Nanoparticle platform for antibody and vaccine delivery

Abstract

To provide a nanoparticle subunit fusion protein, vaccines comprising nanoparticles and pharmaceutical compositions.SOLUTION: A fusion protein comprises: (1) a nanocage monomer; and (2) an antibody fragment comprising a single chain Fc (scFc). Preferably, the nanocage monomer is ferritin, more preferably, the fusion protein comprises a linker between the nanocage monomer and the scFc.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to nanoparticles. In particular, the present invention relates to nanoparticle subunit fusion proteins, vaccines comprising nanoparticles, and related compositions and methods.

Background Art

[0002] Nanoparticles have contributed to the progress of various academic fields. The use of nanoparticles enables targeted delivery; also, it enables the design of regular microarrays, sustained release, and a microenvironment caged for catalytic processes.

[0003] Nanoparticles can be synthesized from various materials such as polysaccharides, liposomes, or inorganic nanomaterials. However, these delivery platforms are associated with important limitations in fusing biomolecules, such as reduced protein activity due to harsh manufacturing conditions, undesirable degradation products, and low encapsulation efficiency. Inappropriate conditions or formulations can inhibit the desired function because they can have a destructive effect on the structure. For example, the trimeric gp120 glycoprotein (the most heavily glycosylated known glycoprotein) and antibody domains have a narrow buffer range for obtaining optimal activity.

[0004] Protein nanoparticles are attractive alternatives to the above technologies; their constituent units are amino acids, and genetic engineering enables exquisite control of composition, molecular weight, and function. The self-assembly of proteins is the optimal method for producing nanoparticles containing highly sensitive metastable proteins. In fact, self-assembled nanoparticles can ensure the generation of nanocapsules that are formed by non-covalent interactions under physiological conditions, are often uniform and symmetric. Self-assembling protein nanoparticles have three different surfaces that can all be finely tuned to convey additional functions: an external surface, an internal surface, and an inter-subunit surface.

[0005] Numerous reports exist regarding the fusion of peptides to self - assembling proteins. Peptides that bind titanium or gold can be used to selectively attach nanoparticles to these metals. However, biological interactions often require tertiary and quaternary structures, and thus folded proteins generally confer more extended functions than peptides. Furthermore, it is estimated that 50% of human proteins are glycosylated, and these post - translational modifications play important roles in maintaining protein structure, transmitting stability, and providing function. There are only a few examples of genetic fusions of glycoproteins and protein nanoparticles.

[0006] Nanocages decorated on the surface with antigens for use in vaccines are described, for example, in U.S. Patent No. 8,546,337, U.S. Patent Application Publication No. 2015 / 0110825, International Patent Application Publication No. 2016 / 109792, Kanekiyo et al. (Nature, 2013, 499:102 - 106), and Sliepen et al. (Retrovirology, 2015, 12:82). Previously, the inventors have shown that it is possible to genetically fuse cargo outside of a self - assembling lumazine synthase protein for multimeric (60 - mer) presentation (Jardine et al., Science, May 10, 2013; 340(6133):711 - 6).

[0007] International Patent Application Publication No. 2010 / 0222501 describes a method for producing composite nanoparticles capable of attaching moieties such as antibodies to organic groups protruding from the surface of the nanoparticles.

[0008] Choe et al. (Materials 2016, 9(12):994) provide an overview of several methods for isolating and targeting antibodies using smart biomaterials that mimic the binding of Fc receptors to antibodies. Fc-binding peptides are applied, for example, to localize antibodies on nanomaterials and to extend the half-life of proteins in serum. This overview presents the recent development of Fc-binding peptides and examines their binding properties and diverse applicability.

[0009] Khoshnejad et al. (Bioconjugate Chem., 2016, 27(3):628-637) describe a study in which monoclonal antibodies against ICAM-1 and PECAM-1 or their single-chain antigen-binding fragments (scFv) were conjugated to ferritin nanoparticles. Ferritin nanoparticles are suggested to offer a platform for targeting endothelial adhesion molecules with a carrier in the 20 nm size range.

[0010] Kang et al. (Fourth International Conference on Multifunctional, Hybrid and Nanomaterials, Poster Program, 2015, P1.048) describe a chimeric protein nanocage of trastuzumab and an scFv variant of human ferritin.

[0011] Carter et al. (Science., 1992, 256(5053):105-7) have shown that the B cell response to an antigen is enhanced when CD19 is co-internalized with the antigen.

[0012] There is a need to develop products, compositions and / or methods that provide useful alternatives to the public. SUMMARY OF THE INVENTION

[0013] According to one embodiment, A nanocage monomer, and a fusion protein comprising an antibody or a fragment thereof linked to the nanocage monomer, wherein the antibody or the fragment thereof comprises a first component of a binding pair, and a plurality of the fusion proteins self-assemble to form a nanocage, and a plurality of the antibodies or fragments thereof decorate the outer surface of the nanocage, whereby the first component of the binding pair is exposed to interact with a second component of the binding pair. A fusion protein.

[0014] In one embodiment, the first component of the binding pair is the Fc portion of an antibody or a fragment thereof, and the second component of the binding pair is an Fc receptor.

[0015] In one embodiment, the first component of the binding pair is an antigen-binding epitope, and the second component of the binding pair is an antigen.

[0016] In one embodiment, the nanocage comprises about 3 to about 100 nanocage monomers, such as 24 monomers or 60 monomers.

[0017] In one embodiment, the nanocage monomer is selected from ferritin, encapsulin, SOR, lumazine synthase, pyruvate dehydrogenase, carboxysome, bolt protein, GroEL, heat shock protein, E2P, MS2 coat protein, fragments thereof, and variants thereof.

[0018] In one embodiment, the fusion protein further comprises a linker between the nanocage monomer and the antibody or a fragment thereof.

[0019] In one embodiment, the linker is flexible or rigid and comprises about 1 to about 30 amino acid residues.

[0020] In one embodiment, the linker comprises about 8 to about 16 amino acid residues.

[0021] In one embodiment, the linker comprises a GGS repeat.

[0022] In one embodiment, the linker comprises four GGS repeats.

[0023] In one embodiment, the fusion protein further comprises the antigen.

[0024] In one embodiment, the antigen comprises a repeat domain.

[0025] In one embodiment, the antigen is a malaria antigen.

[0026] In one embodiment, the antigen is a fragment of the malaria CSP protein.

[0027] In one embodiment, the antigen is a fragment of the NANP repeat domain of the malaria CSP protein.

[0028] In one embodiment, the antigen comprises 5.5 NANP repeats.

[0029] In one embodiment, the antigen is NPNANPNANPNANPNANPNANP.

[0030] In one embodiment, the fusion protein is an Fc domain.

[0031] In one embodiment, the antibody or fragment thereof is specific for a repeat domain.

[0032] In one embodiment, the antibody or fragment thereof is specific for a malaria antigen.

[0033] In one embodiment, the antibody or fragment thereof is specific for the malaria CSP protein.

[0034] In one embodiment, the antibody or fragment thereof is specific for the NANP repeat domain of the malaria CSP protein.

[0035] In one embodiment, the antibody or fragment thereof comprises a sequence having at least 90% sequence identity to the following sequence or a fragment thereof:

Chemical formula

[0036] In one embodiment, the antibody or fragment thereof comprises the following sequence:

Chemical formula

[0037] In one embodiment, the antibody or fragment thereof consists of the following sequence:

Chemical formula

[0038] In one embodiment, the antibody or fragment thereof is specific for a tumor antigen.

[0039] In one embodiment, the antibody or fragment thereof is specific for a self-antigen.

[0040] In one embodiment, the antibody or fragment thereof is specific for CD19, CD22, CD79, BCMA, or CD20.

[0041] In one embodiment, the antibody or fragment thereof is specific for a target organ.

[0042] In one embodiment, the antibody or fragment thereof comprises the heavy chain and / or light chain of a Fab fragment.

[0043] In one embodiment, the antibody or fragment thereof comprises a scFv.

[0044] In one embodiment, the fusion protein further comprises a Fab light chain and / or a Fab heavy chain.

[0045] In one embodiment, the fusion protein is associated with separately produced Fab light chain and / or Fab heavy chain.

[0046] In one embodiment, the fusion protein further comprises a detectable moiety.

[0047] In one embodiment, the detectable moiety is a fluorescent protein such as GFP, EGFP, amethystin, and / or a flavin-based fluorescent protein such as LOV protein like iLOV.

[0048] According to one embodiment, a nanocage comprising at least one fusion protein described herein is provided.

[0049] In one embodiment, each nanocage monomer comprises the fusion protein described herein.

[0050] In one embodiment, about 20% to about 80% of the nanocage monomers comprise the fusion protein described herein.

[0051] In one embodiment, the nanocage is multivalent.

[0052] In one embodiment, the nanocage carries cargo molecules such as pharmaceuticals, diagnostic agents, and / or contrast agents.

[0053] In one embodiment, the cargo molecule is a protein and is fused to the fusion protein such that the cargo molecule is encapsulated within the nanocage.

[0054] In one embodiment, the cargo molecule is a fluorescent protein such as GFP, EGFP, amethystin, and / or a flavin-based fluorescent protein such as LOV protein like iLOV.

[0055] In one embodiment, the cargo molecule is not fused to the fusion protein and is encapsulated in the nanocage.

[0056] In one embodiment, the cargo molecule provides a T cell epitope by being contained inside, but may not provide a B cell epitope in some cases.

[0057] In one embodiment, the cargo molecule is fused to the fusion protein and provides a T cell epitope by being contained inside, but may not provide a B cell epitope in some cases.

[0058] In one embodiment, the cargo molecule is a small molecule, a radioisotope, or a magnetic particle.

[0059] In one embodiment, the fusion protein further contains an antigen on the surface.

[0060] In one embodiment, the antigen is expressed as a fusion protein with the nanocage monomer.

[0061] According to one embodiment, a vaccine comprising the nanocage described herein is provided.

[0062] According to one embodiment, a nucleic acid molecule encoding the fusion protein described herein is provided.

[0063] According to one embodiment, a vector containing the nucleic acid molecule described herein is provided.

[0064] According to one embodiment, a host cell containing the vector described in c herein and producing the fusion protein described herein is provided.

[0065] According to one embodiment, a method of immunizing a subject is provided, comprising administering the nanocage described herein or the vaccine described herein.

[0066] According to one embodiment, a method of treating and / or preventing a disease or condition is provided, comprising administering the nanocage described herein or the vaccine described herein.

[0067] In one embodiment, the disease or condition is cancer, HIV, malaria, or an autoimmune disease.

[0068] According to one embodiment, a diagnostic imaging method is provided, comprising administering the nanocage described herein to a subject, tissue, or sample, and imaging the subject, tissue, or sample, wherein the nanocage comprises a diagnostic label such as a fluorescent protein or a magnetic imaging moiety.

[0069] According to one embodiment, the use of the fusion protein described herein or the nanocage described herein as a research tool such as FACS or ELISA is provided.

[0070] The novel features of the invention will become apparent to those skilled in the art upon examination of the following detailed description of the invention. However, while the detailed description and the specific examples presented herein illustrate certain embodiments of the invention, it is to be understood that the invention is not limited thereto, as various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from the detailed description and the claims that follow, and the examples are provided for illustrative purposes only.

[0071] The invention will be further understood from the following description when taken in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0072]

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Mode for Carrying Out the Invention

[0073] Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes V (ISBN 0-19-854287-9), published by Oxford University Press in 1994; Kendrew et al. (eds.), The Encyclopedia of Molecular Biology (ISBN 0-632-02182-9), published by Blackwell Science Ltd. in 1994; and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference (ISBN 1-56081-569-8), published by VCH Publishers, Inc. in 1995. All methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, but typical materials and methods are described herein. The following terms are used in the description and claims of the present invention.

[0074] Also, it should be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting. The understanding of the described embodiments is facilitated by reference to many patent applications, patents, and publications herein. Each of these references is hereby incorporated by reference in its entirety.

[0075] In understanding the scope of the present application, the articles "a", "an", "the", and "said" shall be taken to mean that there is one or more elements. Further, the term "comprising" as used herein and its derivatives are intended to be open-ended terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but do not exclude the presence of other features, elements, components, groups, integers, and / or steps not recited. The above also applies to terms having similar meanings such as the terms "including", "having", and their derivatives.

[0076] Also, all embodiments described as "comprising" a particular component may also "consist of" or "consist essentially of" that component. "Consist of" has a closed or restrictive meaning, and "consist essentially of" means including the stated components, but excluding other components such as materials present as impurities, inevitable materials resulting from the processes used to provide the components, and components added for purposes other than achieving the technical effects of the present invention. For example, a composition defined using the phrase "consisting essentially of" includes known acceptable additives, excipients, diluents, carriers, etc. Typically, a composition consisting essentially of a set of components will contain non-specified component(s) in a proportion of less than 5% by weight, typically less than 3% by weight, more typically less than 1% by weight, and even more typically less than 0.1% by weight.

[0077] It will be understood that any component defined as being included herein may, by disclaimer or negative limitation, be explicitly excluded from the claimed invention.

[0078] Furthermore, all ranges recited herein, whether or not expressly stated, include all endpoints and points of intermediate ranges within the recited ranges.

[0079] As used herein, terms of degree such as “substantially,” “about,” and “approximately” mean a reasonable amount of deviation of the modified term such that the end result is not materially changed. These terms of degree should be construed to include a deviation of at least ±5% of the modified term, provided that such deviation does not negate the meaning of the term being modified.

[0080] Furthermore, all base sizes or amino acid sizes given for nucleic acids or polypeptides, and all molecular weight or molecular mass values are approximate values, and it goes without saying that they are provided for illustrative purposes. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The abbreviation “e.g.” is derived from the Latin exempli gratia and is used herein to indicate non-limiting examples. Thus, the abbreviation “e.g.” is synonymous with the phrase “for example.” The word “or” shall be construed to include “and” unless the context clearly indicates otherwise.

[0081] The terms "protein nanoparticle" and "nanocage" are used interchangeably herein and refer to a multi-subunit, protein-based polyhedral-shaped structure. The subunit or nanocage monomer is composed of a protein or polypeptide (e.g., a glycosylated polypeptide), and in some cases may also be composed of one or more of the following: nucleic acids, families of missing molecules, organic compounds, and inorganic compounds. Non-limiting examples of protein nanoparticles include ferritin nanoparticles (see, e.g., Zhang, Y. Int. J. Mol. Sci. (International Journal of Molecular Sciences), 12:5406-5421, 2011, which is incorporated herein by reference), encapsulin nanoparticles (see, e.g., Sutter et al., Nature Struct, and Mol. Biol. (Nature Structural and Molecular Biology), 15:939-947, 2008, which is incorporated herein by reference), sulfur oxygenase reductase (SOR) nanoparticles (see, e.g., Urich et al., Science, 311:996-1000, 2006, which is incorporated herein by reference), lumazine synthase nanoparticles (see, e.g., Zhang et al., J. Mol. Biol. (Journal of Molecular Biology), 306:1099-1114, 2001), or pyruvate dehydrogenase nanoparticles (see, e.g., Izard et al., PNAS (Proceedings of the National Academy of Sciences of the United States of America) 96:1240-1245, 1999, which is incorporated herein by reference). Ferritin, encapsulin, SOR, lumazine synthase, and pyruvate dehydrogenase are monomeric proteins that self-assemble into globular protein complexes composed of 24, 60, 24, 60, and 60 protein subunits, respectively, in some cases. Carboxysomes, vault proteins, GroEL, heat shock proteins, E2P, and MS2 coat proteins also generate nanocages contemplated for use herein. In addition, fully or partially synthetic self-assembling monomers are also contemplated for use herein.

[0082] A "vaccine" is a pharmaceutical composition that induces a prophylactic or therapeutic immune response in a subject. In some cases, the immune response is a defensive immune response. Typically, a vaccine induces an antigen-specific immune response against a pathogen, such as an antigen of a viral pathogen, or a cellular component correlated with a diseased state. A vaccine can include polynucleotides (such as nucleic acids encoding the disclosed antigens), peptides or polypeptides (such as the disclosed antigens), viruses, cells, or one or more cellular components. In one specific non-limiting example, the vaccine induces an immune response that reduces the severity of symptoms associated with malaria infection and / or reduces the parasite burden as compared to a control. In another non-limiting example, the vaccine induces an immune response that reduces and / or prevents malaria infection as compared to a control.

[0083] As used herein, the term "antibody," also referred to in the art as "immunoglobulin" (Ig), refers to a protein constructed from paired heavy and light polypeptide chains; there are various Ig isotypes, such as IgG, including IgA, IgD, IgE, IgG1, IgG2, IgG3, IgG4, and IgM. It will be understood that an antibody can be derived from any species, including humans, mice, rats, monkeys, llamas, or sharks. When an antibody is correctly folded, each chain is folded into several distinct globular domains that are joined by additional linear polypeptide sequences. For example, an immunoglobulin light chain is folded into a variable (V L ) domain and a constant (CL) domain, and a heavy chain is folded into a variable (V H ) domain and three constant (C H , C H2 , C H3 ) domains. The interaction of the variable domains (V H and V L ) of the heavy and light chains forms an antigen-binding region (Fv). Each domain has a well-established structure well known to those skilled in the art.

[0084] The variable regions of the light and heavy chains can exhibit considerable sequence diversity among antibodies because they are involved in binding to the target antigen. The constant regions have less sequence diversity, are involved in binding to some native proteins, and induce important immunological events. The variable region of an antibody contains the antigen-binding determinants of the molecule and thus determines the specificity of the antibody for its target antigen. Most of the sequence variability occurs in three segments each for the variable heavy and light chains, i.e., six hypervariable regions; the hypervariable regions combine to form the antigen-binding site and contribute to the binding and recognition of antigenic determinants. The specificity and affinity of an antibody for an antigen are determined by the structure of the hypervariable regions, as well as the size, shape, and chemical nature of the surface presented by the antigen.

[0085] As used herein, the term “antibody fragment” can include any suitable antigen-binding antibody fragment known in the art. An antibody fragment may be a naturally occurring antibody fragment or may be obtained by manipulating a naturally occurring antibody or by using recombinant methods. For example, antibody fragments include, but are not limited to, Fv, single-chain Fv (scFv; a molecule consisting of V L linked to V H by a peptide linker), Fc, single-chain Fc, Fab, F(ab’)2, single-domain antibody (sdAb; a fragment consisting of a single V L or V H ), and those presenting multivalency of any of these.

[0086] As used herein, the term “synthetic antibody” means an antibody produced using recombinant DNA technology. This term should also be construed to mean a DNA molecule encoding such an antibody, a DNA molecule that expresses the antibody protein, or an antibody produced by the synthesis of an amino acid sequence that specifies such an antibody, wherein the DNA or amino acid sequence was obtained using synthetic DNA or amino acid sequence techniques that are available and well known in the art.

[0087] The term "epitope" refers to an antigenic determinant. An epitope is a specific chemical group or peptide sequence on a molecule that is antigenic, i.e., that elicits a specific immune response. Antibodies specifically bind to particular antigenic epitopes, for example, on a polypeptide. Epitopes can be formed from both contiguous and non-contiguous amino acids juxtaposed by the three-dimensional folding of a protein. Epitopes formed from contiguous amino acids are usually retained when exposed to a denaturing solvent, whereas epitopes formed by three-dimensional folding are usually lost upon treatment with a denaturing solvent. Epitopes typically contain at least 3, more usually at least 5, about 9, about 11, or about 8 to about 12 amino acids in a unique spatial conformation. Methods for determining the spatial conformation of an epitope include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, "Epitope Mapping Protocols" in Methods in Molecular Biology, Vol. 66, edited by Glenn E. Morris (1996).

[0088] As used herein, the term "antigen" is defined as a molecule that elicits an immune response. This immune response can include antibody production, activation of specific immunocompetent cells, or both. One of ordinary skill in the art should understand that virtually any macromolecule, including substantially all proteins or peptides, can serve as an antigen. Furthermore, an antigen can be derived from recombinant DNA or genomic DNA. One of ordinary skill in the art should thus understand that DNA containing a nucleotide sequence or partial nucleotide sequence that encodes a protein that elicits an immune response would encode the term "antigen" as used herein. Additionally, one of ordinary skill in the art should understand that an antigen need not be encoded only by the full-length nucleotide sequence of a gene. Embodiments described herein include, but are not limited to, the use of partial nucleotide sequences of two or more genes, and it is readily apparent that these nucleotide sequences can be arranged in various combinations to elicit a desired immune response. Moreover, one of ordinary skill in the art should understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that an antigen can be synthesized or derived from a biological sample. Such biological samples can include, but are not limited to, tissue samples, cells, or biological fluids.

[0089] Accordingly, the compositions described herein may be suitable for the protection or treatment of vertebrate subjects against various disease states such as, for example, infections by viruses, bacteria, fungi or parasites, cancer, and autoimmune disorders. It should be recognized that these specific disease states are mentioned by way of example only and are not intended to be limiting.

[0090] Suitable antigens useful in combination with the compositions described herein include any antigen as defined herein. Antigens are commercially available or can be generated by those skilled in the art. Antigens can be modified live or killed microorganisms, or tumor cells, synthetic products, genetically engineered proteins, peptides, polysaccharides or similar products, or natural products purified from microorganisms or other cells including but not limited to allergens. The antigenic portion can also be a subunit of a protein, peptide, polysaccharide or similar product. Antigens can also be genetic antigens, i.e., DNA or RNA that elicits an immune response.

[0091] Representative examples of antigens that can be used include natural, recombinant or synthetic products derived from viruses, bacteria, fungi, parasites, and other infectious agents, in addition to prophylactic or therapeutic vaccines and autoimmune diseases, hormones, or tumor antigens that may be used in allergens, but are not limited thereto. In one embodiment, the antigen comprises virus-like particles (VLPs) from various viruses such as influenza, HIV, RSV, Newcastle disease virus (NDV). See International Application US2006 / 40862, International Application US2004 / 022001, U.S. Patent Application No. 11 / 582,540, U.S. Patent Application No. 60 / 799,343, U.S. Patent Application No. 60 / 817,402, U.S. Patent Application No. 60 / 859,240, all of which are hereby incorporated by reference in their entirety. In another embodiment, the antigen comprises chimeric VLPs. "Chimeric VLPs" refers to VLPs that contain proteins or portions thereof from at least two different sources (organisms). Usually, one protein is derived from a virus that can drive the formation of VLPs from host cells. Thus, in one embodiment, the chimeric VLP comprises the RSV M protein. In another embodiment, the chimeric VLP comprises the NDV M protein. In another embodiment, the chimeric VLP comprises the influenza virus M protein.

[0092] Viral or bacterial products can be components produced by organisms through enzymatic cleavage or components of organisms generated by recombinant DNA techniques well-known to those skilled in the art.

[0093] Some specific examples of antigens are hepatitis viruses A, B, C, D and E3, human immunodeficiency virus (HIV), herpes viruses 1, 2, 6 and 7, cytomegalovirus, varicella zoster, papillomavirus, Epstein - Barr virus, parainfluenza virus, adenovirus, bunyavirus (such as hantavirus), coxsackievirus, picornavirus, rotavirus, respiratory syncytial virus, rhinovirus, rubella virus, papovavirus, mumps virus, measles virus, poliovirus (multiple types), adenovirus (multiple types), parainfluenza virus (multiple types), avian influenza or pandemic influenza (various types), seasonal influenza, shipping fever virus, western and eastern equine encephalomyelitis, Japanese encephalitis B, Russian spring - summer encephalitis, swine cholera virus, Newcastle disease virus, fowl pox, rabies, viruses such as cat and dog distemper, slow virus, Rous sarcoma virus (RSV), papovaviridae, parvoviridae, picornaviridae, poxviridae (such as smallpox or vaccinia), reoviridae (e.g., rotavirus), retroviridae (HTLV - I, HTLV - II, lentivirus), and togaviridae (e.g., rubivirus) - derived antigens from viral infections caused by viruses belonging to these families. Viruses belonging to these families can cause various diseases and symptoms such as arthritis, bronchiolitis, encephalitis, eye infections (such as conjunctivitis, keratitis), chronic fatigue syndrome, Japanese encephalitis B, Junin, chikungunya, Rift Valley fever, yellow fever, meningitis, opportunistic infections (such as AIDS), pneumonia, Burkitt lymphoma, chickenpox, hemorrhagic fever, measles, mumps, parainfluenza, rabies, colds, polio, leukemia, rubella, sexually transmitted infections, skin diseases (e.g., Kaposi, warts), and viremia, but are not limited to these.

[0094] Antigens can also be derived from bacterial and fungal infections, such as: mycobacteria that cause tuberculosis and leprosy, pneumocci, aerobic gram negative bacilli, infections caused by mycoplasma, staphylococcal infections, streptococcal infections, salmonellae and chlamydiae, Bordetella pertussis (whooping cough bacterium), Leptospira pomona and Leptospira icterohaemorrhagiae-derived antigens. Certain embodiments are Salmonella paratyphi A and B, Corynebacterium diphtheriae (diphtheria bacterium), Clostridium tetani (tetanus bacterium), Clostridium botulinum, Clostridium perfringens, Clostridium feseri and other gas gangrene bacteria, Bacillus anthracis, Yersinia pestis, Pasteurella multocida, Neisseria meningitidis (meningococcus), Neisseria gonorrhoeae (gonococcus), Hemophilus influenzae, Actinomyces (e.g., Nocardia), Acinetobacter, Bacillaceae (e.g., Bacillus anthrasis anthrax bacterium), Bacteroides (e.g., Bacteroides fragilis), Blastomycosis, Bordetella, Borrelia (e.g., Borreliaburgdorferi), Brucella, Candida, Campylobacter, Chlamydia, Coccidioides, Corynebacterium (e.g., Corynebacterium diptheriae), Cryptococcus, Dermatocycoses, Escherichia coli (E. coli, e.g., Enterotoxigenic E. coli and Enterohemorrhagic E. coli), Enterobacter (e.g., Enterobacter aerogenes), Enterobacteriaceae (Klebsiella, Salmonella (e.g., Salmonella typhi, typhoid bacillus, Salmonella enteritidis, enteritis bacterium), Serratia, Yersinia, Shigella (dysentery bacillus)), Erysipelothrix, Haemophilus (e.g., Haemophilus influenzae type B), Helicobacter, Legionella (e.g., Legionella pneumophila), Leptospira, Listeria (e.g., Listeria monocytogenes), Mycoplasma, Mycobacterium (e.g., Mycobacterium leprae and Mycobacterium tuberculosis), Vibrio (e.g., Vibrio cholerae), Pasteurellacea, Proteus, Pseudomonas (e.g., Pseudomonas aeruginosa), Rickettsiaceae, Spirochetes (e.g., Treponema spp, Leptospiraspp), Borrelia spp, Shigella spp, Meningiococcus, Pneumococcus and Streptococcus (e.g., Streptococcus pneumoniae and group A, B, and C Streptococcus), Ureaplasmas, Treponema pollidum, etc.; Staphylococcus aureus, Plasmodium sp, Plasmodium falciparum, Plasmodium vivax, etc., Aspergillus sp, Candida albicans, Pasteurella haemolytica, Corynebacterium diptheriae toxoid, Meningococcal polysaccharide, Bordetella pertusis, Streptococcus pneumoniae polysaccharide, Tetanus toxoid, Mycobacterium bovis, Salmonella typhi, Cryptococcus neoformans, and dead cells of Aspergillus.

[0095] Antigens can also be derived from parasitic malaria, leishmaniasis, trypanosomiasis, toxoplasmosis, schistosomiasis, filariasis malaria, amoebiasis, babesiosis, coccidiosis, cryptosporidiosis, binucleate amoebiasis, dourine, ectoparasites, giardia, helminthiasis, theileriosis, trichomonas and sporozoans (e.g., Plasmodium virax, Plasmodium fakiparium, Plasmodium malariae and Plasmodium ovale). These parasites can cause a variety of diseases and symptoms including scabies, scrub typhus, eye infections, intestinal diseases (such as dysentery, giardiasis), liver diseases, lung diseases, opportunistic infections (such as AIDS-related), malaria, pregnancy complications, and toxoplasmosis, but are not limited to these.

[0096] Tumor-associated antigens suitable for use in the compositions described herein include both mutant and non-mutant molecules that can indicate a single tumor type, can be shared among several types of tumors, and / or can be expressed or overexpressed exclusively in tumor cells as compared to normal cells. In addition to proteins and glycoproteins, tumor-specific patterns of expression of carbohydrates, gangliosides, glycolipids, and mucins have also been demonstrated. Exemplary tumor-associated antigens for use in a cancer vaccine for a subject include protein products of cancer genes, tumor suppressor genes, and other genes having mutations or rearrangements specific to tumor cells, reactivated fetal gene products, cancer fetal antigens, tissue-specific (but not tumor-specific) differentiation antigens, growth factor receptors, cell surface carbohydrate residues, foreign viral proteins, and several other self-proteins. Specific embodiments of tumor-associated antigens include, for example, protein products of the Ras p21 oncogene, the tumor suppressor p53 and the HER-2 / neu and BCR-ab1 cancer genes, and mutant antigens such as CDK4, MUM1, caspase 8, and beta-catenin; overexpressed antigens such as galectin 4, galectin 9, carbonic anhydrase, aldolase A, PRAME, Her2 / neu, ErbB-2, and KSA, cancer fetal antigens such as alpha-fetoprotein (AFP), human chorionic gonadotropin (hCG); self-antigens such as carcinoembryonic antigen (CEA), and melanocyte differentiation antigens such as Mart 1 / Melan A, gp100, gp75, tyrosinase, TRP1, and TRP2; prostate-associated antigens such as PSA, PAP, PSMA, PSM-P1, PSM-P2; reactivated fetal gene products such as MAGE 1, MAGE 3, MAGE 4, GAGE 1, GAGE 2, BAGE, RAGE, and other cancer testicular antigens such as NY-ESO1, SSX2, and SCP1; mucins such as Muc-1 and Muc-2; gangliosides such as GM2, GD2, GD3, neutral glycolipids, and glycoproteins such as Lewis(y) and globo-H; and glycoproteins such as Tn, the Thompson-Freidenreich antigen (TF), sTn.Also included in the present specification as tumor - associated antigens are whole cells and tumor cell lysates and their immunogenic portions, as well as immunoglobulin idiotypes expressed by monoclonal proliferation of B lymphocytes for use against B - cell lymphoma. Tumor - associated antigens and their respective tumor cell targets include, for example, cytokeratins as cancer tumor antigens, particularly cytokeratins 8, 18, and 19. Epithelial membrane antigen (EMA), EphA1, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA10, EphB1, EphB2, EphB3, EphB4, EphB6, human fetal antigen (HEA - 125), human milk fat globule, MBr1, MBr8, Ber - EP4, 17 - 1A, C26, and T16 are also known cancer antigens. Desmin and muscle - specific actin are antigens of myogenic sarcoma. Placental alkaline phosphatase, beta - human chorionic gonadotropin, and alpha - fetoprotein are antigens of trophoblastic tumors and germ cell tumors. Prostate - specific antigen is an antigen of prostate cancer and a cancer - fetal antigen of colon adenocarcinoma. HMB - 45 is an antigen of melanoma. In cervical cancer, useful antigens may be encoded by the human papillomavirus. Chromogranin A and synaptophysin are antigens of neuroendocrine tumors and neuroectodermal tumors. Particularly interesting are aggressive tumors that form solid tumor masses with necrotic regions. Lysis of such necrotic cells is a rich source of antigens for antigen - presenting cells and, thus, may find advantageous use in combination with conventional chemotherapy and / or radiotherapy for the treatment of a subject. Antigens can be derived from any tumor or malignant cell line.

[0097] The antigen may be derived from common allergens that cause allergies. Allergens include organic or inorganic materials derived from various artificial or natural resources such as plant materials, metals, components of cosmetics or detergents, latex, etc. Suitable classes of allergens for use in the compositions and methods described herein include, but are not limited to, pollen, animal dander, grass, mold, dust, antibiotics, venom of stinging insects, and various environmental (including chemicals and metals) drugs and food allergens.Common tree allergens include pollen from box elder, poplar, mulberry, horse chestnut, maple, oak, elm, hickory, and pecan trees; common plant allergens include allergens from rye grass, ragweed, tobacco, spinach dock, and ragweed; plant contact allergens include those derived from poison oak, poison ivy, and nettles; common grass allergens include allergens from timothy, Johnson, Bermuda, goosegrass, and bluegrass; common allergens are also available from molds and fungi such as Alternaria, Fusarium, Hormodendrum, Aspergillus, Micropolyspora, Mucor, and thermophilic actinomycetes; penicillin and tetracycline are common antibiotic allergens; epidermal allergens can be obtained from house dust or organic dust (usually of fungal origin), insects such as house dust mites (Dermalphagoides pterosinyssis), or animal sources such as feathers, dandruff from cats and dogs; common food allergens include allergens from milk and cheese (dairy products), eggs, wheat, nuts (such as peanuts), seafood (such as crustaceans), lentils, beans, and gluten; common environmental allergens include allergens from metals (nickel and gold), chemicals (formaldehyde, trinitrophenol, and turpentine), latex, rubber, fibers (such as cotton or wool), jute, hair dyes, cosmetics, detergents, and perfumes; common drug allergens include allergens from local anesthetics and salicylic acid; antibiotic allergens include penicillin and sulfonamide allergens; common insect allergens include the poisons of bees, wasps, and ants, and the allergen from the calyx of the cockroach mushroom.Among the allergens that have been particularly well-characterized are the major and potential epitopes of the Der pI allergen (Hoyne et al. (1994) Immunology 83, 190-195), bee venom phospholipase A2 (PLA) (Akdis et al. (1996) J. Clin. Invest. 98, 1676-1683), birch pollen allergen Bet v 1 (Bauer et al. (1997) Clin. Exp. Immunol. 107, 536-541), and the multi-epitope recombinant grass allergen rKBG8.3 (Cao et al. (1997) Immunology 90, 46-51), but are not limited thereto. These and other suitable allergens are commercially available and / or can be readily prepared as extracts according to known techniques.

[0098] The antigen can be in purified antigen form or partially purified antigen form and can be derived from any of the above antigens, antigenic peptides, proteins known and available in the art, and other proteins that can be identified using conventional techniques. The antigen typically has its toxicity or virulence properties reduced or destroyed and, when introduced into a suitable subject, will take a form that induces an immune response against a particular microorganism, an extract or product of the microorganism used in the preparation of the antigen, or, in the case of an allergen, will serve to alleviate the symptoms of allergy to a particular allergen. The antigens can be used alone or in combination; for example, by combining a plurality of bacterial antigens, a plurality of viral antigens, a plurality of bacterial antigens, a plurality of parasite antigens, a plurality of bacterial and viral toxoids, a plurality of tumor antigens, a plurality of allergens or any combination of the aforementioned products with an adjuvant composition, a multivalent antigenic composition and / or a vaccine can be created. In the compositions described herein, the antigen can be an antigen captured, adsorbed, or mixed with the vesicular structural components of the composition.

[0099] In one embodiment, antigens suitable for use with the compositions described herein include antigens with low immunogenicity, such as malaria antigens, dengue antigens, and HIV antigens, or antigens intended to confer immunity against pandemic diseases, such as influenza antigens.

[0100] "Encoding" refers to the property of a specific nucleotide sequence (e.g., rRNA, tRNA, and mRNA) or a specific amino acid sequence and other polymers and macromolecules in a biological process resulting therefrom to serve as a template for the synthesis of other polymers and macromolecules. Thus, when a protein is produced in a cell or other biological system by transcription and translation of the mRNA corresponding to that gene, the gene encodes the protein. Both the nucleotide sequence that is identical to the mRNA sequence and usually listed in the sequence listing, the coding strand, and the non-coding strand used as a template for transcription of the gene or cDNA can be said to encode the protein or other product of that gene or cDNA.

[0101] As used herein, the term "expression" is defined as the transcription and / or translation of a specific nucleotide sequence driven by that promoter.

[0102] "Isolated" means altered or removed from its natural state. For example, a nucleic acid or peptide that naturally exists in a living animal is not "isolated", but the same nucleic acid or peptide that is partially or completely separated from its coexisting materials in its natural state is "isolated". An isolated nucleic acid or protein can exist in a substantially purified form or, for example, in a non-natural environment such as a host cell.

[0103] Unless otherwise specified, the term "nucleotide sequence encoding an amino acid sequence" includes degenerate versions of each other and encompasses all nucleotide sequences encoding the same amino acid sequence. A phrase nucleotide sequence encoding a protein or RNA may also include introns to the extent that the nucleotide sequence encoding the protein may include introns in some version.

[0104] As used herein, the term "modulate" means to effect a detectable increase or decrease in the response level of a subject as compared to the response level of the subject in the absence of treatment or compound and / or as compared to the response level of an otherwise identical but untreated subject. The term includes perturbing and / or affecting the original signal or response such that a beneficial therapeutic response in a subject, typically a human, is modulated.

[0105] The term "operably linked" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence, resulting in the expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence if the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, DNA sequences that are operably linked are contiguous and, where necessary to join two protein-coding regions, are in the same reading frame.

[0106] "Parenteral" administration of an immunogenic composition includes, for example, subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrasternal injection or infusion techniques.

[0107] As used herein, the term "polynucleotide" is defined as a chain of nucleotides. Further, a nucleic acid is a polymer of nucleotides. Thus, the nucleic acids and polynucleotides used herein are interchangeable. One of ordinary skill in the art should have the general knowledge that a nucleic acid is a polynucleotide and can be hydrolyzed into monomeric "nucleotides". Monomeric nucleotides can be hydrolyzed to become nucleosides. The polynucleotides used herein include, but are not limited to, all nucleic acid sequences obtained by any means available in the art, including recombinant means, i.e., cloning of nucleic acid sequences from recombinant libraries or cell genomes using conventional cloning techniques and PCR, etc., and synthetic means.

[0108] As used herein, the terms "peptide", "polypeptide", and "protein" are used interchangeably and refer to a compound containing amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can constitute the sequence of a protein or peptide. A polypeptide includes a peptide or protein containing two or more amino acids linked to each other by peptide bonds. As used herein, this term refers to both short chains, which are generally referred to as peptides, oligopeptides, and oligomers in the art, and long chains, which are generally referred to as proteins in the art, and there are many types. "Polypeptide" includes, for example, among others, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, and fusion proteins. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0109] As used herein with respect to an antibody, the term "specifically binds" means an antibody that recognizes a specific antigen but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. However, such cross-reactivity does not change the classification of the antibody as being specific in itself. In another example, an antibody that specifically binds to an antigen may also bind to different genotypes of that antigen. However, such cross-reactivity itself does not change the classification of the antibody as being specific. In some cases, the terms "specific binding" or "specifically binds" are used with respect to the interaction of an antibody, protein, or peptide with a second chemical species, such that the interaction is dependent on the presence of a specific structure (e.g., antigenic determinant or epitope) on the chemical species; for example, an antibody generally does not bind to a protein but rather recognizes and binds to a specific protein structure. If an antibody is specific for epitope "A", the presence of a molecule containing epitope A (or free unlabeled A) in a reaction containing the antibody and labeled "A" will result in a decrease in the amount of labeled A bound to the antibody.

[0110] The terms "therapeutically effective amount," "effective amount," or "sufficient amount" mean an amount sufficient to achieve a desired result, such as an amount effective to induce a protective immune response, when administered to a subject, such as a mammal including a human. The effective amount of the compounds described herein may vary depending on factors such as the immunogen, age, sex, and weight of the subject. As will be understood by those skilled in the art, the dosage or treatment regimen may be adjusted to achieve an optimal therapeutic response. For example, administration of a therapeutically effective amount of a fusion protein described herein is sufficient in embodiments to enhance immunity against pathogens such as Plasmodium. In other embodiments, administration of a therapeutically effective amount of a fusion protein described herein is sufficient to treat a disease or condition such as cancer, HIV, malaria, or an autoimmune disease. In yet other embodiments, administration of a therapeutically effective amount of a fusion protein described herein is sufficient to act as an adjuvant to enhance the effectiveness of a vaccine.

[0111] Furthermore, a treatment regimen for a subject at a therapeutically effective amount may consist of a single administration or may include a series of applications. The length of the treatment period varies depending on various factors such as the immunogen, the age of the subject, the concentration of the drug, the responsiveness of the patient to the drug, or a combination thereof. It will also be understood that the effective dosage of the drug used in the treatment may increase or decrease during the course of a particular treatment regimen. Changes in dosage may occur and may be revealed by standard diagnostic assays known in the art. The fusion proteins described herein may, in embodiments, be administered before, during, or after treatment with conventional therapies for a problem disease or disorder such as malaria, HIV, or cancer. For example, specific uses may be found for the fusion proteins described herein in combination with immunotherapy for treating cancer.

[0112] As used herein, the terms "transfected", "transformed", or "transduced" refer to the process by which exogenous nucleic acid is introduced or transferred into a host cell. A "transfected", "transformed", or "transduced" cell is a cell that has been transfected, transformed, or transduced with exogenous nucleic acid. Cells include the primary subject cells and their progeny.

[0113] As used herein, the phrases "under transcriptional control" or "operably linked" mean that the promoter is in the correct position and orientation with respect to the polynucleotide to control the initiation of transcription by RNA polymerase and the expression of the polynucleotide.

[0114] A "vector" is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid into the interior of a cell. A number of vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. This term should also be interpreted to include non-plasmid and non-viral compounds, such as, for example, polylysine compounds, liposomes, that facilitate the uptake of nucleic acids into cells. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and the like.

[0115] As used herein, the term "subject" refers to any member of the animal kingdom, typically a mammal. The term "mammal" refers to any animal classified as a mammal, including humans, other higher primates, domestic and farm animals such as dogs, cats, cows, horses, sheep, pigs, goats, rabbits, and zoo, sports, or pet animals. Usually, the mammal is a human.

[0116] Administration “in combination with” one or more additional therapeutic agents includes co - administration (simultaneous administration) and sequential administration in any order.

[0117] The term “pharmaceutically acceptable” means that a compound or combination of compounds is compatible with the remaining ingredients of a pharmaceutical formulation and is generally safe for administration to humans in accordance with established governmental standards, including those promulgated by the United States Food and Drug Administration.

[0118] The term “pharmaceutically acceptable carrier” includes, but is not limited to, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and / or absorption delaying agents, etc. The use of pharmaceutically acceptable carriers is well known.

[0119] The term "adjuvant" refers to a compound or mixture that is present in a vaccine and enhances the immune response to an antigen present in the vaccine. For example, an adjuvant may enhance the immune response to a polypeptide present in the vaccine under consideration herein, or to an immunogenic fragment or variant thereof under consideration herein. An adjuvant may serve as a tissue depot that slowly releases the antigen, or as a lymphoid activating factor that non-specifically enhances the immune response. Examples of adjuvants that may be used include MPL-TDM adjuvant (monophosphoryl lipid A / synthetic trehalose dicorynomycolate, available, for example, from GSK Biologics). Another suitable adjuvant is the immunostimulatory adjuvant AS021 / AS02 (GSK). These immunostimulatory adjuvants are formulated to give a strong T cell response and contain QS-21, a saponin from Quillay saponaria, a TLR4 ligand, and monophosphoryl lipid A together in a lipid or liposome carrier. Other adjuvants include non-ionic block copolymer adjuvants (e.g., CRL 1005), aluminum phosphate (e.g., AIPO₄), R-848 (a Th1-like adjuvant), imiquimod, PAM3CYS, poly(I:C), loxoribine, BCG (bacille Calmette-Guerin), Corynebacterium parvum, CpG oligodeoxynucleotides (ODN), cholera toxin-derived antigens (e.g., CTA 1-DD), lipopolysaccharide adjuvants, Freund's complete adjuvant, Freund's incomplete adjuvant, saponin, mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oil-in-water or hydrocarbon emulsions (e.g., MF59 or Montanide ISA 720 available from Novartis Vaccines), keyhole limpet hemocyanin, and dinitrophenol, but are not limited thereto.

[0120] A "variant" is a biologically active fusion protein, antibody, or fragment thereof that has an amino acid sequence different from a reference sequence due to the insertion, deletion, modification, and / or substitution of one or more amino acid residues within a comparison array. Variants generally have less than 100% sequence identity with the comparison sequence. However, typically, a biologically active variant will have an amino acid sequence with at least about 70% amino acid sequence identity with the comparison sequence, such as at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. Variants include peptide fragments of at least 10 amino acids that retain some level of the biological activity of the reference sequence. Variants also include polypeptides in which one or more amino acid residues are added to the N-terminus or C-terminus of the comparison sequence, or within the comparison sequence. Variants also include polypeptides in which some amino acid residues are deleted and optionally substituted by one or more amino acid residues. Variants may also be covalently modified, for example, by substitution with moieties other than naturally occurring amino acids, or by modifying amino acid residues to produce non-naturally occurring amino acids.

[0121] "Percent amino acid sequence identity" is defined herein as the percentage of amino acid residues in a candidate sequence that are identical to residues in a sequence of interest, such as a polypeptide of the present invention, without considering conservative substitutions as part of sequence identity, after aligning the sequences and introducing gaps if necessary to achieve the maximum percent sequence identity. Neither N-terminal, C-terminal, or internal extensions, deletions, or insertions into the candidate sequence are to be construed as affecting sequence identity or homology. Methods and computer programs for alignment are well known in the art, such as "BLAST".

[0122] For the purposes of this specification, "active" or "activity" refers to the biological and / or immunological activity of the fusion proteins described herein, and "biological" activity refers to the biological function (inhibitory or stimulatory) elicited by the fusion protein.

[0123] The fusion proteins described herein may include modifications. Such modifications include, but are not limited to, conjugation to effector molecules such as anti-malarial agents or adjuvants. Modifications further include, but are not limited to, conjugation to detectable reporter moieties. Modifications that extend the half-life (e.g., pegylation) are also included. Proteins and non-protein drugs can be conjugated to the fusion proteins by methods known in the art. Conjugation methods include direct binding, binding via a covalently linked linker, and specific binding pair members (e.g., avidin-biotin). Such methods include, for example, the methods described by Greenfield et al., Cancer Research 50, 6600-6607 (1990), which is incorporated herein by reference, and the methods described by Amon et al., Adv. Exp. Med. Biol. 303, 79-90 (1991) and Kiseleva et al., Mol. Biol. (USSR) 25, 508-514 (1991), both of which are incorporated herein by reference.

[0124] Fusion protein Described herein are fusion proteins. The fusion proteins include nanocage monomers and an antibody or fragment thereof bound to the nanocage monomer, and the antibody or fragment thereof includes an antigen-binding epitope. Multiple fusion proteins self-assemble to form a nanocage, and multiple antibodies or fragments thereof decorate the outer surface of the nanocage, thereby exposing the antigen-binding epitopes for interaction with antigens.

[0125] In other embodiments, the fusion protein comprises a nanocage monomer and an antibody or fragment thereof bound to the nanocage monomer, and the antibody or fragment thereof comprises the Fc portion of the antibody or fragment thereof. A plurality of fusion proteins self-assemble to form a nanocage, and a plurality of antibodies or fragments thereof decorate the outer surface of the nanocage, whereby the Fc portion of the antibody or fragment thereof is exposed to interact with the Fc receptor.

[0126] In typical embodiments, the nanocage comprises from about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 55, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, or 98 to about 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 55, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, or 100 nanocage monomers, such as 24 monomers or 60 monomers, from about 3 to about 100 nanocage monomers. The nanocage monomer may be any known natural, synthetic, or semi-synthetic nanocage monomer, and in embodiments is selected from ferritin, encapsulin, SOR, lumazine synthase, pyruvate dehydrogenase, carboxysome, bolt protein, GroEL, heat shock protein, E2P, MS2 coat protein, fragments thereof, and variants thereof. Figures 1A and 1B show images of self-assembled nanocages.

[0127] In certain embodiments, the fusion proteins described herein include a linker between the nanocage monomer and an antibody or a fragment thereof. This linker allows both the nanocage monomer and the antibody or fragment thereof to adopt conformations that are favorable for self-assembly and antibody function when the protein is expressed. The linker can be flexible or rigid.

[0128] The linker is generally of a length sufficient to impart some flexibility to the fusion protein, although it will be understood that the length of the linker can vary depending on the sequences of the nanocage monomer and antibody and the three-dimensional conformation of the fusion protein. Thus, the linker is typically from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid residues, such as from about 8 to about 16 amino acid residues, for example 8, 10, or 12 amino acid residues, or from about 1 to about 30 amino acid residues.

[0129] The linker can be of any amino acid sequence so long as it does not interfere with the binding of the antigen to the antigen-binding site of the antibody. In one typical example, the linker contains GGS repeats, and more typically, the linker contains about 2, 3, 4, 5, or 6 GGS repeats, such as about 4 GGS repeats.

[0130] Typically, the antibody includes the heavy chain and / or the light chain of the Fab fragment, although it will be understood that any antibody or fragment thereof, such as one of the antibodies described above, can be used in the fusion proteins described herein. In other typical embodiments, the antibody or fragment thereof includes an scFv or scFc.

[0131] In certain embodiments, the fusion protein may further comprise an antigen. Such embodiments are described in U.S. Patent Application No. ______ (Julien et al., filed concurrently with this specification, docket number 3206-5005), which is hereby incorporated by reference in its entirety. Briefly, in such embodiments, the antigen has at least first and second antibody-binding epitopes; and at least an antibody or a fragment thereof specific for the first antigenic epitope. Binding of the antibody or its fragment to the first antigenic epitope presents a second antigenic epitope for binding to the antigen-binding portion, and / or the first antibody-binding epitope binds to the antibody or its fragment, and said binding presents the second antibody-binding epitope in the context of the antibody or its fragment.

[0132] In embodiments, the antigen typically comprises a repeat domain. This facilitates the inclusion of two identical antibody-binding epitopes in a single entity. Of course, the antigen may have different antibody-binding epitopes as described above, in which case a repeat domain would not be appropriate. In related embodiments, the antibody is specific for the repeat domain.

[0133] In typical embodiments, the antigen is a malaria antigen such as a fragment of the malaria CSP protein. More typically, the antigen is a fragment of the NANP repeat domain of the malaria CSP protein and contains 5.5 NANP repeats. In a typical embodiment, the antigen is NPNANPNANPNANPNANPNANP. In related embodiments, the antibody is specific for a malaria antigen such as the malaria CSP protein, and more typically, is specific for the NANP repeat domain of the malaria CSP protein.

[0134] Needless to say, the malaria CSP protein may have other repeating amino acids in addition to, or instead of, NANP, including NPDP, NVDP, and NANA. These may be repeated alone or in combination, with or without NANP, to form an antigen or part of an antigen, and for clarity, shall be included in the term "NANP repeat domain" even in the absence of NANP. The unique position of the NPDP motif at the junction between the N-terminal domain and the central repeat region is conserved in almost all Pf isolates (>99.8%) (Kisalu et al., 2018). In contrast, the NANP-NVDP alternating sequence is generally located immediately after the NPDP motif, but the NANP motif can be repeated more than 40 times and the length can vary greatly among Pf wild isolates. Therefore, repeat-targeting mAbs have been shown to bind to multiple copies of the epitope even within a single PfCSP molecule. mAbs MGG4 and CIS43 bind indiscriminately to NPDP, NVDP, and NANP, but also show a unique preference for specific repeating motifs. Importantly, the described mAbs have the ability to engage the NPDP motif (KQPADGNPDPNANPNVDPN, called the junction epitope), which may enhance their efficacy in inhibiting Pf sporozoites. The paratopes of mAbs MGG4 and CIS43 have the ability to accommodate the interchangeability of certain amino acids within the repeating motif (NPDP vs NVDP vs NANP).

[0135] Typically, the antibody or fragment thereof has a sequence identity of at least 90% to the sequence:

Number

[0136] In other embodiments, the antibody or fragment thereof is an anti-CD22 antibody or anti-CD19 antibody such as epratuzumab or denintuzumab. In certain embodiments, the antigen may be co-presented on the surface of the nanocage as a separate subunit fusion protein or in a form bound to the surface of the nanocage by other known methods. By co-presenting the antigen and the anti-CD19 antibody or fragment thereof, an adjuvant effect specifically binding to the antigen is provided. The antigen co-presented with anti-CD19 can be directly or indirectly bound to the nanoparticle surface and is particularly contemplated to be presented in the context of the above-described antibody or fragment thereof.

[0137] In other embodiments, the antibody or fragment thereof can be directed to any antigen such as those listed above. Typically, the antigen is derived from a cancer or an infectious agent such as hepatitis A, B, or C, HIV, mycobacteria, malaria pathogen, SARS pathogen, herpes virus, influenza virus, poliovirus, or a bacterial pathogen such as Chlamydia or Mycobacteria, or autoreactive B cells or any T cells for co-stimulation and cytotoxic killing.

[0138] Generally, the fusion proteins described herein are associated with the Fab light chain and / or heavy chain, which can be produced separately or continuously from the fusion protein.

[0139] Alternatively, uses as a therapeutic or diagnostic agent may be found for the fusion proteins described herein. Thus, the antibody or fragment thereof in an embodiment can be specific for, for example, a tumor antigen or a self-antigen.

[0140] Substantially identical sequences may contain one or more conservative amino acid mutations. It is known in the art that one or more conservative amino acid mutations to a reference sequence may generate mutant peptides that do not involve substantial changes in physiological, chemical, or functional properties compared to the reference sequence; in such cases, the reference sequence and the mutant sequence are considered "substantially identical" polypeptides. Conservative amino acid mutations may include amino acid additions, deletions, or substitutions; conservative amino acid substitutions are defined herein as the substitution of an amino acid residue with another amino acid residue having similar chemical properties (e.g., size, charge, or polarity).

[0141] In non-limiting examples, conservative mutations can be amino acid substitutions. Such conservative amino acid substitutions may substitute another member of the same group with a basic, neutral, hydrophobic, or acidic amino acid. The term "basic amino acid" means a hydrophilic amino acid having a side chain pK value greater than 7 that is typically positively charged at physiological pH. Basic amino acids include histidine (His or H), arginine (Arg or R), and lysine (Lys or K). The term "neutral amino acid" (also known as "polar amino acid") means a hydrophilic amino acid having a side chain with at least one bond in which the electron pair shared by two atoms is pulled closer to one of the atoms and is uncharged at physiological pH. Polar amino acids include serine (Ser or S), threonine (Thr or T), cysteine (Cys or C), tyrosine (Tyr or Y), asparagine (Asn or N), and glutamine (Gln or Q). The term "hydrophobic amino acid" (also known as "non-polar amino acid") means an amino acid having a hydrophobicity greater than zero according to the normalized consensus hydrophobicity scale of Eisenberg (1984). Hydrophobic amino acids include proline (Pro or P), isoleucine (Ile or I), phenylalanine (Phe or F), valine (Val or V), leucine (Leu or L), tryptophan (Trp or W), methionine (Met or M), alanine (Ala or A), and glycine (Gly or G).

[0142] "Acidic amino acids" refer to hydrophilic amino acids with a side-chain pK value of less than 7 that are typically negatively charged at physiological pH. Acidic amino acids include glutamic acid (Glu or E) and aspartic acid (Asp or D).

[0143] Sequence identity is used to assess the similarity of two sequences; it is determined by calculating the percentage of identical residues when the two sequences are aligned to obtain the maximum correspondence between residue positions. Any known method may be used to calculate sequence identity; for example, computer software can be used to calculate sequence identity. Without wishing to be limiting, sequence identity can be calculated by software such as the NCBI BLAST2 service maintained by the Swiss Institute of Bioinformatics (and available at ca.expasy.org / tools / blast / ), BLAST-P, Blast-N, or FASTA-N, or other suitable software known in the art.

[0144] Substantially identical sequences of the present invention can be at least 85% identical; in another example, substantially identical sequences can be at least 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% (or any percentage in between) identical to the sequences described herein at the amino acid level. In certain embodiments, substantially identical sequences retain the activity and specificity of the reference sequence. In non-limiting embodiments, differences in sequence identity can be due to multiple conservative amino acid mutations.

[0145] The polypeptides or fusion proteins of the present invention may also contain additional sequences that assist in their expression, detection, or purification. Such sequences or tags known to those skilled in the art can be used. For example, without wishing to be limiting, the fusion protein may contain a targeting or signal sequence (e.g., but not limited to ompA), a detection tag, and as a specific example, the tag cassette may include a Strep tag or any variant thereof; see, for example, U.S. Patent No. 7,981,632, His tag, Flag tag having the sequence motif DYKDDDDK, Xpress tag, Avi tag, calmodulin tag, polyglutamate tag, HA tag, Myc tag, Nus tag, S tag, SBP tag, Softag 1, see Softag 3, V5 tag, CREB-binding protein (CBP), glutathione S-transferase (GST), maltose-binding protein (MBP), green fluorescent protein (GFP), thioredoxin tag, or any combination thereof; a purification tag (e.g., but not limited to, His5 or His6), or a combination thereof.

[0146] In another example, the additional sequence may be a biotin recognition site such as those described by Cronan et al. in WO 95 / 04069 or Voges et al. in WO 2004 / 076670. Also, as known to those skilled in the art, a linker sequence may be used in combination with the additional sequence or tag.

[0147] More specifically, the tag cassette may include an extracellular component that can specifically bind to an antibody with high affinity or binding strength. Within the single-chain fusion protein structure, the tag cassette can be located (a) immediately adjacent to the amino terminus relative to the connector region, (b) sandwiched between and linked by linker modules, (c) immediately adjacent to the carboxy terminus relative to the binding domain, (d) sandwiched between and linked by the binding domain (e.g., scFv) and the effector domain, (e) sandwiched between and linked by subunits of the binding domain, or (f) located at the amino terminus of the single-chain fusion protein. In certain embodiments, one or more junction amino acids may be disposed between the tag cassette and the hydrophobic moiety to link them, between the tag cassette and the connector region to link them, between the tag cassette and the linker module to link them, or between the tag cassette and the binding domain to link them.

[0148] Also included herein are isolated or purified fusion proteins, polypeptides, or fragments thereof immobilized on a surface using various methodologies; for example, without wishing to be limiting, the polypeptide may be bound or coupled to the surface via His-tag coupling, biotin binding, covalent bonding, adsorption, etc. The solid surface can be any suitable surface, such as, without limitation, the well surface of a microtiter plate, the channel of a surface plasmon resonance (SPR) sensor chip, a membrane, beads (magnetic-based or sepharose-based beads or other chromatography resins), glass, film, or other useful surface.

[0149] In other embodiments, the fusion protein can be bound to a cargo molecule; the fusion protein can deliver the cargo molecule to a desired site and can be bound to the cargo molecule using any method known in the art (recombinant technology, chemical conjugation, chelation, etc.). The cargo molecule can be any type of molecule such as a therapeutic or diagnostic agent. For example, without wishing to limit in any way, the therapeutic agent can be a radioisotope that may be used in radioimmunotherapy; a toxin such as an immunotoxin; a cytokine such as an immunocytokine; a cytotoxin; an apoptosis inducer; an enzyme; an anti-cancer antibody for immunotherapy; or any other suitable therapeutic molecule known in the art. On the other hand, the diagnostic agent can include, but is not limited to, a radioisotope, a paramagnetic label such as gadolinium or iron oxide, a fluorophore, a near-infrared (NIR) fluorescent dye or dye (such as Cy3, Cy5.5, Alexa680, Dylight680, or Dylight800), an affinity label (such as biotin, avidin, etc.) fused to a detectable protein-based molecule, or any other suitable agent that can be detected by an imaging method. In certain non-limiting examples, the fusion protein may be bound to a fluorescent agent such as FITC or may be gene-fused to an enhanced green fluorescent protein (EGFP).

[0150] In some embodiments, the cargo molecule is a protein and is fused to the fusion protein such that the cargo molecule is encapsulated within the nanocage. In other embodiments, the cargo molecule is not fused to the fusion protein and is encapsulated within the nanocage. The cargo molecule is typically a protein, a small molecule, a radioisotope, or a magnetic particle.

[0151] The fusion proteins described herein specifically bind to their targets. The antibody specificity, which refers to the selective recognition of an antibody for a specific epitope of an antigen by an antibody or fragment described herein, can be determined based on affinity and / or binding strength. Affinity is the equilibrium dissociation constant (K D) is represented by, and measures the binding strength between an antigenic determinant (epitope) and an antibody binding site. Binding force is a measure of the binding strength between an antibody and its antigen. An antibody typically binds with a K of 10 -5 ~10 -1 1 M. K values exceeding 10 D are generally considered to exhibit non-specific binding. As the value of K -4 decreases, the binding strength between the antigenic determinant and the antibody binding site increases. In an embodiment, the antibodies described herein have a K D of less than 10 D M, 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, or 10 -9 M. D

[0152] Also described herein are nanocages comprising at least one fusion protein described herein. It goes without saying that nanocages can self-assemble from multiple identical fusion proteins, multiple different fusion proteins (thus being multivalent), combinations of fusion proteins and wild-type proteins, and any combination thereof. For example, a nanocage may be decorated with at least one fusion protein described herein in combination with at least one anti-cancer antibody for immunotherapy. In a typical embodiment, about 20% to about 80% of the nanocage monomers comprise the fusion proteins described herein.

[0153] Also described herein are nucleic acid molecules encoding the fusion proteins and polypeptides described herein, as well as vectors containing the nucleic acid molecules and host cells containing the vectors.

[0154] The polynucleotides encoding the fusion proteins described herein include polynucleotides having a nucleic acid sequence substantially the same as the nucleic acid sequence of the polynucleotides of the present invention. A "substantially the same" nucleic acid sequence is herein defined as a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% identity with another nucleic acid sequence when the two sequences are optimally aligned (with appropriate nucleotide insertions or deletions) and compared to determine the exact nucleotide match between the two sequences.

[0155] Suitable sources of DNA encoding antibody fragments include cells such as hybridomas and spleen cells that express full-length antibodies. The fragments may be used by themselves as antibody equivalents or may be recombinantly incorporated into equivalents as described above. The DNA deletions and recombinations described in this section can be carried out by known methods such as those described in the published patent applications listed above in the section entitled "Functional Equivalents of Antibodies" and / or other standard recombinant DNA techniques such as those described below. Another source of DNA is single-chain antibodies produced from phage display libraries, as is known in the art.

[0156] Furthermore, expression vectors are provided that contain the aforementioned polynucleotide sequences operably linked to expression, promoter, and enhancer sequences. A variety of expression vectors for the efficient synthesis of antibody polypeptides in prokaryotes, such as bacterial and eukaryotic systems, including but not limited to yeast and mammalian cell culture systems, have been developed. The vectors of the present invention can contain segments of chromosomal, episomal, and synthetic DNA sequences.

[0157] Any suitable expression vector can be used. For example, prokaryotic cloning vectors include plasmids derived from Escherichia coli (E. coli) such as colE1, pCR1, pBR322, pMB9, pUC, pKSM, and RP4. Prokaryotic vectors also include derivatives of phage DNA such as M13 and other filamentous single-stranded DNA phages. An example of a vector useful in yeast is the 2μ plasmid. Vectors suitable for expression in mammalian cells include well-known derivatives of SV-40, adenoviruses, DNA sequences derived from retroviruses, shuttle vectors derived from combinations of functional mammalian vectors such as those described above, and functional plasmids and phage DNA.

[0158] Additional eukaryotic expression vectors are known in the art (e.g., P J. Southern and P. Berg, J. Mol. Appl. Genet (Journal of Molecular and Applied Genetics), 1:327-341 (1982); Subramani et al., Mol. Cell. Biol (Molecular and Cellular Biology), 1:854-864 (1981); Kaufmann and Sharp, “Amplification And Expression of Sequences Cotransfected with a Modular Dihydrofolate Reductase Complementary DNA Gene” (Amplification and Expression of Sequences Cotransfected with a Modular Dihydrofolate Reductase Complementary DNA Gene), J. Mol. Biol (Journal of Molecular Biology), 159:601-621 (1982); Kaufhiann and Sharp, Mol. Cell. Biol (Molecular and Cellular Biology), 159:601-664 (1982); Scahill et al., “Expression And Characterization Of The Product Of A Human Immune Interferon DNA Gene In Chinese Hamster Ovary Cells” (Expression and Characterization of the Product of a Human Immune Interferon DNA Gene in Chinese Hamster Ovary Cells), Proc. Nat’l Acad. Sci USA (Proceedings of the National Academy of Sciences of the United States of America), 80:4654-4659 (1983); Urlaub and Chasin, Proc. Nat’l Acad. Sci USA (Proceedings of the National Academy of Sciences of the United States of America), 77:4216-4220, (1980), all of which are hereby incorporated by reference).

[0159] An expression vector typically contains at least one expression control sequence that is operably linked to the DNA sequence or fragment to be expressed. To control and regulate the expression of the cloned DNA sequence, the control sequence is inserted into the vector. Examples of useful expression control sequences are the lac, trp, tac, trc systems, the major operator and promoter regions of phage lambda, the control region of fd coat protein, glycolytic promoter of yeast, such as the promoter of 3-phosphoglycerate kinase, the promoter of yeast acid phosphatase, such as Pho5, the promoter of yeast alpha mating factor, and promoters derived from polyoma, adenovirus, retrovirus, and simian virus, such as early and late promoters or SV40, and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells and their viruses or combinations thereof.

[0160] Also described herein are recombinant host cells containing the aforementioned expression vectors. The fusion proteins described herein can be expressed in cell lines other than hybridomas. Nucleic acids containing sequences encoding the polypeptides of the present invention can be used for the transformation of appropriate mammalian host cells.

[0161] Particularly highly preferred cell lines are selected based on high levels of expression, constitutive expression of the protein of interest, and minimal contamination from host proteins. Mammalian cell lines available as hosts for expression are well known in the art and include, but are not limited to, many immortalized cell lines such as Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells, and many others. Suitable additional eukaryotic cells include yeast and other fungi. Useful prokaryotic hosts include, for example, Escherichia coli such as E. coli SG-936, E. coli HB 101, E. coli W3110, E. coli X1776, E. coli X2282, E. coli DHI, and E. coli MRC1, Pseudomonas, Bacillus such as Bacillus subtilis, and Streptomyces.

[0162] These recombinant host cells can be used to produce the fusion protein by culturing the cells under conditions that allow expression of the polypeptide and purifying the polypeptide from the host cells or the medium surrounding the host cells. Targeting of the expressed polypeptide for secretion in the recombinant host cell can be facilitated by inserting a sequence encoding a signal or secretion leader peptide at the 5' end of the gene encoding the antibody of interest (see Shokri et al., (2003) Appl Microbiol Biotechnol. 60(6):654-664; Nielsen et al., Prot. Eng. 10:1-6 (1997); von Heinje et al., Nucl. Acids Res. 14:4683-4690 (1986), all of which are incorporated herein by reference). These secretion leader peptide elements can be derived from either prokaryotic or eukaryotic sequences. Thus, preferably, a secretion leader peptide, which is an amino acid linked to the N-terminus of the polypeptide, is used to direct movement of the polypeptide from the cytosolic compartment of the host cell and secretion into the medium.

[0163] The fusion proteins described herein can be fused to additional amino acid residues. Such amino acid residues can be, for example, peptide tags that facilitate isolation. Other amino acid residues for homing of the antibody to a particular organ or tissue are also contemplated.

[0164] It goes without saying that Fab nanocages can be generated by co - transfection of HC ferritin and LC. As an alternative, as shown in Figure 1C, single - chain Fab - ferritin nanocages that require transfection of only one plasmid can be used. This can be done with linkers of different lengths between LC and HC, for example, 60 or 70 amino acids. Using single - chain Fab can ensure that the heavy and light chains pair up. Tags (e.g., Flag, HA, myc, His6x, Strep, etc.) can also be added to the N - terminus of the construct or within the linker to facilitate the above purification. Furthermore, by using a tag system, when co - transfecting different Fab - nanoparticle plasmids, it can be confirmed that many different Fabs are present on the same nanoparticle using sequential / additional affinity chromatography steps. This provides multi - specificity to the nanoparticles. Protease sites (TEV, 3C, etc.) can be inserted to cleave the linker and tag after expression and / or purification if necessary. An example of such a construct is for the anti - HIV broad - neutralizing Fab 10E8: [Chemical formula]

[0165] In another embodiment, methods are described herein for vaccinating a subject by administering a therapeutically effective amount of the fusion proteins described herein to a mammal in need thereof, typically a young, juvenile, or neonatal mammal. "Therapeutically effective" means an amount effective to produce a desired therapeutic effect, such as providing a protective immune response against the antigen in question.

[0166] Any suitable method or route can be used to administer the fusion proteins and vaccines described herein. Routes of administration include, for example, oral, intravenous, intraperitoneal, subcutaneous, or intramuscular administration.

[0167] When the fusion proteins described herein are used in mammals for prophylactic or therapeutic purposes, it goes without saying that they are administered in the form of a composition further comprising a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include, for example, one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, etc., and combinations thereof. The pharmaceutically acceptable carrier may further contain small amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers that enhance the shelf life or effectiveness of the binding protein. Injectable compositions can be formulated, as is well known in the art, to achieve rapid, sustained or delayed release of the active ingredient after administration to a mammal.

[0168] Human antibodies are particularly useful for administration to humans, but can also be administered to other mammals. As used herein, the term "mammal" is intended to include, but is not limited to, humans, laboratory animals, domesticated pets, and farm animals.

[0169] Also included herein are vaccination kits containing a therapeutically or prophylactically effective amount of the fusion proteins described herein. The kit can further contain, for example, any suitable adjuvant. The kit may contain instructions.

[0170] The above disclosure generally describes the present invention. A more complete understanding can be obtained by referring to the following specific examples. These examples are provided for illustrative purposes only and are not intended to be limiting, unless otherwise specified. Accordingly, the present invention should in no way be construed as limited to the following examples, but rather should be construed to include any and all variations that become apparent as a result of the teachings provided herein.

[0171] The following examples do not include a detailed description of conventional methods such as those used for the construction of vectors and plasmids, the insertion of genes encoding polypeptides into such vectors and plasmids, or the introduction of plasmids into host cells. Such methods are well known to those skilled in the art and are described in numerous publications including Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989), Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, which is hereby incorporated by reference into this specification.

[0172] Without further elaboration, it is believed that one skilled in the art can, using the foregoing description and the examples that follow as a guide, make and utilize the compounds of the present invention and practice the claimed methods. Accordingly, the following examples are illustrative of typical embodiments of the invention and should in no way be construed as limiting the remainder of the disclosure.

Example

[0173] Example 1: Construction, Cloning, Expression and Purification of Nanoparticle Constructs of the Present Invention Construct Design and Cloning The amino acid sequence of human ferritin light chain (Uniprot: P02792) was obtained and GGS of 12 amino acids was added to the N-terminus and C-terminus. Upstream of the N-terminal linker, a StrepTag II affinity tag was added to facilitate affinity purification, and AflII and XbaI restriction sites were added to facilitate downstream cloning. Furthermore, NheI and KpnI restriction sites were added downstream of the C-terminal linker (Figure 2A). Similarly, the amino acid sequence of Thermotoga maritima lumazine synthase (Uniprot: Q9X2E5) was obtained, and GGS at the N-terminus and C-terminus 4x was added. 4xThe linker was made adjacent. After adding the AgeI restriction site and the NheI restriction site to the N-terminus of this construct, and adding the AflII site and the XbaI site to the C-terminus, a Strep Tag II was added (Figure 2B). Both constructs were codon-optimized, synthesized, and cloned into the pHLsec expression vector for human expression. The heavy chain of the epratuzumab Fab (epratuzumab HC) or the heavy chain of the dinutuximab Fab (dinutuximab HC) was cloned using the above restriction sites at the N-terminus of the ferritin (Figure 2A) construct and the luciferase synthase (Figure 2B) construct. Furthermore, the NheI restriction site and the KpnI restriction site were used to clone eGFP and iLOV to the C-terminus of ferritin.

[0174] Expression and purification of nanoparticles Fab HC nanoparticle constructs, Fab LC, and unconjugated nanoparticles (Fab is denintuzumab or epratuzumab as an example, and the nanoparticle is ferritin (Figure 2A) or luciferase synthase (Figure 2B)) were co-transfected into HEK293F (Thermo Fisher Scientific) cells temporarily at a ratio of 1:1:1. The cells were split into 200 mL of culture medium at 0.8 x 10 6 cells / mL -1 . 50 μg of DNA was filtered, mixed with the transfection reagent FectoPRO (Polyplus Transfections) at a ratio of 1:1, and incubated at room temperature for 10 minutes. Next, the DNA:FectoPRO solution was added directly to the cells, and the cells were incubated in a Multitron Pro shaker (Infors HT) at 37 °C, 180 rpm, and 8% CO2 for 6 - 7 days.

[0175] Cells were harvested by centrifugation at 6,371 x g for 20 minutes, the supernatant was retained, and filtered using a 0.22 μm Steritop filter (EMD Millipore). The supernatant was passed through a StrepTrap affinity column (GE Healthcare) at 4 mL / min. Before eluting with 20 mM Tris pH 9.0, 150 mM NaCl, 1 mM EDTA, and 10 mM desthiobiotin, the column was washed with 20 mM Tris pH 9.0, 150 mM NaCl, 1 mM EDTA buffer. Fractions containing the eluted nanoparticles were pooled, concentrated, and separated on a Superose 6 Increase size exclusion column (GE Healthcare) at 0.5 mL / min in 20 mM Tris pH 9.0, 150 mM NaCl buffer to achieve size homogeneity. Data showing the purity of antibody Fabs expressing ferritin (Figure 3A) nanoparticles and luciferase (Figure 3B) nanoparticles are shown by the elution profile (top) and Western blot (bottom).

[0176] Using the same protocol, ferritin-GFP / iLOV particles were produced, with the exception that HEK293F cells were transfected with only the ferritin-GFP / iLOV construct.

[0177] Example 2: Negative staining electron microscopy of antibody Fabs expressing ferritin nanoparticles and luciferase nanoparticles The preparation of electron micrographs of antibody Fabs expressing ferritin (Figure 4A) nanoparticles and luciferase (Figure 4B) nanoparticles of the present invention will be described. The purified nanoparticles were stained with 2% uranyl formate. A dataset consisting of 20 - 50 images was acquired using a field emission FEI Tecnai F20 electron microscope operating at 200 kV and 30 e-Å -2They were manually collected by electron beam irradiation. Images were acquired using an Orius charge-coupled device (CCD) camera (Gatan Inc.) at a calibration magnification of 34,483×. As a result, the pixel size in the specimen was 2.61 Å, and a defocus range of approximately 0.75 - 2 μm was used. A total of approximately 1,000 particle images were manually selected using EMAN2. Two-dimensional classification of the particle images was performed with 50 classes permitted.

[0178] Example 3: Binding affinity of antibody Fabs expressing ferritin nanoparticles and lumazine synthase nanoparticles The binding affinity of epratuzumab Fab (Figure 5A), epratuzumab-ferritin (Figure 5B), and epratuzumab-lumazine synthase (Figure 5C) to CD22 was measured by biolayer interferometry (BLI) using an Octet RED96 BLI system (Pall ForteBio). The Ni-NTA biosensor was hydrated with 1x kinetics buffer (1X PBS, pH 7.4, 0.002% Tween, 0.01% BSA), and 25 ng / μL of CD22 (Uniprot: P20273) was loaded at 1,000 rpm for 300 seconds. Next, the biosensor was transferred to a well containing 1x kinetics buffer to reach the baseline for 60 seconds and then transferred to a well containing serial dilutions of the Fab / nanoparticles. Subsequently, after an 180-second association phase, an 180-second dissociation phase in 1x kinetics followed. The analysis was performed using the Octet software with a 1:1 fitting model.

[0179] Example 4: Receptor-mediated endocytosis of antibodies expressing Fab nanoparticles Nanoparticles of antibody-ferritin (Figures 6A and 6B) or ferritin alone (Figure 6C) (0.5 mg / ml - 1 mg / mL) were labeled with Alexa Fluor-647 (4 mg / mL) (Thermo Fisher Scientific) at a ratio of 10:1 v / v for 1 hour. Next, the nanoparticles were dialyzed against 2 L of 1x PBS for 8 hours, and the dialysis buffer was exchanged three times. Using 5 μg / mL of the dialyzed labeled nanoparticles, human Bjab cells (1x10 6Cells / mL) were treated. After the desired internalization time, the cells were washed three times and dispensed into Lab-Tek II chambers (Nalge Nunc International). Images were captured using a WaveFX-XI spinning disk confocal microscope (Quorum Technologies) equipped with a 63x oil immersion objective lens and an EM-CCD camera (Hamamatsu Photonics). Images of the central plane of the cells were acquired and the images were processed and analyzed using Volocity software (Improvision).

[0180] Example 5. Nanoparticles can be made fluorescent by internal conjugation of a fluorescent protein Cloning, expression, purification and EM were the same as above. Furthermore, eGFP and iLOV were cloned into the C-terminus of ferritin using NheI and KpnI restriction sites. The protocol for the production of ferritin-GFP / iLOV particles was the same as above, except that HEK293F cells were transfected with only the ferritin-GFP / iLOV construct. Staining was performed as described above in Example 2. The fluorescence of ferritin-GFP / iLOV nanoparticles was measured with a transilluminator at a wavelength of 365 nm (Figure 7).

[0181] Example 6: Expression and purification of the CSP-NPNA5.5-linker-1210 fusion protein The fusion protein was constructed and purified as follows. First, 5.5x CSP NPNA repeats, followed by a flexible GGS linker of 8, 10, or 12 residues, were cloned into the N-terminus of the 1210-HC-Fab sequence in the pcDNA3.4 TOPO expression vector. CSP-NPNA5.5-8x-1210 Fab (Figure 10A), CSP-NPNA5.5-10x-1210 Fab (Figure 10B), and CSP-NPNA5.5-12x-1210 Fab (Figure 10C) were produced by transient expression in HEK293F cells by co-transfection with the 1210-LC gene (Figure 10D) in the pcDNA3.4 TOPO expression vector using the FectoPRO (Polyplus) transfection reagent. Purification was performed by KappaSelect affinity chromatography (GE Healthcare). The Fab was further purified by size exclusion chromatography (see Superdex 200 Increase 10 / 300 GL, GE Healthcare, Figures 11 and 12).

[0182] Example 7: The fusion protein does not bind to CSP but is recognized and bound by the wild-type antibody The measurement of the binding to CSP was carried out as follows. To determine whether CSP-NPNA5.5-linker-1210 Fab can recognize CSP or whether the CSP binding site is blocked by NPNA5.5, a biolayer interferometry (Octet RED96, ForteBio) experiment was performed (Figure 13). Recombinant CSP was diluted to 10 μg / mL with kinetics buffer (PBS, pH 7.4, 0.01% (w / v) BSA, 0.002% Tween-20) and immobilized on a Ni-NTA (NTA) biosensor (ForteBio). After loading the ligand into the kinetics buffer to establish a stable baseline, the biosensor was immersed in wells containing 1210 Fab, CSP-NPNA5.5-8x-1210 Fab, CSP-NPNA5.5-10x-1210 Fab, and CSP-NPNA5.5-12x-1210 Fab. Then, to monitor the dissociation rate, the chip was immersed in the kinetics buffer.

[0183] The measurement of the binding affinity of the wild-type antibody to the fusion protein was carried out as follows using isothermal titration calorimetry (ITC). The calorimetric titration experiment was performed at 25 °C using an Auto-iTC200 MicroCalorimeter (MicroCal). The protein was dialyzed overnight at 4 °C against 20 mM Tris, 150 mM NaCl pH 8.0. CSP-NPNA5.5-8x-1210 Fab, CSP-NPNA5.5-10x-1210 Fab, and CSP-NPNA5.5-12x-1210 Fab (10 μM) in the calorimetric cell were titrated with 1210 Fab (92 μM) by 15 consecutive injections of 2.5 μl. The experimental data were analyzed according to the 1:1 binding model of Origin 7.0 and are shown in Figures 14 to 16.

[0184] Example 8: Size Exclusion Chromatography Multi-Angle Light Scattering (SEC-MALS) The measurement of the absolute mass of the antibody-fusion protein interaction was carried out as follows. The 1210 Fab / CSP-NPNA5.5-linker-1210 Fab co-complex recovered from ITC was loaded onto a Superdex 200 Increase 10 / 300 GL (GE Healthcare) coupled in-line to an AKTA Pure chromatography system (GE Healthcare) equipped with the following calibrated detection systems: (i) MiniDawn Treos MALS detector (Wyatt); (ii) quasi-elastic light scattering (QELS) detector (Wyatt); and (iii) Optilab T-reX refractive index (RI) detector (Wyatt). Data processing was performed using ASTRA software (Wyatt) and is shown in Fig. 18A.

[0185] Example 9. Antibody-expressing Fab nanoparticles promote B cell activation when co-presented with an antigen The BG505 Env SOSIP trimer was cloned into the N-terminus of ferritin using the AgeI restriction site and the XbaI restriction site. The amino acid sequence of eOD-GT6 was obtained and added to the C-terminus of lumazine synthase, GGS 4xIt was separated by a linker and an NheI restriction site. A Strep Tag II was added to the C-terminus of the construct to facilitate affinity purification. The entire construct was codon-optimized for mammalian expression, synthesized, and cloned into the pHLsec expression vector using the restriction enzymes AgeI and XhoI (Figure 19). Fab HC nanoparticles, Fab LC, and antigen nanoparticles (Fab is denintuzumab, the antigen is BG505 SOSIP or eODGT6, and the nanoparticles are either ferritin or lumazine synthase) were transiently co-transfected into HEK293F (Thermo Fisher Scientific) in the manner described in the previous example and purified using the same protocol as above, except that BG505-containing nanoparticles were also purified by Galanthus Nivalis (GNL) lectin affinity using 500 mM sodium chloride washing and 1 M α-methyl mannoside elution. Negative stain electron microscopy was the same as described in the previous example. Biolayer interferometry was the same as described in the previous example, and CD19mVenus and VRC01 Fab were used as ligands coated on Ni-NTA biosensors and anti-human Fab biosensors to detect binding to denintuzumab nanoparticles and BG505 / eODGT6-nanoparticles, respectively. For the calcium flux assay (Figure 23), Bjab cells (1x10 6 cells) were incubated with 1 μM Fluo-4 dye (Life Technologies) in HBSS for 30 minutes. The cells were washed twice with 5 mL of 1X PBS and resuspended in 500 μl of RPMI on ice. Prior to acquisition, the cells were warmed in a 37 °C bath for 5 minutes and acquired at high values for 30 seconds in the FITC channel of a BD LSR Fortess Cell Analyzer to establish a baseline. Next, the indicated amount of nanoparticles was added to the cells and mixed quickly, and then data was acquired for 5 - 10 minutes or until the signal returned to baseline. The data was analyzed in FlowJo to establish the mean intensity and plotted over time.

[0186] Example 10: Design of single-chain Fc nanoparticles Single-chain Fc nanoparticles were designed using the following sequences. In the sequences, bold indicates the Fc domain, normal font indicates the linker, and underlined indicates ferritin:

Chemical formula

[0187] As shown by the ability to bind to a protein A column, the Fc domain of the antibody is correctly folded. As shown in Figure 24, it can be eluted by low pH or 3 M MgCl2. As shown by the monodisperse peak in size exclusion chromatography, the single-chain Fc nanoparticles are correctly assembled.

[0188] Example 4: Human B cell responses to repetitive epitopes are improved by anti-isotype affinity maturation Overview Through affinity maturation, B cells expressing somatic mutated antibody variants with improved antigen-binding properties are selected to protect against pathogen invasion. The inventors investigated the molecular mechanisms underlying the clonal selection and affinity maturation of human B cells expressing protective antibodies against the circumsporozoite protein (PfCSP) of the malaria parasite, Plasmodium falciparum. Due to the repeatability of PfCSP, direct isotypic interactions between two PfCSP repeat-binding monoclonal antibodies are promoted, molecular details showing that antigen affinity and B cell activation are thereby improved. These data provide a mechanistic explanation for the strong selection of somatic mutations that transmit isotypic antibody interactions after repeated parasite exposure in humans. The inventors' findings indicate different ways of antigen-mediated affinity maturation to improve antibody responses against PfCSP and possibly other repetitive antigens.

[0189] Materials and methods Genotyping This study was approved by the Ethics Committee of the Medical Faculty and University Clinic of the University of Tubingen and adhered to the standards for the conduct of clinical trials of pharmaceuticals and the principles of the Declaration of Helsinki. The clinical trials from which samples were obtained were registered under https: / / clinicaltrials.gov / ct2 / show / NCT02115516 and the number 2013-003900-38 in the EudraCT database and were conducted under approval from the Paul-Ehrlich-Institute under FDA IND 15862 (8, 9). Genomic DNA was extracted from whole blood. IGHV3 gene family segments were amplified using barcoded primers. Amplicons were pooled and prepared for sequencing using the TruSeq PCR-Free Library Preparation Kit (Illumina). Sequencing was performed on a MiSeq sequencer using a 300–300 bp paired-end protocol. Sequencing reads were assembled using PandaSeq (24) and assigned to donors by barcode identification.

[0190] Site-directed mutagenesis Site-directed mutagenesis in the antibody-encoding plasmid was performed using the Q5 Site-Directed Mutagenesis Kit (Qiagen).

[0191] Antibody and Fab production For IgG production, the IGH variable region and the IGK variable region were cloned into expression vectors upstream of the human IGK constant region and the IGG1 constant region, respectively, as previously described (25). Recombinant monoclonal antibodies were expressed in HEK293F cells (ThermoFisher Scientific), and the antibody concentration of the protein G Sepharose (GE healthcare) purified antibody was measured by ELISA as described above (9, 10). Fab was generated by papain digestion of IgG and purified by protein A chromatography, followed by cation exchange chromatography (MonoS, GE Healthcare) and size exclusion chromatography (Superdex 200 Increase 10 / 300 GL, GE Healthcare). For ITC studies, the IGH variable region and the IGK variable region were cloned into the pcDNA3.4 TOPO expression vector immediately upstream of the human IGK constant region and the CH1 constant region, respectively. Fab was transiently expressed in HEK293F cells (ThermoFisher Scientific) and purified by KappaSelect affinity chromatography (GE Healthcare) and size exclusion chromatography (Superdex 200 Increase 10 / 300 GL, GE Healthcare).

[0192] Antigen production ELISA was performed against NANP5 (Alpha Diagnostic International), NANP3 (PSL GmbH, Heidelberg), or PfCSP in the form of the N-terminally truncated form expressed in Escherichia coli (E. coli) as previously described (10, 26). For BLI, SEC-MALS, and single particle negative stain EM, full-length PfCSP (NF54 strain) was cloned into pcDNA3.4-TOPO for transient expression in HEK293F cells. PfCSP was purified by HisTrap Ni / NTA (GE Healthcare) and size exclusion chromatography (Superdex 200 Increase 10 / 300 GL, GE Healthcare).

[0193] Surface plasmon resonance Surface plasmon resonance measurements were performed on a BIACORE T200 instrument (GE Healthcare) docked to a Series S sensor chip CM5 (GE Healthcare). 10 millimolar HEPES containing 150 mM NaCl at pH 7.4 was used as the running buffer as described (9). Anti-human IgG antibodies were immobilized on the chip using an amine coupling-based human antibody capture kit. Equal concentrations of sample antibody and isotype control were captured in the sample flow cell and reference flow cell, respectively. To equilibrate the flow cell, the running buffer was injected at a rate of 10 μL / min for 20 minutes. 0.015 μM, 0.09 μM, 0.55 μM, 3.3 μM, and 20 μM of NANP3 in the running buffer were injected at a rate of 30 μL / min. The flow cell was regenerated with 3 M MgCl2. Data were fit by steady-state kinetics analysis using BIACORE T200 software V2.0.

[0194] Crystallization and structure determination Purified 1210 Fab and chimeric H.2140 / K.1210 Fab were concentrated to 12 mg / mL and diluted to 10 mg / mL with NANP5 (10 mg / mL) and NANP3 (10 mg / mL) at a 1:5 molar ratio, respectively, before crystallization trials. Purified 1450 Fab was mixed with NANP5 at a 3:1 molar ratio and purified by size-exclusion chromatography (Superdex 200 Increase 10 / 300 GL, GE Healthcare) to remove excess 1450 Fab. Then, purified 1450-NANP5 was concentrated to 6 mg / mL before crystallization trials. The 1210-NANP5 co-crystal grew in 20% (w / v) PEG 3350 and 0.2 M sodium citrate and was cryoprotected with 15% (w / v) ethylene glycol. The co-crystal of chimeric H.2140 / K.1210 Fab that formed a complex with NANP3 grew in 20% (w / v) PEG 4000, 0.6 M sodium chloride, and 0.1 M MES pH 6.5 and was cryoprotected with 15% (w / v) glycerol. The 1450-NANP5 co-crystal grew in 22.5% (w / v) PEG 3350 and 0.2 M diammonium hydrogen citrate and was cryoprotected with 15% (w / v) ethylene glycol. Data were collected at the 08ID-1 beamline of the Canadian Light Source (CLS) or the 23-ID beamline of the Advanced Photon Source (APS) and processed and scaled using XDS (27). The structure was determined by molecular replacement using Phaser (28). Structure refinement was performed using phenix.refine (29), and refinement iterations were performed using Coot (30). Software was accessed via SBGrid (31).

[0195] Isothermal titration calorimetry The calorimetric titration experiments were performed at 25 °C using an Auto-iTC200 instrument (Malvern). The protein was dialyzed overnight at 4 °C against 20 mM Tris pH 8.0 and 150 mM sodium chloride. The NANP5 and NANP3 peptides were diluted to 2 - 3 μM in dialysis buffer and added to the calorimetric cell and titrated with 1210, 1210_GL, 1210 H.D100Ymut_K.N92Ymut (1210_YY) Fab, and 1210_H.K56_Nrev_K.N93_Srev (1210_NS) Fab (100 μM) in 2.5 μl 15 consecutive injections. The experiments were performed at least three times and the mean values and standard errors of the mean were recorded (Figure 30). The experimental data were analyzed according to a 1:1 binding model using Origin 7.0. Statistical analysis was performed using a one-sided Mann-Whitney test in Prism.

[0196] Biolayer interferometry binding assay BLI (Octet RED96, ForteBio) experiments were performed to measure the binding affinity of 1210 and 1210_YY IgG to full-length PfCSP. Full-length PfCSP was diluted to 10 μg / mL in kinetics buffer (PBS, pH 7.4, 0.01% (w / v) BSA, and 0.002% Tween20) and immobilized on a Ni / NTA (NTA) biosensor (ForteBio). After loading the ligand in the kinetics buffer to establish a stable baseline, the biosensor was immersed into wells containing a two-fold dilution series of IgG. Subsequently, the chip was immersed in the kinetics buffer to monitor the dissociation rate. The kinetic data were analyzed using ForteBio's data analysis software 9.0 and the curves were fitted to a 1:1 binding model.

[0197] Size exclusion chromatography - multi-angle light scattering (SEC / MALS) The NANP5 peptide was complexed with a 3-fold molar excess of 1210 Fab and loaded onto a Superdex 200 Increase 10 / 300 GL (GE Healthcare) in-line coupled to an AKTA Pure chromatography system (GE Healthcare) equipped with the following calibrated detection systems: (i) MiniDawn Treos MALS detector (Wyatt); (ii) quasi-elastic light scattering (QELS) detector (Wyatt); and (iii) Optilab T-reX refractive index (RI) detector (Wyatt). 330 micrograms of full-length PfCSP was loaded onto a Superdex 200 Increase 10 / 300 GL (GE Healthcare) in-line coupled to an Agilent Technologies 1260 Infinity II HPLC equipped with the above detection system. The full-length PfCSP (5 μM) was complexed with a 20-fold molar excess of 1210 Fab (100 μM), and 100 μL or 400 μL was loaded onto a Superose 6 Increase 10 / 300 GL (GE Healthcare) in-line coupled to an Agilent Technologies 1260 Infinity II HPLC equipped with the above detection system. Data processing was performed using ASTRA software (Wyatt).

[0198] Negative stain transmission electron microscopy A 400-mesh Cu grid was coated with collodion, and a thin continuous layer of carbon was deposited onto the grid. The carbon grid was glow-discharged according to a standard protocol. A 3-μL droplet of 1210 Fab that formed a complex with full-length PfCSP was applied to the glow-discharged carbon grid. After 20 s, the grid was blotted, and 3 μL of 1% (w / v) uranyl formate solution was added three times in two lots, blotting in the middle for 5 s and for the final 18 s. Data were collected with an FEI Tecnai 20 operating at 200 kV. 120 images with a defocus value of 1–3 μm were collected. First, a total of 1080 particle images were manually selected with Relion 2.0 (32), and 2D classification of the particle images was performed allowing 10 classes. Subsequently, using the top 6 2D classes containing 947 particle images, 13,146 particle images were automatically selected from 120 micrographs, and 2D classification was performed allowing 50 classes.

[0199] Retroviral transduction of TKO-EST cells Pre-B cells from triple Rag2, λ5, and SLP-65 TKO-EST-deficient mice lacking endogenous BCR expression were reconstituted with genes for Ig heavy and light chains by retroviral transduction (33). For the generation of virus particles, full-length IGHM and IG KConstructs encoding variable regions were cloned into the pMIZCC and pMIZYN vector backbones (34). Phoenix-Eco virus packaging cells at 1.8 x 105 per well were seeded into 6-well culture plates in complete Iscove's modified Dulbecco's medium (IMDM, 5% FCS, 2 mM glutamine, 0.5 mL β-mercaptoethanol, and containing penicillin / streptomycin). After 24 hours, 0.5 μg of heavy chain and 0.5 μg of light chain plasmids were transfected into the cells using 3 μl of GeneJuice reagent in 100 μl of pure IMDM and incubated at 37 °C and 8% CO2 for 48 hours. The supernatant was collected and virus particles were purified using a 0.45 μm filter. 1 μl / mL of polybrene was added to the virus particle suspension. In parallel, 2 x 105 TKO-EST cells were transferred into 1.5 mL tubes and centrifuged (366 xg, 4 °C, 5 minutes). The supernatant was discarded and the cell pellet was resuspended in 800 μl of the virus particle suspension. Spin transduction was performed on TKO-EST cells at 366 xg and 37 °C. After 3 hours, the medium was replaced with fresh complete IMDM supplemented with IL-7 and the cells were seeded into 6-well plates.

[0200] Ca2+ Flux Measurement Ca2+ flux was measured as described in (33). After viral transduction, 1×106 TKO-EST cells were loaded with the calcium-sensitive dye Indo-1 AM (Molecular Probes) at 37 °C for 45 minutes. The Indo-1 staining solution was prepared by mixing 25 μl of Indo-1 stock solution (prepared by diluting 50 μg of Indo-1 in 25 μl of DMSO), 25 μl of pluronic acid F-127, and 113 μl of FCS, and incubated (5 minutes, in the dark, RT). The Indo-loaded cells were washed with 5 mL of 1% FCS IMDM, resuspended in 500 μl of 1% FCS IMDM, and transferred to a FACS tube. Each sample was preheated individually on a hot plate at 37 °C for 10 minutes before measurement. After the baseline of Ca2+ flux was recorded for 30 seconds with an LSR cytometer, 5 μl of antigen solution containing 4-hydroxytamoxifen (4-OHT, final concentration: 2 μM) was added, and the Ca2+ flux in response to the antigen was recorded for 6 minutes. Surface Ig expression in different cell lines was equally low when measured by FACS by binding of fluorescently labeled antibodies against IgM and IgK. Equivalent functions were confirmed in all cell lines when stimulated with 4-OHT and α-Igκ antibody (1 μg / mL).

[0201] Pf cross-section assay The Pf cross-section assay was performed in 96-well plate format as described (9, 10). Briefly, 75,000 Pf sporozoites obtained from the salivary glands of female Anopheles coluzzii mosquitoes were pre-incubated with monoclonal antibodies at different concentrations for 30 minutes and then incubated with HC-04 human hepatocytes in the presence of 0.5 mg / mL dextran / rhodamine (Molecular Probes). Untreated sporozoites and dextran / rhodamine alone were used as positive controls respectively to determine the background signal of the experiment. After fixation with 1% paraformaldehyde (PFA), the percentage of dextran-positive (i.e., cross-sectioned cells) was measured using an LSR II flow cytometer. The background signal was subtracted from all measurements. Cross-section inhibition was measured based on the cross-section rate observed with untreated sporozoites. Data for each antibody were pooled from at least three independent experiments and titration curves were fitted using a 3-parameter Hill function.

[0202] Immunization and infection of mice All animal experiments were approved by LAGeSo (H0027 / 12) in Berlin, Germany. Immunization and infection were performed as previously described (9, 10). Briefly, 8-week-old C57BL / 6 female mice (5 per group) were given passive immunization intraperitoneally with 100 μg or 30 μg of monoclonal human anti-PfCSP antibody or isotype control (mGO53 (35)) in 100 μl of PBS. Twenty-four hours after passive immunization, the mice were infected by subcutaneous injection of 5,000 PfCSP transgenic rodent malaria parasites (Plasmodium berghei) (Pb-PfCSP) (10) sporozoites into the tail base. From 3 to 12 days after infection, Giemsa-stained blood smear specimens were analyzed daily. At least 100 microscopic fields were counted to declare positive for parasites.

[0203] Results and discussion Sporozoites of Plasmodium falciparum (Pf), a human malaria parasite, express circumsporozoite protein (PfCSP), a surface protein with an immunodominant central NANP repeat region (1-3). When animal models are protected from Pf infection, antibodies against the repeat may be involved (4-6). However, protection mediated by anti-NANP antibodies is not easily achieved by vaccination. Therefore, induction of protective PfCSP NANP antibodies is a major goal in pre-erythrocytic vaccine development (7). We recently showed that the anti-NANP PfCSP memory B cell response in Pf-naïve volunteers after repeated exposure to live Pf sporozoites under chloroquine prophylaxis mainly matures by clonal selection and expansion of potent Pf-inhibitory IGHV3-33 and IGKV1-5 germline-encoded antibodies with an 8-amino acid (aa) long immunoglobulin (Ig) κ complementarity-determining region (CDR) 3 (KCDR3:8) (8, 9).

[0204] Here, we analyzed five representative germline or low-mutated antibodies with reported affinities of 10 -6 ~10 -9 M for the NANP pentamer peptide (NANP5) (Fig. 25A and Table 1) (9). Antigen binding was abrogated when the original IgVκ1-5 was replaced with Vκ2-28, or when the native Ig heavy chain (IgH) was paired with a Vκ1-5 light chain with a 9-aa long KCDR3 (Fig. 25B), demonstrating the importance of the characteristics of these specific Ig genes in antigen recognition.

Table 1

[0205] All VH3-33 / Vk1-5 / KCDR3:8 antibodies were encoded by the IGHV3-33*01 allele (9). IGHV3-33 *Position 52 of IgH CDR (HCDR) 2, which precisely encodes tryptophan rather than serine or arginine, differs from three gene segments (IGHV3-30, IGHV3-30-3, and IGHV3-30-5) that are otherwise very similar (Tables 2 and 3). The H.W52_A variant in antibody 2140, and the IGHV3-30 * 02 and IGHV3-30-5 * All of the H.W52_S and H.W52_R variants of the selected antibodies, including the double mutant (H.W52_R, H.V50_F) that mimics the IGHV3-30

Table 2

Table 3

[0206] Most of the NANP-reactive VH3-33 / Vκ1-5 / KCDR3:8 B cells belong to a clonal expansion and somatic hypermutation (SHM) diversification cell cluster that strongly selected for substitution mutations at HCDR1 (H.S31) and HCDR2 (H.V50, H.N56), as well as at KCDR3 (K.S93), which was presumably the result of affinity maturation (Figure 25, E and F) (9). As shown for the germline antibody 2163 and the low-mutation antibody 1210, the introduction of a defective mutation (mut) or revertant (rev) at position H.V50 and to a lesser extent at position H.S31 revealed a role in the binding to the minimal NANP3 peptide (10, 11) (Figure 25, G and H, and Table 4). In contrast, exchanges at positions H.N56 and K.S93, alone (1210_H.K56_N rev 、1210_K.N93_S rev 、2163_H.N56_K mut) However, neither the combination (1210_NS, 2163_KN) showed a significant effect (Figure 25, G and H, and Table 4). Therefore, affinity maturation to the repeat explained the strong selection of only two of the four characteristic substitution mutations of the VH3-33 / VK1-5 / KCDR3:8 anti-NANP antibody.

Table 4

[0207] Next, the inventors determined the co-crystal structure of the 1210 antigen-binding fragment (Fab) with NANP5 (Figure 26, Figure 27A, and Tables 5-7). The NANP core epitope contained a type I β-turn and an extended conformation (Figure 26, A and C, Figure 27B) that was consistent with previous observations and similar to NANP bound to the IgH 2140 / Igκ1210 chimeric antibody (Figure 27C and Tables 5 and 8) (10-14). The backbone atoms in KCDR3 were optimally arranged to mediate H-bonds with the repeat and are thought to contribute to the strong selection of the 8-amino acid-long KCDR3 (Figure 26, B and C, Tables 3, 6, and 11). The VH3-33 germline residues were involved in most of the antigen contacts, particularly H.V50 and H.W52 (residues encoded in a unique form by the IGHV3-33 allele), as well as H.Y52A and H.Y58 in HCDR2 (Table 6 and Figure 28) (15). Affinity maturation at H.V50 and H.S31 can be explained by the strengthening of van der Waals interactions with the repeat (Figure 26C).

Table 5

Table 6-1

Table 6-2

Table 6-3

Table 7

Table 8

Table 9-1

Table 9-2

Table 10

Table 11

[0208] In particular, the inventors' crystal structure also revealed that two 1210 Fabs (referred to as 1210 Fab-A and Fab-B) bind to one NANP5 peptide in a head-to-head conformation at an angle of 133° (Figures 26D and 29). This unique binding mode results in a buried surface area (BSA) of 263 Å2 between the two Fabs and six isotypic antibody-antibody H-bonds that yield an additional ~120 Å2 of BSA between the Fab and the repeat (Figures 26, E and F, and Tables 6, 7, and 11). Two highly selected mutations, H.N56_K and K.S93_N (Figures 25, E and F), form H-bonds with H.Y52A and H.S99 on the opposite Fab, thereby stabilizing the head-to-head conformation (Figures 26, G and H). Another explanation for the length constraint of KCDR3 was provided by the optimal contact of the 8-amino acid long KCDR3 with the HCDR3 of the opposite 1210 molecule.

[0209] To examine homotypic interactions, the inventors next measured the Fab affinities to NANP5 and NANP3 for 1210, 1210_NS (lacking the selected mutations involved in homotypic binding), 1210 H.D100_Ymut / K.N92_Ymut mutant (1210_YY, designed to disrupt head-to-head binding by steric clash), and 1210 germline (1210_GL) (FIGS. 26I and 30). Compared to 1210, 1210_YY and 1210_NS had significantly weaker affinities to NANP5 but not to NANP3, whereas in the case of 1210_GL, the binding of both peptides was severely deteriorated (FIGS. 26I and 30)(16). These data suggest that only 1210 efficiently recognized the repeat in the high-affinity homotypic head-to-head binding conformation. This hypothesis was confirmed by the analysis of full-length PfCSP with 38 NANP repeats. Approximately 12 1210 Fabs bound to PfCSP and recognized the NANP repeats in a head-to-head binding conformation similar to the 1210 Fab-NANP5 crystal structure (FIGS. 26, J and K, and FIG. 29D)(11, 17). Furthermore, 1210_YY, which has a limited ability to participate in homotypic antibody interactions, showed lower binding affinity to full-length PfCSP than 1210 (FIG. 31). Thus, through affinity maturation, mutations that improve homotypic antibody interactions are selected, thereby indirectly increasing the NANP binding of PfCSP.

[0210] To better understand the selection of SHM at the cellular level, the inventors measured the degree of B cell activation in response to NANP5 in transgenic B cell lines expressing 1210 or variant B cell receptors (BCRs) (FIGS. 32, A-D). BCR signaling was delayed in cells expressing 1210_GL compared to 1210. This effect was even more pronounced in 1210_YY mutant cells. As expected, particularly at low antigen concentrations, 1210_V50Imut, which has a high repeat affinity, transmitted a stronger signal than 1210, while 1210_NS showed no significant difference (FIG. 32D). Thus, B cell activation is promoted by both direct NANP binding and isotype antibody interactions. Despite a 2 log difference in NANP3 affinity (FIGS. 25, G and H) and the various possibilities that these antibodies are involved in isotype interactions, all showed a similar ability to inhibit Pf sporozoites in vitro (FIGS. 32E and FIG. 33). Similarly, all antibodies conferred a similar level of dose-dependent protection from blood-stage parasite development after passive immunization in mice, presumably due to a strong binding effect (FIG. 32F). These data provide a mechanistic explanation for the strong in vivo selection of anti-isotype antibody variants by affinity maturation, independent of the protective effect as a soluble antibody.

[0211] VH3 antibodies have a profound impact on the anti-PfCSP memory response (9, 11, 14). In addition to VH3-33 / Vκ1-5 / KCDR3:8, the inventors observed a cluster of affinity-matured VH3-23 / Vκ1-5 NANP-reactive memory B cell antibodies that were highly mutated upon selection (Figures 34, A and B) (9). The NANP5 binding mode of the representative VH3-23 / Vκ1-5 antibody 1450 is different from 1210, but it also recognizes NANP5 in a head-to-head conformation where the HCDRs are directly juxtaposed and the affinity-matured K.N30 residue forms an H-bond between Fab-A and Fab-B (Figures 34, C to E, Figure 35, A and B, Tables 5, 9, and 10). Sequence analysis of the VH3-23 / Vk1-5 antibody cluster confirmed the enrichment of amino acid exchanges that are directly involved in antibody-antigen interactions, antibody-antibody contacts, or are favorable for the 1450 paratope conformation optimal for NANP epitope recognition (Figure 34B).

[0212] After PfSPZ-CVac immunization of malaria-naive individuals, approximately 15% of PfCSP-reactive memory B cells exhibited the sequence characteristics of VH3-33 / Vκ1-5 / KCDR3:8 or VH3-23 / Vκ1-5 (Figure 34F) (18). Furthermore, these cells were strongly enriched in the expanded anti-PfCSP memory B cell pool compared to the non-expanded population (Figure 34G). Thus, anti-isotype affinity maturation is observed after repeated Pf sporozoite exposure with both low-affinity antibodies using combinations of low-mutation high-affinity VH3-33 antibodies and other genes (9). This phenomenon may also occur in the B cell response induced by RTS,S malaria vaccination (Figure 36) (11).

[0213] Thus, in addition to conventional antibody-antigen affinity maturation, anti-isotype affinity maturation promotes robust clonal expansion and competitive selection of PfCSP-reactive B cells in humans. Even in the absence of affinity maturation, VH3-33 / Vκ1-5 / KCDR3:8 antibodies are moderately strong NANP binders and potent Pf inhibitors. This is highly dependent on H.W52 of HCDR2. Since IGHV3-33 is located in a region of structural polymorphism of the IGH locus, particularly in Pf endemic regions, haplotype frequency may determine the efficient induction of protective humoral anti-PfCSP repeat responses upon vaccination (19). Indeed, one donor in our study was IGHV3-33 negative (Figure 37). We propose that anti-isotype affinity maturation may be a generalizable property of B cell responses when repetitive antigens (such as malaria) bring two antibodies into proximity to optimize binding and promote clustering of surface immunoglobulin molecules through isotypic interactions (20, 21).

[0214] References 1. F. Zavala, A. H. Cochrane, E. H. Nardin, R. S. Nussenzweig, V. Nussenzweig, Circumsporozoite proteins of malaria parasites contain a single immunodominant region with two or more identical epitopes (Malaria parasite circumsporozoite proteins contain a single immunodominant region with two or more identical epitopes). J. Exp. Med. (Journal of Experimental Medicine), 157, 1947-1957 (1983). 2. J.B. Dame, J.L. Williams, T.F. McCutchan, J.L. Weber, R.A. Wirtz, W.T. Hockmeyer, W.L. Maloy, J.D. Haynes, I. Schneider, D. Roberts, G.S. Sanders, E.P. Reddy, C.L. Diggs, L.M. Miller, Structure of the gene encoding the immunodominant surface antigen on the sporozoite of the human malaria parasite Plasmodium falciparum (Structure of the gene encoding the immunodominant surface antigen on the sporozoite of the human malaria parasite Plasmodium falciparum). Science 225, 593 - 599 (1984). 3. V. Enea, J. Ellis, F. Zavala, D.E. Arnot, A. Asavanich, A. Masuda, I. Quakyi, R.S. Nussenzweig, DNA cloning of Plasmodium falciparum circumsporozoite gene: Amino acid sequence of repetitive epitope (DNA cloning of Plasmodium falciparum circumsporozoite gene: Amino acid sequence of repetitive epitope). Science 225, 628 - 630 (1984). 4. P. Potocnjak, N. Yoshida, R.S. Nussenzweig, V. Nussenzweig, Monovalent fragments (Fab) of monoclonal antibodies to a sporozoite surface antigen (Pb44) protect mice against malarial infection. (Monovalent fragments (Fab) of monoclonal antibodies to a sporozoite surface antigen (Pb44) protect mice against malarial infection.) J. Exp. Med. 151, 1504 - 1513 (1980). 5. N. Yoshida, R. S. Nussenzweig, P. Potocnjak, V. Nussenzweig, M. Aikawa, Hybridoma produces protective antibodies directed against the sporozoite stage of malaria parasite. (Hybridoma produces protective antibodies directed against the sporozoite stage of malaria parasite.). Science (Science) 207, 71 - 73 (1980). 6. L. Foquet, C. C. Hermsen, G.-J. van Gemert, E. Van Braeckel, K. E. Weening, R. Sauerwein, P. Meuleman, G. Leroux-Roels, Vaccine-induced monoclonal antibodies targeting circumsporozoite protein prevent Plasmodium falciparum infection. (Vaccine-induced monoclonal antibodies targeting circumsporozoite protein prevent Plasmodium falciparum infection.). J. Clin. Invest. (Journal of Clinical Investigation) 124, 140 - 144 (2014). 7. E. M. Riley, V. A. Stewart, Immune mechanisms in malaria: New insights in vaccine development. (Immune mechanisms in malaria: New insights in vaccine development.). Nat. Med. (Nature Medicine) 19, 168 - 178 (2013). 8. B. Mordmuller, G. Surat, H. Lagler, S. Chakravarty, A. S. Ishizuka, A. Lalremruata, M. Gmeiner, J. J. Campo, M. Esen, A. J. Ruben, J. Held, C. L. Calle, J. B. Mengue, T. Gebru, J. Ibanez, M. Sulyok, E. R. James, P. F. Billingsley, K. C. Natasha, A. Manoj, T. Murshedkar, A. Gunasekera, A. G. Eappen, T. Li, R. E. Stafford, M. Li, P. L. Felgner, R. A. Seder, T. L. Richie, B. K. L. Sim, S. L. Hoffman, P. G. Kremsner, Sterile protection against human malaria by chemoattenuated PfSPZ vaccine. (Chemical attenuation of PfSPZ vaccine provides sterile protection against human malaria). Nature 542, 445 - 449 (2017). 9. R. Murugan, L. Buchauer, G. Triller, C. Kreschel, G. Costa, G. Pidelaserra Marti, K. Imkeller, C. E. Busse, S. Chakravarty, B. K. L. Sim, S. L. Hoffman, E. A. Levashina, P. G. Kremsner, B. Mordmuller, T. Hofer, H. Wardemann, Clonal selection drives protective memory B cell responses in controlled human malaria infection. (Clonal selection promotes protective memory B cell responses in controlled human malaria infection). Sci. Immunol. 3, eaap8029 (2018). 10. G. Triller, S. W. Scally, G. Costa, M. Pissarev, C. Kreschel, A. Bosch, E. Marois, B. K. Sack, R. Murugan, A. M. Salman, C. J. Janse, S. M. Khan, S. H. I. Kappe, A. A. A. Adegnika, B. Mordmuller, E. A. Levashina, J.-P. Julien, H. Wardemann, Natural parasite exposure induces protective human anti-malarial antibodies. Immunity 47, 1197-1209.e10 (2017). 11. D. Oyen, J. L. Torres, U. Wille-Reece, C. F. Ockenhouse, D. Emerling, J. Glanville, W. Volkmuth, Y. Flores-Garcia, F. Zavala, A. B. Ward, C. R. King, I. A. Wilson, Structural basis for antibody recognition of the NANP repeats in Plasmodium falciparum circumsporozoite protein. Proc. Natl. Acad. Sci. U.S.A 114, E10438-E10445 (2017). 12. A. Ghasparian, K. Moehle, A. Linden, J. A. Robinson, Crystal structure of an NPNArepeat motif from the circumsporozoite protein of the malaria parasite Plasmodium falciparum. Chem. Commun. 14, 174-176 (2006). 13. N.K. Kisalu, A.H. Idris, C. Weidle, Y. Flores-Garcia, B.J. Flynn, B.K. Sack, S. Murphy, A. Schon, E. Freire, J.R. Francica, A.B. Miller, J. Gregory, S. March, H.-X. Liao, B.F. Haynes, K. Wiehe, A.M. Trama, K.O. Saunders, M.A. Gladden, A. Monroe, M. Bonsignori, M. Kanekiyo, A.K. Wheatley, A.B. McDermott, S.K. Farney, G.-Y. Chuang, B. Zhang, N. Kc, S. Chakravarty, P.D. Kwong, P. Sinnis, S.N. Bhatia, S.H.I. Kappe, B.K.L. Sim, S.L. Hoffman, F. Zavala, M. Pancera, R.A. Seder, A human monoclonal antibody prevents malaria infection by targeting a new site of vulnerability on the parasite. (A human monoclonal antibody prevents malaria infection by targeting a new site of vulnerability on the parasite.) Nat. Med. (Nature Medicine) 24, 408-416 (2018). 14. J. Tan, B. K. Sack, D. Oyen, I. Zenklusen, L. Piccoli, S. Barbieri, M. Foglierini, C. S. Fregni, J. Marcandalli, S. Jongo, S. Abdulla, L. Perez, G. Corradin, L. Varani, F. Sallusto, B. K. L. Sim, S. L. Hoffman, S. H. I. Kappe, C. Daubenberger, I. A. Wilson, A. Lanzavecchia, A public antibody lineage that potently inhibits malaria infection through dual binding to the circumsporozoite protein. (A public antibody lineage that potently inhibits malaria infection through dual binding to the circumsporozoite protein). Nat. Med. (Nature·Medicine) 24, 401 - 407 (2018). 15. The importance of H.Y52A and H.Y58 for repeat reactivity was confirmed by alanine mutations of antibodies 1210, 2140, and 2219 (Figure 29). 16. All antibodies recognized NANP5 and NANP3 with binding stoichiometries of approximately 2 and approximately 1, respectively, demonstrating that NANP5, rather than the shorter NANP3, enables the binding of two Fabs. 17. C.R. Fisher, H.J. Sutton, J.A. Kaczmarski, H.A. McNamara, B. Clifton, J. Mitchell, Y. Cai, J.N. Dups, N.J. D’Arcy, M. Singh, A. Chuah, T.S. Peat, C.J. Jackson, I.A. Cockburn, T-dependent B cell responses to Plasmodium induce antibodies that form a high-avidity multivalent complex with the circumsporozoite protein. (Malaria parasite-induced T-dependent B cell responses induce antibodies that form a high-avidity multivalent complex with the circumsporozoite protein). PLOS Pathog. 13, e1006469 (2017). 18. B.J. DeKosky, T. Kojima, A. Rodin, W. Charab, G.C. Ippolito, A.D. Ellington, G. Georgiou, In-depth determination and analysis of the human paired heavy-and light-chain antibody repertoire. (In-depth determination and analysis of the human paired heavy- and light-chain antibody repertoire). Nat. Med. 21, 86-91 (2015). 19. C. T. Watson, K. M. Steinberg, J. Huddleston, R. L. Warren, M. Malig, J. Schein, A. J. Willsey, J. B. Joy, J. K. Scott, T. A. Graves, R. K. Wilson, R. A. Holt, E. E. Eichler, F. Breden, Complete haplotype sequence of the human immunoglobulin heavy chain variable, diversity, and joining genes and characterization of allelic and copy-number variation. (Complete haplotype sequence of the human immunoglobulin heavy chain variable, diversity, and joining genes and characterization of allelic and copy-number variation). Am. J. Hum. Genet. (American Journal of Human Genetics) 92, 530-546 (2013). 20. T. Hattori, D. Lai, I. S. Dementieva, S. P. Montano, K. Kurosawa, Y. Zheng, L. R. Akin, K. M. Swist-Rosowska, A. T. Grzybowski, A. Koide, K. Krajewski, B. D. Strahl, N. L. Kelleher, A. J. Ruthenburg, S. Koide, Antigen clasping by two antigen-binding sites of an exceptionally specific antibody for histone methylation. (Antigen clasping by two antigen-binding sites of an exceptionally specific antibody for histone methylation). Proc. Natl. Acad. Sci. U.S.A (Proceedings of the National Academy of Sciences of the United States of America) 113, 2092-2097 (2016). 21. H.M. Davies, S.D. Nofal, E.J. McLaughlin, A.R. Osborne, Repetitive sequences in malaria parasite proteins. (Repetitive sequences in malaria parasite proteins), FEMS Microbiol. Rev. (Federation of European Microbiological Societies Microbiology Reviews) 41, 923 - 940 (2017). 22. G. Yaari, J.A. Vander Heiden, M. Uduman, D. Gadala - Maria, N. Gupta, J.N.H. Stern, K.C. O’Connor, D.A. Hafler, U. Laserson, F. Vigneault, S.H. Kleinstein, Models of somatic hypermutation targeting and substitution based on synonymous mutations from high - throughput immunoglobulin sequencing data. (Models of somatic hypermutation targeting and substitution based on synonymous mutations from high - throughput immunoglobulin sequencing data). Front. Immunol. (Frontiers in Immunology) 4, 358 (2013). 23. N.T. Gupta, J.A. Vander Heiden, M. Uduman, D. Gadala - Maria, G. Yaari, S.H. Kleinstein, Change - O: A toolkit for analyzing large - scale B cell immunoglobulin repertoire sequencing data. (Change - O: A toolkit for analyzing large - scale B cell immunoglobulin repertoire sequencing data). Bioinformatics (Bioinformatics) 31, 3356 - 3358 (2015). 24. A. P. Masella, A. K. Bartram, J. M. Truszkowski, D. G. Brown, J. D. Neufeld, PANDAseq: Paired-end assembler for Illumina sequences. (PANDAseq: Paired-end assembler for Illumina sequences). BMC Bioinformatics 13, 31 (2012). 25. T. Tiller, E. Meffre, S. Yurasov, M. Tsuiji, M. C. Nussenzweig, H. Wardemann, Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning. (Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning). J. Immunol. 329, 112 - 124 (2008). 26. K. Tewari, B. J. Flynn, S. B. Boscardin, K. Kastenmueller, A. M. Salazar, C. A. Anderson, V. Soundarapandian, A. Ahumada, T. Keler, S. L. Hoffman, M. C. Nussenzweig, R. M. Steinman, R. A. Seder, Poly(I:C) is an effective adjuvant for antibody and multi-functional CD4+ T cell responses to Plasmodium falciparum circumsporozoite protein (CSP) and αDEC-CSP in non human primates. (Poly(I:C) is an effective adjuvant for antibody and multi-functional CD4+ T cell responses to Plasmodium falciparum circumsporozoite protein (CSP) and αDEC-CSP in non human primates). Vaccine 28, 7256 - 7266 (2010). 27. W. Kabsch, XDS. Acta Crystallogr. D 66, 125 - 132 (2010). 28. A. J. McCoy, R. W. Grosse-Kunstleve, P. D. Adams, M. D. Winn, L. C. Storoni, R. J. Read, Phaser crystallographic software. J. Appl. Crystallogr. 40, 658 - 674 (2007). 29. P. D. Adams, P. V. Afonine, G. Bunkoczi, V. B. Chen, I. W. Davis, N. Echols, J. J. Headd, L.-W. Hung, G. J. Kapral, R. W. Grosse-Kunstleve, A. J. McCoy, N. W. Moriarty, R. Oeffner, R. J. Read, D. C. Richardson, J. S. Richardson, T. C. Terwilliger, P. H. Zwart, PHENIX: A comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213 - 221 (2010). 30. P. Emsley, B. Lohkamp, W. G. Scott, K. Cowtan, Features and development of Coot. Acta Crystallogr. D 66, 486 - 501 (2010). 31. A. Morin, B. Eisenbraun, J. Key, PC Sanschagrin, MATimony, M. Ottaviano, P. Sliz, Collaboration gets the most out of software. Elife 2, e01456 (2013). 32. S.H.W. Scheres, A Bayesian view on cryo-EM structure determination. J. Mol. Biol. 415, 406-418 (2012). 33. S. Meixlsperger, F. Kohler, T. Wossning, M. Reppel, M. Muschen, H. Jumaa, Conventional light chains inhibit the autonomous signaling capacity of the B cell receptor. Immunity 26, 323-333 (2007). 34. F. Kohler, E. Hug, C. Eschbach, S. Meixlsperger, E. Hobeika, J. Kofer, H. Wardemann, H. Jumaa, Autoreactive B cell receptors mimic autonomous pre-B cell receptor signaling and induce proliferation of early B cells. Immunity 29, 912-921 (2008). 35. H. Wardemann, S. Yurasov, A. Schaefer, J. W. Young, E. Meffre, M. C. Nussenzweig, Predominant autoantibody production by early human B cell precursors. (Initial human B cell precursors produce dominant autoantibodies). Science 301, 1374 - 1377 (2003)

[0215] Example 5: Immunization experiment Figure 38 shows that the malaria vaccine antigen (CSP - NANP5.5 - linker antibody) elicits an IgG titer that can recognize the full - length PfCSP antigen. As expected, the response is boostable and increases with three administrations. In these two examples, the malaria vaccine is displayed on two different nanoparticles, and one elicits a stronger immune response than the other. Figure 39 shows the activity / function of the anti - PfCSP serum induced from the immunization in Figure 38. This is measured by the sporozoite traversal inhibition assay. At a given serum dilution, depending on the method of presenting the malaria vaccine on the nanoparticles, the inhibitory activity varies between 50% and 80%. These results show that 1) the malaria vaccine described herein induces an anti - malaria immune response, and 2) the immune serum obtained has an inhibitory ability against sporozoites.

Sequence Listing Free - Text

[0216] Sequence Listing 1 <223> Antigen Sequence Listing 2 <223> Antibody Sequence Listing 3 <223> Epitope Sequence Listing 4 <223> Flag tag Sequence Listing 5 <223> Antibody Sequence Listing 6 <223> Nanoparticle Sequence Listings 7 - 25 <223> Antibody

Claims

1. nanocage monomers, and A fusion protein comprising an antibody or fragment thereof linked to the nanocage monomer, said antibody or fragment thereof comprising a first member of a binding pair; A fusion protein, wherein a plurality of said fusion proteins self-assemble to form a nanocage, and a plurality of said antibodies or fragments thereof decorate the outer surface of said nanocage, thereby exposing a first component of said binding pair for interaction with a second component of said binding pair.

2. The fusion protein of claim 1 , wherein the first component of the binding pair is an Fc portion of an antibody or fragment thereof and the second component of the binding pair is an Fc receptor.

3. The fusion protein of claim 1 , wherein the first member of the binding pair is an antigen-binding epitope and the second member of the binding pair is an antigen.

4. The fusion protein of any one of claims 1 to 3, wherein the nanocage comprises about 3 to about 100 nanocage monomers, such as 24 or 60 monomers.

5. The fusion protein of any one of claims 1 to 4, wherein the nanocage monomer is selected from ferritin, encapsulin, SOR, lumazine synthase, pyruvate dehydrogenase, carboxysome, vault protein, GroEL, heat shock proteins, E2P, MS2 coat protein, fragments thereof, and variants thereof.

6. The fusion protein of any one of claims 1 to 5, further comprising a linker between the nanocage monomer and the antibody or fragment thereof.

7. The fusion protein of claim 6, wherein the linker is flexible or rigid and comprises from about 1 to about 30 amino acid residues.

8. The fusion protein of claim 7, wherein the linker comprises from about 8 to about 16 amino acid residues.

9. The fusion protein of any one of claims 6 to 8, wherein the linker comprises a GGS repeat.

10. The fusion protein of claim 9 , wherein the linker comprises four GGS repeats.

11. The fusion protein of any one of claims 3 to 10, further comprising an antigen.

12. The fusion protein of any one of claims 3 to 11, wherein the antigen comprises a repeat domain.

13. The fusion protein according to any one of claims 3 to 12, wherein the antigen is a malaria antigen.

14. The fusion protein of claim 13, wherein the antigen is a fragment of a malarial CSP protein.

15. The fusion protein of claim 14, wherein the antigen is a fragment of the NANP repeat domain of the malarial CSP protein.

16. The fusion protein of claim 15, wherein the antigen comprises 5.5 NANP repeats.

17. The fusion protein of claim 16, wherein the antigen is NPNANPNANANPNANPNANPNANPNANP.

18. The fusion protein of any one of claims 1 to 17, wherein the antibody or fragment thereof is specific for the repeat domain.

19. The fusion protein of any one of claims 1 to 18, wherein the antibody or fragment thereof is specific for a malaria antigen.

20. 20. The fusion protein of claim 19, wherein the antibody or fragment thereof is specific for a malarial CSP protein.

21. 21. The fusion protein of claim 20, wherein the antibody or fragment thereof is specific for the NANP repeat domain of the malarial CSP protein.

22. The antibody or fragment thereof has the following sequence: [0010] The fusion protein according to any one of claims 1 to 21, comprising a sequence having at least 90% sequence identity to, or a fragment thereof.

23. The antibody or fragment thereof has the following sequence: [0025] 23. The fusion protein of claim 22, comprising:

24. The antibody or fragment thereof has the sequence: [0030] The fusion protein of claim 23, comprising:

25. The fusion protein of any one of claims 1 to 28, wherein the antibody or fragment thereof is specific for a tumor antigen.

26. The fusion protein of any one of claims 1 to 28, wherein the antibody or fragment thereof is specific for an autoantigen.

27. The fusion protein of any one of claims 1 to 10, wherein the antibody or fragment thereof is specific for CD19, CD22, CD79, BCMA, or CD20.

28. The fusion protein according to any one of claims 1 to 10, wherein the antibody or fragment thereof is specific for a target organ.

29. The fusion protein of any one of claims 1 to 28, wherein the antibody or fragment thereof comprises a heavy and / or light chain of a Fab fragment.

30. The fusion protein of any one of claims 1 to 28, wherein the antibody or fragment thereof comprises an Fc fragment.

31. The fusion protein of any one of claims 1 to 28, wherein the antibody or fragment thereof comprises an scFv.

32. 32. The fusion protein of claim 31 further comprising a Fab light chain and / or a heavy chain.

33. 32. The fusion protein of claim 31 , which is associated with a separately produced Fab light chain and / or a Fab heavy chain.

34. The fusion protein of any one of claims 1 to 33, further comprising a detectable moiety.

35. 35. The fusion protein of claim 34, wherein the detectable moiety is a fluorescent protein such as GFP, EGFP, ametrine, and / or a flavin-based fluorescent protein such as an LOV protein such as iLOV.

36. A nanocage comprising at least one fusion protein according to any one of claims 1 to 35.

37. The nanocage of claim 36, wherein each nanocage monomer comprises a fusion protein of any one of claims 1 to 32.

38. The nanocage of claim 36, wherein about 20% to about 80% of the nanocage monomers comprise the fusion protein of any one of claims 1-26.

39. The nanocage of any one of claims 36-38, wherein the nanocage is multivalent.

40. The nanocage of any one of claims 36 to 39, carrying a cargo molecule, such as a pharmaceutical, diagnostic and / or imaging agent.

41. 41. The nanocage of claim 40, wherein the cargo molecule is a protein and is fused to the fusion protein such that the cargo molecule is encapsulated within the nanocage.

42. 42. The nanocage of claim 41, wherein the cargo molecule is a flavin-based fluorescent protein such as a fluorescent protein, such as GFP, EGFP, ametrine, and / or an LOV protein, such as iLOV.

43. 43. The nanocage of claim 42, wherein the cargo molecule is not fused to the fusion protein and is encapsulated within the nanocage.

44. The nanocage of any one of claims 36 to 43, wherein the cargo molecule contained therein provides a T cell epitope, but optionally does not provide a B cell epitope.

45. The nanocage of any one of claims 36 to 43, wherein the cargo molecule is fused to the fusion protein and is contained therein to provide a T cell epitope, but optionally not a B cell epitope.

46. 44. The nanocage of claim 43, wherein the cargo molecule is a small molecule, a radioisotope, or a magnetic particle.

47. The nanocage of any one of claims 36 to 46, further comprising an antigen on the surface.

48. 48. The nanocage of claim 47, wherein the antigen is expressed as a fusion protein with a nanocage monomer.

49. A vaccine comprising the nanocages of any one of claims 36 to 48.

50. A nucleic acid molecule encoding a fusion protein according to any one of claims 1 to 35.

51. A vector comprising the nucleic acid molecule of claim 50.

52. A host cell comprising the vector according to claim 51 and producing the fusion protein according to any one of claims 1 to 35.

53. A method of conferring immunity to a subject, comprising administering the nanocage of any one of claims 36 to 48 or the vaccine of claim 59.

54. A method of treating and / or preventing a disease or condition comprising administering a nanocage according to any one of claims 36 to 48 or a vaccine according to claim 49.

55. 55. The method of claim 54, wherein the disease or condition is cancer, HIV, malaria, or an autoimmune disease.

56. A method of diagnostic imaging comprising administering a nanocage according to any one of claims 36 to 48 to a subject, tissue or sample, and imaging the subject, tissue or sample, wherein the nanocage comprises a diagnostic label, such as a fluorescent protein or a magnetic imaging moiety.

57. Use of the fusion protein of any one of claims 1 to 35 or the nanocage of any one of claims 36 to 48 as a research tool, such as in FACS or ELISA.

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