Recombinant spirulina expressing a scaffold and its use.

Abstract

This disclosure provides a scaffold containing heterologous moiety and spirulina expressing it. It also provides recombinant spirulina expressing the scaffold and methods for utilizing it to treat diseases and conditions. This specification provides recombinant spirulina expressing a mutant small membrane-bound A kinase anchoring protein (smAKAP) peptide sequence. In some embodiments, the spirulina contains at least 2, 3, 4, 5, 6, or 7 mutant residues in the mutant smAKAP peptide sequence compared to the wild-type smAKAP peptide sequence.

Description

Technical Field

[0001] Cross - References to Related Applications This application claims priority to U.S. Patent Application No. 63 / 511,050, filed on June 29, 2023, the content of which is hereby incorporated by reference in its entirety for all purposes.

[0002] Reference to Electronic Sequence Listing The content of the electronic sequence listing (LUBI_038_01WO_SeqList_ST26.xml, size: 82,287 bytes, and creation date: June 28, 2024) is hereby incorporated by reference in its entirety into this specification.

Background Art

[0003] Antigen - binding domains such as VHH derived from single - chain antibodies of camelids can be expressed in prokaryotes such as Spirulina. However, various limitations still exist, such as the requirements for affinity maturation to achieve improved target binding.

[0004] The present disclosure provides a solution to the limitations of the current state - of - the - art through improved compositions including scaffolds.

[0005] The present disclosure relates to scaffolds and methods of using them. The present disclosure also relates to constructs comprising scaffolds that express binding agents.

Summary of the Invention

[0006] This specification provides recombinant spirulina expressing a mutant small membrane-bound A kinase anchoring protein (smAKAP) peptide sequence. In one embodiment, the spirulina contains at least 2, 3, 4, 5, 6, or 7 mutant residues in the mutant smAKAP peptide sequence compared to the wild-type smAKAP peptide sequence. In another embodiment, one residue in the smAKAP peptide sequence is mutant compared to the wild-type smAKAP peptide sequence. In yet another embodiment, two residues in the smAKAP peptide sequence are mutant compared to the wild-type smAKAP peptide sequence. In yet another embodiment, three residues in the smAKAP peptide sequence are mutant compared to the wild-type smAKAP peptide sequence. In yet another embodiment, the mutant smAKAP peptide exhibits resistance to protease cleavage, determined by a decrease in the detection of cleavage products when exposed to a protease-containing solvent for one hour. In one embodiment, up to approximately 5%, 10%, 20%, 30%, 40%, 50%, or 60% of the smAKAP peptide sequence is cleaved. In another embodiment, the mutation is of a hydrophobic residue. In another embodiment, if recombinant spirulina is immersed in a solvent, the mutation residue is exposed to the solvent. In another embodiment, the mutation residue is selected from the group consisting of C16S, C24S, E5D, Y6H, W22S, C24G, L4E, R9E, L4I, L10I, L19I, and combinations thereof, based on SEQ ID NO: 2. In another embodiment, the mutation residues are C16S and C24S compared to SEQ ID NO: 2. In another embodiment, the mutation residues are E5D, Y6H, C16S, W22S, and C24G compared to SEQ ID NO: 2. In one embodiment, the mutant residues are L4E, E5D, Y6H, R9E, C16S, W22S, and C24G compared to SEQ ID NO: 2. In another embodiment, the mutant residues are L4E, C16S, W22S, and C24G compared to SEQ ID NO: 2. In another embodiment, the mutant residues are R9E, C16S, and C24G compared to SEQ ID NO: 2. In another embodiment, the mutant residues are R9E, C16S, W22S, and C24G compared to SEQ ID NO: 2. In another embodiment, the mutant smAKAP peptide sequence is linked to a heterologous moiety in monomeric form. In another embodiment, the mutant smAKAP peptide sequence is linked to a second heterologous moiety. In another embodiment, the heterologous moiety and the second heterologous moiety are identical.In one embodiment, the heterologous moiety and the second heterologous moiety are different. In one embodiment, recombinant spirulina expresses a different exogenous polypeptide sequence. In one embodiment, the exogenous polypeptide sequence is selected from the group consisting of the oligomerized domain of C4b-binding protein (C4BP), the cholera toxin b subunit, the oligomerized domain of an extracellular matrix protein, TRX, 5HVZ, cTRP, SP651, SP737, and 4B0F. In one embodiment, the exogenous polypeptide sequence is 5HVZ. In one embodiment, 5HVZ is linked to the third heterologous moiety. In one embodiment, the third heterologous moiety has a homodimer configuration. In one embodiment, the heterologous moiety, the second heterologous moiety, and the third heterologous moiety are identical. In one embodiment, the heteromoiety, the second heteromoiety, and the third heteromoiety are different. In one embodiment, the heteromoiety is a binder. In one embodiment, the binder is selected from the group consisting of fab', F(ab')2, fv, domain antibody (dAb), complementarity-determining region (CDR) fragment, CDR graft antibody, single-chain antibody (scFv), single-chain antibody fragment, chimeric antibody, diabody, triabody, tetrabody, minibody, linear antibody, intrabody, nanobody (single-domain antibody), small modular immunotherapy (SMIP), antigen-binding domain immunoglobulin fusion protein, and VHH. In one embodiment, the binder is VHH. In one embodiment, VHH binds to pathogens. In one embodiment, VHH binds to cancer cells. In one embodiment, VHH binds to human cells. In one embodiment, VHH binds to pathogens selected from the group consisting of bacteria, fungi, and viruses.In this context, the pathogens include Escherichia coli (E. coli), enterotoxigenic Escherichia coli (ETEC), anthrax, EHEC, EAEC, Shigella, Mycobacterium, Streptococcus, Staphylococcus, Shigella, Campylobacter, Salmonella, Clostridium, Corynebacterium, Pseudomonas, Neisseria, Listeria, Vibrio, Bordetella, Legionella, bacteriophages, RNA bacteriophages (e.g., MS2, AP205, PP7, and Qβ), Helicobacter pylori, infectious hematopoietic necrosis virus, parvovirus, herpes simplex virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, measles virus, mumps virus, rubella virus, HIV, influenza virus, rhinovirus, and Rotavirus A, Rotavirus B, Rotavirus C, Respiratory Syncytial Virus (RSV), Varicella-zoster virus, Poliovirus, Norovirus, Zika virus, Dengue virus, Rabies virus, Newcastle disease virus, Vitiligo virus, Coronavirus, SARS-related coronavirus, MERS-related coronavirus, SARS-CoV-2, Aspergillus, Candida, Blastomyces, Coccidioides, Cryptococcus, Histoplasma, Plasmodium, Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, Plasmodium vivax, Trypanosoma, Toxoplasma, Giardia, Leishmania, Cryptosporidium, Hellworm parasite: Trichuris The group is selected from the following genus: spp. (whipworms), Enterobius spp. (pinworms), Ascaris spp. (roundworms), Ancylostoma spp. (hookworms), Necatro spp. (American hookworms), Strongyloides spp. (strongyloides), Dracunculus spp. (dracunculus), Onchocerca spp. (onchocerca), Wuchereria spp. (filamentous worms), Taenia spp. (taeniae), Echinococcus spp. (hydatids), Diphyllobothrium spp. (diphyllobothrium), Fasciola spp. (fasciola), and Schistosoma spp. (blood flukes).In one embodiment, the pathogen is a bacterium, and the bacterium is selected from the group consisting of Mycobacterium, Streptococcus, Staphylococcus, Shigella, Campylobacter, Salmonella, Clostridium, Corynebacterium, Pseudomonas, Neisseria, Listeria, Vibrio, Bordetella, and Legionella. In one embodiment, the bacterium is Campylobacter. In one embodiment, the bacterium is Clostridium. In one embodiment, VHH contains a sequence having at least 85% identity to the sequences of SEQ ID NOs. 25-67. In one embodiment, VHH contains the sequences of SEQ ID NOs. 25-67. In one embodiment, recombinant spirulina expresses a scaffold having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequences of SEQ ID NOs. 6, 14, 16, 18, 20, 22, 23, and 24.

[0007] Furthermore, recombinant spirulina expressing a mutant small membrane-bound A kinase anchoring protein (smAKAP) peptide sequence is also provided, the mutations including residue substitutions based on Sequence ID No. 2.

[0008] Recombinant spirulina expressing a mutant small membrane-bound A kinase anchoring protein (smAKAP) peptide sequence is also provided, the mutant being selected from the group consisting of C16S, C24S, E5D, Y6H, W22S, C24G, L4E, R9E, L4I, L10I, L19I, and combinations thereof, based on SEQ ID NO: 2. In one embodiment, smAKAP is ligated to one or more VHH antibodies at its terminus. In another embodiment, smAKAP is ligated to two VHH antibodies, the first VHH at the C-terminus and the second VHH at the N-terminus. In another embodiment, the recombinant spirulina expresses 5HVZ. In another embodiment, 5HVZ is ligated to two VHH antibodies in a homodimer configuration.

[0009] A polynucleotide sequence containing a mutant small membrane-bound A kinase anchoring protein (smAKAP) sequence is also provided, in which the mutation is a single residue substitution compared to the wild-type smAKAP sequence.

[0010] A vector containing a polynucleotide sequence is also provided.

[0011] A method for producing recombinant spirulina is also provided, which includes contacting spirulina cells with the vector of this disclosure.

[0012] Pharmaceutical compositions comprising recombinant spirulina and excipients are also provided.

[0013] Kits are also provided that include recombinant spirulina, polynucleotide sequences, vectors, or pharmaceutical compositions, and instructions for their use.

[0014] A therapeutic method is also provided, which includes administering the pharmaceutical composition to a subject in need, the subject including a bacterial or viral infection.

[0015] Vectors containing nucleotide sequences encoding polypeptides of sequence numbers 5-7 or 14-24 are also provided.

[0016] Recombinant spirulina containing the vectors of this disclosure are also provided.

[0017] Recombinant spirulina expressing a mutant small membrane-bound A kinase anchoring protein (smAKAP) peptide sequence is provided. In one embodiment, the spirulina contains at least 2, 3, 4, 5, 6, or 7 mutant residues in the mutant smAKAP peptide sequence compared to the wild-type smAKAP peptide sequence. In another embodiment, one residue in the smAKAP peptide sequence is mutant compared to the wild-type smAKAP peptide sequence. In yet another embodiment, two residues in the smAKAP peptide sequence are mutant compared to the wild-type smAKAP peptide sequence. In yet another embodiment, three residues in the smAKAP peptide sequence are mutant compared to the wild-type smAKAP peptide sequence. In yet another embodiment, the mutant smAKAP peptide exhibits resistance to protease cleavage, determined by a decrease in the detection of cleavage products when exposed to a protease-containing solvent for one hour. In one embodiment, up to approximately 5%, 10%, 20%, 30%, 40%, 50%, or 60% of the smAKAP peptide sequence is cleaved. In another embodiment, the mutation is of a hydrophobic residue. In another embodiment, if recombinant spirulina is immersed in a solvent, the mutation residue is exposed to the solvent. In another embodiment, the mutation residue is selected from the group consisting of C16S, C24S, E5D, Y6H, W22S, C24G, L4E, R9E, L4I, L10I, L19I, and combinations thereof, based on SEQ ID NO: 2. In another embodiment, the mutation residues are C16S and C24S compared to SEQ ID NO: 2. In another embodiment, the mutation residues are E5D, Y6H, C16S, W22S, and C24G compared to SEQ ID NO: 2. In one embodiment, the mutant residues are L4E, E5D, Y6H, R9E, C16S, W22S, and C24G compared to SEQ ID NO: 2. In another embodiment, the mutant residues are L4E, C16S, W22S, and C24G compared to SEQ ID NO: 2. In another embodiment, the mutant residues are R9E, C16S, and C24G compared to SEQ ID NO: 2. In another embodiment, the mutant residues are R9E, C16S, W22S, and C24G compared to SEQ ID NO: 2. In another embodiment, the mutant residues are L4I, R9E, C16S, and C24G compared to SEQ ID NO: 2. In another embodiment, the mutant smAKAP peptide sequence is linked to a heterologous moiety in monomeric form. In another embodiment, the mutant smAKAP peptide sequence is linked to a second heterologous moiety.In one embodiment, the heterologous moiety and the second heterologous moiety are identical. In another embodiment, the heterologous moiety and the second heterologous moiety are different. In another embodiment, recombinant spirulina expresses a different exogenous polypeptide sequence. In another embodiment, the exogenous polypeptide sequence is selected from the group consisting of the oligomerized domain of C4b-binding protein (C4BP), the cholera toxin b subunit, the oligomerized domain of an extracellular matrix protein, TRX, 5HVZ, cTRP, SP651, SP737, and 4B0F. In another embodiment, the exogenous polypeptide sequence is 5HVZ. In another embodiment, 5HVZ is linked to the third heterologous moiety. In another embodiment, the third heterologous moiety is dimeric. In another embodiment, the heterologous moiety, the second heterologous moiety, and the third heterologous moiety are identical. In one embodiment, the heteromoiety, the second heteromoiety, and the third heteromoiety are different. In one embodiment, the heteromoiety is a binder. In one embodiment, the binder is selected from the group consisting of fab', F(ab')2, fv, domain antibody (dAb), complementarity-determining region (CDR) fragment, CDR graft antibody, single-chain antibody (scFv), single-chain antibody fragment, chimeric antibody, diabody, triabody, tetrabody, minibody, linear antibody, intrabody, nanobody (single-domain antibody), small modular immunotherapy (SMIP), antigen-binding domain immunoglobulin fusion protein, and VHH. In one embodiment, the binder is VHH. In one embodiment, VHH binds to pathogens. In one embodiment, VHH binds to cancer cells. In one embodiment, VHH binds to human cells. In one embodiment, VHH binds to pathogens selected from the group consisting of bacteria, fungi, and viruses.In this context, the pathogens include Escherichia coli (E. coli), enterotoxigenic Escherichia coli (ETEC), anthrax, EHEC, EAEC, Shigella, Mycobacterium, Streptococcus, Staphylococcus, Shigella, Campylobacter, Salmonella, Clostridium, Corynebacterium, Pseudomonas, Neisseria, Listeria, Vibrio, Bordetella, Legionella, bacteriophages, RNA bacteriophages (e.g., MS2), AP205, PP7, and Qβ), Helicobacter pylori, infectious hematopoietic necrotic virus, parvovirus, herpes simplex virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, measles virus, mumps virus, rubella virus, HIV, influenza virus, rhinovirus, rotavirus A, rotavirus B, rotavirus C, respiratory syncytial virus (RSV), varicella-zoster virus Viruses, poliovirus, norovirus, Zika virus, dengue virus, rabies virus, Newcastle disease virus, vitiligo virus, coronavirus, SARS-related coronavirus, MERS-related coronavirus, SARS-CoV-2, Aspergillus, Candida, Blastomyces, Coccidioides, Cryptococcus, Histoplasma, Plasmodium, Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, Plasmodium vivax, Trypanosoma, Toxoplasma, Giardia, Leishmania, Cryptosporidium, Helminth parasites: selected from the group consisting of the genera Whipworm, Pinworm, Ascaris, Hookworm, and American Hookworm, Strongyloides, Dracunculus, Onchocerca, Filamentosa, Tapeworm, Hydatidiformis, Diphyllobothrium, Fasciola, and Schistosoma. In one embodiment, the pathogen is a bacterium, and the bacterium is selected from the group consisting of Mycobacterium, Streptococcus, Staphylococcus, Shigella, Campylobacter, Salmonella, Clostridium, Corynebacterium, Pseudomonas, Neisseria, Listeria, Vibrio, Bordetella, and Legionella. In one embodiment, the bacterium is Campylobacter. In one embodiment, the bacterium is Clostridium. In one embodiment, VHH contains a sequence having at least 85% identity with the sequences of SEQ ID NOs. 25-67. In one embodiment, VHH contains the sequences of SEQ ID NOs. 25-67.In one embodiment, recombinant spirulina expresses a scaffold having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequences of sequence numbers 4, 6, 14, 16, 18, 20, 22, 23, 24, and 76.

[0018] Recombinant spirulina expressing a mutant small membrane-bound A kinase anchoring protein (smAKAP) peptide sequence is provided, the mutation involving residue substitutions based on SEQ ID NO: 2.

[0019] Recombinant spirulina expressing a mutant small membrane-bound A kinase anchoring protein (smAKAP) peptide sequence is provided, the mutant being selected from the group consisting of C16S, C24S, E5D, Y6H, W22S, C24G, L4E, R9E, L4I, L10I, L19I, and combinations thereof, based on SEQ ID NO: 2. In one embodiment, smAKAP is ligated to one or more VHH antibodies at its terminus. In another embodiment, smAKAP is ligated to two VHH antibodies, the first VHH at the C-terminus and the second VHH at the N-terminus. In another embodiment, the recombinant spirulina expresses 5HVZ. In another embodiment, 5HVZ is ligated to two VHH antibodies in a homodimer configuration.

[0020] A polynucleotide sequence containing a mutant small membrane-bound A kinase anchoring protein (smAKAP) sequence is provided, the mutation involving one or more residue substitutions compared to the wild-type smAKAP sequence.

[0021] A vector containing the disclosed polynucleotide sequence is provided.

[0022] A method for producing recombinant spirulina is also provided, which includes contacting spirulina cells with the vector of this disclosure.

[0023] A pharmaceutical composition comprising recombinant spirulina and one or more excipients is provided.

[0024] A kit is provided that includes a recombinant Spirulina, a polynucleotide sequence, a vector, or a pharmaceutical composition, and instructions for its use.

[0025] A therapeutic method is provided that includes administering the pharmaceutical composition of the present disclosure to a subject that needs it, where the subject includes a bacterial infection or a viral infection.

[0026] Vectors are also provided that include nucleotide sequences encoding the polypeptides of SEQ ID NOs: 2-7, 14-24, 74, and / or 76. In an embodiment, the vector includes about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 75.

[0027] Recombinant Spirulina is also provided that includes the vector of the present disclosure.

Brief Description of the Drawings

[0028] The accompanying drawings are incorporated herein and form a part thereof, showing some exemplary embodiments and / or features, but are not limited thereto. The embodiments and drawings disclosed herein are intended to be exemplary rather than limiting.

[0029] [Figure 1-1] FIG. 1A FIGS. 1A-1D show protease susceptibility data for constructs: PP917 2xS3b-C8 (FIG. 1A), PP1895 2xS3b-C8 (FIG. 1B), PP1895 2xRN-29 (FIG. 1C), and PP2130 2xRN-29 + 2xS3b-C8 (FIG. 1D). [Figure 1-2] FIG. 1B FIGS. 1A-1D show protease susceptibility data for constructs: PP917 2xS3b-C8 (FIG. 1A), PP1895 2xS3b-C8 (FIG. 1B), PP1895 2xRN-29 (FIG. 1C), and PP2130 2xRN-29 + 2xS3b-C8 (FIG. 1D). [Figure 1-3]Figures 1A to 1D show protease sensitivity data for the constructs: PP917 2xS3b-C8 (Figure 1A), PP1895 2xS3b-C8 (Figure 1B), PP1895 2xRN-29 (Figure 1C), and PP2130 2xRN-29 + 2xS3b-C8 (Figure 1D). [Figure 1-4] Figures 1A to 1D show protease sensitivity data for the constructs: PP917 2xS3b-C8 (Figure 1A), PP1895 2xS3b-C8 (Figure 1B), PP1895 2xRN-29 (Figure 1C), and PP2130 2xRN-29 + 2xS3b-C8 (Figure 1D).

[0030] [Figure 2] Figure 2 shows the hydrophobic residues on smAKAP that are involved in complex formation with 5HVZ.

[0031] [Figure 3-1] Figure 3A is a diagram of the smAKAP peptide, showing the arginine residues that, when exposed to a solvent, can function as potential cleavage sites for trypsin (see SEQ ID NO: 1). The PP1895 amino acid sequence of SEQ ID NO: 1 is also shown. [Figure 3-2] Figure 3B shows the normalized frequencies for various cleavages of the smAKAP peptide. [Figure 3-3] Figure 3C shows additional Lue and His residues on the smAKAP peptide, which are potential trypsin cleavage sites.

[0032] [Figure 4] Figure 4 shows an example of smAKAP with mutations that confer trypsin and chymotrypsin resistance.

[0033] [Figure 5] Figure 5 shows a Western blot of the mutant smAKAP clone expressed in E. coli.

[0034] [Figure 6-1]Figures 6A-6C show trypsin digestion of clones containing the R63E substitution: PP2451 (Figure 6A), PP1895 (Figure 6B), and PP2451 (Figure 6C). The R63 substitution confers trypsin resistance in the smAKAP peptide. [Figure 6-2] Figures 6A-6C show trypsin digestion of clones containing the R63E substitution: PP2451 (Figure 6A), PP1895 (Figure 6B), and PP2451 (Figure 6C). The R63 substitution confers trypsin resistance in the smAKAP peptide. [Figure 6-3] Figures 6A–6C show trypsin digestion of clones containing the R63E substitution: PP2451 (Figure 6A), PP1895 (Figure 6B), and PP2451 (Figure 6C). The R63 substitution confers trypsin resistance in the smAKAP peptide.

[0035] [Figure 7] Figure 7 shows the size-based analysis of complex formation between smAKAP and 5HVZ dimers.

[0036] [Figure 8] Figure 8 shows the results of complex formation analysis using mutant constructs PP2451 and PP2456 exposed to 0.1 mg / mL and 0.01 mg / mL of trypsin digestion and chymotrypsin digestion.

[0037] [Figure 9] Figure 9 shows the results of chymotrypsin resistance assays using clones PP2451, PP1895, PP2455, PP2456, PP2454, PP2452, and PP2453.

[0038] [Figure 10] Figure 10 shows the results of complex formation assays using clones PP2451, PP1895, PP2456, PP2454, PP2452, and PP2453.

[0039] [Figure 11]Figure 11 shows the SDS-PAGE gels obtained from comparative analysis of smAKAP mutant scaffolds.

[0040] [Figure 12] Figure 12 shows an SDS-PAGE gel demonstrating that the construct containing the smAKAP linker is well expressed in spirulina.

[0041] [Figure 13] Figure 13 shows the SDS-PAGE gels of PP1895 at 0, 4, and 7 days after purification. The purified protein shows increased degradation and instability after storage at 4C. This is in contrast to the results in Figure 11, which show higher stability.

[0042] [Figure 14] Figure 14 shows the results of complex formation analysis using the mutant construct PP6510. [Modes for carrying out the invention]

[0043] A composition comprising recombinant spirulina is provided. Also provided are a scaffold containing a binding domain expressed by recombinant spirulina, and a method for improving it for the production of orally delivered therapeutic proteins. For example, homomerous and heteromerized scaffolds containing a VHH antibody linked via a smAKAP linker can be used to increase binding affinity compared to the VHH antibody alone.

[0044] Also provided are methods comprising administering recombinant spirulina to a subject in need to prevent, treat, or alleviate an infection, disease, or condition.

[0045] definition The following terms are expected to be easily understood by those skilled in the art, but their definitions are provided below to facilitate the explanation of the subject matter of this disclosure.

[0046] All technical and scientific terms used herein are intended to have the same meaning as those commonly understood by those skilled in the art, unless otherwise defined below. References to technologies adopted herein are intended to refer to technologies commonly understood in the art, including variations and / or substitutions of those technologies that are obvious to those skilled in the art.

[0047] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise.

[0048] The term “approximately” or “about” preceding a number means a range (e.g., plus or minus 10% of that value). For example, unless the context of this disclosure indicates otherwise, or unless such interpretation is inconsistent, “approximately 50” may mean 45 to 55, and “approximately 25,000” may mean 22,500 to 27,500. For example, in a list of numbers such as “approximately 49, approximately 50, approximately 55,…”, “approximately 50” means a range extending to less than half the interval between the preceding and succeeding values, e.g., greater than 49.5 and less than 52.5. Furthermore, the phrases “values ​​less than approximately” or “values ​​greater than approximately” should be understood in consideration of the definition of the term “approximately” provided herein. Similarly, the term “approximately” preceding a set of numbers or a range of values ​​(e.g., “approximately 10, 20, 30,” or “approximately 10 to 30”) refers to all the values ​​in the set, or the two extreme values ​​of the range, respectively.

[0049] As used herein, the term “subject” refers to any subject for which diagnosis, prognosis, or therapy is desired, e.g., a human or non-human mammal. The term “subject” may mean a human or non-human mammal that is affected, likely to be affected, or suspected to be affected by a disease. The terms “subject” and “patient” are used interchangeably herein. In aspects, the subject is a mammal. Mammals include primates such as humans, monkeys, chimpanzees, and apes, as well as non-primates such as domesticated animals including laboratory animals (rabbits and rodents, e.g., guinea pigs, rats, mice), household pets and farm animals (e.g., cats, dogs, pigs, cows, sheep, goats, horses, rabbits), and non-domesticated animals such as wild animals, birds, reptiles, and fish.

[0050] As used herein, the term “subjects in need of treatment” includes subjects who can or would benefit from the methods described herein. Subjects in need of treatment include, but are not limited to, subjects who already have a condition or disorder, subjects who are prone to developing a condition or disorder, subjects suspected of having a condition or disorder, and subjects whose condition or disorder is to be prevented, mitigated, or reversed.

[0051] When referring to nucleic acid sequences or protein sequences, the term "identity" is used to indicate the similarity between two sequences. Unless otherwise stated, the percentage identity described herein is determined using the BLAST algorithm, available on the website blast.ncbi.nlm.nih.gov / Blast.cgi, with default parameters.

[0052] Antibody fragments include the antigen-binding portion of an antibody (i.e., "antigen-binding fragment"), and in particular include fab', F(ab')2, fv, domain antibodies (dAb), complementarity-determining region (CDR) fragments, CDR graft antibodies, single-chain antibodies (scFv), single-chain antibody fragments, chimeric antibodies, diabodies, triabodies, tetrabodies, minibodies, linear antibodies, chelated recombinant antibodies, triple-chain antibodies (tribodies) or diabodies (bibodies), intrabodies (intrabodies), nanobodies (nanobodies), small modular immunopharmaceuticals (SMIPs), antigen-binding domain immunoglobulin fusion proteins, single-domain antibodies (including camelized antibodies), antibodies containing VHH, or variants or derivatives thereof, as well as polypeptides containing a portion of immunoglobulin sufficient to bind to a specific antigen (e.g., one, two, three, four, five, or six CDR sequences), insofar as the fragment retains the desired biological activity.

[0053] scaffold This specification provides scaffolds. In some embodiments, the scaffolds may be monomers, dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, nocumers, or decapers. For expression in spirulina, the scaffolds are used to multimerize heterologous moieties of this disclosure. For example, the scaffolds can be used to multimerize VHH for expression in spirulina. Multimerization can achieve increased target binding affinity compared to the affinity of a single monomeric VHH or compared to a scaffold with reduced multimerization (e.g., trimer scaffold vs. dimer scaffold).

[0054] In some embodiments, the scaffold is monomeric. A monomeric scaffold may contain heterologous moieties bound to or ligated to the scaffold. In some embodiments, the scaffold contains maltose-binding protein (MBP). In some embodiments, the scaffold contains thioredoxin (TRX).

[0055] In some embodiments, the scaffold is a dimer. The dimeric scaffold may contain 5HVZ.

[0056] In some embodiments, the scaffold is a trimer. The trimer scaffold may contain cTRP. The trimer scaffold may contain SP651.

[0057] In some embodiments, the scaffold is a pentamer. The pentamer scaffold may contain SP737.

[0058] In some embodiments, the scaffold is a heptamer. The heptamer scaffold may contain 4B0F.

[0059] In one embodiment, the scaffold is selected from the group consisting of the oligomerized domain of C4b-binding protein (C4BP), the cholera toxin b subunit, the oligomerized domain of an extracellular matrix protein, TRX, 5HVZ, cTRP, SP651, SP737, 4B0F, smAKAP, and combinations thereof.

[0060] In some embodiments, any of the scaffolds of this disclosure can be mutated to improve performance. In some embodiments, mutations are introduced to reduce protease sensitivity (e.g., trypsin or chymotrypsin). smAKAP In some embodiments, the scaffold comprises the smAKAP peptide. smAKAP is a small (i.e., 11 kDa) PKA-RI specific protein kinase A anchoring protein, referred herein as small membrane-bound AKAP (smAKAP). smAKAP is tethered to the cell membrane via double acylation (myristoylation and palmitoylation, respectively) of its N-terminal Met-Gly-Cys motif. In some embodiments, smAKAP is expressed in spirulina as part of the scaffold of the present disclosure. smAKAP can function as a linker in the scaffold of the present disclosure.

[0061] The provided scaffold may further contain a 5HVZ domain. In one embodiment, 5HVZ may spontaneously form dimers within spirulina cells. In another embodiment, a homodimeric protein containing two protein molecules (5HVZ-VHH and 5HVZ-VHH) is provided. In another embodiment, the scaffold contains a maltose-binding protein (MBP) at one end and VHH at the other end. In this configuration, the homodimer has two VHHs associated with each other. Dimeric VHHs often enable tighter binding to their targets in a phenomenon known as avidity. This strategy may result in four associated VHH copies when VHHs are at both ends of the 5HVZ scaffold.

[0062] Furthermore, smAKAP is useful because it spontaneously forms trimer complexes with 5HVZ homodimers. This makes it possible to further enhance the multimerity of the scaffold complex. For example, depending on the specific design, these trimers may have 3, 4, or 6 VHHs per trimer complex. In addition, smAKAP can also have VHHs different from those fused to 5HVZ, thereby enabling the combinatorial association of different complexes containing unique VHH pairs.

[0063] In scaffolds containing smAKAP and 5HVZ (e.g., cerberbodies and / or hydrabodies), hydrophobic residues on smAKAP are involved in complex formation with 5HVZ (see Figure 2). In some embodiments, the smAKAP peptide contains residues that may be sensitive to protease cleavage (see Figure 3B and Keil, B. Specificity of proteolysis. Springer-Verlag Berlin-Heidelberg-NewYork, pp. 335. (1992), which are incorporated herein by reference in their entirety). Thus, compositions containing mutant smAKAP peptides exhibiting resistance to proteases or reduced protease cleavage are also provided.

[0064] Any methodology may be used to generate mutant smAKAP peptides for conferring trypsin and chymotrypsin resistance. In one embodiment, the trypsin-sensitive site is removed. In another embodiment, residues may be substituted so that the active site is more basic or more acidic compared to the wild type. Examples of residues that can be mutated are shown in Figure 4, but any other residues can also be mutated. In one embodiment, one or more residues are mutated. In another embodiment, peptides with two or more mutations show increased resistance to proteases compared to peptides with a single residue mutation. In another embodiment, combining different hydrophobic mutations improves resistance to proteases.

[0065] In one embodiment, the smAKAP peptide contains a mutation selected from the group consisting of C16S, C24S, E5D, Y6H, W22S, C24G, L4E, R9E, L4I, L10I, L19I, and combinations thereof, based on SEQ ID NO: 2. In another embodiment, the mutant smAKAP contains the C16S and C24S mutations compared to SEQ ID NO: 2. In another embodiment, the mutant smAKAP contains the C16S, E5D, Y6H, C16S, W22S, and C24G mutations compared to SEQ ID NO: 2. In another embodiment, the mutant smAKAP contains the C16S, L4E, E5D, Y6H, R9E, C16S, W22S, and C24G mutations compared to SEQ ID NO: 2. In one embodiment, the mutant smAKAP includes the mutations C16S, L4E, C16S, W22S, and C24G compared to SEQ ID NO: 2. In another embodiment, the mutant smAKAP includes the mutations C16S, R9E, C16S, and C24G compared to SEQ ID NO: 2. In another embodiment, the mutant smAKAP includes the mutations C16S, R9E, C16S, W22S, and C24G compared to SEQ ID NO: 2. In another embodiment, the mutant smAKAP includes the mutations L4I, L10I, C16S, L19I, and C78A compared to SEQ ID NO: 2. In another embodiment, the mutant smAKAP includes the mutations L4I, R9E, L10I, C16S, L19I, W22S, and C24G compared to SEQ ID NO: 2. In one embodiment, the variant smAKAP contains the L4I, R9E, C16S, and C24G mutations compared to SEQ ID NO: 2.

[0066] In one embodiment, the mutant smAKAP contains an amino acid sequence having at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequences of SEQ ID NOs. 5, 7, 15, 17, 19, 21, 72, 73, and 74.

[0067] In some embodiments, mutant smAKAP exhibits increased protease resistance compared to wild-type smAKAP. For example, compositions comprising mutant smAKAP of the present disclosure may exhibit a reduction in activity and / or cleavage events of up to approximately 0.5, 1, 3, 5, 10, 15, 20, 25, 30, 40, 60, 80, 100, 120, 140, 160, 180, 200, 250, 300, or up to 350 times compared to wild-type. For example, compositions comprising the mutant smAKAP of this disclosure may exhibit a reduction in activity and / or cleavage events of up to approximately 0.5%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34%, 36%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, and 80% compared to the wild type. Activity can be determined after incubation with a protease. For example, the mutant smAKAP of this disclosure may show a reduction in activity of up to approximately 0.5%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34%, 36%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, or 60% compared to wild-type smAKAP after 1 hour incubation with a protease.

[0068] In some embodiments, the scaffold comprises a Kerberos body or hydra body complex as described herein and hereafter, comprising a mutant smAKAP peptide.

[0069] Cerberus Body In some embodiments, the Kerberos body construct may be formed when a Spirulina strain (either two independent strains or a single strain derived from different ORFs) expresses a 5HVZ-mediated homodimer molecule and a smAKAP-linked monomer VHH. These independent complexes further form a complex structure having three antigen-binding sites. In some embodiments, another heterologous moiety of the Disclosure may substitute one or more VHHs in the Kerberos body. Performance can be improved by mutating any of the scaffolds as described herein. For example, smAKAP may be mutated as described herein to reduce protease sensitivity.

[0070] The increase in antigen-binding reagents (VHHs) results in increased binding affinity due to avidity-based interactions of the disclosed Kerberos body constructs. The three binding VHHs may originate from the same VHH that forms a homotrimer. This design also makes it possible to mix diverse VHHs with desirable binding and target-neutralizing properties. Such molecular complexes can bind to multiple epitopes and mutant variants of the targets described herein (e.g., pathogens and their variants) that cannot be captured by conventional immunosurveillance or conventional therapies. In some embodiments, the Kerberos body complex exhibits increased binding compared to dimeric or monomeric forms. For example, the bonding may increase by at least, or up to approximately 5 times, 15 times, 25 times, 35 times, 45 times, 55 times, 65 times, 75 times, 85 times, 95 times, 105 times, 115 times, 125 times, 135 times, 145 times, 155 times, 165 times, 175 times, 185 times, 195 times, 205 times, 215 times, 225 times, 235 times, 245 times, or 300 times compared to scaffolds of other equivalent dimers or monomers.

[0071] Hydra Body In some embodiments, a hydra-body complex is also provided. In some embodiments, the hydra-body comprises at least four heterologous moieties. In some embodiments, the hydra-body comprises at least four antigen-binding VHHs.

[0072] Hydrabody complexes can be formed when a spirulina strain (either two independent strains or a single strain derived from different ORFs) expresses a 5HVZ-mediated homodimer molecule and a smAKAP-linked dimer VHH. The smAKAP-mediated dimer contains VHH at both the N-terminus and C-terminus of smAKAP. The VHH fused to smAKAP can form a homodimer (the same VHH) or a heterodimer (two different VHHs at both ends). Such configurations may allow a single complex to contain multiple therapeutically effective VHHs.

[0073] In some embodiments, the hydra-body complex exhibits increased binding compared to the trimer, dimer, or monomeric form. For example, the binding may increase by at least, or up to approximately 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, 105, 115, 125, 135, 145, 155, 165, 175, 185, 195, 205, 215, 225, 235, 245, or 300 times compared to other equivalent trimer, dimer, or monomeric scaffolds.

[0074] In addition to increased apparent binding based on avidity, the higher-order complex of the Kerberos body and hydra body enables the formation of highly potent and cross-reactive therapeutic complexes that can bind to and neutralize a variety of targets. For example, in some embodiments, the scaffold of the present disclosure is administered to a target in need to neutralize two or more targets. For instance, a hydra body containing one dimer of two VHH dimers that bind to and neutralize a single target may bind better and neutralize both targets than if each were used separately. Such designs allow for easy combination of multiple components and result in reduced target evasion (e.g., viral evasion) due to the involvement of diverse epitopes.

[0075] heterodimer In one embodiment, a form of the 5HVZ scaffold forms an essential heterodimer instead of a homodimer. This enables the assembly of a dimeric 5HVZ complex containing four different functional domains. In one embodiment, the four different functional domains include two different functional domains associated with each of the 5HVZ scaffolds.

[0076] In some embodiments, a heterodimer complex is provided herein. In some embodiments, the heterodimer complex comprises a dimer 5HVZ complex. In some embodiments, the dimer 5HVZ complex comprises at least two, three, four, five, or six different functional domains. In some embodiments, the dimer 5HVZ complex comprises at least four different functional domains. In some embodiments, the dimer 5HVZ complex comprises four different functional domains. In some embodiments, the dimer 5HVZ complex comprises the same functional domain.

[0077] In one embodiment, the functional domain includes a functional domain associated with each of the 5HVZ scaffolds. In another embodiment, the functional domain includes at least one, two, three, four, five, or six functional domains associated with each of the 5HVZ scaffolds. In another embodiment, the functional domain includes at least two functional domains associated with each of the 5HVZ scaffolds. In another embodiment, the functional domain includes two functional domains associated with each of the 5HVZ scaffolds. In another embodiment, the functional domains associated with each of the 5HVZ scaffolds are the same. In another embodiment, the functional domains associated with each of the 5HVZ scaffolds are different. In another embodiment, the dimer 5HVZ complex includes at least two, three, four, five, or six different functional domains associated with each of the 5HVZ scaffolds. In another embodiment, the dimer 5HVZ complex includes at least two different functional domains associated with each of the 5HVZ scaffolds. In one embodiment, the dimer 5HVZ complex contains two distinct functional domains associated with each of the 5HVZ scaffolds. In another embodiment, the dimer 5HVZ complex contains four distinct functional domains, two of which associate with each of the 5HVZ scaffolds. In yet another embodiment, the dimer 5HVZ complex contains six distinct functional domains, three of which associate with each of the 5HVZ scaffolds. In yet another embodiment, the dimer 5HVZ complex contains eight distinct functional domains, four of which associate with each of the 5HVZ scaffolds.

[0078] In one embodiment, the 5HVZ scaffold includes a functional domain including 1-5HVZ1-domain 2. In another embodiment, the 5HVZ scaffold includes a functional domain including 3-5HVZ-domain 4. In yet another embodiment, the 5HVZ scaffold includes a functional domain including 1-5HVZ1-domain 2 and a functional domain including 3-5HVZ-domain 4.

[0079] In some embodiments, the heterodimer complex exhibits increased binding compared to the monomeric form. In some embodiments, the binding may be increased by at least, or up to about 5 times, 15 times, 25 times, 35 times, 45 times, 55 times, 65 times, 75 times, 85 times, 95 times, 105 times, 115 times, 125 times, 135 times, 145 times, 155 times, 165 times, 175 times, 185 times, 195 times, 205 times, 215 times, 225 times, 235 times, 245 times, or 300 times compared to other equivalent monomer scaffolds.

[0080] In addition to increased apparent binding based on avidity, heterodimers enable the formation of highly potent and cross-reactive therapeutic conjugates that can bind to and neutralize a variety of targets. For example, in some embodiments, the scaffolds of the present disclosure are administered to a subject in need to neutralize two or more targets.

[0081] Different species of moiety In one embodiment, the scaffold is bound to a heterologous moiety. In another embodiment, the heterologous moiety comprises a therapeutic agent. In another embodiment, the therapeutic agent comprises a biological agent. In another embodiment, the therapeutic agent comprises a binder. In another embodiment, the heterologous moiety is linked to the N-terminus or C-terminus of the scaffold protein, or inside the scaffold protein.

[0082] In one embodiment, the therapeutic agent is a conjugate containing an antibody or a functional fragment thereof. In another embodiment, the conjugate contains an isolated fully human antibody, a humanized antibody, or a chimeric antibody, or a fragment thereof. In yet another embodiment, the conjugate molecule is an antibody fragment or a single variable domain antibody, preferably Fab, Fab', F(ab')2, scFv, dAb, or VHH.

[0083] In one embodiment, the heterologous antibody comprises a conjugate that is a VHH. VHH can be produced economically in unlimited quantities, is more stable when exposed to heat and solvents compared to conventional antibodies, and is suitable for genetic engineering for a variety of applications, including scaffolding, labeling, and modification of specific amino acids. Furthermore, VHH is 1 / 10 the size of conventional antibodies. Because of its small size, VHH allows for a high density of the binding domain, which provides a significant advantage over conventional antibodies in terms of increased signal and, consequently, improved sensitivity.

[0084] Exemplary VHH contains an antigen-binding fragment of a heavy-chain-only antibody. In one embodiment, VHH is derived from a camelid single-chain antibody. In one embodiment, VHH may be expressed intracellularly. In one embodiment, VHH may be expressed extracellularly. In one embodiment, VHH is expressed as a monomer, dimer, trimer, and / or heptamer. In one embodiment, VHH is expressed as a monomer. In one embodiment, VHH is expressed as a dimer. In one embodiment, VHH is expressed as a trimer. In one embodiment, VHH is expressed as a heptamer. Expression may be constitutive.

[0085] In some embodiments, VHH targets antigens associated with a disease or condition. Illustrative diseases or conditions may be autoimmune, metabolic, neurological, carcinogenic, and parasitic, bacterial, viral, or any combination thereof. In some embodiments, VHH targets autoimmune targets. Autoimmune targets may be associated with diseases selected from the group consisting of inflammatory bowel disease (IBD), celiac disease, ulcerative colitis, and Crohn's disease. In some embodiments, the disease or condition is metabolic. Illustrative metabolic diseases or conditions may include cardiovascular metabolic diseases (CMDs). CMDs are a cluster of associated conditions including obesity, diabetes, and cardiovascular disease. In some embodiments, the disease or condition is bacterial. Bacterial conditions may include diarrhea caused by bacterial pathogens. Illustrative bacterial pathogens include Escherichia coli, Campylobacter jejunum, and enterotoxigenic Escherichia coli.

[0086] In some embodiments, the heteromorphic moisture is selected from the group consisting of small molecules (e.g., drugs), peptides (e.g., ligands), and nucleic acids (e.g., siRNA, DNA, modified RNA, RNA). In some embodiments, the heteromorphic moisture has at least one effector activity selected from the group consisting of regulating biological activity, binding to regulatory proteins, regulating enzyme activity, regulating substrate binding, regulating receptor activation, regulating protein stability / degradation, and regulating transcript stability / degradation. In some embodiments, the heteromorphic moisture has at least one target function selected from the group consisting of regulating function, regulating molecules (e.g., enzymes, proteins, or nucleic acids), and localizing to a specific location. In some embodiments, the heteromorphic moisture is, for example, a cleavable tag or label. In some embodiments, the heterogeneous moiety is selected from the group comprising epigenetic modifiers, epigenetic enzymes, bicyclic peptides, transcription factors, DNA or protein modifying enzymes, DNA insertion agents, efflux pump inhibitors, nuclear receptor activators or inhibitors, proteasome inhibitors, competitive inhibitors of enzymes, protein synthesis inhibitors, nucleases, protein fragments or domains, tags, antigens, antibodies or antibody fragments, ligands or receptors, synthetic peptides or analog peptides derived from naturally occurring bioactive peptides, antimicrobial peptides, pore-forming peptides, targeted peptides or cytotoxic peptides, degrading peptides or self-destructive peptides, CRISPR systems or their components, DNA, RNA, artificial nucleic acids, nanoparticles, oligonucleotide aptamers, peptide aptamers, and pharmacokinetic or pharmacodynamic (PK / PD) agents.

[0087] In some embodiments, the heterologous moisture is selected from the group consisting of drugs, toxins, cytotoxic agents, contrast agents, radionuclides, radiocompounds, organic polymers, inorganic polymers, polyethylene glycol (PEG), biotin, albumin, ligands, receptors, binding peptides, epitope tags, recombinant polypeptide polymers, cytokines, and combinations of two or more of the aforementioned parts. In some embodiments, the heterologous moisture is albumin, and the albumin includes human serum albumin. In some embodiments, the heterologous moisture is an antibody or a fragment thereof, and includes an antibody domain, an antibody fragment, a single-chain antibody, a domain antibody, or any combination thereof.

[0088] In one embodiment, the heterologous moiety includes an antibody. In another embodiment, the antibody is selected from the group consisting of the antibody's Fc domain, antibody fragments, and single-chain antibodies.

[0089] In some embodiments, the scaffold of the Disclosure may include the heterogeneous moiety of the Disclosure. In some embodiments, the heterogeneous moiety includes VHHs. In some embodiments, the Kerberos body or Hydra body may include multiple VHHs of the same or different sequences.

[0090] VHH, derived from single-chain antibodies of the camelid family, is ideal for expression in prokaryotes such as Spirulina because it does not require intracellular disulfide bond formation or specific glycosylation for the synthesis of a bioactive protein. In one embodiment, VHH is constitutively expressed in Spirulina. Expression can be regulated using any promoter. In one embodiment, the promoter comprises Pcpc600. As described herein, VHH can be expressed on scaffolds in various formats, including monomers, dimers, trimers, and heptamers.

[0091] Since VHHs are readily expressed in prokaryotes, they can be rapidly isolated from highly diverse, naive phage display libraries. These typically possess medium nM affinity for their antigenic targets, and therefore, further mutagenesis can be performed to achieve higher affinity. As described herein, expression of VHHs as high-avidity multimers bypasses this and thus accelerates product development.

[0092] In some embodiments, the VHH of the Disclosure binds to a virus or a portion thereof, including, but not limited to, bacteriophages, RNA bacteriophages (e.g., MS2, AP205, PP7, and Qβ), Helicobacter pylori, infectious hematopoietic necrosis virus (IHNV), parvovirus, herpes simplex virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, measles virus, mumps virus, rubella virus, human immunodeficiency virus (HIV), influenza virus, rhinovirus, rotavirus A, rotavirus B, rotavirus C, respiratory syncytial virus (RSV), varicella-zoster virus, poliovirus, norovirus, Zika virus, dengue virus, rabies virus, Newcastle disease virus, vitiligo virus, coronavirus, MERS-related coronavirus, SARS-related coronavirus, and SARS-CoV-2.

[0093] In one embodiment, VHH is anti-Campylobacter VHH. In one embodiment, Campylobacter is C. jejuni. In one embodiment, VHH binds to components of Campylobacter. In one embodiment, VHH binds to flagellin. In one embodiment, administration increases Campylobacter shedding. In one embodiment, administration reduces the level of a biomarker. In one embodiment, the biomarker is an inflammatory biomarker.

[0094] In one embodiment, recombinant spirulina contains VHH that binds to anti-Clostridium toxin. In another embodiment, Clostridium is C. difficile. In another embodiment, VHH binds to a component of Clostridium, toxin A, or toxin B. In another embodiment, VHH contains any amino acid sequence or fragment thereof of SEQ ID NOs. 25-37.

[0095] In one embodiment, recombinant spirulina contains anti-Campylobacter VHH. In one embodiment, Campylobacter is C. jejuni. In one embodiment, VHH binds to components of Campylobacter. In one embodiment, VHH binds to flagellin. In one embodiment, administration increases Campylobacter shedding. In one embodiment, administration reduces the level of a biomarker. In one embodiment, the biomarker is an inflammatory biomarker.

[0096] In one embodiment, recombinant spirulina comprises a polypeptide or fragment thereof that binds to a norovirus polypeptide or antigen. In another embodiment, recombinant spirulina comprises a polypeptide or fragment thereof that binds to the norovirus P domain. In another embodiment, the polypeptide or fragment thereof is VHH. In another embodiment, recombinant spirulina comprises a VHH that binds to a norovirus polypeptide. In another embodiment, recombinant spirulina comprises a VHH that binds to the norovirus P domain. In another embodiment, recombinant spirulina comprises a polypeptide or fragment thereof that binds to the GII genotype, G1 genotype, G11, and G10 genotypes. In another embodiment, recombinant spirulina comprises a polypeptide or fragment thereof that binds to polypeptides derived from two or more norovirus genotypes. In another embodiment, recombinant spirulina comprises a VHH comprising a Nano85 nanobody, a Nano26 nanobody, a Nano94 nanobody, a K922 antibody, or a modified sequence or fragment thereof. In another embodiment, recombinant spirulina comprises a VHH comprising a Nano85 and / or its loop graft modification.

[0097] In one embodiment, spirulina comprises a scaffold containing VHH that binds to a malaria polypeptide or its antigen or fragment thereof. In another embodiment, malaria comprises the genus Plasmodium. In another embodiment, malaria comprises Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale curtisi, Plasmodium ovale wallikeri, Plasmodium quartanum, or Plasmodium malariae. In another embodiment, malaria is Plasmodium falciparum. In another embodiment, recombinant spirulina comprises a polypeptide that binds to a malaria-related protein or fragment thereof in the liver or blood. In another embodiment, recombinant spirulina comprises a polypeptide that binds to a malaria-related protein or fragment thereof during the extracellular phase, at the time of hepatocyte rupture, or during the erythrocyte phase. In another embodiment, recombinant spirulina comprises a polypeptide that binds to a malaria-related protein or fragment thereof during the extracellular phase. In one embodiment, recombinant spirulina comprises a polypeptide that binds to a malaria-related protein or fragment thereof derived from a stage including sporozoites, merozoites, trophozoites, schizonts, or gametocytes. In another embodiment, recombinant spirulina comprises a polypeptide that binds to a malaria-related protein or fragment thereof on the surface of a sporozoite. In another embodiment, recombinant spirulina comprises a polypeptide that binds to a perisporozoite protein (CSP) or fragment thereof. In another embodiment, recombinant spirulina comprises a polypeptide that binds to a Plasmodium falciparum perisporozoite protein (PfCSP) or fragment thereof. In another embodiment, the binding disclosed herein can block hepatocyte infection by sporozoites and protect against malaria. In another embodiment, VHH binds to one or more NANP repeats. In another embodiment, VHH binds to a CSP polypeptide. In another embodiment, VHH binds to a polypeptide comprising one or more NANP repeats.

[0098] Table 0 provides exemplary VHHs of the present disclosure. In some embodiments, the scaffolds of the present disclosure are linked to VHHs that have at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with respect to the VHHs in Table 0. In some embodiments, the scaffolds of the present disclosure are linked to VHHs that have at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with respect to SEQ ID NOs. 25-67. In some embodiments, the scaffolds of the present disclosure are linked to VHHs that have at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with respect to SEQ ID NOs. 25-37. In some embodiments, the scaffold of the present disclosure is linked to a VHH that has at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with respect to Sequence IDs 38-67. [Table 1-1] [Table 1-2] [Table 1-3]

[0099] The VHH-containing scaffolds of this disclosure neutralize or block the activity of the target. In some embodiments, administration of the disclosure neutralizes or blocks the activity of the target by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, or about 100%.

[0100] In some embodiments, VHH can be expressed as a fusion protein having a solubility-enhancing chaperone such as Escherichia coli maltose-binding protein (MBP).

[0101] In some embodiments, compositions provided herein, including a scaffold, can be bonded by heterogeneous moisture to about 1, 2, 3, 4, 5, 6, 7, or up to about 8 different targets. In some embodiments, the scaffold of the Disclosure bonded to 2 targets. In some embodiments, the scaffold of the Disclosure bonded to 3 targets. In some embodiments, the scaffold of the Disclosure bonded to 4 targets. In some embodiments, the scaffold of the Disclosure bonded to 5 targets. In some embodiments, the scaffold of the Disclosure bonded to 6 targets. In some embodiments, the scaffold of the Disclosure bonded to 7 targets. In some embodiments, the scaffold of the Disclosure bonded to 8 targets.

[0102] Also provided are nucleic acids encoding polypeptides of the Disclosure, such as heterologous moieties and / or Kerberos bodies and / or hydra bodies. Such nucleic acids are hereafter referred to as “nucleic acids of the Disclosure” and may, for example, be in the form of gene constructs.

[0103] In some embodiments, the scaffolds of the present disclosure, such as Kerberos bodies or hydra bodies, are more effective at neutralizing targets compared to monomeric, dimeric, or trimeric constructs. In some embodiments, the effectiveness is at least about 1x, 10x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 1000x, 5000x, 10,000x, 15,000x, 100,000x, 200,000x, 300,000x, 500,000x, 800,000x, or 1,000,000x more effective compared to non-multimericic constructs. In some embodiments, the scaffolds of the present disclosure, such as Kerberos bodies or hydra bodies, are more effective at reducing or removing targets compared to monomeric or dimericic constructs. In some embodiments, the effectiveness is at least about 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1,000, 5,000, 10,000, 15,000, 100,000, 200,000, 300,000, 500,000, 800,000, or 1,000,000 times more effective than that of non-multimer constructs.

[0104] In some embodiments, the scaffolds of the present disclosure exhibit increased binding to the target compared to non-multimeric scaffolds or the corresponding binders. In some embodiments, the binding is at least, or at most, about 5%, 15%, 25%, 35%, 45%, 55%, 65%, 75%, 85%, 95%, 105%, 115%, 125%, 135%, 145%, 155%, 165%, 175%, 185%, 195%, 205%, 215%, 225%, 235%, 245%, Increases by 255%, 265%, 275%, 285%, 295%, 305%, 315%, 325%, 335%, 345%, 355%, 365%, 375%, 385%, 395%, 405%, 415%, 425%, 435%, 445%, 455%, 465%, 475%, 485%, 495%, or 500%.

[0105] In some embodiments, multimerized constructs such as Kerberos bodies or hydra bodies are more effective than monomeric or dimerized constructs in reducing symptoms or diseases in subjects requiring them. In some embodiments, the effectiveness in reducing symptoms or diseases is at least about 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1,000, 5,000, 10,000, 15,000, 100,000, 200,000, 300,000, 500,000, 800,000, or 1,000,000 times more effective than non-multimerized constructs.

[0106] In some embodiments, multimerized constructs such as cerberobody or hydrabody are more effective than monomeric or dimerized constructs in neutralizing or reducing pathogens or their variants (e.g., viral / bacterial / cancer variants) that have evaded conventional therapies. In some embodiments, the efficacy is at least about 1x, 10x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 1000x, 5000x, 10,000x, 15,000x, 100,000x, 200,000x, 300,000x, 500,000x, 800,000x, or 1,000,000x more effective than conventional therapies. In some embodiments, efficacy is at least about or at most about 5%, 15%, 25%, 35%, 45%, 55%, 65%, 75%, 85%, 95%, 105%, 115%, 125%, 135%, 145%, 155%, 165%, 175%, 185%, 195%, 205%, 215%, 225%, 235%, 245%, Increases by 255%, 265%, 275%, 285%, 295%, 305%, 315%, 325%, 335%, 345%, 355%, 365%, 375%, 385%, 395%, 405%, 415%, 425%, 435%, 445%, 455%, 465%, 475%, 485%, 495%, or 500%.

[0107] Linker In some embodiments, linkers are also provided that can be used in the compositions and methods provided herein. Any linker can be used in the compositions of the Disclosure to, for example, bond one or more dissimilar mosies of the Disclosure. In some embodiments, a linker can also be used to bond Kerberos bodies and / or Hydra bodies. In some embodiments, the linker includes smAKAP.

[0108] In some embodiments, heterologous mosies can be conjugated directly to the scaffold or via a linker. In some embodiments, two or more heterologous mosies can be conjugated directly or via a linker. Suitable linkers include, for example, cleavable and non-cleavable linkers. Cleavable linkers are typically susceptible to cleavage under intracellular conditions. Suitable cleavable linkers include, for example, peptide linkers that can be cleaved by intracellular proteases such as lysosomal proteases or endosomal proteases. In some embodiments, the linker may be a dipeptide linker such as a valine-citrulline (val-cit) or phenylalanine-lysine (phe-lys) linker. Other suitable linkers include linkers that can be hydrolyzed at pH less than 5.5, such as hydrazone linkers. Additional suitable cleavable linkers include disulfide linkers.

[0109] In some embodiments, the linker is a rigid linker. In some embodiments, the linker is a flexible linker. In some embodiments, the linker links two or more VHH sequences. In some embodiments, the linker links one or more VHH sequences to another heterologous moiety of the present disclosure. In some embodiments, the heterologous moiety is selected from the group consisting of chaperone proteins, targeting proteins, scaffolds, oligomerized domains, enzymes, or lysine.

[0110] In some embodiments, the linker comprises a smAKAP peptide. In some embodiments, the smAKAP peptide may be modified. Modifications may be made, for example, to increase soluble protein expression. In some embodiments, one or more residues in the linker may be mutated. In some embodiments, in an exemplary linker, for example, two Cys residues in the smAKAP peptide are mutated to serine. In some embodiments, the cleavable linker may also be used in compositions provided herein.

[0111] In one embodiment, the linker has at least, or at most about 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, and 100% identity with respect to linkers selected from the group consisting of: AEAAAAKAS (Helix 1 linker, SEQ ID NO: 68), AEAAAKEAAAKAS (Helix 2 linker, SEQ ID NO: 69), AEAAAKEAAAKEAAAKEAAAKAS (Helix 4 linker, SEQ ID NO: 70), APAPSPAPSPAS (PA5 linker, SEQ ID NO: 71), and PA10 lin25.

[0112] In one embodiment, the linker includes a mutant smAKAP having an amino acid sequence that is at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences of SEQ ID NOs. 5, 7, 15, 17, 19, 21, 72, 73, and 74. The linker may also include a wild-type smAKAP sequence based on SEQ ID NO. 2.

[0113] In some embodiments, the linker can be of any length. In some embodiments, the linker is of a length of approximately 1-3, 2-6, 1-8, 3-10, 5-20, 3-15, 10-30, 15-35, 20-35, or 25-35 residues. In some embodiments, the linker is of a length of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids.

[0114] Spirulina Spirulina is synonymous with "Arthrospira." The genus Arthrospira includes 57 species, of which 22 are currently taxonomically recognized. In embodiments, the Spirulina species is selected from the group consisting of: A. amethystine, A. ardissoni, A. argentina, A. balkrishnanii, A. baryana, A. boryana, A. braunii, A. brevi. articulata, A.brevis, A.curta, A.desikacharyiensis, A.funiformis, A.fusiformis, A.ghannae, A.gigantean, A.gomontiana, A.gomontiana var.crassa, A.indica, A.jenneri var.platensis, A.jenneri Stizenberger, A.jenneri f.purpurea, A.joshii, A.khannae, A.laxa, A.laxissima, A.laxissima, A.leopoliensis, A.major, A.margaritae, A.massartii, A.massartii var.indica, A.maxima, A.meneghiniana, A.miniata var.constricta, A.miniata, A.miniata f.acutissima, A.neapolitana, A.nordstedtii, A.oceanica, A.okensis, A.pellucida, A.platensis, A.platensis var.non-constricta, A.platensis f.granulate, A.platensis f.minor, A.platensis var.tenuis, A.santannae, A.setchellii, A.skujae, A.spirulinoides f.tenuis, A.spirulinoides, A.subsalsa, A.subtilissima, A.tenuis, A.tenuissima, and A.versicolor. In embodiments, the spirulina is A. platensis.

[0115] In one embodiment, the recombinant spirulina is non-viable. In another embodiment, the recombinant spirulina is dried, spray-dried, freeze-dried, or freeze-dried.

[0116] Any suitable means for transforming spirulina may be used in this disclosure. An exemplary method for transforming spirulina to express heterologous proteins is described in U.S. Patent No. 10,131,870, which is incorporated herein by reference in its entirety.

[0117] In some embodiments, a method for preparing a spirulina composition expressing the scaffold of the present disclosure comprises introducing an expression vector having a nucleic acid sequence encoding the scaffold into spirulina cells. In some embodiments, the vector is not integrated into the spirulina genome. In some embodiments, the vector is a high-copy or high-expression vector. In some embodiments, the nucleic acid sequence encoding the scaffold is under the control of a strong promoter. In some embodiments, the nucleic acid sequence encoding the scaffold is under the control of a constitutive promoter. In some embodiments, the nucleic acid sequence encoding the scaffold is under the control of an inducible promoter.

[0118] In one embodiment, the method for preparing the composition includes introducing a vector having nucleic acid sequences encoding homology arms and scaffolds (for example, via homologous recombination) into spirulina cells.

[0119] In one embodiment, a vector having nucleic acid sequences encoding homology arms and scaffolds can be introduced into spirulina using electroporation. Electroporation is preferably carried out in the presence of a suitable osmotic stabilizer.

[0120] Before introducing the vector into spirulina, the spirulina may be cultured in any suitable medium for cyanobacteria growth, such as SOT medium. SOT medium contains 1.68 g of NaHCO3, 450 mg of K2HPO, 250 mg of NaNO3, 100 mg of K2504, 100 mg of NaCl, 20 mg of MgSO4.7H2O, 4 mg of CaCl2.2H2O, 1 mg of FeSO4.7H2O, 8 mg of Na2EDTA.2H2O, 0.1 mL of A5 solution, and 99.9 mL of distilled water. A5 solution contains 286 mg of H3BO3, 217 mg of MnSO4.5H2O, 22.2 mg of ZnSO4.7H2O, 7.9 mg of CuSO4.5H2O, 2.1 mg of Na2MoO4.2H2O, and 100 mL of distilled water. The culture may be carried out at a temperature higher than room temperature (e.g., 25-37°C) with shaking (e.g., 100-300 rpm), and with continuous irradiation (e.g., 20-2,000, 50-500, or 100-200 μmol photons m). -2 s -1 ) can be carried out under certain conditions. Growing cells have an optical density at a wavelength of 750 nm that is below a predetermined threshold (e.g., OD). 750 The cells may be collected when the pH reaches 0.3–2.0, 0.5–1.0, or 0.6–0.8. The collected volume of cells may be concentrated by centrifugation and then resuspended in a solution of pH adjuster and salt. The pH adjuster may be any suitable buffer that maintains the viability of spirulina while maintaining the pH of the medium at 6–9 pH, 6.5–8.5 pH, or 7–8 pH. Suitable pH adjusters include HEPES, HEPES-NaOH, sodium phosphate buffer or potassium phosphate buffer, and TES. The salt solution may be NaCl at concentrations of 50 mM–500 mM, 100 mM–400 mM, or 200 mM–300 mM. In some embodiments, the pH may be neutralized using 1–50 mL of 1–100 mM pH adjuster.

[0121] Cells recovered by centrifugation may be washed with an osmotic stabilizer and optionally with a salt solution (e.g., 1 to 50 mL of 0.1 to 100 mM NaCl). Any amount of culture may be concentrated by centrifugation. In some embodiments, 5 to 500 mL of culture may be centrifuged. The osmotic stabilizer may be any type of osmotic regulator that stabilizes the integrity of the spirulina cells during electroporation. In some embodiments, the osmotic stabilizer may be a sugar such as glucose or sucrose (e.g., w / v 0.1 to 25%). In some embodiments, the osmotic stabilizer may be a simple polyol containing glycerin (glycerine, glycerin, or glycerol) (e.g., v / v 1 to 25%). In some embodiments, the osmotic stabilizer may be a polyether containing polyethylene glycol (PEG), poly(oxyethylene), or poly(ethylene oxide) (PEO) (e.g., w / v 0.1 to 20%). PEG or PEO may have molecular weights of 200 to 10,000, 1,000 to 6,000, or 2,000 to 4,000. In some embodiments, pH adjusters or buffers may be used instead of, or in addition to, osmotic stabilizers.

[0122] A vector containing nucleic acid sequences encoding homology arms and scaffolds can be introduced into spirulina cells cultured as described above and washed with an osmotic stabilizer. The vector can be introduced using electroporation.

[0123] Electroporation may be carried out in an electroporation cuvette of 0.1, 0.2, or 0.4 cm with a voltage of 0.6–10 kV / cm, 2.5–6.5 kV / cm, or 4.0–5.0 kV / cm, a capacitance of 1–100 μF, 30–70 μF, or 45–55 μF, and a impedance of 10–500 mΩ, 50–250 mΩ, or 90–110 mΩ. In one embodiment, electroporation may be carried out at 4.5 kV / cm, 50 μF, and 100 mΩ.

[0124] After electroporation, cells can be grown in the presence of one or more antibiotics selected based on the resistance given through successful plasmid-assisted transformation. Post-electroporation cultures can be grown at reduced irradiation levels (e.g., 5-500, 10-100, or 30-60 μmol of photons). -2 s -1 The culture may be carried out using ) or by shaking (e.g., 100-300 rpm). The level of antibiotic in the medium may be 5-100 μg / mL. The culture after electroporation may be continued for 1-5 days or longer. Successful transformants identified by antibiotic resistance may be selected on a plate or in 5-100 mL of SOT medium supplemented with 0.1-2.0 μg of the appropriate antibiotic over a period of 1 week to 1 month.

[0125] The vectors used in this method may be plasmids, bacteriophages, or viral vectors, and may contain insertions or cloning of at least one exogenous polypeptide, antigen, and / or nucleic acid sequences encoding the antigen. The vectors may contain one or more specific sequences that enable recombination to specific desired sites on the spirulina chromosome. These specific sequences may be homologous to sequences present in wild-type spirulina. The vector system may consist of a single vector or plasmid, or two or more vectors or plasmids, some of which may increase the efficiency of targeted mutagenesis or may contain transpositions. Vector selection typically depends on the compatibility of the vector with the spirulina cells into which the vector is introduced. The vectors may contain a reporter gene, such as green fluorescent protein (GFP), which may be fused in-frame to one or more encoded antigenic epitopes or expressed separately. The vectors may also contain positive selection markers, such as antibiotic resistance genes, which can be used for selecting suitable transformants. The vector may also contain negative selection markers such as the type II thioesterase (tesA) gene or the Bacillus subtilis structural gene (sacB). The use of a reporter or marker allows for the identification of those cells that have been successfully transformed with the vector.

[0126] In some embodiments, the vector includes one or two homology arms homologous to the DNA sequence of the spirulina genome adjacent to the target locus. The sequences of the homology arms may be partially or fully complementary to the region of the spirulina genome adjacent to the target locus.

[0127] The homology arms may be of any length that enables site-specific homologous recombination. The homology arms may be of any length between approximately 2000 bp and 500 bp. For example, the homology arms may be approximately 2000 bp, approximately 1500 bp, approximately 1000 bp, or approximately 500 bp. In embodiments having two homology arms, the homology arms may be the same or different lengths. Therefore, each of the two homology arms may be of any length between approximately 2000 bp and 500 bp. For example, each of the two homology arms may be approximately 2000 bp, approximately 1500 bp, approximately 1000 bp, or approximately 500 bp.

[0128] A portion of a vector adjacent to one homology arm, or flanked by two homology arms, modifies a target locus in the spirulina genome through homologous recombination. The modification may alter the length of the target locus, including nucleotide deletions or additions. The additions or deletions may be of any length. The modification may also alter the sequence of nucleotides within the target locus without changing its length. The target locus may be any part of the spirulina genome, including coding regions, non-coding regions, and regulatory sequences.

[0129] How to use The compositions of this disclosure can be used in therapeutic methods. In some embodiments, the compositions of this disclosure may be used to reduce the severity of a disease or disorder in a subject requiring its use. In some embodiments, the compositions may be used to prevent a disease or disorder in a subject. In some embodiments, the compositions may be used to prevent the onset of a disease or disorder in a subject. In some embodiments, the compositions may be used to reduce the severity of a disease or disorder in a subject. In some embodiments, the compositions may be used to prevent or delay the recurrence of a disease in a subject. In some embodiments, the compositions may be used to treat, prevent the recurrence of, or delay the recurrence of cancer in a subject. In some embodiments, the compositions may be used to treat, prevent the recurrence of, or delay the recurrence of systemic pathogens. In some embodiments, the compositions may be used to treat, prevent the recurrence of, or delay the recurrence of systemic parasites. In some embodiments, the compositions may be used to treat, prevent the recurrence of, or delay the recurrence of malaria. In some embodiments, the compositions may be used to treat, prevent the recurrence of, or delay the recurrence of mucosal pathogens.

[0130] In some embodiments, a therapeutic method comprises administering a composition disclosed for the treatment of an enteropathogen. Exemplary enteropathogens include Campylobacter infection, enterotoxigenic Escherichia coli, norovirus, C. difficil, and SARS-CoV-2.

[0131] In some embodiments, the compositions of the present disclosure may be used to treat any disease or condition related to or derived from any microorganism, or to treat or prevent infection by any microorganism, including, but not limited to, the following: Escherichia coli, enterotoxigenic Escherichia coli (ETEC), anthrax, EHEC, EAEC, Shigella, Mycobacterium, Streptococcus, Staphylococcus, Shigella, Campylobacter, Salmonella, Clostridium, Corynebacterium, Shoe Domonas, Neisseria, Listeria, Vibrio, Bordetella, Legionella, bacteriophages, RNA bacteriophages (e.g., MS2, AP205, PP7, and Qβ), Helicobacter pylori, infectious hematopoietic necrotic virus, parvovirus, herpes simplex virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, measles virus, mumps virus, rubella virus, HIV, influenza virus, rhinovirus, rotavirus Rotavirus A, Rotavirus B, Rotavirus C, Respiratory Syncytial Virus (RSV), Varicella-zoster virus, Poliovirus, Norovirus, Zika virus, Dengue virus, Rabies virus, Newcastle disease virus, Vitiligo virus, Coronavirus, SARS-related coronavirus, MERS-related coronavirus, SARS-CoV-2, Aspergillus, Candida, Blastomyces, Coccidioides, Cryptococcus, Histoplasma, Malari Plasmodium, Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, Plasmodium vivax, Trypanosoma, Toxoplasma, Giardia, Leishmania, Cryptosporidium, Hellworm parasites: Whipworms, Pinworms, Ascaris, Hookworms, and American Hookworms, Strongyloides, Dracunculus, Onchocerca, and Filamentosa, Tapeworms, Hydatids, and Diphyllobothrium, Fasciola, and Schistosoma, or combinations thereof.

[0132] In some embodiments, the compositions described herein may be used to treat and / or reduce the severity of infections caused by bacteria including, but not limited to, Mycobacterium, Streptococcus, Staphylococcus, Shigella, Campylobacter, Salmonella, Clostridium, Corynebacterium, Pseudomonas, Neisseria, Listeria, Vibrio, Bordetella, and Legionella.

[0133] In some embodiments, the compositions described herein may be used to treat or reduce the severity of infections caused by parasites, including but not limited to: Plasmodium malariae, Trypanosoma, Toxoplasma, Giardia, and Leishmania, Cryptosporidium, Helminth parasites: Whipworms, Pinworms, Roundworms, Hookworms and American Hookworms, Strongyloides, Dracunculus, Onchocerca and Filamentosa, Tapeworms, Hydatidiformes and Diphyllobothrium (human and animal tapeworms), Fasciola and Schistosoma.

[0134] In some embodiments, the compositions described herein may be used to treat or reduce the severity of infections caused by malaria parasites. In some embodiments, the compositions of the present disclosure may be used to induce an immune response to and / or reduce the severity of infections caused by malaria parasites selected from the group consisting of: Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, and Plasmodium vivax.

[0135] In some embodiments, the compositions described herein may be used to treat or reduce the severity of infections caused by fungi, including but not limited to Aspergillus, Candida, Blastomyces, Coccidioides, Cryptococcus, and Histoplasma. In some embodiments, the compositions may be used to induce an immune response to and / or reduce the severity of Candida albicans or Candida auris infections.

[0136] In some embodiments, the compositions described herein may be used to treat or reduce breast cancer cells, colon cancer cells, brain cancer cells, pancreatic cancer cells, lung cancer cells, cervical cancer cells, uterine cancer cells, prostate cancer cells, ovarian cancer cells, melanoma cancer cells, lymphoma cancer cells, myeloma cancer cells, and leukemia cancer cells.

[0137] In some embodiments, the compositions described herein may be used to treat or reduce symptoms of autoimmune diseases, including but not limited to ulcerative colitis, rheumatoid arthritis, systemic lupus erythematosus (SLE), celiac disease, inflammatory bowel disease, Hashimoto's disease, Addison's disease, Graves' disease, type 1 diabetes mellitus, autoimmune thrombocytopenic purpura (ATP), idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), Crohn's disease, multiple sclerosis, and myasthenia gravis.

[0138] Other GI targets, such as inflammatory and metabolic diseases, as well as microbiome manipulation, are also intended. In some embodiments, the compositions described herein can alter the abundance of microorganisms present in the microbiome. In some embodiments, the compositions described herein can alter the abundance of bacteria present in the microbiome. In some embodiments, the compositions disclosed herein can alter the abundance of Firmicutes, Bacteroides, Proteobacteria, and Actinobacteria. In some embodiments, the compositions described herein can reduce the abundance of pro-inflammatory bacteria in the microbiome. In some embodiments, the compositions described herein may be used to reduce the abundance of bacteria including Bacteroides, Proteobacteria, Enterobacteriaceae, Porphyromonas, Clostridiales, Pasteurellaceae, Veillonellaceae, Neisseriaceae, Clostridiidae, Proetobacteria, Ruminococcus, Drea, Actinomyces, and Fusobacteriaceae. In some embodiments, the compositions described herein can be used to reduce the abundance of bacteria including Escherichia coli, Shigella, Rhodococcus, Veillonella, Stonotrhophomonas maltophilia, Prevotella, Clostridium difficile, Clostridium ramosum, Klebsiella pneumoniae, Proteus mirabilis, Staphylococcus aureus, Ruminococcus turkes, Ruminococcus gunavas, and Helicobacter hepaticus. In some embodiments, the compositions described herein can be used to increase the abundance of anti-inflammatory bacteria in the microbiome. In some embodiments, the compositions described herein can be used to increase butyrate-producing bacteria. In some embodiments, the compositions described herein may be used to increase bacteria including Firmicutes, Lactobacillus, Acanthopagrus, Eubacterium, Bifidobacterium, Faecalibacterium, Rosebria, Coprococcus eutaxus, Rosebria, Akkermansia, and Clostridiales.In some embodiments, the compositions described herein may be used to increase bacteria including Faecalibacterium prausnitzii, Bifidobacterium longum, Eubacterium rectore, Rosbria intestinalis, and Ackermansia muciniphila.

[0139] Pharmaceutical composition In some embodiments, the compositions of the present disclosure are formulated as pharmaceutical compositions. For example, a scaffold may be included in the pharmaceutical composition. The pharmaceutical compositions of the present disclosure can be administered to subjects requiring it. Administration can be carried out by any means. In some embodiments, administration is by oral, intravenous, intradermal, via the airway, intranasal, and combinations thereof.

[0140] In some embodiments, the pharmaceutical composition is administered orally. As used herein, the terms “oral composition” or “oral delivery composition” include compositions administered or delivered to the gastrointestinal tract (e.g., compositions administered orally or to the stomach via a feeding tube). Any suitable area of ​​the gastrointestinal tract may be targeted by the compositions of this disclosure.

[0141] In some embodiments, the compositions of the present disclosure are administered via the airway. In some embodiments, the compositions of the present disclosure are administered by inhalation. In some embodiments, the compositions of the present disclosure are administered intranasally. In some embodiments, the compositions of the present disclosure are administered by nebulizer, inhaler, or mist. In some embodiments, the compositions of the present disclosure are lyophilized and delivered as powder, or as powder resuspended in liquid. In some embodiments, the compositions of the present disclosure are formulated for administration by nebulizer, inhaler, dry powder inhaler, or mist.

[0142] In some embodiments, the pharmaceutical composition comprises an adjuvant. Most adjuvants contain substances designed to protect proteins or their fragments from rapid catabolism, such as aluminum hydroxide or mineral oil, and substances that stimulate an immune response, such as proteins derived from Bordetella pertussis or Mycobacterium tuberculosis. In some embodiments, the adjuvant includes, but is not limited to, Toll-like receptor (TLR) agonists, monophosphoryl lipid A (MPL), synthetic lipid A, lipid A mimetic or analog, aluminum salts, cytokines, saponins, muramyl dipeptide (MDP) derivatives, CpG oligos, lipopolysaccharides (LPS) of Gram-negative bacteria, polyphosphazenes, emulsions, virosomals, cocreates, poly(lactide-co-glycolide) (PLG) microparticles, poloxamer particles, microparticles, montanaides, and liposomes. In some embodiments, the adjuvant is a montanaide adjuvant.

[0143] In some embodiments, the composition comprises at least one adjuvant. In some embodiments, the adjuvant is present in solution. In some embodiments, the adjuvant is present in a concentration of approximately 1 μg / mL to approximately 85 mg / mL (e.g., 1 μg / mL, 50 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL, 250 μg / mL, 300 μg / mL, 350 μg / mL, 400 μg / mL, 450 μg / mL, 500 μg / mL, 550 μg / mL, 600 μg / mL, 650 μg / mL, 700 μg / mL, 750 μg / mL, 800 μg / mL, 850 μg / mL, It is present in concentrations of 900 μg / mL, 950 μg / mL, 1 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, 75 mg / mL, 80 mg / mL, or 85 mg / mL (including all values ​​and ranges contained therein).

[0144] In one embodiment, the composition survives in a gastrointestinal tract or simulated stomach environment. In another embodiment, the composition survives in a gastrointestinal tract or simulated stomach environment for at least 5 minutes. In yet another embodiment, the composition survives overnight in a digestive tract or simulated stomach environment.

[0145] In one embodiment, the composition survives in the nasal cavity. In another embodiment, the composition survives in the upper respiratory tract. In another embodiment, the composition survives in the airway. In another embodiment, the composition survives in the nasal cavity, upper respiratory tract, and / or airway for at least 5 minutes. In another embodiment, the composition survives overnight in the nasal cavity, upper respiratory tract, and / or airway.

[0146] The compositions of the disclosed herein may be administered daily, weekly, twice a week or every two weeks, every other week, monthly, etc. In some embodiments, the compositions of the disclosed herein are administered to a subject for about 1 day to about 1 year. In some embodiments, the compositions of the disclosed herein are administered to a subject for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or longer. In some embodiments, the compositions of the disclosed herein are administered on consecutive days. In some embodiments, the compositions of the disclosed herein are administered on non-consecutive days. In some embodiments, the compositions of the disclosed herein are administered once a day. In some embodiments, the compositions of the disclosed herein are administered multiple times a day. In some embodiments, the compositions of the disclosed herein are administered twice a day, 3 times a day, 4 times a day, or more. In some embodiments, the compositions of the disclosed herein are administered continuously (for example, via a feeding tube). In some embodiments, the compositions of the present disclosure are administered with a meal. In some embodiments, the compositions of the present disclosure are administered when the subject is fasting.

[0147] In some embodiments, the compositions of the present disclosure may comprise one or more pharmaceutically acceptable excipients. Examples of pharmaceutically acceptable carriers include, but are not limited to, physiological saline, buffered physiological saline, dextrose, water, glycerol, sterile isotonic aqueous buffers, and combinations thereof. In some embodiments, the pharmaceutically acceptable excipient is sodium bicarbonate.

[0148] In some embodiments, the composition further comprises excipients. In some embodiments, the composition comprises at least one excipient. In some embodiments, the composition comprises at least two or more excipients. In some embodiments, the composition comprises three or more excipients. In some embodiments, the composition comprises four or more excipients. In some embodiments, the composition comprises five or more excipients. In some embodiments, the composition comprises six or more excipients. In some embodiments, the composition comprises seven or more excipients. Exemplary excipients include, but are not limited to, stabilizers, buffers, surfactants, fillers, sugars, and salts.

[0149] In some embodiments, the excipient is selected from the group comprising stabilizers, buffers, surfactants, fillers, sugars, and salts. Exemplary stabilizers include, but are not limited to, lactose, gelatin, sucrose, sorbitol, human serum albumin, aluminum salts, monosodium glutamate, sodium chloride, mannitol, L-histidine, and dextran. Exemplary buffers include, but are not limited to, hydrogen chloride, general buffers, pH counter buffers, phosphate-buffered saline (PBS), phosphate buffers, acetate buffers, Tris buffers, HEPES buffers, glycine buffers, citrate buffers, and histidine buffers. Exemplary surfactants include, but are not limited to, polyethylene glycol tert-octylphenyl ether, polysorbate-80, sorbitan monooleate, sodium dodecyl sulfate, poloxamer 188, cetyltrimethylammonium bromide, and polysorbate-20. Exemplary bulking agents include, but are not limited to, sucrose, lactose, mannitol, sodium chloride, gelatin, aluminum salts, and hydrolyzed gelatin. Exemplary sugars include, but are not limited to, sucrose, lactose, mannitol, dextrose, fructose, and trehalose. Exemplary salts include, but are not limited to, sodium chloride, potassium chloride, sodium phosphate, potassium phosphate, potassium citrate, aluminum salts, calcium chloride, magnesium chloride, sodium sulfate, ammonium sulfate, and sodium citrate. In some embodiments, the excipients further include histidine, arginine, and glycine.

[0150] In some embodiments, the excipient is present in the solution. In some embodiments, the excipient is present in concentrations of approximately 1 μg / mL to approximately 1 g / mL (for example, 1 μg / mL, 50 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL, 250 μg / mL, 300 μg / mL, 350 μg / mL, 400 μg / mL, 450 μg / mL, 500 μg / mL, 550 μg / mL, 600 μg / mL, 650 μg / mL, 700 μg / mL, 750 μg / mL, 800 μg / mL, 850 μg / mL, 900 μg / mL, 950 μg / mL, 1,000 μg / mL, 50 mg / mL, It is present in concentrations of 100 mg / mL, 150 mg / mL, 200 mg / mL, 250 mg / mL, 300 mg / mL, 350 mg / mL, 400 mg / mL, 450 mg / mL, 500 mg / mL, 550 mg / mL, 600 mg / mL, 650 mg / mL, 700 mg / mL, 750 mg / mL, 800 mg / mL, 850 mg / mL, 900 mg / mL, 950 mg / mL, 1 g / mL, 1.5 g / mL, 2 g / mL, or up to 2.5 g / mL (including all values ​​and ranges contained therein).

[0151] In some embodiments, the excipient is present in an amount of about 0.1% to about 0.5% of the composition. The excipient may be present in an amount of about 0.1%, 0.2%, 0.3%, 0.4%, or up to about 0.5% of the composition.

[0152] kit This specification also discloses kits comprising the disclosed compositions. Kits for the treatment or prevention of cancer, pathogen infection, or immunodeficiency are also disclosed herein. In aspects, a kit may comprise a therapeutic or prophylactic composition comprising an effective amount of the composition comprising recombinant spirulina (e.g., transformed in a scaffold) in unit dosage forms. In aspects, a kit may comprise a sterile container capable of containing the therapeutic composition of spirulina, such containers may be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister packs, or other suitable container forms known in the art. Such containers may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding pharmaceuticals. In aspects, the transformed spirulina may be provided with instructions for administering the spirulina to subjects who have or are at risk of developing cancer, pathogen infection, or immunodeficiency.

[0153] Numbered Embodiments Notwithstanding the attached claims, the following numbered embodiments also form part of this disclosure.

[0154] Embodiment Set 1 1. Recombinant spirulina expressing a mutant small membrane-bound A kinase anchoring protein (smAKAP) peptide sequence.

[0155] 2. Recombinant spirulina according to Embodiment 1, wherein the spirulina contains at least 2, 3, 4, 5, 6, or 7 mutant residues in the mutant smAKAP peptide sequence compared to the wild-type smAKAP peptide sequence.

[0156] 3. Recombinant spirulina according to Embodiment 1, wherein one residue in the smAKAP peptide sequence is mutated compared to the wild-type smAKAP peptide sequence.

[0157] 4. Recombinant spirulina according to any one of Embodiments 2 to 3, wherein two residues in the smAKAP peptide sequence are mutated compared to the wild-type smAKAP peptide sequence.

[0158] 5. Recombinant spirulina according to any one of Embodiments 2 to 4, wherein three residues in the smAKAP peptide sequence are mutated compared to the wild-type smAKAP peptide sequence.

[0159] 6. Recombinant spirulina according to any one of Embodiments 1 to 5, wherein the mutant smAKAP peptide exhibits resistance to protease cleavage, determined by a decrease in the detection of cleavage products, when exposed to a solvent containing a protease for one hour.

[0160] 7. Recombinant spirulina according to Embodiment 6, wherein up to approximately 5%, 10%, 20%, 30%, 40%, 50%, or 60% of the smAKAP peptide sequence is cleaved.

[0161] 8. Recombinant spirulina according to any one of Embodiments 1 to 7, wherein the mutation is in a hydrophobic residue.

[0162] 9. Recombinant spirulina according to any one of Embodiments 1 to 8, wherein when the recombinant spirulina is immersed in a solvent, the mutant residues are exposed to the solvent.

[0163] 10. Recombinant spirulina according to any one of Embodiments 1 to 9, wherein the mutant residue is selected from the group consisting of C16S, C24S, E5D, Y6H, W22S, C24G, L4E, R9E, L4I, L10I, L19I, and combinations thereof, based on Sequence ID No. 2.

[0164] 11. Recombinant spirulina according to Embodiment 10, wherein the mutant residues are C16S and C24S compared to SEQ ID NO: 2.

[0165] 12. Recombinant spirulina according to Embodiment 10, wherein the mutant residues are E5D, Y6H, C16S, W22S, and C24G compared to SEQ ID NO: 2.

[0166] 13. Recombinant spirulina according to Embodiment 10, wherein the mutant residues are L4E, E5D, Y6H, R9E, C16S, W22S, and C24G compared to SEQ ID NO: 2.

[0167] 14. Recombinant spirulina according to Embodiment 10, wherein the mutant residues are L4E, C16S, W22S, and C24G compared to SEQ ID NO: 2.

[0168] 15. Recombinant spirulina according to Embodiment 10, wherein the mutant residues are R9E, C16S, and C24G compared to SEQ ID NO: 2.

[0169] 16. Recombinant spirulina according to Embodiment 10, wherein the mutant residues are R9E, C16S, W22S, and C24G compared to SEQ ID NO: 2.

[0170] 17. Recombinant spirulina according to any one of Embodiments 1 to 16, wherein a mutant smAKAP peptide sequence is linked to a monomeric heterologous moiety.

[0171] 18. Recombinant spirulina according to Embodiment 17, wherein a mutant smAKAP peptide sequence is linked to a second heterologous moiety.

[0172] 19. Recombinant spirulina according to Embodiment 18, wherein the heterologous moiety and the second heterologous moiety are identical.

[0173] 20. Recombinant spirulina according to any one of embodiments 17 to 18, wherein the heterologous moiety and the second heterologous moiety are different.

[0174] 21. Recombinant spirulina according to any one of Embodiments 1 to 20, wherein the recombinant spirulina expresses another exogenous polypeptide sequence.

[0175] 22. Recombinant spirulina according to Embodiment 21, wherein the exogenous polypeptide sequence is selected from the group consisting of the oligomerization domain of C4b-binding protein (C4BP), cholera toxin b subunit, oligomerization domain of extracellular matrix protein, TRX, 5HVZ, cTRP, SP651, SP737, and 4B0F.

[0176] 23. Recombinant spirulina according to Embodiment 22, wherein the exogenous polypeptide sequence is 5HVZ.

[0177] Recombinant spirulina according to Embodiment 23, wherein 24.5HVZ is linked to a third heterogeneous moiety.

[0178] 25. Recombinant spirulina according to Embodiment 24, wherein the third heterogeneous moiety is homodimerized.

[0179] 26. Recombinant spirulina according to any one of embodiments 17 to 25, wherein the heterologous moiety, the second heterologous moiety, and the third heterologous moiety are identical.

[0180] 27. Recombinant spirulina according to any one of embodiments 17 to 25, wherein the heterologous moiety, the second heterologous moiety, and the third heterologous moiety are different.

[0181] 28. Recombinant spirulina according to any one of embodiments 17 to 27, wherein a different moiety is used as a binder.

[0182] 29. Recombinant spirulina according to Embodiment 28, wherein the binder is selected from the group consisting of fab', F(ab')2, fv, domain antibody (dAb), complementarity-determining region (CDR) fragment, CDR graft antibody, single-chain antibody (scFv), single-chain antibody fragment, chimeric antibody, diabody, triabody, tetrabody, minibody, linear antibody, intrabody, nanobody (single-domain antibody), small modular immunotherapy product (SMIP), antigen-binding domain immunoglobulin fusion protein, and VHH.

[0183] 30. Recombinant spirulina according to Embodiment 29, wherein the binder is VHH.

[0184] 31. Recombinant spirulina according to Embodiment 30, wherein VHH binds to a pathogen.

[0185] 32. Recombinant spirulina according to Embodiment 30, wherein VHH binds to cancer cells.

[0186] 33. Recombinant spirulina according to Embodiment 30, wherein VHH binds to human cells.

[0187] 34. Recombinant spirulina according to Embodiment 31, wherein VHH binds to a pathogen selected from the group consisting of bacteria, fungi, and viruses.

[0188] 35. Pathogens include Escherichia coli (E. coli), enterotoxigenic Escherichia coli (ETEC), anthrax, EHEC, EAEC, Shigella, Mycobacterium, Streptococcus, Staphylococcus, Shigella, Campylobacter, Salmonella, Clostridium, Corynebacterium, Pseudomonas, Neisseria, Listeria, Vibrio, Bordetella, Legionella, bacteriophages, RNA bacteriophages (e.g., MS2, AP205), PP7 and Qβ), Helicobacter pylori, infectious hematopoietic necrosis virus, parvovirus, herpes simplex virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, measles virus, mumps virus, rubella virus, HIV, influenza virus, rhinovirus, rotavirus A, rotavirus B, rotavirus C, respiratory syncytial virus (RSV), varicella-zoster virus, poliovirus, norovirus, Zika virus, dengue virus, rabies virus, Newcastle disease virus, vitiligo virus, coronavirus, SARS-related coronavirus, MERS-related coronavirus, SARS-CoV-2, Aspergillus, Candida, Blastomyces, Coccidioides, Cryptococcus, Histoplasma, Plasmodium, Plasmodium falciparum, Plasmodium malariae Recombinant spirulina according to Embodiment 34, selected from the group consisting of worms, malaria parasites (oval malaria parasites), Plasmodium vivax, Trypanosoma, Toxoplasma, Giardia, Leishmania, Cryptosporidium, Hellworm parasites: Whipworms, Pinworms, Ascaris, Hookworms, and American hookworms, Strongyloides, Dracunculus, Onchocerca, Filamentosa, Tapeworms, Hydatidiformes, Diphyllobothrium, Fasciola, and Schistosoma.

[0189] 36. Recombinant spirulina according to Embodiment 34, wherein the pathogen is a bacterium, and the bacterium is selected from the group consisting of Mycobacterium, Streptococcus, Staphylococcus, Shigella, Campylobacter, Salmonella, Clostridium, Corynebacterium, Pseudomonas, Neisseria, Listeria, Vibrio, Bordetella, and Legionella.

[0190] 37. Recombinant spirulina according to Embodiment 36, wherein the bacterium is Campylobacter.

[0191] 38. Recombinant spirulina according to Embodiment 36, wherein the bacterium is Clostridium.

[0192] 39. Recombinant spirulina according to any one of embodiments 35 to 38, wherein VHH contains a sequence having at least 85% identity with the sequences of sequence numbers 25 to 67.

[0193] 40. Recombinant spirulina according to any one of embodiments 35 to 38, wherein VHH contains the sequence of sequence numbers 25 to 67.

[0194] 41. Recombinant spirulina according to any one of embodiments 23 to 40, wherein the recombinant spirulina expresses a scaffold having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequences of SEQ ID NOs. 6, 14, 16, 18, 20, 22, 23, and 24.

[0195] 42. Recombinant spirulina expressing a mutant small membrane-bound A kinase anchoring protein (smAKAP) peptide sequence, wherein the mutation includes a residue substitution based on Sequence ID No. 2.

[0196] 43. Recombinant spirulina expressing a mutant small membrane-bound A kinase anchoring protein (smAKAP) peptide sequence, wherein the mutation is selected from the group consisting of C16S, C24S, E5D, Y6H, W22S, C24G, L4E, R9E, L4I, L10I, L19I, and combinations thereof, based on Sequence ID No. 2.

[0197] 44. Recombinant spirulina according to any one of embodiments 42 to 43, wherein smAKAP is ligated to one or more VHH antibodies at its terminus.

[0198] 45. Recombinant spirulina according to Embodiment 44, wherein smAKAP is ligated to two VHH antibodies, with the first VHH at the C-terminus and the second VHH at the N-terminus.

[0199] 46. ​​Recombinant spirulina according to any one of embodiments 42 to 45, wherein the recombinant spirulina expresses 5HVZ.

[0200] Recombinant spirulina according to Embodiment 46, wherein 47.5HVZ is linked to two VHH antibodies in a homodimer configuration.

[0201] 48. A polynucleotide sequence containing a mutant small membrane-bound A kinase anchoring protein (smAKAP) sequence in which the mutation is a single residue substitution compared to the wild-type smAKAP sequence.

[0202] 49. A vector comprising the polynucleotide sequence described in Embodiment 48.

[0203] 50. A method for producing recombinant spirulina, comprising contacting spirulina cells with the vector described in Embodiment 49.

[0204] 51. A pharmaceutical composition comprising recombinant spirulina according to any one of Embodiments 1 to 47, and an excipient.

[0205] 52. A kit comprising recombinant spirulina according to any one of Embodiments 1 to 47, a polynucleotide sequence according to Embodiment 48, a vector according to Embodiment 49, or a pharmaceutical composition according to Embodiment 51, and instructions for use thereof.

[0206] 53. A method of treatment comprising administering the pharmaceutical composition described in Embodiment 51 to a subject in need thereof, wherein the subject is infected with a bacterial or viral infection.

[0207] 54. A vector containing nucleotide sequences encoding polypeptides of sequence numbers 5-7 or 14-24.

[0208] 55. Recombinant spirulina comprising the vector described in Embodiment 54.

[0209] Embodiment Set 2 1. Recombinant spirulina expressing a mutant small membrane-bound A kinase anchoring protein (smAKAP) peptide sequence.

[0210] 2. Recombinant spirulina according to Embodiment 1, wherein the spirulina contains at least 2, 3, 4, 5, 6, or 7 mutant residues in the mutant smAKAP peptide sequence compared to the wild-type smAKAP peptide sequence.

[0211] 3. Recombinant spirulina according to Embodiment 1, wherein one residue in the smAKAP peptide sequence is mutated compared to the wild-type smAKAP peptide sequence.

[0212] 4. Recombinant spirulina according to any one of Embodiments 2 to 3, wherein two residues in the smAKAP peptide sequence are mutated compared to the wild-type smAKAP peptide sequence.

[0213] 5. Recombinant spirulina according to any one of Embodiments 2 to 4, wherein three residues in the smAKAP peptide sequence are mutated compared to the wild-type smAKAP peptide sequence.

[0214] 6. Recombinant spirulina according to any one of Embodiments 1 to 5, wherein the mutant smAKAP peptide exhibits resistance to protease cleavage, determined by a decrease in the detection of cleavage products, when exposed to a solvent containing a protease for one hour.

[0215] 7. Recombinant spirulina according to Embodiment 6, wherein up to approximately 5%, 10%, 20%, 30%, 40%, 50%, or 60% of the smAKAP peptide sequence is cleaved.

[0216] 8. Recombinant spirulina according to any one of Embodiments 1 to 7, wherein the mutation is in a hydrophobic residue.

[0217] 9. Recombinant spirulina according to any one of Embodiments 1 to 8, wherein when the recombinant spirulina is immersed in a solvent, the mutant residues are exposed to the solvent.

[0218] 10. Recombinant spirulina according to any one of Embodiments 1 to 9, wherein the mutant residue is selected from the group consisting of C16S, C24S, E5D, Y6H, W22S, C24G, L4E, R9E, L4I, L10I, L19I, and combinations thereof, based on Sequence ID No. 2.

[0219] 11. Recombinant spirulina according to Embodiment 10, wherein the mutant residues are C16S and C24S compared to SEQ ID NO: 2.

[0220] 12. Recombinant spirulina according to Embodiment 10, wherein the mutant residues are E5D, Y6H, C16S, W22S, and C24G compared to SEQ ID NO: 2.

[0221] 13. Recombinant spirulina according to Embodiment 10, wherein the mutant residues are L4E, E5D, Y6H, R9E, C16S, W22S, and C24G compared to SEQ ID NO: 2.

[0222] 14. Recombinant spirulina according to Embodiment 10, wherein the mutant residues are L4E, C16S, W22S, and C24G compared to SEQ ID NO: 2.

[0223] 15. Recombinant spirulina according to Embodiment 10, wherein the mutant residues are R9E, C16S, and C24G compared to SEQ ID NO: 2.

[0224] 16. Recombinant spirulina according to Embodiment 10, wherein the mutant residues are R9E, C16S, W22S, and C24G compared to SEQ ID NO: 2.

[0225] 17. Recombinant spirulina according to Embodiment 10, wherein the mutant residues are L4I, R9E, C16S, and C24G compared to SEQ ID NO: 2.

[0226] 18. Recombinant spirulina according to any one of Embodiments 1 to 17, wherein a mutant smAKAP peptide sequence is linked to a monomeric heterologous moiety.

[0227] 19. Recombinant spirulina according to Embodiment 18, wherein a mutant smAKAP peptide sequence is linked to a second heterologous moiety.

[0228] 20. Recombinant spirulina according to Embodiment 19, wherein the heterologous moiety and the second heterologous moiety are identical.

[0229] 21. Recombinant spirulina according to any one of embodiments 18 to 19, wherein the heterologous moiety and the second heterologous moiety are different.

[0230] 22. Recombinant spirulina according to any one of Embodiments 1 to 21, wherein the recombinant spirulina expresses another exogenous polypeptide sequence.

[0231] 23. Recombinant spirulina according to Embodiment 22, wherein the exogenous polypeptide sequence is selected from the group consisting of the oligomerized domain of C4b-binding protein (C4BP), cholera toxin b subunit, oligomerized domain of extracellular matrix protein, TRX, 5HVZ, cTRP, SP651, SP737, and 4B0F.

[0232] 24. Recombinant spirulina according to Embodiment 23, wherein the exogenous polypeptide sequence is 5HVZ.

[0233] Recombinant spirulina according to Embodiment 24, wherein 25.5HVZ is linked to a third heterogeneous moiety.

[0234] 26. Recombinant spirulina according to Embodiment 25, wherein the third heterogeneous moiety is in a dimer configuration.

[0235] 27. Recombinant spirulina according to any one of embodiments 18 to 26, wherein the heterologous moiety, the second heterologous moiety, and the third heterologous moiety are identical.

[0236] 28. Recombinant spirulina according to any one of embodiments 18 to 26, wherein the heterologous moiety, the second heterologous moiety, and the third heterologous moiety are different.

[0237] 29. Recombinant spirulina according to any one of embodiments 18 to 28, wherein a different species of moiety is used as a binder.

[0238] 30. Recombinant spirulina according to Embodiment 29, wherein the binder is selected from the group consisting of fab', F(ab')2, fv, domain antibody (dAb), complementarity-determining region (CDR) fragment, CDR graft antibody, single-chain antibody (scFv), single-chain antibody fragment, chimeric antibody, diabody, triabody, tetrabody, minibody, linear antibody, intrabody, nanobody (single-domain antibody), small modular immunotherapy product (SMIP), antigen-binding domain immunoglobulin fusion protein, and VHH.

[0239] 31. Recombinant spirulina according to Embodiment 30, wherein the binder is VHH.

[0240] 32. Recombinant spirulina according to Embodiment 31, wherein VHH binds to a pathogen.

[0241] 33. Recombinant spirulina according to Embodiment 31, wherein VHH binds to cancer cells.

[0242] 34. Recombinant spirulina according to Embodiment 31, wherein VHH binds to human cells.

[0243] 35. Recombinant spirulina according to Embodiment 32, wherein VHH binds to a pathogen selected from the group consisting of bacteria, fungi, and viruses.

[0244] 36. Pathogens include Escherichia coli (E. coli), enterotoxigenic Escherichia coli (ETEC), anthrax, EHEC, EAEC, Shigella, Mycobacterium, Streptococcus, Staphylococcus, Shigella, Campylobacter, Salmonella, Clostridium, Corynebacterium, Pseudomonas, Neisseria, Listeria, Vibrio, Bordetella, Legionella, bacteriophages, RNA bacteriophages (e.g., MS2, AP205), PP7 and Qβ), Helicobacter pylori, infectious hematopoietic necrosis virus, parvovirus, herpes simplex virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, measles virus, mumps virus, rubella virus, HIV, influenza virus, rhinovirus, rotavirus A, rotavirus B, rotavirus C, respiratory syncytial virus (RSV), varicella-zoster virus, poliovirus, norovirus, Zika virus, dengue virus, rabies virus, Newcastle disease virus, vitiligo virus, coronavirus, SARS-related coronavirus, MERS-related coronavirus, SARS-CoV-2, Aspergillus, Candida, Blastomyces, Coccidioides, Cryptococcus, Histoplasma, Plasmodium, Plasmodium falciparum, Plasmodium malariae Recombinant spirulina according to Embodiment 35, selected from the group consisting of worms, ovoid malaria parasites, Plasmodium vivax, Trypanosoma, Toxoplasma, Giardia, Leishmania, Cryptosporidium, Hellworm parasites: Whipworms, Pinworms, Ascaris, Hookworms, and American Hookworms, Strongyloides, Dracunculus, Onchocerca, Filamentosa, Tapeworms, Hydatidiformes, Diphyllobothrium, Fasciola, and Schistosoma.

[0245] 37. Recombinant spirulina according to Embodiment 35, wherein the pathogen is a bacterium, and the bacterium is selected from the group consisting of Mycobacterium, Streptococcus, Staphylococcus, Shigella, Campylobacter, Salmonella, Clostridium, Corynebacterium, Pseudomonas, Neisseria, Listeria, Vibrio, Bordetella, and Legionella.

[0246] 38. Recombinant spirulina according to Embodiment 37, wherein the bacterium is Campylobacter.

[0247] 39. Recombinant spirulina according to Embodiment 37, wherein the bacterium is Clostridium.

[0248] 40. Recombinant spirulina according to any one of embodiments 36 to 39, wherein VHH contains a sequence having at least 85% identity with the sequences of sequence numbers 25 to 67.

[0249] 41. Recombinant spirulina according to any one of embodiments 36 to 39, wherein VHH contains the sequence of sequence numbers 25 to 67.

[0250] 42. Recombinant spirulina according to any one of embodiments 24 to 41, wherein the recombinant spirulina expresses a scaffold having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequences of SEQ ID NOs: 4, 6, 14, 16, 18, 20, 22, 23, 24, and 76.

[0251] 43. Recombinant spirulina expressing a mutant small membrane-bound A kinase anchoring protein (smAKAP) peptide sequence, wherein the mutation includes a residue substitution based on Sequence ID No. 2.

[0252] 44. Recombinant spirulina expressing a mutant small membrane-bound A kinase anchoring protein (smAKAP) peptide sequence, wherein the mutation is selected from the group consisting of C16S, C24S, E5D, Y6H, W22S, C24G, L4E, R9E, L4I, L10I, L19I, and combinations thereof, based on Sequence ID No. 2.

[0253] 45. Recombinant spirulina according to any one of embodiments 43 to 44, wherein smAKAP is ligated to one or more VHH antibodies at its terminus.

[0254] 46. ​​Recombinant spirulina according to Embodiment 45, wherein smAKAP is ligated to two VHH antibodies, with the first VHH at the C-terminus and the second VHH at the N-terminus.

[0255] 47. Recombinant spirulina according to any one of embodiments 43 to 46, wherein the recombinant spirulina expresses 5HVZ.

[0256] Recombinant spirulina according to Embodiment 47, wherein 48.5HVZ is linked to two VHH antibodies in a homodimer configuration.

[0257] 49. A polynucleotide sequence comprising a mutant small membrane-bound A kinase anchoring protein (smAKAP) sequence, wherein the mutation comprises one or more residue substitutions compared to the wild-type smAKAP sequence.

[0258] 50. A vector comprising the polynucleotide sequence described in Embodiment 49.

[0259] 51. A method for producing recombinant spirulina, comprising contacting spirulina cells with the vector described in Embodiment 50.

[0260] 52. A pharmaceutical composition comprising recombinant spirulina according to any one of Embodiments 1 to 48, and an excipient.

[0261] 53. A kit comprising recombinant spirulina according to any one of Embodiments 1 to 48, a polynucleotide sequence according to Embodiment 49, a vector according to Embodiment 50, or a pharmaceutical composition according to Embodiment 52, and instructions for use thereof.

[0262] 54. A method of treatment comprising administering the pharmaceutical composition described in Embodiment 52 to a subject in need thereof, wherein the subject is infected with a bacterial or viral infection.

[0263] 55. A vector containing nucleotide sequences encoding polypeptides of sequence numbers 2-7, 14-24, 74, and / or 76.

[0264] 56. The vector according to Embodiment 55, wherein the vector contains approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with respect to Sequence ID No. 75.

[0265] 56. Recombinant spirulina comprising the vector described in any one of embodiments 55 to 56. [Examples]

[0266] Example 1 - Evaluation of protease sensitivity Purpose of the clinical trial To evaluate the protease sensitivity of exemplary scaffold constructs, the hydra-body complex PP2130 (a complex of PP1113 and PP1895) was compared to the protease sensitivity of the following constructs: PP917 2xS3b-C8, PP1895 2xS3b-C8, and PP1895 2xRN-29.

[0267] In short, to evaluate the in vitro protease stability of clones, purified protein samples were prepared to 0.2 mg / mL in protease digestion buffer (20 mM Bis-Tris, 150 mM NaCl, 3 mM CaCl2 pH 6.0). The protease enzymes trypsin and chymotrypsin were diluted from stock concentration of 1 mg / mL to 0.2 mg / mL in protease digestion buffer. Both proteins and enzymes were diluted 1:10 in protease digestion buffer. The digestion reaction was carried out by mixing 30 μL of diluted protein with 30 μL of diluted protease. The reaction mixture was mixed by centrifugation at high speed for 30 seconds and incubated in an Eppendorf ThermoMixer at 37°C for 60 minutes with shaking at 900 rpm. The reaction was stopped with an equal volume of stop buffer containing 2 mM PMSF and 2× Pierce protease inhibitor minitablets in PBS. The final concentration of each VHH homodimer after protease neutralization was 0.1 mg / mL. The samples were kept on ice for the ELISA assay.

[0268] The digested protein samples were analyzed for binding activity via ELISA. High-binding ELISA plates were coated by adding 1 μg / mL of SARS-COV-2 RBD in CBC binding buffer at 100 μl / well each and incubated overnight at 4°C. The plates were washed three times with PBS supplemented with 0.05% Tween®-20 (PBS-tw), and then blocked for 1 hour using blocking buffer (PBS-tw supplemented with 5% non-fat dry milk) at 300 μl / well each. Dilutions of undigested and digested proteins were prepared in low-binding 96-well plates by serial dilution with blocking buffer. 100 μL of each protein dilution was added to ELISA plate wells and incubated for 1 hour at room temperature on a plate shaker. The plates were washed and assayed for binding. The HRP-conjugated anti-camelid VHH antibody cocktail (GenScript catalog number A02016) was diluted 10,000-fold in blocking buffer and 100 μl / well was added to the ELISA plates. The plates were incubated for 30 minutes with shaking. The ELISA plates were washed twice with PBS-tw and once with PBS. The HRP reaction was initiated by adding 100 μl / well of TMB Ultra ELISA substrate (Thermo Fisher) to each well. The ELISA plates were incubated at room temperature for 5 minutes to develop color and quenched with 50 μL of 1 M hydrochloric acid. Absorbance was measured at 450 nm using a Spectra Max M5 plate reader.

[0269] The results show that the PP1895 construct is sensitive to both trypsin protease and chymotrypsin protease, with approximately 99% of its activity lost after 1 hour of incubation. On the other hand, the hydrabody complex, PP2130, restores stability under trypsin conditions (see Figures 1A - 1D).

[0270] Example 2 - Mutations in smAKAP to Confer Trypsin and Chymotrypsin Resistance After the protease sensitivity study of Example 1 showing protease sensitivity in smAKAP, mutations in smAKAP were evaluated to determine whether trypsin and chymotrypsin resistance could be engineered into smAKAP.

[0271] To generate mutant smAKAP clones, protein - protein interaction analysis using structural analysis of the complex crystal structure (PDBID 5HVZ) and PISA analysis was performed. For the side chains of arginine (R) and lysine (K) predicted to be trypsin protease substrates, the importance of complex formation was evaluated using structural tools. Similarly, for hydrophobic side chains predicted to be chymotrypsin substrates, evaluation was carried out and potential mutations to maintain complex formation were proposed. Various mutants were generated, including the following mutations: C16S, C24S, E5D, Y6H, W22S, C24G, L4E, R9E, L4I, L10I, L19I and various combinations thereof, compared to the wild - type sequence based on SEQ ID NO: 2. [Table 2]

[0272] Exemplary mutant smAKAP linkers were utilized in the scaffold constructs (see Table 2. See also Figure 4). [Table 3 - 1] [Table 3 - 2] [Table 3 - 3] [Table 3 - 4]

[0273] In the PP1895 construct, since any small amino acid is tolerated at the position, C78S can be exchanged with C78G.

[0274] Example 3 - Mutant smAKAP clone is expressed in an E. coli system. smAKAP variants were generated according to the mutations listed in Table 1, and their expressibility in the E. coli system was evaluated.

[0275] In short, the construct was subcloned into a modified pET28b(+) vector in which the kanamycin bacterial resistance gene was replaced with an ampicillin bacterial resistance gene. The sequence-validated plasmid was transformed into BL21(DE3) E. coli cells (New England Biolabs) and seeded on LB medium supplemented with 100 μg mL-1 of ampicillin. Proteins were expressed using a self-induction protocol in which single colonies of individual transformants were inoculated into 100 mL of Super Broth medium containing appropriate selective agents, shaken at 37°C for 8 hours, followed by shaking at 19°C for 36 hours. The cultures were centrifuged, and the pellet was stored at -20°C until purification. The frozen pellet was thawed on ice and resuspended in 30 mL of 300 mM NaCl at 50 mM Tris pH 8.0 supplemented with Pierce protease inhibitor tablets and 1 mM PMSF. Cells were lysed by high-pressure homogenization at 12,000 psi (Microfluidics LM20). The lysates were centrifuged at 18,500 g for 30 minutes at 4°C using a benchtop centrifuge (Eppendorf Centrifuge 5810R). The supernatant was used for further processing. Recombinant proteins were purified from the clarified lysates by affinity chromatography using a 5 mL HisTrap column (Cytiva, catalog no. 17524802) equilibrated with 50 mM Tris pH 8.0, 300 mM NaCl, and 20 mM imidazole (Thermo Fisher Scientific, catalog no. O3196-500). Samples purified by hexahistidine tagging and HisTrap were further purified using AKTA Pure FPLC-based size exclusion chromatography Superdex 200 increase 10 / 300 (Cytiva, catalog no. 28990944). Protein purity and size were assayed by size exclusion chromatography using an analytical Superdex 200 increase 10 / 300 (Cytiva, catalog number 28990944). 100 μL of purified protein with a purity of approximately 1 mg / mL was analyzed in 1x PBS buffer at room temperature.The purified protein samples were also evaluated by SDS-PAGE under reducing and non-reducing conditions. 3 μg of protein sample was incubated at 70°C in LDS loading buffer with or without 50 mM dithiothreitol (DTT) reducing agent (Thermo Fisher Scientific, catalog no. R0862).

[0276] The results show that all mutant smAKAP clones were expressed in E. coli (see Figure 5).

[0277] Example 4: Evaluation of smAKAP variants and protease resistance Trypsin resistance Following the method of Example 1, smAKAP mutants with the R63E substitution were generated, and it was evaluated whether the mutation conferred trypsin resistance to the mutant smAKAP peptide.

[0278] Clones PP2451, PP1895, and PP2455 were evaluated for trypsin resistance using the same method as described in Example 1.

[0279] The results show that the R63E mutation restores trypsin sensitivity to wild-type smAKAP (see Figures 6A-6C).

[0280] Chymotrypsin resistance The smAKAP mutants generated according to Example 1 were also evaluated for their resistance to chymotrypsin cleavage. Specifically, clones PP2451, PP1895, PP2455, PP2456, PP2454, PP2452, and PP2453 were subjected to assays using the same method as described in Example 1.

[0281] The results show that resistance is best improved by combining hydrophobic mutations such as PP2453 (see Figure 9). Furthermore, in cases where chymotrypsin sensitivity is detected, it is mainly attributable to the presence of the smAKAP linker. However, alternative linkers can be used with the compositions of this disclosure.

[0282] Example 5 - Complex Formation Analysis Generation of Dimer Complexes The ability to generate dimers of smAKAP and 5HVZ and form scaffold complexes was evaluated.

[0283] Dimer formation via the 5HVZ motif is due to protein - protein interactions between three α - helix strands, arranging monomeric polypeptides in an antiparallel structure. Furthermore, the formation of asymmetric disulfide bonds between protomers further strengthens the dimer structure. The dimeric protein is expressed from a clone that genetically encodes a single polypeptide with the 5HVZ dimerization motif fused to the target protein. The dimerization motif (5HVZ) can be fused to both the N - terminus and C - terminus to generate homodimer or heterodimer protein constructs. Designing VHH constructs using the 5HVZ dimerization motif for Spirulina expression has been shown to increase the binding avidity for the target antigen. Dimer formation by the 5HVZ motif results in a protein fold that functions as a docking platform to facilitate the interaction between PKA and membrane - bound A - kinase - anchoring protein (AKAP). A small alpha - helix peptide within AKAP, smAKAP, docks to the 5HVZ platform.

[0284] Complex Formation Evaluation To verify whether smAKAP mutants retain the ability to form scaffold complexes, complex formation analysis was completed using the PP2451, PP1895, PP2456 (W76S), and PP2455 (R63E mutant) clones.

[0285] To evaluate higher-order complex formation (hydra-body formation), purified dimeric proteins (PP917 and PP1113) whose homodimerization is achieved via a 5HVZ scaffold were analyzed using a Superdex 200 increase 10 / 300 (Cytiva, catalog number 28990944). Proteins were analyzed alone or with smAKAP mutant and control clone proteins in a 1:2 molar ratio. Complex formation efficiency was compared by comparing the retention volume and protein absorbance when evaluating the dimers and smAKAP-containing constructs alone. The smAKAP-containing construct that complexes with the 5HVZ dimeric scaffold-containing construct shows a shift in retention volume toward a smaller value and an increase in absorbance toward the complex. Furthermore, the complex-forming construct shows a significant decrease in the height of the absorbance peak at the retention volume where the smAKAP-containing construct is expected to elute.

[0286] The results indicate that the R63E and W76S substitutions did not affect the complex formation of the scaffold containing the mutant smAKAP (see Figure 8). Therefore, not only did it not affect complex formation, but the mutant smAKAP showed improved protease resistance compared to the control.

[0287] Using a method similar to that described above, we completed additional studies using the following alternative mutant clones: PP2451, PP1895, PP2456, PP2454, PP2452, and PP2453.

[0288] Specifically, the crystal structures of smAKAP and the dimer interface were calculated based on the crystal structure (PDB ID 5HVZ) using the web-based interactive analysis tool PDBePISA (PISA). The interface formed between the dimerization motif "5HVZ" and the smAKAP docking peptide was evaluated and scored in terms of physicochemical properties contributing to stabilization. The PISA analysis tool predicts the strength of complex formation using physicochemical properties including solvation energy, interface area, hydrogen bonding, free energy of formation, and salt bridge formation. The summary data presented in Tables 3-4 shows hydrogen bonding or salt bridge formation by the indicated amino acid chain (HSDC column), solvent-accessible surface area (ASA), buried surface area (BSA), buried area percentage (one bar per 10%), and solvation energy effect (ΔiG). The analysis was used to determine the effect of mutations on complex formation. [Table 4] [Table 5]

[0289] These results indicate that the Y60H mutation has the greatest impact on complex formation (see also Figure 10).

[0290] Example 6 - Increased complex stability due to smAKAP mutation The ability of additional mutations to stabilize the smAKAP complex was evaluated. Clones PP2451–PP2456 were subjected to stability testing. To assess the stability of purified proteins, protein samples were evaluated by SDS-PAGE gel under non-reducing or reducing (50 mM DTT) conditions. Proteins from the same lot were stored at 4°C for up to 6 weeks and their stability was evaluated by SDS-PAGE gel under non-reducing or reducing (50 mM DTT) conditions. Comparative analysis of SDS-PAGE gels showed reduced fragmentation in some smAKAP mutant linker clones.

[0291] The results indicate that clones PP2453 and PP2455 containing the R63E mutant are more stable (see Figure 11 vs. Figure 13).

[0292] Example 7 - The smAKAP linker-containing construct is expressed in spirulina. smAKAP variants were generated according to the mutations listed in Table 1, and their expressibility in a spirulina system was evaluated.

[0293] In short, the construct was subcloned into a modified pET28b(+) vector in which the kanamycin bacterial resistance gene was replaced with an ampicillin bacterial resistance gene. The sequence-validated plasmid was transformed into spirulina. The protein was expressed and isolated and assayed by size exclusion chromatography using Superdex 200 increase 10 / 300 (Cytiva, catalog no. 28990944). 100 μL of purified protein at approximately 1 mg / mL was analyzed in 1x PBS buffer at room temperature. The purified protein samples were also evaluated by SDS-PAGE under reducing and non-reducing conditions. 3 μg of protein sample was incubated at 70°C in LDS loading buffer with or without 50 mM dithiothreitol (DTT) reducing agent (Thermo Fisher Scientific, catalog no. R0862). [Table 6]

[0294] The results show that the smAKAP linker-containing construct is expressed in spirulina (see Figure 12).

[0295] Example 8 - Higher-order complex formation via smAKAP mutant Higher-order complex formation using the 5HVZ dimerization domain can be achieved by complexing with a protein linked to the smAKAP peptide. The effect of amino acid substitutions in the smAKAP peptide on complex formation was evaluated through a study comparing the wild-type smAKAP peptide (SEQ ID NO: 2) and the PP6510 mutant (SEQ ID NO: 74).

[0296] In short, the protein linked via the PP6510 mutant was cloned into a pET28 expression vector (SEQ ID NO: 75) and expressed in E. coli cells. The purified protein was evaluated by size exclusion chromatography (SEC). To analyze complex formation, purified dimerized proteins (PP6510 and PP1116), in which homodimerization was achieved via a 5HVZ scaffold, were analyzed using a Superdex 200 increase 10 / 300 (Cytiva, catalog number 28990944). The proteins were analyzed alone or with the smAKAP mutant and control clone proteins in a 1:1.2 molar ratio. Complex formation efficiency was compared by comparing the retention volume and protein absorbance when evaluating the dimers and smAKAP-containing constructs alone.

[0297] The results indicate that the applicant successfully formed a 5HVZ-mediated homodimeric protein and a higher-order oligomer using the PP6510 mutant (see Figure 14).

[0298] Built-in by reference All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entirety for all purposes. However, no reference to any reference, article, publication, patent, patent publication, or patent application cited herein constitutes, or is suggested to constitute, prior art in any country of the world, or part of common knowledge, nor should it be construed as such.

[0299] The provided composition may contain amino acid sequences described in international patent applications PCT / US2020 / 040794 and PCT / US2021 / 065138.

Claims

1. Recombinant spirulina expressing a mutant small membrane-bound A kinase anchoring protein (smAKAP) peptide sequence.

2. The recombinant spirulina according to claim 1, wherein the spirulina comprises at least 2, 3, 4, 5, 6, or 7 mutant residues in the mutant smAKAP peptide sequence compared to the wild-type smAKAP peptide sequence.

3. The recombinant spirulina according to claim 1, wherein one residue in the smAKAP peptide sequence is mutated compared to the wild-type smAKAP peptide sequence.

4. Recombinant spirulina according to any one of claims 2 to 3, wherein two residues in the smAKAP peptide sequence are mutated compared to the wild-type smAKAP peptide sequence.

5. Recombinant spirulina according to any one of claims 2 to 4, wherein three residues in the smAKAP peptide sequence are mutated compared to the wild-type smAKAP peptide sequence.

6. The recombinant spirulina according to any one of claims 1 to 5, wherein the mutant smAKAP peptide exhibits resistance to protease cleavage, as determined by a decrease in the detection of cleavage products, when exposed to a solvent containing a protease for one hour.

7. The recombinant spirulina according to claim 6, wherein up to about 5%, 10%, 20%, 30%, 40%, 50%, or 60% of the smAKAP peptide sequence is cleaved.

8. Recombinant spirulina according to any one of claims 1 to 7, wherein the mutation is in a hydrophobic residue.

9. The recombinant spirulina according to any one of claims 1 to 8, wherein when the recombinant spirulina is immersed in a solvent, the mutant residues are exposed to the solvent.

10. Recombinant spirulina according to any one of claims 1 to 9, wherein the mutant residue is selected from the group consisting of C16S, C24S, E5D, Y6H, W22S, C24G, L4E, R9E, L4I, L10I, L19I, and combinations thereof, based on SEQ ID NO:

2.

11. The recombinant spirulina according to claim 10, wherein the mutant residues are C16S and C24S compared to SEQ ID NO:

2.

12. The recombinant spirulina according to claim 10, wherein the mutant residues are E5D, Y6H, C16S, W22S, and C24G, compared to SEQ ID NO:

2.

13. The recombinant spirulina according to claim 10, wherein the mutant residues are L4E, E5D, Y6H, R9E, C16S, W22S, and C24G compared to SEQ ID NO:

2.

14. The recombinant spirulina according to claim 10, wherein the mutant residues are L4E, C16S, W22S, and C24G compared to SEQ ID NO:

2.

15. The recombinant spirulina according to claim 10, wherein the mutant residues are R9E, C16S, and C24G compared to SEQ ID NO:

2.

16. The recombinant spirulina according to claim 10, wherein the mutant residues are R9E, C16S, W22S, and C24G compared to SEQ ID NO:

2.

17. The recombinant spirulina according to claim 10, wherein the mutant residues are L4I, R9E, C16S, and C24G compared to SEQ ID NO:

2.

18. The recombinant spirulina according to any one of claims 1 to 17, wherein the mutant smAKAP peptide sequence is linked to a monomeric heterologous moiety.

19. The recombinant spirulina according to claim 18, wherein the mutant smAKAP peptide sequence is linked to a second heterologous moiety.

20. Recombinant spirulina according to claim 19, wherein the aforementioned different species of moiety and the second different species of moiety are the same.

21. Recombinant spirulina according to any one of claims 18 to 19, wherein the aforementioned heterologous moiety and the aforementioned second heterologous moiety are different.

22. The recombinant spirulina according to any one of claims 1 to 21, wherein the recombinant spirulina expresses another exogenous polypeptide sequence.

23. The recombinant spirulina according to claim 22, wherein the exogenous polypeptide sequence is selected from the group consisting of the oligomerized domain of C4b-binding protein (C4BP), the cholera toxin b subunit, the oligomerized domain of an extracellular matrix protein, TRX, 5HVZ, cTRP, SP651, SP737, and 4B0F.

24. The recombinant spirulina according to claim 23, wherein the exogenous polypeptide sequence is 5HVZ.

25. The recombinant spirulina according to claim 24, wherein the 5HVZ is connected to a third different moiety.

26. The recombinant spirulina according to claim 25, wherein the third heterogeneous moiety is in a dimer configuration.

27. Recombinant spirulina according to any one of claims 18 to 26, wherein the aforementioned heterogeneous moiety, the second heterogeneous moiety, and the third heterogeneous moiety are the same.

28. Recombinant spirulina according to any one of claims 18 to 26, wherein the aforementioned heterogeneous moiety, the second heterogeneous moiety, and the third heterogeneous moiety are different.

29. Recombinant spirulina according to any one of claims 18 to 28, wherein the heterologous moiety is a binder.

30. Recombinant spirulina according to claim 29, wherein the binder is selected from the group consisting of fab', F(ab')2, fv, domain antibody (dAb), complementarity-determining region (CDR) fragment, CDR graft antibody, single-chain antibody (scFv), single-chain antibody fragment, chimeric antibody, diabody, triabody, tetrabody, minibody, linear antibody, intrabody, nanobody (single-domain antibody), small modular immunotherapy product (SMIP), antigen-binding domain immunoglobulin fusion protein, and VHH.

31. The recombinant spirulina according to claim 30, wherein the binder is VHH.

32. The recombinant spirulina according to claim 31, wherein the VHH binds to the pathogen.

33. The recombinant spirulina according to claim 31, wherein the VHH binds to cancer cells.

34. The recombinant spirulina according to claim 31, wherein the VHH binds to human cells.

35. The recombinant spirulina according to claim 32, wherein the VHH binds to a pathogen selected from the group consisting of bacteria, fungi, and viruses.

36. The aforementioned pathogens include Escherichia coli (E. coli), enterotoxigenic Escherichia coli (ETEC), anthrax, EHEC, EAEC, Shigella, Mycobacterium, Streptococcus, Staphylococcus, Shigella, Campylobacter, Salmonella, Clostridium, Corynebacterium, Pseudomonas, Neisseria, Listeria, Vibrio, Bordetella, Legionella, bacteriophages, RNA bacteriophages (e.g., MS2, AP205, P P7 and Qβ), Helicobacter pylori, infectious hematopoietic necrosis virus, parvovirus, herpes simplex virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, measles virus, mumps virus, rubella virus, HIV, influenza virus, rhinovirus, rotavirus A, rotavirus B, rotavirus C, respiratory syncytial virus (RSV), varicella-zoster virus, poliovirus, norovirus, Zika virus, dengue virus, rabies virus, Newcastle disease virus, vitiligo virus, coronavirus, SARS-related coronavirus, MERS-related coronavirus, SARS-CoV-2, Aspergillus, Candida, Blastomyces, Coccidioides, Cryptococcus, Histoplasma, Plasmodium malariae, Plasmodium falciparum, Plasmodium malariae Recombinant spirulina according to claim 35, selected from the group consisting of worms, ovoid malaria parasites, Plasmodium vivax, Trypanosoma, Toxoplasma, Giardia, Leishmania, Cryptosporidium, Hellworm parasites: Whipworms, Pinworms, Ascaris, Hookworms, and American Hookworms, Strongyloides, Dracunculus, Onchocerca, Filamentosa, Tapeworms, Hydatidiformes, Diphyllobothrium, Fasciola, and Schistosoma.

37. Recombinant spirulina according to claim 35, wherein the pathogen is a bacterium, and the bacterium is selected from the group consisting of Mycobacterium, Streptococcus, Staphylococcus, Shigella, Campylobacter, Salmonella, Clostridium, Corynebacterium, Pseudomonas, Neisseria, Listeria, Vibrio, Bordetella, and Legionella.

38. The recombinant spirulina according to claim 37, wherein the bacterium is Campylobacter.

39. The recombinant spirulina according to claim 37, wherein the bacterium is Clostridium.

40. The recombinant spirulina according to any one of claims 36 to 39, wherein the VHH comprises a sequence having at least 85% identity with the sequences of sequence numbers 25 to 67.

41. The recombinant spirulina according to any one of claims 36 to 39, wherein the VHH comprises the sequence of sequence numbers 25 to 67.

42. The recombinant spirulina according to any one of claims 24 to 41, wherein the recombinant spirulina expresses a scaffold having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequences of SEQ ID NOs: 4, 6, 14, 16, 18, 20, 22, 23, 24, and 76.

43. Recombinant spirulina expressing a mutant small membrane-bound A kinase anchoring protein (smAKAP) peptide sequence, wherein the mutation includes a residue substitution based on Sequence ID No.

2.

44. Recombinant spirulina expressing a mutant small membrane-bound A kinase anchoring protein (smAKAP) peptide sequence, wherein the mutant is selected from the group consisting of C16S, C24S, E5D, Y6H, W22S, C24G, L4E, R9E, L4I, L10I, L19I, and combinations thereof, based on Sequence ID No.

2.

45. The recombinant spirulina according to any one of claims 43 to 44, wherein the smAKAP is linked at its terminus to one or more VHH antibodies.

46. The recombinant spirulina according to claim 45, wherein the smAKAP is linked to two VHH antibodies, the first VHH being at the C-terminus and the second VHH being at the N-terminus.

47. The recombinant spirulina according to any one of claims 43 to 46, wherein the recombinant spirulina expresses 5HVZ.

48. The recombinant spirulina according to claim 47, wherein the 5HVZ is linked to two VHH antibodies in a homodimer configuration.

49. A polynucleotide sequence comprising a mutant small membrane-bound A kinase anchoring protein (smAKAP) sequence, wherein the mutation comprises one or more residue substitutions compared to the wild-type smAKAP sequence.

50. A vector comprising the polynucleotide sequence described in claim 49.

51. A method for producing recombinant spirulina, comprising contacting spirulina cells with the vector described in claim 50.

52. A pharmaceutical composition comprising recombinant spirulina according to any one of claims 1 to 48 and an excipient.

53. A kit comprising recombinant spirulina according to any one of claims 1 to 48, a polynucleotide sequence according to claim 49, a vector according to claim 50, or a pharmaceutical composition according to claim 52, and instructions for use thereof.

54. A method of treatment comprising administering the pharmaceutical composition according to claim 52 to a subject in need thereof, wherein the subject includes a bacterial or viral infection.

55. A vector comprising nucleotide sequences encoding polypeptides of SEQ ID NOs. 2-7, 14-24, 74, and / or 76.

56. The vector according to claim 55, wherein the vector has approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with respect to sequence number 75.

57. Recombinant spirulina comprising the vector described in any one of claims 55 to 56.

Images