Immunogenic scaffolds and use of same in antigenic peptide display

Abstract

The present invention provides a recombinant protein including: (i) the amino acid sequence QLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILA (SEQ ID NO: 6) or an analog thereof having at least 90% identity thereto; (ii) a first peptide of interest: (iii) the amino acid sequence HTTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLE (SEQ ID NO: 7) or an analog thereof having at least 90% identity thereto; and (iv) a second peptide of interest. Further provided is a method for treating a subject afflicted with a neurological disease or condition associated therewith by administration of the recombinant protein.

Description

[0001] IMMUNOGENIC SCAFFOLDS AND USE OF SAME IN ANTIGENIC PEPTIDE DISPLAY

[0002] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0003]

[0001] The contents of the electronic sequence listing (BGU-P-0147-PCT.xml; size: 40,781 bytes; and date of creation: June 25, 2025) are herein incorporated by reference in its entirety.

[0004] CROSS REFERENCE TO RELATED APPLICATIONS

[0005]

[0002] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 667,151, entitled “6-Helix Protein As Immunogenic Scaffold For Antigen Peptide Display”, filed July 3, 2024, the contents of which are incorporated herein by reference in their entirety.

[0006] FIELD OF INVENTION

[0007]

[0003] The present disclosure relates to immunogenic scaffolds for antigenic peptide display, and more particularly to a 6-helix protein scaffold for presenting antigenic peptides to elicit an immune response.

[0008] BACKGROUND

[0009]

[0004] Immunogenic scaffolds have emerged as a promising approach for presenting antigenic peptides to the immune system in order to elicit targeted immune responses. These scaffolds provide a structural framework for displaying multiple copies of antigenic epitopes in a spatially defined manner. By mimicking the natural presentation of antigens on pathogens, immunogenic scaffolds aim to enhance the immunogenicity of peptide antigens and generate more robust and specific immune responses.

[0010]

[0005] Various protein-based scaffolds have been explored for antigen display, including viruslike particles, self-assembling protein nanoparticles, and engineered protein domains. These scaffolds offer advantages such as multivalent antigen presentation, stability, and the ability to incorporate T cell epitopes to further boost immunogenicity. However, challenges remain in optimizing scaffold design to properly orient antigens, maintain their native conformation, and elicit the desired type of immune response.

[0011]

[0006] The development of effective immunogenic scaffolds has implications for vaccine design against infectious diseases, cancer immunotherapy, and treatment of protein misfolding disorders. For neurodegenerative diseases characterized by accumulation of misfolded proteins, such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis, immunogenic scaffolds may provide a means to generate antibodies targeting pathogenic protein conformations. However, careful antigen selection and scaffold engineering are required to avoid potentially harmful autoimmune responses.

[0012]

[0007] Advances in protein engineering, structural biology, and immunology continue to drive innovation in the design of immunogenic scaffolds. Computational approaches are increasingly being applied to predict optimal epitope display and scaffold stability. Additionally, high- throughput screening methods enable rapid evaluation of scaffold variants. Despite progress, further research is needed to develop scaffolds that can reliably elicit potent, long-lasting, and appropriately targeted immune responses for diverse biomedical applications.

[0013]

[0008] As the field evolves, considerations such as manufacturability, stability, and in vivo trafficking of immunogenic scaffolds will be important for clinical translation. Continued interdisciplinary efforts combining expertise in immunology, protein engineering, and disease biology hold promise for realizing the full potential of immunogenic scaffolds as a versatile platform technology.

[0014] SUMMARY

[0015]

[0009] According to the first aspect, there is provided a recombinant protein comprising: (i) the amino acid sequence QLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILA (SEQ ID NO: 6) or an analog thereof having at least 90% identity thereto; (ii) a first peptide of interest: and (iii) the amino acid sequence HTTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLE (SEQ ID NO: 7) or an analog thereof having at least 90% identity thereto; and (iv) a second peptide of interest.

[0016]

[0010] According to another aspect, there is provided a composition comprising a therapeutically effective amount of the recombinant protein of the invention, and a pharmaceutically acceptable carrier.

[0017] [Oil] According to another aspect, there is provided an artificial DNA molecule encoding the recombinant protein of the invention.

[0018]

[0012] According to another aspect, there is provided a recombinant cell comprising the artificial DNA molecule of the invention.

[0013] According to another aspect, there is provided a method for preparing a recombinant protein, the method comprising culturing the recombinant cell of the invention, such that a recombinant protein encoded by the artificial DNA molecule is expressed.

[0019]

[0014] According to another aspect, there is provided a method for treating a subject afflicted with a neurological disease or condition associated therewith, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the recombinant protein of the invention, thereby treating a subject afflicted with the neurological disease or condition associated therewith.

[0020]

[0015] In some embodiments, (i) to (iv) form a unit repeating at least three times in the recombinant protein.

[0021]

[0016] In some embodiments, the recombinant protein comprises the amino acid sequence set forth in any one of SEQ ID Nos: 22-31.

[0022]

[0017] In some embodiments, the recombinant protein further comprises (v) a peptide linker located between any one of: the (i) and the (ii), between the (ii) and the (iii), between the (iii) and the (iv), and any combination thereof.

[0023]

[0018] In some embodiments, the peptide linker is of 1 to 8 amino acids.

[0024]

[0019] In some embodiments, the peptide linker is a glycine residue, or comprises the amino acid sequence GSSGG (SEQ ID NO: 3) or GGSGG (SEQ ID NO: 4).

[0025]

[0020] In some embodiments, (i) to (iv) are organized sequentially from the N’ -terminal end to the C’ -terminal end of said repeating unit in the recombinant protein.

[0026]

[0021] In some embodiments, (i) to (v) are organized as (i) - (v) - (ii) - (v) - (iii) - (v) - (iv) from the N’ -terminal end to the C’ -terminal end of the repeating unit in the recombinant protein.

[0027]

[0022] In some embodiments, the recombinant protein further comprises the linker located between each of the at least three units repeating in the recombinant protein.

[0028]

[0023] In some embodiments, the recombinant protein further comprises at least one third peptide of interest.

[0029]

[0024] In some embodiments, the at least one third peptide of interest is positioned: N’ terminally to the amino acid sequence set forth in SEQ ID NO: 6 or the analog thereof having at least 90% identity thereto; C’ terminally to the second peptide of interest; or both.

[0030]

[0025] In some embodiments, the at least one third peptide of interest comprises two copies of the third peptide of interest positioned C’ terminally to the second peptide of interest.

[0026] In some embodiments, the first, second, and at least one third peptides of interest are identical or comprise different amino acid sequences.

[0031]

[0027] In some embodiments, any one of the first, second, and at least one third peptides of interest is an immunogenic peptide.

[0032]

[0028] In some embodiments, the immunogenic peptide is a fragment of a protein selected from the group consisting of: superoxide dismutase 1 (SOD1), alpha-synuclein, amyloid-beta (AP), tubulin associated unit (TAU), transactive response DNA binding protein 43 (TDP-43), and any combination thereof.

[0033]

[0029] In some embodiments, the fragment is of 8 to 30 amino acids.

[0034]

[0030] In some embodiments, the immunogenic peptide is a fragment of any one of: (a) SOD1 comprising the amino acid sequence set forth in SEQ ID Nos: 2, 14, or both; (b) alpha-synuclein the amino acid sequence set forth in SEQ ID NO: 32; (c) AP comprising the amino acid sequence set forth in SEQ ID NO: 33; (d) TDP-43 comprising the amino acid sequence set forth in SEQ ID Nos: 34, 35, or both; and (e) TAU comprising the amino acid sequence set forth in SEQ ID NO: 36, 37, or both.

[0035]

[0031] In some embodiments, the recombinant protein further comprises a detection tag, a purification tag, or both, and optionally wherein the detection or purification tag is connected to the amino acid set forth in SEQ ID NO: 7 of the C’-terminus of the recombinant protein via the linker.

[0036]

[0032] In some embodiments, the recombinant protein further comprises a signal peptide.

[0037]

[0033] In some embodiments, the signal peptide is the N-term tPA secretory signal comprising the amino acid sequence set forth in SEQ ID NO: 5.

[0038]

[0034] In some embodiments, the recombinant protein further comprises a transmembrane domain.

[0039]

[0035] In some embodiments, the transmembrane domain is the transmembrane domain of CD28 comprising the amino acid sequence set forth in SEQ ID NO: 9.

[0040]

[0036] In some embodiments, the recombinant protein comprises the amino acid sequence set forth in SEQ ID Nos: 10-13, and 15-21.

[0041]

[0037] In some embodiments, the composition is a vaccine.

[0042]

[0038] In some embodiments, the composition is for use in treatment of a neurological disease or condition associated therewith in a subject in need thereof.

[0039] In some embodiments, the method further comprises a step before the culturing comprising introducing or transfecting a cell with the artificial DNA molecule of the invention, thereby providing the recombinant cell of the invention.

[0043]

[0040] In some embodiments, the treating comprises delaying the appearance of at least one symptom, progression, or both, of the neurological disease or condition associated therewith.

[0044]

[0041] The foregoing general description of the illustrative embodiments and the following detailed description thereof are merely exemplary aspects of the teachings of this disclosure and are not restrictive.

[0045] BRIEF DESCRIPTION OF FIGURES

[0046]

[0042] Non-limiting and non-exhaustive examples are described with reference to the following figures.

[0047]

[0043] Fig. 1 includes a sequence of a 6-Helix protein (SEQ ID NO: 1). Interhelical loops are bolded, and glycosylation sites are underlined.

[0048]

[0044] Fig. 2 includes a model of 6-Helix-based chimeric antigenic protein structure generated by AlphaFold. In the chimeric antigen structure, the N peptide and C peptide segments are observed as thin ribbons / coils and the pathogenic P6 / |37 loop epitopes of SOD1 (SE21 peptide sequence (as SEQ ID NO: 2)) are observed as dark thick ribbons.

[0049]

[0045] Figs. 3A-3C includes a non-limiting schematic representation of three 6-Helix-based antigenic constructs of the invention, in some embodiments. (3A) Cytosolic; (3B) Secreted; and (3C) Transmembrane. SE21 - SOD1 derived sequence containing P6 / P7 loop epitope.

[0050]

[0046] Fig. 4 includes fluorescent micrographs showing immunofluorescence assay of mRNA constructs expressed in SHSY-5Y cells. Three channels were used, including Alexa fluor 488 channel for Wheat germ agglutinin (WGA) to stain cell skeleton (outer membrane and the Golgi); Alexa fluor 555 channel for anti-V5 tag (D3H8Q) to stain Helix-6 antigenic protein; and DAPI channel for nuclei. The same microscopic settings were reused across the samples.

[0051]

[0047] Figs. 5A-5C include a non-limiting scheme and vertical bar graphs showing that 9SE- Helx-6His (Agl) construct is highly immunogenic and results in the production of specific antibodies against misfolded SOD1. The sera from mice immunized according to the scheme in (5A) were tested by ELISA using apo-SODlG93Aprotein as coating antigen and holo-SODlWTas specificity control (5B-5C). Data presented are means ± SEM of the readings from three mice (in duplicates) after background subtraction.

[0048] Fig. 6 includes a vertical bar graph showing vaccination of SOD1G93AALS mice with Ag2 / Alhydrogel produced stable immunity against P6 / |37 loop epitope. The immunized mice were bled at the indicated time points, and sera (the numbers indicate dilution factor) were analyzed by ELISA using recombinant SOD1G93Aas antigen (0.5 pg).

[0052]

[0049] Figs. 7A-7B include fluorescent micrographs showing that vaccination of SOD1G93AALS mice with Ag2 / Alhydrogel produced Abs that selectively recognized disease-relevant conformations of misfolded SOD1 in vivo. Lumbar spinal cord sections of WT (7A) and endstage SOD1G93A(7B) mice stained with the serum (1: 100 dilution) from immunized SOD1G93AALS mice.

[0053]

[0050] Fig. 8 includes a vertical bar graph showing that the SOD1G93AALS mice immunized with Ag2 / Alhydrogel produced Abs that specifically recognize misfolded SOD1 mutants. The sera (1: 1,000 dilution) were analyzed by ELISA using recombinant purified SOD1 variants as coating antigens (0.5 pg).

[0054]

[0051] Figs. 9A-9I include graphs showing that vaccination with Ag2 delays the onset of clinical symptoms in hSODlG93AALS mouse model - males. (9A) Disease progression. Average NeuroScore is calculated among the mice of the same age in cohort and plotted against the mice age. (9B-9C) Disease onset defined as time of the appearance of first motor abnormalities (NeuroScore 1). (9D), Grip strength - forelimbs. (9E-9F) Grip strength - hindlimbs. The IHC analysis of the lumbar spinal cord sections using B8H10 antibody (9G), anti-ChAT (9H) or anti- GFAP (91) antibody, (n [mice] =5). Results represent means ± SEM. An unpaired parametric t- test was used to evaluate the significance of the differences between the indicated groups.

[0055]

[0052] Figs. 10A-10F include graphs showing that vaccination with Ag2 delays the onset of clinical symptoms in hSODlG93AALS mouse model - females. The legend of 10A-10F is as in Figs. 9A-9F.

[0056] DETAILED DESCRIPTION

[0057]

[0053] The following description sets forth exemplary aspects of the present disclosure. It should be recognized, however, that such description is not intended as a limitation on the scope of the present disclosure. Rather, the description also encompasses combinations and modifications to those exemplary aspects described herein.

[0058] Recombinant protein

[0059]

[0054] According to one aspect, there is provided a recombinant protein comprising: (i) the amino acid sequence QLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILA (SEQ ID NO: 6) or an analog thereof having at least 80%, 90%, 95%, 97%, or 99% identity thereto, or any value and range therebetween. In some embodiments, the recombinant protein comprises: (i) the amino acid sequence QLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILA (SEQ ID NO: 6) or an analog thereof having 80-100%, 90-100%, 95-100%, 97-100%, or 99- 100% identity thereto. Each possibility represents a separate embodiment of the invention.

[0060]

[0055] According to another aspect, there is provided a recombinant protein comprising: (ii) a first peptide of interest.

[0061]

[0056] According to another aspect, there is provided a recombinant protein comprising: (iii) the amino acid sequence HTTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLE (SEQ ID NO: 7) or an analog thereof having at least 90% identity thereto.

[0062]

[0057] According to another aspect, there is provided a recombinant protein comprising: (iv) a second peptide of interest.

[0063]

[0058] According to another aspect, there is provided a recombinant protein comprising: (i) the amino acid sequence QLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILA (SEQ ID NO: 6) or an analog thereof having at least 90% identity thereto; (ii) a first peptide of interest; (iii) the amino acid sequence HTTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLE (SEQ ID NO: 7) or an analog thereof having at least 90% identity thereto; and (iv) a second peptide of interest.

[0064]

[0059] As used herein, the term “recombinant protein” refers to protein that is produced using genetic engineering techniques. These proteins are typically created by inserting genes encoding the desired protein into a host organism or cell line, which then expresses the protein. Recombinant proteins may be identical to naturally occurring proteins or may be modified versions with altered properties. The production of recombinant proteins involves the use of expression vectors, selection of appropriate host systems, and optimization of growth and purification conditions. Recombinant proteins have diverse applications in research, medicine, and biotechnology, including the production of therapeutic proteins, enzymes for industrial processes, and protein-based materials.

[0065]

[0060] As used herein, the terms “protein”, “polypeptide”, and “peptide” are used herein interchangeably and refer to a molecule composed of a chain of amino acids linked by peptide bonds. These molecules vary in length from a few amino acids to thousands, and possess diverse structures and functions within biological systems. Proteins, polypeptides, and peptides are naturally occurring, synthetically produced, or recombinantly expressed, and can undergo various post-translational modifications. They serve numerous roles in organisms, including catalyzing biochemical reactions, providing structural support, facilitating cellular signaling, and participating in immune responses. In the context of this disclosure, these terms may encompass both native and engineered amino acid sequences, including those used in immunogenic scaffolds or as antigenic epitopes.

[0066]

[0061] As used herein, the term “analog” refers to a compound or substance that is structurally similar to another compound, e.g., any one of SEQ ID Nos: 6 and / or 7, but differs slightly in composition. Analogs share similar chemical and / or biological properties with the original compound, e.g., any one of SEQ ID Nos: 6 and / or 7.

[0067]

[0062] In some embodiments, an analog comprises or is a functional analog.

[0068]

[0063] As used herein, the term “functional analog” refers to a compound, molecule, or substance that performs a similar function or exhibits comparable biological activity to another compound, e.g., any one of SEQ ID Nos: 6 and / or 7 despite potentially having a different chemical structure. Functional analogs mimic the effects, interactions, and / or mechanisms of action of the original compound, e.g., any one of SEQ ID Nos: 6 and / or 7 within a biological system.

[0069]

[0064] In some embodiments, the recombinant protein further comprises: (v) a peptide linker located between: (i) and (ii), between (ii) and (iii), between (iii) and (iv), or any combination thereof. In some embodiments, the linker is a peptide linker. In some embodiments, the linker is a flexible linker. In some embodiments, the linker comprises 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 tolO, 2 to 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 3 to 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, 3 to 10, 4 to 5, 4 to 6, 4 to 7, 4 to 8, 4 to 9, 4 to 10, 5 to 6, 5 to 7, 5 to 8, 5 to 9, 5 to 10, 6 to 7, 6 to 8, 6 to 9, 6 to 10, 7 to 8, 7 to 9, 7 to 10, 8 to 9, 8 to 10, or 9 to 10 amino acids. Each possibility represents a separate embodiment of the invention. In some embodiments, the peptide linker is of 1 to 8 amino acids. In some embodiments, the peptide linker is a glycine residue, or comprises the amino acid sequence GSSGG (SEQ ID NO: 3) or GGSGG (SEQ ID NO: 4). In some embodiments, the peptide linker comprises a plurality of peptide linkers comprising a glycine residue, the amino acid sequence GSSGG (SEQ ID NO: 3) or GGSGG (SEQ ID NO: 4), or any combination thereof.

[0070]

[0065] As used herein, the term “linker” refers to a chemical group, molecule, or structure that connects two or more components or functional groups within a larger molecular system. Linkers serve various purposes, including spacing, flexibility, reactivity, or modulation of properties.

[0071]

[0066] As used herein, the term “plurality” refers to any integer being equal to or greater than 2.

[0067] In some embodiments, (i) to (v) form a unit repeating at least two times in the recombinant protein. In some embodiments, (i) to (v) form a unit repeating at least three times in the recombinant protein. In some embodiments, (i) to (v) form a unit repeating at four times in the recombinant protein. In some embodiments, (i) to (v) form a unit repeating two to five, two to four, two to three, three to five, three to four, or four to five times in the recombinant protein. Each possibility represents a separate embodiment of the invention.

[0072]

[0068] In some embodiments, (i) to (iv) are organized sequentially from the N’ -terminal end to the C’ -terminal end of repeating unit in recombinant protein. In some embodiments, (i) to (iv) are organized as (i) - (ii) - (iii) - (iv) from the N’ -terminal end to the C’ -terminal end of the repeating unit in the recombinant protein. In some embodiments, (i) to (v) are organized as (i) - (v) - (ii) - (v) - (iii) - (v) - (iv) from the N’ -terminal end to the C’ -terminal end of said repeating unit in said recombinant protein.

[0073]

[0069] In some embodiments, the recombinant protein further comprises a linker located between each of the at least three units repeating in the recombinant protein.

[0074]

[0070] In some embodiments, the recombinant protein further comprises at least one third peptide of interest. In some embodiments, the at least one third peptide of interest comprises a plurality of a third peptide of interest. In some embodiments, the at least one third peptide of interest or the plurality of a third peptide of interest comprise two to three, two to four, two to five, three to four, three to five, or four to five copies of the third peptide of interest. Each possibility represents a separate embodiment of the invention.

[0075]

[0071] In some embodiments, the recombinant protein comprises an amino acid sequence having at least 90%, 95%, or 99% identity to any one of SEQ ID Nos: 10-13, and 15-31. Each possibility represents a separate embodiment of the invention.

[0076]

[0072] In some embodiments, the recombinant protein comprises an amino acid sequence having at least 90%, 95%, or 99% identity to any one of SEQ ID Nos: 10-13, and 15-21. Each possibility represents a separate embodiment of the invention. In some embodiments, the recombinant protein comprises the amino acid sequence set forth in SEQ ID Nos: 10, 11, 12, 15, 16, 17, 18, 19, 20, or 21. Each possibility represents a separate embodiment of the invention. In some embodiments, the recombinant protein consists of the amino acid sequence set forth in SEQ ID Nos: 10, 11, 12, 15, 16, 17, 18, 19, 20, or 21. Each possibility represents a separate embodiment of the invention.

[0077]

[0073] In some embodiments, the recombinant protein comprises an amino acid sequence having at least 90%, 95%, or 99% identity to any one of SEQ ID Nos: 22-31. Each possibility represents a separate embodiment of the invention. In some embodiments, the recombinant protein comprises the amino acid sequence set forth in SEQ ID Nos: 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31. Each possibility represents a separate embodiment of the invention. In some embodiments, the recombinant protein consists of the amino acid sequence set forth in SEQ ID Nos: 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31. Each possibility represents a separate embodiment of the invention.

[0078]

[0074] In some embodiments, the recombinant protein of the invention further comprises a methionine residue located in the N’ terminus of the recombinant protein. In some embodiments, the recombinant protein of the invention further comprises a methionine residue as the first amino acid of the N’ terminus of the recombinant protein. In some embodiments, any one of SEQ ID Nos: 10-13, 15-21, and 23-31, further comprises a methionine residue as the first amino acid of the N’ terminus of the recombinant protein.

[0079]

[0075] In some embodiments, the repeating unit of the recombinant protein of the invention comprises the amino acid sequence set forth in SEQ ID NO: 22. In some embodiments, the repeating unit of the recombinant protein of the invention consists of the amino acid sequence set forth in SEQ ID NO: 22.

[0080]

[0076] In some embodiments, the at least one third peptide of interest is positioned N’ terminally to the amino acid sequence set forth in SEQ ID NO: 6 or the analog thereof having at least 90% identity thereto. In some embodiments, the at least one third peptide of interest is positioned C’ terminally to the second peptide of interest. In some embodiments, the at least one third peptide of interest comprises at least two third peptides of interest, the first of which is positioned N’ terminally to the amino acid sequence set forth in SEQ ID NO: 6 or the analog thereof having at least 90% identity thereto, and the second of which is positioned C’ terminally to the second peptide of interest. In some embodiments, the at least one third peptide of interest comprises two copies of the third peptide of interest positioned C’ terminally to the second peptide of interest. In some embodiments, the at least one third peptide of interest comprises at least three third peptides of interest, the first of which is positioned N’ terminally to the amino acid sequence set forth in SEQ ID NO: 6 or the analog thereof having at least 90% identity thereto, and the second and third of which are positioned C’ terminally to the second peptide of interest.

[0081]

[0077] In some embodiments, the first, second, and at least one third peptides of interest are identical. In some embodiments, the first, second, and at least one third peptides of interest comprise different amino acid sequences. In some embodiments, the first, second, and at least one third peptides of interest are fragments of or derived from different proteins. In some embodiments, the first, second, and at least one third peptides of interest are fragments of or derived from the same protein. In some embodiments, the first, second, and at least one third peptides of interest are fragments of or derived from the same protein and comprise different amino acid sequences. In some embodiments, the first, second, and at least one third peptides of interest are different antigens being fragments of or derived from the same protein.

[0082]

[0078] In some embodiments, the first, second, and / or at least one third peptides of interest is an immunogenic peptide.

[0083]

[0079] As used herein, the term “immunogenic peptide” refers to a short sequence of amino acids that can stimulate an immune response when introduced into a biological system. These peptides may be derived from pathogens, tumor cells, or other sources, and typically contain epitopes that are recognized by the immune system. Immunogenic peptides vary in length, ranging from about 5 to 30 amino acids, and often used in vaccine development, immunotherapy, or as research tools to study immune responses.

[0084]

[0080] In some embodiments, the immunogenic peptide is a fragment of a protein selected from: superoxide dismutase 1 (SOD1), alpha-synuclein, amyloid-beta (AP), tubulin associated unit (TAU), transactive response DNA binding protein 43 (TDP-43), or any combination thereof.

[0085]

[0081] In some embodiments, the fragment is of: 8 to 30, 10 to 30, 15 to 30, 20 to 30, 8 to 20, 8 to 15, 8 to 12, 10 to 20, 10 to 18, or 10 to 15 amino acids. Each possibility represents a separate embodiment of the invention.

[0086]

[0082] In some embodiments, the immunogenic peptide is a fragment of SOD1 comprising the amino acid sequence set forth in SEQ ID Nos: 2, 14, or both. In some embodiments, the immunogenic peptide is a fragment of alpha-synuclein the amino acid sequence set forth in SEQ ID NO: 32. In some embodiments, the immunogenic peptide is a fragment of AP comprising the amino acid sequence set forth in SEQ ID NO: 33. In some embodiments, the immunogenic peptide is a fragment of TDP-43 comprising the amino acid sequence set forth in SEQ ID Nos: 34, 35, or both. In some embodiments, the immunogenic peptide is a fragment of TAU comprising the amino acid sequence set forth in SEQ ID NO: 36, 37, or both.

[0087]

[0083] In some embodiments, the immunogenic peptide is a combination of two immunogenic peptides as disclosed herein. In some embodiments, the combination of two immunogenic peptides comprises SEQ ID Nos: 2 and 14. In some embodiments, the combination of two immunogenic peptides comprises SEQ ID Nos: 34 and 35. In some embodiments, the combination of two immunogenic peptides comprises SEQ ID Nos: 36 and 37.

[0084] In some embodiments, the recombinant protein further comprises a detection tag, a purification tag, or both.

[0088]

[0085] In some embodiments, the detection or purification tag is connected to the amino acid set forth in SEQ ID NO: 7 of the C’-terminus of the recombinant protein. In some embodiments, the detection or purification tag is connected to the amino acid set forth in SEQ ID NO: 7 of the C’-terminus of the recombinant protein, via the linker.

[0089]

[0086] In some embodiments, detection or detected is based on an immune reaction. In some embodiments, an immune reaction comprises an immunological assay, such as using an antibody. In some embodiments, purification and / or detection is (performed) in vitro.

[0090]

[0087] In some embodiments, the detection or purification tag comprises a labeling moiety. In some embodiments, the labeling moiety is or comprises a fluorophore. In some embodiments, the purification tag has specific binding affinity to a counterpart agent. In some embodiments, the counterpart agent is a solid. In some embodiments, the purification tag comprises a poly histidine tag, e.g., at least 6 or 8 histidine residues. In some embodiments, the counterpart agent includes Ni. In some embodiments, the purification tag is suitable for affinity-based purification methodology.

[0091]

[0088] As used herein, the term “affinity-based purification” refers to a technique used to isolate and purify specific molecules, such as proteins or peptides, from a complex mixture based on their binding affinity to a particular ligand or molecule. This method typically involves immobilizing a ligand on a solid support, allowing the target molecule to bind to the ligand, washing away unbound components, and then eluting the purified target molecule. Affinitybased purification utilizes various types of interactions, including antibody-antigen, enzymesubstrate, or receptor-ligand interactions, and is employed in applications such as protein isolation, antibody production, or vaccine development.

[0092]

[0089] In some embodiments, the recombinant protein further comprises a signal peptide. In some embodiments, signal peptide is a signal peptide for secretion and / or insertion into the secretory pathway.

[0093]

[0090] As used herein, the term “signal peptide” refers to a short amino acid sequence typically found at the N-terminus of newly synthesized proteins that are destined for secretion or membrane insertion. Signal peptides generally include about 16-30 amino acids and play a crucial role in protein targeting and translocation. These peptides guide proteins to specific cellular compartments, such as the endoplasmic reticulum, mitochondria, or chloroplasts, and are often cleaved off by signal peptidases during or after the protein's translocation. Signal peptides are utilized in biotechnology applications to direct recombinant proteins for secretion or to target them to specific cellular locations.

[0094]

[0091] In some embodiments, the recombinant protein further comprises a transmembrane domain or a membrane anchoring sequence / domain.

[0095]

[0092] As used herein, the term “transmembrane domain” refers to a portion of a protein that spans the entire width of a biological membrane, typically a lipid bilayer. This domain usually consists of hydrophobic amino acid residues that interact with the lipid environment of the membrane. Transmembrane domains play various roles in cellular processes, including anchoring proteins within membranes, facilitating signal transduction across membranes, or forming channels or pores for the passage of molecules. These domains can vary in length and structure, often adopting an alpha-helical conformation within the membrane. Transmembrane domains are important components in the design and engineering of membrane- associated proteins for biotechnological or therapeutic applications.

[0096]

[0093] As used herein, the term “membrane anchoring sequence” refers to a segment of amino acids within a protein that enables its attachment to a biological membrane. This sequence typically includes hydrophobic or amphipathic residues that insert into or associate with the lipid bilayer of cellular membranes. Membrane anchoring sequences include transmembrane domains, lipid-modified motifs such as glycosylphosphatidylinositol (GPI) anchors, or other specialized structures that facilitate membrane association. These sequences play crucial roles in protein localization, membrane organization, and cellular signaling processes. In biotechnology applications, membrane anchoring sequences may be utilized to tether proteins of interest to cell surfaces or artificial membranes for various purposes, including drug delivery, biosensing, or cell engineering.

[0097]

[0094] In some embodiments, the transmembrane domain is the transmembrane domain of CD28 comprising an amino acid sequence having at least 85%, 90%, 95%, or 99% sequence to SEQ ID NO: 9, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the transmembrane domain is the transmembrane domain of CD28 comprising the amino acid sequence set forth in SEQ ID NO: 9, or any human analog thereof. In some embodiments, CD28 is a human CD28.

[0098]

[0095] The full sequence of a human CD28 would be accessible to a person of skill in the art, such as in Genbank accession no. AAA51945.1.

[0099] Compositions

[0096] According to another aspect, there is provided a composition comprising the recombinant protein of the invention. In some embodiments, the composition further comprises an acceptable carrier. In some embodiments, the carrier is a pharmaceutically acceptable carrier.

[0100]

[0097] According to another aspect, there is provided a composition comprising a therapeutically effective amount of the recombinant protein of the invention, and a pharmaceutically acceptable carrier. In some embodiments, the composition is a pharmaceutical composition comprising a therapeutically effective amount of the recombinant protein of the invention. In some embodiments, the composition is a vaccine. In some embodiments, the composition is a pharmaceutical composition.

[0101]

[0098] In some embodiments, the vaccine induces production or synthesis of antibodies exclusively targeting the pathogenic endogenous protein In some embodiments, the vaccine induces production or synthesis of antibodies exclusively targeting the pathogenic endogenous protein in its improper or misfolded state. In some embodiments, the vaccine induces production or synthesis of antibodies exclusively targeting the exposed immunogenic peptide (e.g., the peptide of interest). In some embodiments, the vaccine does not induce production or synthesis of antibodies targeting the endogenous non-pathogenic protein.

[0102]

[0099] In some embodiments, the composition is for use in treatment of a neurological disease or condition associated therewith in a subject in need thereof.

[0103]

[0100] As used herein, the term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents such as acetates, citrates or phosphates. Antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; and agents for the adjustment of tonicity such as sodium chloride or dextrose are also envisioned. The carrier may comprise, in total, from about 0.1% to about 99.99999% by weight of the pharmaceutical compositions presented herein.

[0104]

[0101] As used herein, the term "pharmaceutically acceptable" means suitable for administration to a subject, e.g., a human. For example, the term "pharmaceutically acceptable" can mean approved by a regulatory agency of the Federal or a state government or listed in the U. S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0105]

[0102] In some embodiments, the composition of the invention take the form of solutions, suspensions, emulsions, sustained-release formulations and the like. Examples of suitable pharmaceutical carriers are described in: Remington's Pharmaceutical Sciences" by E.W. Martin, the contents of which are hereby incorporated by reference herein. Such compositions will contain a therapeutically effective amount of the recombinant protein of the invention, preferably in a substantially purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject.

[0106]

[0103] In some embodiments, the composition or a formulation thereof to be administered may contain a quantity of the recombinant protein, according to embodiments of the invention in an amount effective to treat the condition or disease of the subject being treated.

[0107] Artificial DNA molecules and Cells

[0108]

[0104] According to another aspect, there is provided an artificial DNA molecule encoding the recombinant protein of the invention.

[0109]

[0105] As used herein, the term “artificial” refers to something that is created, produced, or designed by human effort rather than occurring naturally. In the context of biological or chemical systems, “artificial” describes synthetic or engineered entities, structures, or processes that are not found in nature but are instead developed through human intervention and technological means. This term encompasses a wide range of constructs, including but not limited to synthetic proteins, engineered genetic sequences, man-made materials, or novel molecular assemblies designed to mimic or improve upon natural systems. Artificial entities or processes are designed to perform specific functions, overcome limitations of natural systems, or explore new possibilities in various fields such as biotechnology, medicine, or materials science.

[0110]

[0106] According to another aspect, there is provided a recombinant cell comprising the artificial DNA molecule of the invention.

[0107] In some embodiments, there is provided a composition comprising: the recombinant protein of the invention, artificial DNA molecule encoding the recombinant protein of the invention, the recombinant cell comprising the artificial DNA molecule of the invention, or any combination thereof.

[0111]

[0108] In some embodiments, the recombinant cell comprises a nucleic acid sequence encoding the recombinant protein of the invention. In some embodiments, the nucleic acid sequence is codon optimized for expression in the recombinant cell.

[0112]

[0109] As used herein, “codon optimization” refers to the process of modifying the genetic sequence of a gene to enhance its expression in a particular host organism without changing the amino acid sequence of the encoded protein. This technique involves replacing rare codons with more frequently used synonymous codons that are preferred by the host's translational machinery. Codon optimization improves protein production efficiency, increase mRNA stability, and reduce translational errors. This process is particularly useful when expressing genes from one organism in a different host species, or when designing synthetic genes for biotechnological applications. Codon optimization is achieved through computational algorithms that consider factors such as codon usage bias, GC content, and mRNA secondary structure.

[0113]

[0110] In some embodiments, the recombinant cell is a microorganism. In some embodiments, the recombinant cell is a bacterium or a fungus. In some embodiments, the bacterium is E. coli. In some embodiments, the fungus is a yeast cell.

[0114] Methods

[0115]

[0111] According to another aspect, there is provided a method for preparing a recombinant protein. In some embodiments, the recombinant protein is the recombinant protein of the invention. In some embodiments, the method comprises culturing the recombinant cell of the invention, such that a recombinant protein encoded by the artificial DNA molecule is expressed.

[0116]

[0112] In some embodiments, the method further comprises a step before the culturing comprising introducing or transfecting a cell with the artificial DNA molecule of the invention, thereby providing the recombinant cell of the invention.

[0117]

[0113] In some embodiments, introducing or transfecting comprises transferring an artificial nucleic acid molecule or vector comprising the nucleic acid molecule encoding the recombinant protein of the invention into a cell; or modifying the genome of a cell to include the nucleic acid molecule encoding the recombinant protein of the invention. In some embodiments, transferring comprises transfection. In some embodiments, transferring comprises transformation. In some embodiments, transferring comprises lipofection. In some embodiments, transferring comprises nucleofection. In some embodiments, transferring comprises viral infection.

[0118]

[0114] In some embodiments, artificial nucleic acid molecule or vector comprises a plasmid, an expression vector, or both.

[0119]

[0115] As used herein, the terms “transfecting” and “introducing” are interchangeable.

[0120]

[0116] According to another aspect, there is provided a method for treating a subject afflicted with a neurological disease or condition associated therewith. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the recombinant protein of the invention, thereby treating a subject afflicted with the neurological disease or condition associated therewith.

[0121]

[0117] In some embodiments, administering comprises a single administration. In some embodiments, administering comprises multiple administrations. In some embodiments, administering comprises multiple administrations comprise at least one administration per week. In some embodiments, multiple administrations comprise repeated administrations. In some embodiments, repeated administrations are provided at an interval sufficient for achieving vaccination.

[0122]

[0118] Interval employed during repeated administration under vaccination protocols would be apparent to one of ordinary skill in the art.

[0123]

[0119] In some embodiments, treating comprises delaying the appearance of at least one symptom of the neurological disease or condition associated therewith. In some embodiments, treating comprises delaying the progression of the neurological disease or condition associated therewith. In some embodiments, treating comprises delaying the appearance of at least one symptom and the progression of the neurological disease or condition associated therewith.

[0124]

[0120] In some embodiments, treating comprises vaccinating the subject against the neurological disease or condition associated therewith.

[0125]

[0121] In some embodiments, vaccinating comprises inducing the production or synthesis of antibodies exclusively targeting the pathogenic endogenous protein In some embodiments, vaccinating comprises inducing the production or synthesis of antibodies exclusively targeting the pathogenic endogenous protein in its improper or misfolded state. In some embodiments, vaccinating comprises inducing the production or synthesis of antibodies exclusively targeting the exposed immunogenic peptide (e.g., the peptide of interest). In some embodiments, vaccinating does not comprise inducing the production or synthesis of antibodies targeting the endogenous non-pathogenic protein.

[0126]

[0122] As used herein, the expression / term “neurological disease or condition associated therewith” refers to a broad category of disorders that affect the central and peripheral nervous systems, including the brain, spinal cord, cranial nerves, peripheral nerves, nerve roots, autonomic nervous system, neuromuscular junction, and muscles. This term encompasses a wide range of conditions, such as neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis), cerebrovascular diseases (e.g., stroke), neurodevelopmental disorders, epilepsy, multiple sclerosis, brain tumors, traumatic brain and spinal cord injuries, and various other disorders that impact neurological function. The phrase “associated therewith” further extends to conditions that are directly or indirectly related to these primary neurological diseases, including complications, secondary effects, or comorbidities that arise as a result of the primary neurological condition.

[0127]

[0123] In some embodiments, the neurological disease or condition associated therewith involves, comprises, is characterized by, relates to, propagated by, enhanced by, any equivalent thereof, or any combination thereof, protein misfolding. In some embodiments, the neurological disease or condition associated therewith is characterized by increased amounts of a misfolded protein. In some embodiments, the misfolded protein is an endogenous protein. In some embodiments, the endogenous protein is non-pathogenic in its proper or native folding state. In some embodiments, the endogenous protein is non-pathogenic when properly folded. In some embodiments, properly folded is natively folded or being in the native protein state or structure. In some embodiments, when misfolded, the endogenous protein is pathogenic. In some embodiments, the endogenous protein is pathogenic in its improper or misfolded state. In some embodiments, the misfolded state or form differs from the native state or form such that the immunogenic peptide (e.g., the peptide of interest), is exposed to the external environment. In some embodiments, the immunogenic peptide (e.g., the peptide of interest), is exposed to the external environment only in the misfolded state or form of the endogenous protein. In some embodiments, the immunogenic peptide (e.g., the peptide of interest), is not exposed to the external environment in the folded or native state or form of the endogenous protein.

[0128]

[0124] In some embodiments, the neurological disease or condition associated therewith is selected from: amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, and any combination thereof.

[0125] In some embodiments, treating comprises preventing. In some embodiments, treating comprises preventing and treating. In some embodiments, treating comprises ameliorating at least one symptom associated with a disease or condition as disclosed herein, e.g., neurological disease or condition associated therewith.

[0129]

[0126] As used herein, the terms “treatment” or “treating” of a disease, disorder, or condition encompasses alleviation of at least one symptom thereof, a reduction in the severity thereof, or inhibition of the progression thereof. Treatment need not mean that the disease, disorder, or condition is totally cured. To be an effective treatment, a useful composition herein needs only to reduce the severity of a disease, disorder, or condition, reduce the severity of symptoms associated therewith, or provide improvement to a patient or subject’s quality of life.

[0130]

[0127] As used herein, the term “prevention” of a disease, disorder, or condition encompasses the delay, prevention, suppression, or inhibition of the onset of a disease, disorder, or condition. As used in accordance with the presently described subject matter, the term "prevention" relates to a process of prophylaxis in which a subject is exposed to the presently described peptides prior to the induction or onset of the disease / disorder process. This could be done where an individual has a genetic pedigree indicating a predisposition toward occurrence of the disease / disorder to be prevented. For example, this might be true of an individual whose ancestors show a predisposition toward certain types of, for example, inflammatory disorders. The term "suppression" is used to describe a condition wherein the disease / disorder process has already begun but obvious symptoms of the condition have yet to be realized. Thus, the cells of an individual may have the disease / disorder, but no outside signs of the disease / disorder have yet been clinically recognized. In either case, the term prophylaxis can be applied to encompass both prevention and suppression. Conversely, the term "treatment" refers to the clinical application of active agents to combat an already existing condition whose clinical presentation has already been realized in a patient.

[0131] General

[0132]

[0128] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0133]

[0129] As used herein, the term “about” when combined with a value refers to plus and minus 10% of the reference value. For example, a length of about 1,000 nanometers (nm) refers to a length of 1,000 nm ± 100 nm.

[0134]

[0130] It is noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a polynucleotide” includes a plurality of such polynucleotides and reference to “the polynucleotide” includes reference to one or more polynucleotides and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely”, “only” and the like in connection with the recitation of claim elements or use of a “negative” limitation.

[0135]

[0131] In those instances where a convention analogous to "at least one of A, B, and C, etc." is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B”.

[0136]

[0132] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0133] Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

[0137]

[0134] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

[0138] EXAMPLES

[0139]

[0135] Generally, the nomenclature used herein, and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8thEdition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference. Other general references are provided throughout this document.

[0140]

[0136] The inventors propose to use 6-Helix protein (Fig. 1) as a scaffold for antigenic peptide display. The sequence of an antigenic peptide is fused in frame with 6-Helix scaffold in place of the interhelical loops. A single copy of an antigenic peptide sequence is inserted, or alternatively multiple copies of the same or different antigenic peptides are inserted (in tandem), to generate a chimeric fusion protein (Fig. 2), which can be produced as a recombinant protein or expressed from an mRNA construct in the target tissue, and used as immunogen for active vaccination. The sequence of the antigenic peptide might be framed, if necessary, by a flexible linker(s) such as GGSGG (SEQ ID NO: 3), to enable flexibility of the target epitope within the chimeric construct.

[0141]

[0137] As predicted by the computational analysis using NetMHCcons method, the sequence of 6-Helix scaffold contains multiple T-cell epitopes, in both mouse and human. These T-cell epitopes in conjunction with the multiple B-cell epitopes presented by the antigenic peptide sequences, are expected to evoke a strong immune response against the antigenic peptide. Insertion in place of the interhelical loops of the 6-Helix bundle provides the antigenic peptide with spatial constraints mimicking its native environment (if the latter is derived from a loop region), facilitating the production of Abs recognizing conformational epitopes. Additional sequences of the antigenic peptide(s) could be introduced at the N- and / or C-terminus of the Helix-6 scaffold to facilitate the formation of Abs against linear epitopes.

[0142]

[0138] The HIV-originated 6-Helix scaffold contains three glycosylation sites (Fig. 1, underlined). Glycosylation at these positions acts to block the recognition of the viral epitopes by the host immune system. When the chimeric antigenic protein is expressed in a tissue from its encoding mRNA, these glycosylation sites may decrease (or prevent) the immunogenicity of the antigen. To prevent this, in mRNA constructs, the corresponding asparagine residues (N) is replaced by glutamine residues (Q).

[0143] Experimental data - Develop an active immunization approach based on mRNA technology to target the pathogenic 16 / [17 loop epitope ofSODl

[0144]

[0139] The inventors generate mRNA constructs to target the expression of an antigen into three different locations: (1) cytosolic; (2) secreted (extracellular); and (3) transmembrane.

[0145]

[0140] The antigen was designed as a chimeric protein, in which sequences corresponding to the pathogenic P6 / P7 loop epitope of SOD1 were implanted in place of the interhelical loops of a soluble scaffold composed of 6 alpha-helical segments forming a bundle (Helix 6) (Fig. 2). Helix 6 is an artificial protein derived from the HIV-1 gp41’s trimer of hairpins. Binding of gpl20 / gp41 to the cell surface receptors (CD4 and a coreceptor, such as CCR5 or CXCR4) induces a sequence of conformational changes in the gpl20 / gp41 oligomer, eventually leading to the creation of a trimer of hairpins structure in gp41. The idea of using such design is to utilize the HIV-derived sequence as the source of T-cell epitopes, essential for an effective humoral immune response, together with the spatial constrains of the 6-Helix bundle to mimic the nativelike conformation of the target SOD1 P6 / P7 loop sequence displayed in multiple copies and expected to provide B-cell epitopes.

[0141] The computation analysis using AlphaFold tool predicted that the incorporation of multiple copies of the P6 / |37 loop epitope is not expected to destabilize the barrel-like helical structure of the scaffold, thus preserving the native-like loop conformation of the target epitopes (as in SOD1). Since the P6 / p7 loop is not exposed in intact properly folded SOD1 protein, but only in misfolded SOD1, the inventors hypothesize that it would not represent a self-antigen for human’s immune system, and would evoke, at its sufficiently high concentration, a substantial immune response.

[0146]

[0142] Three modified mRNA constructs for the targeted expression of the antigenic protein in the three different cellular compartments were designed and synthesized. The 6-Helix protein comprises three N-peptide segments (N40) and three C-peptide segments (C38) alternately linked (N-C-N-C-N-C) by short Gly / Ser sequences (Figs. 2-3). The 6Helx-SE21 (Construct 1) was designed to express the antigenic protein in cytosol. The tPA-6Helx-SE21 (Construct 2), which contains N-term tPA secretory signal, was designed to express a secretable version of the antigen. The tPA-6Helx-SE21-CD28TM (Construct 3), in addition to tPA, contains CD28 transmembrane domain at its C-term, to express the antigen as transmembrane protein. All three constructs have V5 tag for detection.

[0147]

[0143] DNA sequence of the three antigenic constructs: tPA - MDAMKRGLCCVLLLCGAVFVSAR (SEQ ID NO: 4)

[0148] SE21 - SOD1 P6 / P7 loop epitope - DSVISLSGDHDII (SEQ ID NO: 2) x7

[0149]

[0144] In this construct, the SE21 sequence contains CH ID mutation, which is intended to mimic the oxidized state of Cys 111, which is assumed to contribute to ALS pathogenesis.

[0150]

[0145] N40, HIV-derived sequence, Six-helix bundle -

[0151] QLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILA (SEQ ID NO: 6) x3

[0152]

[0146] C38, HIV-derived sequence, Six-helix bundle -

[0153] HTTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLE (SEQ ID NO: 7) x3

[0154]

[0147] Linker - GGSGG (SEQ ID NO: 3) and / or GSSGG (SEQ ID NO: 4).

[0155]

[0148] V5-tag - GKPIPNPLLGLDST (SEQ ID NO: 8)

[0156]

[0149] CD28, an extracellular region consisting of 40 residues (solid purple) and a cytoplasmic region of 13 residues (purple vertical stripes) flank the 27-residue TM domain (horizontal purple stripes) -

[0157] PMLVAYDNAVNLSYNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYS LLVTVAFIIFWVRSKRSRLLHSDYM (SEQ ID NO: 9) 6Helx-SE21 ( Construct 1 )

[0158]

[0150] MDSVISLSGDHDIIQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILA GGSGGDSVISLSGDHDIIGHTTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEGS SGGDSVISLSGDHDIIGSSGGQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARI LAGGSGGDSVISLSGDHDIIGHTTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLE GSSGGDSVISLSGDHDIIGSSGGQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQA RILAGGSGGDSVISLSGDHDIIGHTTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELL EDSVISLSGDHDIIGKPIPNPLLGLDST (SEQ ID NO: 10) tPA-6Helx-SE21 ( Construct 2)

[0159]

[0151] MDAMKRGLCCVLLLCGAVFVSARDSVISLSGDHDIIQLLSGIVQQQNNLLRAIE AQQHLLQLTVWGIKQLQARILAGGSGGDSVISLSGDHDIIGHTTWMEWDREINNYTSL IHSLIEESQNQQEKNEQELLEGSSGGDSVISLSGDHDIIGSSGGQLLSGIVQQQNNLLRAI EAQQHLLQLTVWGIKQLQARILAGGSGGDSVISLSGDHDIIGHTTWMEWDREINNYTS LIHSLIEESQNQQEKNEQELLEGSSGGDSVISLSGDHDIIGSSGGQLLSGIVQQQNNLLR AIEAQQHLLQLTVWGIKQLQARILAGGSGGDSVISLSGDHDIIGHTTWMEWDREINNY TSLIHSLIEESQNQQEKNEQELLEDSVISLSGDHDIIGKPIPNPLLGLDST (SEQ ID NO: 11) tPA-6Helx-SE21 -CD28TM ( Construct 3 )

[0160]

[0152] MDAMKRGLCCVLLLCGAVFVSARDSVISLSGDHDIIQLLSGIVQQQNNLLRAIE AQQHLLQLTVWGIKQLQARILAGGSGGDSVISLSGDHDIIGHTTWMEWDREINNYTSL IHSLIEESQNQQEKNEQELLEGSSGGDSVISLSGDHDIIGSSGGQLLSGIVQQQNNLLRAI EAQQHLLQLTVWGIKQLQARILAGGSGGDSVISLSGDHDIIGHTTWMEWDREINNYTS LIHSLIEESQNQQEKNEQELLEGSSGGDSVISLSGDHDIIGSSGGQLLSGIVQQQNNLLR AIEAQQHLLQLTVWGIKQLQARILAGGSGGDSVISLSGDHDIIGHTTWMEWDREINNY TSLIHSLIEESQNQQEKNEQELLEDSVISLSGDHDIIGKPIPNPLLGLDSTPMLVAYDNAV NLSYNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWV RSKRSRLLHSDYM (SEQ ID NO: 12)

[0161]

[0153] In these constructs, the SOD1 P6 / |37 loop epitope sequence (DSVISLSGDHDII; SEQ ID NO: 2) contains a CH ID substitution (underlined). This substitution in needed to mimic the oxidized state of oxidation-prone Cysl l l, whose oxidation was suggested contribute to ALS pathogenesis in humans. In mice, Ser substitutes for Cys at position 111, but the transgenic ALS animals overexpressing human SOD1 variants congenitally contain Cysl l l. Therefore, CH ID substitution may help to break the potential immune tolerance to SOD1 self-antigen in ALS mouse models and in human patients.

[0154] The expression efficiency of the three mRNA constructs was checked in SHSY-5Y cells transfected using jetMESSENGER® (Polyplus) mRNA transfection reagent. The level of expression was analyzed by immunofluorescence and images were acquired by a confocal microscope. All of the tested constructs appeared to be expressed in the specified regions of the cell (Fig. 4). The expression of the tested constructs was also demonstrated in the cells and the conditioned media using immunoblotting (data not shown).

[0162] Recombinant protein vaccine

[0163]

[0155] In addition, 9SE-Helx-6His (Agl), a 6-Helix scaffold with 5 pathogenic P6 / p7 loop sequences of misfolded SOD1 implanted in place of the interhelical loops, 1 at the N-term, and 3 in tandem at the C-term (altogether 9 copies) was produced as a 6-His tagged protein in E. coli, purified (Ni2+affinity chromatography + SEC + endotoxin removal), and used, in a combination with the appropriate adjuvant (AddaVax (MF59) + CpG ODN 1826), for active vaccination of WT and ALS mice transgenic for human SOD1-G93A fALS variant.

[0164]

[0156] 9SE-Helx-6His (Agl):

[0165] MDSVISLSGDHDIIQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAGGSGG DSVISLSGDHDIIGHTTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEGSSGGDS VIS LSGDHDIIGSSGGQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQ ARIL AGGS GGDSVISLSGDHDIIGHTTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEGSSGG DSVISLSGDHDIIGSSGGQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAG GSGGDSVISLSGDHDIIGHTTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLEDSVI SLSGDHDIIGDSVISLSGDHDIIGDSVISLSGDHDIIGHHHHHH (SEQ ID NO: 13)

[0166]

[0157] The WT mice were immunized intramuscularly with 50 pl suspension containing 50% (v / v) of Agl (9SE-Helx-6His (SEQ ID NO: 12), 30 pg) + 10 pg CpG ODN 1826 and 50% (v / v) AddaVax adjuvant. The primary immunization was followed after 3 weeks with a booster immunization of the same composition (Fig. 5A). Another adjuvant, CDN-A001E (Magic Mouse Adjuvant, Enhanced, Creative Diagnostics, USA) was tested (50 pl, 1: 1 (v / v) ratio, Ag (30 pg): Adjuvant), and found similarly effective. The Ab titter in mouse sera was evaluated by ELISA using purified misfolded fALS apo-SODl-G93A protein as antigen (Ag) for plate coating. To test selectivity of Ab recognition of misfolded SOD1, a properly folded holo-SODl- WT protein was used as specificity control.

[0167]

[0158] After the booster vaccination, the titer of the anti-SODl Ab in the sera reached the level of l: 106(Fig. 5B). The immunogenicity of 9SE-Helx-6His construct appears to be very consistent, since all the mice in group (6 mice) developed similar Ab titer. Five weeks after the booster, no reduction in the Ab titer was observed (Fig. 5B). The antibodies generated appear to be specific for misfolded SOD1 species, since no significant binding was observed to holo- S0D1-WT in ELISA (Fig. 5C). The antibodies generated appeared to be purely conformational, as they recognized neither the isolated P6 / |37 loop-derived peptide nor the denatured SOD1- G93A protein (as in WB) (data not shown).

[0168]

[0159] In parallel, a different version of 9SE-Helx-6His (9SE-Helx-6His-v2) was constructed (Ag2), which includes a longer SODl-derived antigenic peptide (DSVISLSGDHDIIGRT; SEQ ID NO: 13) and extra linkers for its insertion into 6Helix scaffold.

[0169]

[0160] 9SE-Helx-6His (Ag2):

[0170] MDSVISLSGDHDIIGRTGQLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILAG GSGGDSVISLSGDHDIIGRTGGSGGHTTWMEWDREINNYTSLIHSLIEESQNQQEKNEQ ELLEGSSGGDSVISLSGDHDIIGRTGSSGGQLLSGIVQQQNNLLRAIEAQQHLLQLTVW GIKQLQARILAGGSGGDSVISLSGDHDIIGRTGGSGGHTTWMEWDREINNYTSLIHSLIE ESQNQQEKNEQELLEGSSGGDSVISLSGDHDIIGRTGSSGGQLLSGIVQQQNNLLRAIE AQQHLLQLTVWGIKQLQARILAGGSGGDSVISLSGDHDIIGRTGGSGGHTTWMEWDR EINNYTSLIHSLIEESQNQQEKNEQELLEGDSVISLSGDHDIIGRTGDSVISLSGDHDIIGR TGDSVISLSGDHDIIGRTGHHHHHH (SEQ ID NO: 15)

[0171]

[0161] The Ag2 was similarly effective in inducing Ab immune response in WT mice as Agl, but it had a better yield as recombinant protein, thus it was used for further studies using SOD1G93Atransgenic ALS mice. For vaccination of SOD1G93Atransgenic ALS mice the inventors used Th2-specific adjuvant (Aluminium hydroxide, Alhydrogel, Invivogen), since the use of Thl adjuvants for active immunization against neurodegenerative diseases carries significant risks, including exacerbating neuroinflammation through overactivation of microglia and astrocytes, CNS infiltration of activated peripheral immune cells (cytotoxic T cells) due to weakened BBB integrity, and the induction of autoimmune responses, which could potentially worsen disease progression. The 50 pl of 1: 1 (v / v) mixture of 30 pg Ag2 and Alhydrogel was injected i.m. into SOD1G93Amice, followed by a booster of the same composition 2 weeks post priming. The Ab titer produced was similar to that found in WT mice (1 : 106), and it remained stable up to the predefined end point of the experiment (120 d; Fig. 6). Stable immunity in ALS mice could be explained by a sustained stimulation of the memory immune cells generated during the immunization by misfolded SOD1 produced in the course of the disease.

[0172]

[0162] In the immunocytochemistry experiments, sera from the Ag2 immunized mice stained numerous intra- and extra-cellular inclusions in the spinal cord sections of the paralyzed familial ALS (fALS) hSODl-G93A transgenic mice, but not in those of WT mice (Fig. 7), demonstrating that the antibodies produced against Ag2 selectively recognized disease-relevant conformations of misfolded SOD1 in vivo.

[0173]

[0163] When tested by ELISA, the Abs in the sera of Ag2 immunized mice exhibited a unique specificity profile, reacting strongly with mutated versions of SOD1, but only minimally with any form of WT SOD1 — either intact holo-SODl-WT or demetalated apo-SODl-WT, despite its exposed P6 / |37 loop4(Fig. 8).

[0174]

[0164] For comparison, the SE-21 mAb used in the inventors’ earlier studies was generated using a KLH-conjugated unstructured peptide derived from the P6 / p7 loop as antigen. The SE- 21 mAb reacted with a linear epitope and recognized, to the same extent, fALS SOD1 mutants and apo-SODl-WT, but not holo-SODl-WT. The current findings support the idea that the conformation of the exposed P6 / p7 loop in apo-SODl-WT differs from that observed in fALS SOD1 mutants. The demetalated S0D1-WT is a physiological intermediate in the process of proper folding and dimerization of the enzyme. SOD1 misfolding, however, is a multistep process facilitated by SOD1 demetalation, and results in the formation of atypical apo-SODl species deficient in their ability to bind metal cofactors. In their previous work, the inventors demonstrated that apo-SODl-WT has P6 / P7 loop epitope reversibly exposed, while the addition of metal cofactors completely restores the native-like conformation as found in holo-SODl-WT. Prolonged exposure of demetalated apo-SODl-WT to misfolding-promoting conditions results in an irreversible structural transition, forming soluble species of misfolded SOD1 unable to bind metal cofactors, which have their P6 / P7 loop epitope permanently exposed. The antibodies produced in the response to Ag2 appear to not recognize the “physiological” demetalated forms of S0D1-WT, which is a highly favorable phenomenon in the context of their use as potential therapeutics. Whether these antibodies recognize the potentially pathogenic conformations of irreversibly misfolded apo-SODl-WT remains to be tested. Altogether, the conformational factor highlights the importance of using structural mimetics of the pathogenic sequences to be targeted (such as P6 / P7 loop of SOD1) as antigens in active immunization.

[0175]

[0165] Although only about 0.1-0.4% of peripherally circulating Abs are estimated to enter the CNS, the very long plasma half-life of Abs results in an appreciable brain exposure. The inventors measured the antibody titer in the CNS tissue (combined brain and spinal cord (SC) extract) from Ag2 vaccinated WT and SOD1G93Amice and found it to be -0.2% of the level found in serum (data not shown). Although, it has been demonstrated that the blood-SC barrier (BSCB) disruption occurs in ALS, thus possibly increasing the influx of therapeutic Abs into the affected tissue, the inventors did not detect a significant difference in the CNS Ab titer between WT and SOD1G93Amice at the current experimental end point (120 d).

[0176] Evaluation of clinical efficacy of immunization

[0177]

[0166] The experimental cohort comprised 16 gender-balanced SOD1G93Atransgenic ALS mice (strain B6.Cg-Tg(SODl*G93A)lGur / J, Jackson Laboratory), immunized at age 22-25 days with 1: 1 mixture (v / v) of 30 pg Ag2 and Alhydrogel adjuvant (i.m., 50 pl) followed by a booster of the same composition 2 weeks post priming (as in Fig. 5A), and a similar number of animals for adjuvant-only control.

[0178]

[0167] After immunization, mice were assessed for disease pathology by using several behavioral and motor performance tests, acceptable in the field. The disease onset was determined based on NeuroScore, which specifically assesses motor and behavioral deficits (Figs. 9A-9E). NeuroScore is focused on hindlimb function because hindlimb deficits are the earliest reported neurological sign of disease in ALS mice. The protocol was developed by the ALS Therapy Development Institute (ALS TDI), and it “provides an unbiased assessment of onset of paresis (slight or partial paralysis), progression and severity of paralysis and it is sensitive enough to identify drug-induced changes in disease progression”. In addition, the inventors used the grip strength test to monitor disease progression, which is commonly applied to study the motor and neuronal functions of organisms and any disorders associated with them. It measures the neuromuscular function as maximal muscle strength of forelimbs and combined forelimbs and hind limbs, and it is known for “the excellent sensitivity in detecting motor dysfunction in dystrophic mice”, supported by numerous studies.

[0179]

[0168] In a separate experiment, ALS mice were injected (priming + booster) with Alhydrogel adjuvant alone and compared to untreated mice. No differences in the behavioral parameters of disease pathology were detected between these two groups (data not shown).

[0180]

[0169] The results of the in vivo study are summarized in Figs. 9-10, for males and females, respectively. In both males and females, the immunization delayed the onset of clinical motor symptoms by ~ 20 days, which represents an improvement of >20% relatively to untreated animals (Figs. 9A-9C and 10A-10C). At the predefined experimental 120 d end-point, in both male and female mice, vaccination significantly improved the grip strength of hindlimbs, although the effect was more pronounced in male mice (Figs. 9D-9F and 10D-10F).

[0181]

[0170] A molecular pathology analysis was performed at the time point of 90 days, after the appearance of first motor abnormalities in control animals. Immunohistochemistry analysis of the spinal cord tissue using B8H10 antibody, that is specific for misfolded SOD1 species, revealed that the immunization with Ag2 significantly delayed the accumulation of intra- and extra-cellular inclusions of misfolded SOD1 (Fig. 9G). Staining with anti-ChAT antibody indicated the preservation of spinal motor neurons (Fig. 9H). The vaccination also reduced the level of neuroinflammation in the CNS (‘gliosis’) known to be associated with motor neurons (MN) pathology in ALS, manifested in the decreased expression of both GFAP - the marker of activated astrocytes (Fig. 91).

[0182]

[0171] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

Claims

CLAIMSWhat is claimed is:

1. A recombinant protein comprising: (i) the amino acid sequence QLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARILA (SEQ ID NO: 6) or an analog thereof having at least 90% identity thereto; (ii) a first peptide of interest: and (iii) the amino acid sequence HTTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLE (SEQ ID NO: 7) or an analog thereof having at least 90% identity thereto; and (iv) a second peptide of interest.

2. The recombinant protein of claim 1, wherein said (i) to (iv) form a unit repeating at least three times in said recombinant protein.

3. The recombinant protein of claim 1 or 2, comprising the amino acid sequence set forth in any one of SEQ ID Nos: 22-31.

4. The recombinant protein of any one of claims 1 to 3, further comprising (v) a peptide linker located between any one of: said (i) and said (ii), between said (ii) and said (iii), between said (iii) and said (iv), and any combination thereof.

5. The recombinant protein of claim 4, wherein said peptide linker is of 1 to 8 amino acids.

6. The recombinant protein of claim 4 or 5, wherein said peptide linker is a glycine residue, or comprises the amino acid sequence GSSGG (SEQ ID NO: 3) or GGSGG (SEQ ID NO: 4).

7. The recombinant protein of any one of claims 1 to 6, wherein said (i) to (iv) are organized sequentially from the N’ -terminal end to the C’ -terminal end of said repeating unit in said recombinant protein.

8. The recombinant protein of any one of claims 4 to 7, wherein said (i) to (v) are organized as (i) - (v) - (ii) - (v) - (iii) - (v) - (iv) from the N’ -terminal end to the C’ -terminal end of said repeating unit in said recombinant protein.

9. The recombinant protein of any one of claims 4 to 8, further comprising said linker located between each of said at least three units repeating in said recombinant protein.

10. The recombinant protein of any one of claims 1 to 9, further comprising at least one third peptide of interest.

11. The recombinant protein of claim 10, wherein said at least one third peptide of interest is positioned: N’ terminally to said amino acid sequence set forth in SEQ ID NO: 6 or said analog thereof having at least 90% identity thereto; C’ terminally to said second peptide of interest; or both.

12. The recombinant protein of claim 11, wherein said at least one third peptide of interest comprises two copies of said third peptide of interest positioned C’ terminally to said second peptide of interest.

13. The recombinant protein of any one of claims 10 to 12, wherein said first, second, and at least one third peptides of interest are identical or comprise different amino acid sequences.

14. The recombinant protein of any one of claim 10 to 13, wherein any one of said first, second, and at least one third peptides of interest is an immunogenic peptide.

15. The recombinant protein of claim 14, wherein said immunogenic peptide is a fragment of a protein selected from the group consisting of: superoxide dismutase 1 (SOD1), alpha-synuclein, amyloid-beta (AP), tubulin associated unit (TAU), transactive response DNA binding protein 43 (TDP-43), and any combination thereof.

16. The recombinant protein of claim 15, wherein said fragment is of 8 to 30 amino acids.

17. The recombinant protein of claim 15 or 16, wherein said immunogenic peptide is a fragment of any one of: (a) SOD1 comprising the amino acid sequence set forth in SEQ ID Nos: 2, 14, or both; (b) alpha-synuclein the amino acid sequence set forth in SEQ ID NO: 32; (c) AP comprising the amino acid sequence set forth in SEQ ID NO: 33; (d) TDP-43 comprising the amino acid sequence set forth in SEQ ID Nos: 34, 35, or both; and (e) TAU comprising the amino acid sequence set forth in SEQ ID NO: 36, 37, or both.

18. The recombinant protein of any one of claims 1 to 17, further comprising a detection tag, a purification tag, or both, and optionally wherein said detection or purification tag is connected to said amino acid set forth in SEQ ID NO: 7 of the C’-terminus of said recombinant protein via said linker.

19. The recombinant protein of any one of claims 1 to 18, further comprising a signal peptide.

20. The recombinant protein of claim 19, wherein said signal peptide is the N-term tPA secretory signal comprising the amino acid sequence set forth in SEQ ID NO: 5.

21. The recombinant protein of claim 19 or 20, further comprising a transmembrane domain.

22. The recombinant protein of claim 21, wherein said transmembrane domain is the transmembrane domain of CD28 comprising the amino acid sequence set forth in SEQ ID NO: 9.

23. The recombinant protein of any one of claims 4 to 22, comprising the amino acid sequence set forth in SEQ ID Nos: 10-13, and 15-21.

24. A composition comprising a therapeutically effective amount of the recombinant protein of any one of claims 1 to 22, and a pharmaceutically acceptable carrier.

25. The composition of claim 23, being a vaccine.

26. The composition of claim 23 or 24, for use in treatment of a neurological disease or condition associated therewith in a subject in need thereof.

27. An artificial DNA molecule encoding the recombinant protein of any one of claims 1 to 22.

28. A recombinant cell comprising the artificial DNA molecule of claim 26.

29. A method for preparing a recombinant protein, the method comprising culturing the recombinant cell of claim 27, such that a recombinant protein encoded by said artificial DNA molecule is expressed.

30. The method of claim 28, further comprising a step before the culturing comprising introducing or transfecting a cell with the artificial DNA molecule of claim 26, thereby providing the recombinant cell of claim 27.

31. A method for treating a subject afflicted with a neurological disease or condition associated therewith, the method comprising administering to said subject a therapeutically effective amount of a pharmaceutical composition comprising the recombinant protein of any one of claims 1 to 22, thereby treating a subject afflicted with the neurological disease or condition associated therewith.

32. The method of claim 30, wherein said treating comprises delaying the appearance of at least one symptom, progression, or both, of said neurological disease or condition associated therewith.

33. The method of claim 30 or 31, wherein said treating comprises vaccinating said subject against said neurological disease or condition associated therewith.

34. The method of any one of claims 30 to 32, wherein said neurological disease or condition associated therewith is selected from the group consisting of: amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, and any combination thereof.

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