EP-4735014-A2 - RECOMBINANT SPIRULINA EXPRESSING SCAFFOLDS AND METHODS OF UTILIZING THE SAME

Abstract

The present disclosure provides scaffolds comprising heterologous moieties and Spirulina expressing the same. Also provided are recombinant Spirulina expressing scaffolds and methods of utilizing the same for the treatment of diseases and conditions.

Inventors

• ROBERTS, JAMES MICHAEL
• KHUONG, Nhi Yen
• Gewe, Mesfin Mulugeta
• JESTER, BENJAMIN

Assignees

• Lumen Bioscience, Inc.

Dates

Publication Date

20260506

Application Date

20240628

Claims (1)

1. Attorney Docket No.: LUBI-038/01WO 330572-2193 CLAIMS 1. A recombinant Spirulina, that expresses a mutated small-membrane A-kinase anchoring protein (smAKAP) peptide sequence. 2. The recombinant Spirulina of claim 1, wherein the Spirulina comprises at least 2, 3, 4, 5, 6, or 7 mutated residues in the mutant smAKAP peptide sequence as compared to a WT smAKAP peptide sequence. 3. The recombinant Spirulina of claim 1, wherein 1 residue in the smAKAP peptide sequence is mutated as compared to a WT smAKAP peptide sequence. 4. The recombinant Spirulina of any one of claims 2-3, wherein 2 residues in the smAKAP peptide sequence are mutated as compared to a WT smAKAP peptide sequence. 5. The recombinant Spirulina of any one of claims 2-4, wherein 3 residues in the smAKAP peptide sequence are mutated as compared to a WT smAKAP peptide sequence. 6. The recombinant Spirulina of any one of claims 1-5, wherein the mutated smAKAP peptide exhibits resistance to protease cleavage as determined by reduced detection of cleavage products when exposed to a solvent comprising a protease for one hour. 7. The recombinant Spirulina of claim 6, wherein at most about 5%, 10%, 20%, 30%, 40%, 50%, or 60% of the smAKAP peptide sequence is cleaved. 8. The recombinant Spirulina of any one of claims 1-7, wherein the mutation is of a hydrophobic residue. 9. The recombinant Spirulina of any one of claims 1-8, wherein when the recombinant Spirulina is submerged in a solvent, the mutated residue is exposed to the solvent. 10. The recombinant Spirulina of any one of claims 1-9, wherein the mutated residue is selected from the group consisting of: C16S, C24S, E5D, Y6H, W22S, C24G, L4E, R9E, L4I, L10I, L19I, and combination thereof of SEQ ID NO: 2. 11. The recombinant Spirulina of claim 10, wherein the mutated residue is C16S and C24S as compared to SEQ ID NO: 2. 12. The recombinant Spirulina of claim 10, wherein the mutated residue is E5D, Y6H, C16S, W22S, and C24G as compared to SEQ ID NO: 2. 13. The recombinant Spirulina of claim 10, wherein the mutated residue is L4E, E5D, Y6H, R9E, C16S, W22S, and C24G as compared to SEQ ID NO: 2. 14. The recombinant Spirulina of claim 10, wherein the mutated residue is L4E, C16S, W22S, and C24G as compared to SEQ ID NO: 2. Attorney Docket No.: LUBI-038/01WO 330572-2193 15. The recombinant Spirulina of claim 10, wherein the mutated residue is R9E, C16S, and C24G as compared to SEQ ID NO: 2. 16. The recombinant Spirulina of claim 10, wherein the mutated residue is R9E, C16S, W22S, and C24G as compared to SEQ ID NO: 2. 17. The recombinant Spirulina of claim 10, wherein the mutated residue is L4I, R9E, C16S, C24G as compared to SEQ ID NO: 2. 18. The recombinant Spirulina of any one of claims 1-17, wherein the mutant smAKAP peptide sequence is linked to a heterologous moiety in a monomeric configuration. 19. The recombinant Spirulina of claim 18, wherein the mutant smAKAP peptide sequence is linked to a second heterologous moiety. 20. The recombinant Spirulina of claim 19, wherein the heterologous moiety and the second heterologous moiety are the same. 21. The recombinant Spirulina of any one of claims 18-19, wherein the heterologous moiety and the second heterologous moiety are the different. 22. The recombinant Spirulina of any one of claims 1-21, wherein the recombinant Spirulina expresses another exogenous polypeptide sequence. 23. The recombinant Spirulina of claim 22, wherein the exogenous polypeptide sequence is selected from the group consisting of: oligomerization domain of C4b-binding protein (C4BP), cholera toxin b subunit, oligomerization domains of extracellular matrix proteins, TRX, 5HVZ, cTRP, SP651, SP737, and 4B0F. 24. The recombinant Spirulina of claim 23, wherein the exogenous polypeptide sequence is 5HVZ. 25. The recombinant Spirulina of claim 24, wherein the 5HVZ is linked to a third heterologous moiety 26. The recombinant Spirulina of claim 25, wherein the third heterologous moiety is in a dimeric configuration. 27. The recombinant Spirulina of any one of claims 18-26, wherein the heterologous moiety, the second heterologous moiety, and the third heterologous moieties are the same. 28. The recombinant Spirulina of any one of claims 18-26, wherein the heterologous moiety, the second heterologous moiety, and the third heterologous moieties are the different. 29. The recombinant Spirulina of any one of claims 18-28, wherein the heterologous moiety is a binding agent. Attorney Docket No.: LUBI-038/01WO 330572-2193 30. The recombinant Spirulina of 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-grafted antibody, single chain antibodies (scFv), single chain antibody fragment, chimeric antibody, diabody, triabody, tetrabody, minibody, linear antibody, intrabody, nanobody (single domain antibody), small Modular Immunopharmaceuticals (SMIPs), antigen-binding domain immunoglobulin fusion protein, and VHH. 31. The recombinant Spirulina of claim 30, wherein the binding agent is the VHH. 32. The recombinant Spirulina of claim 31, wherein the VHH binds a pathogen. 33. The recombinant Spirulina of claim 31, wherein the VHH binds a cancer cell. 34. The recombinant Spirulina of claim 31, wherein the VHH binds a human cell. 35. The recombinant Spirulina of claim 32, wherein the VHH binds a pathogen selected from the group consisting of: bacteria, fungi, and virus. 36. The recombinant Spirulina of claim 35, wherein the pathogen is selected from the group consisting of: E. coli, Enterotoxigenic E. coli (ETEC), anthrax, EHEC, EAEC, Shigella, Mycobacterium, Streptococcus, Staphylococcus, Shigella, Campylobacter, Salmonella, Clostridium, Corynebacterium, Pseudomonas, Neisseria, Listeria, Vibrio, Bordetella, Legionella, bacteriophage, RNA bacteriophage (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, Poliovirus, Norovirus, Zika Virus, Dengue Virus, Rabies Virus, Newcastle Disease Virus, White Spot Syndrome Virus, a coronavirus, SARS, MERS, SARS- CoV-2, Aspergillus, Candida, Blastomyces, Coccidioides, Cryptococcus, Histoplasma, Plasmodium, P. falciparum, P. malariae, P. ovale, P. vivax , Trypanosoma, Toxoplasma, Giardia, Leishmania Cryptosporidium, helminthic parasites: Trichuris spp., Enterobius spp., Ascaris spp., Ancylostoma spp. and Necatro spp., Strongyloides spp., Dracunculus spp., Onchocerca spp. Wuchereria spp., Taenia spp., Echinococcus spp., and Diphyllobothrium spp., Fasciola spp., and Schistosoma spp. 37. The recombinant Spirulina of claim 35, wherein the pathogen is bacteria and the bacteria 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 of claim 37, wherein the bacteria is Campylobacter. Attorney Docket No.: LUBI-038/01WO 330572-2193 39. The recombinant Spirulina of claim 37, wherein the bacteria is Clostridium. 40. The recombinant Spirulina of any one of claims 36-39, wherein the VHH comprises a sequence with at least 85% identity to a sequence of SEQ ID NO: 25-67. 41. The recombinant Spirulina of any one of claims 36-39, wherein the VHH comprises a sequence SEQ ID NO: 25-67. 42. The recombinant Spirulina of any one of claims 24-41, wherein the recombinant Spirulina expresses a scaffold having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a sequence of SEQ ID NO: 4, 6, 14, 16, 18, 20, 22, 23, 24, and 76. 43. A recombinant Spirulina, that expresses 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, 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 combination thereof of SEQ ID NO: 2. 45. The recombinant Spirulina of any one of claims 43-44, wherein the smAKAP is linked to one or more VHH antibodies at a terminal end. 46. The recombinant Spirulina of claim 45, wherein the smAKAP is linked to two VHH antibodies, wherein a first VHH is at a C-terminus and the second VHH is at an N-terminus. 47. The recombinant Spirulina of any one of claims 43-46, wherein the recombinant Spirulina expresses a 5HVZ. 48. The recombinant Spirulina of 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 one or more residue substitutions as compared to a WT smAKAP sequence. 50. A vector comprising the polynucleotide sequence of claim 49. 51. A method of making a recombinant Spirulina comprising contacting a Spirulina cell with the vector of claim 50. 52. A pharmaceutical composition comprising the recombinant Spirulina of any one of claims 1-48, and an excipient. Attorney Docket No.: LUBI-038/01WO 330572-2193 53. A kit comprising: the recombinant Spirulina of any one of claims 1-48, the polynucleotide sequence of claim 49, the vector of claim 50, or the pharmaceutical composition of claim 52, and instructions for use thereof. 54. A method of treatment comprising administering the pharmaceutical composition of claim 52 to a subject in need thereof, wherein the subject comprises 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 vector of claim 55, wherein the vector comprises from about 80%, 85%, 90%, 95% 96% 97%, 98%, 99%, or 100% identity to SEQ ID NO: 75. 57. A recombinant Spirulina comprising the vector of any one of claims 55-56.

Description

Attorney Docket No.: LUBI-038/01WO 330572-2193 RECOMBINANT SPIRULINA EXPRESSING SCAFFOLDS AND METHODS OF UTILIZING THE SAME CROSS REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Application No. 63/511,050 filed on June 29, 2023, the contents of which is incorporated by reference herein in its entirety for all purposes. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING The contents of the electronic sequence listing (LUBI_038_01WO_SeqList_ST26.xml; Size: 82,287 bytes; and Date of Creation: June 28, 2024) are herein incorporated by reference in its entirety. BACKGROUND Antigen-binding domains such as VHHs from camelid single-chain antibodies are expressible in prokaryotes like Spirulina. However, various limitations remain such as the requirement for affinity maturation to achieve improved target binding. The present disclosure provides a resolution to the limitations of the current state-of- the-art via improved compositions comprising scaffolds. FIELD The present disclosure relates to scaffolds and methods of utilizing the same. The disclosure also relates to constructs comprising scaffolds expressing binding agents. BRIEF SUMMARY Provided herein are recombinant Spirulina, that expresses a mutated small-membrane A-kinase anchoring protein (smAKAP) peptide sequence. In aspects, the Spirulina comprises at least 2, 3, 4, 5, 6, or 7 mutated residues in the mutant smAKAP peptide sequence as compared to a WT smAKAP peptide sequence. In aspects, 1 residue in the smAKAP peptide sequence is mutated as compared to a WT smAKAP peptide sequence. In aspects, 2 residues in the smAKAP peptide sequence are mutated as compared to a WT smAKAP peptide sequence. In aspects, 3 residues in the smAKAP peptide sequence are mutated as compared to a WT smAKAP peptide sequence. In aspects, the mutated smAKAP peptide exhibits resistance to protease cleavage as determined by reduced detection of cleavage products when exposed to a solvent comprising a protease for one hour. In aspects, at most about 5%, 10%, 20%, 30%, Attorney Docket No.: LUBI-038/01WO 330572-2193 40%, 50%, or 60% of the smAKAP peptide sequence is cleaved. In aspects, the mutation is of a hydrophobic residue. In aspects, when the recombinant Spirulina is submerged in a solvent, the mutated residue is exposed to the solvent. In aspects, the mutated residue is selected from the group consisting of: C16S, C24S, E5D, Y6H, W22S, C24G, L4E, R9E, L4I, L10I, L19I, and combination thereof of SEQ ID NO: 2. In aspects, the mutated residue is C16S and C24S as compared to SEQ ID NO: 2. In aspects, the mutated residue is E5D, Y6H, C16S, W22S, and C24G as compared to SEQ ID NO: 2. In aspects, the mutated residue is L4E, E5D, Y6H, R9E, C16S, W22S, and C24G as compared to SEQ ID NO: 2. In aspects, the mutated residue is L4E, C16S, W22S, and C24G as compared to SEQ ID NO: 2. In aspects, the mutated residue is R9E, C16S, and C24G as compared to SEQ ID NO: 2. In aspects, the mutated residue is R9E, C16S, W22S, and C24G as compared to SEQ ID NO: 2. In aspects, the mutant smAKAP peptide sequence is linked to a heterologous moiety in a monomeric configuration. In aspects, the mutant smAKAP peptide sequence is linked to a second heterologous moiety. In aspects, the heterologous moiety and the second heterologous moiety are the same. In aspects, the heterologous moiety and the second heterologous moiety are the different. In aspects, the recombinant Spirulina expresses another exogenous polypeptide sequence. In aspects, the exogenous polypeptide sequence is selected from the group consisting of: oligomerization domain of C4b-binding protein (C4BP), cholera toxin b subunit, oligomerization domains of extracellular matrix proteins, TRX, 5HVZ, cTRP, SP651, SP737, and 4B0F. In aspects, the exogenous polypeptide sequence is 5HVZ. In aspects, the 5HVZ is linked to a third heterologous moiety. In aspects, the third heterologous moiety is in a homodimeric configuration. In aspects, the heterologous moiety, the second heterologous moiety, and the third heterologous moieties are the same. In aspects, the heterologous moiety, the second heterologous moiety, and the third heterologous moieties are the different. In aspects, the heterologous moiety is a binding agent. In aspects, the binding agent is selected from the group consisting of: fab', F (ab') 2, fv, domain antibody (dAb), complementarity Determining Region (CDR) fragment, CDR-grafted antibody, single chain antibodies (scFv), single chain antibody fragment, chimeric antibody, diabody, triabody, tetrabody, minibody, linear antibody, intrabody, nanobody (single domain antibody), small Modular Immunopharmaceuticals (SMIPs), antigen-binding domain immunoglobulin fusion protein, and VHH. In aspects, the binding agent is the VHH. In aspects, the VHH binds a pathogen. In aspects, the VHH binds a cancer cell. In aspects, the VHH binds a human cell. In aspects, the VHH binds a pathogen selected from the group consisting of: bacteria