Recombinant spirulina expressing scaffolds and methods of use thereof

Abstract

The present disclosure provides a scaffold comprising a heterologous moiety and Spirulina spp. That expresses the scaffold. Also provided are recombinant Spirulina expressing the scaffolds and methods of treating diseases and conditions using the same. Homologous and heterologous scaffolds comprising VHH antibodies linked by modified smAKAP linkers can be used to increase binding affinity as compared to VHH antibodies alone.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Application No. 63 / 511,050, filed June 29, 2023, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0003] Reference to the electronic sequence list

[0004] The contents of the electronic sequence list (LUBI_038_01WO_SeqList_ST26.xml; size: 82,287 bytes; and creation date: June 28, 2024) are incorporated herein by reference in their entirety. Background Technology

[0005] Antigen-binding domains (such as VHH from camel-derived single-chain antibodies) are expressible in prokaryotes, such as the genus *Spirulina*. However, various limitations remain, such as the need for affinity maturation to achieve improved target binding.

[0006] This disclosure provides a solution to the limitations of current prior art through an improved composition comprising a stent. Technical Field

[0007] This disclosure relates to scaffolds and methods of using them. This disclosure also relates to constructs comprising scaffolds expressing binding agents. Summary of the Invention

[0008] This document provides a recombinant Spirulina genus expressing a mutated small membrane A-kinase anchoring protein (smAKAP) peptide sequence. In each embodiment, the mutated smAKAP peptide sequence contains at least 2, 3, 4, 5, 6, or 7 mutated residues compared to the WT smAKAP peptide sequence. In each embodiment, 1 residue in the smAKAP peptide sequence is mutated compared to the WT smAKAP peptide sequence. In each embodiment, 2 residues in the smAKAP peptide sequence are mutated compared to the WT smAKAP peptide sequence. In each embodiment, 3 residues in the smAKAP peptide sequence are mutated compared to the WT smAKAP peptide sequence. In each embodiment, when exposed to a solvent containing a protease for one hour, the mutated smAKAP peptide exhibits resistance to protease cleavage, as determined by a reduction in the detection of cleavage products. In each embodiment, at most about 5%, 10%, 20%, 30%, 40%, 50%, or 60% of the smAKAP peptide sequence is cleaved. In each embodiment, the mutation is a hydrophobic residue. In all respects, when the recombinant Spirulina is immersed in a solvent, the mutated residues are exposed to the solvent. In all respects, the mutated residues are selected from the group consisting of C16S, C24S, E5D, Y6H, W22S, C24G, L4E, R9E, L4I, L10I, L19I, and combinations thereof of SEQ ID NO: 2. In all respects, compared with SEQ ID NO: 2, the mutated residues are C16S and C24S. In all respects, compared with SEQ ID NO: 2, the mutated residues are E5D, Y6H, C16S, W22S, and C24G. In all respects, compared with SEQ ID NO: 2, the mutated residues are L4E, E5D, Y6H, R9E, C16S, W22S, and C24G. In all respects, compared with SEQ ID NO: 2, the mutated residues are L4E, C16S, W22S, and C24G. In all respects, compared with SEQ ID NO: 2, the mutated residues are R9E, C16S, and C24G. In all respects, compared with SEQ ID NO: 2, the mutated residues are R9E, C16S, W22S, and C24G. In all respects, the mutated smAKAP peptide sequence is linked to a heterologous portion in a monomeric configuration. In all respects, the mutated smAKAP peptide sequence is linked to a second heterologous portion. In all respects, the heterologous portion and the second heterologous portion are the same. In all respects, the heterologous portion and the second heterologous portion are different. In all respects, the recombinant Spirulina expresses another exogenous polypeptide sequence. In all respects,The exogenous polypeptide sequence is selected from the group consisting of: oligomerization domains of C4b-binding protein (C4BP), cholera toxin b subunits, oligomerization domains of extracellular matrix proteins, TRX, 5HVZ, cTRP, SP651, SP737, and 4BOF. In all respects, the exogenous polypeptide sequence is 5HVZ. In all respects, the 5HVZ is linked to a third heteromeric moiety. In all respects, the third heteromeric moiety is in a homodimeric configuration. In all respects, the heteromeric moiety, the second heteromeric moiety, and the third heteromeric moiety are identical. In all respects, the heteromeric moiety, the second heteromeric moiety, and the third heteromeric moiety are different. In all respects, the heteromeric moiety is a binding agent. In all respects, the binding agent is selected from the group consisting of: fab', F(ab')2, fv, domain antibody (dAb), complementarity-determining region (CDR) fragment, CDR transplanted antibody, single-chain antibody (scFv), single-chain antibody fragment, chimeric antibody, bifunctional antibody, trifunctional antibody, tetrafunctional antibody, microantibody, linear antibody, intracellular antibody, nanobody (single-domain antibody), small modular immunopharmaceutical (SMIP), antigen-binding domain immunoglobulin fusion protein, and VHH. In all respects, the binding agent is the VHH. In all respects, the VHH binds to pathogens. In all respects, the VHH binds to cancer cells. In all respects, the VHH binds to human cells. In all respects, the VHH binds to pathogens selected from the group consisting of: bacteria, fungi, and viruses. In all respects, the pathogens are selected from the group consisting of: *Escherichia coli*, *Enterotoxigenic Escherichia coli* (ETEC), anthrax, EHEC, EAEC, *Shigella*, *Mycobacterium*, *Streptococcus*, *Staphylococcus*, *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, and vitiligo syndrome virus. Coronavirus, SARS, MERS, SARS-CoV-2, Aspergillus, Candida, Blastomyces, Coccidioides, Cryptococcus, Histoplasma, Plasmodium, P. falciparum, P. malariae. malariae), Plasmodium ovale, Plasmodium vivax, Trypanosoma, Toxoplasma, Giardia, Cryptosporidium leishmaniae, helminthic parasites: Trichuris spp., Enterobius spp., Ascaris spp., Ancylostomaspp., Necatro spp., Strongyloides spp., Dracunculus spp., Onchocerca spp., Wuchereria spp., Taenia spp., Echinococcus The pathogens are bacteria selected from the group consisting of: Mycobacterium, Streptococcus, Staphylococcus, Shigella, Campylobacter, Salmonella, Clostridium, Corynebacterium, Pseudomonas, Neisseria, Listeria, Vibrio, Bordetella, and Legionella. In each aspect, the bacteria are Campylobacter. In each aspect, the bacteria are Clostridium. In each aspect, the VHH comprises a sequence having at least 85% identity with the sequence of SEQ ID NO: 25-67. In each aspect,The VHH comprises sequences SEQ ID NO: 25-67. In all respects, the recombinant Spirulina expresses a scaffold with at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequences SEQ ID NO: 6, 14, 16, 18, 20, 22, 23, and 24.

[0009] Also provided is a recombinant Spirulina genus that expresses a mutated small membrane A-kinase anchoring protein (smAKAP) peptide sequence, wherein the mutation includes a substitution of the residue of SEQ ID NO: 2.

[0010] Also provided is a recombinant Spirulina genus expressing a mutated small membrane 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 of SEQ ID NO: 2. In each aspect, smAKAP is linked to one or more VHH antibodies at its terminal. In each aspect, the smAKAP is linked to two VHH antibodies, wherein the first VHH is located at the C-terminus and the second VHH is located at the N-terminus. In each aspect, the recombinant Spirulina genus expresses 5HVZ. In each aspect, the 5HVZ is linked to two VHH antibodies in a homodimeric configuration.

[0011] It also provides a polynucleotide sequence containing a mutated small membrane A-kinase anchoring protein (smAKAP) sequence, wherein the mutation is a single residue substitution compared to the WT smAKAP sequence.

[0012] It also provides vectors containing polynucleotide sequences.

[0013] A method for preparing recombinant Spirulina is also provided, the method comprising contacting Spirulina cells with a carrier disclosed herein.

[0014] Pharmaceutical compositions comprising recombinant Spirulina and excipients are also provided.

[0015] A kit is also provided, which contains: recombinant Spirulina, polynucleotide sequence, vector or pharmaceutical composition and instructions for use.

[0016] Treatment methods are also provided, which involve administering the subject pharmaceutical composition to a subject in need, wherein the subject has a bacterial or viral infection.

[0017] A vector containing the nucleotide sequence encoding a polypeptide of SEQ ID NO: 5-7 or 14-24 is also provided.

[0018] Recombinant Spirulina species containing the vector disclosed herein are also provided.

[0019] A recombinant *Spirulina* species is provided that expresses a mutated small membrane A-kinase anchoring protein (smAKAP) peptide sequence. In each embodiment, the mutated smAKAP peptide sequence contains at least 2, 3, 4, 5, 6, or 7 mutated residues compared to the WT smAKAP peptide sequence. In each embodiment, one residue in the smAKAP peptide sequence is mutated compared to the WT smAKAP peptide sequence. In each embodiment, two residues in the smAKAP peptide sequence are mutated compared to the WT smAKAP peptide sequence. In each embodiment, three residues in the smAKAP peptide sequence are mutated compared to the WT smAKAP peptide sequence. In each embodiment, when exposed to a solvent containing a protease for one hour, the mutated smAKAP peptide exhibits resistance to protease cleavage, as determined by a reduction in the detection of cleavage products. In each embodiment, at most about 5%, 10%, 20%, 30%, 40%, 50%, or 60% of the smAKAP peptide sequence is cleaved. In each embodiment, the mutation is a hydrophobic residue. In each respect, the recombinant Spirulina is immersed in a solvent, and the mutated residues are exposed to the solvent. In each respect, the mutated residues are selected from the group consisting of C16S, C24S, E5D, Y6H, W22S, C24G, L4E, R9E, L4I, L10I, L19I, and combinations thereof of SEQ ID NO: 2. In each respect, compared with SEQ ID NO: 2, the mutated residues are C16S and C24S. In each respect, compared with SEQ ID NO: 2, the mutated residues are E5D, Y6H, C16S, W22S, and C24G. In each respect, compared with SEQ ID NO: 2, the mutated residues are L4E, E5D, Y6H, R9E, C16S, W22S, and C24G. In each respect, compared with SEQ ID NO: 2, the mutated residues are L4E, C16S, W22S, and C24G. In all respects, compared with SEQ ID NO: 2, the mutated residues are R9E, C16S, and C24G. In all respects, compared with SEQ ID NO: 2, the mutated residues are R9E, C16S, W22S, and C24G. In all respects, compared with SEQ ID NO: 2, the mutated residues are L4I, R9E, C16S, and C24G. In all respects, the mutated smAKAP peptide sequence is linked to a heterologous portion in a monomeric configuration. In all respects, the mutated smAKAP peptide sequence is linked to a second heterologous portion. In all respects, the heterologous portion and the second heterologous portion are identical. In all respects, the heterologous portion and the second heterologous portion are different. In all respects, the recombinant Spirulina expresses another exogenous polypeptide sequence.In all respects, the exogenous polypeptide sequence is selected from the group consisting of: oligomerization domains of C4b-binding protein (C4BP), cholera toxin b subunits, oligomerization domains of extracellular matrix proteins, TRX, 5HVZ, cTRP, SP651, SP737, and 4BOF. In all respects, the exogenous polypeptide sequence is 5HVZ. In all respects, 5HVZ is linked to a third heteromeric moiety. In all respects, the third heteromeric moiety is in a dimer configuration. In all respects, the heteromeric moiety, the second heteromeric moiety, and the third heteromeric moiety are identical. In all respects, the heteromeric moiety, the second heteromeric moiety, and the third heteromeric moiety are different. In all respects, the heteromeric moiety is a binding agent. In all respects, the binding agent is selected from the group consisting of: fab', F(ab')2, fv, domain-specific antibodies (dAb), complementarity-determining region (CDR) fragments, CDR-transplanted antibodies, single-chain antibodies (scFv), single-chain antibody fragments, chimeric antibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, microantibodies, linear antibodies, intracellular antibodies, nanobodies (single-domain antibodies), small modular immunopharmaceuticals (SMIPs), antigen-binding domain immunoglobulin fusion proteins, and VHH. In all respects, the binding agent is VHH. In all respects, VHH binds to pathogens. In all respects, VHH binds to cancer cells. In all respects, VHH binds to human cells. In all respects, VHH binds to pathogens selected from the group consisting of: bacteria, fungi, and viruses. In all respects, pathogens are selected from the following groups: *Escherichia coli*, enterotoxigenic *E. coli* (ETEC), anthrax, *EHEC*, *EAEC*, *Shigella* spp., *Mycobacterium* spp., *Streptococcus* spp., *Staphylococcus* spp., *Shigella* spp., *Campylobacter* spp., *Salmonella* spp., *Clostridium* spp., *Corynebacterium* spp., *Pseudomonas* spp., *Neisseria* spp., *Listeria* spp., *Vibrio* spp., *Bordezoella* spp., *Legionella* spp., 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, Leukoplakia Syndrome Virus, Coronavirus, SARS, MERS, SARS-CoV-2, Aspergillus, Candida, Blastomyces, Coccidioides, Cryptococcus, Histoplasma, Plasmodium, Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, Plasmodium vivax, Trypanosoma, Toxoplasma, Giardia, Cryptosporidium leishmaniasis, Helminthus, Trichuris, Pinworm, Ascaris, Hookworm and Isopoda, Strongyloides stercoralis, Gnatifida, Onchocerca salina, Wucetella, Tapeworm, Echinococcus and Spirodactylus, Fasciola hepatica and Schistosoma.In all respects, the pathogen is a bacterium, and said bacterium is selected from the group consisting of: Mycobacterium, Streptococcus, Staphylococcus, Shigella, Campylobacter, Salmonella, Clostridium, Corynebacterium, Pseudomonas, Neisseria, Listeria, Vibrio, Bordetella, and Legionella. In all respects, the bacterium is Campylobacter. In all respects, the bacterium is Clostridium. In all respects, VHH contains a sequence having at least 85% identity with the sequence of SEQ ID NO: 25-67. In all respects, VHH contains the sequence of SEQ ID NO: 25-67. In all respects, 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 NO: 4, 6, 14, 16, 18, 20, 22, 23, 24, and 76.

[0020] A recombinant Spirulina genus is provided that expresses a mutated small membrane A-kinase anchoring protein (smAKAP) peptide sequence, wherein the mutation includes a substitution of the residue of SEQ ID NO: 2.

[0021] A recombinant Spirulina genus is provided that expresses a mutated small membrane 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 of SEQ ID NO: 2. In each aspect, the smAKAP is linked to one or more VHH antibodies at its terminal. In each aspect, the smAKAP is linked to two VHH antibodies, wherein the first VHH is located at the C-terminus and the second VHH is located at the N-terminus. In each aspect, the recombinant Spirulina genus expresses 5HVZ. In each aspect, the 5HVZ is linked to two VHH antibodies in a homodimeric configuration.

[0022] A polynucleotide sequence containing a mutated small membrane A-kinase anchoring protein (smAKAP) sequence is provided, wherein the mutation involves the substitution of one or more residues compared to the WT smAKAP sequence.

[0023] A vector containing the disclosed polynucleotide sequence is provided.

[0024] A method for preparing recombinant Spirulina is also provided, the method comprising contacting Spirulina cells with a carrier disclosed herein.

[0025] A pharmaceutical composition comprising recombinant Spirulina and one or more excipients is provided.

[0026] A kit is provided, which contains: recombinant Spirulina, polynucleotide sequence, vector or pharmaceutical composition and instructions for use.

[0027] A treatment method is provided, the method comprising administering the disclosed pharmaceutical composition to a subject in need, wherein the subject has a bacterial or viral infection.

[0028] A vector is also provided which contains the nucleotide sequence encoding the polypeptide of SEQ ID NO: 2-7, 14-24, 74 and / or 76. In all respects, the vector contains approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 75.

[0029] Recombinant Spirulina species containing the vector disclosed herein are also provided. Attached Figure Description

[0030] The accompanying drawings, which are incorporated herein and form part of the specification, illustrate some, but not only or exclusively, exemplary aspects and / or features. It is intended that the aspects and drawings disclosed herein be considered illustrative rather than limiting.

[0031] Figures 1A-1D The following protease sensitivity data for the construct is shown: PP917 2xS3b-C8 ( Figure 1A ), PP1895 2xS3b-C8 ( Figure 1B ), PP1895 2xRN-29 ( Figure 1C ) and PP2130 2xRN-29+2xS3b-C8 ( Figure 1D ).

[0032] Figure 2 This is a diagram depicting the hydrophobic residues on smAKAP that participate in the formation of the complex with 5HVZ.

[0033] Figure 3A This is a diagram of the smAKAP peptide, depicting the arginine residues exposed to the solvent and potentially serving as trypsin cleavage sites, see SEQ ID NO: 1. The amino acid sequence of PP1895 of SEQ ID NO: 1 is also described. Figure 3B The normalized frequencies of various cleavages of the smAKAP peptide are shown. Figure 3C This diagram shows additional Lue and His residues on the smAKAP peptide that serve as potential trypsin cleavage sites.

[0034] Figure 4 The image is a diagram of smAKAP, which depicts exemplary mutations that confer resistance to trypsin and chymotrypsin.

[0035] Figure 5 The Western blot of the mutant smAKAP clone expressed in the E. coli system is shown.

[0036] Figures 6A-6C The following is an example of trypsin digestion of a clone containing an R63E substitution: PP2451 ( Figure 6A ), PP1895 ( Figure 6B ) and PP2451 ( Figure 6C R63 substitution confers trypsin resistance in the smAKAP peptide.

[0037] Figure 7 Size-determined complex formation analysis of the smAKAP and 5HVZ dimers is shown.

[0038] Figure 8 The results of complex formation analysis performed by exposing mutant constructs PP2451 and PP2456 to trypsin and chymotrypsin digestion at 0.1 mg / mL and 0.01 mg / mL, respectively, are shown.

[0039] Figure 9 Results of chymotrypsin resistance assays using clones PP2451, PP1895, PP2455, PP2456, PP2454, PP2452, and PP2453 are shown.

[0040] Figure 10 Results of complex formation assays using clones PP2451, PP1895, PP2456, PP2454, PP2452, and PP2453 are shown.

[0041] Figure 11 SDS-PAGE gels showing comparative analysis of smAKAP mutant scaffolds are presented.

[0042] Figure 12 An SDS-PAGE gel is shown, which demonstrates that the construct containing the smAKAP linker is well expressed in Spirulina.

[0043] Figure 13 The SDS-PAGE gels of PP1895 purified protein on days 0, 4, and 7 are shown. After storage at 4°C, the purified protein showed degradation and increased instability. This contrasts with the protein that showed greater stability. Figure 11 The result was the opposite.

[0044] Figure 14 The results of complex formation analysis using the mutant construct PP6510 are shown. Detailed Implementation

[0045] Compositions comprising recombinant Spirulina spp. are provided. Also provided are scaffolds comprising binding domains expressed by the recombinant Spirulina spp., and methods for modifying said scaffolds to prepare orally delivered therapeutic proteins. For example, homopolymeric and heteropolymeric scaffolds comprising VHH antibodies linked via smAKAP linkers can be used to increase binding affinity compared to VHH antibodies alone.

[0046] It also provides methods for administering recombinant Spirulina to subjects in need to prevent, treat, or improve infections, diseases, or symptoms.

[0047] definition

[0048] While it is believed that the following terms will be well understood by those skilled in the art, the following definitions are set forth in order to facilitate the interpretation of the subject matter currently disclosed.

[0049] Unless otherwise defined below, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art. References to techniques used herein are intended to refer to techniques commonly understood in the art, including variations of those techniques and / or substitutions for equivalent techniques that would be obvious to one of ordinary skill in the art.

[0050] As used herein, unless the content expressly indicates otherwise, the singular forms “a / an” and “the” include plural indicators.

[0051] When immediately preceding a numerical value, the term “about” or “approximately” refers to a range (e.g., plus or minus 10% of the value). For example, “about 50” can mean 45 to 55, “about 25,000” can mean 22,500 to 27,500, etc., unless the context of this disclosure otherwise indicates or is inconsistent with such interpretation. For example, in a list of numerical values ​​such as “about 49, about 50, about 55, …”, “about 50” means a range extending to less than half the interval between the previous and subsequent values, such as greater than 49.5 to less than 52.5. Furthermore, given the definition of the term “about” provided herein, the phrases “less than about” or “greater than about” should be understood. Similarly, when preceding a series of numerical values ​​or a range of values ​​(e.g., “about 10, 20, 30” or “about 10-30”), the term “about” refers to all values ​​in the series or the endpoints of the range, respectively.

[0052] As used herein, the term “subject” means any subject, such as a human or non-human mammal, who is required to be diagnosed, diagnosed, or treated. The term “subject” may also mean a human or non-human mammal that has, may have, or is suspected of having a disease. The terms “subject” and “patient” are used interchangeably herein. In all respects, a subject is a mammal. Mammals include primates such as humans, monkeys, chimpanzees, and apes, as well as non-primates such as livestock, including laboratory animals (such as rabbits and rodents such as guinea pigs, rats, or mice) and domesticated pets and farm animals (such as cats, dogs, pigs, cattle, sheep, goats, horses, and rabbits), and non-domesticated animals such as wild animals, birds, reptiles, fish, etc.

[0053] As used herein, the term “subject in need” includes subjects who can or will benefit from the methods described herein. Subjects in need of treatment include, but are not limited to, subjects who already have a condition or illness, subjects who are predisposed to having a condition or illness, subjects who suspect they have a condition or illness, and subjects who need to prevent, improve, or reverse a condition or illness.

[0054] When referring to nucleic acid or protein sequences, the term "identity" is used to indicate the similarity between two sequences. Unless otherwise stated, the percentage of identity described herein was determined using the BLAST algorithm with default parameters, available at the World Wide Web address blast.ncbi.nlm.nih.gov / Blast.cgi.

[0055] Antibody fragments include antigen-binding portions of antibodies (i.e., "antigen-binding fragments"), particularly including fab', F(ab')2, fv, domain antibodies (dAb), complementarity-determining region (CDR) fragments, CDR-transplanted antibodies, single-chain antibodies (scFv), single-chain antibody fragments, chimeric antibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, microantibodies, linear antibodies; chelated recombinant antibodies, triple-chain antibodies (trisomy antibodies) or bifunctional antibodies (biantibodies), intracellular antibodies (intracellular antibodies), nanobodies (nanobodies), small modular immunopharmaceuticals (SMIPs), antigen-binding domain immunoglobulin fusion proteins, single-domain antibodies (including camelid antibodies), antibodies containing VHH or variants or derivatives thereof, and polypeptides containing at least a portion of an immunoglobulin sufficient to bind a specific antigen to the polypeptide (e.g., one, two, three, four, five, or six CDR sequences), provided that the fragment retains the desired biological activity.

[0056] support

[0057] This document provides scaffolds. In various respects, the scaffolds can be monomeric, dimeric, trimeric, tetrameric, pentameric, hexameric, heptameric, octameric, nonameric, or decameric. The scaffolds are used to polymerize the heterologous motifs of this disclosure for expression in the genus *Spirulina*. For example, scaffolds can be used to polymerize VHH for expression in the genus *Spirulina*. Polymerization can achieve increased target-binding affinity compared to the affinity of a single monomeric VHH, or compared to a scaffold with reduced polymerization (e.g., trimer versus dimer scaffolds).

[0058] In all respects, the scaffold is monolithic. Monolithic scaffolds may contain heterologous portions attached to or connected to the scaffold. In all respects, the scaffold contains maltose-binding protein (MBP). In all respects, the scaffold contains thioredoxin (TRX).

[0059] In all respects, the stent is dimer. Dimeric stents can contain 5 HVZ.

[0060] In all respects, the stent is trimer. Trimeric stents may contain cTRP. Trimeric stents may contain SP651.

[0061] In all respects, the stent is pentamer. Pentamer stents may contain SP737.

[0062] In all respects, the stent is heptamer. Heptamer stents can contain 4BOF.

[0063] In all respects, the scaffold is selected from the following groups: oligomerization domains of C4b-binding protein (C4BP), cholera toxin b subunit, oligomerization domains of extracellular matrix proteins, TRX, 5HVZ, cTRP, SP651, SP737, 4B0F, smAKAP, and combinations thereof.

[0064] In all respects, any scaffold of this disclosure can be mutated to improve performance. In all respects, mutations can be made to reduce protease sensitivity (e.g., trypsin or chymotrypsin).

[0065] smAKAP

[0066] In all respects, the scaffold contains the smAKAP peptide. smAKAP is a small (i.e., 11 kDa) PKA-RI specific protein kinase A-anchored protein, referred to herein as small membrane AKAP (smAKAP). smAKAP is attached to the plasma membrane via double acylation (myristoylation and palmitoylation, respectively) of its N-terminal Met-Gly-Cys-motif. In all respects, smAKAP is expressed in the genus *Spirulina* as part of the scaffold of this disclosure. smAKAP can act as a linker in the scaffold of this disclosure.

[0067] The provided scaffold can further include a 5HVZ domain. In all respects, 5HVZ can spontaneously assemble into a dimer within Spirulina cells. In all respects, a homodimeric protein containing two protein molecules (5HVZ-VHH and 5HVZ-VHH) is provided. In all respects, the scaffold contains a maltose-binding protein (MBP) at one end and a VHH at the other end. In this conformation, the homodimer has two related VHHs. Dimeric VHHs generally allow for tighter binding to their targets, a phenomenon known as affinity. If the VHHs are located at both ends of the 5HVZ scaffold, this strategy can generate four related copies of the VHH.

[0068] Additionally, smAKAP is useful because it spontaneously forms trimeric complexes with the 5HVZ homodimer. This allows for further increases in the multimerization of the scaffold complex. For example, depending on a specific design, each of these trimeric complexes can have 3, 4, or 6 VHHs. Furthermore, smAKAP can also have VHHs different from those fused with 5HVZ, which allows for the combined assembly of different complexes containing unique VHH pairs.

[0069] In scaffolds containing smAKAP and 5HVZ (e.g., Cerberbody and / or Hydrabody), hydrophobic residues on smAKAP participate in complex formation with 5HVZ, see [link to relevant documentation]. Figure 2 In various respects, the smAKAP peptide contains residues that may be sensitive to protease cleavage; see [link to relevant documentation]. Figure 3B See Keil, B. Specificity of proteolysis. Springer-Verlag-Berlin-Heidelberg-NewYork, p. 335. (1992), the entire reference of which is incorporated by reference. Therefore, compositions comprising a mutant smAKAP peptide exhibiting resistance or reduced protease cleavage are also provided.

[0070] To generate a mutant smAKAP peptide conferring resistance to trypsin and chymotrypsin, any method can be used. In each respect, the trypsin-sensitive site is removed. In each respect, residues can be substituted to make the active site more basic or more acidic compared to WT. Exemplary residues that can be mutated are... Figure 4As shown, however, any other residues can also be mutated. In each respect, one or more residues are mutated. In each respect, peptides with two or more mutations show increased resistance to proteases compared to peptides with a single residue mutation. In each respect, different combinations of hydrophobic mutations improve resistance to proteases.

[0071] In all respects, the smAKAP peptide contains mutations selected from the group consisting of: C16S, C24S, E5D, Y6H, W22S, C24G, L4E, R9E, L4I, L10I, L19I, and combinations thereof of SEQ ID NO: 2. In all respects, compared with SEQ ID NO: 2, the mutant smAKAP contains mutations at C16S and C24S. In all respects, compared with SEQ ID NO: 2, the mutant smAKAP contains mutations at C16S, E5D, Y6H, C16S, W22S, and C24G. In all respects, compared with SEQ ID NO: 2, the mutant smAKAP contains mutations at C16S, L4E, E5D, Y6H, R9E, C16S, W22S, and C24G. In all respects, compared with SEQ ID NO:2, the mutant smAKAP contains mutations at C16S, L4E, C16S, W22S, and C24G. In all respects, compared with SEQ ID NO:2, the mutant smAKAP contains mutations at C16S, R9E, C16S, and C24G. In all respects, compared with SEQ ID NO:2, the mutant smAKAP contains mutations at C16S, R9E, C16S, W22S, and C24G. In all respects, compared with SEQ ID NO:2, the mutant smAKAP contains mutations at L4I, L10I, C16S, L19I, and C78A. In all respects, compared with SEQ ID NO:2, the mutant smAKAP contains mutations at L4I, R9E, L10I, C16S, L19I, W22S, and C24G. In all respects, compared with SEQ ID NO: 2, the mutated smAKAP contains L4I, R9E, C16S and C24G mutations.

[0072] In all respects, the mutant smAKAP contains 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 NO: 5, 7, 15, 17, 19, 21, 72, 73, and 74.

[0073] In all respects, mutant smAKAP exhibits increased protease resistance compared to WT smAKAP. For example, compositions containing mutant smAKAP of this disclosure can exhibit up to about 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 reduced activity and / or cleavage events compared to WT. For example, compositions containing the mutant smAKAP of this disclosure can exhibit reduced activity and / or cleavage events by up to about 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%, or 80% compared to WT. Activity can be determined after incubation with a protease. For example, compared with WT smAKAP incubated with protease for 1 hour, the mutant smAKAP of this disclosure can exhibit up to about 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% reduced activity.

[0074] In all respects, the scaffold comprises a cyboloid or hypodermic complex containing a mutant smAKAP peptide, as described herein and below.

[0075] Seber

[0076] In all respects, a Sebersome construct can be formed when Spirulina strains (two independent strains or a single strain from different ORFs) express a 5HVZ-mediated homodimer molecule and a smAKAP-linked monomeric VHH. These independent complexes form a further complex structure with three antigen-binding sites. In all respects, another heterologous portion of this disclosure can replace one or more VHHs in the Sebersome. As described herein, any scaffold can be mutated to improve performance. For example, smAKAP can be mutated to reduce protease sensitivity, as described herein.

[0077] The presence of the added antigen-binding agent (VHH) results in increased binding affinity due to affinity-based interactions of the disclosed Ceberson construct. The three binding VHHs can originate from the same VHH forming a homotrimer. The design also allows for the mixing of different VHHs with desired binding and target-neutralizing properties. Such molecular complexes will be able to bind to multiple epitopes and mutant variants of the targets described herein (e.g., pathogens and their variants) that may not be captured by routine immune surveillance or conventional therapies. In all respects, the Cebersonson complex exhibits increased binding compared to the dimer or monomeric forms. For example, compared to dimer or monomeric scaffolds that are otherwise comparable, the binding can increase by at least about 5 times or at most about 300 times.

[0078] Hydra

[0079] In all aspects, a hypodermic complex is also provided. In all aspects, the hypodermic body comprises at least four heterologous parts. In all aspects, the hypodermic body comprises at least four antigen-binding VHHs.

[0080] Hydra body constructs can be formed when Spirulina strains (two independent strains or a single strain from different ORFs) express a 5HVZ-mediated homodimer and a smAKAP-linked dimeric VHH. The smAKAP-mediated dimer contains VHHs at both the N-terminus and C-terminus of the smAKAP. VHHs fused to smAKAP can form either homodimers (identical VHHs) or heterodimers (two distinct VHHs at either end). This configuration may enable the inclusion of multiple therapeutically effective VHHs within a single complex.

[0081] In all respects, the Hydralin complex exhibits increased binding compared to its trimer, dimer, or monomeric forms. For example, the binding can be increased by at least about 5 times or at most about 300 times compared to trimer, dimer, or monomeric scaffolds that are otherwise comparable.

[0082] Beyond the increased epigenetic binding based on affinity, higher-order complexes of serratus and hydralisons can form super-effective and cross-reactive therapeutic complexes capable of binding to and neutralizing different targets. For example, in various aspects, the scaffolds of this disclosure can be administered to subjects in need to neutralize two or more targets. For instance, a hydralison containing a dimer of two VHHs that bind to and neutralize a single target may be better than binding to and neutralizing two targets individually. Such designs readily allow for the combination of multiple components, where different epitopes can bind, thereby reducing target escape (e.g., viral escape).

[0083] Heterodimer

[0084] In all respects, one version of the 5HVZ scaffold forms a specific heterodimer rather than a homodimer. This allows for the assembly of a dimer 5HVZ complex containing four distinct functional domains. In all respects, the four distinct functional domains comprise two distinct functional domains associated with each 5HVZ scaffold.

[0085] In various respects, this paper provides heterodimeric complexes. In various respects, the heterodimeric complexes comprise a 5HVZ dimer complex. In various respects, the 5HVZ dimer complex comprises at least two, three, four, five, or six different functional domains. In various respects, the 5HVZ dimer complex comprises at least four different functional domains. In various respects, the 5HVZ dimer complex comprises four different functional domains. In various respects, the 5HVZ dimer complex comprises the same functional domains.

[0086] In each aspect, the functional domain comprises a functional domain associated with each of the 5HVZ stents. In each aspect, the functional domain comprises at least one, two, three, four, five, or six functional domains associated with each of the 5HVZ stents. In each aspect, the functional domain comprises at least two functional domains associated with each of the 5HVZ stents. In each aspect, the functional domain comprises two functional domains associated with each of the 5HVZ stents. In each aspect, the functional domains associated with each of the 5HVZ stents are identical. In each aspect, the functional domains associated with each of the 5HVZ stents are different. In each aspect, the dimer 5HVZ complex comprises at least two, three, four, five, or six different functional domains associated with each of the 5HVZ stents. In each aspect, the dimer 5HVZ complex comprises at least two different functional domains associated with each of the 5HVZ stents. In each aspect, the dimer 5HVZ complex comprises two different functional domains associated with each of the 5HVZ stents. In each aspect, the dimer 5HVZ complex comprises four distinct functional domains, two of which are associated with each of the 5HVZ scaffolds. In each aspect, the dimer 5HVZ complex comprises six distinct functional domains, three of which are associated with each of the 5HVZ scaffolds. In each aspect, the dimer 5HVZ complex comprises eight distinct functional domains, four of which are associated with each of the 5HVZ scaffolds.

[0087] In all aspects, the 5HVZ stent includes functional structural domains including 1-5HVZ1-structural domain 2. In all aspects, the 5HVZ stent includes functional structural domains including 3-5HVZ-structural domain 4. In all aspects, the 5HVZ stent includes functional structural domains including 1-5HVZ1-structural domain 2 and functional structural domains including 3-5HVZ-structural domain 4.

[0088] In all respects, the heterodimeric complex exhibits increased binding compared to the monomeric form. In all respects, the binding can be increased by 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, or 300 times compared to monomeric scaffolds that are otherwise comparable.

[0089] In addition to increased epigenetic binding based on affinity, heterodimers can form highly effective and cross-reactive therapeutic complexes that can bind to and neutralize different targets. For example, in various aspects, the stents of this disclosure can be administered to subjects in need to neutralize two or more targets.

[0090] heterogeneous part

[0091] In various aspects, the scaffold binds to the heterologous portion. In various aspects, the heterologous portion contains a therapeutic agent. In various aspects, the therapeutic agent contains a biologic. In various aspects, the therapeutic agent contains a binding agent. In various aspects, the heterologous portion is located at the N-terminus or C-terminus of the scaffold protein, or is attached to the scaffold protein in vivo.

[0092] In all respects, the therapeutic agent is a conjugate comprising an antibody or a functional fragment thereof. In all respects, the conjugate comprises an isolated fully human, humanized, or chimeric antibody or a fragment thereof. In all respects, the binding molecule is an antibody fragment or a single variable domain antibody, preferably Fab, Fab', F(ab')2, scFv, dAb, or VHH.

[0093] In all respects, the heterologous portion contains a binding agent as the VHH. VHHs can be produced economically and in unlimited quantities, are more stable when exposed to heat and solvents compared to conventional antibodies, and are suitable for a wide range of genetic manipulation applications, including scaffolding, labeling, and alteration of specific amino acids. Furthermore, VHHs are only 1 / 10 the size of conventional antibodies. Due to their smaller size, VHHs offer a higher density of binding domains, which provides a significant advantage in signal enhancement and thus higher sensitivity compared to conventional antibodies.

[0094] An exemplary VHH comprises an antigen-binding fragment of a heavy-chain antibody only. In all respects, the VHH is derived from a camel-derived single-chain antibody. In all respects, the VHH can be expressed intracellularly. In all respects, the VHH can be expressed extracellularly. In all respects, the VHH is expressed as a monomer, dimer, trimer, and / or heptamer. In all respects, the VHH is expressed as a monomer. In all respects, the VHH is expressed as a dimer. In all respects, the VHH is expressed as a trimer. In all respects, the VHH is expressed as a heptamer. Expression can be constitutive.

[0095] In all respects, VHH targets antigens associated with a disease or symptom. Exemplary diseases or symptoms can be autoimmune diseases, metabolic diseases, neurological diseases, cancer and parasitic diseases, bacterial diseases, viral diseases, and any combination thereof. In all respects, VHH targets autoimmune targets. Autoimmune targets can be associated with diseases selected from the group consisting of inflammatory bowel disease (IBD), celiac disease, ulcerative colitis, and Crohn's disease. In all respects, the disease or symptom is metabolic. An exemplary metabolic disease or symptom can be cardiometabolic disease (CMD). CMD is a range of associated symptoms including obesity, diabetes, and cardiovascular disease. In all respects, the disease or symptom is bacterial. Bacterial symptoms can include diarrhea caused by bacterial pathogens. Exemplary bacterial pathogens include Escherichia coli, Campylobacter jejuni, and enterotoxigenic Escherichia coli.

[0096] In all respects, the heterologous portion 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 all respects, the heterologous portion 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 all respects, the heterologous portion has at least one targeting function selected from the group consisting of: regulatory function, regulatory molecule (e.g., enzyme, protein, or nucleic acid), and localization to a specific site. In all respects, the heterologous portion is a tag or label, such as cleavable. In all respects, the heterologous component is selected from the group consisting of: epigenetic modifiers, epigenetic enzymes, bicyclic peptides, transcription factors, DNA or protein modifying enzymes, DNA intercalators, 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, bioactive peptides derived from natural sources, antimicrobial peptides, pore-forming peptides, targeted or cytotoxic peptides, degradation or self-destructive peptides, CRISPR systems or components thereof, DNA, RNA, artificial nucleic acids, nanoparticles, oligonucleotide aptamers, peptide aptamers, and synthetic or mimicking peptides from pharmacokinetic or pharmacodynamic (PK / PD) agents.

[0097] In all respects, the heterologous portion is selected from the group consisting of: drugs, toxins, cytotoxic agents, imaging agents, radionuclides, radioactive compounds, 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 portions. In all respects, the heterologous portion is albumin, and albumin includes human serum albumin. In all respects, the heterologous portion is an antibody or a fragment thereof, and includes antibody domains, antibody fragments, single-chain antibodies, domain antibodies, or any combination thereof.

[0098] In all respects, the heterologous portion contains antibodies. In all respects, antibodies are selected from the group consisting of: the Fc domain of the antibody, antibody fragments, and single-chain antibodies.

[0099] In various respects, the scaffold of this disclosure may include a heterologous portion of this disclosure. In various respects, the heterologous portion includes a VHH. In various respects, the seberon or hydra body may include multiple VHHs with the same or different sequences.

[0100] VHH, derived from a camelid single-chain antibody, is ideal for expression in prokaryotes such as *Spirulina* because the synthesis of bioactive proteins does not require intracellular disulfide bond formation or specific glycosylation. VHH is constitutively expressed in *Spirulina*. Expression can be regulated using any promoter. The promoter includes Pcpc600. As described herein, VHH can be expressed on a scaffold in various forms, including monomers, dimers, trimers, and heptamers.

[0101] Because they are readily expressed in prokaryotes, VHHs can be rapidly isolated from highly diverse, primordial phage display libraries. These typically have moderate nM affinity for their antigenic targets and are therefore susceptible to further mutagenesis to achieve higher affinity. As described in this paper, the expression of VHHs as a high-affinity multimer bypasses this limitation and thus accelerates product development.

[0102] In all respects, the VHH of this disclosure is combined with 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, white spot syndrome virus, coronavirus, MERS, SARS, and SARS-CoV-2.

[0103] In all aspects, VHH is anti-Campylobacter. In all aspects, Campylobacter is Campylobacter jejuni. In all aspects, VHH binds to Campylobacter components. In all aspects, VHH binds to flagellin. In all aspects, administration increases Campylobacter shedding. In all aspects, administration reduces the level of biomarkers. In all aspects, the biomarker is an inflammatory biomarker.

[0104] In all respects, the recombinant Spirulina contains a VHH that binds to Clostridium toxins. In all respects, the Clostridium species is Clostridium difficile. In all respects, the VHH binds to a Clostridium component, toxin A, or toxin B. In all respects, the VHH contains the amino acid sequence or a fragment thereof of any of SEQ ID NO:25-37.

[0105] In all aspects, recombinant Spirulina contains VHH against Campylobacter. In all aspects, Campylobacter is Campylobacter jejuni. In all aspects, VHH binds to Campylobacter components. In all aspects, VHH binds to flagellin. In all aspects, application increases Campylobacter shedding. In all aspects, application reduces the level of biomarkers. In all aspects, the biomarker is an inflammatory biomarker.

[0106] In all respects, the recombinant *Spirulina* genus contains a polypeptide or fragment thereof that binds to a norovirus polypeptide or antigen. In all respects, the recombinant *Spirulina* genus contains a polypeptide or fragment thereof that binds to the norovirus P domain. In all respects, the polypeptide or fragment thereof is a VHH. In all respects, the recombinant *Spirulina* genus contains a VHH that binds to a norovirus polypeptide. In all respects, the recombinant *Spirulina* genus contains a VHH that binds to the norovirus P domain. In all respects, the recombinant *Spirulina* genus contains a polypeptide or fragment thereof that binds to the GII genotype, G1 genotype, or G11.10 genotype. In all respects, the recombinant *Spirulina* genus contains a polypeptide or fragment thereof that binds to polypeptides from two or more norovirus genotypes. In all respects, the recombinant *Spirulina* genus contains a VHH that comprises Nano85 nanobodies, Nano26 nanobodies, Nano94 nanobodies, K922 antibodies, or modified sequences or fragments thereof. In all respects, the recombinant *Spirulina* genus contains a VHH comprising Nano85 and / or its loop-grafted modifications.

[0107] In all aspects, the genus *Spirulina* includes a scaffold containing VHH bound to a malaria polypeptide or antigen or a fragment thereof. In all aspects, malaria includes the genus *Plasmodium*. In all aspects, malaria includes *Plasmodium falciparum*, *Plasmodium vivax*, *Plasmodium ovale*, *Plasmodium vannamei*, *Plasmodium malariae*, or *Plasmodium norotri*. In all aspects, malaria is *Plasmodium falciparum*. In all aspects, recombinant *Spirulina* contains polypeptides bound to malaria-associated proteins or fragments thereof in the liver or blood. In all aspects, recombinant *Spirulina* contains polypeptides bound to malaria-associated proteins or fragments thereof during the extraerythrocyte cycle, hepatocyte rupture, or erythrocyte cycle. In all aspects, recombinant *Spirulina* contains polypeptides bound to malaria-associated proteins or fragments thereof during the extraerythrocyte cycle. In all aspects, recombinant *Spirulina* contains polypeptides bound to malaria-associated proteins or fragments thereof, said malaria-associated proteins or fragments thereof derived from stages including sporozoites, merozoites, trophozoites, merozoites, or gametophytes. In all aspects, recombinant *Spirulina* contains polypeptides bound to malaria-associated proteins or fragments thereof on the surface of sporozoites. In all respects, the recombinant Spirulina genus contains a polypeptide that binds to cyclosporin (CSP) or fragments thereof. In all respects, the recombinant Spirulina genus contains a polypeptide that binds to Plasmodium falciparum cyclosporin (PfCSP) or fragments thereof. In all respects, the binding disclosed herein can block sporozoite infection of hepatocytes and prevent malaria. In all respects, VHH binds to one or more NANP repeat sequences. In all respects, VHH binds to the CSP polypeptide. In all respects, VHH binds to a polypeptide containing one or more NANP repeat sequences.

[0108] Exemplary VHHs of this disclosure are provided in Table 0. In all respects, the brackets of this disclosure and the VHHs of Table 0 contain VHH connections of at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity. In all respects, the brackets of this disclosure and SEQ ID NO: 25-67 contain VHH connections of at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity. In all respects, the brackets of this disclosure and SEQ ID NO: 25-37 contain VHH connections of at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity. In all respects, the brackets of this disclosure and SEQ ID NO: 38-67 contain VHH connections of at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity.

[0109] Table 0. Exemplary VHH sequences

[0110]

[0111]

[0112]

[0113]

[0114]

[0115] The scaffold containing the VHH of this disclosure neutralizes or blocks the activity of the target. In various respects, application of this 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%.

[0116] In various aspects, VHH can be expressed as a fusion protein with solubility-enhancing chaperone proteins, such as E. coli maltose-binding protein (MBP).

[0117] In various aspects, the scaffold-containing compositions provided herein can bind to about one, two, three, four, five, six, seven, or up to about eight different targets via heterologous portions. In various aspects, the scaffolds of this disclosure bind to two targets. In various aspects, the scaffolds of this disclosure bind to three targets. In various aspects, the scaffolds of this disclosure bind to four targets. In various aspects, the scaffolds of this disclosure bind to five targets. In various aspects, the scaffolds of this disclosure bind to six targets. In various aspects, the scaffolds of this disclosure bind to seven targets. In various aspects, the scaffolds of this disclosure bind to eight targets.

[0118] Nucleic acids encoding any heterologous portions and / or polypeptides of this disclosure, such as cybelian and / or hydralian bodies, are also provided. Such nucleic acids are also referred to hereinafter as “nucleic acids of this disclosure” and may be, for example, in the form of gene constructs.

[0119] In all respects, the scaffolds of this disclosure, such as cyboloids or hypodermics, are more effective in neutralizing targets compared to monomeric, dimeric, or trimeric constructs. In all respects, their effectiveness is at least about 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000, 5000, 10,000, 15,000, 100,000, 200,000, 300,000, 500,000, 800,000, or 1,000,000 times higher than that of non-mericarmic constructs. In all respects, the scaffolds of this disclosure, such as cyboloids or hypodermics, are more effective in reducing or eliminating targets compared to monomeric or dimeric constructs. In all aspects, its effectiveness is at least approximately 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000, 5000, 10,000, 15,000, 100,000, 200,000, 300,000, 500,000, 800,000, or 1,000,000 times higher than that of non-polymeric constructs.

[0120] In all respects, the scaffolds of this disclosure exhibit increased binding to the target compared to non-polymeric scaffolds or corresponding binders. The binding increase is at least 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%, 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%.

[0121] In all respects, multimeric constructs, such as selenosomers or hypodermiforms, are more effective in alleviating symptoms or disease in subjects in need compared to monomeric or dimer constructs. In some respects, their effectiveness in alleviating symptoms or disease is at least approximately 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000, 5000, 10,000, 15,000, 100,000, 200,000, 300,000, 500,000, 800,000, or 1,000,000 times greater than that of nonmeric constructs.

[0122] In all respects, multimeric constructs, such as cyperoids or hypodermics, are more effective than monomeric or dimeric constructs in neutralizing or reducing pathogens or their variants (e.g., viral / bacterial / cancer variants) that evade conventional therapies. In all respects, their effectiveness is at least approximately 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000, 5000, 10,000, 15,000, 100,000, 200,000, 300,000, 500,000, 800,000, or 1,000,000 times greater than conventional therapies. In all respects, effectiveness increased by at least approximately or at most 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%, 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%.

[0123] connector

[0124] In various aspects, connectors are also provided that can be used in the compositions and methods provided herein. Any connector can be used in the compositions of this disclosure, for example, for connecting one or more heterogeneous portions of this disclosure. In various aspects, the connector can also be used to connect Seber bodies and / or Hydra bodies. In various aspects, the connector comprises smAKAP.

[0125] In all respects, the heterologous portion can be conjugated to the scaffold directly or via a linker. In all respects, two or more heterologous portions can be conjugated directly or via a linker. Suitable linkers include, for example, cleavable and non-cleavable linkers. Cleavable linkers are generally readily cleavable 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 all respects, the linker can be a dipeptide linker, such as valine-citrulline (val-cit), phenylalanine-lysine (phe-lys), or a linker. Other suitable linkers include linkers that are hydrolyzable at pH less than 5.5, such as hydrazone linkers. Additionally, suitable cleavable linkers include disulfide linkers.

[0126] In all respects, the adapter is a rigid adapter. In all respects, the adapter is a flexible adapter. In all respects, the adapter attaches two or more VHH sequences. In all respects, the adapter attaches one or more VHH sequences to another heterologous portion of this disclosure. In all respects, the heterologous portion is selected from the group consisting of: chaperone proteins, target proteins, scaffolds, oligomerizing domains, enzymes, or lysins.

[0127] In all respects, the linker comprises the smAKAP peptide. In all respects, the smAKAP peptide can be modified. Modifications can be made, for example, to increase the expression of soluble proteins. In all respects, one or more residues in the linker can be mutated. In all respects, two Cys residues in an exemplary linker (e.g., the smAKAP peptide) are mutated to serine residues. In all respects, cleavable linkers can also be used in the compositions provided herein.

[0128] In all respects, the connectors selected from the group consisting of the following have at least about or at most about 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 100% identity with the connectors: AEAAAAKAS (Screw 1 connector; SEQ ID NO: 68), AEAAAAKEAAAKAS (Screw 2 connector; SEQ ID NO: 69), AEAAAAKEAAAKEAAAKEAAAKAS (Screw 4 connector; SEQ ID NO: 70), APAPSPAPSPAS (PA5 connector; SEQ ID NO: 71), and PA10 lin25.

[0129] In all respects, the adapter comprises a mutant smAKAP, which 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 NO: 5, 7, 15, 17, 19, 21, 72, 73, and 74. The adapter may also comprise the WT smAKAP sequence of SEQ ID NO: 2.

[0130] In all respects, the joint can have any length. In all respects, the length of the joint is approximately: 1-3, 2-6, 1-8, 3-10, 5-20, 3-15, 10-30, 15-35, 20-35 or 25-35 residues. In all aspects, the length of the connector is 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.

[0131] Spirulina

[0132] The genus *Spirulina* is synonymous with the genus *Arthrospira*. *Arthrospira* comprises 57 species, of which 22 are currently taxonomically accepted. In all respects, *Spirulina* species are selected from the following groups: *Arthrospira amethystica*, *Arthrospira adisonensis*, *Arthrospira argentina*, *Arthrospira barakriensis*, *Arthrospira bayana*, *Arthrospira boyana*, *Arthrospira brycei*, *Arthrospira short-segmented*, *Arthrospira short-segmented*, *Arthrospira septacea*, *Arthrospira desicachari*, *Arthrospira fasciata*, *Arthrospira fusiformis*, *Arthrospira gana*, *Arthrospira giantis*, *Arthrospira gomonte*, *Arthrospira gomonte* (coarse variety), *Arthrospira indica*, *Arthrospira prata* (Plata variety), *Arthrospira fasciata stitzenberger*, *Arthrospira fasciata purple*, *Arthrospira josie*, *Arthrospira canna*, *Arthrospira broad*, *Arthrospira loose*, *Arthrospira pine*, *Arthrospira leviana*, *Arthrospira levofloxas*, *Arthrospira macrophylla*, *Arthrospira pearlifolia*. Spirulina, *Spirulina massatus*, *Spirulina massatus* Indian variant, *Spirulina macrocarpa*, *Spirulina menekinensis*, *Spirulina vermilion* constricted variant, *Spirulina microcarpa*, *Spirulina microcarpa* cuspidatum variant, *Spirulina naples*, *Spirulina nordsteade*, *Spirulina marinea*, *Spirulina occa*, *Spirulina hyaluronica*, *Spirulina plata*, *Spirulina plata* non-constricted variant, *Spirulina plata* granular variant, *Spirulina plata* miniature variant, *Spirulina plata* petit-variant variant, *Spirulina Santana*, *Spirulina squalis*, *Spirulina skua*, *Spirulina genus-like* petit-variant, *Spirulina genus-like*, *Spirulina saline*, *Spirulina fine*, *Spirulina fine-detail*, *Spirulina microcarpa*, and *Spirulina variegata*. In one aspect, the genus *Spirulina* is *Blunt-topped*.

[0133] In all respects, recombinant Spirulina is non-living. In all respects, recombinant Spirulina is dried, spray-dried, freeze-dried, or lyophilized.

[0134] Any suitable method of transforming Spirulina can be used in this disclosure. Exemplary methods for transforming Spirulina to express heterologous proteins are described in U.S. Patent No. 10,131,870, which is incorporated herein by reference in its entirety.

[0135] In each aspect, the method for preparing a Spirulina composition expressing the scaffold of this disclosure comprises introducing an expression vector having a nucleic acid sequence encoding the scaffold into Spirulina cells. In each aspect, the vector is not integrated into the Spirulina genome. In each aspect, the vector is a high-copy or high-expression vector. In each aspect, the nucleic acid sequence encoding the scaffold is under the control of a strong promoter. In each aspect, the nucleic acid sequence encoding the scaffold is under the control of a constitutive promoter. In each aspect, the nucleic acid sequence encoding the scaffold is under the control of an inducible promoter.

[0136] In various respects, methods for preparing the composition involve introducing a vector (e.g., by homologous recombination) having a homologous arm and a nucleic acid sequence encoding a scaffold into Spirulina cells.

[0137] Electroporation can be used to introduce nucleic acid sequences with homologous arms and coding scaffolds into Spirulina. Electroporation is preferably performed in the presence of a suitable osmotic stabilizer.

[0138] Before introducing the vector into the *Spirulina* genus, *Spirulina* can be cultured in any medium suitable for cyanobacterial growth, such as SOT medium. SOT medium consists of 1.68 g NaHCO3, 50 mg K2HPO4, 250 mg NaNO3, 100 mg K25O4, 100 mg NaCl, 20 mg MgSO4·7H2O, 4 mg CaCl2·2H2O, 1 mg FeSO4·7H2O, 8 mg Na2EDTA·2H2O, 0.1 mL A5 solution, and 99.9 mL distilled water. A5 solution consists of 286 mg H3BO3, 217 mg MnSO4·5H2O, 22.2 mg ZnSO4·7H2O, 7.9 mg CuSO4·5H2O, 2.1 mg Na2MoO4·2H2O, and 100 mL distilled water. Culture can be carried out at temperatures above room temperature (e.g., 25-37°C) and under continuous irradiation (e.g., 20-2,000, 50-500, or 100-200 μmol photons). -2 s -1 The oscillation is performed at a speed of 100-300 rpm. The OD is reached when the optical density at 750 nm reaches a predetermined threshold (e.g., 0.3-2.0, 0.5-1.0, or 0.6-0.8). 750During this process, growing cells can be collected. A specific volume of the collected cells can be concentrated by centrifugation and then resuspended in a pH balancer and salt solution. The pH balancer can be any suitable buffer that maintains the viability of *Spirulina* while keeping the culture medium pH between 6 and 9, 6.5 and 8.5, or 7 and 8. Suitable pH balancers include HEPES, HEPES-NaOH, sodium or potassium phosphate buffer, and TES. The salt solution can be NaCl with concentrations between 50 mM and 500 mM, 100 mM and 400 mM, or 200 mM and 300 mM. In all cases, 1-50 mL of 1-100 mM pH balancing solution can be used to neutralize the pH.

[0139] Cells collected by centrifugation can be washed with an osmotic stabilizer and optionally a salt solution (e.g., 1-50 mL of 0.1-100 mM NaCl). Any volume of culture can be concentrated by centrifugation. In all cases, 5-500 mL of culture can be centrifuged. The osmotic stabilizer can be any type of osmotic balancer that stabilizes the cell integrity of Spirulina during electroporation. In all cases, the osmotic stabilizer can be a sugar (e.g., w / v 0.1-25%), such as glucose or sucrose. In all cases, the osmotic stabilizer can be a simple polyol (e.g., v / v 1-25%), including glycerol, glycerol, or glycerol. In all cases, the osmotic stabilizer can be a polyether, including (e.g., w / v 0.1-20%) polyethylene glycol (PEG), polyethylene oxide, or polyethylene oxide (PEO). PEG or PEO can have any molecular weight of 200 to 10,000, 1,000 to 6,000, or 2,000 to 4,000. In all respects, pH balancers or buffers can be used in place of osmotic stabilizers or in addition to osmotic stabilizers.

[0140] Vectors containing nucleic acid sequences with homologous arms and coding scaffolds can be introduced into Spirulina cells, as described above, by culturing and washing the Spirulina cells with an osmotic stabilizer. Electroporation can be used to introduce the vectors.

[0141] Electroporation can be performed in 0.1 cm, 0.2 cm, or 0.4 cm electroporation cuvettes at voltages of 0.6 kV / cm to 10 kV / cm, 2.5 kV / cm to 6.5 kV / cm, or 4.0 kV / cm to 5.0 kV / cm; 1 μF to 100 μF, 30 μF to 70 μF, or 45 μF to 55 μF; and 10 mΩ to 500 mΩ, 50 mΩ to 250 mΩ, or 90 mΩ to 110 mΩ. In all respects, electroporation can be performed at 4.5 kV / cm, 50 μF, and 100 mΩ.

[0142] Following electroporation, cells can be grown in the presence of one or more antibiotics, selected based on resistance conferred by successful plasmid transformation. Post-electroporation culture can be performed at reduced irradiation levels (e.g., 5-500, 10-100, or 30-60 μmol photons). -2 s -1 Culture can also be performed with shaking (e.g., 100–300 rpm). The antibiotic level in the medium can be between 5 μg / mL and 100 μg / mL. Culture after electroporation can continue for 1–5 days or longer. Successful transformants identified by antibiotic resistance can be selected on plates or in 5–100 mL of SOT medium supplemented with 0.1–2.0 μg of appropriate antibiotic within 1 week to 1 month.

[0143] The vector used in the method can be a plasmid, bacteriophage, or viral vector, in which a nucleic acid sequence encoding at least one exogenous polypeptide, antigen, and / or antigen can be inserted or cloned. The vector can contain one or more specific sequences that allow recombination into specific desired sites on the Spirulina chromosome. These specific sequences may be homologous to sequences present in wild-type Spirulina. The vector system can contain a single vector or plasmid, two or more vectors or plasmids, some of which increase the efficiency of directed mutagenesis or transposition. The choice of vector typically depends on its compatibility with the Spirulina cells to which it is to be introduced. The vector may include a reporter gene, such as green fluorescent protein (GFP), which may be fused within a frame to one or more encoded antigenic epitopes or expressed alone. The vector may also include a positive selection marker, such as an antibiotic resistance gene that can be used to select suitable transformants. The vector may also include a negative selection marker, such as a type II thioesterase (tesA) gene or a Bacillus subtilis structural gene (sacB). The use of reporter molecules or markers allows identification of cells that have been successfully transformed with the vector.

[0144] In all respects, the vector includes one or two homologous arms that are homologous to the DNA sequence of the Spirulina genome at the adjacent target locus. The sequences of the homologous arms may be partially or completely complementary to regions of the Spirulina genome at the adjacent target locus.

[0145] Homologous arms can have any length that allows site-specific homologous recombination. Homologous arms can be any length from approximately 2000 bp to 500 bp. For example, homologous arms can be approximately 2000 bp, approximately 1500 bp, approximately 1000 bp, or approximately 500 bp. In cases with two homologous arms, the homologous arms can be the same or different lengths. Therefore, each of the two homologous arms can be any length between approximately 2000 bp and 500 bp. For example, each of the two homologous arms can be approximately 2000 bp, approximately 1500 bp, approximately 1000 bp, or approximately 500 bp.

[0146] A portion of a vector adjacent to or flanked by one homologous arm can be modified via homologous recombination to target loci in the Spirulina genome. The modification can alter the length of the target locus, including the deletion or addition of nucleotides. Additions or deletions can be of any length. The modification can also alter the sequence of nucleotides within the target locus without changing its length. The target locus can be any part of the Spirulina genome, including coding regions, non-coding regions, and regulatory sequences.

[0147] How to use

[0148] The compositions disclosed herein can be used in treatment methods. In various aspects, the compositions disclosed herein can be used to reduce the severity of a disease or condition in a subject in need. In various aspects, the compositions can be used to prevent a disease or condition in a subject. In various aspects, the compositions can be used to prevent the onset of a disease or condition in a subject. In various aspects, the compositions can be used to reduce the severity of a disease or condition in a subject. In various aspects, the compositions can be used to prevent or delay the recurrence of a disease in a subject. In various aspects, the compositions can be used to treat, prevent, or delay the recurrence of cancer in a subject. In various aspects, the compositions can be used to treat, prevent, or delay the recurrence of systemic pathogens. In various aspects, the compositions can be used to treat, prevent, or delay the recurrence of systemic parasites. In various aspects, the compositions can be used to treat, prevent, or delay the recurrence of malaria. In various aspects, the compositions can be used to treat, prevent, or delay the recurrence of mucosal pathogens.

[0149] In various aspects, the treatment methods include administering the disclosed composition to treat intestinal pathogens. Exemplary intestinal pathogens include: Campylobacter infection, enterotoxigenic Escherichia coli, norovirus, Clostridium difficile, and SARS-CoV-2.

[0150] In all respects, the compositions disclosed herein are intended for the treatment of diseases or symptoms associated with or originating from any microorganism, or for the treatment or prevention of infections caused by any microorganism, including but not limited to *Escherichia coli*, enterotoxigenic *E. coli* (ETEC), anthrax, *EHEC*, *EAEC*, *Shigella* spp., *Mycobacterium* spp., *Streptococcus* spp., *Staphylococcus* spp., *Shigella* spp., Campylobacter spp., *Salmonella* spp., *Clostridium* spp., *Corynebacterium* spp., *Pseudomonas* spp., *Neisseria* spp., *Listeria* spp., *Vibrio* spp., *Bordeaux* spp., *Legionella* spp., 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, and 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, leukoplakia syndrome virus, coronavirus, SARS, MERS, SARS-CoV-2, Aspergillus, Candida, blastomycetes Genus: Coccidioides, Cryptococcus, Histoplasma, Plasmodium, Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, Plasmodium vivax, Trypanosoma, Toxoplasma, Giardia, Cryptosporidium leishmaniasis, helminths: whipworms, pinworms, roundworms, hookworms and nematodes, strongyloides stercoralis, dragon nematodes, onchocerca, nematodes, tapeworms, echinococcosis and sparganosis, fascioliasis, and schistosomes or combinations thereof.

[0151] In all respects, the compositions described herein can be used to treat and / or reduce the severity of infections caused by bacteria, including but not limited to: Mycobacterium spp., Streptococcus spp., Staphylococcus spp., Shigella spp., Campylobacter spp., Salmonella spp., Clostridium spp., Corynebacterium spp., Pseudomonas spp., Neisseria spp., Listeria spp., Vibrio spp., Bordetella spp., and Legionella spp.

[0152] In all respects, the compositions described herein can be used to treat or reduce the severity of infections caused by parasites, including but not limited to Plasmodium, Trypanosoma, Toxoplasma, Giardia and Leishmania, Cryptosporidium, helmintic parasites: whipworms, pinworms, roundworms, hookworms and nematodes, strongyloides stercoralis, draconis, onchocerciasis and filariasis, tapeworms, echinococcosis and sparganosis (human and animal tapeworms), liver flukes, and schistosomes.

[0153] In all respects, the compositions described herein can be used to treat or reduce the severity of infections caused by Plasmodium spp. In all respects, the compositions disclosed herein can be used to induce an immune response to and / or reduce the severity of infections caused by Plasmodium spp., selected from the group consisting of Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, and Plasmodium vivax.

[0154] In all respects, the compositions described herein can 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 all respects, the compositions can be used to induce an immune response to Candida albicans or Candida auris infections and / or reduce their severity.

[0155] In all respects, the compositions described herein can 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.

[0156] In all respects, the compositions described herein can be used to treat or alleviate 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 I diabetes, autoimmune thrombocytopenic purpura (ATP), idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), Crohn's disease, multiple sclerosis, and myasthenia gravis.

[0157] Other GI targets, such as inflammation and metabolic diseases, as well as microbiome manipulation, have also been considered. In various respects, the compositions described herein can alter the abundance of microorganisms present in the microbiome. In various respects, the compositions described herein can alter the abundance of bacteria present in the microbiome. In various respects, the compositions disclosed herein can alter the abundance of Firmicutes, Bacteroidetes, Proteobacteria, and Actinomycetes. In various respects, the compositions described herein can alter the abundance of Firmicutes, Bacteroidetes, Proteobacteria, and Actinomycetes. In various respects, the compositions described herein can be used to reduce the abundance of bacteria comprising Bacteroidetes, Proteobacteria, Enterobacteriaceae, Porphyromonas, Clostridium, Pasteurella, Veillonella, Neisseriaceae, Clostridium, Protobacter, Ruminococcus, Polyspora, Actinomycetes, and Fusobacterialaceae. In various respects, the compositions described herein can be used to reduce the abundance of bacteria comprising *Escherichia coli*, *Shigella*, *Rhodococcus*, *Veillonella*, *Stenotrophomonas maltophilia*, *Prevotella*, *Clostridium difficile*, *Clostridium polycladus*, *Klebsiella pneumoniae*, *Proteus mirabilis*, *Staphylococcus aureus*, *Ruminococcus truncatella*, *Active Ruminococcus*, and *Helicobacter hepatica*. In various respects, the compositions described herein can be used to increase the abundance of anti-inflammatory bacteria in the microbiome. In various respects, the compositions described herein can be used to increase butyrate-producing bacteria. In various respects, the compositions described herein can be used to increase bacteria comprising *Firmwallis*, *Lactobacillus*, *Ruminococci*, *Eubacterium*, *Bifidobacterium*, *Femtobacter*, *Roseidon*, *E. coli*, *Roseidon*, *Ackermania*, and *Clostridium*. In various respects, the compositions described herein can be used to increase bacteria comprising *Prevotella foetida*, *Bifidobacterium longum*, *Eubacterium rectum*, *Roseidon*, and *Ackermania*.

[0158] Pharmaceutical Composition

[0159] In all respects, the compositions of this disclosure are formulated as pharmaceutical compositions. For example, a stent may be included within the pharmaceutical composition. The pharmaceutical compositions of this disclosure can be administered to a subject in need. Administration can be performed in any manner. In all respects, administration is oral, intravenous, intradermal, via airway, intranasal, and combinations thereof.

[0160] In all respects, the pharmaceutical compositions are administered orally. As used herein, the terms "oral composition" or "oral delivery composition" include compositions administered or delivered to the gastrointestinal tract (e.g., oral, oral via a feeding tube, etc.). The compositions disclosed herein may target any suitable area of ​​the gastrointestinal tract.

[0161] In various aspects, the compositions of this disclosure are administered via the airway. In various aspects, the compositions of this disclosure are administered by inhalation. In various aspects, the compositions of this disclosure are administered via intranasal administration. In various aspects, the compositions of this disclosure are administered via a nebulizer, inhaler, or inhaler. In various aspects, the compositions of this disclosure are lyophilized and delivered as a powder or powder resuspended in a liquid. In various aspects, the compositions of this disclosure are formulated for administration via a nebulizer, inhaler, dry powder inhaler, or inhaler.

[0162] In all respects, the pharmaceutical composition contains an adjuvant. Most adjuvants contain substances designed to protect proteins or fragments thereof from rapid catabolic metabolism, such as aluminum hydroxide or mineral oil, and immune response stimulants, such as proteins derived from Bordetella pertussis or Mycobacterium tuberculosis. In all respects, adjuvants include, but are not limited to, Toll-like receptor (TLR) agonists, monophospholipid A (MPL), synthetic lipid A, lipid A mimics or analogs, aluminum salts, cytokines, saponins, muramyl dipeptide (MDP) derivatives, CpG oligomers, lipopolysaccharides (LPS) of Gram-negative bacteria, polyphosphazenes, emulsions, virions, cochlea, poly(lactide-co-glycolic acid) (PLG) microparticles, poloxamer particles, microparticles, montanide, and liposomes. In all respects, the adjuvant is a montanide adjuvant.

[0163] In all respects, the composition comprises at least one adjuvant. In all respects, the adjuvant is present in solution. In various aspects, the adjuvant is present at about 1 µg / mL to about 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, 900 µg / mL, 950 µg / mL, 1mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40mg / mL, 45 mg / mL, 50 mg / mL, 55 The concentrations of 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 thereof, are present.

[0164] In all respects, the composition survives in the gastrointestinal tract or a simulated gastric environment. In all respects, the composition survives in the gastrointestinal tract or a simulated gastric environment for at least 5 minutes. In all respects, the composition survives overnight in the gastrointestinal tract or a simulated gastric environment.

[0165] In all respects, the composition remains in the nasal cavity. In all respects, the composition remains in the upper respiratory tract. In all respects, the composition remains in the airway. In all respects, the composition remains in the nasal cavity, upper respiratory tract, and / or airway for at least 5 minutes. In all respects, the composition remains in the nasal cavity, upper respiratory tract, and / or airway overnight.

[0166] The compositions of this disclosure can be administered daily, weekly, bi-weekly, every other week, monthly, etc. In each aspect, the compositions of this disclosure are administered to subjects for approximately 1 day to approximately 1 year. In each aspect, the compositions of this disclosure are administered to subjects for approximately 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 each aspect, the compositions of this disclosure are administered for several consecutive days. In each aspect, the compositions of this disclosure are administered over several non-consecutive days. In each aspect, the compositions of this disclosure are administered once daily. In each aspect, the compositions of this disclosure are administered multiple times a day. In each aspect, the compositions of this disclosure are administered twice daily, three times a day, four times a day, or more times a day. In each aspect, the compositions of this disclosure are administered continuously (e.g., via feeding tube). In each aspect, the compositions of this disclosure are administered with meals. In each aspect, the compositions of this disclosure are administered when the subject is fasting.

[0167] In all respects, the compositions disclosed herein may comprise one or more pharmaceutically acceptable excipients. Pharmaceutically acceptable carriers include, but are not limited to, saline, buffered saline, glucose, water, glycerol, sterile isotonic buffer, and combinations thereof. In all respects, a pharmaceutically acceptable excipient is sodium bicarbonate.

[0168] In various aspects, the composition further comprises excipients. In various aspects, the composition comprises at least one excipient. In various aspects, the composition comprises at least two or more excipients. In various aspects, the composition comprises three or more excipients. In various aspects, the composition comprises four or more excipients. In various aspects, the composition comprises five or more excipients. In various aspects, the composition comprises six or more excipients. In various aspects, the composition comprises seven or more excipients. Exemplary excipients include, but are not limited to, stabilizers, buffers, surfactants, fillers, sugars, and salts.

[0169] In all respects, excipients are selected from the group consisting of 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 lauryl sulfate, poloxamer 188, hexadecyltrimethylammonium bromide, and polysorbate-20. Exemplary fillers 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, glucose, 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 each respect, the excipients further include histidine, arginine, and glycine.

[0170] In all respects, excipients are present in solution. In various aspects, the excipients are present in an amount of from about 1 µg / mL to about 1 g / 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, 750µg / mL, 800 µg / mL, 850 µg / mL, 900 µg / mL, 950 µg / mL, 1,000 µg / mL, 50 mg / mL, 100 mg / mL, 150 mg / mL, 200 mg / mL, 250 mg / mL, 300 mg / mL, 350 mg / mL, 400 mg / mL, 450 Concentrations of 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 thereof, are present.

[0171] In all respects, the excipients comprise about 0.1% to about 0.5% of the composition. The excipients may comprise about 0.1%, 0.2%, 0.3%, 0.4% or up to about 0.5% of the composition.

[0172] Reagent test kit

[0173] This document also discloses kits comprising the disclosed compositions. Kits for treating or preventing cancer, pathogen infection, or immune disorders are also disclosed. In various aspects, the kit may include a therapeutic or prophylactic composition containing an effective amount of the composition comprising recombinant Spirulina spp. (e.g., converted with a scaffold). In some embodiments, the kit comprises a sterile container containing a therapeutic composition of Spirulina spp.; such a container may be a box, ampoule, bottle, vial, tube, bag, pouch, blister pack, 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 containing the drug. In various aspects, converted Spirulina spp., along with instructions for administering Spirulina spp. to subjects who have or are at risk of having cancer, pathogen infection, or immune disorders, may be provided.

[0174] Numbered Examples

[0175] Despite the appended claims, the following numbered embodiments also form part of this disclosure.

[0176] Example Group 1

[0177] 1. A recombinant Spirulina species expressing a mutated small membrane A-kinase anchoring protein (smAKAP) peptide sequence.

[0178] 2. The recombinant Spirulina genus according to Example 1, wherein the Spirulina genus contains at least 2, 3, 4, 5, 6 or 7 mutated residues in the mutated smAKAP peptide sequence compared to the WT smAKAP peptide sequence.

[0179] 3. The recombinant Spirulina genus according to Example 1, wherein one residue in the smAKAP peptide sequence is mutated compared to the WT smAKAP peptide sequence.

[0180] 4. The recombinant Spirulina genus according to any one of Examples 2 to 3, wherein two residues in the smAKAP peptide sequence are mutated compared to the WT smAKAP peptide sequence.

[0181] 5. The recombinant Spirulina according to any one of Examples 2 to 4, wherein three residues in the smAKAP peptide sequence are mutated compared to the WT smAKAP peptide sequence.

[0182] 6. The recombinant Spirulina according to any one of Examples 1 to 5, wherein the mutant smAKAP peptide exhibits resistance to protease cleavage, as determined by the reduction of cleavage products, when exposed to a solvent containing a protease for one hour.

[0183] 7. The recombinant Spirulina genus according to Example 6, wherein up to about 5%, 10%, 20%, 30%, 40%, 50% or 60% of the smAKAP peptide sequence is cleaved.

[0184] 8. The recombinant Spirulina genus according to any one of Examples 1 to 7, wherein the mutation belongs to a hydrophobic residue.

[0185] 9. The recombinant Spirulina genus according to any one of Examples 1 to 8, wherein when the recombinant Spirulina genus is immersed in a solvent, the mutated residues are exposed to the solvent.

[0186] 10. The recombinant Spirulina genus according to any one of Examples 1 to 9, wherein the mutated residues are selected from the group consisting of: C16S, C24S, E5D, Y6H, W22S, C24G, L4E, R9E, L4I, L10I, L19I of SEQ ID NO: 2 and combinations thereof.

[0187] 11. The recombinant Spirulina genus according to Example 10, wherein the mutated residues are C16S and C24S compared to SEQ ID NO: 2.

[0188] 12. The recombinant Spirulina genus according to Example 10, wherein the mutated residues compared with SEQ ID NO: 2 are E5D, Y6H, C16S, W22S and C24G.

[0189] 13. The recombinant Spirulina genus according to Example 10, wherein the mutated residues compared with SEQ ID NO: 2 are L4E, E5D, Y6H, R9E, C16S, W22S and C24G.

[0190] 14. The recombinant Spirulina genus according to Example 10, wherein the mutated residues compared with SEQ ID NO: 2 are L4E, C16S, W22S and C24G.

[0191] 15. The recombinant Spirulina genus according to Example 10, wherein the mutated residues compared to SEQ ID NO: 2 are R9E, C16S and C24G.

[0192] 16. The recombinant Spirulina genus according to Example 10, wherein the mutated residues compared with SEQ ID NO: 2 are R9E, C16S, W22S and C24G.

[0193] 17. The recombinant Spirulina genus according to any one of Examples 1 to 16, wherein the mutant smAKAP peptide sequence is linked to a heterologous portion in a monomeric configuration.

[0194] 18. The recombinant Spirulina genus according to Example 17, wherein the mutant smAKAP peptide sequence is linked to a second heterologous portion.

[0195] 19. The recombinant Spirulina genus according to Example 18, wherein the heterologous portion is the same as the second heterologous portion.

[0196] 20. The recombinant Spirulina genus according to any one of Examples 17 to 18, wherein the heterologous portion is different from the second heterologous portion.

[0197] 21. The recombinant Spirulina genus according to any one of Examples 1 to 20, wherein the recombinant Spirulina genus expresses another exogenous polypeptide sequence.

[0198] 22. The recombinant Spirulina genus according to Example 21, wherein the exogenous polypeptide sequence is selected from the group consisting of: the oligomerization domain of C4b binding protein (C4BP), the cholera toxin b subunit, the oligomerization domain of extracellular matrix protein, TRX, 5HVZ, cTRP, SP651, SP737 and 4BOF.

[0199] 23. The recombinant Spirulina genus according to Example 22, wherein the exogenous polypeptide sequence is 5HVZ.

[0200] 24. The recombinant Spirulina genus according to Example 23, wherein the 5HVZ is connected to the third heterologous portion.

[0201] 25. The recombinant Spirulina genus according to Example 24, wherein the third heterologous portion is in a homodimer configuration.

[0202] 26. The recombinant Spirulina genus according to any one of Examples 17 to 25, wherein the heterologous portion, the second heterologous portion, and the third heterologous portion are identical.

[0203] 27. The recombinant Spirulina genus according to any one of Examples 17 to 25, wherein the heterologous portion, the second heterologous portion, and the third heterologous portion are different.

[0204] 28. The recombinant Spirulina genus according to any one of Examples 17 to 27, wherein the heterologous portion is a binder.

[0205] 29. The recombinant Spirulina genus according to Example 28, wherein the binding agent is selected from the group consisting of: fab', F(ab')2, fv, domain antibody (dAb), complementarity-determining region (CDR) fragment, CDR transplantation antibody, single-chain antibody (scFv), single-chain antibody fragment, chimeric antibody, bifunctional antibody, trifunctional antibody, tetrafunctional antibody, microantibody, linear antibody, intracellular antibody, nanobody (single-domain antibody), small modular immunopharmaceutical (SMIP), antigen-binding domain immunoglobulin fusion protein, and VHH.

[0206] 30. The recombinant Spirulina genus according to Example 29, wherein the binder is the VHH.

[0207] 31. The recombinant Spirulina genus according to Example 30, wherein the VHH binds to the pathogen.

[0208] 32. The recombinant Spirulina genus according to Example 30, wherein the VHH binds to cancer cells.

[0209] 33. The recombinant Spirulina genus according to Example 30, wherein the VHH binds to human cells.

[0210] 34. The recombinant Spirulina genus according to Example 31, wherein the VHH binds to pathogens selected from the group consisting of bacteria, fungi, and viruses.

[0211] 35. The recombinant Spirulina genus according to Example 34, wherein the pathogen is selected from the group consisting of: Escherichia coli, enterotoxigenic Escherichia coli (ETEC), anthrax, EHEC, EAEC, Shigella spp., Mycobacterium spp., Streptococcus spp., Staphylococcus spp., Shigella spp., Campylobacter spp., Salmonella spp., Clostridium spp., Corynebacterium spp., Pseudomonas spp., Neisseria spp., Listeria spp., Vibrio spp., Bordetella spp., Legionella spp., 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, and 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, Leukoplakia syndrome virus, Coronavirus, SARS, MERS, SARS-CoV-2, Aspergillus, Candida, Blastomyces, Coccidioides, Cryptococcus, Histoplasma, Plasmodium, Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, Plasmodium vivax, Trypanosoma, Toxoplasma, Giardia, Cryptosporidium leishmaniasis, Helminthus, Trichuris trichiura, Pinworm, Ascaris, Hookworm and Isopoda, Strongyloides stercoralis, Gnatifida, Onchocerca salina, Wucetella, Tapeworm, Echinococcus granulosus and Sparganum, Fasciola hepatica and Schistosoma.

[0212] 36. The recombinant Spirulina genus according to Example 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.

[0213] 37. The recombinant Spirulina genus according to Example 36, wherein the bacteria is Campylobacter.

[0214] 38. The recombinant Spirulina genus according to Example 36, wherein the bacteria is Clostridium genus.

[0215] 39. The recombinant Spirulina genus according to any one of Examples 35 to 38, wherein the VHH comprises a sequence having at least 85% identity with the sequence of SEQ ID NO: 25-67.

[0216] 40. The recombinant Spirulina genus according to any one of Examples 35 to 38, wherein the VHH comprises the sequence SEQ ID NO: 25-67.

[0217] 41. The recombinant Spirulina genus according to any one of Examples 23 to 40, wherein the recombinant Spirulina genus expresses a scaffold having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequences of SEQ ID NO: 6, 14, 16, 18, 20, 22, 23, and 24.

[0218] 42. A recombinant Spirulina species expressing a mutated small membrane A-kinase anchoring protein (smAKAP) peptide sequence, wherein the mutation comprises a substitution of a residue of SEQ ID NO: 2.

[0219] 43. A recombinant Spirulina species expressing a mutated small membrane 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 of SEQ ID NO: 2 and combinations thereof.

[0220] 44. The recombinant Spirulina genus according to any one of Examples 42 to 43, wherein smAKAP is linked to one or more VHH antibodies at the terminal.

[0221] 45. The recombinant Spirulina genus according to Example 44, wherein the smAKAP is linked to two VHH antibodies, wherein the first VHH is located at the C-terminus and the second VHH is located at the N-terminus.

[0222] 46. ​​The recombinant Spirulina genus according to any one of Examples 42 to 45, wherein the recombinant Spirulina genus expresses 5HVZ.

[0223] 47. The recombinant Spirulina genus according to Example 46, wherein the 5HVZ is linked to two VHH antibodies in a homodimeric configuration.

[0224] 48. A polynucleotide sequence comprising a mutated small membrane A-kinase anchoring protein (smAKAP) sequence, wherein the mutation is a single residue substitution compared to the WT smAKAP sequence.

[0225] 49. A vector comprising the polynucleotide sequence according to Example 48.

[0226] 50. A method for preparing recombinant Spirulina, the method comprising contacting Spirulina cells with a carrier according to Example 49.

[0227] 51. A pharmaceutical composition comprising recombinant Spirulina according to any one of Examples 1 to 47 and an excipient.

[0228] 52. A kit comprising: a recombinant Spirulina genus according to any one of Examples 1 to 47, a polynucleotide sequence according to Example 48, a vector according to Example 49 or a pharmaceutical composition according to Example 51, and instructions for use thereof.

[0229] 53. A treatment method comprising administering the pharmaceutical composition according to Example 51 to a subject in need, wherein the subject has a bacterial or viral infection.

[0230] 54. A vector comprising a nucleotide sequence encoding a polypeptide of SEQ ID NO: 5-7 or 14-24.

[0231] 55. A recombinant Spirulina species comprising the vector described in Example 54.

[0232] Example Group 2

[0233] 1. A recombinant Spirulina species expressing a mutated small membrane A-kinase anchoring protein (smAKAP) peptide sequence.

[0234] 2. The recombinant Spirulina genus according to Example 1, wherein the Spirulina genus contains at least 2, 3, 4, 5, 6 or 7 mutated residues in the mutated smAKAP peptide sequence compared to the WT smAKAP peptide sequence.

[0235] 3. The recombinant Spirulina genus according to Example 1, wherein one residue in the smAKAP peptide sequence is mutated compared to the WT smAKAP peptide sequence.

[0236] 4. The recombinant Spirulina genus according to any one of Examples 2 to 3, wherein two residues in the smAKAP peptide sequence are mutated compared to the WT smAKAP peptide sequence.

[0237] 5. The recombinant Spirulina according to any one of Examples 2 to 4, wherein three residues in the smAKAP peptide sequence are mutated compared to the WT smAKAP peptide sequence.

[0238] 6. The recombinant Spirulina according to any one of Examples 1 to 5, wherein the mutant smAKAP peptide exhibits resistance to protease cleavage, as determined by the reduction of cleavage products, when exposed to a solvent containing a protease for one hour.

[0239] 7. The recombinant Spirulina genus according to Example 6, wherein up to about 5%, 10%, 20%, 30%, 40%, 50% or 60% of the smAKAP peptide sequence is cleaved.

[0240] 8. The recombinant Spirulina genus according to any one of Examples 1 to 7, wherein the mutation belongs to a hydrophobic residue.

[0241] 9. The recombinant Spirulina genus according to any one of Examples 1 to 8, wherein when the recombinant Spirulina genus is immersed in a solvent, the mutated residues are exposed to the solvent.

[0242] 10. The recombinant Spirulina genus according to any one of Examples 1 to 9, wherein the mutated residues are selected from the group consisting of: C16S, C24S, E5D, Y6H, W22S, C24G, L4E, R9E, L4I, L10I, L19I of SEQ ID NO: 2 and combinations thereof.

[0243] 11. The recombinant Spirulina genus according to Example 10, wherein the mutated residues are C16S and C24S compared to SEQ ID NO: 2.

[0244] 12. The recombinant Spirulina genus according to Example 10, wherein the mutated residues compared with SEQ ID NO: 2 are E5D, Y6H, C16S, W22S and C24G.

[0245] 13. The recombinant Spirulina genus according to Example 10, wherein the mutated residues compared with SEQ ID NO: 2 are L4E, E5D, Y6H, R9E, C16S, W22S and C24G.

[0246] 14. The recombinant Spirulina genus according to Example 10, wherein the mutated residues compared with SEQ ID NO: 2 are L4E, C16S, W22S and C24G.

[0247] 15. The recombinant Spirulina genus according to Example 10, wherein the mutated residues compared to SEQ ID NO: 2 are R9E, C16S and C24G.

[0248] 16. The recombinant Spirulina genus according to Example 10, wherein the mutated residues compared with SEQ ID NO: 2 are R9E, C16S, W22S and C24G.

[0249] 17. The recombinant Spirulina genus according to Example 10, wherein the mutated residues compared with SEQ ID NO: 2 are L4I, R9E, C16S, and C24G.

[0250] 18. The recombinant Spirulina genus according to any one of Examples 1 to 17, wherein the mutant smAKAP peptide sequence is linked to a heterologous portion in a monomeric configuration.

[0251] 19. The recombinant Spirulina genus according to Example 18, wherein the mutant smAKAP peptide sequence is linked to a second heterologous portion.

[0252] 20. The recombinant Spirulina genus according to Example 19, wherein the heterologous portion is the same as the second heterologous portion.

[0253] 21. The recombinant Spirulina genus according to any one of Examples 18 to 19, wherein the heterologous portion is different from the second heterologous portion.

[0254] 22. The recombinant Spirulina genus according to any one of Examples 1 to 21, wherein the recombinant Spirulina genus expresses another exogenous polypeptide sequence.

[0255] 23. The recombinant Spirulina genus according to Example 22, wherein the exogenous polypeptide sequence is selected from the group consisting of: the oligomerization domain of C4b binding protein (C4BP), the cholera toxin b subunit, the oligomerization domain of extracellular matrix protein, TRX, 5HVZ, cTRP, SP651, SP737 and 4BOF.

[0256] 24. The recombinant Spirulina genus according to Example 23, wherein the exogenous polypeptide sequence is 5HVZ.

[0257] 25. The recombinant Spirulina genus according to Example 24, wherein the 5HVZ is connected to the third heterologous portion.

[0258] 26. The recombinant Spirulina genus according to Example 25, wherein the third heterologous portion is in a dimer configuration.

[0259] 27. The recombinant Spirulina genus according to any one of Examples 18 to 26, wherein the heterologous portion, the second heterologous portion, and the third heterologous portion are identical.

[0260] 28. The recombinant Spirulina genus according to any one of Examples 18 to 26, wherein the heterologous portion, the second heterologous portion, and the third heterologous portion are different.

[0261] 29. The recombinant Spirulina genus according to any one of Examples 18 to 28, wherein the heterologous portion is a binder.

[0262] 30. The recombinant Spirulina genus according to Example 29, wherein the binding agent is selected from the group consisting of: fab', F(ab')2, fv, domain antibody (dAb), complementarity-determining region (CDR) fragment, CDR transplantation antibody, single-chain antibody (scFv), single-chain antibody fragment, chimeric antibody, bifunctional antibody, trifunctional antibody, tetrafunctional antibody, microantibody, linear antibody, intracellular antibody, nanobody (single-domain antibody), small modular immunopharmaceutical (SMIP), antigen-binding domain immunoglobulin fusion protein, and VHH.

[0263] 31. The recombinant Spirulina genus according to Example 30, wherein the binder is the VHH.

[0264] 32. The recombinant Spirulina genus according to Example 31, wherein the VHH binds to the pathogen.

[0265] 33. The recombinant Spirulina genus according to Example 31, wherein the VHH binds to cancer cells.

[0266] 34. The recombinant Spirulina genus according to Example 31, wherein the VHH binds to human cells.

[0267] 35. The recombinant Spirulina genus according to Example 32, wherein the VHH binds to pathogens selected from the group consisting of bacteria, fungi, and viruses.

[0268] 36. The recombinant Spirulina genus according to Example 35, wherein the pathogen is selected from the group consisting of: Escherichia coli, enterotoxigenic Escherichia coli (ETEC), anthrax, EHEC, EAEC, Shigella spp., Mycobacterium spp., Streptococcus spp., Staphylococcus spp., Shigella spp., Campylobacter spp., Salmonella spp., Clostridium spp., Corynebacterium spp., Pseudomonas spp., Neisseria spp., Listeria spp., Vibrio spp., Bordetella spp., Legionella spp., 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, and 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, Leukoplakia syndrome virus, Coronavirus, SARS, MERS, SARS-CoV-2, Aspergillus, Candida, Blastomyces, Coccidioides, Cryptococcus, Histoplasma, Plasmodium, Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, Plasmodium vivax, Trypanosoma, Toxoplasma, Giardia, Cryptosporidium leishmaniasis, Helminthus, Trichuris trichiura, Pinworm, Ascaris, Hookworm and Isopoda, Strongyloides stercoralis, Gnatifida, Onchocerca salina, Wucetella, Tapeworm, Echinococcus granulosus and Sparganum, Fasciola hepatica and Schistosoma.

[0269] 37. The recombinant Spirulina genus according to Example 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.

[0270] 38. The recombinant Spirulina genus according to Example 37, wherein the bacteria is Campylobacter.

[0271] 39. The recombinant Spirulina genus according to Example 37, wherein the bacteria is Clostridium genus.

[0272] 40. The recombinant Spirulina genus according to any one of Examples 36 to 39, wherein the VHH comprises a sequence having at least 85% identity with the sequence of SEQ ID NO: 25-67.

[0273] 41. The recombinant Spirulina genus according to any one of Examples 36 to 39, wherein the VHH comprises the sequence SEQ ID NO: 25-67.

[0274] 42. The recombinant Spirulina genus according to any one of Examples 24 to 41, wherein the recombinant Spirulina genus expresses a scaffold having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequences of SEQ ID NO: 4, 6, 14, 16, 18, 20, 22, 23, 24, and 76.

[0275] 43. A recombinant Spirulina species expressing a mutated small membrane A-kinase anchoring protein (smAKAP) peptide sequence, wherein the mutation comprises a substitution of a residue of SEQ ID NO: 2.

[0276] 44. A recombinant Spirulina species expressing a mutated small membrane 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 of SEQ ID NO: 2 and combinations thereof.

[0277] 45. The recombinant Spirulina genus according to any one of Examples 43 to 44, wherein smAKAP is linked to one or more VHH antibodies at the terminal.

[0278] 46. ​​The recombinant Spirulina genus according to Example 45, wherein the smAKAP is linked to two VHH antibodies, wherein the first VHH is located at the C-terminus and the second VHH is located at the N-terminus.

[0279] 47. The recombinant Spirulina genus according to any one of Examples 43 to 46, wherein the recombinant Spirulina genus expresses 5HVZ.

[0280] 48. The recombinant Spirulina genus according to Example 47, wherein the 5HVZ is linked to two VHH antibodies in a homodimeric configuration.

[0281] 49. A polynucleotide sequence comprising a mutated small membrane A-kinase anchoring protein (smAKAP) sequence, wherein the mutation comprises substitution of one or more residues relative to the WT smAKAP sequence.

[0282] 50. A vector comprising the polynucleotide sequence according to Example 49.

[0283] 51. A method for preparing recombinant Spirulina, the method comprising contacting Spirulina cells with a carrier according to Example 50.

[0284] 52. A pharmaceutical composition comprising recombinant Spirulina according to any one of Examples 1 to 48 and an excipient.

[0285] 53. A kit comprising: a recombinant Spirulina species according to any one of Examples 1 to 48, a polynucleotide sequence according to Example 49, a vector according to Example 50 or a pharmaceutical composition according to Example 52, and instructions for use thereof.

[0286] 54. A treatment method comprising administering the pharmaceutical composition according to Example 52 to a subject in need, wherein the subject has a bacterial or viral infection.

[0287] 55. A vector comprising a nucleotide sequence encoding a polypeptide of SEQ ID NO: 2-7, 14-24, 74 and / or 76.

[0288] 56. The carrier according to Example 55, wherein the carrier is approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 75.

[0289] 56. A recombinant Spirulina species comprising a vector according to any one of Examples 55 to 56.

[0290] Example

[0291] Example 1 - Assessment of protease sensitivity

[0292] Research Objectives

[0293] To evaluate the protease sensitivity of exemplary scaffold constructs, the protease sensitivity of the hypodrama complex PP2130 (a complex of PP1113 and PP1895) was compared with that of the following constructs: PP917 2xS3b-C8, PP1895 2xS3b-C8, and PP1895 2xRN-29.

[0294] In summary, to assess the in vitro stability of the cloned proteases, purified protein samples were prepared at 0.2 mg / mL in protease digestion buffer (20 mM Bis-Tris, 150 mM NaCl, 3 mM CaCl2, pH 6.0). The proteases trypsin and chymotrypsin were diluted from a stem concentration of 1 mg / mL to 0.2 mg / mL in protease digestion buffer. Both the protein and enzyme were diluted 1:10 in protease digestion buffer. The digestion reaction was performed 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 at 37°C for 60 minutes with shaking at 900 rpm in an Eppendorf Thermo Mixer. The reaction mixture was quenched with an equal volume of stop buffer containing 2 mM PMSF and 2x Pierce protease inhibitor microparticles in PBS. The final concentration of each VHH homodimer after protease neutralization was 0.1 mg / mL. Samples were kept on ice for ELISA assays.

[0295] The binding activity of digested protein samples was analyzed by ELISA. High-binding ELISA plates were coated with 1 μg / mL SARS-COV-2 RBD in 100 μl / well of CBC binding buffer 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 with 300 μl / well of blocking buffer (PBS-tw supplemented with 5% skim milk powder). Dilutes 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 each well of the ELISA plate and incubated on a plate shaker at room temperature for 1 hour. The plates were washed and binding was measured. A mixture of HRP-conjugated anti-cameloid VHH antibodies (GenScript catalog number A02016) was diluted 10,000-fold in blocking buffer, and 100 μl / well was added to each well of the ELISA plate. Incubate the plate with shaking for 30 minutes. Wash the ELISA plate twice with PBS-tw and once with PBS. Initiate the HRP reaction by adding 100 μl / well of TMB Ultra ELISA substrate (Thermo Fisher Scientific) to each well. Develop the ELISA plate by incubating at room temperature for 5 minutes and quenching with 50 μL of 1 M hydrochloric acid. Read the absorbance at 450 nm using a Spectra Max M5 plate reader.

[0296] The results showed that the PP1895 construct was sensitive to both trypsin and chymotrypsin: approximately 99% of its activity was lost after 1 hour of incubation. In contrast, the Hydrasome complex PP2130 regained stability under trypsin conditions (see [link to relevant documentation]). Figures 1A-1D .

[0297] Example 2 - Mutations in smAKAP that confer resistance to trypsin and chymotrypsin

[0298] Following the protease sensitivity study in Example 1 that demonstrated protease sensitivity in smAKAP, mutations in smAKAP were evaluated to determine whether trypsin and chymotrypsin resistance could be engineered into smAKAP.

[0299] To generate mutant smAKAP clones, protein-protein interactions were performed using structural and PISA analyses of the complex crystal structure (PDBID 5HVZ). The importance of the arginine (R) and lysine (K) side chains (predicted as trypsin substrates) in complex formation was assessed using structural tools. Side chains predicted as chymotrypsin substrates with similar hydrophobicity were evaluated, and potential mutations maintaining complex formation were proposed. Various mutants were generated, containing the following mutations compared to the WT sequence of SEQ ID NO: 2: C16S, C24S, E5D, Y6H, W22S, C24G, L4E, R9E, L4I, L10I, L19I, and various combinations thereof.

[0300] Table 1. Exemplary mutant smAKAP sequences

[0301]

[0302] An exemplary mutant smAKAP connector is used for scaffold constructs; see Table 2, and also see... Figure 4 .

[0303] Table 2: Exemplary mutant smAKAP clones, adapters, seber bodies, and hydra bodies constructs

[0304]

[0305]

[0306]

[0307]

[0308]

[0309] In the PP1895 construct, C78S can be interchanged with C78G because the position allows for any small amino acid.

[0310] Example 3 - Expression of mutant smAKAP clone in E. coli system

[0311] The smAKAP mutants were generated based on the mutations described in Table 1, and their expression in the E. coli system was evaluated.

[0312] In short, the construct was subcloned into a modified pET28b(+) vector, in which the kanamycin resistance gene was replaced with an ampicillin resistance gene. The sequence-validated plasmid was transformed into BL21(DE3) *E. coli* cells (New England Biolabs) and plated on LB medium supplemented with 100 μg mL⁻¹ ampicillin. Protein expression was performed using an automated induction protocol, in which a single colony of the transformant was inoculated into 100 mL of super broth medium containing an appropriate selected drug, shaken at 37°C for 8 h, followed by shaking at 19°C for 36 h. The culture was centrifuged, and the precipitate was stored at -20°C until purification. The frozen precipitate was thawed on ice and resuspended in 30 mL of 50 mM Tris pH 8.0, 300 mM NaCl 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 min at 4 °C on an Eppendorf 5810R benchtop centrifuge. 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 17524802) equilibrated with 50 mM Tris pH 8.0, 300 mM NaCl, and 20 mM imidazole (Thermo Fisher Scientific, catalog 03196-500). The hexahistine-labeled and HisTrap-purified samples were further purified using size exclusion chromatography based on AKTA-pure FPLC with a Superdex 200 increase 10 / 300 (Cytiva, catalog 28990944). Protein purity and size were analyzed by size exclusion chromatography on an analytical Superdex 200 increase 10 / 300 (Stenofan, catalog 28990944), with 100 μL of purified protein (approximately 1 mg / mL) run at room temperature in 1xPBS buffer. Purified protein samples were also evaluated on SDS-PAGE under reducing and non-reducing conditions. 3 µg protein samples were incubated at 70 °C in LDS loading buffer with or without 50 mM dithiothreitol (DTT) reducing agent (Thermo Fisher Scientific, catalog R0862).

[0313] The results showed that all mutant smAKAP clones were expressed in E. coli. (See [link to relevant documentation]) Figure 5 .

[0314] Example 4 - Assessment of smAKAP mutant and protease resistance

[0315] trypsin resistance

[0316] According to the method of Example 1, smAKAP mutants with R63E substitution were generated to assess whether the mutation confers trypsin resistance to the mutant smAKAP peptide.

[0317] The trypsin resistance of clones PP2451, PP1895 and PP2455 was assessed using a method similar to that described in Example 1.

[0318] The results showed that the R63E mutation rescued the sensitivity of WT smAKAP to trypsin. (See [link to relevant documentation]). Figures 6A-6C .

[0319] chymotrypsin resistance

[0320] The ability of the smAKAP mutant generated according to Example 1 to resist chymotrypsin cleavage was also evaluated. Specifically, clones PP2451, PP1895, PP2455, PP2456, PP2454, PP2452, and PP2453 were assayed using a method similar to that described in Example 1.

[0321] The results showed that combining hydrophobic mutants (such as in PP2453) produced the highest level of improved resistance. See [link to relevant documentation]. Figure 9 Furthermore, in cases where chymotrypsin sensitivity is detected, this is primarily due to the presence of the smAKAP linker. However, alternative linkers can be used with the compositions disclosed herein.

[0322] Example 5 - Complex Formation Analysis

[0323] The formation of dimer complexes

[0324] Dimers of smAKAP and 5HVZ were generated, and their ability to form scaffold complexes was evaluated.

[0325] Dimer formation via the 5HVZ motif is due to protein-protein interactions between the three α-helical chains, resulting in an antiparallel conformation of the monomeric polypeptide. Furthermore, the formation of asymmetric disulfide bonds between protomers further enhances the dimer structure. The dimer protein is expressed from a clone genetically encoding a single polypeptide containing a 5HVZ dimerization motif fused to the protein of interest. The dimerization motif (5HVZ) can be fused at both the N-terminus and C-terminus to generate homo- or hetero-dimeric constructs. Engineering VHH constructs using the 5HVZ dimerization motif for Spirulina expression showed increased binding affinity to the antigen of interest. The dimer formed by the 5HVZ motif leads to protein folding, acting as a docking platform to facilitate the interaction between PKA and membrane-attached A-kinase anchored protein (AKAP). The small α-helical peptide smAKAP in AKAP docks to the 5HVZ platform.

[0326] Complex formation assessment

[0327] To verify whether the smAKAP mutant retained the ability to form a scaffold complex, complex formation analysis was performed using PP2451, PP1895, PP2456 (W76S), and PP2455 (R63E mutant) clones.

[0328] To evaluate higher-order complex formation (hydra bodies), purified dimeric proteins (PP917 and PP1113) were run on an analytical Superdex 200 increase 10 / 300 (Stenofan, catalog number 28990944) to achieve homodimerization in the dimeric proteins using a 5HVZ scaffold. Proteins were run alone or in a 1:2 molar ratio with proteins from smAKAP mutants and control clones. Complex formation efficiency was compared by comparing the retention volume and protein absorbance of the dimers and smAKAP-containing constructs evaluated individually. The smAKAP-containing construct that formed a complex with the construct containing the 5HVZ dimeric scaffold showed a change towards a smaller retention volume and an increase in absorbance for the complex. Additionally, the absorbance peak height of the construct forming the complex was significantly reduced at the expected retention volume of the smAKAP-containing construct after elution.

[0329] The results showed that R63E and W76S substitutions did not affect the formation of the complex in scaffolds containing the mutant smAKAP. See [link to relevant documentation]. Figure 8 Therefore, compared with the control, not only was the formation of the complex unaffected, but the mutant smAKAP also exhibited enhanced protease resistance.

[0330] Using a similar method as described above, additional studies were conducted using the following alternative mutant clones: PP2451, PP1895, PP2456, PP2454, PP2452, and PP2453.

[0331] Specifically, the crystal structure of the smAKAP and dimer interface was calculated using the web-based interactive analysis tool PDBePISA (PISA) based on the crystal structure (PDB ID5HVZ). The interface formed between the dimerizing motif “5HVZ” and the smAKAP docking peptide was evaluated, and its stability physicochemical properties were scored. The PISA analysis tool used physicochemical properties, including solvation energy, interfacial area, hydrogen bonding, formation free energy, and salt bridge formation, to predict the strength of complex formation. The summary data presented in Tables 3-4 show the hydrogen bonding or salt bridge formation of the indicated amino acid chains (HSDC column), solvent-accessible surface area (ASA), embedded surface area (BSA), percentage of embedded area (one bar per 10%), and solvation energy effect (ΔiG). The analysis was used to determine the role of mutations in complex formation.

[0332] Table 3. Interaction between smAKAP peptide and 5HVZ dimer promerogeneic A

[0333]

[0334]

[0335] Table 4. Interaction between smAKAP peptide and 5HVZ dimer promerogeneic B

[0336]

[0337]

[0338] These results indicate that the Y60H mutation plays a maximal role in complex formation. See also: Figure 10 .

[0339] Example 6 - Increasing complex stability through smAKAP mutation

[0340] The ability of other mutations to stabilize the smAKAP complex was evaluated. Stability assays were performed on clones PP2451-PP2456. To assess the stability of the purified proteins, protein samples were evaluated by SDS-PAGE gel under both non-reducing and reducing (50 mM DTT) conditions. Proteins from the same batch were also stored at 4°C for up to 6 weeks, and their stability was assessed by SDS-PAGE gel under both non-reducing and reducing (50 mM DTT) conditions. Comparative analysis of the SDS-PAGE gels showed reduced fragmentation in some smAKAP mutant adapter clones.

[0341] The results showed that clones PP2453 and PP2455, containing the R63E mutation, were more stable. (See [link to documentation]) Figure 11 Compared to Figure 13 .

[0342] Example 7 - Expression of constructs containing smAKAP linkers in Spirulina

[0343] The smAKAP mutant was generated based on the mutations described in Table 1, and the ability of the mutant to be expressed in the Spirulina system was evaluated.

[0344] In short, the construct was subcloned into a modified pET28b(+) vector, in which the kanamycin resistance gene was replaced with the ampicillin resistance gene. The sequence-validated plasmid was transformed into *Spirulina*. Proteins were expressed, isolated, and determined by size exclusion chromatography on an analytical Superdex 200 increase 10 / 300 (Stenofan, catalog 28990944), with 100 μL of purified protein at approximately 1 mg / mL run in 1xPBS buffer at room temperature. The purified protein samples were also evaluated on SDS-PAGE under reducing and non-reducing conditions. 3 µg of protein samples were incubated at 70 °C in LDS loading buffer with or without 50 mM dithiothreitol (DTT) reducing agent (Thermo Fisher Scientific, catalog R0862).

[0345] Table 5. Expression of constructs containing the smAKAP linker in Spirulina.

[0346]

[0347] The results showed that constructs containing the smAKAP linker were expressed in the genus Spirulina. See [link to previous section]. Figure 12 .

[0348] Example 8 - Formation of higher-order complexes via smAKAP mutants

[0349] The formation of higher-order complexes using the 5HVZ dimerization domain can be achieved by complexing with proteins linked by the smAKAP peptide. The effect of amino acid substitutions of the smAKAP peptide on complex formation was evaluated by comparing the WT smAKAP peptide (SEQ ID NO: 2) with the PP6510 mutant (SEQ ID NO: 74).

[0350] In short, the protein linked via the PP6510 mutant was cloned into the pET28 expression vector (SEQ ID NO:75) and expressed in *E. coli* cells. The purified proteins were evaluated by size exclusion chromatography (SEC). To analyze complex formation, the purified dimeric proteins (PP6510 and PP1116) were run on an analytical Superdex 200 increase 10 / 300 (Stopfan, catalog number 28990944), where homodimerization was achieved via a 5HVZ scaffold. Proteins were run alone or in combination with proteins from the smAKAP mutant and control clones at a molar ratio of 1:1.2. Complex formation efficiency was compared by comparing the retention volume and protein absorbance of the dimeric constructs evaluated individually and those containing the smAKAP construct.

[0351] The results showed that the applicant successfully used the PP6510 mutant to form higher-order oligomers of a 5HVZ-mediated homodimeric protein. See [link to relevant documentation]. Figure 14 .

[0352] By incorporating via reference

[0353] For all purposes, all references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated herein by full reference. However, any mention of any reference, article, publication, patent, patent publication, or patent application cited herein is not and should not be construed as an admission or in any way an implication that it constitutes valid prior art or is part of common general knowledge in any country of the world.

[0354] The provided composition may contain the amino acid sequences described in International Patent Applications No. PCT / US2020 / 040794 and No. PCT / US2021 / 065138.

Claims

1. A recombinant Spirulina species expressing a mutated small membrane A-kinase anchoring protein (smAKAP) peptide sequence.

2. The recombinant Spirulina genus according to claim 1, wherein the Spirulina genus contains at least 2, 3, 4, 5, 6 or 7 mutated residues in the mutated smAKAP peptide sequence compared to the WT smAKAP peptide sequence.

3. The recombinant Spirulina according to claim 1, wherein one residue in the smAKAP peptide sequence is mutated compared to the WT smAKAP peptide sequence.

4. The recombinant Spirulina genus according to any one of claims 2 to 3, wherein two residues in the smAKAP peptide sequence are mutated compared to the WT smAKAP peptide sequence.

5. The recombinant Spirulina genus according to any one of claims 2 to 4, wherein three residues in the smAKAP peptide sequence are mutated compared to the WT 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 the reduction in 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. The recombinant Spirulina genus according to any one of claims 1 to 7, wherein the mutation belongs to a hydrophobic residue.

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

10. The recombinant Spirulina genus according to any one of claims 1 to 9, wherein the mutated residues are selected from the group consisting of: C16S, C24S, E5D, Y6H, W22S, C24G, L4E, R9E, L4I, L10I, L19I of SEQ ID NO: 2 and combinations thereof.

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

2.

12. The recombinant Spirulina genus according to claim 10, wherein the mutated residues compared to SEQ ID NO: 2 are E5D, Y6H, C16S, W22S and C24G.

13. The recombinant Spirulina genus according to claim 10, wherein the mutated residues compared to SEQ ID NO: 2 are L4E, E5D, Y6H, R9E, C16S, W22S and C24G.

14. The recombinant Spirulina genus according to claim 10, wherein the mutated residues compared to SEQ ID NO: 2 are L4E, C16S, W22S and C24G.

15. The recombinant Spirulina genus according to claim 10, wherein the mutated residues compared to SEQ ID NO: 2 are R9E, C16S, and C24G.

16. The recombinant Spirulina genus according to claim 10, wherein the mutated residues compared to SEQ ID NO: 2 are R9E, C16S, W22S and C24G.

17. The recombinant Spirulina genus according to claim 10, wherein the mutated residues compared to SEQ ID NO: 2 are L4I, R9E, C16S, and C24G.

18. The recombinant Spirulina genus according to any one of claims 1 to 17, wherein the mutant smAKAP peptide sequence is linked to a heterologous portion in a monomeric configuration.

19. The recombinant Spirulina genus according to claim 18, wherein the mutant smAKAP peptide sequence is linked to the second heterologous portion.

20. The recombinant Spirulina genus according to claim 19, wherein the heterologous portion is the same as the second heterologous portion.

21. The recombinant Spirulina genus according to any one of claims 18 to 19, wherein the heterologous portion is different from the second heterologous portion.

22. The recombinant Spirulina genus according to any one of claims 1 to 21, wherein the recombinant Spirulina genus 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 oligomerization domain of C4b binding protein (C4BP), the cholera toxin b subunit, the oligomerization domain of extracellular matrix protein, TRX, 5HVZ, cTRP, SP651, SP737 and 4BOF.

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

25. The recombinant Spirulina genus according to claim 24, wherein the 5HVZ is connected to the third heterologous portion.

26. The recombinant Spirulina genus according to claim 25, wherein the third heterologous portion is in a dimer configuration.

27. The recombinant Spirulina genus according to any one of claims 18 to 26, wherein the heterologous portion, the second heterologous portion, and the third heterologous portion are identical.

28. The recombinant Spirulina genus according to any one of claims 18 to 26, wherein the heterologous portion, the second heterologous portion, and the third heterologous portion are different.

29. The recombinant Spirulina genus according to any one of claims 18 to 28, wherein the heterologous portion is a binder.

30. The recombinant Spirulina according to claim 29, wherein the binding agent is selected from the group consisting of: fab', F(ab')2, fv, domain antibody (dAb), complementarity-determining region (CDR) fragment, CDR transplantation antibody, single-chain antibody (scFv), single-chain antibody fragment, chimeric antibody, bifunctional antibody, trifunctional antibody, tetrafunctional antibody, microantibody, linear antibody, intracellular antibody, nanobody (single-domain antibody), small modular immunopharmaceutical (SMIP), antigen-binding domain immunoglobulin fusion protein, and VHH.

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

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

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

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

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

36. The recombinant Spirulina genus according to claim 35, wherein the pathogen is selected from the group consisting of: Escherichia coli, enterotoxigenic Escherichia coli (ETEC), anthrax, EHEC, EAEC, Shigella, Mycobacterium, Streptococcus, Staphylococcus, 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, leukoplakia syndrome virus, coronavirus, SARS, MERS, SARS-CoV-2, Aspergillus, Candida, Blastomyces, Coccidioides, Cryptococcus, Histoplasma, Plasmodium, P. falciparum, P. malariae. malariae), Plasmodium ovale, Plasmodium vivax, Trypanosoma, Toxoplasma, Giardia, Cryptosporidium leishmaniae, helminthic parasites: Trichuris spp., Enterobius spp., Ascaris spp., Ancylostomaspp., and Necatro spp.The following are listed: *Strongyloides stercoralis* spp., *Dracunculus spp.*, *Onchocerca spp.*, *Wuchereria spp.*, tapeworms (*Taenia spp.*), *Echinococcus spp.*, *Diphyllobothrium spp.*, *Fasciola spp.*, and *Schistosoma spp.* 37. The recombinant Spirulina genus 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 genus according to claim 37, wherein the bacteria is Campylobacter.

39. The recombinant Spirulina genus according to claim 37, wherein the bacteria is Clostridium genus.

40. The recombinant Spirulina genus according to any one of claims 36 to 39, wherein the VHH comprises a sequence having at least 85% identity with the sequence of SEQ ID NO: 25-67.

41. The recombinant Spirulina genus according to any one of claims 36 to 39, wherein the VHH comprises the sequence SEQ ID NO: 25-67.

42. The recombinant Spirulina genus according to any one of claims 24 to 41, wherein the recombinant Spirulina genus expresses a scaffold having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequences of SEQ ID NO: 4, 6, 14, 16, 18, 20, 22, 23, 24, and 76.

43. A recombinant Spirulina species expressing a mutated small membrane A-kinase anchoring protein (smAKAP) peptide sequence, wherein the mutation comprises a substitution of a residue of SEQ ID NO:

2.

44. A recombinant Spirulina species expressing a mutated small membrane 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 of SEQ ID NO: 2 and combinations thereof.

45. The recombinant Spirulina genus according to any one of claims 43 to 44, wherein smAKAP is linked to one or more VHH antibodies at the terminal.

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

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

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

49. A polynucleotide sequence comprising a mutated small membrane A-kinase anchoring protein (smAKAP) sequence, wherein the mutation comprises substitution of one or more residues relative to the WT smAKAP sequence.

50. A vector comprising the polynucleotide sequence according to claim 49.

51. A method for preparing recombinant Spirulina, the method comprising contacting Spirulina cells with a carrier according to claim 50.

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

53. A kit comprising: a recombinant Spirulina genus 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 treatment method comprising administering the pharmaceutical composition of claim 52 to a subject in need, wherein the subject has a bacterial or viral infection.

55. A vector comprising a nucleotide sequence encoding a polypeptide of SEQ ID NO: 2-7, 14-24, 74 and / or 76.

56. The carrier according to claim 55, wherein the carrier is approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:

75.

57. A recombinant Spirulina species comprising a vector according to any one of claims 55 to 56.

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