide composition, and (ii) Recovering modified antibodies from the culture. Regarding methods that include

[0027] In another embodiment, the present invention is: (i) VH region and CH1 region; or (ii) VL region and CL region This invention relates to an antibody chain comprising and fused to a glycomodule motif (GM) (hereinafter referred to as "the antibody chain of the present invention").

[0028] In another embodiment, the present invention relates to a polynucleotide encoding an antibody chain of the present invention (hereinafter referred to as "the second polynucleotide of the present invention").

[0029] In another embodiment, the present invention relates to a vector comprising a second polynucleotide of the present invention (hereinafter referred to as "the second vector of the present invention").

[0030] In another embodiment, the present invention relates to a host cell comprising a second vector of the present invention.

[0031] In another embodiment, the present invention provides an in vitro method for detecting a target antigen present in a sample (hereinafter referred to as "the second method of the present invention"), (i) Contacting the sample with the modified antibody of the present invention, wherein the modified antibody can specifically bind to the target antigen under conditions sufficient for binding of the target antigen to the modified antibody; and (ii) Determine the presence of a complex containing the target antigen and a modified antibody. Regarding methods that include

[0032] In another embodiment, the present invention relates to an in vitro method for purifying a target antigen present in a sample (hereinafter referred to as "the third method of the present invention"), (i) Contacting the sample with the modified antibody of the present invention, wherein the modified antibody can specifically bind to the target antigen under conditions sufficient for binding of the target antigen to the modified antibody; and (ii) Recover the complex containing the target antigen and the modified antibody. Regarding methods that include [Brief explanation of the drawing]

[0033] [Figure 1] Screening of antibody expression by direct ELISA. In this study, plates were coated with 0.5 μg / mL human TNFα, incubated with culture medium derived from independent clones, and detected with HRP peroxidase-conjugated anti-human IgG antibody (Fab-specific). Values ​​are shown as the increase ratio relative to the wild-type signal. [Figure 2]Non-reductive immunoblots of ranibizumab fused to GM at various positions. Equal volumes of culture medium from independent clones (referred to as c1-13) were added. A) Detection of independent clones expressing AF using anti-human IgG antibody (Fab-specific). B) Detection of independent clones expressing GM-AF using anti-human IgG antibody (Fab-specific). C) Detection of independent clones expressing AF using anti-OLLAS antibody. Full-length human monoclonal antibody (human IgG) and OLLAS-tagged protein (C+OLLAS protein) were diluted in PBX 0.1% BSA and used as controls. AF: antibody fragment, HC: heavy chain, LC: light chain. [Figure 3] Antigen recognition of ranibizumab and GB-AF-010 (SEQ ID NOs. 15 and 16). Antigen recognition was evaluated by direct ELISA: 96-well plates were coated with 0.5 μg / mL VEGF and incubated with ranibizumab (commercial, purified) or GB-AF-010, and detected with HRP-conjugated anti-human IgG (Fab-specific). Results were normalized by all Fabs. [Figure 4] Non-reductive immunoblotting of certolizumab fused to GM at various positions. Equal volumes of culture medium were added to independent clones expressing AF (referred to as c1-c4). The test cassettes were as follows: (SP)10-certolizumab has (SP)10 at the N-terminus of certolizumab in both the heavy and light chains (SEQ ID NOs. 19 and 20); certolizumab-(SP)10 has (SP)10 at the C-terminus of certolizumab in both the heavy and light chains (SEQ ID NOs. 21 and 22); certolizumab-HC-(SP)10 has (SP)10 at the C-terminus of the certolizumab heavy chain (SEQ ID NOs. 21), the light chain lacks a glucomodorum (SEQ ID NOs. 24), and certolizumab is not fused to GM (SEQ ID NOs. 23 and 24). Detection by anti-human IgG (Fab-specific). A full-length human monoclonal antibody (human IgG) prepared with PBX 0.1% BSA was used as a positive control. [Figure 5]Antigen recognition of certolizumab and certolizumab-GM. Antigen recognition was evaluated by direct ELISA: 96-well plates were coated with 0.5 μg / mL human TNFα (hTNFα), incubated with certolizumab (commercial, purified) or GB-AF-011, and detected with HRP peroxidase-conjugated anti-human IgG-conjugated anti-human IgG antibody (Fab-specific). Results were normalized by all Fabs. [Figure 6] Antigen recognition specificity of GB-AF-011. Antigen recognition was evaluated by direct ELISA: 96-well plates were coated with 0.5 μg / mL human TNFα (hTNFα), mouse TNFα, or PBS as a negative control (blank), incubated with GB-AF-011, and detected with HRP peroxidase-conjugated anti-human IgG antibody (Fab-specific). [Figure 7] Comparison of TNFα neutralization by several anti-TNFα agents. Neutralization ability was evaluated (measured as the percentage inhibition rate of hTNFα binding for etanercept) by direct ELISA: 96-well plates were coated with 1 μg / mL etanercept, certolizumab (commercial, purified), infliximab (commercial, purified), or GB-AF-011, pre-incubated with biotinylated TNFα, and then the mixture of biotinylated hTNFα and the anti-TNFα agent was added to the 96-well plates and detected with conjugated streptavidin-HRP. [Figure 8A] Temperature stability of GB-AF-011 compared to commercially available certolizumab. Certolizumab and GB-AF-011 were incubated at 37°C and monitored over time. The concentrations were 8 μg / mL for GB-AF-011 (lyophilized and dialyzed against PBS) and 34 μg / mL for certolizumab (purified product). Results were analyzed by A) direct ELISA (average of 2 replicates); B) reductive immunoblotting using anti-human IgG (Fab-specific) for detection; and C) non-reductive immunoblotting using anti-human IgG (Fab-specific) for detection. [Figure 8B]Temperature stability of GB-AF-011 compared to commercially available certolizumab. Certolizumab and GB-AF-011 were incubated at 37°C and monitored over time. The concentrations were 8 μg / mL for GB-AF-011 (lyophilized and dialyzed against PBS) and 34 μg / mL for certolizumab (purified product). Results were analyzed by A) direct ELISA (average of 2 replicates); B) reductive immunoblotting using anti-human IgG (Fab-specific) for detection; and C) non-reductive immunoblotting using anti-human IgG (Fab-specific) for detection. [Figure 8C] Temperature stability of GB-AF-011 compared to commercially available certolizumab. Certolizumab and GB-AF-011 were incubated at 37°C and monitored over time. The concentrations were 8 μg / mL for GB-AF-011 (lyophilized and dialyzed against PBS) and 34 μg / mL for certolizumab (purified product). Results were analyzed by A) direct ELISA (average of 2 replicates); B) reductive immunoblotting using anti-human IgG (Fab-specific) for detection; and C) non-reductive immunoblotting using anti-human IgG (Fab-specific) for detection. [Figure 9] The stability of certolizumab-GM compared to commercially available reference samples when faced with colonic conditions. Initial concentrations were 8 μg / mL for GB-AF-011, 32 μg / mL for certolizumab (purified product), and 24 μg / mL for infliximab (purified product). The anti-TNFα agents were diluted 1 / 5 in colonic contents, incubated at 37°C, and collected at various time intervals. The results were analyzed by A) reductive immunoblotting using anti-human IgG (Fab-specific) for detection, and B) non-reductive immunoblotting using anti-human IgG (Fab-specific) for detection. [Figure 10] Analysis of GB-AF-011 production in heterotrophic bioreactors. A) Monitoring of microalgae-producing strain growth in a 1L bioreactor. Growth was tracked by optical density (OD) at 750 nm. B) Non-reductive immunoblotting using anti-human IgG (Fab-specific) for detection. Culture medium isolated from cells was added. [Figure 11]Structure of a novel antibody fragment. AF: Antibody fragment. LC: Light chain. HC: Heavy chain. GM: Glycomodule. CL: Enterokinase cleavage sequence. SS-1: Metalloproteinase gametricin secretion signal. SS-2: Carbonic anhydrase 1 secretion sequence. [Modes for carrying out the invention]

[0034] Detailed description of the invention modified antibody The authors of this invention have found that modified antibodies fused to a glycomodule motif confer enhanced efficacy by improving stability and / or activity. In particular, the authors have found that modified antibodies fused to a glycomodule motif are more pronounced than unmodified antibodies in terms of potency, stability, and agglutination resistance.

[0035] Given the properties of modified antibodies fused to glycomodule motifs, these novel antibodies enable applications beyond injection routes and facilitate antibody use across many different regions. Due to their improved stability under physiological conditions, the AFs described herein can be used alone or in combination with other proteins such as growth factors or cytokines as therapeutic agents for inflammatory diseases, infectious diseases, gastrointestinal diseases, and / or diseases associated with undesirable angiogenesis, particularly in parenteral formulations (e.g., oral, topical, or inhaled).

[0036] Therefore, in a first embodiment, the present invention relates to a modified antibody (hereinafter, "the modified antibody of the present invention") comprising a first antibody chain comprising a VH region and a CH1 region and a second antibody chain comprising a VL region and a CL region, wherein at least one of the antibody chains is fused to at least one glycomodule motif (GM).

[0037] As used herein, “modified antibody” means any isotype of immunoglobulin that can compete with an intact antibody through specific binding to a target antigen, and includes, for example, chimeric, humanized, and fully human modified antibodies.

[0038] As used herein, “isotype” refers to an antibody class encoded by a heavy chain constant region gene (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibodies).

[0039] The modified antibodies of the present invention refer to (i) antibodies comprising a complete heavy chain (VH region and CH1, CH2, and CH3 regions) and a complete light chain (VL region and CL region), and (ii) shortened forms of antibodies comprising a first antibody chain comprising a VH region and a CH1 region and a second antibody chain comprising a VL region and a CL region. The modified antibodies may simply originate from a single source, or they may be "chimeras" in which different parts of the modified antibody may originate from two different antibodies. In some embodiments, the modified antibody is an antibody fragment in which the heavy chain does not contain the constant domain CH2 and / or the constant domain CH3.

[0040] The modified antibody of the present invention comprises a first chain, which refers to a heavy chain. In certain embodiments, the heavy chain consists of a variable domain, VH, and three constant domains CH1, CH2, and CH3. In another particular embodiment of the present invention, the modified antibody comprises only the variable domain (VH) and the first constant domain (CH1). In certain embodiments, the CH1 region is located at the C-terminus of the VH region. In yet another particular embodiment, the heavy chain of the modified antibody of the present invention does not include the constant domains CH2 and / or CH3.

[0041] The modified antibody of the present invention comprises a second chain, which refers to a light chain. In certain embodiments, the light chain consists of a variable domain, VL, and a constant domain, CL. The modified antibody of the present invention comprises a VL region and a CL region. The modified antibody according to the present invention may comprise a complete light chain or a fragment thereof, insofar as the fragment comprises a VL region and a CL region. In certain embodiments, the CL region is located at the C-terminus of the VL region.

[0042] The variable regions of the heavy chain and light chain of the modified antibody according to the present invention (VH and VL, respectively) include an antigen-binding site of an immunoglobulin (Ig) molecule.

[0043] As used herein, the term "antigen-binding site" refers to the portion of a modified antibody that determines the specific antigen to which it can bind. The variable regions (VH and VL) of the heavy and light chains of the modified antibody of the present invention can be called hypervariable regions because they can bind to a variety of antigens. These variable regions include a region at the top called the antigen-binding site. The antigen-binding site is also called a paratope. Each paratope consists of six complementarity-determining regions (CDRs), three from the light chain and three from the heavy chain, extending from a folded antiparallel β-sheet. As used herein, the term "CDR" refers to a complementarity-determining region within the antibody variable sequence, corresponding to an antibody region having a structure complementary to the target antigen or epitope.

[0044] In the modified antibody of the present invention, at least one antibody chain is fused to at least one glycomodule motif (GM).

[0045] As used herein, a “glycomodule motif (GM)” refers to an amino acid sequence comprising at least one residue capable of either hydroxylation or glycosylation, or a glycosylated residue. As used herein, the term “glycosylation site” refers to an amino acid that acts as a target site for glycosylation. In preferred embodiments, a glycosylation site is an amino acid sequence that acts as a target for glycosylation in microalgae. Glycosylation is a reaction catalyzed by glucosyltransferase that adds a sugar chain to another molecule, preferably a protein, in a site-specific manner. Protein glycosylation can occur in various forms, such as N-links, O-links, and phosphocelling glycosylation. Not limited examples of glycosylated amino acids include proline, serine, threonine, hydroxylysine, hydroxyproline, arginine, asparagine, and any variant of glycosylated native amino acids. Thus, within a glycosylation site, a proline residue can be hydroxylated to form hydroxyproline (Hyp). In preferred embodiments, glycosylation occurs at any serine (Ser) or hydroxyproline (Pro) of the glycomodule motif. The glycosylation site can be located at either or both ends of the glycomodule motif, and / or optionally inside the glycomodule. Preferably, the glycosylation of the glycomodule motif is O-glycosylation.

[0046] There are generally two types of hydroxyproline O-glycosylation: 1) an arabinogalactan glycomodule, which consists of clustered, discontinuous hydroxyproline (Hyp) residues, where the Hyp residue is O-glycosylated with an arabinogalactan adduct; and 2) an arabinosylated glycomodule, which consists of a continuous sequence of Hyp residues, where some or all of the Hyp residue is arabinosylated (O-glycosylated) with an arabinose chain of approximately 1 to 5 residues in length. O-glycosylation can occur after one or more hydroxylations of the residue at that site.

[0047] In some embodiments, the modified antibody of the present invention comprises only one glycomodule motif, which may be in the first antibody chain or the second antibody chain, and these glycomodule motifs may be located at the C-terminus or N-terminus of the antibody chain.

[0048] Therefore, in certain embodiments, the modified antibody of the present invention comprises one glycomodule motif located at the C-terminus of the first antibody chain.

[0049] In another specific embodiment, the modified antibody of the present invention comprises a single glycomodule motif located at the N-terminus of the first antibody chain.

[0050] In another specific embodiment, the modified antibody of the present invention comprises one glycomodule motif located at the C-terminus of a second antibody chain.

[0051] In another specific embodiment, the modified antibody of the present invention comprises one glycomodule motif located at the N-terminus of a second antibody chain.

[0052] In some embodiments, the modified antibody of the present invention comprises two or more glycomodule motifs, particularly two glycomodule motifs, one of which is located on a first antibody chain and the other on a second antibody chain. These glycomodule motifs may be located at the C-terminus or N-terminus of the antibody chain.

[0053] In certain embodiments, the glycomodule motif is located at the C-terminus of the first antibody chain and at the C-terminus of the second antibody chain.

[0054] In another specific embodiment, the glycomodule motif is located at the N-terminus of the first antibody chain and at the N-terminus of the second antibody chain.

[0055] In another specific embodiment, the glycomodule motif is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and its functionally equivalent variants, and (SP) n It comprises an amino acid sequence selected from the group consisting of the following.

[0056] Disclosed herein (SP) n This refers to nucleic acid constructs that encode n-time serine-proline repeat units, such as those disclosed in US9006410B2.

[0057] In certain embodiments, the number of n repeating units is 5 to 30. In preferred embodiments, the number of n repeating units is 10 or 20. Thus, in certain embodiments, the Glycomodule motif is (SP) 10 (Sequence ID 6) or (SP) 20 It comprises an amino acid sequence selected from the group consisting of (Sequence ID 7).

[0058] "Functionally equivalent variants of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5" means, as used herein, all sequences resulting from modifications, insertions, and / or deletions of one or more amino acids in the above sequences, provided that the function of the glycomodule motif is substantially maintained.

[0059] A suitable assay for determining whether a polypeptide can be considered a functionally equivalent variant of a glycomodule comprises expressing a fusion protein comprising the glycomodule variant and a marker protein, and detecting whether the addition of the glycomodule to the marker protein results in glycosylation of the fusion protein. The presence of glycosylation in the protein can be determined by any method known in the art, including, but not limited to, staining of glycoproteins (e.g., methods based on periodate Schiff staining), enzymatic or chemical removal of glycans bound to the protein, and detection of molecular weight shifts by Western blotting and / or mass spectrometry. A suitable assay for determining whether a given sequence can function as a glycan module and be considered a functionally equivalent variant of the glycomodule used in the present invention is described in Ramos-Martinez et al. (Plant Biotechnol J. 2017, 15: 1214-1224).

[0060] Preferably, variants of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 are polypeptides that (i) have one or more amino acid residues replaced with conserved or unconserved amino acid residues (preferably conserved amino acid residues) (such substituted amino acids may or may not be encoded by the genetic code), (ii) have one or more modified amino acid residues, e.g., residues modified by substituent bonding, (iii) are polypeptides resulting from the selective processing of similar mRNAs, (iv) are polypeptide fragments, and / or (v) are polypeptides resulting from the fusion of a polypeptide defined in (i) to (iii) with another polypeptide, such as a secretion reader sequence or a sequence used for purification (e.g., a His tag) or a sequence used for detection (e.g., an Sv5 epitope tag). Fragments include polypeptides produced by proteolytic cleavage of the original sequence (including multi-site proteolysis). Variants may be post-translationally modified or chemically modified. Such variants should be obvious to those skilled in the art.

[0061] Those skilled in the art will recognize that the values ​​of nucleotide sequence identity can be appropriately adjusted by taking into account codon degeneracy, conserved amino acid substitutions, and the arrangement of the reading frame in order to determine the corresponding sequence identity of two nucleotide sequences encoding the polypeptide of the present invention.

[0062] In the context of this invention, "conservative amino acid mutation" and "conservative amino acid substitution" are used synonymously. "Conservative amino acid substitution" refers to the interchangeability of residues having similar side chains, and means replacing one or more amino acids in a natural amino acid sequence with another amino acid having a similar side chain to obtain a silent mutation that does not alter the function of the protein. Conservative substitutions of amino acids in a natural amino acid sequence can be selected from other members of the group to which the natural amino acid belongs. For example, the group of amino acids with aliphatic side chains includes glycine, alanine, valine, leucine, and isoleucine; the group of amino acids with aliphatic-hydroxyl side chains includes serine and threonine; the group of amino acids with amide-containing side chains includes asparagine and glutamine; the group of amino acids with aromatic side chains includes phenylalanine, tyrosine, and tryptophan; the group of amino acids with basic side chains includes lysine, arginine, and histidine; and the group of amino acids with sulfur-containing side chains includes cysteine ​​and methionine. In some embodiments of the present invention, preferred conserved amino acid substitutions are valine-leucine, valine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine. Thus, the present invention refers to functionally equivalent variants of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, and those having an amino acid sequence in which one or more amino acids differ from the sequence given as a result of one or more conserved amino acid substitutions. It is well known in the art that one or more amino acids in a polypeptide sequence can be substituted with at least one other amino acid having similar charge and polarity, and that such substitutions result in silent mutations in the modified polypeptide that do not alter the function of the unmodified sequence.The present invention refers to any polypeptide sequence that differs by one or more amino acids, insofar as the further provided polypeptide sequence has the same, similar, or equivalent glycomodule motif as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, as a result of a conserved or non-conserved substitution to the sequence indicated by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, and / or as a result of a sequence insertion or deletion.

[0063] In the context of two or more amino acid or nucleotide sequences, the terms “identity,” “identical,” or “identity percentage” refer to two or more sequences or subsequences that are identical, or have a specified percentage of identical amino acid or nucleotide residues, when compared and aligned (with gaps introduced if necessary) to obtain the greatest possible match, without considering conservative amino acid substitutions as part of sequence identity. The identity percentage can be measured using sequence comparison software or algorithms, or by visual inspection. Various algorithms and software are known in the art that can be used to obtain alignment of amino acid or nucleotide sequences.

[0064] The percentage of sequence identity can be determined by comparing two optimally aligned sequences across a comparison window. The sequences to be aligned may be polynucleotide sequences or polypeptide sequences. For optimal alignment of the two sequences, the polynucleotide or amino acid sequences in the comparison window may contain insertions or deletions (i.e., gaps) compared to a reference sequence (which may not contain insertions or deletions). The percentage of sequence identity is calculated by determining the number of positions in both sequences being compared where identical nucleotide residues or identical amino acid residues are present, obtaining the number of matching positions, then dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the quotient by 100 to obtain the percentage of sequence identity. Sequence identity between two polypeptide sequences or two polynucleotide sequences can be verified, for example, using the Gap program in Genetics Computer Group, Inc.'s WISCONSIN PACKAGE version 10.0-UNIX®, based on the method of Needleman and Wunsch (J. Mol. Biol. 48:443-453, 1970), using a default parameter set for pairwise comparison (Gap Creation Penalty=8, Gap Extension Penalty=2 for amino acid sequence comparison; Gap Creation Penalty=50, Gap Extension Penalty=3 for nucleotide sequence comparison), or using the BLAST 2.2.1 software suite (Altschul et al.), using the BLOSUM62 matrix (Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919, 1992) and a default parameter set for pairwise comparison (gap creation cost=11, gap extension cost=1). This can be determined by using the TBLASTN program (al., Nucleic Acids Res. 25:3389-3402).

[0065] Functionally equivalent variants of SEQ ID NOs. 1, 2, 3, 4, and 5 also contain at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, and 65% of the sequences of SEQ ID NOs. This also includes sequences having sequence identity of 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0066] In a preferred embodiment, a functionally equivalent variant of sequence number 1, 2, 3, 4, or 5 has at least 50% sequence identity with the corresponding sequence of sequence number 1, 2, 3, 4, or 5, and the sequence identity is determined over the entire length of the sequence of sequence number 1, 2, 3, 4, or 5.

[0067] In certain embodiments, the first antibody chain (heavy chain) and / or second antibody chain (light chain) of the modified antibody of the present invention comprises at least one glycomodule motif, at least two glycomodule motifs, at least three glycomodule motifs, at least four glycomodule motifs, at least five glycomodule motifs, at least six glycomodule motifs, at least seven glycomodule motifs, at least eight glycomodule motifs, at least nine glycomodule motifs, at least ten glycomodule motifs, or more.

[0068] In some embodiments, the first antibody chain and / or the second antibody chain comprises two or more glycomodule motifs, all of which may be located at the C-terminus or N-terminus of the antibody chain. In other embodiments, some of these glycomodule motifs may be located at the C-terminus of the antibody chain and others at the N-terminus.

[0069] In certain embodiments, the glycomodule motif is linked to the first antibody chain by a linker sequence.

[0070] In another specific embodiment, the glycomodule motif is linked to a second antibody chain by a sequence linker.

[0071] As used herein, the term “linker” means a suitable peptide that enables two or more functional domains in a fusion protein to be linked together. The linker may be a flexible linker or a rigid linker. In preferred embodiments, the linker is a flexible linker. “Flexible linker” as used herein means that the linked domains require some degree of movement or interaction. These generally consist of small, nonpolar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. The small size of these amino acids makes them flexible, allowing for the mobility of the functional domains being linked. By incorporating Ser or Thr, hydrogen bonds with water molecules can be formed, maintaining the stability of the linker in aqueous solution, thus reducing unfavorable interactions between the linker and the protein moiety.

[0072] In certain embodiments, the linker is a peptide containing 1 to 25 amino acid residues, 1 to 20 amino acid residues, 2 to 15 amino acid residues, 3 to 10 amino acid residues, 3 to 7 amino acid residues, 4 to 25 amino acid residues, 4 to 20 amino acid residues, 4 to 15 amino acid residues, 4 to 10 amino acid residues, 5 to 25 amino acid residues, 5 to 20 amino acid residues, 5 to 15 amino acid residues, or 5 to 10 amino acid residues.

[0073] Exemplary linkers include glycine and serine-rich linkers, such as (GGP)n or (GGGS)n, where n is 1 to 5. The most commonly used flexible linkers have sequences consisting primarily of stretches of Gly and Ser residues ("GS" linkers). By adjusting the copy number "n", the length of this GS linker can be optimized to achieve appropriate separation of functional domains or to maintain necessary interdomain interactions. In a preferred embodiment, the linker sequence that ligates the glycomodule motif to the first antibody chain is (GGGS) n or (GGGGS) n It consists of including.

[0074] In another specific embodiment, the modified antibody of the present invention further comprises a detection tag. As used herein, the term “tag” means a polypeptide useful for facilitating the detection, isolation, and / or purification of a protein. Generally, the label sequence is placed on a portion of the target protein that does not adversely affect its functionality. In a more specific embodiment, the detection tag is selected from the group consisting of the OLLAS tag (SEQ ID NO: 8), the Flag tag (SEQ ID NO: 26), and the His tag (SEQ ID NO: 25, SEQ ID NOs: 29-34). In a more specific embodiment, the detection tag is the OLLAS tag (SEQ ID NO: 8).

[0075] In another specific embodiment, the modified antibody of the present invention comprises a processing site between the detection tag and the rest of the chain. In a more specific embodiment, the processing site is a protease recognition site. As used herein, the term “protease recognition site” refers to an amino acid sequence that is readily cleaved by enzymes that perform proteolysis, which is the catabolism of a protein by hydrolysis of peptide bonds, where the protein has been translated. Suitable processing sites for use in the modified antibody of the present invention include amino acid sequences that can be cleaved by proteases such as enterokinase, Arg-C endoprotease, Glu-C endoprotease, Lys-C endoprotease, factor Xa, and SUMO protease (Tauseef et al., 2005 Protein Expr. Purif. 43:1-9). In a more specific embodiment, the processing site is an enterokinase cleavage sequence (SEQ ID NO: 9) or a TEV protease (SEQ ID NO: 28), preferably an enterokinase cleavage sequence.

[0076] The modified antibody of the present invention may be derived from different antibodies. In certain embodiments, the modified antibody is derived from neutralizing antitumor necrosis factor antibody (TNFα).

[0077] Tumor necrosis factor (TNFα) is a pleiotropic cytokine that has beneficial functions in immunomodulation and host defense, but also harmful pro-inflammatory and cytotoxic functions during inflammation. TNFα is a key mediator of autoimmune processes that play a vital role in several inflammatory diseases, including rheumatoid arthritis (RA), ulcerative colitis, and Crohn's disease. Inhibition of TNFα has been achieved by anti-TNFα biologics such as etanercept, antibodies like infliximab and adalimumab, or the modified antibody certolizumab, which is used to treat autoimmune diseases.

[0078] In more specific embodiments, the modified antibody of the present invention is derived from a neutralizing antitumor necrosis factor α (TNFα) selected from the group consisting of adalimumab, infliximab, certolizumab, or golimumab. In even more specific embodiments, the modified antibody of the present invention is derived from certolizumab.

[0079] Adalimumab is a monoclonal antibody used to treat rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease, ulcerative colitis, psoriasis vulgaris, hidradenitis suppurativa, uveitis, and juvenile idiopathic arthritis.

[0080] Infliximab is a monoclonal antibody used to treat Crohn's disease, ulcerative colitis, rheumatoid arthritis, ankylosing spondylitis, psoriasis, psoriatic arthritis, and Behçet's disease.

[0081] Certolizumab is a Fab' fragment of recombinant humanized antibody used to treat Crohn's disease, rheumatoid arthritis, psoriatic arthritis, and ankylosing spondylitis.

[0082] Golimumab is a monoclonal antibody used to treat rheumatoid arthritis, psoriatic arthritis, and ankylosing spondylitis.

[0083] In certain embodiments, the modified antibody of the present invention is derived from certolizumab and comprises a heavy chain as defined in SEQ ID NO: 19 or SEQ ID NO: 21 and / or a light chain as defined in SEQ ID NO: 20 or SEQ ID NO: 22.

[0084] Sequence ID 19 disclosed herein is an amino acid sequence (SP) 10 This relates to a modified antibody derived from certolizumab, comprising (SEQ ID NO: 6) and including a glycomodule motif at the N-terminus of the first antibody chain (heavy chain).

[0085] Sequence ID 20 disclosed herein is an amino acid sequence (SP) 10It relates to a modified antibody derived from certolizumab, comprising It relates to a modified antibody derived from certolizumab, comprising (SEQ ID NO: 6) and one glycomodule motif located at the C-terminus of the first antibody chain (heavy chain).

[0086] SEQ ID NO: 21 disclosed herein is the amino acid sequence (SP) 10 It relates to a modified antibody derived from certolizumab, comprising (SEQ ID NO: 6) and one glycomodule motif located at the C-terminus of the first antibody chain (heavy chain).

[0087] SEQ ID NO: 22 disclosed herein is the amino acid sequence (SP) 10 It relates to a modified antibody derived from certolizumab, comprising (SEQ ID NO: 6) and one glycomodule motif located at the C-terminus of the second antibody chain (light chain).

[0088] In another specific embodiment, the modified antibody of the present invention is derived from a neutralizing anti-vascular endothelial growth factor (VEGF). In a more specific embodiment, the modified antibody of the present invention is derived from bevacizumab or ranibizumab, preferably ranibizumab.

[0089] VEGF Vascular endothelial growth factor (VEGF) is a potent mitogen for vascular endothelial cells that has been reported as a central regulator of both normal and abnormal angiogenesis.

[0090] Bevacizumab is a modified antibody used in colon cancer, lung cancer, glioblastoma, and renal cell carcinoma. Ranibizumab is a modified antibody against VEGF that is applicable for the treatment of intravascular (exudative) age-related macular degeneration, macular edema after retinal vein occlusion, diabetic macular edema, diabetic retinopathy, and myopic choroidal neovascularization.

[0091] In another specific embodiment, the modified antibody of the present invention is derived from ranibizumab and comprises a heavy chain defined by SEQ ID NO: 15 or SEQ ID NO: 17 and / or a light chain defined by SEQ ID NO: 16 or SEQ ID NO: 18.

[0092] Sequence ID 15 disclosed herein is an amino acid sequence (SP) 20 This relates to a modified antibody derived from ranibizumab, comprising (SEQ ID NO: 7) and including a glycomodule motif at the C-terminus of the first antibody chain (heavy chain). The sequence of SEQ ID NO: 15 comprises an enterokinase cleavage sequence.

[0093] Sequence ID 16 disclosed herein is an amino acid sequence (SP) 20 This relates to a modified antibody derived from ranibizumab, comprising (SEQ ID NO: 7) and a glycomodule motif located at the C-terminus of the second antibody chain (light chain). The sequence of SEQ ID NO: 16 comprises an enterokinase cleavage sequence.

[0094] Sequence ID 17 disclosed herein is an amino acid sequence (SP) 10 This relates to a modified antibody derived from ranibizumab, comprising (SEQ ID NO: 6) and including a glycomodule motif at the N-terminus of the first antibody chain (heavy chain). The sequence of SEQ ID NO: 17 comprises an enterokinase cleavage sequence.

[0095] Sequence ID 18 disclosed herein is an amino acid sequence (SP) 10 This relates to a modified antibody derived from ranibizumab, comprising (SEQ ID NO: 6) and containing a glycomodule motif at the N-terminus of the second antibody chain (light chain). The sequence of SEQ ID NO: 18 comprises an enterokinase cleavage sequence.

[0096] In another specific embodiment, the modified antibody of the present invention is derived from a neutralizing antiintegrin. In a more specific embodiment, the modified antibody of the present invention is derived from natalizumab or vedolizumab.

[0097] Natalizumab is used to treat multiple sclerosis and Crohn's disease, while vedolizumab is used to treat ulcerative colitis or Crohn's disease.

[0098] In another specific embodiment, the modified antibody of the present invention is derived from a neutralizing anti-IL23 / IL-12. In a more specific embodiment, the modified antibody of the present invention is derived from a neutralizing anti-IL23 / IL-12 selected from the group consisting of ustekinumab, guselkumab, tildrakizumab, or risankizumab.

[0099] Ustekinumab is used to treat Crohn's disease, ulcerative colitis, psoriasis vulgaris, and psoriatic arthritis, while guselkumab, tildrakizumab, and risankizumab are used to treat psoriasis and have potential applications for Crohn's disease and ulcerative colitis.

[0100] Polynucleotides, vectors, and host cells In a second embodiment, the present invention relates to a polynucleotide comprising a polynucleotide encoding the antibody chain of the modified antibody of the present invention, hereinafter referred to as "the first polynucleotide of the present invention," or a polynucleotide composition comprising a first polynucleotide encoding the first antibody chain of the modified antibody of the present invention and a second polynucleotide encoding the second antibody chain of the modified antibody of the present invention, hereinafter referred to as the polynucleotide composition of the present invention.

[0101] The terms “nucleic acid,” “polynucleotide,” and “nucleotide sequence,” when used interchangeably herein, refer to nucleotides of any length and any polymer form composed of ribonucleotides or deoxyribonucleotides. These terms include both single-stranded and double-stranded polynucleotides, as well as modified polynucleotides (e.g., methylated, protected). Generally, as used herein, nucleic acid is a “coding sequence” referring to a DNA sequence that, under the control of appropriate regulatory sequences, is transcribed and translated into polypeptides in a host cell. The boundaries of a coding sequence are determined by a 5' (amino)-terminus start codon and a 3' (carboxy)-terminus translation termination codon. Codex sequences may include, but are not limited to, prokaryotic sequences, cDNA derived from eukaryotic mRNA, genomic DNA sequences derived from eukaryotic (e.g., mammalian) DNA, and synthetic DNA sequences. The transcription termination sequence is typically located at 3' of the coding sequence.

[0102] In some embodiments, each of the polynucleotides forming part of the first polynucleotide of the present invention or a composition of the present invention further comprises a nucleotide sequence encoding a secretion signal peptide, the secretion signal peptide being fused in-frame to the N-terminuses of the first and second antibody chains.

[0103] As used herein, the terms “signal peptide” or “secretionary signal peptide” refer to relatively short peptides, typically 5–40 amino acid residues in length, that direct intracellularly synthesized proteins into the secretory pathway. Signal peptides typically contain a series of hydrophobic amino acids that form a secondary α-helix structure. Furthermore, many peptides contain a series of positively charged amino acids that can contribute to the protein adopting a topology suitable for its translocation. Signal peptides tend to have a motif at their carboxyl terminus that is recognized by peptidases, which can hydrolyze the signal peptide to produce a free signal peptide and a mature protein.

[0104] In the present invention, any secreted signal peptide can be used, such as, as exemplary non-limiting examples, a signal peptide derived from Chlamydomonas reinhardtii carbonic anhydrase (CAH1) having the nucleotide sequence shown in SEQ ID NO: 10, a signal peptide derived from Chlamydomonas periplasmic aryl sulfatase 1 (ARS1) having the nucleotide sequence shown in SEQ ID NO: 11, or a signal peptide derived from Chlamydomonas gametricin containing Chlamydia having the nucleotide sequence shown in SEQ ID NO: 12.

[0105] It will be understood that, for the polynucleotides of the present invention to be expressed in the host of the target cell, the polynucleotides may be provided operably ligated with a regulatory region. Those skilled in the art will understand that an appropriate regulatory region may be used based on the host cell in which the polynucleotides can be expressed. The nature of the regulatory region is not particularly limited in the present invention.

[0106] In another embodiment, the present invention relates to a vector comprising the first polynucleotide of the present invention, hereinafter referred to as "the first vector of the present invention," or a vector composition in which each vector comprises one of the polynucleotides of the polynucleotide composition of the present invention, hereinafter referred to as "the vector composition of the present invention."

[0107] As used herein, the terms “vector” or “expression vector” refer to a replicable DNA construct used to express a first polynucleotide or the polynucleotide composition of the present invention in cells, preferably eukaryotic cells. The choice of expression vector depends on the choice of host. A variety of expression host / vector combinations are available. Useful expression vectors for eukaryotic hosts include, for example, vectors containing expression regulatory sequences derived from SV40, bovine papillomavirus, adenovirus, and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids such as E. coli plasmids containing pCR 1, pBR322, pMB9, and their derivatives, plasmids with a broader host range such as M13, and filamentous single-stranded DNA phages.

[0108] In certain embodiments, the vector is suitable for expression in microalgae. Preferred vectors for the present invention are vectors commonly known to those skilled in the art, such as the pChlamy_4 vector (Invitrogen), or vectors developed for algae, such as those available from the Chlamydomonas Center.

[0109] In another embodiment, the present invention relates to a host cell comprising the first vector or vector composition of the present invention, hereinafter referred to as "the host cell of the present invention."

[0110] The term “host cell” is used to refer not only to a specific target cell but also to the offspring or potential offspring of such a cell. Such offspring may not be identical to the parent cell in practice, as certain modifications may occur in the next generation due to either mutation or environmental influences, but they are still included in the scope of this term when used herein. A host cell can be any prokaryotic cell (e.g., *E. coli*) or eukaryotic cell (e.g., yeast or plant cells).

[0111] In certain embodiments, the host cell is a microalga. When used herein, microalgae refer to a large and diverse group of simple, typically autotrophic microalgae, ranging from unicellular to multicellular forms, typically found in freshwater and marine systems. Suitable examples of microalgae include those of the Cyanobacteria, Chlorophyta, Rhodophyta, Heterokonta, and Haptophyta divisions. Cyanobacteria may include Spirulina (Arthrospira), Aphanizomenon flos-aquae, Anabaena cylindrica, or Lyngbya majuscule. Algae of the division Chlorophyta may include Chlorella, Scenedesmus, Dunaliella, Tetraselmis, Haematococcus, Ulva, Codium, Botryococcus, or species of Caulerpa. Algae of the division Rhodophyta may include Porphyridium cruentum, species of the genera Gracilaria, Grateloupia, Palmaria, Corallina, Chondrus crispus, Porphyra, or Rhodosorus. Algae of the division Heterokonta may include Nannochloropsis oculate, Odontella aurita, Phaeodactylum tricornutum, species of Fucus, Sargassum, Padina, Undaria pinnatifida, or Laminaria.Algae of the haptophyta division may include species of the genera Isochrysis sp., Tisochrysis sp., or Pavlova sp. Algae may include Crypthecodinium cohnii, Schizochytrium, Ulkenia, or Euglena gracilis. Algae may include Chlorella, Ipomoea quamoclit, Dunaliella, Haematococcus and Bracteacoccus; microalgae of the haptophyta division such as Isochrysis; and heterokontophyta microalgae such as Phaeodactylum, Ochromonas and Odontella.

[0112] In more specific embodiments, the microalgae are green algae. Suitable examples of green algae include Chlorella or Haematococcus, Botryococcus, or Chlamydomonas. In even more specific embodiments, the microalgae belong to the genus Chlamydomonas.

[0113] When used herein, Chlamydomonas is a genus of green algae consisting of approximately 325 species, all of which are cellular flagellated algae, and are found in stagnant water, moist soil, freshwater, seawater, and even in snow as "snow algae." In preferred embodiments, the microalgae are derived from the species Chlamydomonas reinhardtii.

[0114] Chlamydomonas, as used herein, is a single-celled green alga about 10 micrometers in diameter that swims using two flagella. Chlamydomonas has a cell wall made of hydroxyproline-rich glycoproteins, large cup-shaped chloroplasts, a large pyrenoid, and a light-sensing "eye spot."

[0115] In another specific embodiment, the host cell is a plant cell. The term “plant cell,” as used herein, refers to a plant expression system capable of undergoing glycosylation as described in the definition of “glycomodule motif.”

[0116] Pharmaceutical composition In another embodiment, the present invention relates to a pharmaceutical composition comprising a modified antibody of the present invention, a first polynucleotide or polynucleotide composition of the present invention, a first vector or vector composition of the present invention, or a host cell of the present invention or one pharmaceutically acceptable excipient, hereinafter referred to as "the pharmaceutical composition of the present invention."

[0117] The term "pharmaceutical composition" refers to a form in which the biological activity of the active ingredients contained herein is effective and the composition is not unacceptably toxic to the subject to which it is administered. The term "pharmaceutical composition" also includes veterinary compositions. As used herein, the term "veterinary composition" refers to a substance or combination of substances that can be administered to an animal for the purpose of treating or preventing a disease of the animal, or for the purpose of restoring, modifying or altering physiological function by exerting pharmacological, immunological or metabolic effects, or for the purpose of making a veterinary diagnosis. A premixture of medicinal feed prepared for incorporation into feed shall also be considered a "veterinary composition."

[0118] The term "excipient" refers to a substance that assists in the absorption of any of the components or compounds of the pharmaceutical composition of the present invention, or a substance that stabilizes any of the components or compounds, and / or a substance that assists in the preparation of the pharmaceutical composition in the sense of giving consistency to the pharmaceutical composition or giving flavor to improve the palatability of the pharmaceutical composition. Accordingly, excipients have, as an example, but not limited to, the function of binding components (e.g., starch, sugar, or cellulose), a sweetening function, a coloring function, a function of protecting the active substance (e.g., a function of blocking from air and / or moisture), a function of filling pills, capsules or any other dosage form, or a disintegration function that promotes the dissolution of components, but this does not exclude other excipients not listed in this paragraph. Accordingly, the term "excipient" is defined as a material that is included in a dosage form, added to an active substance or its aggregate, enabling their preparation and stability, altering their organic properties, or determining the physical and chemical properties of the pharmaceutical composition as well as their bioavailability.

[0119] When used herein, the expression "pharmaceutically acceptable excipients" includes any solvent, dispersion medium, coating agent, antimicrobial agent and antifungal agent, isotonic agent and absorption retardant that is physiologically compatible with the modified antibody of the present invention, the first polynucleotide or polynucleotide composition of the present invention, the first vector or vector composition of the present invention, or the first host cell of the present invention.

[0120] A "dosage form" is a configuration in which the active ingredient and excipients are adapted to provide a pharmaceutical composition or pharmaceutical preparation. A dosage form is defined by the combination of the form in which the pharmaceutical composition is provided by the manufacturer and the form in which the pharmaceutical composition is administered.

[0121] The pharmaceutical composition of the present invention comprises a therapeutically effective amount of the modified antibody of the present invention. “Therapeutically effective amount” is any amount of the component or compound of the composition sufficient to produce the desired effect when administered to a subject. Therefore, the component or compound of the composition refers to the modified antibody of the present invention. The therapeutically effective amount may vary, for example, with age, weight, overall health, sex, and the subject's diet, as well as the mode and timing of administration, excretion rate, and any possible concomitant therapy with other drugs.

[0122] In certain embodiments, the modified antibody or pharmaceutical composition of the present invention is administered orally, topically, respiratoryly, or by eye.

[0123] As used herein, the term "topical" refers to a method of administration that has a local effect on the body surface, and therefore, topical routes of administration may also include enteral administration of drugs that are poorly absorbed by the gastrointestinal tract, or inhaled formulations for airway delivery.

[0124] As used herein, the term "enteral administration" refers to the administration of a drug through the human gastrointestinal tract. Enteral administration involves the esophagus, stomach, small intestine, and large intestine (i.e., the gastrointestinal tract). Methods of administration include oral administration, sublingual administration (dissolving the drug under the tongue), and rectal administration.

[0125] The modified antibodies of the present invention, or the pharmaceutical compositions of the present invention, may be in forms suitable for oral use, such as tablets, lozenges, aqueous or oily suspensions, dispersed powders or granules, emulsions, solutions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to any method known in the art for the manufacture of pharmaceutical compositions, and such compositions may contain one or more agents selected from the group consisting of sweeteners, flavoring agents, coloring agents and preservatives in order to provide a pharmaceutically refined and palatable preparation.

[0126] Oral formulations may also be provided as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, such as calcium carbonate, calcium phosphate, or kaolin, or as soft gelatin capsules in which the active ingredient is mixed with water or an oil medium, such as peanut oil, liquid paraffin, or olive oil.

[0127] The aqueous suspension contains the active material mixed with a mixture suitable for the production of the aqueous suspension. Such excipients may be antiprecipitation agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, tragacanth gum, and gum arabic; dispersants or wetting agents may be natural phosphatidyl, such as lecithin, or condensation products of alkylene oxide and fatty acids, such as polyoxyethylene stearate, or condensation products of ethylene oxide and long-chain aliphatic alcohols, such as heptadecaethyleneoxyethanol, or condensation products of ethylene oxide and a partial ester derived from a fatty acid and hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide and a partial ester derived from a fatty acid and hexitol anhydride, such as polyethylene sorbitan monooleate. The aqueous suspension may also contain one or more preservatives, such as ethyl or n-propyl, p-hydroxybenzoate, one or more colorants, one or more flavoring agents, and one or more sweeteners, such as sucrose or saccharin.

[0128] Oily suspensions can be formulated by suspending the active ingredient in a vegetable oil, such as arachis oil, olive oil, sesame oil, or coconut oil, or in a mineral oil such as liquid paraffin. Oily suspensions may contain thickeners such as beeswax, hard paraffin, or cetyl alcohol. Sweeteners and flavorings, such as those mentioned above, may be added to provide a palatable oral formulation. These compositions can be preserved by adding antioxidants such as ascorbic acid.

[0129] Furthermore, various systems are known that can be used for sustained-release administration of the pharmaceutical composition of the present invention, and these include, but are not limited to, encapsulation in liposomes, microbubbles, microparticles, or microcapsules. Suitable sustained-release forms, as well as materials and methods for their preparation, are well known in the prior art. Accordingly, the orally administered form of the pharmaceutical composition of the present invention is a sustained-release form further comprising at least one coating or matrix. The sustained-release coating or matrix may include, but is not limited to, semi-synthetic or synthetic, water-insoluble or modified natural polymers, waxes, fats, fatty alcohols, fatty acids, natural, semi-synthetic, or synthetic plasticizers, or two or more combinations thereof. Enteric coatings can be applied by conventional processes known to those skilled in the art.

[0130] For topical application, creams, ointments, jellies, solutions, or suspensions containing the modified antibody of the present invention can be used. Similarly, transdermal patches can also be used for local administration.

[0131] As used herein, the term "respiratory administration" refers to the delivery of a drug via the respiratory system. This is an effective route of administration, particularly for drugs with poor oral bioavailability. For respiratory use, inhalation devices, dry powders, nebulizer aerosols, medium-dose inhalers (MDIs), and soft mist inhalers (SMIs) containing the modified antibodies of the present invention are employed.

[0132] As used herein, the term "eye drops" refers to liquid eye drops that are applied directly to the surface of the eye in small amounts, usually one or a few drops. Eye drops containing the modified antibody of the present invention are employed. The eye drops may contain saline solution and / or a lubricant to match the salinity of the eye. The eye drops can be administered using an eye dropper or a glass pipette with a rubber bulb.

[0133] However, it should be understood that the specific dose level and frequency of administration for a particular patient can be modified and will vary depending on various factors, including the activity of the particular compound used, its metabolic stability and duration of action, age, weight, health status, sex, diet, mode and timing of administration, excretion rate, drug combinations, severity of the particular condition, and the host during treatment.

[0134] In addition to the foregoing, the present invention also encompasses the possibility that the pharmaceutical compositions of the present invention may be administered to a subject together with other components or compounds, even if they do not form part of the pharmaceutical compositions of the present invention.

[0135] therapeutic use In another embodiment, the present invention relates to a modified antibody of the present invention, a first polynucleotide or polynucleotide composition of the present invention, a first vector or vector composition of the present invention, a host cell of the present invention, or a pharmaceutical composition of the present invention for use in pharmaceuticals. The pharmaceutical uses referred to in the present invention may be for human or veterinary use.

[0136] In another embodiment, the present invention relates to a modified antibody of the present invention, a first polynucleotide or polynucleotide composition of the present invention, a first vector or vector composition of the present invention, a host cell of the present invention, or a pharmaceutical composition of the present invention, for use in a pharmaceutical in which the antibody is administered topically. The term “topical” is used as described or explained above, and this definition also applies to the therapeutic use of the present invention.

[0137] In another embodiment, the present invention relates to a modified antibody of the present invention, a TNFα-neutralizing modified antibody for use in the treatment of inflammatory diseases, a first polynucleotide or polynucleotide composition of the present invention, a first vector or vector composition of the present invention, a host cell of the present invention, or a pharmaceutical composition of the present invention.

[0138] As used herein, the term “treat” refers to a process that involves slowing, interrupting, stopping, controlling, halting, mitigating, or reversing the progression or severity of an existing symptom, disorder, condition, or disease, but does not necessarily include the complete elimination of all disease-related symptoms, conditions, or disorders. Treatment of a disorder or disease may result, for example, cessation of the progression of the disorder or disease (e.g., no worsening of symptoms) or delay of the progression of the disorder or disease (only if the cessation of progression is transient). “Treatment” of a disorder or disease may also result in a partial response (e.g., improvement of symptoms) or a complete response (e.g., disappearance of symptoms) in the subject / patient affected by the disorder or disease. Thus, “treatment” of a disorder or disease may also refer to improvement of the disorder or disease, which may, for example, result in cessation or delay of the progression of the disorder or disease. Relapse may occur after such a partial or complete response. It should be understood that subjects / patients may experience broad responses to treatment.

[0139] The term "subject" as used herein refers to individuals, plants, or animals, including humans, non-human primates (e.g., chimpanzees and other apes and monkeys); birds, fish, farm animals such as cattle, sheep, pigs, goats, and horses; domestic mammals such as dogs and cats; and laboratory animals such as rodents such as mice, rats, and guinea pigs. This term does not indicate a specific age or sex. In preferred embodiments of the present invention, the subject is human.

[0140] In this invention, the pathological condition or disorder to be treated is an inflammatory disease. Inflammatory diseases encompass a vast number of diseases and pathological conditions characterized by inflammation. Inflammatory diseases include those of the nervous system (examples, but not limited to, encephalitis, myelitis, meningitis, neuritis, dacryodeclamydia, scleritis, episcleritis, keratitis, retinitis, chorioretinitis, blepharitis, conjunctivitis, uveitis, otitis, labyrinthitis, and mastoiditis), and the cardiovascular system (examples, but not limited to, endocarditis, myocarditis, pericarditis, arteritis, phlebitis, and capillary angiitis). ), respiratory system (examples include, but are not limited to, sinusitis, rhinitis, pharyngitis, laryngitis, tracheitis, bronchitis, bronchiolitis, pneumonia and pleurisy), digestive system (examples include, but are not limited to, inflammatory bowel disease including ulcerative colitis and Crohn's disease, stomatitis, gingivitis, gingivostomatitis, glossitis, tonsillitis, sialadenitis / parotitis, cheilitis, esophagitis, gastritis, gastroenteritis, enteritis, gallbladder It may affect the enteritis (including panentotitis, duodenitis, ileitis, colitis, appendicitis, proctitis, hepatitis, ascending cholangitis, cholecystitis, pancreatitis, and peritonitis), the musculoskeletal system (including, but not limited to, arthritis, dermatomyositis, myositis, synovitis, bursitis, tendinitis, esophagitis, osteochondritis, spondylitis, periostitis, and chondritis), the urinary system (including, but not limited to, nephritis, urethritis, cystitis, and urethritis), the reproductive system (including, but not limited to, oophoritis, salpingitis, endometritis, parametritis, cervicitis, vaginitis, vulvitis, mastitis, orchitis, epididymitis, and prostatitis), and the endocrine system (including, but not limited to, isletitis, hypophysitis, thyroiditis, parathyroiditis, and adrenalitis).

[0141] In another embodiment, the present invention relates to a modified antibody of the present invention, a first polynucleotide or polynucleotide composition of the present invention, a first vector or vector composition of the present invention, a host cell of the present invention, or a pharmaceutical composition of the present invention for use in the treatment of infectious diseases. Infectious diseases are caused by pathogens or microorganisms (such as bacteria, viruses, protozoa, or fungi) entering the body and growing and multiplying therein. Infectious diseases differ from mere infection, which is the entry and replication of pathogens or microorganisms (such as bacteria, viruses, protozoa, or fungi) into the body, to a tissue response to their presence or toxins they produce. The most important barriers to the entry of infectious pathogens into the human host are the skin and mucous membranes. If these tissues have been damaged or affected by a previous disease, invasion by infectious pathogens can occur. These infectious pathogens may cause localized infectious diseases such as cysts, or they may enter the bloodstream and be carried throughout the body, causing systemic bloodstream infections (sepsis) or localized infections at distant sites such as meningitis. Infectious diseases can be caused by viruses such as the common cold, influenza, COVID-19, gastroenteritis, hepatitis, or respiratory syncytial virus (RSV); by bacteria such as streptococcus, salmonella, tuberculosis, pertussis, chlamydia, gonorrhea and other sexually transmitted infections (STIs), urinary tract infections (UTIs), Escherichia coli, and Clostridioides difficile; by fungi such as dermatophytes (athlete's foot, etc.), fungal nail infections, vaginal candidiasis (vaginal yeast infection), and oral thrush; or by parasites such as giardiasis, toxoplasmosis, hookworm infection, or pinworm infection.

[0142] In another embodiment, the present invention relates to a modified antibody, a first polynucleotide or polynucleotide composition, a first vector or vector composition, a host cell, or a pharmaceutical composition for use in the treatment of gastrointestinal diseases. Those skilled in the art will understand that the gastrointestinal disease to be treated requires a modified antibody according to the present invention that is specific to the molecule that needs to be targeted in the particular disease. For example, if the disease to be treated is an infection caused by enterotoxigenic Escherichia coli, the modified antibody used is specific to the fimbriae of Escherichia coli and prevents the attachment of the pathogen to the gastrointestinal tract.

[0143] Digestive disorders refer to a variety of diseases of the digestive system. These conditions range from mild to severe. Some common disorders include heartburn, cancer, irritable bowel syndrome, histamine intolerance, and lactose intolerance. Other digestive system disorders include, in particular, gallstones, cholecystitis and cholangitis, rectal disorders (anal fissures, hemorrhoids, proctitis and rectal prolapse), esophageal disorders (strictures, achalasia and esophagitis), gastric disorders (gastritis, gastric ulcers and cancer usually caused by Helicobacter pylori infection), liver disorders (hepatitis B, hepatitis C, cirrhosis, liver failure, alcoholic hepatitis and autoimmune hepatitis), intestinal disorders such as pancreatitis and pancreatic pseudocysts, polyps and cancer, infections, celiac disease, Crohn's disease, ulcerative colitis, diverticulosis, malabsorption, short bowel syndrome and intestinal ischemia, gastroesophageal reflux disease, peptic ulcers, and hiatal hernia.

[0144] In another embodiment, the present invention relates to a modified antibody of the present invention, wherein the modified antibody is a VEGF-neutralizing modified antibody, a first polynucleotide or polynucleotide composition of the present invention, a first vector or vector composition of the present invention, a host cell of the present invention, or a pharmaceutical composition of the present invention, for use in the treatment of diseases associated with unwanted angiogenesis.

[0145] Diseases associated with unwanted angiogenesis refer to any pathological condition that affects the circulatory system. Some common disorders associated with unwanted angiogenesis include, among others, edema, venous occlusion, peripheral vascular disease (PVD), carotid artery disease, ischemia, abdominal aortic aneurysm, chronic venous insufficiency, and deep vein thrombosis.

[0146] In another embodiment, the present invention relates to a modified antibody of the present invention, wherein the modified antibody is a VEGF-neutralizing antibody, a first polynucleotide or polynucleotide composition of the present invention, a first vector or vector composition of the present invention, a first host cell of the present invention, or a pharmaceutical composition of the present invention, for use in the treatment of intravascular age-related macular degeneration, macular edema after retinal vein occlusion, diabetic macular edema, diabetic retinopathy, or myopic choroidal neovascularization.

[0147] Method for producing modified antibodies In another embodiment, the present invention relates to a method for producing the modified antibody of the present invention, hereinafter referred to as "the first method of the present invention," (i) Proliferating cells comprising the first polynucleotide or polynucleotide composition of the present invention under conditions suitable for enabling the expression of modified antibodies from the polynucleotide or from the polynucleotide of the polynucleotide composition, (ii) Recovering modified antibodies from the culture. Regarding methods that include

[0148] In certain embodiments, the cells are plant cells or microalgae. In more specific embodiments, if the cells are microalgae, they are green algae, more specifically, of the genus Chlamydomonas, preferably of the species Chlamydomonas reinhardtii.

[0149] A first method of the present invention comprises a first step of growing cells comprising the first polynucleotide or polynucleotide composition of the present invention. The first polynucleotide or polynucleotide composition of the present invention, comprising the first vector or vector composition of the present invention, can be introduced into cells by known techniques such as transfection, electroporation, particle impaction, and transformation using the isolated first vector of the present invention. In a preferred embodiment, the vector is introduced by transformation or electroporation. Transformed cells can be regenerated on a solid nutrient medium or in a liquid medium.

[0150] Furthermore, the first method of the present invention comprises growing the cells under conditions suitable for enabling the expression of modified antibodies from the first polynucleotide or polynucleotide composition of the present invention. Appropriate culture conditions for the growth of microalgae and the expression of modified antibodies may vary depending on the microalgae species. However, these conditions are well known in the art and can be easily determined.

[0151] In certain embodiments, microalgae are cultured in a suitable medium, without illumination, under mixed or heterotrophic conditions, and at a suitable temperature in a bioreactor. Virtually any medium suitable for microalgae growth can be used, but a non-exclusive example of such a medium is TAP medium. The temperature can be varied, typically between approximately 17°C and 37°C, and particularly between 21°C and 30°C. Culturing can be carried out with or without aeration. Similarly, the maintenance period may vary depending on the amount of microalgae and the amount of modified antibody to be prepared. Again, these conditions are well known and can be easily determined in specific circumstances.

[0152] The second step of the first method of the present invention comprises recovering the modified antibody from the culture.

[0153] In a particular embodiment of the first method of the present invention, the first polynucleotide or polynucleotide composition comprises a nucleotide sequence encoding a secretion signal peptide. Thus, the modified antibody is recovered from the culture supernatant.

[0154] In another specific embodiment of the first method of the present invention, the modified antibody is retained and accumulated within the cell. Thus, the first method of the present invention includes an additional step of extracting the modified antibody from the cell.

[0155] Techniques and conditions for extracting active compounds from cells are widely known in the prior art, and any of them can be used in relation to the present invention.

[0156] "Extraction" refers to the process by which active compounds retained within cells, particularly the modified antibodies of the present invention, are released into the culture medium. This extraction can be carried out, for example, by mechanical means such as pressure or ultrasound.

[0157] GM-modified antibody chain In another embodiment, the present invention is: (i) VH region and CH1 region; or (ii) VL region and CL region This invention relates to an antibody chain comprising and fused to a glycomodule motif (GM), hereinafter referred to as "the antibody chain of the present invention."

[0158] In certain embodiments, if the antibody chain of the present invention comprises a VH region and a CH1 region, the CH1 region is located at the C-terminus of the VH region.

[0159] In another specific embodiment, if the antibody chain of the present invention comprises a VL region and a CL region, the CL region is located at the C-terminus of the VL region.

[0160] The term "glycomodule" is defined or described above, and this definition also applies to the antibody chain of the present invention. The glycomodule motif may be located at the C-terminus or the N-terminus of the antibody chain of the present invention.

[0161] In certain embodiments, the glycomodule motif is located at the C-terminus of an antibody chain comprising a VH region and a CH1 region (heavy chain).

[0162] In another specific embodiment, the glycomodule motif is located at the C-terminus of an antibody chain, which comprises a VL region and a CL region (light chain).

[0163] In another specific embodiment, the glycomodule motif is located at the N-terminus of an antibody chain comprising a VH region and a CH1 region (heavy chain).

[0164] In another specific embodiment, the glycomodule motif is located at the N-terminus of an antibody chain comprising a VL region and a CL region (light chain).

[0165] In another specific embodiment, the glycomodule motif is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or a functionally equivalent variant thereof and (SP) n , especially (SP) 10 (Sequence ID 6) or (SP) 20 It comprises an amino acid sequence selected from the group consisting of (Sequence ID 7).

[0166] In another specific embodiment, the glycomodule motif is linked to the antibody chain of the present invention by a linker.

[0167] "Sequence 1", "Sequence 2", "Sequence 3", "Sequence 4", "Sequence 5", "(SP) n The terms "linker" and "linker" are described or defined above, and these definitions also apply to the antibody chain of the present invention.

[0168] In another specific embodiment, the antibody chain of the present invention further comprises a detection tag. In a more specific embodiment, the detection tag is selected from the group consisting of OLLAS tags (SEQ ID NO: 8), Flag tags (SEQ ID NO: 26), and His tags (SEQ ID NO: 25, SEQ ID NOs: 29-34). In a more specific embodiment, the detection tag is an OLLAS tag (SEQ ID NO: 8).

[0169] The term "tag" is defined or described above, and this definition also applies to the antibody chain of the present invention.

[0170] In another specific embodiment, the antibody chain of the present invention comprises a processing site between the detection tag and the rest of the chain. In a more specific embodiment, the processing site is a protease recognition site. In a more specific embodiment, the processing site is preferably an enterokinase cleavage sequence (SEQ ID NO: 9) or a TEV protease (SEQ ID NO: 28), and is preferably an enterokinase cleavage sequence.

[0171] The term "protease recognition site" is defined or described above, and this definition also applies to the antibody chain of the present invention.

[0172] In another embodiment, the present invention relates to a polynucleotide encoding an antibody chain of the present invention, hereinafter referred to as "the second polynucleotide of the present invention."

[0173] In certain embodiments, the second polynucleotide of the present invention further comprises a nucleotide sequence encoding a secretion signal peptide, the secretion signal peptide being fused in-frame to the N-terminus of the antibody chain of the present invention.

[0174] In more specific embodiments, the signal peptide is selected from the group consisting of carbonic anhydrase 1 (CAH) signal peptide (SEQ ID NO: 10), ARS signal peptide (SEQ ID NO: 11), or gametricin signal peptide (SEQ ID NO: 12).

[0175] The terms “polynucleotide” and “signal peptide” are defined or described above, and these definitions also apply to the second polynucleotide of the present invention.

[0176] In another embodiment, the present invention relates to a vector comprising a second polynucleotide of the present invention, hereinafter referred to as "the second vector of the present invention."

[0177] The term "vector" is defined or described above, and this definition also applies to the second vector of the present invention.

[0178] In another embodiment, the present invention relates to a host cell comprising a second vector of the present invention.

[0179] In certain embodiments, the host cell is a plant cell or a microalgae cell, preferably a microalgae cell of the Chlamydomonas species.

[0180] The term "host cell" is defined or explained above.

[0181] Method for detecting antigens in a sample In another embodiment, the present invention provides an in vitro method for detecting a target antigen present in a sample (hereinafter referred to as "the second method of the present invention"), (i) Contacting the sample with the modified antibody of the present invention, wherein the modified antibody can specifically bind to the target antigen under conditions sufficient for binding of the target antigen to the modified antibody; and (ii) Determine the presence of a complex containing the target antigen and a modified antibody. Regarding methods that include

[0182] In the present invention, the term "in vitro" means that the determination of the presence of a complex containing the target antigen and a modified antibody is performed outside the body of the subject. The term "subject" is defined or described above, and this definition also applies to the second method of the present invention.

[0183] The term “sample” refers to a small portion or quantity that is considered representative of the whole and is collected or isolated for research, analysis, or experimentation purposes. In this invention, such research, analysis, or experimentation refers to the detection of the presence of a complex containing a target antigen and a modified antibody. The term “sample” also includes samples that have been manipulated in any way after collection, such as by treatment with reagents, solubilization, or concentration of specific components. In preferred embodiments, the sample is a biological sample.

[0184] The term “biological sample” includes, but is not limited to, biological tissues and / or bodily fluids of an individual obtained by any method known to those skilled in the art for that purpose. Examples of such samples include, but are not limited to, blood samples and other liquid samples of biological origin, solid tissue samples such as biopsy samples, or tissue cultures or cells and their offspring derived therefrom, such as cells in cell culture, cell supernatants, cell lysates, serum, plasma, biological fluids and tissue samples.

[0185] The term "detect" refers to reporting or identifying the presence of a complex containing a target antigen and a modified antibody in a sample by generating a signal.

[0186] In the first step, the second method of the present invention comprises contacting a sample with the modified antibody of the present invention, the modified antibody capable of specifically binding to a target antigen. The contact between the sample and the modified antibody of the present invention must be carried out under conditions sufficient for binding between the target antigen and the modified antibody of the present invention.

[0187] Contacting a sample with the modified antibody of the present invention for a sufficient period of time under conditions effective for enabling complex formation generally involves simply adding the antibody composition to the sample and incubating the mixture for a period of time sufficient for the modified antibody to form a complex with the target antigen.

[0188] "Under conditions sufficient for complex formation" means that those conditions preferably include diluting the antigen and / or modified antibody with a solution such as BSA, bovine gamma globulin (BGG), or phosphate-buffered saline (PBS) / Tween. These additives also tend to help reduce nonspecific background.

[0189] "Appropriate" or "sufficient" conditions also mean that the incubation is at a temperature or for a period of time sufficient to allow for effective bonding. The incubation process generally consists of about 1 to 2 to 4 hours at a temperature of preferably around 21°C to 37°C, or it may be overnight at about 4°C.

[0190] A second step of the second method of the present invention comprises determining the presence of a complex comprising a target antigen and a modified antibody. Generally, the detection of these complexes is well known in the art and can be achieved by applying a number of approaches. These methods generally rely on the detection of labels or markers, such as radioactive, fluorescent, biological, and enzymatic tags. Naturally, as is known in the art, further advantages can be found by using a second antibody and / or a secondary binding ligand, such as a biotin / avidin ligand binding sequence.

[0191] Those skilled in the art will understand that there are a variety of conventional assays that can be used in the second method of the present invention, including Western blotting or immunoblotting, ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), competitive EIA (competitive enzyme immunoassay), DAS-ELISA (Double Antibody Sandwich-ELISA), immunocytochemistry and immunohistochemistry techniques, flow cytometry, or multiplex detection techniques based on the use of protein microspheres, biochips, or microarrays containing the modified antibodies of the present invention.

[0192] It will also be understood that unlabeled modified antibodies may need to be detected with additional reagents, such as a labeled secondary antibody, which will then be labeled. This is particularly useful for increasing the sensitivity of the detection method, as it can amplify the signal.

[0193] Purification method for target antigen In another embodiment, the present invention relates to an in vitro method for purifying a target antigen present in a sample, hereinafter referred to as "the third method of the present invention," (i) Contacting the sample with the modified antibody of the present invention, wherein the modified antibody can specifically bind to the target antigen under conditions sufficient for binding of the target antigen to the modified antibody; and (ii) Recover the complex containing the target antigen and the modified antibody. Regarding methods that include

[0194] The terms “in vitro,” “sample,” and “biological sample” are defined or described above, and these definitions also apply to the third method of the present invention.

[0195] The phrase "specifically binds to" refers to a binding reaction that determines the presence of the target antigen in the presence of a heterogeneous population of proteins and other biologics. Therefore, under the specified assay conditions, the modified antibody preferentially binds to a specific antigen and does not bind in significant amounts to other components present in the sample.

[0196] In the first step, the third method of the present invention comprises contacting a sample with the modified antibody of the present invention, the modified antibody capable of specifically binding to a target antigen. The contact between the sample and the modified antibody of the present invention must be carried out under conditions sufficient for binding between the target antigen and the modified antibody of the present invention.

[0197] Contacting a sample with the modified antibody of the present invention for a sufficient period of time under conditions effective for enabling complex formation generally involves simply adding the antibody composition to the sample and incubating the mixture for a period of time sufficient for the modified antibody to form a complex with the target antigen.

[0198] "Conditions sufficient for complex formation" means that those conditions preferably include diluting the antigen and / or modified antibody with a solution such as BSA, bovine gamma globulin (BGG), or phosphate-buffered saline (PBS) / Tween. These additives also tend to help reduce nonspecific background.

[0199] "Appropriate" or "sufficient" conditions also mean that the incubation is at a temperature or for a period of time sufficient to allow for effective bonding. The incubation process generally consists of about 1 to 2 to 4 hours at a temperature of preferably around 21°C to 37°C, or it may be overnight at about 4°C.

[0200] The second step of the third method of the present invention comprises recovering a complex containing a target antigen and a modified antibody. Techniques and conditions for recovering these complexes are widely known in the prior art, and any of them can be used in the present invention. Some examples of techniques sufficient for recovering these complexes include affinity chromatography techniques, ligand binding assays, or lectin binding assays. [Examples]

[0201] The present invention will be described below with reference to examples, but this should not be considered to limit the scope of the present invention.

[0202] Example 1. Design, production, and comparison of HTS of various antibody fragments in microalgae. The heavy chain (VH region and CH1 region) and light chain (VL region and CL region) of the reference antibody fragment (AF) were cloned into a co-expression vector (similar to the vector described in Patent Document WO2019215303A1). The codons of the heavy chain (HC) and light chain (LC) sequences were fitted to the Chlamydomonas nuclear codon AF, and heavy and light chains fused with glycomodule motifs (GM) of various sizes at various positions were tested (Figure 11). (SP) 10 (Sequence ID 6), (SP) 20 Various GMs, including (SEQ ID NO: 7), GP1 (SEQ ID NO: 2), PHC21A (SEQ ID NO: 4), and LCL (SEQ ID NO: 1), were investigated. To ensure that AF is targeted to the periplasmic space or secreted into the culture medium, and therefore easily recovered in the culture medium, secretion signal sequences were cloned to the 5' side of the AF sequence. The included signal sequences were the metalloproteinase gametricin secretion signal (SEQ ID NO: 12) or the carbonic anhydrase 1 secretion sequence (SEQ ID NO: 10). Enterokinase cleavage sequences (SEQ ID NO: 9) were added to some of the expression cassettes.

[0203] Chlamydomonas was transformed by electroporation or glass bead transformation with a co-expression vector (similar to the vector described in Patent Document WO2019215303A1) containing a novel AF (as shown in Figure 11) and DNA sequences for the expression of an additional hygromycin or zeosin-resistant cassette. After selecting transformants by growth on a zeosin-containing TAP plate, transgenic microalgae expressing polynucleotides encoding heavy chains (VH and CH1 regions) and light chains (VL and CL regions) fused to the GM motif and targeted to the periplasm or secreted into the culture medium were grown in 96-well plates. Transgenic microalgae expressing fully assembled antibody fragments were selected by a screening method that may be dot blotting, ELISA, or Western blotting. Algae-independent transformants were grown in flasks under mixed or heterotrophic conditions.

[0204] In this example, expression strains were screened by direct ELISA of the culture medium (Figure 1). The screening results show how the yield of functional AF increases by using GM fused to one of the antibody fragment chains.

[0205] Example 2. Production of fully assembled anti-VEGF (ranibizumab) with and without GM. Regarding anti-VEGF (ranibizumab), those without GM (SEQ ID NO: 13 and SEQ ID NO: 14), and those with SP at the C-terminus of both the heavy and light chains. 20 Those fused to (SEQ ID NOs. 15 and 16) or at the N-terminus of both the heavy and light chains (SP) 10 The fused components (SEQ ID NOs: 17 and 18) were cloned. Some of the cassettes contained detection tags such as the OLLAS tag (SEQ ID NOs: 8) and an enterokinase cleavage sequence (SEQ ID NOs: 9). These vectors, containing DNA cassettes for AF expression and additional cassettes for hygromycin or zeosin resistance expression, were used to transform Chlamydomonas by electroporation or glass bead transformation. After selecting transformants by growth on zeosin-containing TAP plates, transgenic microalgae expressing polynucleotides encoding heavy chains (VH and CH1 regions) and light chains (VL and CL regions) fused to the GM motif and targeted to the periplasm or secreted into the culture medium were grown in 96-well plates. Transgenic microalgae expressing fully assembled antibody fragments were selected by non-reductive immunoblotting of the recovered culture medium. Figure 2 shows a comparison of the expression of various cassettes (ranibizumab fused to GM at various positions) by non-reductive immunoblotting. As can be seen in Figure 2, in the absence of GM fusion, there were no detectable fully assembled Fabs. Furthermore, GM fusion results in a higher percentage of Fab and an increased yield of assembled Fabs compared to free chains.

[0206] The novel ranibizumab fused to the GM is hereafter referred to as GB-AF-010 (SEQ ID NOs: 15 and 16). GB-AF-010 was obtained from the culture medium of transgenic microalgae expressing polynucleotides encoding heavy chains (VH and CH1 regions) and light chains (VL and CL regions) that are fused to the GM motif and targeted to the periplasmic space or secreted into the culture medium. The activity of GB-AF-010 (unpurified, in culture medium) was compared with commercially available (purified) ranibizumab by direct ELISA (Figure 3). The results show that GB-AF-010, tested directly in culture medium without the purification step, has an affinity for VEGF equivalent to that of purified commercially available ranibizumab.

[0207] Example 3. Production of fully assembled anti-TNFα (certolizumab) with and without GM. The heavy chain (VH region and CH1 region) and light chain (VL region and CL region) of certolizumab were cloned into a co-expression vector (similar to the vector described in Patent Document WO2019215303A1) fused to the GM at various positions: (SP) 10 -Certolizumab has (SP) at the N-terminus of both the heavy and light chains (SEQ ID NOs. 19 and 20) 10 There is certolizumab-(SP) 10 This is due to the presence of (SP) at the C-terminus of both the heavy and light chains of certolizumab (SEQ ID NO: 21 and SEQ ID NO: 22). 10 There is certolizumab-HC-(SP) 10 This is the C-terminus of the certolizumab heavy chain (SEQ ID NO: 21) (SP) 10 There are cases where certolizumab is not fused with GM (SEQ ID NO: 23 and SEQ ID NO: 24).

[0208] The heavy chain (HC) and light chain (LC) sequences were codon-matched. Similar to Examples 1-Figure 2, after screening by affinity using ELISA, several positive clones were selected and the expression of fully assembled AF was analyzed. Immunoblotting under non-reducing conditions confirmed enhanced Fab expression in constructs containing GM (Figure 5). Small amounts of GM-free AF ​​were insufficient for direct immunoblotting detection of Fab from culture medium.

[0209] Novel certolizumab fused to the GM (SEQ ID NOs: 21 and 22) will be referred to as GB-AF-011. GB-AF-011 was obtained from the culture medium of transgenic microalgae expressing polynucleotides encoding heavy chains (VH and CH1 regions) and light chains (VL and CL regions) that are fused to the GM motif and targeted to the periplasmic space or secreted into the culture medium. The activity of GB-AF-011 (unpurified) was compared with certolizumab purified from a commercially available source by direct ELISA (Figure 5). The results show that GB-AF-011, although tested directly in the culture medium without purification, maintains the same affinity for TNFα as purified certolizumab. did.

[0210] Furthermore, the specificity of GB-AF-011 was tested by direct ELISA (Figure 6). The results showed that GB-AF-011 retained specificity for human TNFα, did not recognize mouse TNF, and was equivalent to unmodified certolizumab (Figure 6).

[0211] The efficacy of GB-AF-011 in neutralizing TNFα was measured by competitive ELISA, and the ability of several anti-TNF agents to inhibit the binding of labeled TNFα to its receptor was tested (using etanercept). Surprisingly, GB-AF-011 inhibited TNFα binding to its receptor significantly (more than 10 times) more effectively than commercially available purified certolizumab (Figure 7).

[0212] Example 4. Improved stability of GB-AF-011 compared to certolizumab. GB-AF-011 was obtained from the culture medium of transgenic microalgae expressing polynucleotides encoding heavy chains (VH and CH1 regions) and light chains (VL and CL regions) fused to a GM motif and targeted to the periplasm or secreted into the culture medium. GB-AF-011 was exposed to various temperatures and monitored over time, and its stability was compared to commercially available purified certolizumab. Results were analyzed by direct ELISA and immunoblotting under both non-reducing and reducing conditions (Figure 8). After 72 hours or more at room temperature or 37°C, certolizumab-GM was more stable than purified certolizumab. Furthermore, certolizumab showed a band with a higher molecular size than the corresponding Fab, associated with aggregate morphology not observed in GB-AF-011.

[0213] Example 5. Stability of certolizumab-GM in the presence of colonic proteases. GB-AF-011 was obtained from the culture of transgenic microalgae expressing polynucleotides encoding heavy chains (VH and CH1 regions) and light chains (VL and CL regions) that are fused to a GM motif and targeted to the periplasmic space or secreted into the culture medium. GB-AF-011 and commercially available purified anti-TNFα agents (certolizumab, infliximab, and etanercept) were incubated under colonic conditions (1 / 5 dilution in colonic contents, 37°C) and monitored over time. The stability of anti-TNF agents in the face of colonic proteases was compared by immunoblotting under reducing conditions. The results show that GB-AF-011 is significantly more resistant to intestinal proteases than current treatment-associated antibodies.

[0214] Example 6. Method for producing GB-AF-011 by fermentation. Algae expressing fully assembled Fab(GB-AF-011) were grown in a bioreactor under heterotrophic conditions until high cell density was reached. A 1 L bioreactor (PSI Photobioreactor FMT150) was used for culturing Chlamydomonas under fed-batch conditions. The bioreactor was filled with 0.9 L of modified TAP medium. The bioreactor, all connecting tubes, filters, glass bottles, and media were then autoclaved at 121°C for 20 minutes before use. A concentrated feed solution mimicking the basal medium was prepared. This feed solution was filtered and sterilized using a 0.22 mm vacuum cup filter and stored at room temperature. Before each fed-batch bioreactor culture, 200 mL of the feed solution was transferred to a 250 mL Erlenmeyer connected to the bioreactor. An apparatus was used to maintain a constant culture temperature (21-25°C), pH (7.30), agitation, and airflow rate (0.6 L / min). Defoaming agents were added as needed.

[0215] To prepare the Chlamydomonas strain inoculum, the target strain was inoculated into a shaking flask containing 80 mL of TAP medium and grown to the stationary phase for 3 days with mixed nutrients using a 150 rpm orbital shaker. Then, 80 mL of the inoculum was aseptically added to a bioreactor pre-filled with 0.9 L of modified TAP medium. Culture growth was monitored by measuring the OD at 750 nm, and cell density was estimated. Since Fab' is secreted directly into the culture control medium and used regardless of prior purification, the medium was separated from the cells and cryopreserved at each culture point for further analysis of Fab' production. Fed-batch cultures were grown for 8 days without illumination.

[0216] Figure 10 shows the growth profile of the Chlamydomonas strain grown in a 1 L bioreactor. Production analysis of GB-AF-011 was performed by non-reductive immunoblotting (Figure 10).

Claims

1. A modified antibody comprising a first antibody chain comprising a VH region and a CH1 region, and a second antibody chain comprising a VL region and a CL region, wherein at least one of the antibody chains is fused to at least one glycomodule motif (GM).

2. (i) The modified antibody comprises one glycomodule motif located at the C-terminus of the first antibody chain; (ii) The modified antibody consists of one glycomodule motif located at the N-terminus of the first antibody chain; (iii) The modified antibody comprises one glycomodule motif located at the C-terminus of the second antibody chain; or (iv) The modified antibody contains one glycomodule motif, which is located at the N-terminus of the second antibody chain. The fusion protein according to claim 1.

3. The fusion protein according to claim 2, wherein the modified antibody comprises a glycomodule motif in the first antibody chain and the second antibody chain.

4. (i) The glycomodule motif is located at the C-terminus of the first antibody chain and at the C-terminus of the second antibody chain; or (ii) The glycomodule motif is located at the N-terminus of the first antibody chain and at the N-terminus of the second antibody chain. The fusion protein according to claim 3.

5. The Glico module motif is sequence number 1, sequence number 2, sequence number 3, sequence number 4, sequence number 5 and (SP) ｎ , especially (SP) １０ (Sequence ID 6) or (SP) ２０ A modified antibody according to any one of claims 1 to 4, comprising an amino acid sequence selected from the group consisting of (Sequence ID 7).

6. The glycomodule motif comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2 or functionally equivalent variants thereof, or (SP) １０ Or (SP) ２０ The modified antibody according to claim 5, wherein the nucleotide sequence encoding the modified antibody is as described in claim 5.

7. A modified antibody according to any one of claims 1 to 6, wherein a glycomodule motif is linked to a first antibody chain by a linker sequence.

8. A modified antibody according to any one of claims 1 to 6, wherein a glycomodule motif is linked to a second antibody chain by a linker sequence.

9. A modified antibody according to any one of claims 1 to 8, wherein the CL region is the C-terminus of the VL region.

10. A modified antibody according to any one of claims 1 to 9, wherein the CH1 region is the C-terminus of the VH region.

11. A modified antibody according to any one of claims 1 to 10, further comprising a detection tag.

12. The modified antibody according to claim 11, wherein the detection tag is the OLLAS tag (SEQ ID NO: 8).

13. The modified antibody according to claim 11 or 12, comprising a processing site between the detection tag and the rest of the chain, wherein the processing site is preferably an enterokinase cleavage sequence (SEQ ID NO: 9).

14. A modified antibody according to any one of claims 1 to 13, preferably derived from neutralizing antitumor necrosis factor antibody α (TNFα), certolizumab.

15. The modified antibody according to claim 14, having a heavy chain as defined in SEQ ID NO: 19 or SEQ ID NO: 21, and / or a light chain as defined in SEQ ID NO: 20 or SEQ ID NO:

22.

16. A modified antibody according to any one of claims 1 to 13, which is derived from neutralizing anti-vascular endothelial growth factor (VEGF), preferably ranibizumab.

17. The modified antibody according to claim 16, having a heavy chain as defined in SEQ ID NO: 15 or SEQ ID NO: 17, and / or a light chain as defined in SEQ ID NO: 16 or SEQ ID NO:

18.

18. A polynucleotide composition comprising a polynucleotide encoding the antibody chain of a modified antibody according to any one of claims 1 to 17, or a first polynucleotide encoding the first antibody chain of a modified antibody according to any one of claims 1 to 17 and a second polynucleotide encoding the second antibody chain of a modified antibody according to any one of claims 1 to 17.

19. The polynucleotide or polynucleotide composition according to claim 18, wherein each polynucleotide of the polynucleotide or polynucleotide composition further comprises a nucleotide sequence encoding a secretion signal peptide, and the secretion signal peptide is fused in frame to the N-terminus of a first antibody chain and a second antibody chain.

20. The polynucleotide or polynucleotide composition according to claim 19, wherein the signal peptide is selected from the group consisting of carbonic anhydrase 1 (CAH) signal peptide (SEQ ID NO: 10), arylsulfatase 1 signal peptide (SEQ ID NO: 11), or gametricin signal peptide (SEQ ID NO: 12).

21. A vector comprising a polynucleotide according to any one of claims 18 to 20, or a vector composition in which each vector comprises one polynucleotide of the polynucleotide composition according to any one of claims 18 to 20.

22. A host cell comprising the vector or vector composition described in claim 21.

23. The host cell according to claim 22, which is a plant cell or a microalgae cell, preferably a microalgae cell of the Chlamydomonas species.

24. A pharmaceutical composition comprising a modified antibody according to any one of claims 1 to 17, a polynucleotide or polynucleotide composition according to any one of claims 18 to 20, a vector or vector composition according to claim 21, or a host cell according to claim 22 or 23, and at least one pharmaceutically acceptable excipient.

25. A modified antibody according to any one of claims 1 to 17, a polynucleotide or polynucleotide composition according to any one of claims 18 to 20, a vector or vector composition according to claim 21, a host cell according to claim 22 or 23, or a pharmaceutical composition according to claim 24, for use in pharmaceuticals.

26. A modified antibody according to any one of claims 1 to 17, a polynucleotide or polynucleotide composition according to any one of claims 18 to 20, a vector or vector composition according to claim 21, a host cell according to claim 22 or 23, or a pharmaceutical composition according to claim 24, for use in a pharmaceutical in which the antibody is administered locally.

27. A modified antibody according to any one of claims 1 to 17, wherein the modified antibody is against TNFα, for use in the treatment of inflammatory diseases; a polynucleotide or polynucleotide composition according to any one of claims 18 to 20; a vector or vector composition according to claim 21; a host cell according to claim 22 or 23; or a pharmaceutical composition according to claim 24.

28. A modified antibody according to any one of claims 1 to 17, wherein the modified antibody is against the pathogen, for use in the treatment of an infectious disease caused by the pathogen; a polynucleotide or polynucleotide composition according to any one of claims 18 to 20; a vector or vector composition according to claim 21; a host cell according to claim 22 or 23; or a pharmaceutical composition according to claim 24.

29. A modified antibody according to any one of claims 1 to 17, a polynucleotide or polynucleotide composition according to any one of claims 18 to 20, a vector or vector composition according to claim 21, a host cell according to claim 22 or 23, or a pharmaceutical composition according to claim 24, for use in the treatment of gastrointestinal diseases.

30. A modified antibody according to any one of claims 1 to 17, wherein the modified antibody is against VEGF, for use in the treatment of diseases related to unwanted angiogenesis; a polynucleotide or polynucleotide composition according to any one of claims 18 to 20; a vector or vector composition according to claim 21; a host cell according to claim 22 or 23; or a pharmaceutical composition according to claim 24.

31. A modified antibody according to any one of claims 1 to 17, wherein the modified antibody is against VEGF, for use in the treatment of intravascular age-related macular degeneration, macular edema after retinal vein occlusion, diabetic macular edema, diabetic retinopathy, or myopic choroidal neovascularization; a polynucleotide or polynucleotide composition according to any one of claims 18 to 20; a vector or vector composition according to claim 21; a host cell according to claim 22 or 23; or a pharmaceutical composition according to claim 24.

32. A modified antibody according to any one of claims 25 to 31, which is administered orally or applied topically.

33. A method for producing a modified antibody according to any one of claims 1 to 12, (i) Proliferating cells comprising a polynucleotide or polynucleotide composition according to any one of claims 18 to 20 under conditions suitable for enabling the expression of modified antibodies from the polynucleotide or from the polynucleotide of the polynucleotide composition, and (ii) Recovering modified antibodies from the culture. A method that includes the following:

34. The method according to claim 33, wherein the modified antibody is recovered from the culture supernatant when the polynucleotide or polynucleotide composition comprises a nucleotide sequence encoding a secretory signal peptide.

35. The method according to claim 33 or 34, wherein the cells are plant cells or microalgae cells, preferably transgenic microalgae of the Chlamydomonas species.

36. (i) VH region and CH1 region; or (ii) VL region and CL region An antibody chain comprising a glycomodule motif (GM) and fused to it.

37. (i) The glycomodule motif is located at the C-terminus of the antibody chain; or (ii) The glycomodule motif is located at the N-terminus of the antibody chain. The antibody chain according to claim 36.

38. The glycomodule motif comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or functionally equivalent variants thereof, or (SP) ｎ , especially (SP) １０ (Sequence ID 6) or (SP) ２０ The antibody chain according to claim 36 or 37, which is a nucleotide sequence encoding (SEQ ID NO: 7).

39. The antibody chain according to claim 38, comprising an amino acid sequence selected from the group consisting of a glycomodule motif and having an amino acid sequence of SEQ ID NO: 2 or a functionally equivalent variant thereof, or (SP) １０ or (SP) ２０ and being a nucleotide sequence encoding the same.

40. The antibody chain according to any one of claims 36 to 39, wherein a glycomodule motif is linked to the antibody chain by a linker.

41. (i) If the antibody comprises a VH region and a CH1 region, the CH1 region is at the C-terminus of the VH region; or (ii) If the antibody chain comprises a VL region and a CL region, the CL region is located at the C-terminus of the VL region. The antibody chain according to any one of claims 36 to 40.

42. The antibody chain according to any one of claims 36 to 41, further comprising a detection tag.

43. The antibody chain according to claim 42, wherein the detection tag is an OLLAS tag (SEQ ID NO: 8).

44. The antibody chain according to claim 42 or 43, comprising a processing site between the detection tag and the rest of the chain, wherein the processing site is preferably an enterokinase cleavage sequence (SEQ ID NO: 9).

45. A polynucleotide encoding an antibody chain fused to a glycomodule motif according to any one of claims 36 to 44.

46. The polynucleotide according to claim 45, wherein the polynucleotide further comprises a nucleotide sequence encoding a secretion signal peptide, and the secretion signal peptide is fused in-frame to the N-terminus of the antibody chain.

47. The polynucleotide according to claim 46, wherein the signal peptide is selected from the group consisting of carbonic anhydrase 1 (CAH) signal peptide (SEQ ID NO: 10), ARS signal peptide (SEQ ID NO: 11), or gametricin signal peptide (SEQ ID NO: 12).

48. A vector comprising the polynucleotide described in any one of claims 45 to 47.

49. A host cell comprising the vector described in claim 48.

50. The host cell according to claim 49, which is a plant cell or a microalgae cell, preferably a microalgae cell of the Chlamydomonas species.

51. An in vitro method for detecting a target antigen present in a sample, (i) Contacting the sample with the modified antibody according to any one of claims 1 to 17, wherein the modified antibody can specifically bind to the target antigen under conditions sufficient for binding of the target antigen to the modified antibody; and (ii) Determining the presence of a complex containing the target antigen and a modified antibody. A method that includes the following:

52. An in vitro method for purifying a target antigen present in a sample, (i) Contacting the sample with the modified antibody according to any one of claims 1 to 17, wherein the modified antibody can specifically bind to the target antigen under conditions sufficient for binding of the target antigen to the modified antibody; and (ii) Recover the complex containing the target antigen and the modified antibody. A method that includes the following:

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