Compositions and methods for inducing immune responses against ESR1, PI3K, HER2, and HER3

Abstract

The present disclosure relates to the field of molecular virology, in particular to nucleic acid molecules encoding viral genomes and self-replicating RNA (srRNA) constructs of modified equine encephalitis viruses, pharmaceutical compositions containing same, and the use of such nucleic acid molecules and compositions for the production of desired products in cell culture or in an organism. Also provided are methods for eliciting an immune response in a subject in need thereof, and methods for preventing and / or treating cancer.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority to U.S. Patent Application No. 17 / 537,199, filed on November 29, 2021. The previously mentioned application is hereby incorporated by reference in its entirety, including any drawings.

[0002] Incorporation of Sequence Listing The contents of the accompanying sequence listing are hereby incorporated by reference into the specification of this application. The accompanying sequence listing file, designated as 058462 - 505001 WO_Sequence Listing_ST26.xml, was created on November 28, 2022 and is 101 KB in size.

[0003] Field The present disclosure relates to the fields of molecular virology and immunology, and in particular, to viral genomes of modified Venezuelan equine encephalitis virus and nucleic acid molecules encoding self - replicating RNA (srRNA), pharmaceutical compositions containing the same, and the use of such nucleic acid molecules and compositions for the production of desired products in cell culture or in vivo. Also provided are methods for inducing an immune response in a subject in need thereof, and methods for preventing and / or treating a health condition.

Background Art

[0004] Background The development of resistance to cancer therapeutic or prophylactic agents is a common problem in the treatment of cancer or pre - cancerous lesions, and the mechanisms of resistance to therapeutic agents are known in various cases. Resistance is often the result of changes in gene expression (overexpression or blockade of protein expression), changes in genes due to mutations, or altered sequences due to altered splicing or translocation, or activation of altered proteins in cells (over - activation or blockade of protein activation).

[0005] One way to address such cancers in which such changes occur in gene expression, alteration, and mutation has been the development of cancer vaccines. Cancer vaccines target antigens expressed by tumors, but the application of these vaccines has not been as effective as once hoped due to the induction of immune tolerance by the chronic overexpression of target proteins in the absence of costimulatory molecules and the induction of an immunosuppressive environment. Preventive cancer vaccines may have been more promising, but cancer is very mutable, with multiple genetic changes but only a few truly universal changes. Thus, it is difficult to predict which antigens are overexpressed in any particular cancer or whether an individual should be vaccinated, and if so, which antigens should be used.

[0006] The disclosure provided herein offers solutions to the problems that have existed with previous attempts to create cancer vaccines and potentially proposes improved methods for cancer treatment and prevention. SUMMARY OF THE INVENTION

[0007] The present disclosure generally relates to the development of immunotherapeutic agents, such as recombinant nucleic acid constructs and pharmaceutical compositions containing the same, for use in the prevention and management of various health conditions, such as cancer. In particular, as described in more detail below, some embodiments of the present disclosure provide nucleic acid constructs containing sequences encoding a modified genome or self-replicating RNA (srRNA) of alphavirus eastern equine encephalitis virus (EEEV), wherein at least a part of the nucleic acid sequence encoding a viral structural protein for the modified EEEV genome or srRNA is replaced by a coding sequence of a polypeptide construct comprising the following: a) a coding sequence of estrogen receptor 1 (ESR1) or a variant thereof; b) a coding sequence of PI3K or a variant thereof; c) a coding sequence of HER2 or a variant thereof; and d) a coding sequence of HER3 or a variant thereof. Also disclosed are recombinant cells engineered to contain one or more of the nucleic acid constructs disclosed herein, methods for producing a molecule of interest, and pharmaceutical compositions comprising one or more of the following: (a) the nucleic acid constructs of the present disclosure, (b) the recombinant cells of the present disclosure, or (c) the pharmaceutical compositions of the present disclosure. In certain aspects of the present disclosure, further provided are compositions and methods for inducing an immune response in a subject in need thereof, and / or for preventing and / or treating various health conditions, including cancer, in a subject in need of prevention and / or treatment. The above summary is merely illustrative and is in no way intended to be limiting. In addition to the exemplary embodiments and features described herein, further aspects, embodiments, objects, and features of the present disclosure will become fully apparent from the drawings, detailed description, and claims.

[0008] In one aspect of the present disclosure, provided herein is a nucleic acid construct comprising a nucleic acid sequence encoding a modified eastern equine encephalitis virus (EEEV) genome or srRNA, wherein at least a part of the nucleic acid sequence encoding the viral structural protein of the modified EEEV genome or srRNA is replaced by a coding sequence of a polypeptide construct including the following: a) a coding sequence of estrogen receptor 1 (ESR1) or a variant thereof; b) a coding sequence of PI3K or a variant thereof; c) a coding sequence of HER2 or a variant thereof, and d) a coding sequence of HER3 or a variant thereof.

[0009] In some embodiments, the modified EEEV genome or srRNA does not contain a nucleic acid sequence encoding a viral structural protein.

[0010] In some embodiments, the nucleic acid sequence encoding the modified EEEV or srRNA is operably linked to a promoter sequence.

[0011] In some embodiments, the coding sequences of (a)-(d) are operably linked to each other within a single open reading frame (e.g., within a polycistronic ORF). In some embodiments, each antigen is under the control of a separate promoter. In some other embodiments, all four antigens are under the control of a single promoter, such as the S26 subgenomic promoter.

[0012] In some embodiments, the coding sequence is operably linked to the coding sequence of a self-cleaving peptide or an internal ribosome entry site (IRES). In some embodiments, the self-cleaving peptide comprises one or more self-cleavable cleavage sequences derived from calcium-dependent serine protease (furin), porcine teschovirus-1 2A (P2A), foot-and-mouth disease virus (FMDV) 2A (F2A), equine rhinitis A virus (ERAV) 2A (E2A), Thosea asigna virus 2A (T2A), cytoplasmic polyhedrosis virus 2A (BmCPV2A), flacherie virus 2A (BmIFV2A), or combinations thereof. In some embodiments, the IRES is derived from Kaposi's sarcoma-associated herpesvirus (KSHV) IRES, hepatitis virus IRES, pestivirus IRES, cripavirus IRES, Rhopalosiphum padi virus IRES, fibroblast growth factor IRES, platelet-derived growth factor IRES, vascular endothelial growth factor IRES, insulin-like growth factor IRES, picornavirus IRES, encephalomyocarditis virus (EMCV) IRES, Pim-1 IRES, p53 IRES, Apaf-1 IRES, TDP2 IRES, L-myc IRES, and c-myc IRES.

[0013] In some embodiments, at least one of the coding sequences of (a)-(d) comprises one or more molecular modifications. In some embodiments, the one or more molecular modifications are configured into a plurality of modification cassettes arranged in tandem along the length of the coding sequence. In some embodiments, the plurality of modification cassettes are operably linked to each other by one or more linkers.

[0014] In some embodiments, the coding sequence of the ESR1 variant of (a) comprises one or more molecular modifications that promote ligand-independent receptor activity. In some embodiments, the one or more molecular modifications comprise activating mutations selected from the group consisting of K303R, E380Q, Y537C, Y537S, Y537N, and D538G.

[0015] In some embodiments, the coding sequence of the PI3K variant of (b) comprises one or more molecular modifications that promote ligand-independent receptor activity. In some embodiments, the one or more molecular modifications comprise activating mutations selected from the group consisting of E542K, E545K, H1047L, and H1047R.

[0016] In some embodiments, the HER2 variant of (c) comprises the coding sequences of the extracellular domain and the transmembrane domain.

[0017] In some embodiments, the HER3 variant of (d) comprises the coding sequence of kinase-inactive HER3.

[0018] In some embodiments, the nucleic acid sequence has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 7-10.

[0019] In some embodiments, the coding sequence of the polypeptide construct, in the 5'- to 3'- direction, comprises: a) the coding sequence of a variant of PI3K comprising one or more activating molecule modifications selected from E542K, H1047L, E545K, and H1047R; b) the coding sequence of the self-cleaving peptide P2A; c) the coding sequence of a variant of HER2 comprising its extracellular domain and transmembrane domain; d) the coding sequence of the self-cleaving peptide P2A; e) the coding sequence of a kinase-inactive variant of HER3; f) the coding sequence of an internal ribosome entry site (IRES); and g) the coding sequence of a variant of ESR1 comprising one or more activating molecule modifications selected from Y537C, E380Q, K303R, Y537S, D538G, and Y537N.

[0020] In one aspect, recombinant cells comprising the nucleic acid constructs disclosed herein are provided herein. In some embodiments, the recombinant cells are mammalian cells or insect cells.

[0021] In yet another aspect, pharmaceutical compositions comprising a pharmaceutically acceptable excipient and the nucleic acid constructs of the present disclosure are provided herein.

[0022] In some embodiments, the composition is formulated into a delivery system using a delivery vehicle, where the delivery system comprises liposomes, virus replicon particles (VRPs), lipid-based nanoparticles (LNPs), polymeric nanoparticles, physiological buffers, microspheres, immunostimulating complexes (ISCOMs), conjugates of bioactive ligands, or any combination thereof. In some embodiments, the lipid is present at a lipid-to-RNA mass ratio of about 100:1 to about 4:1. In some embodiments, the lipid-based nanoparticles have an average diameter of about 25 nm to about 1000 nm. In some embodiments, the composition is formulated as a vaccine.

[0023] In another aspect, provided herein is a method of inducing an immune response in a subject in need thereof or treating a health condition, the method comprising administering to the subject a composition comprising a nucleic acid construct of the present disclosure. In some embodiments, the method is a method of inducing an immune response. In some embodiments, the method is a method of treating cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the composition is administered to the subject individually as monotherapy (single-agent therapy) or as a first therapy in combination with at least one additional therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The features of the present disclosure are described in detail in the appended claims. A more complete understanding of the features and advantages of the present disclosure can be obtained by reference to the following detailed description that illustrates exemplary embodiments in which the principles of the present disclosure are utilized, as well as the following appended drawings:

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[0031] The present disclosure generally relates to nucleic acid constructs that express variants of ESR1, PI3K, HER2, and HER3 for the purpose of both preventing and treating human diseases such as, for example, breast cancer. Since it is difficult to predict which antigens are overexpressed in any particular cancer or whether an individual should be vaccinated and, in that case, which antigens should be used, these constructs address the problem using treatment methods such as, for example, cancer vaccines. In particular, provided herein are gene expression systems having excellent expression potential suitable for expressing the coding sequence of estrogen receptor 1 (ESR1) or a variant thereof, the coding sequence of PI3K or a variant thereof, the coding sequence of HER2 or a variant thereof, and the coding sequence of HER3 or a variant thereof in recombinant cells. For example, some embodiments of the present disclosure relate to nucleic acid constructs such as expression constructs and vectors containing a modified genome or srRNA of eastern equine encephalitis virus (EEEV) (wherein at least a part of the nucleic acid sequence encoding the viral structural protein of the modified EEEV genome or srRNA is replaced by the coding sequence of a polypeptide construct comprising the coding sequence of estrogen receptor 1 (ESR1) or a variant thereof, the coding sequence of PI3K or a variant thereof, the coding sequence of HER2 or a variant thereof, and the coding sequence of HER3 or a variant thereof). Further provided are recombinant cells genetically engineered to incorporate one or more of the nucleic acid molecules disclosed herein. Biological materials and recombinant products derived from such recombinant cells are also within the scope of the application. Also provided are compositions and methods useful for inducing an immune response or treating cancer in a subject in need thereof.

[0032] The headings of the sections used herein are for organizational purposes only and are not to be construed as limiting the described subject matter.

[0033] The various features of the disclosure may also be described in relation to a single embodiment, and the features may also be provided separately or in any suitable combination. Conversely, the disclosure may be described herein in relation to separate embodiments for clarity, but the disclosure may also be practiced in a single embodiment. Definition

[0034] Unless otherwise defined, all technical terms, notations, and other scientific or technical terms used in this specification are intended to have the meanings commonly understood by those of ordinary skill in the art to which this application pertains. In some cases, terms having commonly understood meanings are defined in this specification for clarity and / or for ready reference, and the inclusion of such definitions in this specification should not necessarily be construed as representing a substantial difference from what is commonly understood in the art. Many of the techniques and procedures described or referenced in this specification are well understood by those of ordinary skill in the art and are commonly employed using conventional methodologies.

[0035] The singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. For example, the term "cell" includes one or more cells, including mixtures thereof. "A and / or B" is used herein to include all alternatives of "A", "B", "A or B", and "A and B".

[0036] As used herein, the terms "administer" and "administering" refer to the delivery of a bioactive composition or formulation by a route of administration that includes, without limitation, intranasal, transdermal, intravenous, intraarterial, intramuscular, intraarticular, intraperitoneal, subcutaneous, intramuscular, oral, vaginal, or topical administration, or combinations thereof. The terms include, without limitation, administration by a healthcare provider and self-administration.

[0037] The terms "cell", "cell culture", and "cell line" refer not only to a particular target cell, cell culture, or cell line, but also to the progeny or potential progeny of such cell, cell culture, or cell line, regardless of the number of transfers or passages in culture. It should be understood that not all progeny are exactly the same as the parental cell. This is because certain modifications can occur in later generations due to either mutation (e.g., intentional or inadvertent mutation) or environmental effects (e.g., methylation or other epigenetic modifications), and as a result, the progeny may not actually be identical to the parental cell, but are still included within the scope of the terms used herein as long as the progeny retain the same function as the original cell, cell culture, or cell line.

[0038] The term "construct" refers to a recombinant molecule, e.g., a recombinant nucleic acid or polypeptide that includes one or more nucleic acid sequences or amino acid sequences from a heterologous origin. For example, a polypeptide construct is a chimeric polypeptide molecule in which two or more amino acid sequences of different origins are operably linked to each other in the state of a single peptide construct. Similarly, a nucleic acid construct can be a chimeric nucleic acid molecule in which two or more nucleic acid sequences of different origins are attached to a single nucleic acid molecule. Representative nucleic acid constructs can include any recombinant nucleic acid molecule, linear or circular, single-stranded or double-stranded DNA or RNA nucleic acid molecule, and can be derived from any source capable of genomic integration or autonomous replication, e.g., plasmids, cosmids, viruses, autonomous replication polynucleotide molecules, phages, and nucleic acid molecules in which one or more nucleic acid sequences are operably linked. Two or more nucleic acid constructs can be contained within a single nucleic acid molecule, e.g., a single vector, or can be contained within two or more separate nucleic acid molecules, e.g., two or more separate vectors.

[0039] The terms "effective amount," "therapeutically effective amount," or "pharmaceutically effective amount" of a composition of the present disclosure, such as a nucleic acid construct, srRNA, recombinant cell, and / or pharmaceutical composition, generally refer to an amount sufficient for the composition to achieve a given purpose as compared to in the absence of the composition (e.g., achieve an administered effect, stimulate an immune response, prevent or treat a disease, or reduce one or more symptoms of a disease, disorder, infection, or health condition). An example of an "effective amount" is an amount sufficient to contribute to the treatment, prevention, or reduction of a symptom(s) of a disease, and may also be referred to as a "therapeutically effective amount." "Reduction" of a symptom means a decrease in the severity or frequency of the symptom, or elimination of the symptom. The exact amount of a composition comprising a "therapeutically effective amount" will depend on the purpose of the treatment and will be ascertainable by one of ordinary skill in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0040] As used herein, the term "naked" refers to a nucleic acid substantially free of other macromolecules such as, for example, lipids, polymers, and proteins. A "naked" nucleic acid such as self-replicating RNA is not formulated with other macromolecules to improve cellular uptake. Thus, a naked nucleic acid is not encapsulated in, adsorbed to, or bound to liposomes, microparticles, nanoparticles, cationic emulsions, etc.

[0041] As used herein, the term "operably linked" means a physical or functional linkage between two or more elements, such as polypeptide sequences or polynucleotide sequences, that can operate in their intended manner. For example, when used in the context of a nucleic acid molecule as described herein, or a coding sequence and a promoter sequence within a nucleic acid molecule, the term "operably linked" means that the coding sequence and the promoter sequence are in an appropriate spatial and distance relationship to enable the respective binding effects of transcription factors or RNA polymerase in transcription in-frame. It should be understood that operably linked elements can be continuous or discontinuous (e.g., they can be linked to each other via a linker). In the context of a polypeptide construct, "operably linked" refers to a physical linkage (e.g., directly or indirectly linked) between amino acid sequences (e.g., different segments, portions, regions, or domains) that provides the described activity of the construct. The operably linked segments, portions, regions, and domains of the polypeptides or nucleic acid molecules disclosed herein can be continuous or discontinuous (e.g., they can be linked to each other via a linker).

[0042] As used herein, the term "portion" refers to a fraction. With respect to a particular structure such as a polynucleotide sequence, amino acid sequence or protein, the term "portion" can refer to a continuous or discontinuous fraction of the structure. For example, a portion of an amino acid sequence can include at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, and at least 90% of the amino acids of the amino acid sequence. Additionally or alternatively, when the portion is a discontinuous fraction, the discontinuous fraction can be composed of 2, 3, 4, 5, 6, 7, 8, or more portions of the structure (e.g., domains of a protein), each portion being a continuous element of the structure. For example, a discontinuous fraction of an amino acid sequence can be composed of 2, 3, 4, 5, 6, 7, 8, or more, e.g., 4 or fewer portions of the amino acid sequence, each portion including at least 1, at least 2, at least 3, at least 4, at least 5 consecutive amino acids, at least 10 consecutive amino acids, at least 20 consecutive amino acids, or at least 30 consecutive amino acids of the amino acid sequence.

[0043] When ranges of values are 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 limits of the range, and any other specified value or intervening value within the defined range, is included within the disclosure. The upper and lower limits of these smaller ranges can be independently included within the smaller ranges and are also included within the disclosure, subject to any clearly excluded limits within the defined range. When the defined range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0044] In this specification, a particular range is presented using a numerical value preceded by the term "about". The term "about" is used to literally support the exact number that the term precedes, as well as numbers that are close to, or nearly, the number that the term precedes. When determining whether a number is close to, or nearly, a specifically recited number, a number that is close to or approximates an unrecited number can be a number that provides a value that is substantially equivalent to the specifically recited number in the context in which it is presented. If the degree of approximation is not clear from the context, "about" shall mean within plus or minus 10% of the provided value, or rounded to the nearest significant digit, in all cases in which the provided value is included. In some embodiments, the term "about" indicates plus or minus up to 10%, plus or minus up to 5%, or plus or minus up to 1% of the specified value.

[0045] As used herein in the context of two or more nucleic acids or proteins, the term "percent identity" refers to two or more sequences or subsequences having a specified percentage of nucleotides or amino acids that are identical, or that are identical as measured using the BLAST or BLAST 2.0 sequence comparison algorithms with their default parameters as described below, or as measured by manual alignment and visual inspection (e.g., about 60% sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region when compared and aligned to maximize matches over a comparison window or specified region). See, e.g., the NCBI web site at ncbi.nlm.nih.gov / BLAST. Such sequences are said to be "substantially identical." This definition also refers to, or can be applied to, the complement of a sequence. This definition includes sequences with deletions and / or additions, as well as sequences with substitutions. Percent identity can be calculated using publicly available techniques and widely available computer programs such as the GCS program package (Devereux et al., Nucleic Acids Res. 12:387, 1984), BLASTP, BLASTN, FASTA (Atschul et al., J Mol Biol 215:403, 1990). Percent identity can be measured using sequence analysis software, such as the sequence analysis software of the Genetics Computer Group of the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wis. 53705), using its specified parameters.

[0046] As used herein, the term "pharmaceutically acceptable excipient" refers to any suitable substance that provides a pharmaceutically acceptable carrier, additive, or diluent for the administration of the compound(s) of interest to a subject. In so doing, "pharmaceutically acceptable excipient" can include substances referred to as pharmaceutically acceptable diluents, pharmaceutically acceptable additives, and pharmaceutically acceptable carriers. As used herein, the term "pharmaceutically acceptable carrier" includes, without limitation, saline, solvents, dispersions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., and is compatible with pharmaceutical administration. Auxiliary active compounds (e.g., antibiotics and additional therapeutic agents) can also be incorporated into the composition.

[0047] As used herein, "subject" or "individual" includes animals, such as humans (e.g., human individuals) and non-human animals. In some embodiments, "subject" or "individual" is a patient under the care of a physician. Thus, a subject can be a human patient or individual having, at risk of having, or suspected of having a target health condition (e.g., cancer) and / or one or more symptoms of a health condition. A subject can also be an individual diagnosed with a risk of a target health condition at the time of diagnosis or thereafter. The term "non-human animal" includes all vertebrates, such as mammals, such as rodents, such as mice, non-human primates, and other mammals, such as sheep, dogs, cows, chickens, and non-mammals, such as amphibians, reptiles, etc.

[0048] As used herein, aspects and embodiments of the present disclosure are to be understood to include aspects and embodiments of "comprising", "consisting of", and "consisting essentially of". As used herein, "comprising" is synonymous with "including", "containing", or "characterized by", is inclusive or open-ended, and does not exclude additional, unrecited elements or method steps. As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claims of the composition or method. As used herein, "consisting essentially of" does not exclude materials or steps that do not substantially affect the basic and novel characteristics of the claimed composition or method. Any recitation of the term "comprising" herein, particularly in the description of the components of a composition or the steps of a method, is to be understood to encompass compositions and methods consisting essentially of, and consisting of, the recited components or steps.

[0049] All genes, gene names, and gene products disclosed herein are intended to correspond to homologs from any species to which the compositions and methods disclosed herein are applicable. Thus, the terms include, but are not limited to, genes and gene products from human and mouse. When a gene or gene product from a particular species is disclosed, it is understood that this disclosure is intended only as an example and is not to be construed as a limitation unless clearly indicated in the context in which it is presented. Thus, for example, with respect to a gene or gene product disclosed herein, in some embodiments, it relates to mammalian nucleic acid and amino acid sequences and is intended to include homologous and / or orthologous genes and gene products from other animals including, but not limited to, other mammals, fish, amphibians, reptiles, and birds. In some embodiments, the genes, nucleic acid sequences, amino acid sequences, peptides, polypeptides, and proteins are of human origin. The term "gene" is also intended to include variants thereof.

[0050] It should be understood that certain features of the present disclosure are described in the context of separate embodiments for clarity and can also be provided in combination in a single embodiment. Conversely, various features of the present disclosure are described in the context of a single embodiment for brevity and can also be provided separately or in any suitable partial combination. All combinations of embodiments related to the present disclosure are specifically encompassed by the present disclosure and are disclosed herein as if all combinations were individually and explicitly disclosed. In addition, all partial combinations of various embodiments and their elements are also specifically encompassed by the present disclosure and are disclosed herein as if each such partial combination were individually and explicitly disclosed herein. Self-replicating RNA

[0051] As will be understood by those of skill in the art, the term “self-replicating RNA” refers to an RNA molecule that contains all of the genetic information necessary to direct its own self-amplification or self-replication within a permissive cell. To direct its own replication, an srRNA generally (1) interacts with a polymerase, replicase, or protein, nucleic acid, or ribonucleoprotein from a virus or host cell to encode other proteins that can catalyze the RNA amplification process; and (2) contains cis-acting RNA sequences required for the replication and transcription of the RNA encoded by the subgenomic replicon. These sequences can bind to its self-encoded proteins, or non-self-encoded cell-derived proteins, nucleic acids, or ribonucleoproteins, or complexes between any of these components, during the replication process. In some embodiments of the present disclosure, an alphavirus srRNA construct generally contains the following elements: a 5′ viral or defective interfering RNA sequence that is cis-required for replication, a sequence encoding a biologically active alphavirus nonstructural protein (e.g., nsP1, nsP2, nsP3, and nsP4), a subgenomic promoter (sg) for subgenomic RNA (sgRNA), a 3′ viral sequence that is cis-required for replication, and optionally a polyadenylation domain (poly(A)). In these cases, a subgenomic promoter (sg) that directs the expression of a heterologous sequence can be included within the srRNA constructs of the present disclosure.

[0052] Furthermore, the term srRNA generally refers to a molecule of positive polarity or “message” sense, and the srRNA may be of a length different from that of any known naturally occurring alphavirus. In some embodiments of the present disclosure, the srRNA does not contain at least a part of the coding sequence of one or more alphavirus structural proteins; and / or the sequence encoding the structural gene may be replaced with a heterologous sequence. In these cases where the srRNA is packaged within a recombinant alphavirus particle, it may contain one or more sequences, so-called packaging signals, that function to initiate interaction with alphavirus structural proteins leading to particle formation.

[0053] The srRNA constructs of the present disclosure generally have a length of at least about 2 kb. For example, the srRNA has a length of at least about 2 kb, at least about 3 kb, at least about 4 kb, at least about 5 kb, at least about 6 kb, at least about 7 kb, at least about 8 kb, at least about 9 kb, at least about 10 kb, at least about 11 kb, at least about 12 kb or more than 12 kb. In some embodiments, the srRNA has a length of from about 4 kb to about 20 kb, from about 4 kb to about 18 kb, from about 5 kb to about 16 kb, from about 6 kb to about 14 kb, from about 7 kb to about 12 kb, from about 8 kb to about 16 kb, from about 9 kb to about 14 kb, from about 10 kb to about 18 kb, from about 11 kb to about 16 kb, from about 5 kb to about 18 kb, from about 6 kb to about 20 kb, from about 5 kb to about 10 kb, from about 5 kb to about 8 kb, from about 5 kb to about 7 kb, from about 5 kb to about 6 kb, from about 6 kb to about 12 kb, from about 6 kb to about 11 kb, from about 6 kb to about 10 kb, from about 6 kb to about 9 kb, from about 6 kb to about 8 kb, from about 6 kb to about 7 kb, from about 7 kb to about 11 kb, from about 7 kb to about 10 kb, from about 7 kb to about 9 kb, from about 7 kb to about 8 kb, from about 8 kb to about 11 kb, from about 8 kb to about 10 kb, from about 8 kb to about 9 kb, from about 9 kb to about 11 kb, from about 9 kb to about 10 kb, or from about 10 kb to about 11 kb. In some embodiments, the srRNA has a length of from about 6 kb to about 14 kb. In some embodiments, the srRNA has a length of from about 6 kb to about 16 kb. Eastern equine encephalitis virus (EEEV)

[0054] Eastern equine encephalitis virus (EEEV) is a mosquito-borne virus belonging to the genus Alphavirus, which is included in a group of genetically, structurally, and serologically related viruses of the Togavirus family. Currently, the genus Alphavirus includes Sindbis virus (SINV), Semliki Forest virus (SFV), Ross River virus (RRV), Venezuelan equine encephalitis virus (VEEV), and Eastern equine encephalitis virus (EEEV), all of which are closely related and can infect various vertebrates such as mammals, rodents, fish, and birds, as well as large mammals such as humans and horses, and invertebrates such as insects. In particular, EEEV has been widely studied, and the life cycles, replication patterns, etc. of these viruses have been well characterized. Further information on this point can be found, for example, in Corrin T. et al., Vector-Borne and Zoonotic Diseases, Vol. 21, No. 5, 2021. In addition, since alphaviruses have been shown to replicate very efficiently in animal cells, they are useful as vectors for producing proteins and nucleic acids in such cells. Infection between species and individuals mainly occurs via mosquitoes, contributing to the collection of arboviruses - or arthropod-borne viruses.

[0055] Each of these alphaviruses has a positive-sense single-stranded RNA genome surrounded by a nucleocapsid enclosed in an envelope containing viral spike proteins. Alphavirus particles are enveloped and tend to be spherical (although slightly pleomorphic) and have an isometric nucleocapsid. The alphavirus genome is a positive-sense single-stranded RNA approximately 11–12 kb in length and has two open reading frames, a first frame encoding a 5’ cap, a 3’ polyA tail, and nonstructural proteins with enzymatic functions, and a second frame encoding viral structural proteins (e.g., capsid protein CP, E1 glycoprotein, E2 glycoprotein, E3 protein, and 6K protein). As an example, EEEV has a single-stranded plus-sense RNA of approximately 11.7 kb that is capped at the 5’ end and polyadenylated at the 3’ end. EEEV is transmitted by the bite of an infected mosquito, and most epizootic spread occurs in hardwood bottomlands and wetland areas where mosquito larvae develop. As suggested by its name, EEEV can infect horses and cause fever, behavioral changes, and other symptoms of encephalitis. Wild birds are the main reservoir hosts of EEEV. However, the infection is often fatal for horses.

[0056] The 5’ two-thirds of the alphavirus genome encodes a number of nonstructural proteins required for viral RNA transcription and replication. These proteins are translated directly from the RNA and, together with cellular proteins, form an RNA-dependent RNA polymerase that is essential for viral genome replication and subgenomic RNA transcription. Four nonstructural proteins (nsP1-4) are produced as a single polyprotein and constitute the viral replication machinery. Processing of the polyprotein occurs in a highly regulated manner, and cleavage at the P2 / 3 junction affects the use of the RNA template during genome replication. This site is at the bottom of a narrow depression and is not easily accessible. When cleaved, nsP3 creates a ring structure that surrounds nsP2. These two proteins have an extensive interface. Mutations in nsP2 that produce non-cytopathic viruses or temperature-sensitive phenotypes cluster in the P2 / P3 interface region. P3 mutations on the opposite side of the position of the nsP2 non-cytopathic mutation prevent efficient cleavage of P2 / 3. This can affect RNA infectivity and may alter viral RNA production levels.

[0057] The 3’-proximal third of the genome contains subgenomic RNA that serves as a template for the translation of all the structural proteins required for virus particle formation: the core nucleocapsid protein C, and the envelope proteins P62 and E1 that associate as a heterodimer. The viral membrane-anchor surface glycoproteins are involved in receptor recognition and entry into target cells by membrane fusion. The subgenomic RNA is transcribed from the p26S subgenomic promoter that is present at the 3’ end of the RNA sequence encoding the nsP4 protein. Proteolytic maturation of P62 to E2 and E3 causes changes on the virus surface. The glycoprotein “spikes” of E1, E2, and sometimes E3 together form E1 / E2 dimers or E1 / E2 / E3 trimers, with E2 extending from the center to the apex, E1 filling the apex space, and E3, when present, at the head of the spike. When the virus is exposed to the acidity of the endosome, E1 dissociates from E2 to form E1 homotrimers. This is necessary for the fusion step to drive both the cell membrane and the virus membrane together. The alphavirus glycoprotein E1 is a class II virus fusion protein, structurally different from the class I fusion proteins found in influenza virus and HIV. The E2 glycoprotein functions to interact with the nucleocapsid via its cytoplasmic domain, and its external domain serves to bind to cell receptors. Most alphaviruses lose the peripheral protein E3, but in Semliki virus it remains bound to the virus surface.

[0058] Alphavirus replication has been reported to occur on membranous surfaces within host cells. In the first step of the infectious cycle, the 5’ end of the genomic RNA is translated into a polyprotein (nsP1-4) that has RNA polymerase activity to generate a negative strand complementary to the genomic RNA. In the second step, the negative strand serves as a template for the production of two RNAs, respectively: (1) a positive genomic RNA corresponding to the genomic RNA of a secondary virus that produces other nsP proteins by translation and acts as the virus genome; (2) a subgenomic RNA that encodes the structural proteins of the virus to form infectious particles. The positive genomic RNA / subgenomic RNA ratio is regulated by proteolytic autoproteolysis of the polyprotein into nsP1, nsP2, nsP3, and nsP4. In fact, viral gene expression occurs in two stages. The first stage is mainly the synthesis of the positive genomic strand and the negative strand. In the second stage, since the synthesis of the subgenomic RNA is virtually exclusive, a large amount of structural proteins are produced. Estrogen receptor 1 (ESR1)

[0059] Estrogen is a steroid hormone that functions as the primary female hormone. Receptor 1 (ESR1) encodes estrogen receptor α (ERα), and Estrogen Receptor 2 (ESR2) encodes estrogen receptor β (ERβ). The biological effects of estrogen are mediated almost entirely by its binding to and activation of ERα and ERβ, which are members of the nuclear receptor superfamily of transcription factors characterized by highly conserved DNA- and ligand-binding domains. Previous studies have suggested that estrogen is associated with breast tumorigenesis, ovarian and endometrial carcinogenesis. Approximately 70% of all breast cancers are classified as estrogen receptor positive (ER+); they depend on constitutive estrogen receptor signaling. Various classes of endocrine (anti-estrogen) therapies, including selective estrogen receptor modulators (SERMs), downregulators, and aromatase inhibitors (AIs), are effective treatments for these cancers as adjuvant therapy, but approximately 50% of women will ultimately relapse and die from metastatic ER+ disease. Thus, despite the emergence of newer therapies such as AIs, the recurrence rate in ER+ breast cancer, particularly in cases where metastasis has occurred, remains unchanged. Significantly, all patients who develop metastatic ER+ disease progress to endocrine therapy-resistant disease. At this stage, there is no therapy for ER+ breast cancer. Human epidermal growth factor 2 (HER2)

[0060] The human epidermal growth factor receptor (HER) family, consisting of HER1 (also known as EGFR), HER2, HER3, and HER4, drives the progression of many epithelial malignancies (Roskoski R., Jr The ErbB / HER family of protein-tyrosine kinases and cancer. Pharmacol Res 2014; 79:34-74). HER2, also known as Erb-B2 (erythroblastic leukemia viral oncogene homolog 2), CD340, or p185, is an 185-kD cancer protein encoded by the ERBB2 gene. It consists of three domains, including an intracellular domain with tyrosine kinase properties, a transmembrane domain, and an extracellular domain. HER2 is a preferred dimerization partner for other HER proteins, such as HER3 (which heterodimerizes with it). Dimerization with HER2 leads to autophosphorylation of tyrosine residues within the cytoplasmic region of the receptor and initiates various signaling pathways. HER2 has tumor-promoting functions in several cancers, and amplification or overexpression of HER2 is associated with enhanced disease recurrence and poor prognosis. Treatment of HER2-amplified breast cancer with HER2-targeted tyrosine kinase inhibitors (TKIs) leads to increased HER3 expression and downstream signaling, which results in treatment resistance. Human epidermal growth factor 3 (HER3)

[0061] HER3, which is overexpressed in breast, lung, gastric, head and neck, and ovarian cancers, as well as melanoma, is associated with poor prognosis (Takikita M et al., Membranous expression of Her3 is associated with a decreased survival in head and neck squamous cell carcinoma. J Transl Med 2011; 9:126; Chiu et al, HER-3 overexpression is prognostic of reduced breast cancer survival: A study of 4046 patients. Ann Surg 2010; 251(6):1107-16; Hayashi et al., High expression of HER3 is associated with a decreased survival in gastric cancer. Clin Cancer Res 2008; 14(23):7843-9; Giltnane et al, Quantitative multiplexed analysis of ErbB family coexpression for primary breast cancer prognosis in a large retrospective cohort. Cancer 2009; Begnami et al., Prognostic implications of altered human epidermal growth factor receptors (HERs) in gastric carcinomas: HER2 and HER3 are predictors of poor outcome. J Clin Oncol 2011; 29(22):3030-6; Reschke et al., HER3 is a determinant for poor prognosis in melanoma, Clin Cancer Res2 008; 14(16):5188-97; Lee et al., Assessment of Her-1, Her-2, and Her-3 expression and Her-2 amplification in advanced stage ovarian carcinoma. Int J Gynecol Pathol 2005), However, since it lacks catalytic kinase activity and is not transformed by itself, it is not a proven therapeutic target. However, HER3 is thought to function as a signaling substrate for other HER proteins with which it heterodimerizes (Musgrove et al., Biological determinants of endocrine resistance in breast cancer. Nat Rev Cancer 2009; 9(9):631-43; Tovey et al., Can molecular markers predict when to implement treatment with aromatase inhibitors in invasive breast cancer? Clin Cancer Res 2005; 11(13):4835-42). PIK3CA

[0062] Pathological activation of the PI3K pathway is common to the most frequent signaling events associated with cell transformation, cancer, and metastasis (Cancer Genome Atlas Network. Comprehensive molecular portraits of human breast tumours. Nature 2012; Mollon L, Aguilar A, Anderson E, et al. A systematic literature review of the prevalence of PIK3CA mutations and mutation hotspots in HR+ / HER2-metastatic breast cancer. Cancer Res 2018; 78: Suppl 13:1207-1207. Abstract; Goncalves MD, Hopkins BD, Cantley LC. Phosphatidyl inositol 3-kinase, growth disorders, and cancer. N Engl J Med 2018; 379: 2052-2062). This is exemplified by frequent activating mutations in PIK3CA, as well as loss of PTEN functionality in common cancers such as breast, colon, and ovarian cancers. Approximately 40% of patients with HR-positive, HER2-negative breast cancer have activating mutations in the gene PIK3CA, including hyperactivation of the α isoform (p110α) of phosphatidylinositol 3-kinase (PI3K). The compositions of the present disclosure

[0063] As described in more detail below, one aspect of the present disclosure relates to a nucleic acid construct containing a modified genome of alphavirus eastern equine encephalitis virus (EEEV) or a sequence encoding srRNA, wherein at least a part of the nucleic acid sequence encoding the viral structural protein of the modified EEEV genome or srRNA is replaced by the coding sequence of a polypeptide construct comprising a) a coding sequence of estrogen receptor 1 (ESR1) or a variant thereof; b) a coding sequence of PI3K or a variant thereof; c) a coding sequence of HER2 or a variant thereof; and d) a coding sequence of HER3 or a variant thereof. Recombinant cells and cell cultures genetically engineered to include the nucleic acid constructs disclosed herein are also provided. Nucleic acid construct

[0064] As described in more detail below, one aspect of the present disclosure relates to a nucleic acid construct comprising a nucleic acid sequence encoding a modified genome of eastern equine encephalitis virus (EEEV) or srRNA, wherein at least a part of the nucleic acid sequence encoding the viral structural protein of the modified EEEV genome or srRNA is replaced by the coding sequence of a polypeptide construct comprising a) a coding sequence of ESR1 or a variant thereof; b) a coding sequence of PI3K or a variant thereof; c) a coding sequence of HER2 or a variant thereof; and d) a coding sequence of HER3 or a variant thereof. Recombinant cells and cell cultures genetically engineered to include the nucleic acid constructs disclosed herein are also provided. In some embodiments, the sequence encoding the nucleic acid construct can be operably linked, for example, placed under the control of elements required for expression (e.g., a promoter sequence), and it enables the expression of the srRNA construct in a host cell, or a subject, or an ex vivo cell-free expression system.

[0065] The terms "nucleic acid molecule" and "polynucleotide" are used interchangeably herein and refer to both RNA and DNA molecules, including nucleic acid molecules such as cDNA, genomic DNA, synthetic DNA, and DNA, as well as RNA molecules including nucleic acid analogs. The nucleic acid molecule can be double-stranded or single-stranded (e.g., sense strand or antisense strand). The nucleic acid molecule may contain non-conventional or modified nucleotides. The terms "polynucleotide sequence" and "nucleic acid sequence" are used interchangeably herein to refer to the sequence of a polynucleotide molecule. The terminology regarding nucleotide bases as defined in 37 CFR §1.822 is used herein.

[0066] The nucleic acid molecules of the present disclosure can be of any length, for example, from about 1.5 Kb to about 50 Kb, from about 5 Kb to about 40 Kb, from about 5 Kb to about 30 Kb, from about 5 Kb to about 20 Kb, or from about 10 Kb to about 50 Kb, such as from about 15 Kb to 30 Kb, from about 20 Kb to about 50 Kb, from about 20 Kb to about 40 Kb, from about 5 Kb to about 25 Kb, or from about 30 Kb to about 50 Kb.

[0067] Non-limiting, exemplary embodiments of the nucleic acid constructs of the present disclosure may include one or more of the following features. In some embodiments, the nucleic acid construct includes a nucleic acid sequence encoding a modified EEEV genome or srRNA, where the modified EEEV genome or srRNA lacks at least a portion of the nucleic acid sequence encoding one or more structural proteins of the unmodified EEEV genome or srRNA; for example, the modified EEEV genome or srRNA does not include at least a portion of the coding sequences for one or more EEEV structural proteins, CP, E1, E2, E3, and 6K. Both pathogenic and non-pathogenic EEEV strains are suitable. Non-limiting examples of EEEV strains suitable for the compositions and methods of the present disclosure include EEEV792138, 783372, BeAn5122, BeAr300851, BeAr436087, C-49, FL91-4679, FL93-939, GML903836, MP-9, PE6, and V105-00210. Additional suitable EEEV strains include, but are not limited to, those described on the Virus Pathogen Resource website (ViPR; publicly available at www.viprbrc.org / brc / vipr_genome_search.spg? method= SubmitForm&blockId=868&decorator=toga). In some embodiments, the modified EEEV genome or srRNA is derived from the EEEV strain FL93-939.

[0068] Non-limiting exemplary embodiments of the nucleic acid constructs of the present disclosure can include one or more of the following features. In some embodiments, the modified EEEV genome or srRNA lacks at least a portion of the nucleic acid sequences encoding one or more viral structural proteins CP, E1, E2, E3, and 6K of the unmodified EEEV genome or srRNA. In some embodiments, the modified EEEV genome or srRNA lacks a portion or all of the sequence encoding CP. In some embodiments, the modified EEEV genome or srRNA lacks a portion or all of the sequence encoding E1. In some embodiments, the modified EEEV genome or srRNA lacks a portion or all of the sequence encoding E2. In some embodiments, the modified EEEV genome or srRNA lacks a portion or all of the sequence encoding E3. In some embodiments, the modified EEEV genome or srRNA lacks a portion or all of the sequence encoding 6K. In some embodiments, the modified EEEV genome or srRNA lacks a portion or all of the sequence encoding a combination of CP, E1, E2, E3, and 6K. Some embodiments of the present disclosure provide a modified EEEV genome or srRNA in which the coding sequences of the non-structural proteins nsP1, nsP2, nsP3, and nsP4 of the unmodified EEEV genome or srRNA are present, but the EEEV genome or srRNA of at least a portion or all of the sequences encoding one or more structural proteins (e.g., CP, E1, E2, E3, and 6K) is absent. Some embodiments of the present disclosure provide a modified EEEV genome or srRNA in which the coding sequences of the non-structural proteins nsP1, nsP2, nsP3, and nsP4 of the unmodified EEEV genome or srRNA are present, but the EEEV genome or srRNA of at least a portion or all of the sequences encoding one or more structural proteins (e.g., CP, E1, E2, E3, and 6K) is absent.

[0069] In some embodiments, the modified viral genome or srRNA lacks a substantial portion of the nucleic acid sequence encoding one or more viral structural proteins. One of ordinary skill in the art will understand that a substantial portion of the nucleic acid sequence encoding a viral structural polypeptide can include a viral structural polypeptide-encoding nucleic acid sequence sufficient to enable the putative identification of that polypeptide either by manual evaluation of the sequence by one of ordinary skill in the art or by computer automated sequence comparison and identification using algorithms such as BLAST (see, e.g., “Basic Local Alignment Search Tool”; Altschul SF et al., J. Mol. Biol. 215:403-410, 1993). Thus, a substantial portion of a nucleotide sequence includes a sequence sufficient to enable the specific identification and / or isolation of the nucleic acid fragment containing the sequence. For example, a substantial portion of a nucleic acid sequence can include at least about 20%, such as about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95% of the full-length nucleic acid sequence. As described above, the present disclosure provides nucleic acid molecules and constructs that lack partial or complete nucleic acid sequences encoding one or more viral structural proteins. One of ordinary skill in the art has the benefit of the sequences disclosed herein and can readily use all or substantial portions of the disclosed sequences in the compositions and methods of the present disclosure. Accordingly, the present application includes the complete sequences disclosed herein, such as those set forth in the accompanying Sequence Listing, and substantial portions of those sequences as defined above.

[0070] In some embodiments, the modified EEEV genome or srRNA lacks the entire sequence encoding the viral structural protein, e.g., the modified EEEV genome or srRNA does not include the nucleic acid sequence encoding the structural protein of the unmodified viral genome or srRNA.

[0071] The nucleic acid constructs of the present disclosure further include a coding sequence of a polypeptide construct that replaces at least a part of the nucleic acid sequence encoding the viral structural protein or srRNA of the modified EEEV genome. In principle, the nucleic acid constructs disclosed herein generally include the coding sequences of any number of polypeptide constructs. In some embodiments, the nucleic acid constructs disclosed herein may include the coding sequences of at least 1, at least 2, at least 3, at least 4, at least 5, or at least 6 polypeptide constructs. The coding sequence of a polypeptide construct relates to a construct of genetic material that includes the coding sequence and control information sufficient to direct proper transcription and / or translation of the coding sequence in cells in vivo and / or ex vivo. The coding sequence of a polypeptide construct can be inserted into a vector and / or into a subject for targeting to a desired host cell. Thus, in some embodiments, the term "coding sequence of a polypeptide construct" can be used interchangeably with the term "expression construct". In some embodiments, the coding sequence of a polypeptide construct can be, for example, a nucleic acid construct that includes a gene encoding a protein or functional RNA operably linked to regulatory elements such as a promoter and / or a termination signal, and optionally any one or a combination of other nucleic acid sequences that affect transcription or translation of the gene.

[0072] The nucleic acid constructs described herein include the coding sequences of ESR1 or its variants, PI3K or its variants, HER2 or its variants, and HER3 or its variants, and it encodes a polypeptide containing an epitope capable of eliciting an immune response. Variants of ESR1, PI3K, HER2, and HER3 are the same as those of the reference protein (e.g., ESR1, PI3K, HER2, or HER3), or may include coding sequences of polypeptides having essentially the same amino acid sequence, except that each has at least one amino acid modification, e.g., deletion, insertion, or substitution. Amino acid substitutions may preferably be conservative amino acid substitutions at non-essential amino acid residues within the protein. "Conservative amino acid substitution" means that an amino acid residue is substituted with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are known in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), amino acids having acidic side chains (e.g., aspartic acid, glutamic acid), amino acids having uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids having nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids having β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids having aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Variants of the protein may have an amino acid sequence that is at least about 80%, 90%, 95%, or 99%, preferably at least about 90%, more preferably at least about 95% identical to the amino acid sequence of the protein. Preferably, the variant is a functional variant of the protein that retains the same function as the protein. The term "variant" when used with respect to a nucleic acid sequence refers to a nucleic acid sequence that is different from another, usually the related nucleotide acid sequence, by one or more nucleotides.Thus, the term "variant" can refer to a change in one or more nucleotides of a reference nucleic acid, including the insertion of one or more new nucleotides, the deletion of one or more nucleotides, and the substitution of one or more existing nucleotides. Variants can also include point mutations, multiple mutations, single nucleotide polymorphisms (SNPs), deletions, insertions, and translocations. Thus, variants of the coding sequences described herein include, for example, nucleic acids encoding polypeptides that can be full-length, mutated, truncated, inactive, peptides / epitopes, or combinations thereof of ESR1, PI3K, HER2, and / or HER3.

[0073] The full-length amino acid sequence of ESR1 is defined as SEQ ID NO: 1 as follows: MTMTLHTKASGMALLHQIQGNELEPLNRPQLKIPLERPLGEVYLDSSKPAVYNYPEGAAYEFNAAAAANAQVYGQTGLPYGPGSEAAAFGSNGLGGFPPLNSVSPSPLMLLHPPPQLSPFLQPHGQQVPYYLENEPSGYTVREAGPPAFYRPNSDNRRQGGRERLASTNDKGSMAMESAKETRYCAVCNDYASGYHYGVWSCEGCKAFFKRSIQGHNDYMCPATNQCTIDKNRRKSCQACRLRKCYEVGMMKGGIRKDRRGGRMLKHKRQRDDGEGRGEVGSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEMLDAHRLHAPTSRGGASVEETDQSHLATAGSTSSHSLQKYYITGEAEGFPATV

[0074] In some embodiments, the coding sequence of ESR1 within the nucleic acid constructs described herein encodes the amino acid sequence of SEQ ID NO: 1. In some embodiments, the nucleic acid constructs of the present disclosure comprise a nucleic acid sequence encoding ESR1 having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the coding sequence of ESR1 encodes a smaller portion of the amino acid sequence of SEQ ID NO: 1. These smaller portions can include at least 8, 10, 12, 14, 16, 18, 20, 30 or more amino acids of SEQ ID NO: 1. Exemplary portions of ESR1 useful in the constructs disclosed herein include those in Table 1 below: Table 1

Table 1

[0075] In some embodiments, the nucleic acid constructs of the present disclosure comprise a nucleic acid sequence encoding a portion of ESR1 having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequences of SEQ ID NOS: 11-16.

[0076] In some embodiments, the coding sequences of the polypeptide constructs of the nucleic acids described herein include one or more molecular modifications. Exemplary types of molecular modifications of the coding sequences described herein can be one or more deletions, substitutions, insertions, duplications, mutations, frameshift mutations, splice mutations, or combinations thereof.

[0077] In some embodiments, one or more molecular modifications are configured into a plurality of modification cassettes. In some embodiments, the plurality of modification cassettes are arranged tandemly in the length direction of the coding sequence. In some embodiments, the length and amino acid composition of the modification cassette can be optimized to obtain the desired activity or property of the coding sequence or its variant. In some embodiments, one of the plurality of modification cassettes comprises from about 2 to about 50 amino acid residues, such as from about 5 to about 45, from about 10 to about 40, from about 15 to about 30, from about 20 to about 50, from about 2 to about 30, from about 3 to about 25, from about 4 to about 20, from about 5 to about 15, from about 6 to about 10, from about 3 to about 15, from about 4 to about 10, from about 5 to about 30, from about 2 to about 5, from about 3 to about 5, from about 4 to about 8 amino acid residues, etc. In some embodiments, one of the plurality of modification cassettes comprises 31 amino acid residues. In some embodiments, one of the plurality of modification cassettes comprises 1, 2, 3, 4, 5 or more molecular modifications.

[0078] In some embodiments, the ESR1 variants described herein comprise one or more molecular modifications that promote ligand-independent receptor activity. These variants are activating mutations in the ligand-binding domain of ESR1 that render the estrogen receptor insensitive to hormonal therapy. In some embodiments, the one or more molecular modifications comprise activating mutations selected from the group consisting of K303R, E380Q, Y537C, Y537S, Y537N, and D538G at positions corresponding to the amino acid sequence of SEQ ID NO: 1.

[0079] In some embodiments, one or more molecular modifications are operably linked to each other by a linker. The linker is a peptide linker, and it links two adjacent modification cassettes together as described herein. In some embodiments, the length and amino acid composition of the peptide linker sequence can be optimized to alter the directionality, flexibility, and / or proximity of the modification cassettes relative to each other to achieve the desired activity or property of ESR1 or the ESR1 variant.

[0080] In some embodiments, the polypeptide linker comprises a single-stranded polypeptide sequence comprising from about 1 to about 30 amino acid residues (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acid residues). In some embodiments, the linker sequence comprises from about 2 to 30, from about 3 to 25, from about 4 to 20, from about 5 to 15, from about 6 to 10, from about 3 to 15, from about 4 to 10, from about 5 to 30, from about 2 to 5, from about 3 to 5, from about 4 to 8 amino acid residues.

[0081] In some embodiments, the length and amino acid composition of the linker peptide sequence can be optimized to change the orientation, flexibility, and / or proximity of the modification cassettes relative to each other to achieve the desired activity or properties of the encoded polypeptide. In some embodiments, the orientation, flexibility, and / or proximity of modification cassettes relative to each other can be altered as a "tuning" tool to achieve a tuning effect that will enhance or reduce the activity of the encoding polypeptide or encoded polypeptide variant. In certain embodiments, the linker comprises only glycine and / or serine residues (e.g., a serine-glycine linker). Examples of such polypeptide linkers include Gly, Ser; Gly Ser; Gly Gly Ser; Ser Gly Gly; Gly Gly Gly Ser; Ser Gly Gly Gly; Gly Gly Gly Gly Ser; Ser Gly Gly Gly Gly; Gly Gly Gly Gly Gly Ser; Ser Gly Gly Gly Gly Gly; Gly Gly Gly Gly Gly Gly Ser; Ser Gly Gly Gly Gly Gly Gly; (Gly Gly Gly Gly Ser)n where n is an integer of 1 or more; and (Ser Gly Gly Gly Gly)n where n is an integer of 1 or more. In some embodiments, the polypeptide linker is modified such that the amino acid sequence Gly Ser Gly (GSG) (which occurs at the junction of conventional Gly / Ser linker polypeptide repeats) is absent. In some embodiments, examples of the peptide linker include amino acid sequences selected from the group consisting of SEQ ID NOs: 25-29.

[0082] In some embodiments, the coding sequence of the polypeptide construct of the nucleic acid construct described herein encodes a variant of ESR1 that includes a portion of the ESR1 amino acid sequence operably linked to a (underlined) GGGGS linker. An exemplary amino acid sequence includes the amino acid sequence of SEQ ID NO: 2 as follows: MEHLYSMKCKNVVPLCDLLLEMLDAHRLHAP GGGGS PGFVDLTLHDQVHLLQCAWLEILMIGLVWRS GGGGS AANLWPSPLMIKRSKRNSLALSLTADQMVSA GGGGS MEHLYSMKCKNVVPLSDLLLEMLDAHRLHAP GGGGS MEHLYSMKCKNVVPLYGLLLEMLDAHRLHAP GGGGS MEHLYSMKCKNVVPLNDLLLEMLDAHRLHAP GGGGS An exemplary illustration of this type of configuration is shown in FIG. 4.

[0083] In some embodiments, the nucleic acid constructs of the present disclosure include nucleic acid sequences encoding ESR1 having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 2.

[0084] As described above, the nucleic acid constructs described herein also include the coding sequence of PI3K or a variant thereof.

[0085] The full-length amino acid sequence of PI3K is defined in SEQ ID NO: 3 below:

[0086] In some embodiments, the coding sequence of PI3K of the nucleic acid constructs described herein encodes the amino acid sequence of SEQ ID NO: 3. In some embodiments, the nucleic acid constructs of the present disclosure include nucleic acid sequences encoding PI3K having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the coding sequence of PI3K encodes a smaller portion of the amino acid sequence of SEQ ID NO: 3. These smaller portions can include at least 8, 10, 12, 14, 16, 18, 20 or more amino acids of SEQ ID NO: 3. Exemplary portions of ESR1 useful in the constructs disclosed herein include those in Table 2 below: Table 2

Table 2

[0087] In some embodiments, the nucleic acid constructs of the present disclosure include nucleic acid sequences encoding a portion of PI3K having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequences of SEQ ID NOS: 17-20.

[0088] In some embodiments, the coding sequence of the polypeptide construct in the nucleic acids described herein encodes a PI3K variant that includes one or more molecular modifications that promote ligand-independent receptor activity. These variants are activating mutations in the ligand-binding domain of PI3K. In some embodiments, the one or more molecular modifications include activating mutations selected from the group consisting of E542K, E545K, H1047L, and H1047R of the amino acid sequence of SEQ ID NO: 3.

[0089] In some embodiments, one or more molecular modifications are operably linked to each other by a linker. Linkers suitable for use in the polypeptide constructs described herein are those described above. In some embodiments, the peptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-29.

[0090] In some embodiments, the coding sequence of the polypeptide construct of the nucleic acid construct described herein encodes a variant of PI3K that comprises a portion of the amino acid sequence of PI3K operably linked by a (underlined) GGGGS linker. An exemplary amino acid sequence comprises the amino acid sequence of SEQ ID NO: 4 as follows: MDKEQLKAISTRDPLSKITEQEKDFLWSHRHY GGGGS EQEALEYFMKQMNDALHGGWTTKMDWIFHTIK GGGGS QLKAISTRDPLSEITKQEKDFLWSHRHYCVT GGGGS EQEALEYFMKQMNDARHGGWTTKMDWIFHTIK GGGGS

[0091] In some embodiments, the nucleic acid constructs of the present disclosure comprise a nucleic acid sequence encoding PI3K having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 4.

[0092] As described above, the nucleic acid constructs described herein also comprise a coding sequence of HER2 or a variant thereof.

[0093] In some embodiments, the coding sequence of the polypeptide construct in the nucleic acids described herein encodes a truncated HER2 variant that includes the extracellular and transmembrane domains of HER2 as described in Crosby et al., “Vaccine-Induced Memory CD8+ T Cells Provide Clinical Benefit in HER2 Expressing Breast Cancer: A Mouse to Human Translational Study,” Clinical Cancer Research 25(9):2725-2736 (2019).

[0094] In some embodiments, the truncated HER2 variant includes the amino acid sequence of SEQ ID NO: 5 below: MELAALCRWGLLLALLPPGAASTQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDNGDPLNNTTPVTGASPGGLRELQLRSLTEILKGGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACPYNYLSTDVGSCTLVCPLHNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFETLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQPCPINCTHSPLTSIISAVVGILLVVVLGVVFGILIKRRQQKIRK

[0095] In some embodiments, the nucleic acid constructs of the present disclosure include nucleic acid sequences encoding HER2 variants having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 5.

[0096] As described above, the nucleic acid constructs described herein also include coding sequences for HER3 or variants thereof.

[0097] In some embodiments, the coding sequence of the polypeptide construct in the nucleic acids described herein encodes a kinase-inactive HER3 variant as described in Osada et al., “Vaccination Targeting Human HER3 Alters the Phenotype of Infiltrating T cells and Responses to Immune Checkpoint Inhibition,” Oncoimmunology 6(6):e1315495 (2017).

[0098] In some embodiments, the kinase-inactive HER3 variant comprises the amino acid sequence of SEQ ID NO: 6 below:

[0099] In some embodiments, the nucleic acid construct of the present disclosure includes a nucleic acid sequence encoding a HER3 variant having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 6.

[0100] In some embodiments, the coding sequences of ESR1 or a variant thereof, PI3K or a variant thereof, HER2 or a variant thereof, and HER3 or a variant thereof include the coding sequence of a single peptide (e.g., a single gene construct). In some embodiments, the coding sequences of ESR1 or a variant thereof, PI3K or a variant thereof, HER2 or a variant thereof, and HER3 or a variant thereof include the coding sequences of multiple polypeptides, e.g., multiple genes (e.g., two or three genes). In some embodiments, each of the coding sequences of ESR1 or a variant thereof, PI3K or a variant thereof, HER2 or a variant thereof, and HER3 or a variant thereof is operably linked to a separate promoter sequence. In some embodiments, the coding sequences of ESR1 or a variant thereof, PI3K or a variant thereof, HER2 or a variant thereof, and HER3 or a variant thereof are operably linked to each other within a single open reading frame (e.g., within a polycistronic ORF). In some embodiments, the coding sequence of the polycistronic ORF is operably linked to a promoter sequence. In some embodiments, at least one of the promoter sequences is a subgenomic (sg) promoter. In some embodiments, the sg promoter is the 26S genomic promoter.

[0101] In some embodiments, the coding sequences of ESR1 or a variant thereof, PI3K or a variant thereof, HER2 or a variant thereof, and HER3 or a variant thereof can be directly or indirectly linked to each other (e.g., via one or more linker sequences). For example, in some embodiments, the coding sequences can be directly linked to each other, e.g., adjacent to each other. In some embodiments, at least two (e.g., two, three, four, or five) of the coding sequences are operably linked to each other by one or more linker sequences. In some embodiments, the length and amino acid composition of the linker sequences can be optimized to alter the directionality, flexibility, and / or proximity of the polypeptides to each other to achieve the desired activity or properties of the encoded protein. In some embodiments, one of the linker sequences of the plurality of linker sequences includes the coding sequence of one or more self-cleaving peptide sequences. Generally, any proteolytic cleavage site known in the art can be incorporated into the nucleic acid molecules of the present disclosure and can be a proteolytic cleavage sequence that is cleaved after production, e.g., by a protease. Further suitable proteolytic cleavage sites also include proteolytic cleavage sequences that are cleavable after the addition of an exogenous protease. As used herein, the term "self-cleaving peptide" refers to a "self-cleaving" peptide that has self-cleaving activity and can cleave itself from a larger polypeptide moiety. Several self-cleaving peptides (e.g., "2A-like" peptides from equine rhinitis A virus (E2A), porcine teschovirus-1 (P2A), and Theiler's murine encephalomyelitis virus (T2A), etc.), first identified in foot-and-mouth disease virus (FMDV), a member of the picornavirus group, have subsequently been identified, and their activity in proteolytic cleavage has been demonstrated in various eukaryotic cell lines in vitro, ex vivo, and in vivo. Thus, the concept of self-cleaving peptides is available to those skilled in the art, and many naturally occurring self-protease systems have been identified.Well-studied self-protease systems include, for example, viral proteases, developmental proteins (e.g., HetR, Hedgehog protein), RumA self-protease domain, UmuD, etc. Non-limiting examples of self-proteolytic peptides suitable for the compositions and methods of the present disclosure include calcium-dependent serine endoprotease (furin), porcine teschovirus-1 2A (P2A), foot-and-mouth disease virus (FMDV) 2A (F2A), equine rhinitis A virus (ERAV) 2A (E2A), Theophila asiatica virus 2A (T2A), cytoplasmic polyhedrosis virus 2A (BmCPV2A), flacherie virus 2A (BmIFV2A), or one or more self-proteolytic cleavage sequences derived from a combination thereof.

[0102] In some embodiments, the coding sequences of ESR1 or a variant thereof, PI3K or a variant thereof, HER2 or a variant thereof, and HER3 or a variant thereof are operably linked to each other by the coding sequence of one or more internal ribosome entry sites (IRESs). An IRES or "internal ribosome entry site" is a sequence located between polycistronic genes that enables the production of an expression product resulting from a second gene by internal initiation of translation of a dicistronic mRNA. It facilitates the direct internal ribosome entry to the start codon, such as ATG, of a cistron (protein-coding region), resulting in cap-independent translation of that gene. See, for example, Jackson et al., 1990. Trends Biochem Sci 15(12):477-83) and Jackson and Kaminski. 1995. RNA 1(10):985-1000. In some embodiments, the IRES can be a viral IRES, a cellular IRES, or an artificial IRES. Examples of IRESs commonly used by those skilled in the art include those described in U.S. Patent No. 6,692,736. In some embodiments, the IRES is selected from Kaposi's sarcoma-associated herpesvirus (KSHV) IRES, hepatitis virus IRES, pestivirus IRES, cribavirus IRES, loparvirus padi IRES, fibroblast growth factor IRES, platelet-derived growth factor IRES, vascular endothelial growth factor IRES, insulin-like growth factor IRES, picornavirus IRES, encephalomyocarditis virus (EMCV) IRES, Pim-1 IRES, p53 IRES, Apaf-1 IRES, TDP2 IRES, L-myc IRES, and c-myc IRES. In some embodiments, the IRES is obtainable from EMCV.

[0103] One of ordinary skill in the art will understand that the coding sequences of ESR1 or variants thereof, PI3K or variants thereof, HER2 or variants thereof, and HER3 or variants thereof, sequences encoding self-cleaving peptides, or various configurations of IRES can be utilized as long as the expression of ESR1 or variants thereof, PI3K or variants thereof, HER2 or variants thereof, and HER3 or variants thereof is appropriately maintained. These sequences are typically configured such that the polypeptide encoded by the gene of interest is released from the protease and other sequences after cleavage by the self-protease.

[0104] As used herein, the term "operably linked" means a functional linkage between two or more sequences. For example, an operable linkage between a polynucleotide of interest and a regulatory sequence (e.g., a promoter) is a functional linkage that enables the expression of the polynucleotide of interest. In this sense, the term "operably linked" refers to the positioning of the regulatory region and the transcribed coding sequence such that the regulatory region is effective to regulate the transcription or translation of the coding sequence of interest. In some embodiments disclosed herein, the term "operably linked" means a configuration in which a regulatory sequence is positioned in an appropriate position relative to a sequence encoding a polypeptide or functional RNA such that the control sequence directs or modulates the expression or cellular localization of the mRNA encoding the polypeptide, the polypeptide, and / or the functional RNA. Thus, a promoter is operably linked to a nucleic acid sequence if it can mediate the transcription of the nucleic acid sequence. The operably linked elements may or may not be adjacent.

[0105] The basic techniques for operably linking two or more sequences of DNA together are well known to those skilled in the art, and such methods are described in many books on standard molecular biology procedures (see, for example, Maniatis et al., “Molecular Cloning: A Laboratory Manual” 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; and Gibson et al., Nature Methods 6:343-45, 2009).

[0106] As shown in Figure 3, T cell responses to various one-gene, two-gene, and four-gene constructs having various sequences of “Gene 1”, “Gene 2”, “Gene 3”, or “Gene 4” were measured (since PI3K did not form a response in BALB / c mice, it is not shown in Figure 3). In some embodiments, it should be understood that “Gene 1”, “Gene 2”, “Gene 3”, or “Gene 4” can be ESR1 or a variant thereof. Similarly, in some embodiments, “Gene 1”, “Gene 2”, “Gene 3”, or “Gene 4” can be HER2 or a variant thereof. In other embodiments, “Gene 1”, “Gene 2”, “Gene 3”, or “Gene 4” can be HER3 or a variant thereof. In some embodiments, “Gene 1”, “Gene 2”, “Gene 3”, or “Gene 4” can be PI3K or a variant thereof. An exemplary composition of the nucleic acid constructs described herein is shown in Table 3. Table 3.

Table 3-1

Table 3-2

Table 3-3

Table 3-4

[0107] In some embodiments, the composition is selected from the group consisting of pRB-136, pRB-146, pRB-151, and pRB-153.

[0108] In some embodiments, the nucleic acid sequence has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 7-10.

[0109] In some embodiments, the coding sequence of the polypeptide construct, in the 5'- to 3'-direction, comprises: a) the coding sequence of a mutant of PI3K comprising one or more molecular modifications selected from E542K, H1047L, E545K, and H1047R, wherein said molecular modification is operably linked by a GGGGS linker; b) the coding sequence of the self-cleaving peptide P2A; c) the coding sequence of a mutant of HER2 comprising its extracellular domain and transmembrane domain; d) the coding sequence of the self-cleaving peptide P2A; e) the coding sequence of a kinase-inactive mutant of HER3; f) the coding sequence of an internal ribosome entry site (IRES); and g) the coding sequence of a mutant of ESR1 comprising one or more molecular modifications selected from Y537C, E380Q, K303R, Y537S, D538G, and Y537N, wherein said molecular modification is operably linked by a GGGGS linker.

[0110] In some embodiments, the coding sequences of ESR1, PI3K, HER2, and / or HER3 are redesigned and / or optimized for desired properties such as enhanced stability, potency, and expression (e.g., translational efficiency), and it can in turn maximize the impact of producing, delivering, and administering the biological therapeutic agent. For example, in some embodiments, the coding sequence is optimized for expression at a higher level than the expression level of the reference coding sequence. With respect to the sequence optimization of nucleotide sequences, the degeneracy of the genetic code provides the possibility of substituting at least one base of the protein encoding the gene sequence with a different base without changing the amino acid sequence of the polypeptide produced from the gene. Thus, the nucleic acid constructs of the present disclosure can also have any base sequence altered from any polynucleotide sequence disclosed herein by substitutions related to the degeneracy of the genetic code. References describing codon usage frequencies are readily publicly available. In some embodiments, polynucleotide sequence variants can be made for various reasons, e.g., to optimize expression for a particular host (e.g., changing the codon usage in alphavirus mRNA to that preferred for other organisms such as humans, non-human primates, hamsters, mice, or monkeys). Thus, in some embodiments, the coding sequence is optimized for expression in the target host cell through the use of codons optimized for expression. Techniques for constructing synthetic nucleic acid sequences encoding a gene using preferred codons optimal for host cell expression can be determined by computational methods that analyze the commonality of codon usage frequencies and their relative abundance for encoding native proteins of the host cell genome by techniques well known in the art. The Codon Usage Database (http: / / www.kazusa.or.jp / codon) can be used to generate codon-optimized sequences in a mammalian cell environment.Furthermore, various software tools are available for converting the sequence of a given organism to the optimal codon usage frequency of another host organism, such as the JCat codon optimization tool (www.jcat.de), the Integrated DNA Technologies (IDT) codon optimization tool (https: / / www.idtdna.com / CodonOpt), and the Optimizer online codon optimization tool (http: / / genomes.urv.es / OPTIMIZER). Such synthetic sequences can be constructed by techniques known in the art for the construction of synthetic nucleic acid molecules and may be obtained from various commercial vendors.

[0111] In some embodiments, the coding sequence of the GOI is optimized for enhanced RNA stability and / or expression. RNA stability generally relates to the "half-life" of the RNA. "Half-life" relates to the period required to remove half of the activity, amount, or number of molecules. In the context of the present disclosure, the half-life of an RNA is an indicator of the stability of said RNA. The half-life of an RNA can affect the "expression period" of the RNA. Additional information regarding principles, strategies, and methods for use in enhancing RNA stability can be found, for example, in Leppek K. et al., Combinatorial optimization of mRNA structure, stability, and translation for RNA-based therapeutics. bioRxiv. (Preprint). Mar 30, 2021. doi: 10.1101 / 2021.03.29.437587. Recombinant cell

[0112] The nucleic acid constructs of the present disclosure can be introduced into host cells to produce recombinant cells containing the nucleic acid molecules. Accordingly, prokaryotic or eukaryotic cells containing nucleic acid constructs encoding the modified EEEV genomes described herein are also a feature of the present disclosure. In related aspects, some embodiments disclosed herein relate to methods of transforming cells, including introducing the nucleic acid constructs provided herein into host cells such as animal cells, and then methods of selecting or screening the transformed cells. Introduction of the nucleic acid constructs of the present disclosure into cells can be accomplished by methods known to those of skill in the art, such as viral infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, nucleofection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun technology, direct microinjection, nanoparticle-mediated nucleic acid delivery, and the like.

[0113] In one aspect, some embodiments of the present disclosure relate to recombinant cells, such as recombinant animal cells containing the nucleic acid constructs disclosed herein. The nucleic acid construct can be stably integrated into the host genome, can be episomally replicated, or can be present within the recombinant host cell as a minicircle expression vector for stable or transient expression. Accordingly, in some embodiments of the present disclosure, the nucleic acid construct is maintained and replicated in the recombinant host cell as an episomal unit. In some embodiments, the nucleic acid construct is stably integrated into the genome of the recombinant cell. Stable integration can be accomplished by use of classical random genome recombination techniques or more precise genome editing techniques, such as guide RNA-directed CRISPR / Cas9 or TALEN genome editing. In some embodiments, the nucleic acid construct is present within the recombinant host cell as a minicircle expression vector for stable or transient expression.

[0114] In some embodiments, the recombinant cell is a prokaryotic cell such as the bacterium Escherichia coli, or a eukaryotic cell such as an insect cell (e.g., a mosquito cell or Sf21 cell), or a mammalian cell (e.g., a COS cell, NIH 3T3 cell, or HeLa cell). In some embodiments, the cell is in vivo. In some embodiments, the cell is ex vivo. In some embodiments, the cell is in vitro. In some embodiments, the recombinant cell is a eukaryotic cell. In some embodiments, the recombinant cell is an animal cell. In some embodiments, the animal cell is a vertebrate cell or an invertebrate cell. In some embodiments, the recombinant cell is a mammalian cell. In some embodiments, the recombinant cell is a simian kidney CV1 cell (COS-7) transformed by SV40, a human embryonic kidney cell (e.g., HEK 293 or HEK 293 cells), a baby hamster kidney cell (BHK), a mouse Sertoli cell (e.g., TM4 cells), a simian kidney cell (CV1), a human cervical cancer cell (HeLa), a dog kidney cell (MDCK), a buffalo rat liver cell (BRL3A), a human lung cell (W138), a human hepatocyte (Hep G2), a mouse mammary tumor (MMT 060562), a TRI cell, an FS4 cell, a Chinese hamster ovary cell (CHO cell), an African green monkey kidney cell (Vero cell), a human A549 cell, a human cervical cell, a human CHME5 cell, a human PER.C6 cell, an NS0 mouse myeloma cell, a human epidermal laryngeal cell, a human fibroblast, a human HUH-7 cell, a human MRC-5 cell, a human muscle cell, a human lymphatic endothelial cell, a human astrocyte cell, a human macrophage cell, a human RAW264.7 cell, a mouse 3T3 cell, a mouse L929 cell, a mouse connective tissue cell, a mouse muscle cell, a rabbit kidney cell, selected from the group consisting of.

[0115] In some embodiments, the recombinant cell is an insect cell, e.g., a cell of an insect cell line. In some embodiments, the recombinant cell is an Sf21 cell. Further suitable insect cell lines include, without limitation, cell lines established from the orders Diptera, Lepidoptera, and Hemiptera of insects and can be derived from different tissue sources. In some embodiments, the recombinant cell is a cell of a Lepidoptera insect cell line. Over the past few decades, the availability of Lepidoptera insect cell lines has increased by approximately 50 lines per decade. More information on available Lepidoptera insect cell lines can be found, for example, in Lynn D.E., Available lepidopteran insect cell lines. Methods Mol Biol. 2007; 388: 117-38, which is incorporated herein by reference. In some embodiments, the recombinant cell is a mosquito cell, e.g., a cell of a mosquito species within the genera Anopheles (An), Aedes (Cx.), and Stegomyia. Exemplary mosquito cell lines suitable for the compositions and methods described herein include cell lines derived from the following mosquito species: Aedes aegypti, Aedes albopictus, Aedes pseudoscutellaris, Aedes triseriatus, Aedes vexans, Anopheles gambiae, Anopheles stephensi, Anopheles albimanus, Culex quinquefasciatus, Culex theileri, Culex tritaeniorhynchus, Culex bitaeniorhynchus, and Toxorhynchites amboinensis. Suitable mosquito cell lines include, without limitation, CCL-125, Aag-2, RML-12, C6 / 26, C6 / 36, C7-10, AP-61, A.t. GRIP-1, A.t. GRIP-2, UM-AVE1, Mos.55, Sua1B, 4A-3B, Mos.43, MSQ43, and LSB-AA695BB. In some embodiments, the mosquito cell is a cell of the C6 / 26 cell line.

[0116] In another aspect, provided herein is a cell culture comprising at least one recombinant cell disclosed herein and a culture medium. Generally, the culture medium can be any suitable culture medium for culturing the cells described herein. A variety of techniques for transforming the above host cells and species are known in the art and are described in technical and scientific literature. Accordingly, a cell culture comprising at least one recombinant cell disclosed herein is also within the scope of this application. Methods and systems suitable for generating and maintaining cell cultures are known in the art.

[0117] Recombinant polypeptides produced by the methods disclosed herein are also within the scope of the present disclosure.

[0118] Non-limiting exemplary embodiments of the disclosed methods for producing recombinant polypeptides can include one or more of the following features. In some embodiments, the method for producing a recombinant polypeptide of the present disclosure further comprises isolating and / or purifying the produced polypeptide. In some embodiments, the method for producing a polypeptide of the present disclosure further comprises structurally modifying the produced polypeptide to increase its half-life. Pharmaceutical composition

[0119] The nucleic acid constructs, recombinant cells, and recombinant polypeptides of the present disclosure can be incorporated into compositions (including pharmaceutical compositions). Such compositions generally include one or more of a nucleic acid construct, a recombinant cell, a recombinant polypeptide described and provided herein, and a pharmaceutically acceptable excipient, such as a carrier. In some embodiments, the compositions of the present disclosure are formulated for the prevention, treatment, or management of a health condition such as cancer. For example, the compositions of the present disclosure can be formulated as a prophylactic composition, a therapeutic composition, or a pharmaceutical composition comprising a pharmaceutically acceptable excipient, or a mixture thereof. In some embodiments, the compositions of the present disclosure are formulated for use as a vaccine. In some embodiments, the compositions of the present application are formulated for use as an adjuvant.

[0120] Thus, in one aspect, provided herein is a pharmaceutical composition comprising a pharmaceutically acceptable excipient and: a) a nucleic acid construct of the present disclosure; b) a recombinant cell of the present disclosure; and / or c) a recombinant polypeptide of the present disclosure.

[0121] Non-limiting exemplary embodiments of the pharmaceutical compositions of the present disclosure may include one or more of the following features. The nucleic acid constructs of the present disclosure can be used in naked form or formulated using a delivery vehicle. Exemplary routes of use in free form include, for example, insertion into a nucleic acid, for example, a vector. For example, as described in more detail below, the nucleic acid constructs described herein can be used as vaccines.

[0122] In some embodiments, provided herein are compositions comprising a nucleic acid construct disclosed herein and a pharmaceutically acceptable excipient. In some embodiments, compositions comprising a recombinant cell disclosed herein and a pharmaceutically acceptable excipient are provided herein. In some embodiments, the composition comprises a recombinant polypeptide disclosed herein and a pharmaceutically acceptable excipient.

[0123] In some embodiments, the compositions of the present disclosure are formulated for the prevention, treatment, or management of a health condition such as cancer. For example, the compositions of the present disclosure can be formulated as a prophylactic composition, a therapeutic composition, or a pharmaceutical composition comprising a pharmaceutically acceptable excipient, or a mixture thereof.

[0124] For use in the pharmaceutical compositions of the present disclosure, the nucleic acids or recombinant cells described herein can be formulated in or with a delivery vehicle. Exemplary delivery vehicles suitable for the compositions and methods of the present disclosure include, but are not limited to, liposomes (e.g., neutral or anionic liposomes), microspheres, immunostimulating complexes (ISCOMs), lipid-based nanoparticles (LNPs), polymeric nanoparticles, virus replicon particles (VRPs), or those conjugated to bioactive ligands, and they facilitate delivery and / or activate an immune response. These compounds are readily available to those skilled in the art; see, for example, Liposomes: A Practical Approach, RCP New Ed, IRL press (1990). Adjuvants other than liposomes and the like are also used and are known in the art. Adjuvants can protect antigens (e.g., srRNA constructs) by sequestering them in local deposits and thereby rapidly spreading, or they can contain substances that stimulate the host to secrete chemotactic agents and other components of the immune system. Appropriate selections are made by those skilled in the art, for example, from those described below.

[0125] Thus, in some embodiments, the compositions of the present disclosure can include one or more of the following: a physiological buffer, liposomes, lipid-based nanoparticles (LNPs), polymeric nanoparticles, virus replicon particles (VRPs), microspheres, immunostimulating complexes (ISCOMs), conjugates of bioactive ligands, or any combination thereof.

[0126] In some embodiments, the nucleic acid constructs of the present disclosure can be delivered to cells or a subject by lipid-based nanoparticles (LNPs). LNPs are generally less immunogenic than viral particles. Many humans have existing immunity to viral particles, but there is no existing immunity to LNPs. In addition, an adaptive immune response to LNPs is unlikely to occur, and this allows for repeated dosing of LNPs.

[0127] Lipids suitable for the compositions and methods described herein are cationic lipids, ionizable cationic lipids, anionic lipids, or neutral lipids.

[0128] In some embodiments, the LNPs of the present disclosure can include one or more ionizable lipids. As used herein, the term "ionizable lipid" means a lipid that is cationic or becomes ionizable (protonated) when the pH drops below the pKa of the ionizable group of the lipid, but is more neutral at higher pH values. At pH values lower than the pKa, the lipid can bind to negatively charged nucleic acids (e.g., oligonucleotides). As used herein, the term "ionizable lipid" includes either a lipid that bears a positive charge upon a drop in pH from physiological pH, and many lipid species that bear a net positive charge at a selected pH such as physiological pH. Permanently cationic lipids such as DOTMA have proven to be too toxic for clinical use. Ionizable lipids can be present in the lipid formulation according to the embodiment, preferably in some embodiments in an amount of about 30 to about 70 Mol%, in other embodiments about 30 Mol%, in other embodiments about 40 Mol%, in other embodiments about 45 Mol%, in other embodiments about 47.5 Mol%, in still other embodiments about 50 Mol%, and in still other cases about 60 Mol% (where "Mol%" means the percentage of the total number of moles of a particular component). The term "about" in this paragraph means a range of plus or minus 5 Mol%. DODMA or 1,2-dioleoyl-oxy-3-dimethylaminopropane is an ionizable lipid such as DLin-MC3-DMA or O-(Z,Z,Z,Z-heptatriaconta-6,9,26,29-tetraen-19-yl)-4-(N,N-dimethylamino) ("MC3").

[0129] Exemplary ionizable lipids suitable for the compositions and methods of the present disclosure include those described in PCT Publication Nos. WO2020252589A1 and WO2021000041A1, U.S. Pat. Nos. 8,450,298 and 10,844,028, and Love K.T. et al., Proc Natl Acad Sci USA, Feb. 2, 2010 107 (5) 1864-1869, which are incorporated herein by reference in their entirety. Accordingly, in some embodiments, the LNPs of the present disclosure include one or more lipid compounds described in Love K.T. et al., 2010, supra, such as C16-96, C14-110, and C12-200. In some embodiments, the LNP includes an ionizable cationic lipid selected from the group consisting of ALC-0315, C12-200, LN16, MC3, MD1, SM-102, and any combination thereof. In some embodiments, the LNP of the present disclosure includes C12-200. The structure of the C12-200 lipid is known in the art and is described, for example, in U.S. Pat. Nos. 8,450,298 and 10,844,028 (incorporated herein by reference in their entirety). In some embodiments, C12-200 is combined with cholesterol, C14-PEG2000, and DOPE. In some embodiments, C12-200 is combined with DSPC and DMG-PEG2000.

[0130] In some embodiments, the LNPs of the present disclosure include one or more cationic lipids. Suitable cationic lipids include, but are not limited to, 98N12-5, C12-200, C14-PEG2000, DLin-KC2-DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1, and 7C1. In some embodiments, the LNPs of the present disclosure include one or more neutral lipids. Non-limiting neutral lipids suitable for the compositions and methods of the present disclosure include DPSC, DPPC, POPC, DOPE, and SM. In some embodiments, the LNPs of the present disclosure include one or more ionizable lipid compounds described in PCT Publication Nos. WO2020252589A1 and WO2021000041A1 (incorporated herein by reference in their entireties).

[0131] Other lipids or combinations of lipids known in the art can be used to produce LNPs. Non-limiting examples of lipids suitable for use in producing LNPs include DOTMA, DOSPA, DOTAP, DMRIE, DC-cholesterol, DOTAP-cholesterol, GAP-DMORIE-DPyPE, and GL67A-DOPE-DMPE-polyethylene glycol (PEG). Non-limiting examples of cationic lipids include 98N12-5, C12-200, C14-PEG2000, DLin-KC2-DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1, 7C1, and any combination thereof. Non-limiting examples of neutral lipids include DPSC, DPPC, POPC, DOPE, and SM. Non-limiting examples of PEG-modified lipids include PEG-DMG, PEG-CerC14, and PEG-CerC20.

[0132] In some embodiments, the LNPs of the present disclosure include at least one lipid selected from the group consisting of C12-200, C14-PEG2000, DOPE, DMG-PEG2000, DSPC, DOTMA, DOSPA, DOTAP, DMRIE, DC-cholesterol, DOTAP-cholesterol, GAP-DMORIE-DPyPE, and GL67A-DOPE-DMPE-polyethylene glycol (PEG). In some embodiments, C12-200 is combined with cholesterol, C14-PEG2000, and DOPE. In some embodiments, C12-200 is combined with DSPC and DMG-PEG2000.

[0133] In some embodiments, the mass ratio of lipid to nucleic acid in the LNP delivery system is from about 100:1 to about 3:1, from about 70:1 to about 10:1, or from 16:1 to 4:1. In some embodiments, the mass ratio of lipid to nucleic acid in the LNP delivery system is from about 16:1 to about 4:1. In some embodiments, the mass ratio of lipid to nucleic acid in the LNP delivery system is about 20:1. In some embodiments, the mass ratio of lipid to nucleic acid in the LNP delivery system is about 8:1. In some embodiments, the lipid-based nanoparticles have an average diameter of less than about 1000 nm, about 500 nm, about 250 nm, about 200 nm, about 150 nm, about 100 nm, about 75 nm, about 50 nm, or about 25 nm. In some embodiments, the LNP has an average diameter ranging from about 70 nm to 100 nm. In some embodiments, the LNP has an average diameter ranging from about 88 nm to about 92 nm, from 82 nm to about 86 nm, or from about 80 nm to about 95 nm.

[0134] In some embodiments, the compositions of the present disclosure are formulated in liposomes. In some embodiments, the compositions of the present disclosure are formulated in lipid-based nanoparticles (LNPs). In some embodiments, the compositions of the present disclosure are formulated in polymer nanoparticles.

[0135] As described above, neutral lipids, also known as "structural lipids" or "helper lipids," can be incorporated into lipid formulations and lipid particles in some embodiments. The lipid formulations and lipid particles can contain one or more structural lipids at about 10-40 Mol% of the composition. Suitable structural lipids support the formation of particles during manufacture. Structural lipids refer to any one of many lipid species that exist in either anionic, uncharged, or neutral zwitterionic form at physiological pH. Representative structural lipids include diacyl phosphatidylcholine, diacyl phosphatidylethanolamine, diacyl phosphatidylglycerol, ceramide, sphingomyelin, dihydrosphingomyelin, kephalin, and cerebroside.

[0136] Exemplary structural lipids include zwitterionic lipids such as distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyl oleoyl phosphatidylcholine (POPC), palmitoyl oleoyl-phosphatidylethanolamine (POPE), and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), and 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (trans DOPE).

[0137] In another embodiment, the structural lipid can be any lipid that is negatively charged at physiological pH. These lipids include phosphatidylglycerol, such as dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylglycerol (POPG), cardiolipin, phosphatidylinositol, diacylphosphatidylserine, diacylphosphatidic acid, and other anionic modifying groups attached to neutral lipids. Other suitable structural lipids include glycolipids (e.g., monosialoganglioside GM1).

[0138] A stabilizer can be included in embodiments of the lipid formulation to ensure the integrity of the mixture. A stabilizer is a type of molecule that disrupts or aids in the formation of intermolecular hydrophobic-hydrophilic interactions. Suitable stabilizers include, but are not limited to, polysorbate 80 (also known as Tween 80, IUPAC name 2-[2-[3,4-bis(2-hydroxyethoxy)oxolan-2-yl]-2-(2-hydroxyethoxy)ethoxy]ethyloctadec-9-enoate), Myrj 52 (polyoxyethylene(40) stearate), and Brij(™) S10 (polyoxyethylene(10) stearyl ether). Polyethylene glycol-conjugated lipids can also be used. Stabilizers can be used alone or in combination with each other.

[0139] In some embodiments, the stabilizer is included at about 0.1-3 Mol% of the total lipid mixture. In some embodiments, the stabilizer is included at about 0.5-2.5 Mol% of the total lipid mixture. In some embodiments, the stabilizer is present at more than 2.5 Mol%. In some embodiments, the stabilizer is present at 5 Mol%. In some embodiments, the stabilizer is present at 10 Mol%. In some embodiments, the stabilizer is about 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, etc. In other embodiments, the stabilizer is 2.6-10 Mol% of the lipid mixture. In other embodiments, the stabilizer is present at more than 10 Mol% of the lipid mixture.

[0140] Steroids may also be included in the lipid composition for certain applications, and the lipid particles made therefrom contain steroids such as cholesterol and plant sterols.

[0141] In some embodiments, the therapeutic compositions described herein, e.g., nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions, are incorporated into therapeutic compositions for use in methods of preventing or treating a subject having cancer, suspected of having cancer, or at high risk of developing cancer.

[0142] In some embodiments, the composition is an immunogenic composition, e.g., a composition capable of stimulating an immune response in a subject. In some embodiments, the immunogenic composition is formulated as a vaccine. In some embodiments, the pharmaceutical composition is formulated as an adjuvant. In some embodiments, the immunogenic composition is formulated as a vehicle for gene delivery of biological therapeutics, e.g., various molecules having biological activity. Non-limiting examples of biological therapeutics include cytokines, chemokines, and other soluble immunomodulators, enzymes, peptide and protein agonists, peptide and protein antagonists, hormones, receptors, antibodies and antibody derivatives, growth factors, transcription factors, and gene silencing / editing molecules. In some embodiments, the pharmaceutical composition is formulated as an adjuvant.

[0143] In some embodiments, the immunogenic composition is substantially non-immunogenic or minimally immunogenic (e.g., a composition that minimally stimulates the immune response of a subject). In some embodiments, the non-immunogenic or minimally immunogenic composition is formulated as a biological therapeutic. In some embodiments, the pharmaceutical composition is formulated for one or more of intranasal administration, transdermal administration, intraperitoneal administration, intramuscular administration, intra-articular administration, intratumoral administration, intra-articular administration, intravenous administration, subcutaneous administration, intravaginal administration, and oral administration.

[0144] Pharmaceutical compositions suitable for injection include sterile aqueous solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In the case of intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (trademark) (BASF, Parsippany, N.J.), or phosphate buffered saline (PBS), tris (tromethamine), and HEPES. In these cases, the composition should be sterile and should be fluid to the extent that easy injectability is possible. The composition should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycols, etc.), and suitable mixtures thereof. Suitable fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants such as sodium dodecyl sulfate. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is common to include in the composition isotonicifying agents such as sugars, mannitol, sorbitol, sucrose, trehalose, and / or polyalcohols such as sodium chloride. In some embodiments, the composition includes tris and sucrose. Prolonged absorption of the injectable composition can be brought about by including in the composition agents that delay absorption, for example, aluminum monostearate and gelatin.

[0145] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound in an appropriate solvent with one or a combination of the ingredients enumerated above as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating the active compound in a sterile vehicle that contains a basic dispersion medium and the other required ingredients from those enumerated above.

[0146] In some embodiments, the composition is formulated for one or more of intranasal administration, transdermal administration, intramuscular administration, intratumoral administration, intra-articular administration, intravenous administration, intraperitoneal administration, oral administration, intravaginal, or intracranial administration. The methods of the disclosure

[0147] Administration of any one of the therapeutic compositions described herein, e.g., nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions, can be used, for example, in the treatment of related health conditions such as cancer. In some embodiments, the nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions described herein are useful for inducing an immune response in a subject in need thereof. In some embodiments, the nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions described herein can be incorporated into a therapeutic agent for use in a method of treating a subject having, suspected of having, or at high risk of developing one or more related health conditions. Exemplary health conditions or diseases can include, but are not limited to, breast cancer. In some embodiments, the subject is a patient under the care of a physician.

[0148] Non-limiting examples of breast cancer suitable for the methods of the present disclosure include ductal carcinoma of the breast, lobular carcinoma of the breast, undifferentiated carcinoma of the breast, lobosarcoma of the breast, angiosarcoma of the breast, and primary lymphoma of the breast. Breast cancers may include breast cancers of stage I, stage II, stage IIIA, stage IIIB, stage IIIC, and stage IV. Examples of ductal carcinoma of the breast may include intraductal carcinoma with predominant intraductal component, which is an invasive cancer type, inflammatory breast cancer, and ductal carcinoma of the breast. Examples of ductal carcinoma of the breast may include invasive lobular carcinoma with predominant intraductal component, invasive lobular carcinoma, and invasive lobular adenocarcinoma. Breast cancers may include Paget's disease, extramammary Paget's disease, Paget's disease associated with intraductal carcinoma, and Paget's disease associated with invasive ductal carcinoma. Breast cancers may include breast neoplasms having histological and ultrastructural heterogeneity (e.g., mixed cell type). Breast cancers can be classified as basal-like, luminal A, luminal B, ERBB2 / Her2+, or normal breast-like molecular subtypes.

[0149] Accordingly, in one aspect, provided herein is a method for inducing an immune response in a subject in need of inducing an immune response, the method comprising administering to the subject a composition comprising a) a nucleic acid construct of the present disclosure; b) a recombinant cell of the present disclosure; c) a recombinant polypeptide of the present disclosure; and / or d) a pharmaceutical composition of the present disclosure.

[0150] In another aspect, provided herein is a method for preventing and / or treating a health condition in a subject in need thereof, the method comprising prophylactically or therapeutically administering to the subject a composition comprising a) a nucleic acid construct of the present disclosure; b) a recombinant cell of the present disclosure; c) a recombinant polypeptide of the present disclosure; and / or d) a pharmaceutical composition according to any one of the present disclosure.

[0151] In some embodiments, the health condition is cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the subject has or is suspected of having cancer.

[0152] In some embodiments, the disclosed compositions are formulated to be compatible with their intended route of administration. For example, the nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions of the present disclosure can be administered orally, by inhalation, or via a parenteral route. Examples of parenteral routes of administration include, for example, intramuscular administration, intratumoral administration, intraocular administration, intravenous administration, intra-articular administration, intradermal administration, subcutaneous administration, transdermal (topical) administration, transmucosal administration, intravaginal administration, and rectal administration. In some embodiments, the composition is administered intramuscularly. In some embodiments, the composition is administered intratumorally. Solutions or suspensions used for parenteral application can contain the following components: sterile diluents such as water for injection, physiological saline, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetate, citrate, phosphate, tris, sucrose; and tonicity regulators such as sodium chloride or dextrose. The pH can be adjusted with acids or bases such as monobasic and / or dibasic sodium phosphate, hydrochloric acid or sodium hydroxide (for example, a pH of about 7.2 to 7.8, for example, 7.5). Parenteral formulations can be enclosed in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic.

[0153] The therapeutic compositions described herein, such as nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions, can be administered one or more times per day to one or more times per week, including once every other day. Treatment of a subject with a therapeutically effective amount of the subject's nucleic acid construct, recombinant cell, recombinant polypeptide, and / or the pharmaceutical composition of the present disclosure can include a single treatment or a series of treatments. In some embodiments, the composition is administered in 1 to 2, 2 to 3, or 3 to 4 doses administered at intervals of 1 week, for example, at intervals of 1 to 2 weeks, 2 to 3 weeks, or 3 to 4 weeks. This can be followed by additional administrations every 1 month, 2 months, 3 months, or 4 months. In some embodiments, 3 doses can be administered intramuscularly at intervals of 3 to 4 weeks, followed by intramuscular administrations every 3 months. Alternatively, the composition can be administered at shorter intervals, for example, every 8 hours over 5 days, followed by a rest period of 2 to 14 days, for example, 9 days, followed by further administrations every 8 hours over 5 more days. With respect to nucleic acid constructs and recombinant polypeptides, the therapeutically effective amount (e.g., effective dosage) of the nucleic acid construct or recombinant polypeptide of the present disclosure depends on the nucleic acid construct or recombinant polypeptide selected.

[0154] As described above, a therapeutically effective amount includes the amount of a therapeutic composition that is sufficient to promote a particular effect when administered to a subject in a health state, such as a person having cancer, suspected of having cancer, or at risk. In some embodiments, the effective amount includes an amount sufficient to prevent or delay the onset of symptoms of a disease, alter the course of symptoms of a disease (e.g., without limitation, slow the progression of symptoms of a disease), or reverse the symptoms of a disease.

[0155] If at least any one or all of the signs or symptoms of the disease are improved or ameliorated, the treatment is considered an effective treatment. Efficacy can also be measured by the regression of the individual's deterioration, as evaluated by the need for hospitalization or medical intervention (e.g., the progression of the disease stops or at least slows down). Methods for measuring these indicators are known to those of ordinary skill in the art and / or are described herein. Treatment includes any treatment of a disease in a subject or animal (some non-limiting examples include humans or mammals), (1) inhibiting the disease, e.g., stopping or slowing the progression of symptoms; or (2) alleviating the disease, e.g., causing regression of symptoms; and (3) preventing or reducing the likelihood of symptoms occurring.

[0156] In some embodiments, the nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions of the present disclosure can be administered to a subject in a composition having a pharmaceutically acceptable carrier and in an amount effective to stimulate an immune response. Generally, the subject is immunized by an initial series of injections (or administration via one of the other routes described below), and then boosted to enhance the protection provided by the original series of administrations. The initial series of injections and subsequent boosters are administered over such dosages and such periods as are necessary to stimulate an immune response in the subject. In some embodiments of the disclosed methods, the subject is a mammal. In some embodiments, the mammal is a human subject.

[0157] As described above, carriers acceptable as pharmaceutically suitable for injection include sterile aqueous solutions (where water-soluble), or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In these cases, the compositions must be sterile and must be fluid to the extent that injectability is easy. The compositions must further be stable under the conditions of manufacture and storage and must be protected against contamination with microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium including, for example, water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Suitable fluidity can be maintained, for example, by the use of coatings such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.

[0158] Sterile injectable solutions can be prepared by incorporating the nucleic acid construct, recombinant cells, and / or recombinant polypeptide, as required, in an appropriate mount in a suitable solvent, together with one or a combination of the ingredients listed above, followed by filter sterilization.

[0159] When the nucleic acid construct, recombinant cells, recombinant polypeptide, and / or pharmaceutical composition are appropriately protected, as described above, they can be administered orally, for example, with an inert diluent or an absorbable edible carrier. The nucleic acid construct, recombinant cells, recombinant polypeptide, and / or pharmaceutical composition and other ingredients can also be enclosed in hard or soft shell gelatin capsules, compressed into tablets, or incorporated directly into an individual's diet. For oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc. Additional Therapies

[0160] In some embodiments, the compositions described in the present disclosure are administered to a subject individually as a monotherapy (single therapy) or as a first therapy in combination with at least one additional therapy (e.g., a second therapy). In some embodiments, the second therapy is selected from the group consisting of chemotherapy, radiation therapy, immunotherapy, hormone therapy, toxin therapy, targeted therapy, and surgery. In some embodiments, the second therapy is selected from the group consisting of chemotherapy, radiation therapy, immunotherapy, hormone therapy, toxin therapy, or surgery. In some embodiments, the first therapy and the second therapy are administered concomitantly. In some embodiments, the first therapy is administered simultaneously with the second therapy. In some embodiments, the first therapy and the second therapy are administered sequentially. In some embodiments, the first treatment is administered before the second therapy. In some embodiments, the first therapy is administered after the second therapy. In some embodiments, the first therapy is administered before and / or after the second therapy. In some embodiments, the first therapy and the second therapy are administered alternately. In some embodiments, the first therapy and the second therapy are co-administered in a single formulation. Kit

[0161] Also provided herein are various kits for practicing the methods described herein, as well as instructions for their manufacture and use. In particular, some embodiments of the present disclosure provide kits for inducing an immune response in a subject. Some other embodiments relate to kits for methods of treating cancer in a subject in need thereof. For example, in some embodiments, provided herein are kits comprising one or more of the nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions provided and described herein, as well as written instructions for their production and use.

[0162] In some embodiments, the kit of the present disclosure further includes one or more means useful for administering any one of the provided nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions to a subject. For example, in some embodiments, the kit of the present disclosure further includes one or more syringes (including pre-filled syringes) and / or catheters (including pre-filled syringes) that are used for administering any one of the provided nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions to a subject. In some embodiments, the kit can have one or more additional therapeutic agents that can be administered simultaneously or sequentially with the components of other kits for a desired purpose, for example, to diagnose, prevent, or treat the health condition in a subject in need of diagnosis, prevention, or treatment.

[0163] Any of the above-described kits can further include one or more additional reagents, where the additional reagents can be selected from a dilution buffer; a reconstitution solution, a washing buffer, a control reagent, a control expression vector, a negative control, a positive control, reagents suitable for in vitro production of the provided nucleic acid construct, recombinant cells, recombinant polypeptides, and / or the pharmaceutical composition of the present disclosure.

[0164] In some embodiments, the components of the kit can be placed in separate containers. In some other embodiments, the components of the kit can be combined within a single container. Thus, in some embodiments of the present disclosure, the kit includes one or more of the nucleic acid constructs, recombinant cells, recombinant polypeptides, and / or pharmaceutical compositions provided and described herein within one container (e.g., within a sterile glass or plastic vial), and an additional therapeutic agent within another container (e.g., within a sterile glass or plastic vial).

[0165] In another embodiment, the kit includes a combination of the compositions described herein, optionally, within a single common container, in combination with one or more additional therapeutic agents co-formulated with the pharmaceutical composition, one or more of the nucleic acid constructs, recombinant cells, and / or recombinant polypeptides of the present disclosure.

[0166] If the kit includes a pharmaceutical composition for parenteral administration to a subject, the kit can include a device (e.g., an infusion device or a catheter) for performing such administration. For example, the kit can include one or more of the subcutaneous injection needles or other infusion devices described above that contain one or more of the nucleic acid constructs, recombinant cells, and / or recombinant polypeptides of the present disclosure.

[0167] In some embodiments, the kit can further include instructions for using the components of the kit to perform the methods disclosed herein. For example, the kit can include a package insert that contains information regarding the pharmaceutical composition and dosage form within the kit. Typically, such information will assist the patient and physician in using the enclosed pharmaceutical composition and dosage form effectively and safely. For example, the following information regarding the combinations of the present disclosure: pharmacokinetics, pharmacodynamics, clinical trials, efficacy parameters, indications and usage, contraindications, warnings, precautions, adverse reactions, overdose, appropriate usage and dosage, delivery methods, appropriate storage conditions, reference literature, manufacturer / distributor information, and intellectual property information, can be provided in the package insert.

[0168] Instructions for implementing the method are generally recorded on a suitable recording medium. For example, the instructions can be printed on a substrate such as paper or plastic. The instructions can be present in the kit as an attached document, on the label of the kit or a container of its components (e.g., related to packaging or sub-packaging), etc. The instructions can exist as an electronic storage data file on a suitable computer-readable storage medium, such as a CD-ROM, floppy disk, flash drive, etc. In some examples, the actual instructions do not exist in the kit, but means can be provided to obtain the instructions from a remote information source (e.g., via the Internet). An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or downloaded. Similar to the instructions, this means for obtaining the instructions can be recorded on a suitable substrate.

[0169] All publications and patent applications mentioned in this disclosure are hereby incorporated by reference into this specification to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0170] Any reference cited in this specification is not admitted to constitute prior art. The discussion of the references is to state what the author has asserted, and the applicant reserves the right to dispute the accuracy and relevance of the cited references. Although many information sources, including articles in scientific journals, patent documents, and textbooks, are referred to in this specification, it is clearly understood that this reference does not admit that any of these documents form part of the common general knowledge in the art.

[0171] The discussion of the general methods given in this specification is for illustrative purposes only. Other alternative methods and alternatives will be apparent to those skilled in the art upon consideration of this disclosure and should be included within the spirit and scope of this application.

[0172] Further embodiments are disclosed in more detail in the following examples provided by way of illustration and are not intended to limit the present disclosure or the claims.

Example

[0173] The practice of the present invention, unless otherwise indicated, employs conventional techniques of molecular biology, microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are well known to those skilled in the art. Such techniques are described, for example, in Sambrook, J., & Russell, D. W. (2012). Molecular Cloning: A Laboratory Manual (4th ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory and Sambrook, J., & Russel, D. W. (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory (collectively referred to herein as "Sambrook"); Ausubel, F. M. (1987). Current Protocols in Molecular Biology. New York, NY: Wiley (including supplements through 2014); Bollag, D. M. et al. (1996). Protein Methods. New York, NY: Wiley-Liss; Huang, L. et al. (2005). Nonviral Vectors for Gene Therapy. San Diego: Academic Press; Kaplitt, M. G. et al. (1995). Viral Vectors: Gene Therapy and Neuroscience Applications. San Diego, CA: Academic Press; Lefkovits, I. (1997). The Immunology Methods Manual: The Comprehensive Sourcebook of Techniques. San Diego, CA: Academic Press; Doyle, A. et al. (1998). Cell and Tissue Culture: Laboratory Procedures in Biotechnology.New York, NY: Wiley; Mullis, K. B., Ferre, F. & Gibbs, R. (1994). PCR: The Polymerase Chain Reaction. Boston: Birkhauser Publisher; Greenfield, E. A. (2014). Antibodies: A Laboratory Manual (2nd ed.). New York, NY: Cold Spring Harbor Laboratory Press; Beaucage, S. L. et al. (2000). Current Protocols in Nucleic Acid Chemistry. New York, NY: Wiley, (including supplements between 2000 and 2014); and Makrides, S. C. (2003). Gene Transfer and Expression in Mammalian Cells. Amsterdam, NL: Elsevier Sciences B.V., etc. are fully described in the literature (the disclosures of the above literature are incorporated herein by reference).

[0174] Further embodiments are disclosed in more detail in the following examples, which are provided by way of illustration and are not intended to limit the present disclosure or the claims. Example 1. Structure of the EEEV Vector

[0175] This example describes the experiments performed to construct a base EEEV vector (e.g., without a heterologous gene) that is subsequently used for the construction of an EEEV vector that expresses the gene(s) of interest (e.g., ESR1 or a variant thereof, PI3K or a variant thereof, HER2 or a variant thereof, and HER3 or a variant thereof).

[0176] The base EEEV vector (i.e., without the heterologous gene of interest) was constructed as follows: The base EEEV vector was de novo synthesized with a 4 - 4 kb portion (Twist Bioscience) from a reference sequence (Genbank EF151502) having several modifications. Silent mutations G301A, A3550C, G4516A, G5725A, and G7399A were incorporated to eliminate restriction enzyme cleavage sites. A unique restriction enzyme cleavage site (SpeI, 5’-A’CTAG,T-3’) was incorporated in place of the coding sequence of the native EEEV structural gene (where 5’A coincides with the position of the ATG start codon of the structural polyprotein, and 3’T coincides with the position of the stop codon TAA of the structural polyprotein). For subsequent Gibson Assembly (registered trademark) procedures (Gibson et al., Nat. Methods 6, 343 - 345, 2009), a 5’ adapter sequence (5’-CTGGAGACGTGGAGGAGAACCCTGGACCT-3’; SEQ ID NO: 21) was inserted upstream of the SpeI site, and a 3’ adapter sequence (5’-GACCGCTACGCCCCAATGACCCGACCAGC-3’; SEQ ID NO: 22) was inserted downstream of the SpeI site. A bacteriophage T7 RNA polymerase promoter (5’-TAATACGACTCACTATAG-3’; SEQ ID NO: 23) was included upstream of the EEEV genomic sequence, and downstream was a poly(A) sequence followed by a SapI site (which cleaves upstream of the recognition site). Immediately downstream of the SapI site was a T7 termination sequence (5’-AACCCCTCTCTAAACGGAGGGGTTTTTTT-3’; SEQ ID NO: 24) followed by a unique restriction enzyme cleavage site (NotI, 5’-GC’GGCC,GC-3’). This portion was combined with a linearized pYL backbone and four synthetic fragments in a 5-piece Gibson Assembly (registered trademark) reaction to obtain the EEEV base vector.

[0177] The construction of the EEEV vector containing heterologous genes was carried out as follows: The base EEEV vector was linearized by digestion with SpeI. The ESR1, PI3K, HER2, and HER3 mutants were codon-optimized / refactored by computer for human expression and synthesized de novo (GeneArt, IDT) together with the EMCV IRES. The synthetic products were amplified using primers with either 5' and 3' adapter sequences added to the ends of the genes, or primers with added P2A sequences and / or sequences homologous to adjacent gene inserts. The digested products and the PCR products were combined by the Gibson Assembly® procedure to obtain the final vector. Example 2. In Vitro Evaluation of the Modified EEEV Vector

[0178] This example describes the results of in vitro experiments conducted to evaluate the expression levels of the synthetic EEEV replicon constructs described in Example 1 above and to investigate any differential behavior (e.g., replication and protein expression).

[0179] In Vitro Transcription: RNA was prepared by in vitro transcription from a SapI-linearized plasmid template using bacteriophage T7 polymerase with either a 5' ARCA cap (HiScribe™ T7 ARCA mRNA kit, NEB) or capless transcription (HiScribe™ T7 High Yield RNA Synthesis Kit, NEB), followed by addition of 5' Cap 1 (Vaccinia Capping System, mRNA Cap 2´-O-Methyltransferase, NEB). The RNA was then purified using phenol / chloroform extraction or column purification (Monarch® RNA Cleanup Kit, NEB). The RNA concentration was determined by absorbance at 260 nm (Nanodrop, Thermo Fisher Scientific).

[0180] Replication: RNA was transfected into BHK-21 or Vero cells (e.g., 4D-Nucleofector™, Lonza) by electroporation. At 15 - 22 hours post-transfection, the cells were fixed and permeabilized (eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set, Invitrogen), stained with a PE-conjugated anti-dsRNA mouse monoclonal antibody (J2, Scicons), and the frequency of dsRNA+ cells and the mean fluorescence intensity (MFI) of dsRNA in individual cells were quantified by fluorescence flow cytometry.

[0181] Protein expression: RNA was transformed into BHK-21 or Vero cells (e.g., 4D-Nucleofector™, Lonza) by electroporation. ESR1: At 15 - 22 hours after transformation, cells were harvested and lysed in RIPA buffer. The lysate protein concentration was normalized and then probed by immunoblot using an anti-ERα rabbit antibody (A300-497A, Bethyl) and imaged using an AF800-conjugated anti-rabbit goat antibody (A32735, Thermo) (Figure 2A). The fluorescence signal from cell samples transformed with a synthetic single-gene EEEV replicon expressing ESR1 was used to normalize the expression levels to evaluate the relative ESR1 expression from a panel of two-gene and four-gene replicons (Figure 2B). PI3K: At 15 - 22 hours after transformation, cells were harvested and lysed in RIPA buffer. The lysate protein concentration was normalized and then probed by immunoblot using an anti-PI3KCA rabbit antibody (PA587398, Thermo) and imaged using an AF800-conjugated anti-rabbit goat antibody (A32735, Thermo) (Figure 2C). The fluorescence signal from cell samples transformed with a synthetic single-gene EEEV replicon expressing PI3K was used to normalize the expression levels to evaluate the relative PI3K expression from a panel of two-gene and four-gene replicons (Figure 2B). HER2: At 15 - 22 hours after transformation, cells were fixed and permeabilized (eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set, Invitrogen) and stained using an AF488-conjugated anti-HER2 mouse monoclonal antibody (24D2, Biolegend). The mean fluorescence intensity (MFI) of AF488 was used as a readout of HER2 expression. The MFI of cells transformed with a synthetic single-gene EEEV replicon expressing HER2 was used to normalize the expression levels to evaluate the relative HER2 expression from a panel of two-gene and four-gene replicons (Figure 2E).HER3: At 15 - 22 hours post - transfection, cells were fixed and permeabilized (eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set, Invitrogen) and stained using an APC - conjugated anti - HER3 mouse monoclonal antibody (2IB4C3, Biolegend). The mean fluorescence intensity (MFI) of APC was used as a readout of HER3 expression. The MFI of cells transformed with a synthetic single - gene EEEV replicon expressing HER3 was used to normalize the expression levels to evaluate the relative HER3 expression from a panel of two - gene and four - gene replicons (Figure 2F). The normalized ESR1, PI3K, HER2, and HER3 expression data from the four - gene replicon were visualized by a spider graph (Figure 2G). Example 3. In Vivo Evaluation of Modified EEEV Vectors

[0182] This example describes the results of in vivo experiments conducted to evaluate any differential immune responses following vaccination with the synthetic EEEV replicon constructs described herein (e.g., both non - formulated and LNP - formulated vectors).

[0183] In these experiments, synthetic replicon constructs derived from the EEEV strain FL93 - 939 were designed and subsequently evaluated.

[0184] Mice and Injections: BALB / c mice were purchased from Charles River Labs, Envigo, or Jackson Laboratories. On the day of administration, 0.01 - 10 μg of material was divided and intramuscularly injected into one or both quadriceps muscles. The vectors were administered as non - formulated or LNP - formulated in saline. Animals were monitored for body weight and other general observations throughout the course of the study. For immunogenicity studies, animals were administered on day 0 only or on days 0 and 21.

[0185] LNP formulation: srRNA was formulated into lipid nanoparticles using a microfluidics mixer, and the particle size and polydispersity were analyzed using dynamic light scattering and encapsulation efficiency. Lipids were suspended in ethanol. For L1, RNA was suspended in 10 mM citrate buffer pH 5.0 at a concentration of 172 μg / ml and mixed at a flow rate of 3:1 (water:organic). For L2, RNA was suspended in 250 mM NaOAc pH 4.0 at a concentration of 82 μg / ml and mixed at a flow rate of 3:1 (water:organic).

[0186] ELISpot. To measure the magnitude of ESR1, HER2, or HER3-specific T cell responses, IFNγ ELISpot assays were performed using a mouse IFNγ ELISpot PLUS kit (HRP) (MabTech) according to the manufacturer's instructions. Briefly, splenocytes were isolated and resuspended in medium containing ESR1, HER2, and HER3 as positive controls, PMA / ionomycin-derived peptides, or DMSO as mock stimulation at a concentration of 5×10 6 cells / mL. Evaluation of Linkers

[0187] The results of the mouse IFNγ detection ELISpot assay, as measured by the number of spot-forming units corresponding to responder spleen T cells, 14 days after intramuscular injection of a single-gene replicon RNA encoding an ESR1 mutation with internal linkage by various sequences and various linkers, are shown in Figure 1. The GGGGS, EAAAK, RVRR, GPGPG, and AAY linkers were tested in various sequences in an ESR1 antigen cassette containing the K303R, E380Q, Y537C, Y537S, Y537N, and D538G mutations. The columns for each cassette correspond to the following stimulation conditions using single peptides in the order of K303R, E380Q, Y537N, Y537S, Y537C, D538G, wild-type ESR1, and medium. (Plotted as the number of counted spot-forming units per million cells) The total T cell response is shown on the Y-axis. The GGGGS linker of Sequence 1 gave the most robust T cell response. Evaluation of the Number and Sequence of Genes

[0188] The results of the mouse IFNγ detection ELISpot assay, measured by the number of spot-forming units corresponding to responder spleen T cells, 35 days after two intramuscular injections of replicon RNA encoding ESR1, HER2, and HER3, are shown in Figure 3. Various constructs, in either one-gene, two-gene, or four-gene forms, with various sequences and linker sequences of ESR1, PI3K, HER2, and HER3 were tested to determine which gene configuration within the construct resulted in the most robust T cell response upon stimulation. The Y-axis shows the total T cell response. The PI3K response was not measured in this experiment because it did not form a response in BALB / c mice. Evaluation of the sequences of gene and lipid formulations

[0189] The results of the mouse IFNγ detection ELISpot assay, measured by the number of spot-forming units corresponding to responder spleen T cells, 35 days after two intramuscular injections of replicon RNA either in physiological saline or formulated in two different LNP compositions, L1 or L2, encoding ESR1, PI3K, HER2, and HER3, are shown in Figure 5. Various four-gene constructs with different replicon vector backbones were tested to determine which RNA replicon vector and formulation resulted in the most robust T cell response upon stimulation. Example 4. Efficacy against estrogen receptor-positive breast cancer

[0190] Two efficacy models are shown in Figure 6 to mimic two clinical scenarios. In the treatment model, a tumor cell line expressing the resistant mutations targeted by the vaccine is first transplanted. Subsequently, vaccination is administered. This simulates the scenario of a treated patient with existing mutations. In the prevention model, vaccination is administered before transplanting a tumor cell line encoding the resistant mutations included in the vaccine. This scenario mimics a treated patient prior to the emergence of acquired mutations. Administration of the replicon RNA encoding the (one or more) mutations expressed by the tumor cell line should elicit a robust T cell response in mice leading to delayed tumor growth. If tumor growth is not delayed, it is likely to indicate that the tumor cell line has evolved to lose the targeted mutations and that the immune system can exert selective pressure for the replicon RNA to lose the activating mutations.

[0191] While specific alternatives of the present disclosure are disclosed, it is to be understood that various modifications and combinations are possible and are contemplated within the true spirit and scope of the appended claims. Accordingly, there is no intention to be limiting to the exact summary and disclosure presented herein.

Claims

**Claim 1** A nucleic acid construct comprising a nucleic acid sequence encoding a modified eastern equine encephalitis virus (EEEV) genome or self-replicating RNA (srRNA), wherein at least a part of the nucleic acid sequence encoding the viral structural protein of the modified EEEV genome or srRNA is as follows: a) a coding sequence of estrogen receptor 1 (ESR1) or a variant thereof; b) a coding sequence of PI3K or a variant thereof; c) a coding sequence of HER2 or a variant thereof; and d) a coding sequence of HER3 or a variant thereof, wherein the nucleic acid construct is replaced by a coding sequence of a polypeptide construct comprising the same. **Claim 2** The nucleic acid construct according to claim 1, wherein the modified EEEV genome or srRNA does not contain a nucleic acid sequence encoding a viral structural protein. **Claim 3** The nucleic acid construct according to claim 1, wherein the nucleic acid sequence encoding the modified EEEV or srRNA is operably linked to a promoter sequence. **Claim 4** The nucleic acid construct according to claim 1, wherein the coding sequences of (a) to (d) are operably linked to each other within a single open reading frame (ORF). **Claim 5** The nucleic acid construct according to claim 1, wherein the coding sequences of (a) to (d) are operably linked to each other by one or more connector sequences encoding a self-proteolytic peptide or an internal ribosome entry site (IRES). **Claim 6** The nucleic acid construct according to claim 5, wherein the self-proteolytic peptide comprises one or more self-proteolytic cleavage sequences derived from calcium-dependent serine endoprotease (furin), porcine teschovirus-1 2A (P2A), foot-and-mouth disease virus (FMDV) 2A (F2A), equine rhinitis A virus (ERAV) 2A (E2A), Theileria equi virus 2A (T2A), cytoplasmic polyhedrosis virus 2A (BmCPV2A), flacherie virus 2A (BmIFV2A), or a combination thereof. **Claim 7** The nucleic acid construct according to claim 5, wherein the internal ribosome entry site (IRES) is derived from Kaposi's sarcoma-associated herpesvirus (KSHV) IRES, hepatitis virus IRES, pestivirus IRES, clipavirus IRES, ropalosiphum padi virus IRES, fibroblast growth factor IRES, platelet-derived growth factor IRES, vascular endothelial growth factor IRES, insulin-like growth factor IRES, picornavirus IRES, encephalomyocarditis virus (EMCV) IRES, Pim-1 IRES, p53 IRES, Apaf-1 IRES, TDP2 IRES, L-myc IRES, and c-myc IRES.

8. The nucleic acid construct according to claim 1, wherein at least one of the coding sequences of (a) to (d) above contains one or more molecular modifications.

9. The nucleic acid according to claim 8, wherein the one or more molecular modifications are composed of a plurality of modified cassettes arranged tandemly in the length direction of the coding sequence.

10. The nucleic acid according to claim 9, wherein the plurality of modified cassettes are operably linked to each other by one or more linkers.

11. The nucleic acid construct according to claim 8, wherein the coding sequence of the ESR1 variant of (a) contains one or more molecular modifications that promote ligand-independent receptor activity.

12. The nucleic acid construct according to claim 11, wherein the one or more molecular modifications include activating mutations selected from the group consisting of K303R, E380Q, Y537C, Y537S, Y537N, and D538G.

13. The nucleic acid construct according to claim 8, wherein the PI3K variant of (b) contains one or more molecular modifications that promote ligand-independent receptor activity.

14. The nucleic acid construct according to claim 13, wherein the one or more molecular modifications include activating mutations selected from the group consisting of E542K, E545K, H1047L, and H1047R.

15. The nucleic acid construct according to claim 1, wherein the HER2 variant of (c) contains the coding sequences of the extracellular domain and the transmembrane domain.

16. The nucleic acid construct according to claim 1, wherein the HER3 variant of (d) contains the coding sequence of kinase-inactive HER3.

17. The nucleic acid construct according to claim 1, wherein the nucleic acid sequence is selected from the group consisting of SEQ ID NOs: 7 to 10.

18. The coding sequence of the polypeptide construct, in the 5'- to 3'- direction, comprises the following: a) the coding sequence of a mutant of PI3K containing one or more activating mutations selected from E542K, H1047L, E545K, and H1047R; b) the coding sequence of the self-cleaving peptide P2A; c) the coding sequence of a mutant of HER2 comprising its extracellular domain and transmembrane domain; d) the coding sequence of the self-cleaving peptide P2A; e) the coding sequence of a kinase-inactive mutant of HER3; f) the coding sequence of an internal ribosome entry site (IRES); and g) the coding sequence of a mutant of ESR1 containing one or more activating mutations selected from Y537C, E380Q, K303R, Y537S, D538G, and Y537N, the nucleic acid construct according to claim 1.

19. A recombinant cell comprising the nucleic acid construct according to claim 1.

20. The recombinant cell according to claim 19, wherein the recombinant cell is a mammalian cell or an insect cell.

21. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and the nucleic acid construct according to claim 1.

22. The pharmaceutical composition according to claim 21, wherein the composition is formulated into a delivery system using a delivery vehicle, wherein the delivery system comprises liposomes, virus replicon particles (VRP), lipid-based nanoparticles (LNP), polymeric nanoparticles, physiological buffer, microspheres, immunostimulating complexes (ISCOM), conjugates of bioactive ligands, or any combination thereof.

23. The pharmaceutical composition according to claim 22, wherein the lipid is present at a lipid-to-RNA mass ratio of 100:1 to 4:

1.

24. The pharmaceutical composition according to claim 22, wherein the lipid-based nanoparticles have an average diameter of 25 nm to 1000 nm.

25. The pharmaceutical composition according to claim 21, wherein the composition is formulated as a vaccine.

26. A pharmaceutical composition for inducing an immune response or treating a health condition in a subject in need thereof, comprising the nucleic acid construct according to claim 1.

27. The pharmaceutical composition according to claim 26, wherein the pharmaceutical composition is for inducing an immune response.

28. The pharmaceutical composition according to claim 26, wherein the pharmaceutical composition is for treating cancer.

29. The pharmaceutical composition according to claim 28, wherein the cancer is breast cancer.

30. The pharmaceutical composition according to claim 25, wherein the composition is administered individually to a subject as a monotherapy (single-agent therapy) or as a first therapy in combination with at least one additional therapy.

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