Self-assembling protein nanocage decorated with antibodies (SAPNA) and parts thereof

Abstract

To provide the production of protein-based antibody scaffolds.SOLUTION: The present invention provides a protein cage polypeptide (or scaffold protein) useful or capable of forming a hollow tetrahedral pyramidal structure, and a "self-assembling protein nanoparticle decorated with antibodies" (SAPNA) which is a chimeric protein assembly comprising: (a) one or more antibodies and (b) the protein cage polypeptide that provides a scaffold upon which to array the antibodies. In some embodiments, the antibody is capable of specifically binding to a pathogenic biological agent or portion thereof.SELECTED DRAWING: Figure 1A

Description

Technical Field

[0001] The present invention relates to the field of production of protein-based antibody scaffolds.

Background Art

[0002] Related Patent Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 838,826, filed Apr. 25, 2019, which is hereby incorporated by reference in its entirety.

[0003] Statement Regarding Government Sponsorship This invention was made with government support under Contract No. DE-AC02-05CH11231 awarded by the U.S. Department of Energy. The government has certain rights in this invention.

[0004] Therapeutic monoclonal antibodies are a major force in the biopharmaceutical industry, while cancer immunotherapy is a rapidly growing area of intensive research. According to Ecker et al. (5), monoclonal antibody products represented nearly $75 billion in 2013, approximately half of the total sales of all biopharmaceutical products. Correctly viewed, as of late 2014, only 47 monoclonal antibody products were approved for use in the United States or Europe. Therefore, there is significant room for growth, and most large pharmaceutical companies are seen to have antibody development programs. In fact, approximately 70 new monoclonal antibody products are expected to be sold by 2020, resulting in worldwide sales of approximately $125 billion (5).

[0005] U.S. Patent No. 6,756,039 (Yeates, Padilla, and Colovos) discloses a fusion protein capable of self-assembling to construct a regular structure and comprising at least two oligomer-forming domains tightly linked together, for example, via an alpha-helical linker.

[0006] U.S. Patent No. 7,608,681 (Dennis, Lowman, and DeLano) discloses a peptide ligand having affinity for IgG or serum albumin.

[0007] U.S. Patent No. 8,969,521 (Baker, King, Sheffler, and Yeates) discloses a general method for designing self - assembling protein nanomaterials and isolated polypeptides that contain a specific 184 - amino - acid sequence and are capable of forming multimeric assemblies.

[0008] U.S. Patent Application Publication No. 20070218547 (Yeates, Padilla, Yoshida, and Colovos) discloses a self - assembling protein that produces an extended material, the self - assembling protein comprising a fusion protein including a first oligomer - forming domain that naturally binds to a homodimer structure and a second oligomer - forming domain that naturally binds to a homotetramer structure, wherein the first oligomer - forming domain and the second oligomer - forming domain are tightly linked to each other. SUMMARY OF THE INVENTION

[0009] The present invention provides a protein cage polypeptide (or scaffold protein) that is useful for forming or capable of forming a hollow tetrahedral pyramid structure and that can specifically bind to an antibody or a portion thereof, or to any chimeric protein, molecule, or compound containing the antibody or a portion thereof.

[0010] In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody moiety is the Fc region of an antibody such as, for example, an IgG, IgA, IgD, IgE, or IgM antibody. In some embodiments, the antibody is a human, chicken, mouse, rabbit, sheep, or goat antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the IgG antibody is a human IgG antibody. In some embodiments, the antibody is part of a chimeric protein, molecule, or compound that includes the antibody or a portion thereof. In some embodiments, the chimeric protein or other molecule or compound includes the Fc region of the antibody. In some embodiments, the antibody or a portion thereof covalently binds to a chimeric protein, molecule, or compound. In some embodiments, the binding affinity K a of the protein cage polypeptide or scaffold protein for the antibody or a portion thereof 7 is -1 10 8 M -1 10 9 M -1 or greater than 10

[0011] In some embodiments, the protein cage polypeptide includes a polypeptide of from about 400 to about 700 amino acid residues. In some embodiments, the protein cage polypeptide includes a polypeptide of from about 450 to about 650 amino acid residues.

[0012] In some embodiments, the protein cage polypeptide includes an amino acid sequence having the following structure:

[0013]

Chemical Structure

[0014] where AHL is an "alpha helix linker" and insert A and / or insert B can each independently specifically bind to the antibody or a portion thereof.

[0015] In some embodiments, Insertion A has a length of from about 17 amino acids to about 25 amino acids. In some embodiments, Insertion B has a length of from about 28 amino acids to about 85 amino acids. In some embodiments, the binding affinity K of Insertion A and / or Insertion B for an antibody or a portion thereof a is, independently of each other, 10 7 M -1 、10 8 M -1 、or 10 9 M -1 or more. In some embodiments, Insertion A and / or Insertion B each independently comprises the amino acid sequence DCAWHLGELVWCT (SEQ ID NO: 41) or GCDCAWHLGELVWCTCG (SEQ ID NO: 42).

[0016] In some embodiments, the protein cage polypeptide comprises an amino acid sequence having the following structure:

[0017]

Chemical formula

[0018] where AHL is an "alpha helix linker", Insertion A has a length of from about 17 amino acids to about 25 amino acids and has the amino acid sequence DCAWHLGELVWCT (SEQ ID NO: 41) or GCDCAWHLGELVWCTCG (SEQ ID NO: 42), Insertion B has a length of from about 28 amino acids to about 85 amino acids and comprises the amino acid sequence DCAWHLGELVWCT (SEQ ID NO: 41) or GCDCAWHLGELVWCTCG (SEQ ID NO: 42). SEQ ID NOs: 41 and 42 can bind to the Fc region of IgG.

[0019] In some embodiments, Polypeptide 1 comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% amino acid homology to the amino acid sequence from the N-terminus of any one of SEQ ID NOs: 1-40 to the AQEAQKQK sequence. In some embodiments, Polypeptide 1 comprises an amino acid sequence comprising: YGTAR, TDD, LXENLGTR, IDV, TGXRT, and / or SA; wherein X is any charged amino acid residue. In some embodiments, Polypeptide 1 comprises from about 278 to about 303 amino acid residues.

[0020] In some embodiments, AHL comprises an amino acid sequence comprising: AQEAQKQK. In some embodiments, AHL comprises from about 5, 6, 7, 8, 9, 10, or 11 amino acid residues.

[0021] In some embodiments, Polypeptide 2 comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% amino acid homology to the amino acid sequence from the C-terminus of the AQEAQKQK sequence to the N-terminus of Insertion A of any one of SEQ ID NOs: 1-40. In some embodiments, Polypeptide 2 comprises an amino acid sequence comprising: LTEVETYVLS (SEQ ID NO: 43). In some embodiments, Polypeptide 2 comprises from about 30 to about 36 amino acid residues. In some embodiments, Polypeptide 2 comprises about 33 amino acid residues.

[0022] In some embodiments, polypeptide 3 comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% amino acid homology to the amino acid sequence from the C-terminus of insertion A to the N-terminus of insertion B of any one of SEQ ID NOs: 1-40. In some embodiments, polypeptide 3 comprises an amino acid sequence comprising: FTLTVPSERGLQR (SEQ ID NO: 44) and / or CATCEQIAD (SEQ ID NO: 45). In some embodiments, polypeptide 3 comprises from about 110 to about 130 amino acid residues. In some embodiments, polypeptide 3 comprises about 121 amino acid residues.

[0023] In some embodiments, polypeptide 4 comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% amino acid homology to the amino acid sequence from the C-terminus of insertion B. In some embodiments, polypeptide 4 comprises an amino acid sequence comprising: EHHHHHH. In some embodiments, polypeptide 4 comprises from about 5 to about 13 amino acid residues. In some embodiments, polypeptide 4 comprises about 8 amino acid residues.

[0024] In some embodiments, the protein cage polypeptide comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% amino acid homology to any one of SEQ ID NOs: 1-40. In some embodiments, the protein cage polypeptide comprises an amino acid sequence comprising any one or more, or all, of a continuous or discrete amino acid residue(s) marked with an asterisk in FIG. 6. In some embodiments, the protein cage polypeptide comprises an amino acid sequence comprising any one or more, or all, of a continuous charged amino acid(s) at the corresponding position marked with a "#" in FIG. 6.

[0025] The present invention provides a hollow tetrahedral pyramid structure comprising 12 protein cage polypeptides of the present invention constructed as a hollow tetrahedral pyramid structure, and the protein cage polypeptides can bind to an antibody or a portion thereof. In some embodiments, the hollow tetrahedral pyramid structure entraps one or more molecules of interest smaller than the structure inside. In some embodiments, the molecules of interest smaller than the structure are therapeutically or detectably relevant.

[0026] The present invention provides "self - assembling protein nanoparticles modified with an antibody" (SAPNA), which is a chimeric protein aggregate, comprising (a) one or more antibodies and (b) a protein cage polypeptide that provides a scaffold for arranging the antibody thereon, wherein the one or more antibodies bind to insertion A and / or insertion B of the protein cage polypeptide.

[0027] The present invention provides a SAPNA which is a chimeric protein aggregate and comprises (a) one or more antibodies and (b) an engineered protein that provides a scaffold on which the antibody is arranged. The scaffold protein forms a hollow tetrahedral pyramid that can aggregate or disassemble according to buffer conditions. Since the scaffold is hollow, the system can confine a molecule of interest smaller than the system inside so that it is released when the antibody localizes the SAPNA to the target. These particles are engineered to bind and display any IgG antibody (or only the Fc region), such as a human or rabbit IgG antibody (or only the Fc region) or a fragment thereof, in a modular manner via a high-affinity interaction with the Fc CH2 / CH3 domain of the antibody. By physically confining 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 antibodies or Fc domains per nanoparticle, activation of any oligomerization-dependent receptor pathway that can utilize the antibody becomes possible. In some embodiments, antibodies recognizing different epitopes can be mounted on the same nanoparticle to provide multifunctionality by individually separating the loading and mixing. In some embodiments, since oligomerization of the Fc receptor is an essential element for activation, the above nanoparticles can be used to stimulate innate immune cells or adaptive immune cells.

[0028] The present invention provides a SAPNA structure comprising (1) one protein cage polypeptide or scaffold protein (or engineered protein cage protein (PC)), or a plurality of protein cage polypeptides or scaffold proteins (or engineered protein cage proteins (PC)) constructed into a three-dimensional aggregate such as a tetrahedral pyramid, (2) optionally one or more human or rabbit IgG antibodies, (3) optionally an IgG binding loop, and (4) optionally a cargo of interest, such as a compound or molecule such as a macromolecule, constrained or encapsulated by the three-dimensional aggregate when a plurality of polypeptides or scaffold proteins (or engineered protein cage proteins (PC)) are constructed into a three-dimensional aggregate including the antibody. One embodiment of the present invention is shown in FIG. 1A.

[0029] Human IgG antibodies recognize and tightly bind to various targets. In some embodiments, the target is a portion of a pathogen. In some embodiments, the target is a natural cellular component. In some embodiments, the IgG binding loop is a protein sequence conjugated to a PC and functioning as a connection between the antibody and the PC. There are several references (1-3) dealing with the PC, however, none of the contexts are related to the antibody. The PC component can self-assemble from 12 copies of itself to construct a hollow tetrahedral pyramid under most physiological conditions. In some embodiments, the SAPNA structure can deliver or carry cargo anywhere the antibody localizes the SAPNA. In some embodiments, the size of the cargo ranges from about 150 kDa to about 20 kDa. Many useful macromolecules fall within this range.

[0030] The SAPNA structure can assemble and disassemble. Using this functionality, it is possible to first capture or release the cargo. In addition, there are many types of antibodies. Therefore, it is possible to mix various antibodies with the PC to create SAPNAs with diverse assemblies of antibodies on the surface of the SAPNA. The ability to alternate the antibodies provides additional functionality.

[0031] In some embodiments, apart from the ability to carry and localize cargo, SAPNA can alter cell behavior without cargo. External stimuli that affect cells often begin with the binding of ligands that bring transmembrane receptors into close contact (oligomerization)(4). This is achieved, for example, by the binding of two or more receptors to a ligand such as a cytokine, but ligands for many receptors are unknown or may be limited to the cell surface of other cells. In some embodiments, the functional power of any IgG antibody developed for any single receptor is significantly enhanced through presentation on the PC (FIGS. 1A, 1B; FIG. 7). This antibody is not greatly limited to inhibiting the receptor, but can activate intracellular signaling pathways and, as a result, can provide much finer control of cell activity. In some embodiments, various types of antibodies are presented on the PC and the protein can affect signals operating through the multi-chain immune recognition receptor (MIRR). Many immune cells utilize MIRR for the control of intracellular signaling(4). MIRR often requires the binding of multiple chains by extracellular ligands for oligomerization and subsequent activation. In some embodiments, SAPNA will give IgG antibodies, which are currently limited to inhibitory mechanisms, activation / signal transduction ability in a modular manner. This will completely open up new therapeutic avenues for existing and newly developed human IgG antibodies against any disease for which modulation of cell signaling is desired.

[0032] SAPNA has great potential without its use being limited to a single or a few diseases. The potential of SAPNA is also not fixed as the number of monoclonal antibody products being developed increases, and thus the potential uses of SAPNA are the same. In some embodiments, in addition to some available therapeutic IgG antibodies (e.g., anti-PD-1 / PD-L1, anti-CTLA4), there are well-defined ligand-receptor interactions that can be modulated, so the SAPNA structure is used to target cancer in immunotherapy. Regarding the list of therapeutic antibodies, their origin and isotype, mode of action, as well as license indication, please refer to reference (6). Furthermore, cancer immunology is a research field that is largely based on the use of flow cytometry with antibody staining, which will enable the testing of a wide range of preclinical candidates. The present invention provides a nucleic acid encoding the protein cage polypeptide of the present invention. In some embodiments, the nucleic acid is a polynucleotide. In some embodiments, the nucleic acid is a vector such as an expression vector. In some embodiments, the nucleic acid encoding the protein cage polypeptide is operably linked to a promoter capable of expressing the protein cage polypeptide in a host cell. In some embodiments, the nucleic acid is a vector capable of stable introduction into and / or stable maintenance in a host cell.

[0033] The present invention provides a host cell comprising a nucleic acid encoding the protein cage polypeptide of the present invention. In some embodiments, the nucleic acid is a vector capable of stable introduction into and / or stable maintenance in a host cell.

[0034] The present invention provides a composition comprising the protein cage polypeptide (or scaffold protein) or the hollow tetrahedral pyramid structure of the present invention, wherein the protein cage polypeptide (or scaffold protein) or the hollow tetrahedral pyramid structure specifically binds to an antibody or a portion thereof, or any chimeric protein, molecule or compound comprising the antibody or a portion thereof.

[0035] The present invention provides a method for producing a protein cage polypeptide, comprising: (a) providing a host cell of the present invention; (b) culturing the host cell under suitable conditions for expressing the protein cage polypeptide; and (c) optionally, recovering the protein cage polypeptide.

[0036] The present invention provides a method for detecting or isolating a pathogenic biological agent or a part thereof, the method comprising: (a) providing a "self-assembling protein nanoparticle modified with an antibody" (SAPNA), wherein the antibody can specifically bind to a pathogenic biological agent or a part thereof; (b) contacting the SAPNA with a sample containing the pathogenic biological agent or a part thereof such that the SAPNA binds to the pathogenic biological agent or a part thereof; (c) detecting the SAPNA, the pathogenic biological agent or a part thereof and / or separating the SAPNA-bound pathogenic biological agent or a part thereof from the remaining part of the sample through detection; and (d) determining the abundance of the pathogenic biological agent or a part thereof.

[0037] In some embodiments, the method further comprises obtaining a sample from a subject suffering from a disease caused by a pathogenic biological agent, a subject diagnosed with the disease, or a subject suspected of suffering from the disease. In some embodiments, the subject is human. In some embodiments, the subject is a mammal or a bird. In some embodiments, the subject is a common pet or livestock animal. In some embodiments, the method further comprises treating the subject's disease, such as administering to the subject a therapeutically effective dose of a medicament known or capable of curing or alleviating the effects of the disease.

[0038] The present invention provides a SAPNA that chemically binds to one or more chemical compounds, such as one or more agents, and is then directed to the biological / cellular site of drug deposition in a manner similar to an antibody-drug conjugate (ADC).

[0039] The foregoing aspects and others will be readily understood by those skilled in the art from the following description of the embodiments to be explained, when read in conjunction with the accompanying drawings.

Brief Description of the Drawings

[0040]

Fig. 1A

Fig. 1B

Fig. 2A

Fig. 2B

Fig. 3A

Fig. 3B

Fig. 4A

Fig. 4B

Fig. 5A

Fig. 5B

Fig. 6

Fig. 7

Fig. 8

Fig. 9

Fig. 10

Fig. 11

Fig. 12

Fig. 13

BEST MODE FOR CARRYING OUT THE INVENTION

[0041] Before explaining the present invention in detail, it should be understood that the present invention is not limited to a specific sequence, expression vector, enzyme, host microorganism, or process, and can vary precisely unless otherwise indicated. It should also be understood that the technical terms used in this specification are for the purpose of merely describing specific embodiments and are not intended to be limiting.

[0042] In this specification and the following claims, reference will be made to several terms defined to have the following meanings:

[0043] As used herein, the terms "optional" or "optionally" mean that the feature or structure described thereafter may or may not be present, or that the event or situation described thereafter may or may not occur, and that the description includes examples where a particular feature or structure is present and examples where the feature or structure is not present, or examples where an event or situation occurs and examples where it does not occur.

[0044] As used in the specification and the appended claims, the singular forms "a", "an", and "the" include the plural unless the context clearly dictates otherwise. That is, for example, reference to a "polypeptide" includes not only a single polypeptide molecule but also a plurality of polypeptides of a particular amino acid sequence.

[0045] As used herein, the terms "optional" or "optionally" mean that the feature or structure described thereafter may or may not be present, or that the event or situation described thereafter may or may not occur, and that the description includes examples where a particular feature or structure is present and examples where the feature or structure is not present, or examples where an event or situation occurs and examples where it does not occur.

[0046] When a range of values is given, unless a different clear indication is given in the context, it should be understood that each value between the upper limit and the lower limit of that range is specifically disclosed down to one-tenth of the unit of the lower limit. Any described value within the described range or a value existing between values within the described range, and a smaller range therebetween than between any other described value within the described range or a value existing between values within the described range, are each encompassed within the scope of the present invention. The upper and lower limit values of such a smaller range may independently be included within or excluded from that range, and also each range within the smaller range that includes any one, none, or both of such limit values is also included within the scope of the present invention depending on any specifically excluded limit value within the described range. When the described range includes one or both of the limit values, ranges excluding any one or both of the included limit values are also included within the present invention.

[0047] The term "about" refers to a value 10% more than the described value and a value 10% less than the described value.

[0048] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the technical field to which this invention belongs. Any methods and materials similar or equivalent to those described in this specification can be used in the methods or tests of the present invention, but the preferred methods and materials will be described hereinafter. All documents mentioned in this specification are hereby incorporated by reference into this specification for the purpose of disclosing and describing the methods and / or materials related to the citation of the documents.

[0049] As used herein, the term "host cell" is used to refer to a living biological cell that can be transformed via insertion of an expression vector.

[0050] The term "expression vector" or "vector" refers to a compound and / or composition that causes a host cell to express nucleic acid and / or protein by transduction, transformation, or infection of the cell, in a manner other than or not derived from the cell. An "expression vector" contains the sequence of nucleic acid (usually RNA or DNA) to be expressed by the host cell. Optionally, the expression vector also contains substances that assist in the delivery of the nucleic acid into the host cell, such as, for example, viruses, liposomes, protein coatings, etc. Expression vectors intended for use in the present invention include expression vectors into which a nucleic acid sequence can be inserted in addition to any preferred or required operative elements. Furthermore, the expression vector must be capable of being introduced into and replicating within a host cell. A particular expression vector is a plasmid, in particular a plasmid with restriction enzyme sites that has been well-characterized and contains operative elements that are preferred or required for transcription of the nucleic acid sequence. Such plasmids as well as other expression vectors are well-known to those skilled in the art.

[0051] The terms "polynucleotide" and "nucleic acid" are used interchangeably and refer to single- or double-stranded polymers of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. The nucleic acids of the invention will generally contain phosphodiester bonds, but in some cases, alternative backbones containing, for example, phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphophoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press); positive backbones; non-ionic backbones, and non-ribose backbones, may be used. That is, the nucleic acid or polynucleotide may also contain modified nucleotides that allow for accurate read-through by polymerases. A "polynucleotide sequence" or "nucleic acid sequence" includes both the sense and antisense strands of a nucleic acid, either as separate single strands or as double strands. As will be understood by those skilled in the art, a description of a single strand also defines the sequence of the complementary strand, i.e., the sequences described herein also provide the complement of the sequences. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses its variants (e.g., degenerate codon substitutions), as well as the complementary sequence and not only the explicitly recited sequence. The nucleic acid can be DNA, RNA, or a hybrid, both genomic DNA and cDNA, and the nucleic acid can contain combinations of deoxyribonucleotides and ribonucleotides, and combinations of bases such as uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, isoguanine, etc.

[0052] As used herein, the term "promoter" refers to a polynucleotide sequence capable of promoting the transcription of a DNA sequence within a cell. That is, the promoters used in the polynucleotide constructs of the present invention include cis-acting and trans-acting transcriptional regulatory factors and regulatory sequences involved in the control or regulation of the timing and / or rate of gene transcription. For example, a promoter can be a cis-acting transcriptional regulatory factor and includes enhancers, promoters, transcription terminators, origins of replication, chromosomal integration sequences, 5' and 3' untranslated regions, or intron sequences, which are involved in transcriptional control. These cis-acting sequences typically interact with proteins or other biomolecules to effect (activate / stop, control, regulate, etc.) gene transcription. A promoter is located 5' to the gene to be transcribed and, as used herein, includes the 5' sequence from the translation start codon (i.e., includes the 5' untranslated region of the mRNA and typically includes 100-200 bp). The core promoter sequence is most often within 1-2 kb of the translation start site, often within 1 kbp of the translation start site, and frequently within 500 bp of the translation start site. By convention, promoter sequences are usually provided as sequences on the coding strand of the gene they control. In the context of the present application, a promoter is typically named after the gene whose expression it naturally controls. The promoters used in the expression constructs of the present invention are named after the genes. A reference to a promoter by name includes not only the wild-type, native promoter but also variants of that promoter that retain the ability to induce expression. A reference to a promoter by name is not limited to a particular species but includes promoters from corresponding genes of other species.

[0053] The term "operatively linked" refers to the functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, it refers to the functional relationship of a transcriptional control sequence to a transcribed sequence. For example, a promoter or enhancer sequence is operatively linked to a DNA or RNA sequence if the sequence stimulates or regulates the transcription of the DNA or RNA sequence in an appropriate host cell or other expression system. Generally, a promoter transcriptional control sequence that is operatively linked to a transcribed sequence is physically adjacent to the transcribed sequence, i.e., they are cis-acting. However, some transcriptional control sequences, such as enhancers, for example, need not be physically adjacent or proximal to the coding sequence whose transcription they enhance.

[0054] The term "cell" refers to any cell of any organism and encompasses the range from single-celled organisms to mammalian cells in vitro or in vivo.

[0055] By various methods according to the methods of the present invention, it is possible to modify the expression of a nucleic acid encoding any protein cage polypeptide taught herein. One skilled in the art will recognize that it is possible to vary the expression level of a protein by increasing the gene copy number, the strength of the ribosome binding site, the strength of the promoter, and various transcriptional control factors.

[0056] The present invention can be used for various purposes (as described, it can be used as a tool in the context of research or for therapeutic use in a clinical setting). In some embodiments, the SAPNA structure is a therapeutic or research tool that can modulate the immune system by binding / inhibiting soluble receptors / ligands on the cell surface in humans or research models. In some embodiments, the SAPNA structure can activate one or more intracellular cell pathways by enhancing the oligomerization of extracellular cell surface receptors / ligands. In some embodiments, the SAPNA structure is labeled with, for example, a fluorescent dye or label and can be used to visualize cell surface target antibodies in immunofluorescence or flow cytometry. In some embodiments, the fluorescent dye is an Alexa Fluor® fluorescent dye. In some embodiments, the SAPNA structure is a tool for testing / screening the feasibility of using any combination of human / rabbit IgG antibodies that cause cell changes or physiological responses in vivo. In some embodiments, the SAPNA structure is useful for opsonization to circulate and penetrate particles in vivo. In some embodiments, the SAPNA structure can target and manipulate viruses / viral particles in an aqueous or semi-aqueous environment. In some embodiments, the SAPNA structure can encapsulate cargo inside and then target the cell surface. In some embodiments, the SAPNA structure can access the intracellular environment via endocytosis (initiation and regulation of endocytosis) with or without cargo. In some embodiments, the SAPNA structure is a vaccine or vaccine adjuvant. In some embodiments, the SAPNA structure is an in vitro immune cell activation tool. In some embodiments, the SAPNA structure is a biodegradable aesthetic product that binds a fluorescent protein to the keratin of hair or skin via the presentation of anti-fluorescent and anti-keratin antibodies on a scaffold thereby. In some embodiments, the SAPNA molecule loaded with an antibody can positively or negatively select a cell population from a pool of mixed cells.

[0057] There is nothing clinically equivalent to the present invention. There is another group working on the manipulation of ferritin (7), but due to the spacing of the ferritin molecules, the antibody occupancy is much more difficult to predict, so it would be a poorer nanocage for immunological regulation via antibody presentation. Since the SAPNA nanocage we are manipulating has larger pores, it would probably be a better carrier for smaller molecules. This group also seems to be more focused on using the ferritin cage for cargo delivery to cells (8, 9). Antibodies with engineered Fc regions and bispecific antibodies are two different but competing types of technology.

[0058] Many antibodies do not pass clinical trials (10), but this has led to research on enhancing antibody-dependent cell-mediated cytotoxicity (ADCC). However, most of these efforts aim to enhance Fc gamma receptors that bind to the antibody Fc region via Fc mutations. ADCC requires the aggregation of Fc-gamma receptors via Fc binding, which will be physically forced in SAPNA, so the SAPNA nanocage would be superior to these methods. In addition, SAPNA can utilize these efforts and actually carry mutated Fc to further increase therapeutic efficacy.

[0059] Bispecific / multispecific antibodies (11 - 15) are inherently antibodies that have two or more different antigen recognition regions, but are linked by various techniques (14, 15). While bispecific / multispecific antibodies have great potential, they have to be designed, tested, and optimized individually as compared to SAPNA, which is modular and available for use with most commercially available IgG antibodies. The major advantage that SAPNA retains is that other non - antibody molecules can be presented simultaneously with the antibody. By mixing various antibodies prior to formulation and then adding SAPNA cages that do not yet carry antibodies, it is possible to load several (about 2 - 12) different antibodies onto the same nanocage. This can then function as a large multispecific nanoparticle, which is a major advantage over current multispecific antibodies. The modular nature and multifunctionality of SAPNA are highly desired characteristics in next - generation biological therapeutics.

[0060] In some embodiments, the protein cage polypeptide (or scaffold protein) specifically binds to an antibody or a portion thereof, or to any chimeric protein, molecule or compound comprising the antibody or a portion thereof, and the antibody or a portion thereof specifically binds to a pathogenic biological agent or a portion thereof.

[0061] In some embodiments, the tetrahedral pyramid structure specifically binds to an antibody or a portion thereof, or to any chimeric protein, molecule or compound comprising the antibody or a portion thereof, and the antibody or a portion thereof specifically binds to a pathogenic biological agent or a portion thereof.

[0062] In some embodiments, the SAPNA molecule can be used as a multivalent detection platform for pathogenic biological agents, including but not limited to viruses, bacteria, and abnormal folded proteins (such as prions and other amyloids) associated with any human / mammalian disease, by loading one or more antibodies specific for an antigen protein or other surface molecules specific for pathogenic biological agents onto the SAPNA molecule. The detection application extends to the isolation and determination of the abundance (i.e., the severity of infection) of pathogenic agents. As described above, for analytical purposes, the SAPNA molecule can be covalently labeled with a molecule such as a fluorophore for detection, while a multivalent His-tag (up to 12 copies) can be used to manipulate and isolate various antigen-binding fractions. The pathogens to be analyzed extend to those of animals (such as common pets, livestock, etc.) that are interesting and influential for human health and well-being, in addition to humans. In some embodiments, common pets include dogs, cats, rabbits, guinea pigs, hamsters, mice, etc. In some embodiments, livestock are mammals such as cows, horses, pigs, sheep, or goats, or birds such as chickens, ducks, or geese.

[0063] The surfaces of viruses and bacteria are covered or modified with proteins or other molecules that are necessary for their biological functions, such as attachment to host cells, entry into the host, and survival under harsh conditions. Since these molecules are important for growth, they tend to be conserved in a given species or strain of virus or bacteria. As a result, such molecules can function as robust targets for identification. The molecules are more specific and characteristic for various viruses and bacteria, and thus are suitable for specific attribution of identification in diagnostic applications. The ability to recognize specific viruses and bacteria by antibodies that bind to the molecules on their surfaces, or by antibodies that may bind to the molecules produced by their lysis, is understood and is actually widely applied. In some embodiments, the SAPNA molecules provide characteristic and advantageous features for identifying and isolating viruses and bacteria, which is due to the ability of SAPNA, in a multivalent and modular manner, to present selected antibodies that provide a specific recognition profile for binding and support for chemical features for isolation and reporter readout, for example by fluorescence.

[0064] Various embodiments of the present invention can present multiple distinct types of antibodies on the SAPNA molecule. For example, the SAPNA molecule can simultaneously present antibodies that are specific for various strains or subtypes of a single virus or bacterium. This enables the easy and effective identification of viruses with known variants or subtypes within a population. A well - understood example is the influenza virus, which obviates the need to design various reagents to detect the various strains of a virus. Presenting multiple types of antibodies can further provide a valuable advantage in distinguishing between pathogens (e.g., different bacteria) that express partially overlapping sets of surface antigens. As an example, if bacterium A expresses surface proteins X and Y, bacterium B expresses proteins Y and Z, and bacterium C expresses proteins X and Z, then a SAPNA molecule presenting antigens directed against proteins Y and Z will preferentially identify bacterium B due to the avidity effect. Of course, there can be other scenarios regarding the preferential detection of combinations of surface antigens, and this applies to both bacteria and viruses.

[0065] Various embodiments of the present invention may have different numbers of a single type of antibody presented on the SAPNA molecule, achieved by adding antibodies to the SAPNA core in different theoretical amounts. Since it is understood that the degree of multivalency of molecular binding strongly affects binding avidity, the ability to tailor the number of antibodies presented on the SAPNA molecule can provide a control (i.e., tunability) that is more valuable than the final binding affinity. Such control provides value in generating reagents with the most desirable window of detection for actively binding the intended target molecule while still providing readout information on negative binding to non-cognate molecules that may be similarly likely to varying degrees to the intended detection target. The narrow range of affinity relative to target specificity is a common challenge for the monovalent or oligovalent reagents that have been used to date for target identification by bacteria.

[0066] Various embodiments of the present invention will be specific to various viral, bacterial, and amyloid marker proteins. The list of potential targets expands and continues to grow with the discovery of new pathogens and requires only that specific antibodies are known or can be established against marker proteins of interest (performance routinely demonstrated in today's industry). Among the medically urgent viruses, spike (S) proteins of various coronaviruses, including SARS-CoV, SARS-CoV-2, and MERS-CoV, will be exemplary targets for identification. For the HIV virus, the gp120 glycoprotein is an exemplary target for identification. For the Ebola virus, the GP surface protein is an exemplary target. For the influenza virus, the hemagglutinin (HA) protein is an exemplary target, and various subtypes of the virus can be identified by various HA variants. Regarding bacterial targets, exemplary embodiments will be directed against diverse surface proteins and polysaccharide molecules. Specific examples of those of value in human pathogenicity will include SAPNA molecules carrying antibodies against capsular polysaccharides (CPS) from Haemophilus influenzae type b (Hib) or group B streptococci, or against any number of other pathogenic bacteria with capsular polysaccharide coatings. Further examples will be SAPNA molecules equipped with antibodies against the outer surface protein A (OspA) of the Lyme disease agent (Borrelia burgdorferi or related species), the capsular antigen of poly(D)glutamic acid of Bacillus anthracis, or the heparin-binding antigen (NHBA) of Neisseria gonorrhea. Prions and other amyloid diseases are often neurodegenerative and can affect both humans and animals. In these pathologies, native proteins are misfolded and then aggregate to form cytotoxic amyloid aggregates, which can be distributed throughout the body and deposited in various organ systems, leading to disease.Relevant to this embodiment of the present invention, unfolded / aggregated toxic forms of prion / amyloid proteins have a conformation different from the native folded form of the protein and generate toxic forms of these pathogenic agents that are distinguishable by antibodies. Examples of protein diseases in which the pathogenic protein is detectable by the SAPNA molecule include human Creutzfeldt-Jakob Disease and bovine bovine spongiform encephalopathy ("mad cow disease"). Detection of pathogenic proteins is extended to the following other amyloid proteins: beta amyloid (A-beta) (involved in Alzheimer's disease), tau protein (involved in various tauopathies), alpha-synuclein (involved in Parkinson's disease), transthyretin (involved in systemic amyloidosis), and others. These are merely selected examples.

[0067] Cited references: 1. Y.T. Lai et al., Designing and defining dynamic protein cage nanoassemblies in solution. Sci Adv 2, e1501855 (2016). 2. Y.T. Lai, K.L. Tsai, M.R. Sawaya, F.J. Asturias, T.O. Yeates, Structure and flexibility of nanoscale protein cages designed by symmetric self-assembly. J. Am. Chem. Soc. 135, 7738 - 7743 (2013). 3. J.E. Padilla, C. Colovos, T.O. Yeates, Nanohedra: using symmetry to design self assembling protein cages, layers, crystals, and filaments. Proc. Natl. Acad. Sci. U.S.A. 98, 2217 - 2221 (2001). 4. A. B. Sigalov, The SCHOOL of nature: I. Transmembrane signaling. Self Nonself 1, 4 - 39 (2010). 5. D. M. Ecker, S. D. Jones, H. L. Levine, The therapeutic monoclonal antibody market. MAbs 7, 9 - 14 (2015). 6. M. Suzuki, C. Kato, A. Kato, Therapeutic antibodies: their mechanisms of action and the pathological findings they induce in toxicity studies. J Toxicol Pathol 28, 133 - 139 (2015). 7. H. J. Kang et al., Developing an antibody - binding protein cage as a molecular recognition drug modular nanoplatform. Biomaterials 33, 5423 - 5430 (2012). 8. W. Choe, T. A. Durgannavar, S. J. Chung, Fc - Binding Ligands of Immunoglobulin G: An Overview of High Affinity Proteins and Peptides. Materials (Basel) 9, (2016). 9. Y. J. Kang et al., Polyvalent display of monosaccharides on ferritin protein cage nanoparticles for the recognition and binding of cell - surface lectins. Macromol.Biosci.14, 619 - 625 (2014). 10.G.A.Lazar et al., Engineered antibody Fc variants with enhanced effector function. Proc. Natl. Acad. Sci. U.S.A. 103, 4005 - 4010 (2006). 11.D.W.LaFleur et al., Monoclonal antibody therapeutics with up to five specificities: functional enhancement through fusion of target - specific peptides. MAbs 5, 208 - 218 (2013). 12.N.Dimasi et al., Development of a Trispecific Antibody Designed to Simultaneously and Efficiently Target Three Different Antigens on Tumor Cells. Mol. Pharm. 12, 3490 - 3501 (2015). 13.J.Stieglmaier, J.Benjamin, D.Nagorsen, Utilizing the BiTE (bispecific T - cell engager) platform for immunotherapy of cancer. Expert Opin Biol Ther 15, 1093 - 1099 (2015). 14.C.Spiess, Q.Zhai, P.J.Carter, Alternative molecular formats and therapeutic applications for bispecific antibodies. Mol. Immunol. 67, 95 - 106 (2015). 15.H. Byrne, P.J. Conroy, J.C. Whisstock, R.J. O’Kennedy, A tale of two specificities: bispecific antibodies for therapeutic and diagnostic applications. Trends Biotechnol. 31, 621-632 (2013).

[0068] The present invention has been described in conjunction with its preferred specific embodiments, but it should be understood that the foregoing description is intended to be illustrative and not limiting of the scope of the present invention. Other aspects, advantages, and modifications within the scope of the present invention will be apparent to those skilled in the art to which the present invention pertains.

[0069] All patents, patent applications, and published publications referred to herein are hereby incorporated by reference in their entirety.

[0070] Although the present invention has been described, the following examples are provided for illustrative purposes and not by way of limitation to explain the inventive subject matter.

Example

[0071] Example 1 Materials and Methods Design of Antibody-Modified Self-Assembling Protein Nanoparticles (SAPNAs) The workflow of SAPNAs was the following iterative process: manipulating a set of DNA constructs, attempting to express the protein, and once the protein was expressed, characterizing the construct and testing it for human Fc (hFc) binding. Site-directed mutagenesis was used to incorporate synthetic DNA fragments into the template scaffold (cloning into the pET22b+ vector), generating subsequent mutations for any novel construct. The template scaffold, a self-assembling tetrahedral protein cage, was cited from studies in the Yeates laboratory at UCLA (1 - 3). Using the unique capabilities of the high-throughput small-angle X-ray scattering (HT-SAXS) beamline developed by our group in recent collaborative research, two scaffold variants were structurally characterized under variable salt and pH conditions in solution (4). These two scaffold variants were used as templates for further functional manipulation. With the possible many applications in mind for presenting antibodies (see above), we aimed to functionalize the scaffold. Potential mutagenic sites were identified from the perspective of the available structure of the template scaffold and the multiple sequence alignment of evolutionarily related homologs. Verification of sequencing for the correct sequence involved expressing and purifying the constructs in parallel. The following buffers were used for purification: 1. Lysis (50 mM Tris pH 8.0, 300 mM NaCl, 10 mM imidazole), 2. Wash (50 mM Tris pH 8.0, 300 mM NaCl, 100 mM imidazole), 3. Elution (50 mM Tris pH 8.0, 300 mM NaCl, 300 mM imidazole), 4. Gel filtration (20 mM Tris pH 7.4 or 8.0, 100 mM NaCl, or PBS pH 7.4, or PBS pH 7.4, 0.05% Triton-X100). Once the His-tagged protein eluted from the Ni-NTA beads, the concentration was measured from the absorbance and the theoretical extinction coefficient. Due to the high valency of the construct (12 monomers, each with a His tag), an increased affinity for the Ni-NTA beads was obtained in a relatively pure fraction.Accordingly, any detected concentration of the protein above the baseline is predicted to be from a fully folded mutant scaffold to a half-folded mutant scaffold. These resulting constructs of the protein were further purified by size exclusion chromatography (SEC) and examined by peak shift assay for hFc binding. This mutagenesis process was repeated until a conformation that binds hFc without forming a detectable amount of scaffold oligomers was discovered (Table 2). The optimal set of conformations was further characterized by structural techniques, size exclusion chromatography - small angle X-ray scattering - multi-angle light scattering (SEC-SAXS-MALS).

[0072] Related research A unique small peptide motif engineered to bind to the Fc region of IgG antibodies was first described in 2000 and named Fc-III [DCAWHLGELVWCT] (5). This motif was discovered through the use of peptide phage display, which is an iterative method of selection for macromolecular binding interactions. Fc-III was further enhanced by adding stabilizing amino acids in the form of a cyclic peptide, called Fc-III-4C [CDCAWHLGELVWCTC] (6). In 2012, the ability of the Fc-III peptide to bind and target antibodies when incorporated within loops of the ferritin protein cage was demonstrated (7). The appearance of this ferritin protein cage is disclosed (International Publication No. WO 2013 / 055058 A9). As described below, the Fc-III and Fc-III-4C sequences were engineered into several sites within the previously mentioned scaffold templates, and we demonstrate that this enables the reproducible binding and presentation of human and rabbit IgG antibodies in solution.

[0073] Results We successfully engineered a scaffold based on self-assembling proteins to bind and display antibodies. The SAPNA structure in Figure 1 is a representative model of the predicted structure and allows sampling in a dynamic system solution when binding to human or rabbit IgG Fc domains or antibodies. To biochemically demonstrate the antibody / Fc binding performance of our scaffold molecules, human IgG1 Fc conjugated to the fluorescent protein PerCP (Fc-PerCP) was added to the scaffold and subjected to SEC (Figures 2A and 2B). The peak absorbance at 280 nm (A280) (Figure 2A), which is the protein readout information, shifts from a retention volume of 13.2 mL to 12.9 mL, indicating an increase in the size of the scaffold. Furthermore, the peak absorbance at 482 nm (A482), which is the fluorescence readout information of PerCP, appears at 12.9 mL, supporting that the increase in the size of the scaffold is due to the binding of Fc-PerCP. Similarly, a peak shift assay with Alexa Fluor®-488-labeled human IgG1 isotype antibody (hIgG1 antibody-488) was performed using the scaffold (Figures 3A and 3B). The A280 peak (Figure 3A) shifts from a retention volume of 13.2 mL to 12.3 mL, indicating an increase in the size of the scaffold. The 488 peak absorbance (A488), which is the fluorescence readout information of the Alexa Fluor®-488 fluorescent dye, appears at 12.3 mL, supporting that the increase in the size of the scaffold is due to the binding of hIgG1 antibody-488. Note that we have evidence suggesting that the chemical conjugation of fluorophores and fluorescent proteins to antibodies / Fc (presumably via the widely used primary amine method) may reduce the ability of functionalized scaffolds to bind antibodies / Fc. For this reason, we did not expect high peaks of A482 and A488 in Figures 2B and 3B, which these data reflect.

[0074] To structurally evaluate the scaffold with respect to the binding of Fc and antibody, solution techniques SEC-SAXS-MALS (Figures 4A, 4B, 5A, and 5B, respectively) were used. For further scattering analysis, the sample peak regions of the scaffold, hFc, and scaffold-hFc complex were selected (Figure 4A). In Figure 4B, all molecules / complexes were compared using the P(r) function, which is a histogram of the average distance of the orientation of the scattering particles (8). That is, the wider the area under these histograms, the greater the scale and number of "end-to-end" distances within the molecules present. Thus, the increase in the diameter of the scaffold due to the addition of hFc and antibody molecules will be easily represented by the P(r) function. In Figure 4B, it is clear that the various scaffold states (X, Y, Z) along the scaffold-hFc peak in Figure 4A represent the mounting of hFc molecules on the scaffold. This ease of mounting is also seen in the increase in the radius of gyration (Rg) and maximum length (Dmax) in Table 1. Further evidence for the mounting of hFc on the scaffold is the MALS data in Table 1, where the average molecular weight of the MALS peak increased from 764 kDa to 1020 kDa due to the addition of hFc to the scaffold. Similar results were seen when characterizing the binding of polyclonal IgG rabbit anti-GFP antibody to the scaffold (scaffold-R-anti-GFP) using SEC-SAXS-MALS in Figures 5A and 5B. Analysis of a single region of the scaffold-R-anti-GFP peak showed an increase in the P(r) function (Figure 5B) as well as in Rg, Dmax, and the average molecular weight of the MALS peak (Table 1).

[0075]

Table 1

[0076] Table 2. Sequences of scaffold variants designed and experimentally investigated so far The initially disclosed template: MPFITVGQENSTSIDLYYEDHGTGTPVVLIHGFPLSGHSWERQSAALLDAGYRVITYDRRGFGQSSQPTTGYDYDTFAADLNTVLETLDLQDAVLVGFSMGTGEVARYVSSYGTARIAAVAFLASLEPFLLKTDDNPDGAAPQEFFDGIVAAVKADRYAFYTGFFNDFYNLDENLGTRISEEAVRNSWNTAASGGFFAAAAAPTTWYTDFRADIPRIDVPALILHGTGDRTLPIENTARVFHKALPSAEYVEVEGAPHGLLWTHAEEVNTALLAFLAKAQEAQKQKLLTEVETYVLSIIPSGPLKAEIAQRLEDVFAGKNTDLEVLMEWLKTRPILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDKAVKLYRKLKREITFHGAKEISLSYSAGALASCMGLIYNRMGAVTTEVAFGLVCATCEQIADSQHRSHRQLEHHHHHH (SEQ ID NO: 40)

[0077] SAPNA_1 MPFITVGQENSTSIDLYYEDHGTGTPVVLIHGFPLSGHSWERQSAALLDAGYRVITYDRRGFGQSSQPTTGYDYDTFAADLNTVLETLDLQDAVLVGFSMGTGEVARYVSSYGTARIAAVAFLASLEPFLLKTGCDCAWHLGELVWCTCGDNPDGAAPQEFFDGIVAAVKADRYAFYTGFFNDFYNLDENLGTRISEEAVRNSWNTAASGGFFAAAAAPTTWYTDFRADIPRIDVPALILHGTGDRTLPIENTARVFHKALPSAEYVEVEGAPHGLLWTHAEEVNTALLAFLAKAQEAQKQKLLTEVETYVLSIIPSGPLKAEIAQRLEDVFAGKNTDLEVLMEWLKTRPILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDKAVKLYRKLKREITFHGAKEISLSYSAGALASCMGLIYNRMGAVTTEVAFGLVCATCEQIADSQHRSHRQLEHHHHHH(SEQ ID NO: 1)

[0078] SAPNA_2 MPFITVGQENSTSIDLYYEDHGTGTPVVLIHGFPLSGHSWERQSAALLDAGYRVITYDRRGFGQSSQPTTGYDYDTFAADLNTVLETLDLQDAVLVGFSMGTGEVARYVSSYGTARIAAVAFLASLEPFLLKTGCDCAWHLGELVWCTCGDNPDGAAPQEFFDGIVAAVKADRYAFYTGFFNDFYNLDENLGTRISEEAVRNSWNTAASGGFFAAAAAPTTWYTDFRADIPRIDVPALILHGTGDRTLPIENTARVFHKALPSAEYVEVEGAPHGLLWTHAEEVNTALLAFLAKAQEAQKQKLLTEVETYVLSIIPSGPLKAEIAQRLEDVFAGKNTDLEVLMEWLKTRPILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDKAVKLYRKLKREITFHGAKEISLSYSAGALASCMGLIYNRMGAVTTEVAFGLVCATCEQIADSQENLYFQGLEHHHHHH(SEQ ID NO: 2)

[0079] SAPNA_3 MPFITVGQENSTSIDLYYEDHGTGTPVVLIHGFPLSGHSWERQSAALLDAGYRVITYDRRGFGQSSQPTTGYDYDTFAADLNTVLETLDLQDAVLVGFSMGTGEVARYVSSYGTARIAAVAFLASLEPFLLKTGGGSGCDCAWHLGELVWCTCGSGGGDNPDGAAPQEFFDGIVAAVKADRYAFYTGFFNDFYNLDENLGTRISEEAVRNSWNTAASGGFFAAAAAPTTWYTDFRADIPRIDVPALILHGTGDRTLPIENTARVFHKALPSAEYVEVEGAPHGLLWTHAEEVNTALLAFLAKAQEAQKQKLLTEVETYVLSIIPSGPLKAEIAQRLEDVFAGKNTDLEVLMEWLKTRPILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDKAVKLYRKLKREITFHGAKEISLSYSAGALASCMGLIYNRMGAVTTEVAFGLVCATCEQIADSQHRSHRQLEHHHHHH(SEQ ID NO: 3)

[0080] SAPNA_4 MPFITVGQENSTSIDLYYEDHGTGTPVVLIHGFPLSGHSWERQSAALLDAGYRVITYDRRGFGQSSQPTTGYDYDTFAADLNTVLETLDLQDAVLVGFSMGTGEVARYVSSYGTARIAAVAFLASLEPFLLKTGGGSGCDCAWHLGELVWCTCGSGGGDNPDGAAPQEFFDGIVAAVKADRYAFYTGFFNDFYNLDENLGTRISEEAVRNSWNTAASGGFFAAAAAPTTWYTDFRADIPRIDVPALILHGTGDRTLPIENTARVFHKALPSAEYVEVEGAPHGLLWTHAEEVNTALLAFLAKAQEAQKQKLLTEVETYVLSIIPSGPLKAEIAQRLEDVFAGKNTDLEVLMEWLKTRPILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDKAVKLYRKLKREITFHGAKEISLSYSAGALASCMGLIYNRMGAVTTEVAFGLVCATCEQIADSQENLYFQGLEHHHHHH(SEQ ID NO: 4)

[0081] SAPNA_5 MPFITVGQENSTSIDLYYEDHGTGTPVVLIHGFPLSGHSWERQSAALLDAGARVITYDRRGFGQSSQPTTGYDYDTFAADLNTVLETLDLQDAVLVGFSMGTGEVARYVSSYGTARIAAVAFLASLEPFLLKTGCDCAWHLGELVWCTCGDNPDGAAPQEFFDGIVAAVKADRYAFYTGFFNDFYNLDENLGTRISEEAVRNSWNTAASGGFFAAAAAPTTWYTDFRADIPRIDVPALILHGTGDRTLPIENTARVFHKALPSAEYVEVEGAPHGLLWTHAEEVNTALLAFLAKAQEAQKQKLLTEVETYVLSIIPSGPLKAEIAQRLEDVFAGKNTDLEVLMEWLKTRPILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDKAVKLYRKLKREITFHGAKEISLSYSAGALASCMGLIYNRMGAVTTEVAFGLVCATCEQIADSQHRSHRQLEHHHHHH(SEQ ID NO: 5)

[0082] SAPNA_6 MPFITVGQENSTSIDLYYEDHGTGTPVVLIHGFPLSGHSWERQSAALLDAGARVITYDRRGFGQSSQPTTGYDYDTFAADLNTVLETLDLQDAVLVGFSMGTGEVARYVSSYGTARIAAVAFLASLEPFLLKTGCDCAWHLGELVWCTCGDNPDGAAPQEFFDGIVAAVKADRYAFYTGFFNDFYNLDENLGTRISEEAVRNSWNTAASGGFFAAAAAPTTWYTDFRADIPRIDVPALILHGTGDRTLPIENTARVFHKALPSAEYVEVEGAPHGLLWTHAEEVNTALLAFLAKAQEAQKQKLLTEVETYVLSIIPSGPLKAEIAQRLEDVFAGKNTDLEVLMEWLKTRPILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDKAVKLYRKLKREITFHGAKEISLSYSAGALASCMGLIYNRMGAVTTEVAFGLVCATCEQIADSQENLYFQGLEHHHHHH (SEQ ID NO: 6)

[0083] SAPNA_7 MPFITVGQENSTSIDLYYEDHGTGTPVVLIHGFPLSGHSWERQSAALLDAGARVITYDRRGFGQSSQPTTGYDYDTFAADLNTVLETLDLQDAVLVGFSMGTGEVARYVSSYGTARIAAVAFLASLEPFLLKTGGGSGCDCAWHLGELVWCTCGSGGGDNPDGAAPQEFFDGIVAAVKADRYAFYTGFFNDFYNLDENLGTRISEEAVRNSWNTAASGGFFAAAAAPTTWYTDFRADIPRIDVPALILHGTGDRTLPIENTARVFHKALPSAEYVEVEGAPHGLLWTHAEEVNTALLAFLAKAQEAQKQKLLTEVETYVLSIIPSGPLKAEIAQRLEDVFAGKNTDLEVLMEWLKTRPILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDKAVKLYRKLKREITFHGAKEISLSYSAGALASCMGLIYNRMGAVTTEVAFGLVCATCEQIADSQHRSHRQLEHHHHHH(SEQ ID NO:7)

[0084] SAPNA_8 MPFITVGQENSTSIDLYYEDHGTGTPVVLIHGFPLSGHSWERQSAALLDAGARVITYDRRGFGQSSQPTTGYDYDTFAADLNTVLETLDLQDAVLVGFSMGTGEVARYVSSYGTARIAAVAFLASLEPFLLKTGGGSGCDCAWHLGELVWCTCGSGGGDNPDGAAPQEFFDGIVAAVKADRYAFYTGFFNDFYNLDENLGTRISEEAVRNSWNTAASGGFFAAAAAPTTWYTDFRADIPRIDVPALILHGTGDRTLPIENTARVFHKALPSAEYVEVEGAPHGLLWTHAEEVNTALLAFLAKAQEAQKQKLLTEVETYVLSIIPSGPLKAEIAQRLEDVFAGKNTDLEVLMEWLKTRPILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDKAVKLYRKLKREITFHGAKEISLSYSAGALASCMGLIYNRMGAVTTEVAFGLVCATCEQIADSQENLYFQGLEHHHHHH (SEQ ID NO: 8)

[0085] SAPNA_9 MPFITVGQENSTSIDLYYEDHGTGTPVVLIHGFPLSGHSWERQSAALLDAGYRVITYDRRGFGQSSQPTTGYDYDTFAADLNTVLETLDLQDAVLVGFSMGTGEVARYVSSYGTARIAAVAFLASLEPFLLKTGRWGCDCAWHLGELVWCTCGWEGDNPDGAAPQEFFDGIVAAVKADRYAFYTGFFNDFYNLDENLGTRISEEAVRNSWNTAASGGFFAAAAAPTTWYTDFRADIPRIDVPALILHGTGDRTLPIENTARVFHKALPSAEYVEVEGAPHGLLWTHAEEVNTALLAFLAKAQEAQKQKLLTEVETYVLSIIPSGPLKAEIAQRLEDVFAGKNTDLEVLMEWLKTRPILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDKAVKLYRKLKREITFHGAKEISLSYSAGALASCMGLIYNRMGAVTTEVAFGLVCATCEQIADSQHRSHRQLEHHHHHH (SEQ ID NO: 9)

[0086] SAPNA_10 MPFITVGQENSTSIDLYYEDHGTGTPVVLIHGFPLSGHSWERQSAALLDAGYRVITYDRRGFGQSSQPTTGYDYDTFAADLNTVLETLDLQDAVLVGFSMGTGEVARYVSSYGTARIAAVAFLASLEPFLLKTGRWDCAWHLGELVWCTWEGDNPDGAAPQEFFDGIVAAVKADRYAFYTGFFNDFYNLDENLGTRISEEAVRNSWNTAASGGFFAAAAAPTTWYTDFRADIPRIDVPALILHGTGDRTLPIENTARVFHKALPSAEYVEVEGAPHGLLWTHAEEVNTALLAFLAKAQEAQKQKLLTEVETYVLSIIPSGPLKAEIAQRLEDVFAGKNTDLEVLMEWLKTRPILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDKAVKLYRKLKREITFHGAKEISLSYSAGALASCMGLIYNRMGAVTTEVAFGLVCATCEQIADSQHRSHRQLEHHHHHH(SEQ ID NO:10)

[0087] SAPNA_11 MPFITVGQENSTSIDLYYEDHGTGTPVVLIHGFPLSGHSWERQSAALLDAGYRVITYDRRGFGQSSQPTTGYDYDTFAADLNTVLETLDLQDAVLVGFSMGTGEVARYVSSYGTARIAAVAFLASLEPFLLKTGGGGRWDCAWHLGELVWCTWEGGGGDNPDGAAPQEFFDGIVAAVKADRYAFYTGFFNDFYNLDENLGTRISEEAVRNSWNTAASGGFFAAAAAPTTWYTDFRADIPRIDVPALILHGTGDRTLPIENTARVFHKALPSAEYVEVEGAPHGLLWTHAEEVNTALLAFLAKAQEAQKQKLLTEVETYVLSIIPSGPLKAEIAQRLEDVFAGKNTDLEVLMEWLKTRPILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDKAVKLYRKLKREITFHGAKEISLSYSAGALASCMGLIYNRMGAVTTEVAFGLVCATCEQIADSQHRSHRQLEHHHHHH(SEQ ID NO:11)

[0088] SAPNA_12 MPFITVGQENSTSIDLYYEDHGTGTPVVLIHGFPLSGHSWERQSAALLDAGYRVITYDRRGFGQSSQPTTGYDYDTFAADLNTVLETLDLQDAVLVGFSMGTGEVARYVSSYGTARIAAVAFLASLEPFLLKTGGGGRWDAAWHLGELVWATWEGGGGDNPDGAAPQEFFDGIVAAVKADRYAFYTGFFNDFYNLDENLGTRISEEAVRNSWNTAASGGFFAAAAAPTTWYTDFRADIPRIDVPALILHGTGDRTLPIENTARVFHKALPSAEYVEVEGAPHGLLWTHAEEVNTALLAFLAKAQEAQKQKLLTEVETYVLSIIPSGPLKAEIAQRLEDVFAGKNTDLEVLMEWLKTRPILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDKAVKLYRKLKREITFHGAKEISLSYSAGALASCMGLIYNRMGAVTTEVAFGLVCATCEQIADSQHRSHRQLEHHHHHH(SEQ ID NO: 12)

[0089] SAPNA_13 MPFITVGQENSTSIDLYYEDHGTGTPVVLIHGFPLSGHSWERQSAALLDAGYRVITYDRRGFGQSSQPTTGYDYDTFAADLNTVLETLDLQDAVLVGFSMGTGEVARYVSSYGTARIAAVAFLASLEPFLLKTGGGSGADCAWHLGELVWCTAGSGGGDNPDGAAPQEFFDGIVAAVKADRYAFYTGFFNDFYNLDENLGTRISEEAVRNSWNTAASGGFFAAAAAPTTWYTDFRADIPRIDVPALILHGTGDRTLPIENTARVFHKALPSAEYVEVEGAPHGLLWTHAEEVNTALLAFLAKAQEAQKQKLLTEVETYVLSIIPSGPLKAEIAQRLEDVFAGKNTDLEVLMEWLKTRPILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDKAVKLYRKLKREITFHGAKEISLSYSAGALASCMGLIYNRMGAVTTEVAFGLVCATCEQIADSQHRSHRQLEHHHHHH(SEQ ID NO: 13)

[0090] SAPNA_14 MPFITVGQENSTSIDLYYEDHGTGTPVVLIHGFPLSGHSWERQSAALLDAGYRVITYDRRGFGQSSQPTTGYDYDTFAADLNTVLETLDLQDAVLVGFSMGTGEVARYVSSYGTARIAAVAFLASLEPFLLKTGGGSGADAAWHLGELVWATAGSGGGDNPDGAAPQEFFDGIVAAVKADRYAFYTGFFNDFYNLDENLGTRISEEAVRNSWNTAASGGFFAAAAAPTTWYTDFRADIPRIDVPALILHGTGDRTLPIENTARVFHKALPSAEYVEVEGAPHGLLWTHAEEVNTALLAFLAKAQEAQKQKLLTEVETYVLSIIPSGPLKAEIAQRLEDVFAGKNTDLEVLMEWLKTRPILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDKAVKLYRKLKREITFHGAKEISLSYSAGALASCMGLIYNRMGAVTTEVAFGLVCATCEQIADSQHRSHRQLEHHHHHH(SEQ ID NO:14)

[0091] SAPNA_15 MPFITVGQENSTSIDLYYEDHGTGTPVVLIHGFPLSGHSWERQSAALLDAGYRVITYDRRGFGQSSQPTTGYDYDTFAADLNTVLETLDLQDAVLVGFSMGTGEVARYVSSYGTARIAAVAFLASLEPFLLKTDDNPDGAAPQEFFDGIVAAVKADRYAFYTGFFNDFYNLDENLGTRISEEAVRNSWNTAASGGFFAAAAAPTTWYTDFRADIPRIDVPALILHGTGDRTLPIENTARVFHKALPSGGGSGCDCAWHLGELVWCTCGSGGGAEYVEVEGAPHGLLWTHAEEVNTALLAFLAKAQEAQKQKLLTEVETYVLSIIPSGPLKAEIAQRLEDVFAGKNTDLEVLMEWLKTRPILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDKAVKLYRKLKREITFHGAKEISLSYSAGALASCMGLIYNRMGAVTTEVAFGLVCATCEQIADSQHRSHRQLEHHHHHH(SEQ ID NO:15)

[0092] SAPNA_16 MPFITVGQENSTSIDLYYEDHGTGTPVVLIHGFPLSGHSWERQSAALLDAGYRVITYDRRGFGQSSQPTTGYDYDTFAADLNTVLETLDLQDAVLVGFSMGTGEVARYVSSYGTARIAAVAFLASLEPFLLKTDDNPDGAAPQEFFDGIVAAVKADRYAFYTGFFNDFYNLDENLGTRISEEAVRNSWNTAASGGFFAAAAAPTTWYTDFRADIPRIGGGSGCDCAWHLGELVWCTCGSGGGVPALILHGTGDRTLPIENTARVFHKALPSAEYVEVEGAPHGLLWTHAEEVNTALLAFLAKAQEAQKQKLLTEVETYVLSIIPSGPLKAEIAQRLEDVFAGKNTDLEVLMEWLKTRPILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDKAVKLYRKLKREITFHGAKEISLSYSAGALASCMGLIYNRMGAVTTEVAFGLVCATCEQIADSQHRSHRQLEHHHHHH(SEQ ID NO:16)

[0093] SAPNA_17 MPFITVGQENSTSIDLYYEDHGTGTPVVLIHGFPLSGHSWERQSAALLDAGYRVITYDRRGFGQSSQPTTGYDYDTFAADLNTVLETLDLQDAVLVGFSMGTGEVARYVSSYGTARIAAVAFLASLEPFLLKTDDNPDGAAPQEFFDGIVAAVKADRYAFYTGFFNDFYNLGGGSGCDCAWHLGELVWCTCGSGGGRISEEAVRNSWNTAASGGFFAAAAAPTTWYTDFRADIPRIDVPALILHGTGDRTLPIENTARVFHKALPSAEYVEVEGAPHGLLWTHAEEVNTALLAFLAKAQEAQKQKLLTEVETYVLSIIPSGPLKAEIAQRLEDVFAGKNTDLEVLMEWLKTRPILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDKAVKLYRKLKREITFHGAKEISLSYSAGALASCMGLIYNRMGAVTTEVAFGLVCATCEQIADSQHRSHRQLEHHHHHH(SEQ ID NO:17)

[0094] SAPNA_18 MPFITVGQENSTSIDLYYEDHGTGTPVVLIHGFPLSGHSWERQSAALLDAGYRVITYDRRGFGQSSQPTTGYDYDTFAADLNTVLETLDLQDAVLVGFSMGTGEVARYVSSYGTARIAAVAFLASLEPFLLKTDDNPDGAAPQEFFDGIVAAVKADRYAFYTGFFNDFYNLDENLGTRISEEAVRNSWNTAASGGFFAAAAAPTTWYTDFRADIPRIDVPALILHGTGGGSGCDCAWHLGELVWCTCGSGGGTLPIENTARVFHKALPSAEYVEVEGAPHGLLWTHAEEVNTALLAFLAKAQEAQKQKLLTEVETYVLSIIPSGPLKAEIAQRLEDVFAGKNTDLEVLMEWLKTRPILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDKAVKLYRKLKREITFHGAKEISLSYSAGALASCMGLIYNRMGAVTTEVAFGLVCATCEQIADSQHRSHRQLEHHHHHH (SEQ ID NO: 18)

[0095] SAPNA_19 MPFITVGQENSTSIDLYYEDHGTGTPVVLIHGFPLSGHSWERQSAALLDAGYRVITYDRRGFGQSSQPTTGYDYDTFAADLNTVLETLDLQDAVLVGFSMGTGEVARYVSSYGGGSGCDCAWHLGELVWCTCGSGGGRIAAVAFLASLEPFLLKTDDNPDGAAPQEFFDGIVAAVKADRYAFYTGFFNDFYNLDENLGTRISEEAVRNSWNTAASGGFFAAAAAPTTWYTDFRADIPRIDVPALILHGTGDRTLPIENTARVFHKALPSAEYVEVEGAPHGLLWTHAEEVNTALLAFLAKAQEAQKQKLLTEVETYVLSIIPSGPLKAEIAQRLEDVFAGKNTDLEVLMEWLKTRPILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDKAVKLYRKLKREITFHGAKEISLSYSAGALASCMGLIYNRMGAVTTEVAFGLVCATCEQIADSQHRSHRQLEHHHHHH (SEQ ID NO:19)

[0096] SAPNA_20 MPFITVGQENSTSIDLYYEDHGTGTPVVLIHGFPLSGHSWERQSAALLDAGYRVITYDRRGFGQSSQPTTGYDYDTFAADLNTVLETLDLQDAVLVGFSMGTGEVARYVSSYGTARIAAVAFLASLEPFLLKTDDNPDGAAPQEFFDGIVAAVKADRYAFYTGFFNDFYNLDENLGTRISEEAVRNSWNTAASGGFFAAAAAPTTWYTDFRADIPRIDVPALILHGTGDRTLPIENTARVFHKALPSAEYVEVEGAPHGLLWTHAEEVNTALLAFLAKAQEAQKQKLLTEVETYVLSIIPSGPLKAEIAQRLEDVFAGGGGSGCDCAWHLGELVWCTCGSGGGDLEVLMEWLKTRPILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDKAVKLYRKLKREITFHGAKEISLSYSAGALASCMGLIYNRMGAVTTEVAFGLVCATCEQIADSQHRSHRQLEHHHHHH(SEQ ID NO: 20)

[0097] SAPNA_21 MPFITVGQENSTSIDLYYEDHGTGTPVVLIHGFPLSGHSWERQSAALLDAGYRVITYDRRGFGQSSQPTTGYDYDTFAADLNTVLETLDLQDAVLVGFSMGTGEVARYVSSYGTARIAAVAFLASLEPFLLKTDDNPDGAAPQEFFDGIVAAVKADRYAFYTGFFNDFYNLDENLGTRISEEAVRNSWNTAASGGFFAAAAAPTTWYTDFRADIPRIDVPALILHGTGDRTLPIENTARVFHKALPSAEYVEVEGAPHGLLWTHAEEVNTALLAFLAKAQEAQKQKLLTEVETYVLSIIPSGPLKAEIAQRLEDVFAGKNTDLEVLMEWLKTRPILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDKAVKLYRKLKREITFHGAKEISLSYSAGALASCMGLIYNRMGAVTTEVAFGLVCATCEQIADSQGGGSGCDCAWHLGELVWCTCGSGGGLEHHHHHH(SEQ ID NO: 21)

[0098] SAPNA_22 MPFITVGQENSTSIDLYYEDHGTGTPVVLIHGFPLSGHSWERQSAALLDAGYRVITYDRRGFGQSSQPTTGYDYDTFAADLNTVLETLDLQDAVLVGFSMGTGEVARYVSSYGTARIAAVAFLASLEPFLLKTDDNPDGAAPQEFFDGIVAAVKADRYAFYTGFFNDFYNLDENLGTRISEEAVRNSWNTAASGGFFAAAAAPTTWYTDFRADIPRIDVPALILHGTGDRTLPIENTARVFHKALPSAEYVEVEGAPHGLLWTHAEEVNTALLAFLAKAQEAQKQKLLTEVETYVLSIIPSGPLKAEIAQRLEDVFAGGGRWGCDCAWHLGELVWCTCGWEGGDLEVLMEWLKTRPILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDKAVKLYRKLKREITFHGAKEISLSYSAGALASCMGLIYNRMGAVTTEVAFGLVCATCEQIADSQHRSHRQLEHHHHHH(SEQ ID NO: 22)

[0099] SAPNA_23 MPFITVGQENSTSIDLYYEDHGTGTPVVLIHGFPLSGHSWERQSAALLDAGYRVITYDRRGFGQSSQPTTGYDYDTFAADLNTVLETLDLQDAVLVGFSMGTGEVARYVSSYGTARIAAVAFLASLEPFLLKTDDNPDGAAPQEFFDGIVAAVKADRYAFYTGFFNDFYNLDENLGTRISEEAVRNSWNTAASGGFFAAAAAPTTWYTDFRADIPRIDVPALILHGTGDRTLPIENTARVFHKALPSAEYVEVEGAPHGLLWTHAEEVNTALLAFLAKAQEAQKQKLLTEVETYVLSIIPSGPLKAEIAQRLEDVFAGRWGSGCDCAWHLGELVWCTCGSGWEDLEVLMEWLKTRPILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDKAVKLYRKLKREITFHGAKEISLSYSAGALASCMGLIYNRMGAVTTEVAFGLVCATCEQIADSQHRSHRQLEHHHHHH(SEQ ID NO: 23)

[0100] SAPNA_24 MPFITVGQENSTSIDLYYEDHGTGTPVVLIHGFPLSGHSWERQSAALLDAGYRVITYDRRGFGQSSQPTTGYDYDTFAADLNTVLETLDLQDAVLVGFSMGTGEVARYVSSYGTARIAAVAFLASLEPFLLKTDDNPDGAAPQEFFDGIVAAVKADRYAFYTGFFNDFYNLDENLGTRISEEAVRNSWNTAASGGFFAAAAAPTTWYTDFRADIPRIDVPALILHGTGDRTLPIENTARVFHKALPSAEYVEVEGAPHGLLWTHAEEVNTALLAFLAKAQEAQKQKLLTEVETYVLSIIPSGPLKAEIAQRLEDVFAGGGGCDCAWHLGELVWCTCGGGDLEVLMEWLKTRPILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDKAVKLYRKLKREITFHGAKEISLSYSAGALASCMGLIYNRMGAVTTEVAFGLVCATCEQIADSQHRSHRQLEHHHHHH(SEQ ID NO: 24)

[0101] SAPNA_25 MPFITVGQENSTSIDLYYEDHGTGTPVVLIHGFPLSGHSWERQSAALLDAGYRVITYDRRGFGQSSQPTTGYDYDTFAADLNTVLETLDLQDAVLVGFSMGTGEVARYVSSYGTARIAAVAFLASLEPFLLKTDDNPDGAAPQEFFDGIVAAVKADRYAFYTGFFNDFYNLDENLGTRISEEAVRNSWNTAASGGFFAAAAAPTTWYTDFRADIPRIDVPALILHGTGDRTLPIENTARVFHKALPSAEYVEVEGAPHGLLWTHAEEVNTALLAFLAKAQEAQKQKLLTEVETYVLSIIPSGPLKAEIAQRLEDVFAGGCDCAWHLGELVWCTCGDLEVLMEWLKTRPILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDKAVKLYRKLKREITFHGAKEISLSYSAGALASCMGLIYNRMGAVTTEVAFGLVCATCEQIADSQHRSHRQLEHHHHHH(SEQ ID NO: 25)

[0102] SAPNA_26 MPFITVGQENSTSIDLYYEDHGTGTPVVLIHGFPLSGHSWERQSAALLDAGYRVITYDRRGFGQSSQPTTGYDYDTFAADLNTVLETLDLQDAVLVGFSMGTGEVARYVSSYGTARIAAVAFLASLEPFLLKTDDNPDGAAPQKFFDGIVAAVKADRYAFYTGFFNDFYNLDENLGTRISEEAVRNSWNTAASGGFFAAAAAPTTWYTDFRADIPRIDVPALILHGTGDRTLPIENTARVFHKALPSAEYVEVEGAPHGLLWTHAEEVNTALLAFLAKAQEAQKQKLLTEVETYVLSIIPSGPLKAEIAQRLEDVFAGKNTDLEVLMEWLKTRPILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDKAVKLYRKLKREITFHGAKEISLSYSAGALASCMGLIYNRMGAVTTEVAFGLVCATCEQIADSQGGGSGCDCAWHLGELVWCTCGSGGGLEHHHHHH(SEQ ID NO: 26)

[0103] SAPNA_27 MPFITVGQENSTSIDLYYEDHGTGTPVVLIHGFPLSGHSWERQSAALLDAGYRVITYDRRGFGQSSQPTTGYDYDTFAADLNTVLETLDLQDAVLVGFSMGTGEVARYVSSYGTARIAAVAFLASLEPFLLKTDDNPDGAAPQEFFDGIVAAVKADRYAFYTGFFNDFYNLKENLGTRISEEAVRNSWNTAASGGFFAAAAAPTTWYTDFRADIPRIDVPALILHGTGDRTLPIENTARVFHKALPSAEYVEVEGAPHGLLWTHAEEVNTALLAFLAKAQEAQKQKLLTEVETYVLSIIPSGPLKAEIAQRLEDVFAGKNTDLEVLMEWLKTRPILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDKAVKLYRKLKREITFHGAKEISLSYSAGALASCMGLIYNRMGAVTTEVAFGLVCATCEQIADSQGGGSGCDCAWHLGELVWCTCGSGGGLEHHHHHH(SEQ ID NO: 27)

[0104] SAPNA_28 MPFITVGQENSTSIDLYYEDHGTGTPVVLIHGFPLSGHSWERQSAALLDAGYRVITYDRRGFGQSSQPTTGYDYDTFAADLNTVLETLDLQDAVLVGFSMGTGEVARYVSSYGTARIAAVAFLASLEPFLLKTDDNPDGAAPQEFFDGIVAAVKADRYAFYTGFFNDFYNLDENLGTRISEEAVRNSWNTAASGGFFAAAAAPTTWYTDFRADIPRIDVPALILHGTGDRTLPIKNTARVFHKALPSAEYVEVEGAPHGLLWTHAEEVNTALLAFLAKAQEAQKQKLLTEVETYVLSIIPSGPLKAEIAQRLEDVFAGKNTDLEVLMEWLKTRPILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDKAVKLYRKLKREITFHGAKEISLSYSAGALASCMGLIYNRMGAVTTEVAFGLVCATCEQIADSQGGGSGCDCAWHLGELVWCTCGSGGGLEHHHHHH(SEQ ID NO:28)

[0105] SAPNA_29 MPFITVGQENSTSIDLYYEDHGTGTPVVLIHGFPLSGHSWERQSAALLDAGYRVITYDRRGFGQSSQPTTGYDYDTFAADLNTVLETLDLQDAVLVGFSMGTGEVARYVSSYGTARIAAVAFLASLEPFLLKTDDNPDGAAPQKFFDGIVAAVKADRYAFYTGFFNDFYNLKENLGTRISEEAVRNSWNTAASGGFFAAAAAPTTWYTDFRADIPRIDVPALILHGTGDRTLPIENTARVFHKALPSAEYVEVEGAPHGLLWTHAEEVNTALLAFLAKAQEAQKQKLLTEVETYVLSIIPSGPLKAEIAQRLEDVFAGKNTDLEVLMEWLKTRPILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDKAVKLYRKLKREITFHGAKEISLSYSAGALASCMGLIYNRMGAVTTEVAFGLVCATCEQIADSQGGGSGCDCAWHLGELVWCTCGSGGGLEHHHHHH(SEQ ID NO:29)

[0106] SAPNA_30 MPFITVGQENSTSIDLYYEDHGTGTPVVLIHGFPLSGHSWERQSAALLDAGYRVITYDRRGFGQSSQPTTGYDYDTFAADLNTVLETLDLQDAVLVGFSMGTGEVARYVSSYGTARIAAVAFLASLEPFLLKTDDNPDGAAPQEFFDGIVAAVKADRYAFYTGFFNDFYNLKENLGTRISEEAVRNSWNTAASGGFFAAAAAPTTWYTDFRADIPRIDVPALILHGTGDRTLPIKNTARVFHKALPSAEYVEVEGAPHGLLWTHAEEVNTALLAFLAKAQEAQKQKLLTEVETYVLSIIPSGPLKAEIAQRLEDVFAGKNTDLEVLMEWLKTRPILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDKAVKLYRKLKREITFHGAKEISLSYSAGALASCMGLIYNRMGAVTTEVAFGLVCATCEQIADSQGGGSGCDCAWHLGELVWCTCGSGGGLEHHHHHH(SEQ ID NO: 30)

[0107] SAPNA_31 MPFITVGQENSTSIDLYYEDHGTGTPVVLIHGFPLSGHSWERQSAALLDAGYRVITYDRRGFGQSSQPTTGYDYDTFAADLNTVLETLDLQDAVLVGFSMGTGEVARYVSSYGTARIAAVAFLASLEPFLLKTDDNPDGAAPQKFFDGIVAAVKADRYAFYTGFFNDFYNLDENLGTRISEEAVRNSWNTAASGGFFAAAAAPTTWYTDFRADIPRIDVPALILHGTGDRTLPIKNTARVFHKALPSAEYVEVEGAPHGLLWTHAEEVNTALLAFLAKAQEAQKQKLLTEVETYVLSIIPSGPLKAEIAQRLEDVFAGKNTDLEVLMEWLKTRPILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDKAVKLYRKLKREITFHGAKEISLSYSAGALASCMGLIYNRMGAVTTEVAFGLVCATCEQIADSQGGGSGCDCAWHLGELVWCTCGSGGGLEHHHHHH(SEQ ID NO: 31)

[0108] SAPNA_32 MPFITVGQENSTSIDLYYEDHGTGTPVVLIHGFPLSGHSWERQSAALLDAGYRVITYDRRGFGQSSQPTTGYDYDTFAADLNTVLETLDLQDAVLVGFSMGTGEVARYVSSYGTARIAAVAFLASLEPFLLKTDDNPDGAAPQKFFDGIVAAVKADRYAFYTGFFNDFYNLKENLGTRISEEAVRNSWNTAASGGFFAAAAAPTTWYTDFRADIPRIDVPALILHGTGDRTLPIKNTARVFHKALPSAEYVEVEGAPHGLLWTHAEEVNTALLAFLAKAQEAQKQKLLTEVETYVLSIIPSGPLKAEIAQRLEDVFAGKNTDLEVLMEWLKTRPILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDKAVKLYRKLKREITFHGAKEISLSYSAGALASCMGLIYNRMGAVTTEVAFGLVCATCEQIADSQGGGSGCDCAWHLGELVWCTCGSGGGLEHHHHHH(SEQ ID NO: 32)

[0109] SAPNA_33 MPFITVGQENSTSIDLYYEDHGTGTPVVLIHGFPLSGHSWERQSAALLDAGARVITYDRRGFGQSSQPTTGYDYDTFAADLNTVLETLDLQDAVLVGFSMGTGEVARYVSSYGTARIAAVAFLASLEPFLLKTDDNPDGAAPQEFFDGIVAAVKADRYAFYTGFFNDFYNLDENLGTRISEEAVRNSWNTAASGGFFAAAAAPTTWYTDFRADIPRIDVPALILHGTGDRTLPIENTARVFHKALPSAEYVEVEGAPHGLLWTHAEEVNTALLAFLAKAQEAQKQKLLTEVETYVLSIIPSGPLKAEIAQRLEDVFAGKNTDLEVLMEWLKTRPILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDKAVKLYRKLKREITFHGAKEISLSYSAGALASCMGLIYNRMGAVTTEVAFGLVCATCEQIADSQGGGSGCDCAWHLGELVWCTCGSGGGLEHHHHHH(SEQ ID NO: 33)

[0110] SAPNA_34 MPFITVGQENSTSIDLYYEDHGTGTPVVLIHGFPLSGHSWERQSAALLDAGYRVITYDRRGFGQSSQPTTGYDYDTFAADLNTVLETLDLQDAVLVGFSMGTGEVARYVSSYGTARIAAVAFLASLEPFLLKTDDNPDGAAPQEFFDGIVAAVKADRYAFYTGFFNDFYNLDENLGTRISEEAVRNSWNTAASGGFFAAAAAPTTWYTDFRADIPRIDVPALILHGTGDRTLPIENTARVFHKALPSAEYVEVEGAPHGLLWTHAEEVNTALLAFLAKAQEAQKQKLLTEVETYVLSIIPSGPLKAEIAQRLEDVFAGKNTDLEVLMEWLKTRPILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDKAVKLYRKLKREITFHGAKEISLSYSAGALASCMGLIYNRMGAVTTEVAFGLVCATCEQIADSQGGGSGGGGCDCAWHLGELVWCTCGSGGGLEHHHHHH(SEQ ID NO: 34)

[0111] SAPNA_35 MPFITVGQENSTSIDLYYEDHGTGTPVVLIHGFPLSGHSWERQSAALLDAGYRVITYDRRGFGQSSQPTTGYDYDTFAADLNTVLETLDLQDAVLVGFSMGTGEVARYVSSYGTARIAAVAFLASLEPFLLKTDDNPDGAAPQEFFDGIVAAVKADRYAFYTGFFNDFYNLDENLGTRISEEAVRNSWNTAASGGFFAAAAAPTTWYTDFRADIPRIDVPALILHGTGDRTLPIENTARVFHKALPSAEYVEVEGAPHGLLWTHAEEVNTALLAFLAKAQEAQKQKLLTEVETYVLSIIPSGPLKAEIAQRLEDVFAGKNTDLEVLMEWLKTRPILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDKAVKLYRKLKREITFHGAKEISLSYSAGALASCMGLIYNRMGAVTTEVAFGLVCATCEQIADSQGGGSGGGSGGGSGGCDCAWHLGELVWCTCGSGGGLEHHHHHH(SEQ ID NO: 35)

[0112] SAPNA_36 MPFITVGQENSTSIDLYYEDHGTGTPVVLIHGFPLSGHSWERQSAALLDAGYRVITYDRRGFGQSSQPTTGYDYDTFAADLNTVLETLDLQDAVLVGFSMGTGEVARYVSSYGTARIAAVAFLASLEPFLLKTDDNPDGAAPQEFFDGIVAAVKADRYAFYTGFFNDFYNLDENLGTRISEEAVRNSWNTAASGGFFAAAAAPTTWYTDFRADIPRIDVPALILHGTGDRTLPIENTARVFHKALPSAEYVEVEGAPHGLLWTHAEEVNTALLAFLAKAQEAQKQKLLTEVETYVLSIIPSGPLKAEIAQRLEDVFAGKNTDLEVLMEWLKTRPILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDKAVKLYRKLKREITFHGAKEISLSYSAGALASCMGLIYNRMGAVTTEVAFGLVCATCEQIADSQGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGCDCAWHLGELVWCTCGSGGGLDHHHHHH(SEQ ID NO: 36)

[0113] SAPNA_37 MPFITVGQENSTSIDLYYEDHGTGTPVVLIHGFPLSGHSWERQSAALLDAGYRVITYDRRGFGQSSQPTTGYDYDTFAADLNTVLETLDLQDAVLVGFSMGTGEVARYVSSYGTARIAAVAFLASLEPFLLKTDDNPDGAAPQEFFDGIVAAVKADRYAFYTGFFNDFYNLDENLGTRISEEAVRNSWNTAASGGFFAAAAAPTTWYTDFRADIPRIDVPALILHGTGDRTLPIENTARVFHKALPSAEYVEVEGAPHGLLWTHAEEVNTALLAFLAKAQEAQKQKLLTEVETYVLSIIPSGPLKAEIAQRLEDVFAGRWGSGADCAWHLGELVWCTAGSGWEDLEVLMEWLKTRPILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDKAVKLYRKLKREITFHGAKEISLSYSAGALASCMGLIYNRMGAVTTEVAFGLVCATCEQIADSQHRSHRQLEHHHHHH(SEQ ID NO: 37)

[0114] SAPNA_38 MPFITVGQENSTSIDLYYEDHGTGTPVVLIHGFPLSGHSWERQSAALLDAGYRVITYDRRGFGQSSQPTTGYDYDTFAADLNTVLETLDLQDAVLVGFSMGTGEVARYVSSYGTARIAAVAFLASLEPFLLKTDDNPDGAAPQEFFDGIVAAVKADRYAFYTGFFNDFYNLDENLGTRISEEAVRNSWNTAASGGFFAAAAAPTTWYTDFRADIPRIDVPALILHGTGDRTLPIENTARVFHKALPSAEYVEVEGAPHGLLWTHAEEVNTALLAFLAKAQEAQKQKLLTEVETYVLSIIPSGPLKAEIAQRLEDVFAGGGRWGADCAWHLGELVWCTAGWEGGDLEVLMEWLKTRPILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDKAVKLYRKLKREITFHGAKEISLSYSAGALASCMGLIYNRMGAVTTEVAFGLVCATCEQIADSQHRSHRQLEHHHHHH(SEQ ID NO: 38)

[0115] SAPNA_39 MPFITVGQENSTSIDLYYEDHGTGTPVVLIHGFPLSGHSWERQSAALLDAGYRVITYDRRGFGQSSQPTTGYDYDTFAADLNTVLETLDLQDAVLVGFSMGTGEVARYVSSYGTARIAAVAFLASLEPFLLKTDDNPDGAAPQEFFDGIVAAVKADRYAFYTGFFNDFYNLDENLGTRISEEAVRNSWNTAASGGFFAAAAAPTTWYTDFRADIPRIDVPALILHGTGDRTLPIENTARVFHKALPSAEYVEVEGAPHGLLWTHAEEVNTALLAFLAKAQEAQKQKLLTEVETYVLSIIPSGPLKAEIAQRLEDVFAGGADCAWHLGELVWCTAGDLEVLMEWLKTRPILSPLTKGILGFVFTLTVPSERGLQRRRFVQNALNGNGDPNNMDKAVKLYRKLKREITFHGAKEISLSYSAGALASCMGLIYNRMGAVTTEVAFGLVCATCEQIADSQHRSHRQLEHHHHHH(SEQ ID NO:39)

[0116] References cited in Example 1: 1.Y.T.Lai,D.Cascio,T.O.Yeates,Structure of a 16-nm cage designed by using protein oligomers. Science 336,1129(2012). 2.Y.T.Lai,K.L.Tsai,M.R.Sawaya,F.J.Asturias,T.O.Yeates,Structure and flexibility of nanoscale protein cages designed by symmetric self-assembly. J.Am.Chem.Soc.135,7738-7743(2013). 3. J.E. Padilla, C. Colovos, T.O. Yeates, Nanohedra: using symmetry to design self-assembling protein cages, layers, crystals, and filaments. Proc. Natl. Acad. Sci. U.S.A. 98, 2217 - 2221 (2001). 4. Y.T. Lai et al., Designing and defining dynamic protein cage nanoassemblies in solution. Sci Adv 2, e1501855 (2016). 5. W.L. DeLano, M.H. Ultsch, A.M. de Vos, J.A. Wells, Convergent solutions to binding at a protein-protein interface. Science 287, 1279 - 1283 (2000). 6. Y. Gong, L. Zhang, J. Li, S. Feng, H. Deng, Development of the Double Cyclic Peptide Ligand for Antibody Purification and Protein Detection. Bioconjug Chem 27, 1569 - 1573 (2016). 7. H.J. Kang et al., Developing an antibody-binding protein cage as a molecular recognition drug modular nanoplatform. Biomaterials 33, 5423 - 5430 (2012). 8.C.D.Putnam,M.Hammel,G.L.Hura,J.A.Tainer,X-ray solution scattering (SAXS) combined with crystallography and computation:defining accurate macromolecular structures, conformations and assemblies in solution. Q.Rev.Biophys.40,191-285(2007).

[0117] Example 2 Materials and Methods Dynamic light scattering (DLS) analysis of SAPNA binding to antibodies Samples were diluted in PBS pH 7.4 and subjected to a DynaPro Plate Reader III. The DLS acquisition time was 5 seconds, and 5 acquisitions were made per sample. The temperature was 20°C.

[0118] Primary human T cell proliferation assay Primary human pan-T cells (including not only CD4 + and CD8 + T cells but also some gamma / delta T cell subsets) isolated from peripheral blood (PB) mononuclear cells (MNCs) of random donors were seeded in 96-well plates. Triplicate wells were treated on day 1 with soluble SAPNA loaded with anti-CD3 / anti-CD28 antibodies at various ratios or with competing techniques. Fresh, foreign-free medium containing exogenous recombinant human IL-2 was added every 3 - 4 days. T cells were stained using: Live / Dead staining, anti-CD3 antibody, anti-CD4 antibody, anti-CD8 antibody, anti-CCR7 antibody, anti-CD45RA antibody, and anti-CD95 antibody. T cell differentiation was evaluated by flow cytometry using the following T cell subset identification staining scheme based on the literature: T CM (CCR7+ CD45RA-), T EM (CCR7- CD45RA-), T EMRA (CCR7- CD45RA+), T SCM (CD45RA+ CCR7+→CD95+), Tnaive (CD45RA+ CCR7+→CD95-). Samples were run on an LSR Fortessa X20 Analyzer flow cytometer and data were analyzed using FlowJo 10.6.1.

[0119] Isolation of CD8+ T cells using SAPNA conjugated with magnetic beads. Primary human pan T cells expanded for 14 days were seeded into 96-well plates. First, SAPNA was incubated with magnetic Ni-NTA (mag) beads at room temperature for 5 minutes, then rabbit anti-CD8 antibody was added and incubated for an additional 20 minutes. A control without SAPNA was prepared from the mixture. This control and mag-SAPNA-CD8 beads were added to the wells in triplicate, and the plate was returned to an incubator at 37 °C and 5% CO2 for 1 hour. The cell-bead solution was resuspended and placed on a magnet for 2 minutes. The components bound to the beads were attracted to the magnet, while the supernatant containing the cell suspension was transferred to a new plate for flow cytometry staining. The cells were stained and evaluated as in the "Primary human T cell proliferation assay" column.

[0120] Immunofluorescence microscopy HeLa cells were cultured in an incubator at 37°C and 5% CO2 and seeded on a coverslip. The cells were fixed in 4% paraformaldehyde + 0.2% Triton X-100 in PBS. Then they were permeabilized in PBS + 0.5% Triton X-100 (PBST) for 30 minutes. The permeabilized cells were blocked in PBST (+5% FBS) for 30 minutes. Control staining was performed using rabbit-anti-ROBO1 antibody and goat-anti-rabbit-A488 secondary antibody. For the experimental group, SAPNA was chemically labeled with Alexa Fluor®-488 and incubated with rabbit-anti-ROBO1 for at least 30 minutes. Then the loaded SAPNA molecules were incubated in PBST (+5% FBS) at room temperature for 1 hour. The coverslip was washed with PBST and then with PBS only. The coverslip was mounted using DNA staining, antifade mounting media containing DAPI.

[0121] Results SAPNA molecules have 12 possible antibody Fc binding sites and can carry any human or rabbit IgG (Figure 7). An example of the ability of SAPNA to bind to rabbit-anti-ROBO1 antibody is shown (Figure 8). We hypothesized that SAPNA could force the physical proximity of cell surface receptors, a process required for T cell activation and proliferation (Figure 9).

[0122] To evaluate and investigate SAPNA, T cell proliferation for 14 days was performed using clinically relevant donor-derived peripheral blood T cells (Figure 10). These data show that SAPNA produces a T cell product that contains the highest number of cytotoxic CD8 + T cells, cells that were intended to be engineered with a chimeric antigen receptor (CAR) that is directed towards cancer cells. cytotoxic CD8 + T cells (Figure 4 - upper left panel). SAPNA, against competing technologies, CD4 +Ranks second in performance regarding T cell proliferation (lower left panel of Figure 10). These are important elements of the final CAR T cell product 1 . Furthermore, it has been found that T cell subsets with a more stem-like phenotype, such as memory T stem (T SCM ) cells, have the highest long-term antitumor effect in vivo. Therefore, increasing the number of these present in the final expanded CAR T cell product is of great therapeutic value (Turtle, C.J. et al. CD19 CAR-T cells of defined CD4+:CD8+ composition in adult B cell ALL patients. J. Clin. Invest. 126, 2123-2138 (2016); Gattinoni, L. et al. Wnt signaling arrests effector T cell differentiation and generates CD8+ memory stem cells. Nat. Med. 15, 808-813 (2009)). By the SAPNA technology, during expansion, the largest number of CD4 + and CD8 + T SCM cells are produced (upper right and lower right figures of Figure 10). Collectively, these data indicate that SAPNA is technically excellent and has the ability to generate a larger number of CAR T cells with more effective antitumor activity

[0123] We hypothesized that the SAPNA molecule binds to magnetic nickel beads and also binds and presents antibodies due to the 12 his tags on the SAPNA molecule (one per monomer). Through this dual action, it was demonstrated that it is possible to isolate (or negatively select) a cell population with a specific cell surface marker, such as CD8, from a mixed group of cells using SAPNA (Figure 11).

[0124] Immunofluorescence microscopy was used to evaluate whether SAPNA could target the surface of cancer cells. After verifying that chemically labeled SAPNA with Alexa Fluor®-488 had little effect on its structure using small-angle X-ray scattering (SAXS) (Figure 12), the same rabbit-anti-ROBO1 antibody used for DLS in Figure 8 was loaded onto this labeled nanoparticle, directing the labeled nanoparticle to the surface of HeLa cervical cancer cells. The 488-labeled SAPNA specifically targeted the surface of the cells (Figure 13).

[0125] Although the invention has been described with reference to specific embodiments thereof, it should be understood by those skilled in the art that various changes can be made and equivalents can be substituted without departing from the true spirit and scope of the invention. Furthermore, numerous modifications may be made to adapt a particular situation, material, composition of matter, process, process step, to the objectives, spirit, and scope of the present invention. All such modifications are intended to be within the scope of the appended claims.

[0126] All of the cited references are hereby specifically incorporated by reference in their entirety.

Claims

1. A protein cage polypeptide or scaffold protein that is useful for or capable of forming a hollow tetrahedral pyramidal structure, wherein the protein cage polypeptide or scaffold protein is capable of specifically binding to an antibody or portion thereof.

2. 2. The protein cage polypeptide or scaffold protein of claim 1, comprising an amino acid sequence having the following structure: Polypeptide 1 - AHL - Polypeptide 2 - Insert A - Polypeptide 3 - Insert B - Polypeptide 4 (chemical structure I), where AHL is an "alpha helical linker" and Insert A and / or Insert B are each independently capable of specifically binding to said antibody or portion thereof.

3. 3. The protein cage polypeptide or scaffold protein of claim 2, wherein Insertion A has a length of about 17 amino acids to about 25 amino acids and / or Insertion B has a length of about 28 amino acids to about 85 amino acids.

4. 4. The protein cage polypeptide or scaffold protein of claim 2 or 3, wherein Insertion A and / or Insertion B each independently comprise the amino acid sequence DCAWHLGELVWCT (SEQ ID NO: 41) or GCDCAWHLGELVWCTCG (SEQ ID NO: 42).

5. 5. The protein cage polypeptide of claim 2, wherein the protein cage polypeptide comprises an amino acid sequence having the following structure: Polypeptide 1 - AHL - Polypeptide 2 - Insert A - Polypeptide 3 - Insert B - Polypeptide 4 (chemical structure I), where AHL is an "alpha helical linker", Insert A has a length of about 17 amino acids to about 25 amino acids and comprises the amino acid sequence DCAWHLGELVWCT (SEQ ID NO: 41) or GCDCAWHLGELVWCTCG (SEQ ID NO: 42), and Insert B has a length of about 28 amino acids to about 85 amino acids and comprises the amino acid sequence DCAWHLGELVWCT (SEQ ID NO: 41) or GCDCAWHLGELVWCTCG (SEQ ID NO: 42).

6. 6. The protein cage polypeptide of claim 2, wherein polypeptide 1 comprises an amino acid sequence with at least about 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% amino acid identity to the amino acid sequence from the N-terminus to the AQEAQKQK sequence of any one of SEQ ID NOs: 1-40.

7. 7. The protein cage polypeptide of claim 2, wherein said polypeptide 1 comprises an amino acid sequence comprising the following: YGTAR, TDD, LXENLGTR, IDV, TGXRT, and / or SA, where X is any charged amino acid residue.

8. 8. The protein cage polypeptide of claim 2, wherein polypeptide 1 comprises from about 278 to about 303 amino acid residues.

9. 9. The protein cage polypeptide of claim 2, wherein the AHL comprises an amino acid sequence comprising AQEAQKQK.

10. 10. The protein cage polypeptide of claim 2, wherein the AHL comprises about 5, 6, 7, 8, 9, 10 or 11 amino acid residues.

11. 11. The protein cage polypeptide of any one of claims 2 to 10, wherein Polypeptide 2 comprises an amino acid sequence with at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% amino acid identity to the amino acid sequence from the C-terminus of the AQEAQKQK sequence to the N-terminus of Insert A in any one of SEQ ID NOs: 1-40.

12. 12. The protein cage polypeptide of claim 2, wherein said polypeptide 2 comprises an amino acid sequence comprising: LTEVETYVLS (SEQ ID NO:43).

13. 13. The protein cage polypeptide of any one of claims 2-12, wherein polypeptide 2 comprises from about 30 to about 36 amino acid residues, and in some embodiments polypeptide 2 comprises about 33 amino acid residues.

14. 14. The protein cage polypeptide of any one of claims 2 to 13, wherein Polypeptide 3 comprises an amino acid sequence with at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% amino acid identity to the amino acid sequence from the C-terminus of Insert A to the N-terminus of Insert B of any one of SEQ ID NOs: 1-40.

15. 15. The protein cage polypeptide of claim 2, wherein the polypeptide 3 comprises an amino acid sequence comprising the following: FTLTVPSERGLQR (SEQ ID NO: 44) and / or CATCEQIAD (SEQ ID NO: 45).

16. 16. The protein cage polypeptide of claim 2, wherein polypeptide 3 comprises from about 110 to about 130 amino acid residues.

17. 17. The protein cage polypeptide of claim 2, wherein polypeptide 3 comprises approximately 121 amino acid residues.

18. 18. The protein cage polypeptide of any one of claims 2 to 17, wherein polypeptide 4 comprises an amino acid sequence with at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% amino acid identity to an amino acid sequence from the C-terminus of Insert B of any one of SEQ ID NOs: 1-40.

19. 19. The protein cage polypeptide of claim 2, wherein polypeptide 4 comprises an amino acid sequence comprising the following: EHHHHHH.

20. 20. The protein cage polypeptide of claim 2, wherein polypeptide 4 comprises from about 5 to about 13 amino acid residues.

21. 21. The protein cage polypeptide of any one of claims 2 to 20, wherein polypeptide 4 comprises about 8 amino acid residues.

22. 22. The protein cage polypeptide of any one of claims 2 to 21, wherein the protein cage polypeptide comprises an amino acid sequence with at least about 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% amino acid identity to any one of SEQ ID NOs: 1-40.

23. 23. The protein cage polypeptide of claim 22, wherein the protein cage polypeptide comprises an amino acid sequence that includes any one or more, or all, of the consecutive or distinct amino acid residues marked with an asterisk in FIG.

6.

24. 24. The protein cage polypeptide of claim 22 or 23, wherein the protein cage polypeptide comprises an amino acid sequence that includes any one or more, or all, of the stretches of charged amino acids at the positions corresponding to those marked with a "#" in FIG.

25. 25. The protein cage polypeptide of any one of claims 1 to 24, wherein the protein cage polypeptide comprises a polypeptide of about 400 to about 700 amino acid residues.

26. 26. The protein cage polypeptide of claim 25, wherein the protein cage polypeptide comprises a polypeptide of about 450 to about 650 amino acid residues.

27. 27. The protein cage polypeptide of any one of claims 1 to 26, wherein the antibody is an IgG antibody.

28. 28. The protein cage polypeptide of any one of claims 1 to 27, wherein the portion of an antibody is an Fc region of an IgG antibody.

29. 29. The protein cage polypeptide of claim 27 or 28, wherein the IgG antibody is a human IgG antibody.

30. 29. The protein cage polypeptide of claim 27 or 28, which is part of the IgG antibody of an Fc chimeric protein.

31. The binding affinity K of the protein cage polypeptide or scaffold protein to the antibody or portion thereof. ａ is 10 ７ M －１ , 10 ８ M －１ , or 10 ９ M －１ 31. A protein cage polypeptide or scaffold protein according to any one of claims 1 to 30, wherein

32. 32. The protein cage polypeptide or scaffold protein of any one of claims 1 to 31, wherein the protein cage polypeptide or scaffold protein specifically binds to the antibody or portion thereof, or to any chimeric protein, molecule or compound comprising the antibody or portion thereof, and the antibody or portion thereof specifically binds to a pathogenic biological agent or portion thereof.

33. A hollow tetrahedral pyramidal structure comprising 12 protein cage polypeptides according to one of claims 1 to 32 constructed as a tetrahedral pyramidal structure.

34. A chimeric protein assembly, an "antibody-modified self-assembling protein nanoparticle" (SAPNA), comprising: (a) one or more antibodies; and (b) a protein cage polypeptide that provides a scaffold on which the antibodies are arranged, wherein the one or more antibodies bind to insert A and / or insert B of the protein cage polypeptide.

35. 35. The SAPNA of claim 34, wherein the antibody is capable of specifically binding to a pathogenic biological agent or a portion thereof.

36. 33. A structure of an "antibody-modified self-assembling protein nanoparticle" (SAPNA), comprising: (1) one protein cage polypeptide or scaffold protein according to any one of claims 1 to 32, or a plurality of such protein cage polypeptides or scaffold proteins assembled into a three-dimensional assembly; (2) optionally, one or more human or rabbit IgG antibodies; (3) optionally, an IgG binding loop; and (4) optionally, a cargo of interest that is constrained or encapsulated by the three-dimensional assembly when the plurality of polypeptides or scaffold proteins (or engineered protein cage proteins (PCs)) are assembled into the three-dimensional assembly.

37. 37. The SAPNA structure of claim 36, wherein the three-dimensional aggregate is a tetrahedral pyramid.

38. A method for detecting or isolating a pathogenic biological agent or a portion thereof, comprising: (a) providing an "antibody-modified self-assembling protein nanoparticle" (SAPNA), wherein the antibody is capable of specifically binding to a pathogenic biological agent or a portion thereof; (b) contacting the SAPNA with a sample containing the pathogenic biological agent or a portion thereof such that the SAPNA binds to the pathogenic biological agent or a portion thereof; (c) detecting the SAPNA, the pathogenic biological agent or a portion thereof, and / or separating the SAPNA that binds the pathogenic biological agent or a portion thereof from the remainder of the sample via detection; and (d) determining the abundance of the pathogenic biological agent or a portion thereof.

Images