Compositions and methods for expression of EGFR

Abstract

The present disclosure relates to modified viral genomes or self-replicating RNAs (srRNAs), and pharmaceutical compositions containing the modified viral genomes or self-replicating RNAs (srRNAs), and the use of such nucleic acid molecules and compositions for producing desired products in cell cultures or living bodies. Also provided are methods for modulating pharmacodynamic effects in a subject in need thereof and methods for preventing and / or treating a variety of health conditions and diseases.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 593,800, filed October 27, 2023. The disclosure of the aforementioned application, including any accompanying drawings, is expressly incorporated herein by reference. Technical Field

[0003] This disclosure relates to the field of immunology, and particularly to modified viral genomes or self-replicating RNA (srRNA), pharmaceutical compositions containing said modified viral genomes or self-replicating RNA (srRNA), and the use of such nucleic acid molecules and compositions for producing desired products in cell cultures or in vivo. Methods for inducing an immune response in a subject in need are also provided, as well as methods for preventing and / or treating various health conditions and diseases. Background Technology

[0004] Resistance to cancer treatments or preventative agents is a common problem in cancer or precancerous treatments, and in some cases, the mechanisms of resistance are known. Resistance often results from changes in gene expression (overexpression or blocked expression of proteins), gene changes due to mutations, or sequence alterations caused by protein splicing or translocation changes or activation changes (excessive or blocked activation of proteins) in cells.

[0005] One approach to treating cancers involving such changes in gene expression, alteration, and mutation is the development of cancer vaccines. Cancer vaccines target antigens expressed by tumors, but their application has not been as effective as previously hoped due to the induction of immune tolerance caused by prolonged overexpression of target proteins in the absence of co-stimulatory molecules, as well as the induction of an immunomodulatory environment. Prophylactic cancer vaccines may be more promising, but cancer is highly variable, with a wide range of genetic variations, but few truly universal ones. Therefore, it is difficult to predict which antigens will be overexpressed in any particular cancer, whether an individual should be vaccinated, and if so, which antigens to use.

[0006] The disclosure provided herein offers solutions to problems encountered in previous attempts to develop cancer vaccines and potentially provides improved approaches for cancer treatment and prevention. Summary of the Invention

[0007] This disclosure generally relates to the development of immunotherapeutic agents, such as recombinant nucleic acid constructs, and pharmaceutical compositions comprising said recombinant nucleic acid constructs 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 this disclosure provide nucleic acid constructs containing sequences encoding modified viral genomes or self-replicating RNA (srRNA) (e.g., replicons or self-amplifying RNA), wherein at least a portion of the nucleic acid sequence encoding a viral structural protein of the modified alphavirus genome or srRNA is replaced by a coding sequence of a polypeptide construct comprising a coding sequence of one or more portions of an epidermal growth factor receptor (EGFR) containing one or more acquired resistance mutations. Recombinant cells engineered to comprise one or more nucleic acid constructs disclosed herein are also disclosed; and pharmaceutical compositions comprising one or more of: (a) nucleic acid constructs of this disclosure and / or (b) recombinant cells of this disclosure. In specific aspects of this disclosure, compositions and methods for modulating at least one pharmacodynamic effect in a subject are further provided, as well as methods for the prevention and / or treatment of various health conditions, including cancer.

[0008] In one aspect of this disclosure, a nucleic acid construct is provided comprising a nucleic acid sequence encoding a modified alphavirus genome or self-replicating RNA (srRNA), wherein at least a portion of the nucleic acid sequence encoding a viral structural protein of the modified alphavirus genome or srRNA is replaced by a coding sequence of a polypeptide construct comprising a coding sequence of an epidermal growth factor receptor (EGFR) containing one or more acquired resistance mutations.

[0009] In some implementations, the modified alphavirus genome or srRNA does not contain nucleic acid sequences encoding viral structural proteins.

[0010] In some embodiments, the nucleic acid sequence encoding the polypeptide construct is operatively linked to a promoter sequence. In some embodiments, the promoter sequence is a 26S subgenomic (sg) promoter.

[0011] In some embodiments, the modified alphavirus genome or srRNA is a modified alphavirus genome or srRNA belonging to the VEEV / EEEV group, or the SFV group, or the SINV group. In some embodiments, the alphavirus is Venezuelan equine encephalitis virus (VEEV), Eastern equine encephalitis virus (EEEV), Madariaga virus (MADV), Chikungunya virus (CHIKV), Western equine encephalitis virus (WEEV), or Sindbis virus (SINV).

[0012] In some embodiments, the srRNA is a capped srRNA containing a 5'-cap. In some embodiments, the capped srRNA is a co-transcribed capped srRNA. In some embodiments, the capped srRNA is an enzymatically capped srRNA.

[0013] In some embodiments, the one or more acquired resistance mutations are configured into a plurality of alteration boxes arranged in series along the length of the coding sequence. In some embodiments, the plurality of alteration boxes are operatively connected to each other via one or more connectors.

[0014] In some embodiments, the one or more acquired resistance mutations include one or more (i) activating mutations, (ii) mutations that enhance the binding affinity of EGFR to adenosine triphosphate (ATP), and / or (iii) mutations that block the binding of EGFR or its variants to an inhibitor. In some embodiments, the one or more activating mutations include in-frame insertions in exon 20. In some embodiments, the in-frame insertions in exon 20 include mutations selected from: A763_Y764insFQEA, S768_D770dup, S768_V769ins, A767_V769dup, D770_N771insX, V769_D770insX, H773_V774insX, H773dup, N771_H773dup, P772_H773insX, and N771_P772insX. In some implementations, the insertions within the exon 20 frame include S768_D770dup, A767_V769dup, and / or H773dup.

[0015] In some implementations, the one or more activating mutations include L858R substitution.

[0016] In some embodiments, the one or more acquired resistance mutations enhance the binding affinity of EGFR to ATP. In some embodiments, the one or more acquired resistance mutations are selected from T790M, G719X, L858R, L718Q, G724S, L861X, S768I / V, E709X, L747S, D761Y, and T854A.

[0017] In some embodiments, the one or more acquired resistance mutations block the binding of EGFR to the inhibitor. In some embodiments, the EGFR inhibitor is selected from osimertinib, lazatinib, erlotinib, gefitinib, CO-1686, HM61713, EGF816, ASP8273, and avitinib, afatinib, morboteinib, icotinib, dacomitinib, poziotinib, cetuximab, ervantumab, morboteinib, vormetinib, DZD9008, CLN-081, STX-721, YK-029A, HS-10376, zipalletinib, and TAK-788. JMT101, ABT-101, BEBT-109, DZG9008, PLB1004, EMB-01, HS-20117, MCLA-129, BLU-945; BDTX-1535; NX-019; JIN-A02; BBT-207; BLU-525; THE-349; STX-241; ABK3376; BI-732; BLU-701; BBT-176, BLU-451, ametinib, and talotinib. In some embodiments, the one or more acquired drug resistance mutations are selected from C797S, C797G, T790M, L858R, L858M, L718V, L718Q, C796S, L798I, L792X, SV768IL, L692V, G719A, G719S, G719C, G719D, S786I, L861Q, L861R, V834X, V843X, G724S, E709K, E709H, E709A, E709G, E709V, D761Y, D761N, R776C, R776H, and T854A.

[0018] In some implementations, the nucleic acid sequence encoding EGFR 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 with nucleic acid sequences selected from SEQ ID NO: 13-32.

[0019] In some embodiments, the coding sequence of the polypeptide construct comprises a coding sequence for EGFR containing one or more acquired resistance mutations selected from the following: S768_D770dup, A767_V769dup, H773dup, T790M, L858R, and C797S. In some embodiments, the coding sequence is oriented from 5' to 3'.

[0020] This article also provides a recombinant cell comprising a nucleic acid construct according to this disclosure.

[0021] In some embodiments, the recombinant cells are mammalian cells or insect cells.

[0022] This document also provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a nucleic acid construct of the present disclosure.

[0023] In some embodiments, the composition is formulated together with a delivery medium into a delivery system, wherein the delivery system comprises liposomes, viral replicon particles (VRPs), lipid-based nanoparticles (LNPs), polymer nanoparticles, physiological buffers, microspheres, immunostimulatory complexes (ISCOMs), conjugates of bioactive ligands, or combinations thereof. In some embodiments, the LNP delivery system comprises cationic lipids, ionizable cationic lipids, anionic lipids, or neutral lipids. In some embodiments, the lipids are 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.

[0024] In some embodiments, the composition is formulated as a vaccine or immunotherapy agent.

[0025] This document also provides a method for inducing an immune response or treating a healthy condition in a subject in need. The method includes administering to the subject a composition comprising a nucleic acid construct containing this disclosure.

[0026] In some implementations, the method is used to induce an immune response.

[0027] In some embodiments, the method is a method for treating cancer. In some embodiments, the cancer is non-small cell lung cancer (NSCLC).

[0028] In some embodiments, the composition is administered to the subject as a monotherapy (single-drug therapy) alone or as a first therapy in combination with at least one other therapy.

[0029] This document also provides a method for inducing at least one pharmacodynamic effect in a subject. The method includes administering to the subject a composition comprising a nucleic acid construct containing this disclosure.

[0030] In some embodiments, the applied composition leads to the induction of one or more of the following: an immune response and a mediator selected from TNF, IL-1b, IL-12, IL-2, IFNa, IFNb, IL-6, and IFNγ.

[0031] In some implementations, the at least one pharmacodynamic effect includes

[0032] One or more of the following: immunogenicity, biomarker response, therapeutic effect, preventive effect, desired effect, undesirable effect, adverse effect, and role in disease models.

[0033] In some embodiments, the applied composition enhances antitumor immunity in the tumor microenvironment. In some embodiments, the subject has cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the lung cancer is NSCLC.

[0034] Unless explicitly or clearly excluded from the context of the implementation or aspect, each aspect and implementation described herein can be used together.

[0035] The foregoing description of the invention is merely illustrative and is not intended to be limiting in any way. Other aspects, embodiments, objects, and features of this disclosure will become fully apparent from the accompanying drawings, detailed description, and claims, in addition to the illustrative embodiments and features described herein. Attached Figure Description

[0036] Figure 1 This is a bar graph showing the relative expression of EGFR transgenic cassettes from VEEV-derived srRNA vectors. ARCA-capped srRNA encoding EGFR containing one or more acquired resistance mutations was transfected into 500,000 BHK-21 cells at 500 ng via nuclear transfection. Cells were collected after 15 hours and stained with H&L of goat anti-rabbit IgG conjugated with rabbit anti-EGFR antibody EPR15348 and secondary AF647. The mean fluorescence intensity (MFI) of transfected cells was measured by flow cytometry (FC).

[0037] Figure 2 This is a bar graph showing the relative expression of EGFR transgenic cassettes from VEEV-derived srRNA vectors. Enzymatically capped srRNA encoding EGFR containing one or more acquired resistance mutations was transfected into 500,000 BHK-21 cells at 500 ng via nuclear transfection. Cells were collected after 15 hours and stained with H&L of goat anti-rabbit IgG conjugated with rabbit anti-EGFR antibody EPR15348 and secondary AF647. The mean fluorescence intensity (MFI) of transfected cells was measured by flow cytometry (FC).

[0038] Figure 3The optimal cassette design for the EGFR single-gene construct was determined using ELISpot. Mice were administered a single intramuscular injection of 10 μg. Fourteen days later, spleens were collected and ELISpot was performed to determine the number of IFNγ-expressing T cells present after restimulation with the C797S mutant peptide. The figures show individual mouse values ​​with geometric mean and 95% confidence intervals. Statistical analysis of one-way ANOVA was performed.

[0039] Figure 4 This is a bar graph showing the in vitro evaluation of EGFR transgenic cassette expression in different srRNA vectors. 500 ng of srRNA encoding EGFR containing one or more acquired resistance mutations was transfected into 500,000 BHK-21 cells via nuclear transfection. Cells were collected after 15 hours and stained with H&L of goat anti-rabbit IgG conjugated with rabbit anti-EGFR antibody EPR15348 and secondary AF647. The mean fluorescence intensity (MFI) of transfected cells was measured by flow cytometry (FC).

[0040] Figure 5 This is a bar graph showing the in vitro evaluation of EGFR transgenic cassette expression in different srRNA vectors. 50 ng of srRNA encoding EGFR containing one or more acquired resistance mutations was transfected into 500,000 BHK-21 cells via nuclear transfection. Cells were collected after 15 hours and stained with H&L of goat anti-rabbit IgG conjugated with rabbit anti-EGFR antibody EPR15348 and secondary AF488. The mean fluorescence intensity (MFI) of transfected cells was measured by flow cytometry (FC).

[0041] Figures 6A-6B Displayed in HLA-A2 ( Figure 6A ) or HLA-A11 ( Figure 6B In transgenic mice, T-cell responses were measured as by IFNγ ELISpot 7 days after injection of 2 x 10 μg doses of srRNA at 3-week intervals. The figure shows individual mouse values ​​with mean and standard deviation. Statistical analysis of one-way ANOVA was performed. Detailed Implementation

[0042] This document provides, in particular, viral expression systems (including self-replicating RNA (srRNA) based on RNA viruses (e.g., alphaviruses)) with excellent expression potential, suitable for expressing heterologous molecules, such as therapeutic peptides, in recombinant cells. For example, some embodiments of this disclosure generally relate to expressing nucleic acid constructs containing one or more acquired resistance mutations in the epidermal growth factor receptor (EGFR) for therapeutic purposes in treating human health conditions or diseases, such as cancer. These constructs address the problems associated with therapeutic modalities (e.g., tyrosine kinase inhibitor administration) due to the development of previously demonstrated acquired resistance mutations. In some embodiments, this document provides gene expression systems with excellent expression potential suitable for expressing coding sequences of EGFR containing one or more acquired resistance mutations in recombinant cells. For example, some embodiments of this disclosure relate to nucleic acid constructs (e.g., expression constructs and vectors) containing a modified alphavirus genome or srRNA, wherein at least a portion of the nucleic acid sequence encoding a viral structural protein of the modified alphavirus genome or srRNA is replaced by a coding sequence of a polypeptide construct, the coding sequence of which includes (a) a coding sequence of EGFR containing one or more acquired drug resistance mutations. In some embodiments, the polypeptide construct does not contain a dimerizing domain. Further provided are recombinant cells genetically engineered to contain one or more nucleic acid constructs disclosed herein. Biomaterials and recombinant products derived from such recombinant cells are also within the scope of this application. Compositions and methods are also provided for (i) modulating pharmacodynamic effects in subjects in need and for preventing and / or treating health conditions in subjects in need.

[0043] As described in more detail below, EGFR inhibitors have been approved or tested for the treatment of a variety of cancers, including non-small cell lung cancer (NSCLC), head and neck cancer, colorectal cancer, and Her2-positive breast cancer, and are increasingly being added to standard of care. EGFR inhibitors that can target either the intracellular tyrosine kinase domain or the extracellular domain of EGFR are often plagued by low population response rates, leading ineffective or suboptimal chemotherapy in many cases, as well as unnecessary drug toxicity and cost. For example, the reported clinical response rate for colorectal cancer with cetuximab (a chimeric monoclonal antibody targeting the extracellular domain of EGFR) is approximately 11% (Cunningham et al., N Engl J Med 2004; 351: 337-45), and the reported clinical response rate for NSCLC with erlotinib is approximately 8.9% (Shepherd FA et al., N Engl J Med 2005; 353:123-132). Because activating mutations in EGFR have been identified in NSCLC (Lynch et al., N Engl J Med 2004; 2004; 350: 2129-2139), patients with tumors showing such mutations have experienced a dramatic increase in clinical response rates when treated with EGFR TKIs (tyrosine kinase inhibitors) (Chong and Janne, Nat Med 2013; 19: 1389-1400). However, resistance mechanisms almost always occur when EGFR inhibitors are used to treat these patients.

[0044] This disclosure provides solutions, in particular, for the development of acquired drug resistance mutations in cancer, thereby offering improved methods for treating health conditions, including cancer. As shown in the examples below, srRNA vectors capable of expressing various EGFR acquired drug resistance mutations can be generated using the compositions and methods disclosed herein.

[0045] definition

[0046] Unless otherwise defined, all technical terms, symbols, and other scientific terms or expressions used herein are intended to have the meaning commonly understood by one of ordinary skill in the art to which this application pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or convenience of reference, and the inclusion of such definitions herein should not necessarily be construed as indicating a material difference from the commonly understood meaning in the art. Many techniques and procedures described or mentioned herein are well understood by one of ordinary skill in the art and are typically employed by one of ordinary skill in the manner of practice.

[0047] Unless the context clearly specifies otherwise, the singular forms “a,” “an,” and “the” include plural indicators. For example, the term “a cell” includes one or more cells, including mixtures thereof. In this document, “A and / or B” is used to include all the following alternative forms: “A,” “B,” “A or B,” and “A and B.”

[0048] As used herein, the terms “administration” and “administering” refer to the delivery of a bioactive composition or formulation via a route of administration, including but not limited to intranasal, transdermal, intravenous, intra-arterial, intramuscular, intraductal, intraperitoneal, subcutaneous, intramuscular, oral, intravaginal, and local administration, or combinations thereof. The terms include, but are not limited to, administration by a medical professional and self-administration.

[0049] The terms “cell,” “cell culture,” and “cell line” refer not only to the specific subject cell, cell culture, or cell line, but also to the progeny or potential progeny of such cells, cell cultures, or cell lines, regardless of the number of transfers or passages in culture. It should be understood that not all progeny are identical to the parent cells. This is because certain modifications may occur in offspring due to mutations (e.g., intentional or unintentional mutations) or environmental influences (e.g., methylation or other epigenetic modifications), making the progeny potentially distinct from the parent cells, but still included within the scope of the terminology used herein, provided that the progeny retains the same function as the original cell, cell culture, or cell line.

[0050] The term "construction" refers to a recombinant molecule, such as a recombinant nucleic acid or polypeptide, containing one or more isolated nucleic acid sequences or amino acid sequences from a heterologous source. For example, a polypeptide construct can be a chimeric polypeptide molecule in which two or more amino acid sequences from different origins are operatively linked together within a single polypeptide construct. Similarly, a nucleic acid construct can be a chimeric nucleic acid molecule in which two or more nucleic acid sequences from different origins are assembled into a single nucleic acid molecule. Thus, a representative nucleic acid construct can contain any recombinant nucleic acid molecule (linear or circular single- or double-stranded DNA or RNA nucleic acid molecule) from any source capable of genome integration or autonomous replication, including nucleic acid molecules in which one or more nucleic acid sequences have been operatively linked. Two or more nucleic acid constructs can be contained within a single nucleic acid molecule (such as a single vector) or can be contained within two or more separate nucleic acid molecules (such as two or more separate vectors).

[0051] In some embodiments of this disclosure, nucleic acid constructs may be incorporated into a vector. The term "vector" is used herein to refer to a nucleic acid molecule or sequence capable of transferring or transporting another nucleic acid molecule. Therefore, the term "vector" encompasses both DNA-based vectors and RNA-based vectors. The term "vector" includes cloning vectors and expression vectors, as well as viral vectors and integration vectors. An "expression vector" is a vector containing a regulatory region that enables the expression of DNA sequences and fragments in vitro, ex vivo, and / or in vivo. In some embodiments, the vector may contain a sequence that directs autonomous replication in the cell, such as plasmids (DNA-based vectors) or self-replicating RNA vectors. In some embodiments, the vector may contain a sequence sufficient to allow integration into the host cell's DNA. Useful vectors include, for example, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, granules, bacterial artificial chromosomes, and viral vectors. In some embodiments, the vectors of this disclosure may be single-stranded vectors (e.g., ssDNA or ssRNA). In some embodiments, the vectors of this disclosure may be double-stranded vectors (e.g., dsDNA or dsRNA). In some embodiments, the vector is a gene delivery vector. In some implementations, vectors are used as gene delivery media to transfer genes into cells.

[0052] In addition to the components of the construct, the vector may also contain, for example, one or more selectable markers, one or more origins of replication (such as prokaryotic and eukaryotic origins), at least one multiple cloning site, and / or elements that facilitate stable integration of the construct into the genome of a cell. As described above, two or more constructs may be incorporated into a single nucleic acid molecule (such as a single vector), or may be incorporated into two or more separate nucleic acid molecules (such as two or more separate vectors). An “expression construct” typically contains at least a control sequence operatively linked to a target nucleotide sequence. In this way, for example, a promoter operatively linked to the nucleotide sequence to be expressed is provided in the expression construct for expression in cells. The compositions and methods for preparing and using the constructs and cells are known to those skilled in the art for practice with respect to this disclosure.

[0053] The terms "effective amount," "therapeutic effective amount," or "pharmaceutical effective amount" for compositions (e.g., nucleic acid constructs (e.g., srRNA constructs), recombinant cell and / or pharmaceutical compositions) in this disclosure generally refer to an amount of the composition sufficient to achieve the intended purpose (e.g., to achieve the effect desired by administering the composition, to stimulate an immune response, to prevent or treat a disease, or to alleviate one or more symptoms of a disease, disorder, infection, or health condition) relative to the absence of the composition. An example of an "effective amount" is an amount sufficient to induce treatment, prevention, or alleviation of one or more symptoms of a disease, which may also be referred to as a "therapeutic effective amount." "Amelioration" of symptoms means a reduction in the severity or frequency of one or more symptoms, or the elimination of one or more symptoms. The exact amount of the composition (including the “therapeutic effective amount”) will depend on the purpose of treatment and will be determined by a person skilled in the art using known techniques (see, for example, 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, edited by Gennaro, Lippincott, Williams & Wilkins).

[0054] When referring to nucleic acids, the term "naked," as used in this article, means essentially free of other macromolecules (such as lipids, polymers, and proteins). Naked nucleic acids (such as self-replicating RNA) are not formulated with other macromolecules to improve cellular uptake. Therefore, naked nucleic acids are not encapsulated in liposomes, microparticles, nanoparticles, cationic emulsions, etc., and are not adsorbed onto or bound to them.

[0055] As used herein, the term “operably linked” refers to a physical or functional connection between two or more elements (e.g., a polypeptide sequence or a polynucleotide sequence) that allows them to operate in the manner they are intended to. For example, when used in the context of a nucleic acid molecule or a coding and promoter sequence within a nucleic acid molecule described herein, the term “operably linked” means that the coding and promoter sequences are within a frame and within appropriate space and distance to allow the corresponding binding of a transcription factor or RNA polymerase to exert an effect on transcription. It should be understood that operably linked elements can be continuous or discontinuous (e.g., linked to each other via a linker). In the context of a polypeptide construct, “operably linked” refers to a physical connection (e.g., direct or indirect) between amino acid sequences (e.g., different segments, portions, regions, or domains) to provide the described activity of the construct. The operably linked segments, portions, regions, and domains of the polypeptide or nucleic acid molecules disclosed herein can be continuous or discontinuous (e.g., linked to each other via a linker).

[0056] As used herein, the term "portion" refers to a fraction. With respect to a particular structure (such as a polynucleotide sequence, an amino acid sequence, or a protein), the term "portion" can refer to a continuous or discontinuous fraction of that structure. For example, a fraction of an amino acid sequence comprises 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 in that amino acid sequence. Alternatively or additionally, if the portion is a discontinuous fraction, then the discontinuous fraction consists of 2, 3, 4, 5, 6, 7, 8, or more parts of a structure (e.g., a domain of a protein), each part being a continuous element of the structure. For example, a discontinuous fraction of an amino acid sequence may be composed of 2, 3, 4, 5, 6, 7, 8 or more (e.g., no more than 4) parts of the amino acid sequence, wherein each part contains at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 20 or at least 30 consecutive amino acids of the amino acid sequence.

[0057] In the context of two or more nucleic acids or proteins, the term "percentage of identity," as used herein, refers to two or more sequences or subsequences that are identical or have a specified percentage of identical nucleotides or amino acids (e.g., approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity in a specified region when comparing and aligning on a comparison window or specified region to obtain the maximum correspondence), as measured by using the BLAST or BLAST 2.0 sequence comparison algorithm with the default parameters described below, or by manual alignment and visual inspection. See, for example, the NCBI website ncbi.nlm.nih.gov / BLAST. Such sequences are then referred to as "substantially identical." This definition also relates to, or can be applied to, complements of query sequences. This definition includes sequence comparisons performed using the BLAST algorithm, where the parameters of the algorithm are selected to give the maximum match between the respective sequences across the full length of their respective reference sequences. This definition also includes sequences with deletions and / or additions, as well as sequences with substitutions. Sequence identity can be calculated over regions of at least about 20 amino acids or nucleotides, or over regions of 10–100 amino acids or nucleotides, or over the entire length of a given sequence. Sequence identity can be calculated using publicly available techniques and widely available computer programs, such as the GCS package (Devereux et al., Nucleic Acids Res (1984) 12:387), BLASTP, BLASTN, and FASTA (Atschul et al., J Mol Biol (1990) 215:403). Sequence identity can be measured using sequence analysis software with its default parameters, such as the sequence analysis software package from the Genetics Computer Group at the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wisconsin 53705). Other methods that may be appropriately used to determine the similarity or identity of amino acid sequences include those that rely on a position-specific structure score matrix (P3SM) incorporating structure prediction scores from Rosetta, and those that are based on length-normalized edit distance, as previously described, for example, in Setcliff et al., Cell Host & Microbe 23(6), May 2018.

[0058] As used herein, the term "pharmaceuticalally acceptable excipient" refers to any suitable substance that provides a pharmaceutically acceptable carrier, additive, or diluent for administration to a subject of one or more target compounds. Therefore, "pharmaceuticalally acceptable excipient" can encompass substances referred to as pharmaceutically acceptable diluents, pharmaceutically acceptable additives, and pharmaceutically acceptable carriers. As used herein, the term "pharmaceuticalally acceptable carrier" includes, but is not limited to, saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents compatible with drug administration. Complementary active compounds (e.g., antibiotics and other therapeutic agents) may also be incorporated into the composition.

[0059] When used in relation to cells, nucleic acids, proteins, or vectors, the term "recombinant" indicates that the cells, nucleic acids, proteins, or vectors have been altered or produced through human intervention, such as modification by laboratory methods or as a result of laboratory methods. Thus, for example, recombinant proteins and nucleic acids include proteins and nucleic acids produced by laboratory methods. Recombinant proteins may contain amino acid residues not found in the natural (non-recombinant or wild-type) form of the protein, or may contain amino acid residues that have been modified (e.g., tagged). The term may include any modification to the sequence of a peptide, protein, or nucleic acid. Such modifications may include: any chemical modification to the sequence of a peptide, protein, or nucleic acid, including any chemical modification to one or more amino acids, deoxyribonucleotides, or ribonucleotides; the addition, deletion, and / or substitution of one or more amino acids in a peptide or protein; the production of fusion proteins (e.g., fusion proteins containing antibody fragments); and the addition, deletion, and / or substitution of one or more nucleic acids in a nucleic acid sequence. When used in relation to cells, the term "recombinant" is not intended to include naturally occurring cells, but covers cells that have been engineered / modified to contain or express peptides or nucleic acids not present in the cells when they are not engineered / modified.

[0060] 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 person who has, is at risk of having, or is suspected of having one or more symptoms of a desired health condition (e.g., an autoimmune disease, an inflammatory disease, or a cardiovascular disease) and / or a health condition. A subject can also be an individual who is diagnosed at or after the diagnosis as being at risk of a desired health condition. The term "non-human animal" includes all vertebrates, such as mammals, such as rodents (e.g., mice), non-human primates and other mammals, such as sheep, dogs, cats, cattle; chickens; and non-mammals, such as amphibians, reptiles, etc.

[0061] It should be understood that the aspects and embodiments described herein include “comprising aspects and embodiments,” “consisting of aspects and embodiments,” and “substantially consisting of aspects and embodiments.” As used herein, “comprising” is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended, not excluding additional, unlisted elements or method steps. As used herein, “consisting of” excludes any element, step, or component not specified in the claimed composition or method. As used herein, “substantially consisting of” does not exclude materials or steps that do not substantially affect the essential and novel features of the claimed composition or method. Any expression of the term “comprising” herein, particularly in the description of the components of a composition or the steps of a method, should be understood to cover compositions and methods that are substantially composed of the listed components or steps and those composed of the listed components or steps.

[0062] Where a range of values ​​is provided, it should be understood that, unless the context explicitly specifies otherwise, every intermediate value between the upper and lower limits of the range (up to one-tenth of the unit of the lower limit), and any other stated or intermediate value within the stated range, is included in this disclosure. The upper and lower limits of these smaller ranges may be independently included within those smaller ranges and are also included in this disclosure, subject to any exact exclusion of any limit within the stated range. Where a stated range includes one or two limits, the range excluding any one or both of those included limits is also included in this disclosure.

[0063] Certain ranges are presented herein with numerical values ​​preceded by the term "about," which, as used herein, has its general meaning of approximate. The term "about" is used herein to provide textual support for precise figures that follow and for figures that are close to or approximate to the number following the term. In determining whether a number is close to or approximate to a specifically listed number, the unlisted number that is close to or approximates may be a number that is a fundamental equivalent of the specifically listed number provided in the context in which it is presented. If the approximation is not readily apparent from the context, "about" means within ±10% of the provided value, or rounded to the nearest significant figure, in all cases including the provided value. In some embodiments, the term "about" indicates a specified value ± at most 10%, at most ± 5%, or at most ± 1%.

[0064] Where ranges of values ​​are provided, those skilled in the art will understand that all ranges disclosed herein encompass any and all possible subranges and combinations thereof. Any range listed can be readily identified as adequately describing the same range and such that the same range can be decomposed into at least two, three, four, five, ten, etc., equal parts. As a non-limiting example, each range discussed herein can be readily decomposed into a lower third, a middle third, and an upper third, etc. As will also be understood by those skilled in the art, all terms such as “at most,” “at least,” “greater than,” “less than,” etc., include the listed numbers and refer to ranges that can subsequently be decomposed into subranges as discussed above. Finally, as will be understood by those skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles means a group having 1, 2, or 3 articles. Similarly, a group having 1-5 articles means a group having 1, 2, 3, 4, or 5 articles, and so on.

[0065] 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. Therefore, the terminology includes, but is not limited to, genes and gene products from humans and mice. It should be understood that when a gene or gene product from a particular species is disclosed, this disclosure is intended to be exemplary only and should not be construed as limiting unless the context in which it appears explicitly indicates otherwise. Thus, for example, genes or gene products disclosed herein (which in some embodiments involve mammalian nucleic acid and amino acid sequences) are intended to cover 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, genes, nucleic acid sequences, amino acid sequences, peptides, polypeptides, and proteins are human. The term "gene" is also intended to include variants thereof.

[0066] The headings (e.g., (a), (b), (i), etc.) are presented solely for ease of reading the specification and claims. The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. The use of headings in the specification or claims does not require steps or elements to be in alphabetical or numerical order or the order in which they are presented.

[0067] It should be understood that certain features of this disclosure described in the context of individual embodiments for clarity may also be provided in combination in a single embodiment. Conversely, various features of this disclosure described in the context of individual embodiments for brevity may also be provided individually or in any suitable sub-combination. All combinations of embodiments belonging to this disclosure are precisely covered by this disclosure and disclosed herein, as if each combination were individually and explicitly disclosed herein. Furthermore, all sub-combinations of various embodiments and their elements are also precisely covered by this disclosure and disclosed herein, as if each such sub-combination were individually and explicitly disclosed herein.

[0068] EGFR

[0069] Epidermal growth factor receptors, also known as EGFR, ErbB, or HER, are receptor protein tyrosine kinases belonging to the ErbB receptor family, including ErbB1 (or HER1 or EGFR), ErbB2 (or HER2), ErbB3 (or HER3), and ErbB4 (or HER4) receptors (Ullrich, 1984). ErbB receptors typically contain an extracellular domain that binds to EGFR ligands; a lipophilic transmembrane domain; a conserved intracellular tyrosine kinase domain; and a carboxyl-terminal signaling domain with several phosphorylated tyrosine residues. Activated by its six structurally related agonists—EGF, tumor growth factor α (TGFα), heparin-binding EGF-like growth factor (HB-EGF), bidirectional regulators, β-cytokinin, and epidermal regulatory factors—the receptors promote pathways that induce proliferation and transformation. Activated EGFR homodimerizes or heterodimerizes, subsequently initiating autophosphorylation of cytoplasmic tyrosine residues. These phosphorylated amino acids represent docking sites for a variety of different proteins (Prenzel 2001). Tyrosine phosphorylation of EGFR leads to the recruitment of a variety of signaling proteins, including adaptors GRB2 (growth factor receptor-binding protein-2) and Nck (Nck adaptor protein), PLC-γ (phospholipase-C-γ), SHC (transforming protein containing the Src homology-2 domain), and STATS (signal transduction and transcription activator protein 5).

[0070] Epidermal growth factor receptor (EGFR) has been identified as a relevant target for the treatment of solid tumors because it is involved in regulating cellular functions important for cancer cell proliferation and survival. EGFR is commonly expressed in a variety of tumors, and high expression is often associated with poor prognosis. A new class of targeted therapies aimed at inhibiting EGFR has emerged, namely tyrosine kinase inhibitors. Two known examples are gefitinib (Iressa) or erlotinib (Tarceva). Although some patients initially respond to these therapies, they eventually progress through mechanisms of "acquired" resistance.

[0071] Self-replicating RNA

[0072] As those skilled in the art will understand, the term “self-replicating RNA” (srRNA) refers to an RNA molecule containing all the genetic information necessary to direct its own amplification or self-replication within a cell. Therefore, srRNA is sometimes also referred to as “self-amplifying RNA” (saRNA). In some embodiments, srRNA is a “replicon,” which can be a linear or circular portion of DNA or RNA that replicates sequentially as units. Non-limiting examples of replicons include “replicon RNA” or “RNA replicon.” To direct its own replication, srRNA typically (1) encodes a polymerase, replicase, or other protein that can interact with viral or host cell-derived proteins, nucleic acids, or ribonucleoproteins to catalyze the RNA amplification process; and (2) contains cis-acting RNA sequences required for the replication and transcription of subgenomic RNA. These sequences can bind during replication to proteins they encode or to non-encoded cell-derived proteins, nucleic acids, or ribonucleoproteins, or complexes between any of these components. In some embodiments of this disclosure, the replicon (e.g., srRNA) is derived from Venezuelan equine encephalitis virus (VEEV). In some embodiments of this disclosure, the VEEV srRNA construct (e.g., srRNA, saRNA, or RNA replicon molecule) typically contains the following elements: one or more cis-5′ viral or defective interfering RNA sequences required for replication, sequences encoding biologically active alphavirus nonstructural proteins (e.g., nsP1, nsP2, nsP3, and nsP4), a subgenomic promoter (sg) of subgenomic RNA (sgRNA), a cis-3′ viral sequence required for replication, and optionally a poly(A) bundle. In some cases, a subgenomic promoter (sg) that directs the expression of a heterologous sequence may be included in the srRNA construct of this disclosure.

[0073] Furthermore, the term srRNA molecule (e.g., srRNA, saRNA, or RNA replicon molecule) generally refers to a positively polar or meaningful (“message” sense) molecule, and the length of srRNA can differ from the length of any known naturally occurring alphavirus. In some embodiments of this disclosure, the srRNA does not contain at least a portion of the coding sequence for one or more alphavirus structural proteins; and / or the sequence encoding the structural gene can be substituted with a heterologous sequence. In those cases, where the srRNA will be packaged into recombinant alphavirus particles, it may contain one or more sequences (so-called packaging signals) for initiating interactions with alphavirus structural proteins to result in particle formation.

[0074] The length of the srRNA construct in this disclosure is typically at least about 2 kb. For example, the length of the srRNA can be 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 implementations, the length of the srRNA can be approximately 4 kb to approximately 20 kb, approximately 4 kb to approximately 18 kb, approximately 5 kb to approximately 16 kb, approximately 6 kb to approximately 14 kb, approximately 7 kb to approximately 12 kb, approximately 8 kb to approximately 16 kb, approximately 9 kb to approximately 14 kb, approximately 10 kb to approximately 18 kb, approximately 11 kb to approximately 16 kb, approximately 5 kb to approximately 18 kb, approximately 6 kb to approximately 20 kb, approximately 5 kb to approximately 10 kb, approximately 5 kb to approximately 8 kb, approximately 5 kb to approximately 7 kb, approximately 5 kb to approximately 6 kb, approximately 6 kb to approximately 12 kb, approximately 6 kb to approximately 11 kb, approximately 6 kb to approximately 10 kb, approximately 6 kb to approximately 9 kb, approximately 6 kb to approximately 8 kb, approximately 6 kb to approximately 7 kb, approximately 7 kb to approximately 11 kb, approximately 7 kb to approximately 10 kb, approximately 7 kb to approximately 9 kb, approximately 7 The lengths are approximately 8 kb to 11 kb, 8 kb to 10 kb, 8 kb to 9 kb, 9 kb to 11 kb, 9 kb to 10 kb, or 10 kb to 11 kb. In some embodiments, the srRNA length can be approximately 6 kb to 14 kb. In some embodiments, the srRNA length can be approximately 6 kb to 16 kb.

[0075] In some embodiments, the srRNA construct of this disclosure may include a 5' cap. As used herein, the term "5' cap" refers to a structure found at the 5' end of some eukaryotic RNA (e.g., RNA transcripts) and typically comprises a dinucleotide or guanosine nucleotide linked to RNA (e.g., mRNA) via a 5'-to-5'-triphosphate linker (also known as Gppp or G(5')ppp(5')). In some embodiments, the guanosine nucleotide contained in the 5' cap may be modified, for example, by methylation at one or more sites (e.g., at position 7) on the base (guanine) and / or by methylation at one or more sites on the ribose. In some embodiments, the guanosine nucleotide contained in the 5' cap includes 2'O methylation at the ribose (2'OMeG). In some embodiments, the guanosine nucleotide contained in the 5' cap includes methylation at position 7 of the guanine (m7G). In some embodiments, the guanosine nucleotide contained in the 5' cap includes methylation at the 7 position of guanine and 2'O methylation at the ribose (m7(2'OMeG)).

[0076] Several different cap structures can be used to generate the 5' cap of synthetic srRNA transcribed in vitro. In some embodiments, srRNA with a 5' cap or a 5' cap analog disclosed herein can be provided via in vitro transcription, wherein the 5' cap is co-transcribed into the RNA strand, or it can be attached to the RNA post-transcriptionally using a capping enzyme. -- Therefore, in some embodiments, the 5' capping of synthetic srRNA can be performed co-transcribed with a chemical cap analog (i.e., capping during in vitro transcription). For example, the CleanCap® technology uses commercially available reagents with an AG initiator to provide highly efficient capping (90%+) in co-transcriptional reactions to provide a natural cap 1 structure with 2'-O-methyl and N7-methyl on a separate guanine component. As another example, the anti-reverse cap analog (ARCA) cap contains a 5'-5'-guanine triphosphate-guanine linker, where one guanine contains an N7-methyl and a 2'-O-methyl. Alternatively, in some embodiments, the synthetic srRNA molecule can also be enzymatically capped post-transcriptionally. These can produce more reliable 5' cap structures that more closely mimic the endogenous 5' cap, either structurally or functionally, which exhibits enhanced binding to cap-binding proteins, increased half-life, reduced susceptibility to 5' endonucleases, and / or reduced 5' uncapping. Numerous synthetic 5' cap analogs have been developed, and these analogs are known in the art to enhance mRNA stability and translateability (see, for example, Grudzien-Nogalska E. et al., Synthetic mRNAs with superior translation and stability properties in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology, Vol. 969 (Rabinovich, PH editor), 2013).

[0077] Therefore, in some embodiments, the srRNA of this disclosure is a co-transcribed capped srRNA. Exemplary co-transcribed capped srRNAs include, but are not limited to, anti-reverse cap analogs (ARCA) and CleanCap® srRNA.

[0078] In other embodiments, the srRNA of this disclosure is an enzymatically capped srRNA. Exemplary capping enzymes include, but are not limited to, vaccinia virus capping enzyme (VCE) and Faustovirus capping enzyme (FCE).

[0079] The composition of this disclosure text

[0080] As described in more detail below, one aspect of this disclosure relates to nucleic acid construct sequences encoding modified alphavirus genomes or srRNA, wherein at least a portion of the nucleic acid sequence encoding viral structural proteins of the modified alphavirus genome or srRNA is replaced by a coding sequence of a polypeptide construct comprising a coding sequence of EGFR containing one or more acquired drug resistance mutations. Recombinant cells and cell cultures, engineered to include nucleic acid constructs as disclosed herein, are also provided.

[0081] A. Nucleic acid constructs

[0082] As described in more detail below, one aspect of this disclosure relates to nucleic acid constructs comprising a nucleic acid sequence encoding a modified alphavirus genome or srRNA, wherein at least a portion of the nucleic acid sequence encoding a viral structural protein of the modified alphavirus genome or srRNA is replaced by a coding sequence of a polypeptide construct comprising a coding sequence of an epidermal growth factor receptor (EGFR) containing one or more acquired resistance mutations. In some embodiments, the sequence encoding the srRNA construct may be operatively linked, for example, under the control of elements required for expression (e.g., promoter sequences) that allow the srRNA construct to be expressed in host cells, in a subject, or in an ex vivo cell-free expression system.

[0083] The terms “nucleic acid molecule” and “polynucleotide” are used interchangeably herein and refer to both RNA and DNA molecules, including nucleic acid molecules comprising cDNA, genomic DNA, synthetic DNA, and DNA or RNA molecules containing nucleic acid analogs. Nucleic acid molecules can be double-stranded or single-stranded (e.g., sense or antisense strands). Nucleic acid molecules can contain unconventional or modified nucleotides. The terms “polynucleotide sequence” and “nucleic acid sequence” as used herein refer interchangeably to the sequence of a polynucleotide molecule. Nucleotide base nomenclature as set forth in 37 CFR §1.822 is used herein.

[0084] The nucleic acid molecules in this disclosure may have any length, including, for example, between about 1.5 Kb and about 50 Kb, between about 5 Kb and about 40 Kb, between about 5 Kb and about 30 Kb, between about 5 Kb and about 20 Kb, or between about 10 Kb and about 50 Kb, for example, between about 15 Kb and about 30 Kb, between about 20 Kb and about 50 Kb, between about 20 Kb and about 40 Kb, between about 5 Kb and about 25 Kb, or between about 30 Kb and about 50 Kb.

[0085] Non-limiting exemplary embodiments of the nucleic acid constructs (e.g., srRNA constructs) of this disclosure may include one or more of the following features. In some embodiments of this disclosure, the coding sequence of EGFR is optimized for one or more desired features. In some embodiments, the coding sequence of EGFR is optimized for one or more of the following: (a) enhanced RNA stability, (b) enhanced expression levels, (c) minimized rare codon usage, (d) minimized secondary structure, (e) facilitated better srRNA replication, and (f) facilitated a better RNA manufacturing process.

[0086] In some embodiments, the modified alphavirus genome or srRNA vector lacks at least a portion of the nucleic acid sequence encoding one or more of the viral structural proteins CP, E1, E2, E3, and 6K. In some embodiments, the modified alphavirus genome or srRNA vector lacks a portion or the entire sequence encoding CP. In some embodiments, the modified alphavirus genome or srRNA vector lacks a portion or the entire sequence encoding E1. In some embodiments, the modified alphavirus genome or srRNA vector lacks a portion or the entire sequence encoding E2. In some embodiments, the modified alphavirus genome or srRNA vector lacks a portion or the entire sequence encoding E3. In some embodiments, the modified alphavirus genome or srRNA vector lacks a portion or the entire sequence encoding 6K. In some embodiments, the modified alphavirus genome or srRNA vector lacks a portion or the entire sequence encoding a combination of CP, E1, E2, E3, and 6K. In some embodiments of this disclosure, there are coding sequences for the non-structural proteins nsP1, nsP2, nsP3, and nsP4 of the modified alphavirus genome or srRNA vector, but there is no sequence encoding at least a portion or the entire sequence of one or more structural proteins (e.g., CP, E1, E2, E3, and 6K) of the modified alphavirus genome or srRNA vector.

[0087] In some embodiments, the modified alphavirus genome or srRNA vector lacks a significant portion of the nucleic acid sequence encoding one or more viral structural proteins. Those skilled in the art will understand that a significant portion of the nucleic acid sequence encoding a viral structural polypeptide may contain sufficient nucleic acid sequence to provide presumed identification of the polypeptide, either manually evaluated by those skilled in the art or through automated computer sequence comparison and identification using algorithms such as BLAST (see, for example, “Basic Local Alignment Search Tool”; Altschul SF et al., J. Mol. Biol. 215:403-410, 1993). Therefore, a significant portion of the nucleotide sequence contains sufficient sequence to provide specific identification and / or isolation of the nucleic acid fragment containing the sequence. For example, a significant portion of the nucleic acid sequence may contain at least about 20%, such as about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% of the full-length nucleic acid sequence.

[0088] In some embodiments, the modified alphavirus genome or srRNA vector of this disclosure lacks the entire sequence encoding viral structural proteins; for example, the modified alphavirus genome or srRNA vector does not contain a nucleic acid sequence encoding viral structural proteins.

[0089] The nucleic acid construct of this disclosure further includes a coding sequence of a polypeptide construct, wherein the coding sequence replaces at least a portion of the nucleic acid sequence encoding a viral structural protein encoding a modified alphavirus genome or srRNA. The coding sequence of the polypeptide construct may be a construct of genetic material containing the coding sequence and sufficient regulatory information to guide the coding sequence to be correctly transcribed and / or translated in vivo and / or in vitro cells. The coding sequence of the polypeptide construct may be inserted into a vector for targeting desired host cells and / or a subject. Therefore, in some embodiments, the term "coding sequence of a polypeptide construct" may be used interchangeably with the term "expression construct." In some embodiments, the coding sequence of the polypeptide construct may be a nucleic acid construct comprising a gene encoding a protein or functional RNA operatively linked to regulatory elements (e.g., promoters and / or termination signals and optionally any other nucleic acid sequence or combination of other nucleic acid sequences that influence gene transcription or translation).

[0090] As described above, the nucleic acid constructs described herein contain the coding sequence of EGFR. In some embodiments, the nucleic acid constructs encode polypeptides containing a peptide / epitope of EGFR or a combination thereof capable of evoking an immune response. Variants of EGFR may cover the coding sequence of polypeptides whose amino acid sequences are identical or substantially identical to the amino acid sequence of a reference protein (e.g., EGFR), except having at least one modified amino acid (e.g., deletion, insertion, or substitution, respectively). Amino acid substitutions may be conserved amino acid substitutions, preferably at non-essential amino acid residues in the protein. "Conserved amino acid substitution" is the substitution of an amino acid residue 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 with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Protein variants may have an amino acid sequence that is at least about 80%, 90%, 95%, or 99%, preferably at least about 90%, and 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. When used with respect to nucleic acid sequences, the term "variant" refers to a nucleic acid sequence that differs from another generally associated nucleotide sequence by one or more nucleotides. Therefore, 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 point mutations, single nucleotide polymorphisms (SNPs), deletions, insertions, and translocations. Therefore, variants of the coding sequence described herein include nucleic acids encoding polypeptides that can be, for example, full-length, mutated, truncated, inactivated peptides / epitopes of EGFR, or combinations thereof.

[0091] The full-length amino acid sequence of human EGFR is shown in SEQ ID NO: 1 (1210 aa):

[0092] MRPSGTAGAA LLALLAALCP ASRALEEKKV CQGTSNKLTQ LGTFEDHFLS LQRMFNNCE VVLGNLEITYV QRNYDLSFLK TIQEVAGYVL IALNTVERIP LENLQIIRGN MYYENSYALA VLSNYDANKT GLKELPMRNL QEILHGAVRF SNNPALCNVE SIQWRDIVSS DFLSNMSMDF QNHLGSCQKC DPSCPNGSCW GAGEENCQKL TKIICAQQCS GRCRGKSPSD CCHNQCAAGC TGPRESDCLV CRKFRDEATC KDTCPPLMLY NPTTYQMDVN PEGKYSFGAT CVKKCPRNYV VTDHGSCVRA CGADSYEMEE DGVRKCKKCE GPCRKVCNGI GIGEFKDSLS INATNIKHFK NCTSISGDLH ILPVAFRGDS FTHTPPLDPQ ELDILKTVKE ITGFLLIQAW PENRTDLHAF ENLEIIRGRT KQHGQFSLAV VSLNITSLGL RSLKEISDGD VIISGNKNLC YANTINWKKL FGTSGQKTKI ISNRGENSCK ATGQVCHALC SPEGCWGPEP RDCVSCRNVS RGRECVDK CNLLEGEPREFV ENSECIQCHP ECLPQAMNIT CTGRGPDNCI QCAHYIDGPH CVKTCPAGVM GENNTLVWKY ADAGHVCHLC HPNCTYGCTG PGLEGCPTNG PKIPSIATGM VGALLLLLV VALGIGLFMR RRHIVRKRTL RRLLQERELV EPLTPSGEAP NQALLRILKE TEFKKIKVLGSG AFGTVYKGL WIPEGEKVKI PVAIKELREA TSPKANKEIL DEAYVMASVD NPHVCRLLGIC LTSTVQLIT QLMPFGCLLD YVREHKDNIG SQYLLNWCVQ IAKGMNYLED RRLVHRDLAAR NVLVKTPQH VKITDFGLAK LLGAEEKEYH AEGGKVPIKW MALESILHRI YTHQSDVWSY GVTVWELMTF GSKPYDGIPA SEISSILEKG ERLPQPPICT IDVYMIMVKC WMIDADSRPK FRELIIEFSK MARDPQRYLV IQGDERMHLP SPTDSNFYRA LMDEEDMDDV VDADEYLIPQ QGFFSPSTS RTPLLSSLSA TSNNSTVACI DRNGLQSCPI KEDSFLQRYS SDPTGALTED SIDDTFLPVP EYINQSVPKR PAGSVQNPVY HNQPLNPAPS RDPHYQDPHS TAVGNPEYLN TVQPTCVNST FDSPAHWAQK GSHQISLDNP DYQQDFFPKE AKPNGIFKGS TAENAEYLRV APQSSEFIGA

[0093] As described above, the coding sequence for EGFR in the nucleic acid constructs of this disclosure may contain one or more molecular alterations, such as mutations. In some embodiments, the coding sequence for EGFR in the nucleic acid construct contains one or more acquired resistance mutations. As used herein, the term "acquired resistance mutation" generally refers to any mutation that causes EGFR to become resistant to the action of a therapeutic drug and / or become substantially unresponsive after a period of exposure to the drug. Exemplary types of molecular alterations (e.g., mutations) in the coding sequences described herein may be one or more of deletions, substitutions, insertions, duplications, mutations, frameshift variants, splice variants, and any combination thereof.

[0094] In some implementations, the therapeutic agent is a tyrosine kinase inhibitor (TKI). This contrasts with neoantigens and intrinsic or primary resistance that occurs when EGFR mutants have little or no objective response to first-line therapy (e.g., TKI).

[0095] As used herein, the amino acid sequence numbering of EGFR and EGFR acquired resistance mutations typically refers to the amino acid sequence and number of wild-type human EGFR as shown in SEQ ID NO: 1. In some embodiments, the coding sequence of the EGFR protein in the nucleic acid construct described herein encodes the amino acid sequence of SEQ ID NO: 1 containing one or more acquired resistance mutations. In some embodiments, the one or more acquired resistance mutations are selected from A763_Y764insFQEA, S768_D770dup, S768_V769ins, A767_V769dup, D770_N771insX, V769_D770insX, H773_V774insX, H773dup, N771_H773dup, P772_H773insX, and N771_P772insX, T790M, G719X, L858R, L718Q, G724S, S768I, G719X, and L861X. , S768I / V, E709X, L747S, D761Y, T854A, C797S, C797G, T790M, L858R, L858M, L718V, L718Q, C796S, L798I, L792X, SV768IL, L692V, G71 9A, G719S, G719C, G719D, S786I, L861Q, L861R, V834X, V843X, G724S, E709K, E709H, E709A, E709G, E709V, D761Y, D761N, R776C and R776H. In some embodiments, the one or more acquired drug resistance mutations are selected from S768_D770dup, A767_V769dup, H773dup, T790M, L858R, and C797S.

[0096] In some embodiments, the nucleic acid constructs of this disclosure comprise a nucleic acid sequence encoding an EGFR protein 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 with the amino acid sequence of SEQ ID NO: 1. In some embodiments, the coding sequence of the EGFR protein encodes a smaller portion of the amino acid sequence of SEQ ID NO: 1. These smaller portions may comprise at least 8, 10, 12, 14, 16, 18, 20, 30, or more amino acids of SEQ ID NO: 1. Exemplary portions of EGFR useful in the constructs disclosed herein include those in Table 1 below:

[0097] Table 1.

[0098]

[0099] In some embodiments, the nucleic acid construct of this disclosure comprises a nucleic acid sequence encoding a portion of an EGFR 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 with the amino acid sequence of SEQ ID NO: 2-7.

[0100] In some embodiments, the one or more mutations are configured into multiple alteration cassettes. In some embodiments, the multiple alteration cassettes are arranged in series along the length of the coding sequence. In some embodiments, the length and amino acid composition of the alteration cassettes can be optimized to achieve a desired activity or property of the coding sequence or a variant thereof. In some embodiments, the alteration cassettes in the multiple alteration cassettes contain about 2 to about 50 amino acid residues, such as about 5 to about 45, about 10 to about 40, about 15 to about 30, about 20 to about 50, about 2 to about 30, about 3 to about 25, about 4 to about 20, about 5 to about 15, about 6 to about 10, about 3 to about 15, about 4 to about 10, about 5 to about 30, about 2 to about 5, about 3 to about 5, or about 4 to about 8 amino acid residues. In some embodiments, the alteration cassettes in the multiple alteration cassettes contain one, two, three, four, five, or more mutations.

[0101] In some embodiments, the EGFR described herein comprises one or more acquired resistance mutations as activating mutations. EGFR activating mutations are typically found in exons 18 to 21 of the EGFR gene, which is part of the gene encoding the tyrosine kinase domain of the EGFR protein. These activating mutations result in ligand-independent activation of tyrosine kinase activity in EGFR and can render EGFR insensitive to one or more tyrosine kinase inhibitors.

[0102] In some embodiments, the one or more activating mutations include in-frame insertions in exon 20. The one or more EGFR exon 20 mutations may be located at one or more residues selected from A763, A767, S768, V769, D770, N771, P772, and H773. Exemplary EGFR exon 20 insertions may include H773_V774insH, A767_v769ASV, N771_P772insH, D770_N771insG, H779_V774insH, N771delinsHH, S768_D770dupDVD, A767_V769dupASV, A767_V769dupASV, P772_H773dup, N771_H773dupNPH, S768_D770dupSVD, N771delinsGY, S768_D770delinsSVD, D770_D770delinsGY, A767_V769dupASV and / or H773dup. In specific respects, exon 20 mutations are A763insFQEA, A767insASV, S768dupSVD, V769insASV, D770insSVD, D770insNPG, H773insNPH, N771del insGY, N771del insFH and / or N771dupNPH.

[0103] In some embodiments, in-frame insertions of exon 20 include mutations selected from: A763_Y764insFQEA, S768_D770dup, S768_V769ins, A767_V769dup, D770_N771insX, V769_D770insX, H773_V774insX, H773dup, N771_H773dup, P772_H773insX, and N771_P772insX. In some embodiments, exon 20 mutations are in-frame insertions selected from S768_D770dup, A767_V769dup, and / or H773dup.

[0104] In some implementations, insertions within exon 20 include S768_D770dup, A767_V769dup, and / or H773dup.

[0105] In some implementations, the activating mutation is located in exon 21 of EGFR. Exemplary acquired resistance mutations in exon 21 of EGFR include, but are not limited to, L858R.

[0106] The one or more EGFR acquired resistance mutations may also include mutations that enhance the binding affinity of EGFR to ATP. Exemplary acquired resistance mutations of this type include those located at the entrance of the hydrophobic pocket in the posterior part of the ATP-binding cleft of EGFR. Substitution of one or more of these residues in EGFR can induce resistance by interfering with TKI binding. The one or more EGFR acquired resistance mutations may be located at one or more residues selected from T790, G719, L858, C797, L718, G724, S768, L861, E709, L747, D761, and T854. Exemplary acquired resistance mutations that enhance the binding affinity of EGFR to ATP include, but are not limited to, T790M, G719X, L858R, C797S, L718Q, G724S, L861X, S768I / V, E709X, L747S, D761Y, and T854A.

[0107] The one or more acquired EGFR resistance mutations may also include one or more acquired resistance mutations that block the binding of EGFR to an inhibitor. An EGFR inhibitor can refer to a molecule with the ability to inhibit the biological function of native EGFR. While the inhibitors preferred herein specifically interact with (e.g., bind to) EGFR, molecules that inhibit EGFR biological activity through interaction with other members of the EGFR signaling pathway are also included in particular. Preferred EGFR biological activity inhibited by an EGFR inhibitor is associated with tumor development, growth, or spread. EGFR inhibitors include, but are not limited to, peptides, non-peptide small molecules, antibodies, antibody fragments, antisense molecules, and oligonucleotide decoys.

[0108] In some implementations, EGFR inhibitors are tyrosine kinase inhibitors. As used herein, the term "tyrosine kinase inhibitor (TKI)" can refer to a small molecule capable of inhibiting the ErbB signaling pathway. Generally, TKIs considered herein can be classified into four groups: (1) ATP-competitive inhibitors, which primarily bind to the ATP-binding site of the kinase when it is in the active conformation; (2) inhibitors that recognize and bind to the inactive conformation of the ATP-binding site of the kinase, thereby activating it in an energy-disadvantaged manner; (3) allosteric inhibitors, which bind outside the ATP-binding site, thereby modifying the three-dimensional structure of the receptor and disrupting the interaction between ATP and the kinase pocket; and (4) covalent inhibitors, which irreversibly bind to the ATP-binding site of the target kinase via covalent bonding. Exemplary EGFR tyrosine kinase inhibitors include, but are not limited to, osimertinib, lazatinib, erlotinib, gefitinib, CO-1686, HM61713, EGF816, ASP8273, avitinib, afatinib, morboteinib, icotinib, dacomitinib, poziotinib, cetuximab, ervantumab, morboteinib, vormetinib, DZD9008, CLN-081, STX-721, YK-029A, HS-10376, zipalletinib, and TAK-788. JMT101, ABT-101, BEBT-109, DZG9008, PLB1004, EMB-01, HS-20117, MCLA-129, BLU-945; BDTX-1535; NX-019; JIN-A02; BBT-207; BLU-525; THE-349; STX-241; ABK3376; BI-732; BLU-701; BBT-176, BLU-451, ametinib, and talotinib.

[0109] In some implementations, one or more acquired resistance mutations that block the binding of EGFR to the inhibitor include, but are not limited to, C797S, C797G, T790M, L858R, L858M, L718V, L718Q, C796S, L798I, L792X, SV768IL, L692V, G719A, G719S, G719C, G719D, S786I, L861Q, L861R, V834X, V843X, G724S, E709K, E709H, E709A, E709G, E709V, D761Y, D761N, R776C, R776H, and T854A.

[0110] In some embodiments, the one or more acquired resistance mutations are operatively linked to each other via a linker. As described herein, the linker may be a peptide linker that connects two adjacent acquired resistance mutation cassettes together. In some embodiments, the length and amino acid composition of the peptide linker sequence may be optimized to alter the orientation, flexibility, and / or proximity of the cassettes relative to each other to achieve the desired activity or properties of EGFR.

[0111] In some embodiments, the peptide linker comprises a single-chain peptide sequence containing about 1 to about 30 amino acid residues (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.). In some embodiments, the linker sequence comprises about 2 to 30, about 3 to 25, about 4 to 20, about 5 to 15, about 6 to 10, about 3 to 15, about 4 to 10, about 5 to 30, about 2 to 5, about 3 to 5, or about 4 to 8 amino acid residues.

[0112] In some embodiments, the length and amino acid composition of the linker peptide sequence can be optimized to alter the orientation, flexibility, and / or proximity of the acquired resistance mutant cassettes relative to each other to achieve the desired activity or properties of the encoded peptide. In some embodiments, the orientation, flexibility, and / or proximity of the acquired resistance mutant cassettes relative to each other can be altered as a “modulation” tool to achieve a regulatory effect that enhances or reduces the activity of the encoded peptide or a variant of the encoded peptide. In some embodiments, the linker contains only glycine and / or serine residues (e.g., a glycine-serine linker). Examples of such peptide linkers include: Gly, Ser; Gly Ser; Gly Gly Ser; Ser Gly Gly; Gly Gly GlySer; 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 greater; and (Ser Gly Gly Gly Gly)n, where n is an integer of 1 or greater. In some embodiments, the peptide linker is modified such that the amino acid sequence Gly Ser Gly (GSG) is absent (which appears at the junction of a conventional Gly / Ser linker peptide repeat sequence). In some embodiments, the peptide linker comprises an amino acid sequence selected from SEQ ID NO: 8-12.

[0113] In some embodiments, the coding sequence of the polypeptide construct of the nucleic acid construct described herein encodes EGFR, comprising a portion of the EGFR amino acid sequence operatively linked by a GGGGS linker (underlined). An exemplary amino acid sequence includes the amino acid sequence of SEQ ID NO: 13 below:

[0114] GICLTSTVQLIMQLMPFGCL GGGGS VKTPQHVKITDFGRAKLLGAEEK GGGGS LITQLMPFGSLLDYVREHKDNIGS GGGGS DEAYVMASVDSVDNPHVCRLLGIC GGGGS DEAYVMASVASVDNPHVCRLLGIC GGGGS DEAYVMASVDNPHHVCRLLGICLTS GGGGS

[0115] In some embodiments, the nucleic acid construct of this disclosure comprises a nucleic acid sequence encoding an EGFR 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 with the amino acid sequence of SEQ ID NO: 13.

[0116] In some embodiments, the amino acid sequence includes the amino acid sequence of SEQ ID NO: 14 as follows:

[0117] DEAYVMASVASVDNPHVCRLLGIC GGGGS DEAYVMASVDSVDNPHVCRLLGIC GGGGS DEAYVMASVDNPHHVCRLLGICLTS GGGGS VKTPQHVKITDFGRAKLLGAEEK GGGGS LITQLMPFGSLLDYVREHKDNIGS GGGGS GICLTSTVQLIMQLMPFGCL GGGGS

[0118] In some embodiments, the nucleic acid construct of this disclosure comprises a nucleic acid sequence encoding an EGFR 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 with the amino acid sequence of SEQ ID NO: 14.

[0119] In some embodiments, the amino acid sequence includes the amino acid sequence of SEQ ID NO: 15 as follows:

[0120] GICLTSTVQLIMQLMPFGCL GGGGS LITQLMPFGSLLDYVREHKDNIGS GGGGS VKTPQHVKITDFGRAKLLGAEEK GGGGS DEAYVMASVDNPHHVCRLLGICLTS GGGGS EAYVMASVDSVDNPHVCRLLGIC GGGGS DEAYVMASVASVDNPHVCRLLGIC GGGGS

[0121] In some embodiments, the nucleic acid construct of this disclosure comprises a nucleic acid sequence encoding an EGFR 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 with the amino acid sequence of SEQ ID NO: 15.

[0122] In some embodiments, the amino acid sequence includes the amino acid sequence of SEQ ID NO: 16 as follows:

[0123] GICLTSTVQLIMQLMPFGCL GGGGS VKTPQHVKITDFGRAKLLGAEEK GGGGS LITQLMPFGSLLDYVREHKDNIGS GGGGS DEAYVMASVASVDNPHVCRLLGIC GGGGS DEAYVMASVDSVDNPHVCRLLGIC GGGGS DEAYVMASVDNPHHVCRLLGICLTS GGGGS

[0124] In some embodiments, the nucleic acid construct of this disclosure comprises a nucleic acid sequence encoding an EGFR 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 with the amino acid sequence of SEQ ID NO: 16.

[0125] In some embodiments, the amino acid sequence includes the amino acid sequence of SEQ ID NO: 17 as follows:

[0126] DEAYVMASVDSVDNPHVCRLLGIC GGGGSDEAYVMASVDNPHHVCRLLGICLTS GGGGS VKTPQHVKITDFGRAKLLGAEEK GGGGS LITQLMPFGSLLDYVREHKDNIGS GGGGS GICLTSTVQLIMQLMPFGCL GGGGS DEAYVMASVASVDNPHVCRLLGIC GGGGS

[0127] In some embodiments, the nucleic acid construct of this disclosure comprises a nucleic acid sequence encoding an EGFR 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 with the amino acid sequence of SEQ ID NO: 17.

[0128] In some embodiments, the amino acid sequence includes the amino acid sequence of SEQ ID NO: 18 as follows:

[0129] LITQLMPFGSLLDYVREHKDNIGS GGGGS DEAYVMASVASVDNPHVCRLLGIC GGGGS DEAYVMASVDSVDNPHVCRLLGIC GGGGS GICLTSTVQLIMQLMPFGCL GGGGS DEAYVMASVDNPHHVCRLLGICLTS GGGGS VKTPQHVKITDFGRAKLLGAEEK GGGGS

[0130] In some embodiments, the nucleic acid construct of this disclosure comprises a nucleic acid sequence encoding an EGFR 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 with the amino acid sequence of SEQ ID NO: 18.

[0131] In some embodiments, the amino acid sequence includes the amino acid sequence of SEQ ID NO: 19 as follows:

[0132] DEAYVMASVDNPHHVCRLLGICLTS GGGGS DEAYVMASVASVDNPHVCRLLGIC GGGGS DEAYVMASVDSVDNPHVCRLLGIC GGGGS LITQLMPFGSLLDYVREHKDNIGS GGGGS VKTPQHVKITDFGRAKLLGAEEK GGGGS GICLTSTVQLIMQLMPFGCL GGGGS

[0133] In some embodiments, the nucleic acid construct of this disclosure comprises a nucleic acid sequence encoding an EGFR 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 with the amino acid sequence of SEQ ID NO: 19.

[0134] In some embodiments, the amino acid sequence includes the amino acid sequence of SEQ ID NO: 20 as follows:

[0135] GICLTSTVQLIMQLMPFGCL GGGGS DEAYVMASVDSVDNPHVCRLLGIC GGGGS DEAYVMASVASVDNPHVCRLLGIC GGGGS VKTPQHVKITDFGRAKLLGAEEK GGGGS DEAYVMASVDNPHHVCRLLGICLTS GGGGS LITQLMPFGSLLDYVREHKDNIGS GGGGS

[0136] In some embodiments, the nucleic acid construct of this disclosure comprises a nucleic acid sequence encoding an EGFR 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 with the amino acid sequence of SEQ ID NO: 20.

[0137] In some embodiments, the amino acid sequence includes the amino acid sequence of SEQ ID NO: 21 as follows:

[0138] DEAYVMASVDSVDNPHVCRLLGIC GGGGS LITQLMPFGSLLDYVREHKDNIGS GGGGS DEAYVMASVASVDNPHVCRLLGIC GGGGS DEAYVMASVDNPHHVCRLLGICLTS GGGGS GICLTSTVQLIMQLMPFGCL GGGGS VKTPQHVKITDFGRAKLLGAEEK GGGGS

[0139] In some embodiments, the nucleic acid construct of this disclosure comprises a nucleic acid sequence encoding an EGFR 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 with the amino acid sequence of SEQ ID NO: 21.

[0140] In some embodiments, the amino acid sequence includes the amino acid sequence of SEQ ID NO: 22 as follows:

[0141] LITQLMPFGSLLDYVREHKDNIGS GGGGS DEAYVMASVDSVDNPHVCRLLGIC GGGGS GICLTSTVQLIMQLMPFGCL GGGGS VKTPQHVKITDFGRAKLLGAEEK GGGGS DEAYVMASVASVDNPHVCRLLGIC GGGGS DEAYVMASVDNPHHVCRLLGICLTS GGGGS

[0142] In some embodiments, the nucleic acid construct of this disclosure comprises a nucleic acid sequence encoding an EGFR 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 with the amino acid sequence of SEQ ID NO: 22.

[0143] In some embodiments, the amino acid sequence includes the amino acid sequence of SEQ ID NO: 23 as follows:

[0144] DEAYVMASVDNPHHVCRLLGICLTS GGGGS GICLTSTVQLIMQLMPFGCL GGGGS DEAYVMASVASVDNPHVCRLLGIC GGGGS VKTPQHVKITDFGRAKLLGAEEK GGGGS DEAYVMASVDSVDNPHVCRLLGIC GGGGS LITQLMPFGSLLDYVREHKDNIGS GGGGS

[0145] In some embodiments, the nucleic acid construct of this disclosure comprises a nucleic acid sequence encoding an EGFR 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 with the amino acid sequence of SEQ ID NO: 23.

[0146] In some embodiments, the amino acid sequence includes the amino acid sequence of SEQ ID NO: 24 as follows:

[0147] VKTPQHVKITDFGRAKLLGAEEK GGGGS DEAYVMASVASVDNPHVCRLLGIC GGGGS GICLTSTVQLIMQLMPFGCL GGGGS LITQLMPFGSLLDYVREHKDNIGS GGGGS DEAYVMASVDNPHHVCRLLGICLTS GGGGS DEAYVMASVDSVDNPHVCRLLGIC GGGGS

[0148] In some embodiments, the nucleic acid construct of this disclosure comprises a nucleic acid sequence encoding an EGFR 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 with the amino acid sequence of SEQ ID NO: 24.

[0149] In some embodiments, the amino acid sequence includes the amino acid sequence of SEQ ID NO: 25 as follows:

[0150] DEAYVMASVDNPHHVCRLLGICLTS GGGGS GICLTSTVQLIMQLMPFGCL GGGGS VKTPQHVKITDFGRAKLLGAEEK GGGGS LITQLMPFGSLLDYVREHKDNIGS GGGGS DEAYVMASVASVDNPHVCRLLGIC GGGGS DEAYVMASVDSVDNPHVCRLLGIC GGGGS

[0151] In some embodiments, the nucleic acid construct of this disclosure comprises a nucleic acid sequence encoding an EGFR 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 with the amino acid sequence of SEQ ID NO: 25.

[0152] In some embodiments, the amino acid sequence includes the amino acid sequence of SEQ ID NO: 26 as follows:

[0153] LITQLMPFGSLLDYVREHKDNIGS GGGGS VKTPQHVKITDFGRAKLLGAEEK GGGGS DEAYVMASVASVDNPHVCRLLGIC GGGGS GICLTSTVQLIMQLMPFGCL GGGGS DEAYVMASVDSVDNPHVCRLLGIC GGGGS DEAYVMASVDNPHHVCRLLGICLTS GGGGS

[0154] In some embodiments, the nucleic acid construct of this disclosure comprises a nucleic acid sequence encoding an EGFR 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 with the amino acid sequence of SEQ ID NO: 26.

[0155] In some embodiments, the amino acid sequence includes the amino acid sequence of SEQ ID NO: 27 as follows:

[0156] GICLTSTVQLIMQLMPFGCL GGGGS DEAYVMASVASVDNPHVCRLLGIC GGGGS VKTPQHVKITDFGRAKLLGAEEK GGGGS DEAYVMASVDSVDNPHVCRLLGIC GGGS LITQLMPFGSLLDYVREHKDNIGS GGGGS DEAYVMASVDNPHHVCRLLGICLTS GGGS

[0157] In some embodiments, the nucleic acid construct of this disclosure comprises a nucleic acid sequence encoding an EGFR 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 with the amino acid sequence of SEQ ID NO: 27.

[0158] In some embodiments, the amino acid sequence includes the amino acid sequence of SEQ ID NO: 28 as follows:

[0159] DEAYVMASVDNPHHVCRLLGICLTS GGGGS GICLTSTVQLIMQLMPFGCL GGGGS LITQLMPFGSLLDYVREHKDNIGS GGGGS DEAYVMASVDSVDNPHVCRLLGIC GGGGS VKTPQHVKITDFGRAKLLGAEEK GGGGS DEAYVMASVASVDNPHVCRLLGIC GGGGS

[0160] In some embodiments, the nucleic acid construct of this disclosure comprises a nucleic acid sequence encoding an EGFR 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 with the amino acid sequence of SEQ ID NO: 28.

[0161] In some embodiments, the amino acid sequence includes the amino acid sequence of SEQ ID NO: 29 as follows:

[0162] VKTPQHVKITDFGRAKLLGAEEK GGGGS DEAYVMASVDSVDNPHVCRLLGIC GGGGS LITQLMPFGSLLDYVREHKDNIGS GGGGS DEAYVMASVASVDNPHVCRLLGIC GGGGS GICLTSTVQLIMQLMPFGCL GGGGS DEAYVMASVDNPHHVCRLLGICLTS GGGS

[0163] In some embodiments, the nucleic acid construct of this disclosure comprises a nucleic acid sequence encoding an EGFR 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 with the amino acid sequence of SEQ ID NO: 29.

[0164] In some embodiments, the amino acid sequence includes the amino acid sequence of SEQ ID NO: 30 as follows:

[0165] DEAYVMASVASVDNPHVCRLLGIC GGGGS GICLTSTVQLIMQLMPFGCL GGGGS LITQLMPFGSLLDYVREHKDNIGS GGGGS DEAYVMASVDNPHHVCRLLGICLTS GGGGS DEAYVMASVDSVDNPHVCRLLGIC GGGGS VKTPQHVKITDFGRAKLLGAEEK GGGGS

[0166] In some embodiments, the nucleic acid construct of this disclosure comprises a nucleic acid sequence encoding an EGFR 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 with the amino acid sequence of SEQ ID NO: 30.

[0167] In some embodiments, the amino acid sequence includes the amino acid sequence of SEQ ID NO: 31 as follows:

[0168] GICLTSTVQLIMQLMPFGCL GGGGS DEAYVMASVASVDNPHVCRLLGIC GGGGS DEAYVMASVDNPHHVCRLLGICLTS GGGGS LITQLMPFGSLLDYVREHKDNIGS GGGGS DEAYVMASVDSVDNPHVCRLLGIC GGGGS VKTPQHVKITDFGRAKLLGAEEK GGGGS

[0169] In some embodiments, the nucleic acid construct of this disclosure comprises a nucleic acid sequence encoding an EGFR 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 with the amino acid sequence of SEQ ID NO: 31.

[0170] In some embodiments, the amino acid sequence includes the amino acid sequence of SEQ ID NO: 32 as follows:

[0171] DEAYVMASVDSVDNPHVCRLLGIC GGGGS DEAYVMASVASVDNPHVCRLLGIC GGGGS VKTPQHVKITDFGRAKLLGAEEK GGGGS LITQLMPFGSLLDYVREHKDNIGS GGGGS DEAYVMASVDNPHHVCRLLGICLTS GGGGS GICLTSTVQLIMQLMPFGCL GGGGS

[0172] In some embodiments, the nucleic acid construct of this disclosure comprises a nucleic acid sequence encoding an EGFR 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 with the amino acid sequence of SEQ ID NO: 32.

[0173] In some embodiments, the nucleic acid sequence encoding the polypeptide construct is operatively linked to a promoter sequence. In some embodiments, the promoter sequence is a subgenomic (sg) promoter. In some embodiments, the sg promoter sequence is a 26S subgenomic promoter. In some embodiments, the subgenomic promoter is heterologous relative to the remainder of the modified viral genome or srRNA. In some embodiments, the subgenomic promoter is an alphavirus subgenomic promoter.

[0174] In some embodiments of this disclosure, at least one non-structural protein (nsP) or a portion thereof of the modified viral genome or srRNA is heterologous relative to the remainder of the modified viral genome or srRNA. In some embodiments, the nucleic acid constructs disclosed herein further comprise a nucleic acid sequence encoding a heterologous nsP or a portion thereof. In some embodiments, the nucleic acid constructs disclosed herein further comprise one or more untranslated regions (UTRs). In some embodiments, at least one of the UTRs is a heterologous UTR.

[0175] In some embodiments of the methods described herein, the recombinant alphavirus srRNA is a recombinant alphavirus srRNA belonging to the genus Alphavirus of the family Togaviridae. In some embodiments of this disclosure, the modified alphavirus genome or srRNA is a modified alphavirus genome or srRNA belonging to the Venezuelan equine encephalitis virus / Eastern equine encephalitis virus (VEEV / EEEV) group, the Semliki Forest Virus (SFV) group, or the Sindbis Virus (SINV) group. In some embodiments, the modified alphavirus genome or srRNA is a modified alphavirus genome or srRNA belonging to the BFV complex, EEEV complex, MIDV complex, NDUV complex, SFV complex, VEEV complex, or WEEV complex. In some implementation schemes, the A virus is Eastern Equine Encephalitis Virus (EEEV), Venezuelan Equine Encephalitis Virus (VEEV), Everglades Virus (EVEV), Mukamb Virus (MUCV), Pixuna Virus (PIXV), Middleburg Virus (MIDV), Chikungunya Virus (CHIKV), Arlangian Virus (ONNV), Ross River Virus (RRV), Bama Forest Virus (BF), Gait Virus (GET), SAGV, Bebaru Virus (BEBV), Mayaro Virus (MAYV), Una Virus (UNAV), Sindbis Virus (SINV), AURAV, Waitarova Virus (WHAV), Babanken Virus (BABV), Zillagachi Virus (KYZV), Western Equine Encephalitis Virus (WEEV), Highland J Virus (HJV), Morganburg Virus (FMV), Ndumou Virus (NDUV), Madariaga Virus (MADV), or Bogi River Virus. In some embodiments, the alpha virus is VEEV, EEEV, CHIKV, or SINV. In some embodiments, the alpha virus is VEEV. In some embodiments, the alpha virus is EEEV. In some embodiments, the alpha virus is Western equine encephalitis virus (WEEV). In some embodiments, the alpha virus is CHIKV. In some embodiments, the alpha virus is SINV.

[0176] In some embodiments, the alphavirus is chikungunya virus (CHIKV). Non-limiting examples of CHIKV strains suitable for the compositions and methods of this disclosure include CHIKV S27, CHIKV LR2006-OPY-1, CHIKVYO123223, CHIKV DRDE, CHIKV 37997, CHIKV 99653, CHIKV Ag41855, and Nagpur (India) 653496 strain. Both virulent and non-virulent CHIKV strains are suitable. Further examples of CHIKV strains suitable for the compositions and methods of this disclosure include, but are not limited to, those described in Afreen et al. Microbiol. Immunol. 2014, 58:688-696, Lanciotti and Lambert ASTMH 2016, 94(4):800-803, and Langsjoen et al. mBio. 2018, 9(2):e02449-17. In some embodiments, the modified CHIKV genomic or replicon RNA (e.g., self-replicating RNA) is derived from CHIKV strain S27. In some embodiments, the modified CHIKV genomic or replicon RNA is derived from CHIKV strain DRDE. In some embodiments, the modified CHIKV genomic or replicon RNA is derived from CHIKV strain DRDE-06. In some embodiments, the modified CHIKV genomic or replicon RNA is derived from CHIKV strain DRDE-07. In some implementations, the modified CHIKV genome or replicon RNA is derived from the CHIKV strain S27.

[0177] In some embodiments, the alphavirus is Eastern Equine Encephalitis Virus (EEEV). Non-limiting examples of EEEV strains suitable for the compositions and methods of this disclosure include EEEV 792138, 783372, BeAn5122, BeAr300851, BeAr436087, C-49, FL91-4679, FL93-939, GML903836, MP-9, PE6, and V105-00210. Both virulent and non-virulent EEEV strains are suitable. Other suitable EEEV strains include, but are not limited to, those available on the Virus Pathogen Resource website (ViPR).

[0178] The strain described in www.viprbrc.org / brc / vipr_genome_search.spg?method=SubmitForm&blockId=868&decorator=toga is publicly available. In some embodiments, the modified EEEV genome or replicon RNA (e.g., self-replicating RNA) is derived from the EEEV strain FL93-939.

[0179] In some embodiments, the alphavirus is a Sindbis virus (SINV). In some embodiments, the modified genome or RNA replicon (e.g., self-replicating RNA) belongs to the SINV strain. Non-limiting examples of SINV strains suitable for the compositions and methods of this disclosure include SINV strains AR339, AR86, and Girdwood. Examples of SINV strains suitable for the compositions and methods of this disclosure include, but are not limited to, those described in Sammels et al. J. Gen. Virol. 1999, 80(3):739-748, Lundström and Pfeffer Vector Borne Zoonotic Dis. 2010, 10(9):889-907, Sigei et al. Arch. of Virol. 2018, 163:2465-2469, and Ling et al. J. Virol. 2019, 93:e00620-19. Other suitable SINV 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). Both virulent and non-virulent SINV strains are suitable. In some embodiments, the modified genome or RNA replicon is a modified genome or RNA replicon of the SINV strain Girdwood. In some embodiments, the modified genome or RNA replicon is a modified genome or RNA replicon of the SINV strain AR86. In some embodiments, the modified SINV genome or replicon RNA is derived from the SINV strain Girdwood. In some embodiments, the modified SINV genome or replicon RNA is derived from the SINV strain AR86. In some embodiments, at least one heterologous nsP or a portion thereof of the modified genome or RNA replicon is derived from the SINV strain AR86. In some embodiments, the at least one heterologous nsP or a portion thereof is a portion of nsP1, nsP3, nsP4, or any combination thereof. In some embodiments, the modified genome or RNA replicon is a modified genome or RNA replicon of the SINV strain AR86.

[0180] In some embodiments, the alphavirus is Western Equine Encephalitis Virus (WEEV). Non-limiting examples of WEEV strains suitable for the compositions and methods of this disclosure include WEEV California, McMillan, IMP181, Imperial, Imperial181, IMPR441, 71V-1658, AG80-646, BFS932, COA592, EP-6, E1416, BFS1703, BFS2005, BSF3060, BSF09997, CHLV53, KERN5547, 85452NM, Montana-64, S8-122, and TBT-235. Further examples of WEEV strains suitable for the compositions and methods described in this disclosure include 5614, 93A27, 93A30, 93A38, 93A79, B628 (Cl 15), CBA87, CNTR34, CO921356, Fleming, Lake43, PV012357A, PV02808A, PV72102, R02PV001807A, R02PV002957B, R02PV003422B, R05PV003422B, R0PV003814A, and R0PV00384A. Both highly toxic and non-toxic WEEV strains are suitable. Other suitable WEEV strains include, but are not limited to, Bergren NA et al., J. Virol. 88(16): 9260-9267, August 2014, and the Virus Pathogen Resource Website (ViPR).

[0181] The link provided is publicly available at https: / / www.viprbrc.org / brc / vipr_genome_search.spg?method=SubmitForm&blockId=57240&decorator=toga. ） Those described herein. In some embodiments, the modified WEEV genome or srRNA is derived from the WEEV strain Imperial. In some embodiments, the modified WEEV genome or srRNA is derived from the WEEV strain McMillan.

[0182] In some embodiments, the alphavirus is Madariaga virus (MADV), formerly known as East South American equine encephalitis virus (SA EEEV). Non-limiting examples of MADV strains suitable for the compositions and methods of this disclosure include ArgLL, ArgB, BeAn-5122, ArgM, 24443 (TR59), 25714 (BG60), BeAr 18205, 900188 (PA62), BeAr 81828, BeAr 126650, 68U231, 77U1104 (PE70), 75V1496, BeAr 300851, 75U40, and ElDelirio (Arrigo NC et al., ibid. 2010). Other examples of MADV strains suitable for the compositions and methods of this disclosure include 76V25343, 77U1 (BR77), BeAr348998, IVICPan57151, BeAn416361, 903836 (PA84), BeAr436087, 435731 (PA86), C49 (CO92), PE-0.0155-96 (0.0155), PE-3.0815-96 (3.0815), PE-16.0050-98 (16.0050), PE-18.0140-99 (18.0140), and PE-18.0172-99 (18.0172) (Arrigo NC et al., ibid. 2010). Other suitable MADV strains include, but are not limited to, those described below: Arrigo NC et al., ibid. 2010, and the Virus Pathogen Resource website (ViPR; which can be found on...) www.viprbrc.org / brc / vipr_genome_search.spg?method= SubmitForm&blockId=868&decorator=toga (Publicly available). In some embodiments, the modified MADV genome or srRNA is derived from the MADV strain BeAr300851.

[0183] In some embodiments of this disclosure, the coding sequence of the polypeptide construct comprises, in the 5' to 3' direction (i.e., in the direction from the N-terminus to the C-terminus of the polypeptide sequence), a coding sequence for an epidermal growth factor receptor (EGFR) containing one or more acquired resistance mutations selected from S768_D770dup, A767_V769dup, H773dup, T790M, L858R, and C797S.

[0184] In some embodiments, the coding sequence of the EGFR protein is redesigned and / or optimized to obtain desired properties, such as increased stability, potency, and expression (e.g., translation efficiency), which in turn can maximize the effects of producing, delivering, and administering the biotherapeutic agent EGFR. For example, in some embodiments, the coding sequence of EGFR is optimized for expression at a level higher than that of a reference coding sequence. In some embodiments, the coding sequence of EGFR is optimized for one or more of the following: (a) enhanced RNA stability, (b) enhanced expression levels, (c) minimized rare codon usage, (d) minimized secondary structure, (e) improved srRNA replication, and (f) improved RNA manufacturing processes.

[0185] Regarding nucleotide sequence optimization, the degeneracy of the genetic code provides the possibility of substituting at least one base of a gene sequence encoding a protein with a different base without altering the amino acid sequence of the polypeptide produced by the gene. Therefore, nucleic acid constructs of this disclosure may also have any base sequence altered by substituting any polynucleotide sequence disclosed herein, based on the degeneracy of the genetic code. References describing codon usage are readily available. In some embodiments, polynucleotide sequence variants may be generated for various reasons, such as to optimize expression in a particular host (e.g., changing codon usage in alphavirus mRNA to codon usage preferred by other organisms, such as humans, non-human primates, hamsters, mice, or monkeys). Thus, in some embodiments, the coding sequence is optimized for expression in target host cells using codons optimized for expression. Techniques for constructing synthetic nucleic acid sequences encoding genes using preferred codons optimal for host cell expression can be determined by analyzing the universality and relative abundance of codon usage of natural proteins encoding the host cell genome using techniques well known in the art. A codon usage database (http: / / www.kazusa.or.jp / codon) can be used to generate codon-optimized sequences for mammalian cellular environments. Furthermore, various software tools are available to convert sequences from one organism into optimal codon usages for different host organisms, such as the JCat codon optimization tool (www.jcat.de), the Integrated DNA Technologies (IDT) codon optimization tool (https: / / www.idtdna.com / CodonOpt), or the Optimizer online codon optimization tool (http: / / genomes.urv.es / OPTIMIZER). Such synthetic sequences can be constructed using techniques known in the art for constructing synthetic nucleic acid molecules and are available from multiple commercial vendors.

[0186] Therefore, in some embodiments, the coding sequence of EGFR is optimized for expression at a higher level than that of a reference coding sequence (e.g., an uncodontized coding sequence). In some embodiments, the codontized sequence of EGFR results in an increase in expression level of at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% compared to the uncodontized reference coding sequence. In some embodiments, the codontized sequence of EGFR results in an increase in expression level of at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold compared to the uncodontized reference coding sequence.

[0187] In some implementations, the coding sequence of EGFR is optimized to enhance RNA stability and / or expression. RNA stability is generally associated with the RNA's "half-life." "Half-life" refers to the time period required to eliminate half of the molecular activity, amount, or quantity. In the context of this disclosure, the half-life of RNA indicates the stability of the RNA. The half-life of RNA can affect the "expression duration" of the RNA. Further information on principles, strategies, and methods for enhancing RNA stability can be found, for example, in Leppek K. et al., Nature Communications, March 22, 2022, 13(1):1536.

[0188] In some embodiments, the nucleic acid construct of this disclosure comprises a nucleic acid sequence encoding a polypeptide construct of this disclosure, wherein 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 with the nucleic acid sequence of SEQ ID NO: 13-32.

[0189] In some embodiments, the nucleic acid constructs of this disclosure comprise nucleic acid sequences encoding polypeptide constructs having 100% sequence identity with the amino acid sequences of SEQ ID NO: 13-32.

[0190] Nucleic acid sequences that have a high sequence identity (e.g., 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%) with the target modified alphavirus genome or srRNA can be identified and / or isolated by genomic sequence analysis, hybridization, and / or PCR using sequences identified herein (e.g., SEQ ID NO: 1) or any other sequence known in the art, with degenerate primers or gene-specific primers from sequences identified in the corresponding alphavirus genome or srRNA.

[0191] The molecular techniques and methods for assembling and characterizing these novel nucleic acid constructs are described more fully in the embodiments of this application. In some embodiments, the nucleic acid molecules are recombinant nucleic acid molecules. As used herein, the term recombinant means any molecule (e.g., DNA, RNA, polypeptide) that is derived from or generated from (however indirectly) human manipulation. As a non-limiting example, cDNA is a recombinant DNA molecule, such as any nucleic acid molecule that has been generated by one or more in vitro polymerase reactions or has been attached to a adapter or integrated into a vector (such as a cloning vector or expression vector). As a non-limiting example, a recombinant nucleic acid molecule: 1) has been synthesized or modified in vitro, for example, by chemical or enzymatic techniques, such as by using chemical nucleic acid synthesis, or by using enzymes to replicate, polymerize, exonucleate, endonucleate, ligate, reverse transcribe, transcribe, modify (including, for example, methylation), or recombine (including homologous recombination and site-specific recombination); 2) contains a linked nucleotide sequence that is not naturally occurring; 3) has been engineered using molecular cloning techniques such that it lacks one or more nucleotides relative to a naturally occurring nucleotide sequence; and / or 4) has been manipulated using molecular cloning techniques such that it has one or more sequence variations or rearrangements relative to a naturally occurring nucleotide sequence.

[0192] In some embodiments, the nucleic acid molecules disclosed herein are produced using recombinant DNA technologies (e.g., polymerase chain reaction (PCR) amplification, cloning, etc.) or chemical synthesis. Nucleic acid molecules disclosed herein include natural nucleic acid molecules and their homologs, including but not limited to natural allelic variants and modified nucleic acid molecules in which one or more nucleotide residues have been inserted, deleted, and / or substituted in such a manner that such modifications provide the desired properties to achieve the biological activities described herein.

[0193] Nucleic acid molecules (including variants of naturally occurring nucleic acid sequences) can be produced using a variety of methods known to those skilled in the art (see, for example, Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989)). The sequence of a nucleic acid molecule can be modified relative to the naturally occurring sequence from which it is derived using a variety of techniques, including but not limited to classical mutagenesis and recombinant DNA techniques, such as, but not limited to, site-directed mutagenesis, chemical treatment of nucleic acid molecules to induce mutations, restriction enzyme cleavage of nucleic acid fragments, ligation of nucleic acid fragments, PCR amplification and / or mutagenesis of selected regions of nucleic acid sequences, recombinant cloning; and chemical synthesis, including the chemical synthesis of mixtures of oligonucleotides and the ligation of mixtures to “build” mixtures of nucleic acid molecules; and combinations thereof. Homologous nucleic acid molecules can be selected from mixtures of modified nucleic acid molecules by screening for the function of proteins or srRNA encoded by the nucleic acid molecules, and / or hybridization with wild-type genes or fragments thereof, or by PCR using primers homologous to target or wild-type nucleic acid molecules or sequences.

[0194] B. Recombinant cells

[0195] The nucleic acid constructs of this disclosure can be introduced into host cells to generate recombinant cells containing nucleic acid molecules. Therefore, prokaryotic or eukaryotic cells containing nucleic acid constructs encoding modified alphavirus genomes or srRNAs as described herein are also characteristic of this disclosure. In related aspects, some embodiments disclosed herein relate to methods for transforming cells, methods including introducing nucleic acid constructs as provided herein into host cells (e.g., animal cells), followed by selection or screening of the transformed cells. Introducing the nucleic acid constructs of this disclosure into cells can be achieved by methods known to those skilled in the art, such as viral infection, transfection, conjugation, protoplast fusion, liposome transfection, electroporation, nuclear transfection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-glucan-mediated transfection, liposome-mediated transfection, particle gun technology, direct microinjection, nanoparticle-mediated nucleic acid delivery, etc.

[0196] In one aspect, some embodiments of this disclosure relate to recombinant cells, such as recombinant animal cells, containing the nucleic acid constructs described herein. The nucleic acid constructs can be stably integrated into the host genome, or can be replicated as episomes, or exist in the recombinant host cell as microcircular expression vectors for stable or transient expression. Therefore, in some embodiments of this disclosure, the nucleic acid constructs are maintained and replicated in the recombinant host cell as episome units. In some embodiments, the nucleic acid constructs are stably integrated into the genome of the recombinant cell. Stable integration can be accomplished using classical random genomic recombination techniques or with more precise genome editing techniques, such as CRISPR / Cas9 or TALEN genome editing guided by guide RNA. In some embodiments, the nucleic acid constructs exist in the recombinant host cell as microcircular expression vectors for stable or transient expression.

[0197] In some embodiments, the recombinant cells are prokaryotic cells, such as the bacteria *Escherichia coli* (E. coli); or eukaryotic cells, such as insect cells (e.g., mosquito cells or Sf21 cells) or mammalian cells (e.g., COS cells, NIH 3T3 cells, or HeLa cells). In some embodiments, the cells are in vivo. In some embodiments, the cells are ex vivo. In some embodiments, the cells are in vitro. In some embodiments, the recombinant cells are eukaryotic cells. In some embodiments, the recombinant cells are animal cells. In some embodiments, the animal cells are vertebrate cells or invertebrate cells. In some embodiments, the recombinant cells are mammalian cells. Non-limiting examples of recombinant cells suitable for the methods and compositions of this disclosure include monkey kidney CV1 cells transformed with SV40 (e.g., COS-7 cells), human embryonic kidney cells (e.g., HEK293 or HEK 293 cells) or derived cells thereof (e.g., BHK-21 or BHK-570 cells), juvenile hamster kidney cells (BHK), mouse Support cells (e.g., TM4 cells), monkey kidney cells (e.g., CV1 cells), human cervical cancer cells (e.g., HeLa cells), canine kidney cells (MDCK cells), buffalo rat hepatocytes (e.g., BRL 3A cells), human lung cells (e.g., W138 cells), human hepatocytes (e.g., Hep G2 cells), and mouse mammary tumor cells (e.g., MMT cells). 060562 cells), TRI cells, FS4 cells, Chinese hamster ovary cells (CHO cells), African green monkey kidney cells (e.g., Vero cells), human A549 cells, human cervical cells, human CHME5 cells, human PER.C6 cells, NSO mouse myeloma cells, human epidermoid laryngeal cells, human fibroblasts, human HUH-7 cells, human MRC-5 cells, human muscle cells, human endothelial cells, human astrocytes, human macrophages, human RAW264.7 cells, mouse 3T3 cells, mouse L929 cells, mouse connective tissue cells, mouse muscle cells, and rabbit kidney cells.

[0198] In some implementations, the recombinant cells are immune cells. In some implementations, the immune cells are B cells, monocytes, natural killer (NK) cells, natural killer T (NKT) cells, basophils, eosinophils, neutrophils, dendritic cells (DCs), macrophages, regulatory T cells, and helper T cells (T cells). H ), cytotoxic T cells (T CTL ), memory T cells, γδ (γδ) T cells, hematopoietic stem cells, or hematopoietic stem cell progenitor cells. In some embodiments, the immune cells are B cells, T cells, macrophages, or dendritic cells (DCs). In some embodiments, the immune cells are B cells. In some embodiments, the immune cells are T cells.

[0199] In some implementations, the recombinant cell is a cell derived from the cell described above (i.e., a derivative cell of the original cell described herein), such as an amplification from a clone of the original cell (an engineered form of the original cell) or a reclassified cell of the original cell after it has undergone extensive passage or has been passaged through another host.

[0200] In some embodiments, the recombinant cells are insect cells, such as cells from insect cell lines. In some embodiments, the recombinant cells are Sf21 cells. Other suitable insect cell lines include, but are not limited to, cell lines established from the orders Diptera, Lepidoptera, and Hemiptera, and may be derived from different tissue sources. In some embodiments, the recombinant cells are cells from Lepidoptera insect cell lines. The availability of Lepidoptera insect cell lines has increased by approximately 50 lines per decade over the past few decades. More information on available Lepidoptera insect cell lines can be found, for example, Lynn DE, Available lepidopteran insect cell lines. MethodsMol Biol. 2007;388:117-38, which is incorporated herein by reference. In some embodiments, the recombinant cells are mosquito cells, such as cells from mosquito species within the genera Anopheles (An.), Culex (Cx.), and Aedes (Stegomyia) (Ae.). Exemplary mosquito cell lines suitable for the compositions and methods described herein include cell lines from the following mosquito species: Aedes aegypti, Aedes albopictus, Aedes pseudoscutellaris, Aedes triseriatus, Aedes vexans, Anopheles gambiae, Anopheles stessae, Anopheles albimanus, Culex quinquefasciatus, Culex theileri, Culex tritaeniorhynchus, Culex bitaeniorhynchus, and Toxorhynchites amboinensis. Suitable mosquito cell lines include, but are not limited to, CCL-125, Aag-2, RML-12, C6 / 26, C6 / 36, C7-10, AP-61, At GRIP-1, At GRIP-2, UM-AVE1, Mos.55, Sua1B, 4a-3B, Mos.43, MSQ43, and LSB-AA695BB. In some embodiments, the mosquito cells are cells from the C6 / 26 cell line.

[0201] On the other hand, this document provides cell cultures comprising at least one recombinant cell as disclosed herein and a culture medium. Generally, the culture medium can be any suitable medium used for culturing the cells described herein. Techniques for transforming the wide variety of host cells and species mentioned above are known in the art and described in the technical and scientific literature. Therefore, cell cultures comprising at least one recombinant cell as disclosed herein are also within the scope of this application. Methods and systems suitable for producing and maintaining cell cultures are known in the art.

[0202] D. Pharmaceutical Composition

[0203] The nucleic acid constructs and recombinant cells of this disclosure can be incorporated into compositions (including pharmaceutical compositions). Such compositions typically contain one or more pharmaceutically acceptable excipients (e.g., carriers) from the nucleic acid constructs and recombinant cells described and provided herein. In some embodiments, the compositions of this disclosure are formulated for the prevention, treatment, or management of health conditions such as autoimmune diseases, inflammatory diseases, or cardiovascular diseases. For example, the compositions of this disclosure can be formulated as prophylactic compositions, therapeutic compositions, or pharmaceutical compositions or mixtures thereof containing pharmaceutically acceptable excipients. In some embodiments, the compositions of this disclosure are formulated for use as vaccines or immunotherapeutic agents. In some embodiments, the compositions of this application are formulated for use as adjuvants.

[0204] Therefore, in one respect, this document provides pharmaceutical compositions comprising pharmaceutically acceptable excipients and: (a) nucleic acid constructs of this disclosure; and / or (b) recombinant cells of this disclosure.

[0205] Non-limiting exemplary embodiments of the pharmaceutical compositions disclosed herein may include one or more of the following features. In some embodiments, compositions are provided herein comprising nucleic acid constructs as disclosed herein and pharmaceutically acceptable excipients. In some embodiments, compositions are provided herein comprising recombinant cells as disclosed herein and pharmaceutically acceptable excipients.

[0206] In some embodiments, the nucleic acid constructs (e.g., vectors or srRNA molecules) of this disclosure may be used in naked form or formulated with delivery media. Exemplary delivery media suitable for the compositions and methods of this disclosure include, but are not limited to, liposomes (e.g., neutral or anionic liposomes), microspheres, immunostimulatory complexes (ISCOM), lipid-based nanoparticles (LNPs), solid lipid nanoparticles (SLNs), polymeric complexes, polymeric nanoparticles, viral replicon particles (VRPs), or conjugates with bioactive ligands that can facilitate delivery and / or enhance immune responses. 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 are also used, and said adjuvants are known in the art. Adjuvants can protect antigens (e.g., nucleic acid constructs, vectors, srRNA molecules) from rapid diffusion by isolating them in a localized deposit, or they may contain substances that stimulate the host to secrete chemotactic factors that are chemotactic to macrophages and other components of the immune system. Those skilled in the art can make appropriate selections from, for example, those described below.

[0207] The compositions of this disclosure can be formulated in forms compatible with their intended route of administration, such as liposomes, lipid-based nanoparticles (LNPs), polymer nanoparticles, polymeric complexes, viral replicon particles (VRPs), microspheres, immunostimulatory complexes (ISCOMs), conjugates of bioactive ligands, or any combination thereof. Therefore, in some embodiments, the compositions of this disclosure can be formulated in liposomes.

[0208] polymer nanoparticles

[0209] In some embodiments, the compositions of this disclosure may be formulated in polymer nanoparticles. In some embodiments, the polymer nanoparticles include cationic polymers, non-cationic polymers, or combinations thereof. In some embodiments, the cationic polymer includes naturally derived cationic polymers. In some embodiments, the naturally derived cationic polymer includes chitosan, gelatin, dextran, cellulose, cyclodextrin, or combinations thereof. In some embodiments, the cationic polymer includes synthetic cationic polymers. In some embodiments, the synthesized cationic polymers include polyethyleneimine (PEI), poly-L-lysine (PLL), poly(amino acid) (PAA), poly(amidoamine) (PAMAM), poly(cystamine bisacrylamide-co-4-amino-1-butanol) (pABOL), poly(amino-co-ester) (PAE), poly(2-N,N-dimethylaminoethyl methacrylate, poly(β-amino ester) (PBAE), imidazole-containing polymers, tertiary amine-containing polymers, poly(2-(dimethylamino)ethyl methacrylate), poly-N-(2-hydroxy-propyl)methacrylamide, polyamidoamine dendritic polymers, cationic sugar polymers, or derivatives thereof.

[0210] In some embodiments, the noncationic polymer is negatively charged (i.e., anionic) or electrically neutral. In some embodiments, the noncationic polymer includes polyethylene glycol (PEG), polyesters (e.g., polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), polyglycolic acid (PGA), polycaprolactone (PCL)), and polysarcosine (pSar) or derivatives thereof. In some embodiments, the polymer is water-soluble and / or biodegradable.

[0211] In some embodiments of this disclosure, the polymer nanoparticles comprise one or more of the following: poly-(γ-L-glutamine) (PGGA), poly-(γ-L-aspartic glutamine) (PGAA), poly-L-lactic acid (PLLA), poly-(lactic acid-co-glycolic acid) (PLGA), polyalkyl cyanoacrylate (PACA), polyanhydride, polyhydroxy acid, polypropyl fumarate, polyamide, polyacetal, polyether, polyester, poly(orthoester), polycyanoacrylate, [N-(2-hydroxypropyl)methacrylamide] (HPMA) copolymer, polyvinyl alcohol, polyurethane, polyphosphazene, polyacrylate, polyurea, polyamine poly(ε-caprolactone) (PCL), and copolymers thereof.

[0212] Lipid-based nanoparticles (LNPs)

[0213] In some embodiments, the compositions of this disclosure can be formulated in lipid-based nanoparticles (LNPs). For example, the nucleic acid constructs of this disclosure can be delivered to cells or subjects via lipid-based nanoparticles (LNPs). The immunogenicity of LNPs is generally lower than that of viral particles. While many people have pre-existing immunity to viral particles, they do not have pre-existing immunity to LNPs. Furthermore, an adaptive immune response against LNPs is unlikely to occur, which allows for repeated administration of LNPs.

[0214] The lipids suitable for use in the compositions and methods described herein can be cationic lipids, ionizable cationic lipids, anionic lipids, or neutral lipids.

[0215] In some embodiments, the LNP of this disclosure may comprise one or more ionizable lipids. As used herein, the term "ionizable lipid" refers to a lipid that is cationic when the pH decreases below the pKa of the ionizable group of the lipid, or becomes ionizable (protonated) but more neutral at higher pH values. At pH values ​​below the pKa, the lipid is then able to associate with negatively charged nucleic acids (e.g., oligonucleotides). As used herein, the term "ionizable lipid" includes lipids that exhibit a positive charge when the pH decreases from physiological pH, as well as any of many lipid classes that carry a net positive charge at selective pH (such as physiological pH). Permanently cationic lipids (such as DOTMA) have been shown to be too toxic for clinical use. Ionizable lipids may be present in lipid formulations according to other embodiments, preferably in proportions of about 30 Mol% to about 70 Mol% in some embodiments, about 30 Mol% in others, about 40 Mol% in others, about 45 Mol% in others, about 47.5 Mol% in still others, about 50 Mol% in yet others, and about 60 Mol% in still others (“Mol%” means the percentage of total moles of a particular component). The LNP of this disclosure may comprise DODMA or 1,2-dioleoyloxy-3-dimethylaminopropane, which are ionizable lipids such as DLin-MC3-DMA or O-(Z,Z,Z,Z-heptadecane-6,9,26,29-tetraen-19-yl)-4-(N,N-dimethylamino) (“MC3”).

[0216] Exemplary ionizable lipids applicable to the compositions and methods of this disclosure include ionizable lipids described in: PCT disclosures WO 2020252589A1 and WO 2021000041A1, U.S. Patents 8,450,298 and 10,844,028, and Love KT et al., Proc Natl Acad Sci USA, February 2, 2010, 107(5) 1864-1869, all of which are incorporated herein by reference in their entirety. Accordingly, in some embodiments, the LNP of this disclosure comprises one or more lipid compounds described above in Love KT et al., 2010, ibid., such as C16-96, C14-110, and C12-200. In some embodiments, the LNP comprises an ionizable cationic lipid selected from ALC-0315, C12-200, LN16, MC3, MD1, SM-102, and any combination thereof. In some embodiments, the LNP of this disclosure comprises C12-200. The structure of the C12-200 lipid is known in the art and described, for example, in U.S. Patent Nos. 8,450,298 and 10,844,028, which are 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.

[0217] In some embodiments, the LNP of this disclosure comprises 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 LNP of this disclosure comprises one or more neutral lipids. Non-limiting neutral lipids suitable for the compositions and methods of this disclosure include DPSC, DPPC, POPC, DOPE, and SM. In some embodiments, the LNP of this disclosure comprises one or more ionizable lipid compounds described in PCT disclosures WO 2020252589 A1 and WO2021000041 A1, which are hereby incorporated herein by reference in their entirety.

[0218] Many other lipids or combinations of lipids known in the art can be used to generate LNPs. Non-limiting examples of lipids suitable for generating 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 combinations 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.

[0219] In some embodiments, the LNP of this disclosure comprises at least one lipid selected from the following: 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.

[0220] In some embodiments, the lipid-to-nucleic acid mass ratio in the LNP delivery system is about 100:1 to about 3:1, about 70:1 to 10:1, or 16:1 to 4:1. In some embodiments, the lipid-to-nucleic acid mass ratio in the LNP delivery system is about 16:1 to 4:1. In some embodiments, the lipid-to-nucleic acid mass ratio in the LNP delivery system is about 20:1. In some embodiments, the lipid-to-nucleic acid mass ratio 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 average diameter of the LNP ranges from about 70 nm to 100 nm. In some implementations, the average diameter of the LNP ranges from about 88 nm to about 92 nm, from 82 nm to about 86 nm, or from about 80 nm to about 95 nm.

[0221] As described above, in some embodiments, neutral lipids (also known as “structural lipids” or “auxiliary lipids”) may also be incorporated into lipid formulations and lipid particles. Lipid formulations and lipid particles may contain one or more structural lipids as about 10 mol% to 40 mol% of the composition. Suitable structural lipids support particle formation during manufacturing. Structural lipids refer to any of many lipid classes that exist in anionic, uncharged, or neutral zwitterionic forms at physiological pH. Representative structural lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, diacylphosphatidylglycerol, ceramides, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides.

[0222] Exemplary structural lipids include zwitterionic lipids such as distearylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylethanolamine (DOPE), palmitoylphosphatidylcholine (POPC), palmitoylphosphatidylethanolamine (POPE), and dioleoylphosphatidylethanolamine 4-(N-maleiminomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearylphosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), and 1,2-ditransoleoyl-sn-glycerol-3-phosphoethanolamine (transDOPE).

[0223] In another embodiment, the structural lipid can be any lipid that is negatively charged at physiological pH. These lipids include phosphatidylglycerols such as dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylglycerol (POPG), cardiolipin, phosphatidylinositol, diacylphosphatidylserine, diacylphosphatidic acid, and other anionic modifying groups linked to neutral lipids. Other suitable structural lipids include glycolipids (e.g., monosialotetrahexosylganglioside GM1).

[0224] Stabilizers can be included in lipid formulation implementations to ensure the integrity of the mixture. Stabilizers are molecules that disrupt or facilitate the formation of hydrophobic-hydrophilic interactions between molecules. Suitable stabilizers include, but are not limited to, polysorbate 80 (also known as Tween 80, IUPAC name 2-[2-[3,4-bis(2-hydroxyethoxy)oxopentane-2-yl]-2-(2-hydroxyethoxy)ethoxy]ethyl octadec-9-enoate), Myrj52 (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.

[0225] In some embodiments, the stabilizer comprises about 0.1 mol% to 3 mol% of the total lipid mixture. In some embodiments, the stabilizer comprises about 0.5 mol% to 2.5 mol% of the total lipid mixture. In some embodiments, the stabilizer is present at a concentration greater than 2.5 mol%. In some embodiments, the stabilizer is present at a concentration of 5 mol%. In some embodiments, the stabilizer is present at a concentration of 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 comprises 2.6 mol% to 10 mol% of the lipid mixture. In other embodiments, the stabilizer is present at a concentration greater than 10 mol% of the lipid mixture.

[0226] Steroids can also be included in lipid compositions for certain applications, and lipid particles prepared from them contain sterols (such as cholesterol and phytosterols).

[0227] 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 agent. In some embodiments, the pharmaceutical composition is formulated for one or more of the following administration methods: intranasal, transdermal, intraperitoneal, intramuscular, intranodal, intratumoral, intra-articular, intravenous, subcutaneous, intravaginal, cardiac, and oral administration.

[0228] Pharmaceutical compositions suitable for injection include sterile aqueous solutions or dispersions and sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, antibacterial water, Cremophor EL™ (BASF, Pasipani, NJ), or phosphate-buffered saline (PBS), tris (tromethamine), and HEPES. In these cases, the composition should be sterile and should be a fluid to a degree that facilitates injection. It should be stable under the conditions of manufacture and storage and be preserved against contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. For example, appropriate flowability can be maintained by using coatings (e.g., lecithin), by maintaining the desired particle size in the case of dispersions, and by using surfactants (e.g., sodium dodecyl sulfate). The prevention of microbial activity can be achieved through various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.). In many cases, isotonic agents, such as sugars, polyols (e.g., mannitol, sorbitol), sucrose, trehalose, and / or sodium chloride, are typically included in the composition. In some embodiments, the composition contains tris and sucrose. Prolonged absorption of injectable compositions can be achieved by including agents that delay absorption (e.g., aluminum monostearate and gelatin) in the composition.

[0229] Sterile injectable solutions can be prepared by incorporating the active compound in the desired amount into a suitable solvent having one or a combination of the components listed above, and then sterilizing by filtration. Typically, dispersions are prepared by incorporating the active compound into a sterile medium containing a base dispersion medium and other desired components from those listed above.

[0230] In some embodiments, the pharmaceutical composition is formulated for one or more of the following administration methods: intranasal administration, transdermal administration, intrathecal administration, intraperitoneal administration, intramuscular administration, intratracheal administration, intratumoral administration, intratumoral administration, intra-articular administration, intravenous administration, subcutaneous administration, intravaginal administration, intraocular administration, rectal administration, intracystic instillation, and oral administration.

[0231] In some embodiments, the pharmaceutical compositions of this disclosure are formulated for inhalation, such as aerosols, sprays, nebulizers, liquids, or powders. Inhalation administration may be in the form of a dry powder or aerosol formulation, which is inhaled by a subject (e.g., a patient) using an inhalation device (e.g., a microneedle, a metered-dose inhaler, or a nebulizer).

[0232] The method of this public text

[0233] The administration of any of the therapeutic compositions described herein (e.g., nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions) may be used to modulate at least one pharmacodynamic effect in a subject or to treat a related health condition (such as cancer).

[0234] Non-limiting examples of cancers to which the methods of this disclosure are applicable include cancer, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of this type of cancer include, but are not limited to: basal cell carcinoma; bile duct cancer; bladder cancer; bone cancer; brain and CNS cancers; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colorectal cancer; connective tissue cancer; digestive system cancers; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; stomach cancer (including gastrointestinal cancers); glioblastoma (GBM); liver cancer; liver tumors; intraepithelial neoplasia; kidney cancer or renal cell carcinoma; laryngeal cancer; leukemia; liver cancer; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma); lymphomas including Hodgkin lymphoma and non-Hodgkin lymphoma; melanoma; myeloma; neuroblastoma; oral cancer (e.g., lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; respiratory system cancers; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; stomach cancer; testicular cancer; thyroid cancer. Cancer; uterine or endometrial cancer; urinary tract cancer; vulvar cancer; and other cancers and sarcomas; and B-cell lymphomas (including low-grade / follicular non-Hodgkin lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-lytic cell NHL; large mass NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenström macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloid leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal angiogenesis associated with nevus hamartomatosis, edema (such as that associated with brain tumors), and Megs syndrome. In some implementations, the cancer is non-small cell lung cancer.

[0235] In some embodiments, nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions as described herein can be used to modulate (e.g., induce or inhibit) pharmacodynamic effects in subjects in need. In some embodiments, pharmacodynamic effects include inducing an immune response in subjects. Non-limiting examples of pharmacodynamic effects include immunogenic effects, biomarker responses, therapeutic effects, prophylactic effects, desired effects, undesired effects, adverse effects, and effects in disease models.

[0236] Therefore, one aspect of this disclosure relates to a method for modulating pharmacodynamic effects in subjects of need, the method comprising administering to a subject a composition comprising one or more of: (a) a nucleic acid construct as described herein; (b) recombinant cells as described herein; and (c) a pharmaceutical composition as described herein. In some embodiments, the pharmacodynamic effect includes one or more of: immunogenic effects, biomarker responses, therapeutic effects, prophylactic effects, desired effects, undesired effects, adverse effects, and effects in disease models. In some embodiments, the pharmacodynamic effect includes inducing an immune response in a subject.

[0237] Therefore, in another aspect, this document provides a method for preventing or treating a health condition in a subject, the method comprising prophylactically or therapeutically administering to the subject a composition comprising one or more of the following: (a) a replicon as described herein, such as a self-replicating RNA construct (srRNA); (b) a nucleic acid as described herein; (c) a recombinant cell as described herein; and (d) a pharmaceutical composition as described herein. In some embodiments, the administered composition elicits an immune response in the subject. In some embodiments, the administered composition induces the production of one or more pro-inflammatory molecules in the subject. In some embodiments, the one or more pro-inflammatory molecules include interleukin-1α (IFNα), interleukin-1β (IFNβ), interleukin-18 (IL-18), interleukin-6 (IL-6), interleukin-1α (IL-1α), interleukin-1β (IL-1β), interleukin-12 (IL-12), interleukin-2 (IL-2), IL-23, IL-27, interferon-γ (IFNγ), cytokines, TNF-α, GM-CSF and MIP1α, granzyme B, granzyme A, perforin, or any combination thereof. In some embodiments, the subject has previously been treated with one or more therapies and has developed at least partial resistance to said one or more therapies.

[0238] As described above, administration of any of the therapeutic compositions described herein (e.g., nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions) can be used to induce at least one pharmacodynamic effect in a subject. In some embodiments, the ability of the nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions described herein to confer at least one pharmacodynamic effect is analyzed in vivo or in vitro. Examples of pharmacodynamic effects that can be analyzed include: immunogenic effects (e.g., inducing an immune response in vivo), biomarker responses, therapeutic effects, preventive effects, desired effects, undesired effects, adverse effects, and effects in disease models. Effects in disease models may include pharmacodynamic effects that lead to the control, mitigation, or reversal of disease and / or its properties, such as prevention or reduction of mortality or morbidity or disease severity. In some embodiments, the assessment of pharmacodynamic effects includes evaluating the induction of an immune response in vivo. In some embodiments, the assessment of pharmacodynamic effects includes evaluating the induction of cytokine pathways that can enhance immune responses and prevent angiogenesis and metastasis.

[0239] In some embodiments, the disclosed compositions are formulated to be compatible with their intended route of administration. For example, the nucleic acid constructs (e.g., srRNA constructs), recombinant cell and / or pharmaceutical compositions of this disclosure may be administered orally or by inhalation, but more likely, they will be administered via a parenteral route. Examples of parenteral administration routes include, for example, intramuscular, intratumoral, intravenous, intranodal, intradermal, subcutaneous, transdermal (topical), transmucosal, intravaginal, and rectal administration. In some embodiments, the composition is administered intramuscularly. In some embodiments, the composition is administered intratumorally or via cardiac administration. Solutions or suspensions intended for parenteral use may contain the following components: sterile diluents, such as water for injection, saline solution, fixative oil, polyethylene glycol, glycerol, 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 agents for toning, such as sodium chloride or dextrose. The pH may be adjusted (e.g., to approximately 7.2–7.8, such as 7.5) with an acid or base (e.g., sodium dihydrogen phosphate and / or disodium hydrogen phosphate, hydrochloric acid, or sodium hydroxide). Parenteral preparations may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0240] The LD50 (lethal dose for 50% of the population) and ED can be determined in cell cultures or laboratory animals, for example, by methods used to determine these parameters. 50Standard pharmaceutical procedures (the dosage effective in 50% of the population) are used to determine the dosage, toxicity, and therapeutic efficacy of nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions of this subject matter. The dose ratio between toxic effects and therapeutic effects is the therapeutic index, and it can be expressed as the ratio LD. 50 / ED 50 Compounds exhibiting a high therapeutic index are generally suitable. While compounds exhibiting toxic side effects can be used, delivery systems for targeting such compounds to affected tissue sites should be carefully designed to minimize potential damage to uninfected cells and thereby reduce side effects.

[0241] For example, data obtained from cell culture assays and animal studies can be used to determine dosage ranges for use in humans. Doses of such compounds are typically within the range of ED (Extra-Active). 50 The dosage is within a range of circulating concentrations with very low or no toxicity. The dosage can vary within this range depending on the dosage form and route of administration used. For any compound used in the methods of this disclosure, the therapeutically effective dose can be initially estimated from cell culture assays. Doses can be established in animal models to achieve IC50 values ​​as determined in cell cultures. 50 (For example, the range of circulating plasma concentrations at which a test compound achieves half-maximal inhibition of symptoms). Such information can be used to more accurately determine the effective dose in humans. Plasma levels can be measured, for example, by high-performance liquid chromatography.

[0242] The therapeutic compositions described herein, such as nucleic acid constructs (e.g., srRNA constructs), recombinant cell and / or pharmaceutical compositions, can be administered once or more daily to once or more weekly (including every other day). Those skilled in the art will understand that certain factors may influence the dosage and timing required for effective treatment of a subject, including but not limited to the severity of the disease, prior treatment, the subject's general health conditions and / or age, and other pre-existing conditions. Furthermore, treatment of a subject with a therapeutically effective amount of the subject matter of this disclosure, such as multivalent peptides and multivalent antibodies, may comprise a single treatment or a series of treatments. In some embodiments, the composition is administered every 8 hours for five consecutive days, followed by a rest period of 2 to 14 days (e.g., 9 days), and then administered every 8 hours for another five days. Regarding nucleic acid constructs (e.g., srRNA constructs), the therapeutically effective amount (e.g., effective dose) of the nucleic acid construct described herein depends on the nucleic acid construct selected.

[0243] As discussed above, a therapeutically effective amount includes an amount sufficient to promote a specific effect when the therapeutic composition is administered to a subject, such as a subject who has, is suspected of having, or is at risk of developing a health condition (e.g., an autoimmune disease, an inflammatory disease, or a cardiovascular disease). In some embodiments, an effective amount includes an amount sufficient to prevent or delay the development of disease symptoms, alter the course of disease symptoms (e.g., but not limited to slowing the progression of disease symptoms), or reverse disease symptoms.

[0244] The efficacy of a treatment, including the disclosed therapeutic compositions, for treating a healthy condition or disease can be determined by a skilled clinician. However, a treatment is considered effective if at least any or all of the signs or symptoms of the disease are improved or alleviated. Efficacy can also be measured by failure of individual deterioration (e.g., cessation or at least slowing of the progression of the healthy condition or disease) as assessed by hospitalization or the need for medical intervention. Methods for measuring these measures are known to those skilled in the art and / or described herein. Treatment includes any treatment of a healthy condition or disease in a subject or animal (some non-limiting examples include humans or mammals) and includes: (1) inhibiting the healthy condition or disease, such as stopping or slowing the progression of symptoms; or (2) alleviating the healthy condition or disease, such as causing symptom resolution; and (3) preventing the development of symptoms or reducing their likelihood.

[0245] In some embodiments, the nucleic acid constructs (e.g., srRNA constructs), recombinant cells, and / or pharmaceutical compositions of this disclosure may be administered to a subject in a composition having a pharmaceutically acceptable carrier and in an amount that effectively stimulates an immune response. Typically, the subject may be immunized via an initial series of injections (or administered via one of the other routes described below), followed by a booster to increase the protection provided by the initial series of administrations. The initial series of injections and subsequent boosters are administered at a dose necessary to stimulate the subject's immune response and for a period of time necessary to stimulate the subject's immune response. In some embodiments, the administered composition results in an increase in interferon production in the subject. In some embodiments of the disclosed methods, the subject is a mammal. In some embodiments, the mammal is a human subject.

[0246] When nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions are properly protected, as described above, they can be administered orally, for example, using an inert diluent or an assimilateable edible carrier. Nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions, along with other ingredients, can also be encapsulated in hard or soft-shell gelatin capsules, compressed into tablets, or directly incorporated into an individual's diet. For oral therapeutic administration, the active compound can be combined with excipients and used in the form of ingestible tablets, sublingual tablets, lozenges, capsules, elixirs, suspensions, syrups, rice paper wafers, etc.

[0247] In some embodiments, the nucleic acid constructs of this disclosure can be delivered to cells or subjects via lipid-based nanoparticles (LNPs). LNPs are generally less immunogenic than viral particles. While many people have pre-existing immunity to viral particles, they do not have pre-existing immunity to LNPs. Furthermore, adaptive immune responses against LNPs are unlikely to occur, which allows for repeated administration of LNPs.

[0248] Other treatments

[0249] In some embodiments, the composition according to this disclosure is administered to a subject as a monotherapy (single-drug therapy) alone or as a first therapy in combination with at least one other therapy (e.g., a second therapy). In some embodiments, the second therapy is selected from chemotherapy, radiotherapy, immunotherapy, hormone therapy, toxin therapy, targeted therapy, and surgery. In some embodiments, the second therapy is selected from chemotherapy, radiotherapy, immunotherapy, hormone therapy, toxin therapy, or surgery. In some embodiments, the first therapy and the second therapy are administered concurrently. In some embodiments, the first therapy and the second therapy are administered simultaneously. In some embodiments, the first therapy and the second therapy are administered sequentially. In some embodiments, the first therapy 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 administered together as a single formulation.

[0250] Reagent test kit

[0251] This document also provides various kits for practicing the methods described herein, as well as written instructions for preparing and using said kits. In particular, some embodiments of this disclosure provide kits for modulating (e.g., inducing, eliciting, or inhibiting) pharmacodynamic effects. Some embodiments of this disclosure provide kits for eliciting an immune response in a subject. Some other embodiments relate to kits for preventing health conditions (e.g., autoimmune diseases) in subjects in need. Some other embodiments relate to kits for methods of treating health conditions (e.g., autoimmune diseases) in subjects in need. For example, in some embodiments, kits are provided herein that comprise one or more of nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions as provided and described herein, along with written instructions for preparing and using them.

[0252] In some embodiments, the kit of this disclosure further includes one or more means for administering any one of the provided nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions to a subject. For example, in some embodiments, the kit of this disclosure further includes one or more syringes (including drug-loaded syringes) and / or catheters (including drug-loaded syringes) for administering any one of the provided nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions to a subject. In some embodiments, the kit may have one or more additional therapeutic agents that can be administered simultaneously or sequentially with other kit components for a desired purpose, such as for diagnosing, preventing, or treating a condition in a subject in need.

[0253] Any of the kits described above may further include one or more additional reagents, wherein such additional reagents may be selected from: dilution buffers, reconstitution solutions, wash buffers, control reagents, control expression vectors, negative controls, positive controls, reagents suitable for in vitro generation of the nucleic acid constructs, recombinant cells and / or pharmaceutical compositions provided herein.

[0254] In some embodiments, the kit components may be in separate containers. In some other embodiments, the kit components may be combined in a single container. Thus, in some embodiments of this disclosure, the kit contains one or more of the nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions provided and described herein in one container (e.g., in a sterile glass or plastic vial) and another therapeutic agent in another container (e.g., in a sterile glass or plastic vial).

[0255] In another embodiment, the kit comprises a combination of the composition described herein (containing one or more nucleic acid constructs, recombinant cells and / or pharmaceutical compositions of this disclosure) and one or more other therapeutic agents, optionally formulated together in a single common container as a pharmaceutical composition.

[0256] If the kit contains a pharmaceutical composition for parenteral administration to a subject, the kit may contain a device for performing such administration (e.g., an injection device or catheter). For example, the kit may contain one or more subcutaneous injection needles or other injection devices as discussed above that contain one or more nucleic acid constructs, recombinant cells, and / or pharmaceutical compositions contained in this disclosure.

[0257] In some implementations, the kit components may be in separate containers. In other implementations, the kit components may be combined in a single container.

[0258] In some implementations, the kit may further include instructions for using the kit components to practice the methods disclosed herein. For example, the kit may include a packaging insert containing information about the pharmaceutical compositions and dosage forms in the kit. Typically, such information helps patients and physicians use the packaged pharmaceutical compositions and dosage forms effectively and safely. For example, the insert may provide information about a combination of the following aspects of this disclosure: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and usage, contraindications, warnings, precautions, adverse reactions, overdose, correct dosage and administration, supply specifications, correct storage conditions, references, manufacturer / distributor information, and intellectual property information.

[0259] Instructions for practicing the methods are typically recorded on a suitable recording medium. For example, the instructions may be printed on a substrate such as paper or plastic. The instructions may be included as a packaging insert in the kit, or on a label on a container of the kit or its components (e.g., related to packaging or sub-packaging). The instructions may also exist as an electronic storage data file on a suitable computer-readable storage medium (e.g., CD-ROM, floppy disk, flash drive, etc.). In some cases, the actual instructions are not included in the kit, but rather a means of obtaining the instructions from a remote source (e.g., via the Internet) is provided. An example of this embodiment is a kit containing a URL where the instructions can be viewed and / or downloaded. Like the instructions themselves, this means of obtaining the instructions can be recorded on a suitable substrate.

[0260] All publications and patent applications mentioned in this disclosure are incorporated herein by reference as if each individual publication or patent application were specifically and individually incorporated by reference.

[0261] No references cited herein are acknowledged to constitute prior art. The discussion of references states the claims of their authors, and the applicant reserves the right to question the accuracy and relevance of the cited documents. It will be clearly understood that although numerous sources of information are mentioned herein, including scientific journal articles, patent documents, and textbooks, such mentions do not constitute an admission that any of these documents constitutes part of common general knowledge in the art.

[0262] The discussion of the general methods presented herein is for illustrative purposes only. Other alternative methods and solutions will be apparent to those skilled in the art upon review of this disclosure and will be included within the spirit and scope of this application.

[0263] Further embodiments are disclosed in detail in the following examples. These embodiments are provided by way of illustration only and are not intended to limit the scope of this disclosure or the claims in any way.

[0264] Example

[0265] Unless otherwise indicated, the practice of this invention will employ conventional techniques of molecular biology, microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology well known to those skilled in the art. Such techniques are well explained in the literature, such as Sambrook, J. and Russell, DW (2012). Molecular Cloning: A Laboratory Manual (4th edition). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory; and Sambrook, J. and Russell, DW (2001). Molecular Cloning: A Laboratory Manual (3rd edition). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory (collectively referred to as "Sambrook" in this article); Ausubel, FM (1987). Current Protocols in Molecular Biology. New York, NY: Wiley (including supplements up to 2014); Bollag, DM 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, MG 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, KB, Ferré, F. and Gibbs, R. (1994).PCR: The Polymerase Chain Reaction. Boston: Birkhauser Publisher; Greenfield, EA (2014). Antibodies: A Laboratory Manual (2nd ed.). New York, NY: Cold Spring Harbor Laboratory Press; Beaucage, SL et al. (2000). Current Protocols in Nucleic Acid Chemistry. New York, NY: Wiley, (including supplements up to 2014); and Makrides, SC (2003). Gene Transfer and Expression in Mammalian Cells. Amsterdam, NL: Elsevier Sciences BV. The published content of these references is incorporated into this paper by citation.

[0266] Further embodiments are disclosed in detail in the following examples. These embodiments are provided by way of illustration only and are not intended to limit the scope of this disclosure or the claims in any way.

[0267] Example 1

[0268] Construction of modified alphavirus vector

[0269] This embodiment describes experiments conducted to construct basic alphavirus vectors (e.g., without heterologous genes), which are then used to construct vectors expressing target genes (e.g., EGFR).

[0270] EEEV basic carrier

[0271] The basic EEEV vector (i.e., without the target heterologous gene) was constructed as follows: The basic EEEV vector was synthesized de novo from a reference sequence with several modifications (Genbank EF151502) in four approximately 4 kb portions (Twist Bioscience). Silent mutations G301A, A3550C, G4516A, G5725A, and G7399A were incorporated to eliminate restriction endonuclease cleavage sites. A unique restriction endonuclease cleavage site (SpeI, 5'-A'CTAG, T-3') was incorporated to replace the coding sequence of the native EEEV structural gene (where 5' A matches the start codon of the structural polyprotein ATG, and 3' T matches the stop codon TAA of the structural polyprotein). A 5' adaptor sequence (5'-CTGGAGACGTGGAGGAGAACCCTGGACCT-3'; SEQ ID NO: 2) was inserted upstream of the SpeI site, and a 3' adaptor sequence (5'-GACCGCTACGCCCCAATGACCCGACCAGC-3'; SEQ ID NO: 3) was inserted downstream of the SpeI site for subsequent Gibson Assembly® procedures (Gibson et al., Nat. Methods 6, 343-345, 2009). The phage T7 RNA polymerase promoter (5'-TAATACGACTCACTATAG-3'; SEQ ID NO: 4) was included upstream of the EEEV genome sequence, and downstream of it was a poly(A) sequence, followed by the SapI site upstream of the cleavage recognition site. Immediately downstream of the SapI site is the T7 terminator sequence (5'-AACCCCTCTCTAAACGGAGGGGTTTTTTT-3'; SEQ ID NO: 5), followed by a unique restriction enzyme site (NotI, 5'-GC'GGCC, GC-3'). These parts are combined in a five-piece Gibson Assembly® reaction (linearized pYL backbone and four synthetic fragments) to generate the EEEV basic vector.

[0272] CHIKV basic carrier

[0273] The basic CHIKV S27 vector was synthesized de novo from a reference sequence (Genbank AF369024) in four approximately 4 kb portions (Twist Bioscience, Thermo Fisher GeneArt). This reference sequence contained a silenced A5366G mutation and a unique restriction endonuclease site (SpeI, 5'-A'CTAG, T-3') replacing the coding sequence of the CHIKV structural gene (where 5'A matches the ATG start codon of the structural multiprotein, and 3'T matches the TAA stop codon of the structural multiprotein). A 5' adaptor sequence (5'-CTGGAGACGTGGAGGAGAACCCTGGACCT-3'; SEQ ID NO: 2) was inserted upstream of the SpeI site, and a 3' adaptor sequence (5'-GACCGCTACGCCCCAATGACCCGACCAGC-3'; SEQ ID NO: 3) was inserted downstream of the SpeI site for subsequent Gibson Assembly® procedures. The phage T7 RNA polymerase promoter (5'-TAATACGACTCACTATAG-3'; SEQ ID NO: 4) is included upstream of the CHIKV genome sequence, and downstream of it is a poly(A) sequence, followed by a SapI site upstream of the cleavage recognition site. Immediately downstream of the SapI site is the T7 terminator sequence (5'-AACCCCTCTCTAAACGGAGGGGTTTTTTT-3'; SEQ ID NO: 5), followed by a unique restriction enzyme site (NotI, 5'-GC'GGCC, GC-3'). These portions are combined in a five-piece Gibson Assembly® reaction (linearized pYL backbone and four synthetic fragments) to generate the CHIKV S27 basic vector.

[0274] The CHIKV DRDE base vector was similarly constructed from a reference sequence (Genbank EF210157), except that the S27 3' UTR was used instead of the DRDE 3' UTR.

[0275] SINV base carrier

[0276] The basic SINV Girdwood vector was synthesized de novo from the Girdwood strain reference sequence (Genbank MF459683) in four approximately 4 kb portions (TwistBioscience, Thermo Fisher GeneArt). The Girdwood strain reference sequence replaces the coding sequence of the SINV structural gene with a unique restriction endonuclease cleavage site (SpeI, 5'-A'CTAG, T-3'). This 5' A is the next nucleotide after the P2A sequence following nucleotide 93 of the structural polyprotein gene, and the 3' T matches the position of the TGA stop codon of the structural polyprotein. The phage T7 RNA polymerase promoter (5'-TAATACGACTCACTATAG-3'; SEQ ID NO: 4) is included upstream of the SINV genome sequence, and downstream of it is a poly(A) sequence followed by a SapI site upstream of the cleavage recognition site. Immediately downstream of the SapI site is the T7 terminator sequence (5'-AACCCCTCTCTAAACGGAGGGGTTTTTTT-3'; SEQ ID NO: 5), followed by a unique restriction enzyme site (NotI, 5'-GC'GGCC, GC-3'). These components are assembled in a five-piece Gibson Assembly® reaction (e.g., a linearized pYL backbone and four synthetic fragments) to generate the SINV Girdwood basic vector.

[0277] The basic SINV AR86 vector is similarly constructed from the reference sequence (Genbank U38305), except that the nsP2 coding sequence is derived from the Girdwood reference sequence.

[0278] VEE Basic Carrier

[0279] The basic VEE vector was synthesized de novo from the TC-83 strain reference sequence (Genbank L01443) in four approximately 4 kb portions (Twist Bioscience, Thermo Fisher GeneArt). The TC-83 strain reference sequence contained a silenced A2087G mutation and a unique restriction endonuclease cleavage site (SpeI, 5'-A'CTAG, T-3') replacing the coding sequence of the VEE structural gene (where 5'A is the next nucleotide after the P2A sequence following nucleotide 93 of the structural polyprotein gene, and 3'T matches the position of the TGA stop codon of the structural polyprotein). A 5' adaptor sequence (5'-CTGGAGACGTGGAGGAGAACCCTGGACCT-3'; SEQ ID NO: 2) was inserted upstream of the SpeI site, and a 3' adaptor sequence (5'-GACCGCTACGCCCCAATGACCCGACCAGC-3'; SEQ ID NO: 3) was inserted downstream of the SpeI site for subsequent Gibson Assembly® procedures. The phage T7 RNA polymerase promoter (5'-TAATACGACTCACTATAG-3'; SEQ ID NO: 4) is included upstream of the VEE genome sequence, and downstream of it is a poly(A) sequence, followed by a SapI site upstream of the cleavage recognition site. Immediately downstream of the SapI site is the T7 terminator sequence (5'-AACCCCTCTCTAAACGGAGGGGTTTTTTT-3'; SEQ ID NO: 5), followed by a unique restriction enzyme site (NotI, 5'-GC'GGCC, GC-3'). These portions are assembled in a five-piece Gibson Assembly® reaction (e.g., a linearized pYL backbone and four synthetic fragments) to produce the VEE basic vector.

[0280] MADV Basic Carrier

[0281] The basic MADV vector (i.e., without the target heterologous gene) was constructed as follows: The basic MADV vector was de novo synthesized from a reference sequence (Genbank KJ469641) with several modifications, using three 869 to 4992 bp portions. Unique restriction endonuclease sites (SpeI, 5'-A'CTAG, T-3') were incorporated to replace the coding sequence of the natural MADV structural gene (where 5' A matches the start codon of the structural polyprotein ATG, and 3' T matches the stop codon of the structural polyprotein TAA). A 5' adaptor sequence (5'-CTGGAGACGTGGAGGAGAACCCTGGACCT-3'; SEQ ID NO: 2) was inserted upstream of the SpeI site, and a 3' adaptor sequence (5'-GACCGCTACGCCCCAATGACCCGACCAGC-3'; SEQ ID NO: 3) was inserted downstream of the SpeI site for subsequent Gibson Assembly® procedures (Gibson et al., Nat. Methods 6, 343-345, 2009). The phage T7 RNA polymerase promoter (5'-TAATACGACTCACTATAG-3'; SEQ ID NO: 4) was included upstream of the MADV genome sequence, and downstream of it was a poly(A) sequence, followed by the SapI site upstream of the cleavage recognition site. Immediately downstream of the SapI site is the T7 terminator sequence (5'-AACCCCTCTCTAAACGGAGGGGTTTTTTT-3'; SEQ ID NO: 5), followed by a unique restriction enzyme site (NotI, 5'-GC'GGCC, GC-3'). These portions are combined in a five-piece Gibson Assembly® reaction (linearized pYL backbone and four synthetic fragments) to generate the MADV basic vector.

[0282] WEEV basic carrier

[0283] The basic WEEV vector (i.e., without the target heterologous gene) was constructed as follows: The basic WEEV Imperial181 vector was de novo synthesized from a reference sequence (Genbank GQ287641) with several modifications in four 200-3875 bp portions. Nucleotides were assigned to undefined bases in the reference sequence: K1655T, W4518C, and M7804A. Silent mutations A6948G and A7242G were incorporated to eliminate restriction endonuclease cleavage sites. A unique restriction endonuclease cleavage site (SpeI, 5'-A'CTAG, T-3') was incorporated to replace the coding sequence of the native WEEV structural gene (where 5' A matches the start codon of the structural polyprotein ATG, and 3' T matches the stop codon TAA of the structural polyprotein). A 5' adaptor sequence (5'-CTGGAGACGTGGAGGAGAACCCTGGACCT-3'; SEQ ID NO: 2) was inserted upstream of the SpeI site, and a 3' adaptor sequence (5'-GACCGCTACGCCCCAATGACCCGACCAGC-3'; SEQ ID NO: 3) was inserted downstream of the SpeI site for subsequent Gibson Assembly® procedures (Gibson et al., Nat. Methods 6, 343-345, 2009). The phage T7 RNA polymerase promoter (5'-TAATACGACTCACTATAG-3'; SEQ ID NO: 4) was included upstream of the WEEV genome sequence, and downstream of it was a poly(A) sequence, followed by the SapI site upstream of the cleavage recognition site. Immediately downstream of the SapI site is the T7 terminator sequence (5'-AACCCCTCTCTAAACGGAGGGGTTTTTTT-3'; SEQ ID NO: 5), followed by a unique restriction enzyme site (NotI, 5'-GC'GGCC, GC-3'). These portions are combined in a five-piece GibsonAssembly® reaction (linearized pYL backbone and four synthetic fragments) to generate the WEEV Imperial181 basic vector. A similar method is used to generate the WEEV McMillan basic vector.

[0284] The EGFR transgene was synthesized using flanking sequences homologous to the 5' and 3' adaptor sequences (IDT) and inserted into a SpeI-linearized base vector via Gibson Assembly® to produce the final vector.

[0285] Example 2

[0286] In vitro evaluation of modified alphavirus vectors

[0287] This embodiment describes the results of in vitro experiments conducted to evaluate the expression level of the synthetic srRNA construct described in Example 1 above and to investigate any differential behavior (e.g., protein expression).

[0288] In vitro transcription RNA was prepared in vitro from a SapI-linearized plasmid template using phage T7 RNA polymerase via in vitro transcription using a 5' ARCA cap (HiScribe™ T7 ARCA mRNA Kit, NEB) or by capless transcription (HiScribe™ T7 High-Yield RNA Synthesis Kit, NEB) followed by the addition of a 5' cap (vaccinia virus capping system, mRNA cap 2'-O-methyltransferase, NEB). RNA was then purified using phenol / chloroform extraction or column purification (Monarch® RNA Clearance Kit, NEB). RNA concentration was determined by absorbance at 260 nm (Nanodrop, Thermo Fisher Scientific).

[0289] copy RNA was transformed into BHK-21 or Vero cells via electroporation (e.g., 4D-Nucleofector™, Lonza). 15–22 hours post-transformation, cells were fixed and permeabilized (eBioscience™ Foxp3 / transcription factor staining buffer, Invitrogen) and stained with a PE-conjugated anti-dsRNA mouse monoclonal antibody (J2, Schicons) to quantify the frequency of dsRNA+ cells by fluorescence flow cytometry.

[0290] Protein expression RNA was transformed into BHK-21 cells via electroporation (e.g., 4D-Nucleofector™, Lonza). Cells were fixed and permeabilized (eBioscience™ Foxp3 / transcription factor staining buffer, Invitrogen) 15–22 hours post-transformation and subsequently stained with H&L using rabbit anti-EGFR antibody EPR15348 followed by either AF647-conjugated or AF488-conjugated goat anti-rabbit IgG. Mean fluorescence intensity (MFI) was used as a reading of EGFR expression. Results are shown in… Figure 1 , Figure 2 , Figure 4 and Figure 5 middle.

[0291] Figure 1The data show that ARCA-capped srRNA vectors containing EGFR transgenic cassettes possess EGFR expression characteristics, and that the identity of the EGFR transgenic cassette can influence the relative level of protein expression. This data allows for the selection of EGFR transgenic cassettes with high EGFR expression levels. Figure 2 The data show that enzymatically capped srRNA vectors containing EGFR transgenic cassettes possess EGFR expression characteristics, and that the identity of the EGFR transgenic cassette can influence the relative level of protein expression. This data allows for the selection of EGFR transgenic cassettes with high EGFR expression levels. Figure 4 and Figure 5 The study showed that different srRNA vectors containing the same EGFR transgenic cassette retained the characteristics of EGFR expression, and that the identity of the srRNA vector could affect the relative level of protein expression.

[0292] Example 3

[0293] In vivo evaluation of modified alphavirus vector

[0294] This example describes the results of in vivo experiments conducted to evaluate the srRNA constructs described herein (e.g., unformed and LNP-formed vectors).

[0295] In these experiments, synthetic srRNA constructs derived from various alphavirus strains were designed and subsequently evaluated.

[0296] Mice and injection :

[0297] HLA-A2 or HLA-A1101 transgenic mice were purchased from Charles River Labs, Envigo, or Jackson Laboratories. On the day of administration, 10 μg of material was injected intramuscularly into one quadriceps femoris muscle or separately into two quadriceps femoris muscles. Animal weight and other comprehensive observations were monitored throughout the study. For immunogenicity studies, animals were administered only on day 0 or on both days 0 and 21.

[0298] LNP formulations srRNA was formulated in lipid nanoparticles using a microfluidic mixer, and particle size, polydispersity (using dynamic light scattering), and encapsulation efficiency (using dye repulsion assay (Ribogreen)) were analyzed. The lipids were suspended in ethanol. Each srRNA was suspended at a concentration of 82 µg / ml in 100 mM NaOAc (pH 4.0) and mixed at a flow rate of 3:1 (aqueous:organic).

[0299] ELISpot.To measure the intensity of the EGFR-specific T cell response, IFNγ ELISpot analysis was performed using the Mouse IFNγ ELISpot PLUS Kit (HRP) (MabTech) according to the manufacturer's instructions. The results of the mouse IFNγ ELISpot assay, measured by speckle-forming units corresponding to the responding splenic T cells, are shown in Figure 6, 14 days after two intramuscular injections of EGFR-encoding srRNA. Total T cell response (plotted as speckle-forming units per million cells) is shown on the Y-axis.

[0300] These data demonstrate the immunogenicity of EGFR mutations in mice expressing human MHC molecules and predict their immunogenicity in humans. The results of this study led to the selection of the optimal vector.

[0301] While certain alternatives to this disclosure have been presented, it should be understood that various modifications and combinations are possible and considered within the true spirit and scope of the appended claims. Therefore, there is no intention to limit the exact abstract and disclosure presented herein.

[0302]

[0303]

[0304]

[0305]

Claims

1. A nucleic acid construct comprising a nucleic acid sequence encoding a modified alphavirus genome or self-replicating RNA (srRNA), wherein at least a portion of the nucleic acid sequence encoding a viral structural protein of the modified alphavirus genome or srRNA is replaced by a coding sequence of a polypeptide construct, the coding sequence of the polypeptide construct comprising a coding sequence of an epidermal growth factor receptor (EGFR) containing one or more acquired drug resistance mutations.

2. The nucleic acid construct according to claim 1, wherein the modified alphavirus genome or srRNA does not contain a nucleic acid sequence encoding a viral structural protein.

3. The nucleic acid construct of claim 1, wherein the nucleic acid sequence encoding the polypeptide construct is operatively linked to a promoter sequence.

4. The nucleic acid construct according to claim 3, wherein the promoter sequence is a 26S subgenome (sg) promoter.

5. The nucleic acid construct according to claim 1, wherein the modified alphavirus genome or srRNA is a modified alphavirus genome or srRNA belonging to the VEEV / EEEV group, or the SFV group, or the SINV group.

6. The nucleic acid construct according to claim 5, wherein the alphavirus is Venezuelan equine encephalitis virus (VEEV), Eastern equine encephalitis virus (EEEV), Madariaga virus (MADV), Chikungunya virus (CHIKV), Western equine encephalitis virus (WEEV), or Sindbis virus (SINV).

7. The nucleic acid construct according to claim 1, wherein the srRNA is a capped srRNA containing a 5'-cap.

8. The nucleic acid construct according to claim 7, wherein the capped srRNA is a co-transcribed capped srRNA.

9. The nucleic acid construct according to claim 7, wherein the capped srRNA is an enzymatically capped srRNA.

10. The nucleic acid construct of claim 1, wherein the one or more acquired drug resistance mutations are configured into a plurality of alteration boxes arranged in series along the length of the coding sequence.

11. The nucleic acid construct of claim 10, wherein the plurality of alteration boxes are operatively connected to each other via one or more adapters.

12. The nucleic acid construct according to claim 1, wherein the one or more acquired drug resistance mutations include one or more (i) activating mutations, (ii) mutations that enhance the binding affinity of EGFR to adenosine triphosphate (ATP), and / or (iii) mutations that block the binding of EGFR or its variants to inhibitors.

13. The nucleic acid construct of claim 12, wherein the one or more activating mutations comprise in-frame insertions in exon 20.

14. The nucleic acid construct of claim 13, wherein the insertion in exon 20 comprises a mutation selected from the group consisting of: A763_Y764insFQEA, S768_D770dup, S768_V769ins, A767_V769dup, D770_N771insX, V769_D770insX, H773_V774insX, H773dup, N771_H773dup, P772_H773insX, and N771_P772insX.

15. The nucleic acid construct according to claim 14, wherein the insertion within the exon 20 frame includes S768_D770dup, A767_V769dup, and / or H773dup.

16. The nucleic acid construct of claim 12, wherein the one or more activating mutations include L858R substitution.

17. The nucleic acid construct according to claim 12, wherein one or more acquired drug resistance mutations enhance the binding affinity of EGFR to ATP.

18. The nucleic acid construct according to claim 17, wherein the one or more acquired drug resistance mutations are selected from T790M, G719X, L858R, L718Q, G724S, L861X, S768I / V, E709X, L747S, D761Y and T854A.

19. The nucleic acid construct according to claim 12, wherein one or more acquired drug resistance mutations block the binding of EGFR to the inhibitor.

20. The nucleic acid according to claim 19, wherein the EGFR inhibitor is selected from osimertinib, lazatinib, erlotinib, gefitinib, CO-1686, HM61713, EGF816, ASP8273, avitinib, afatinib, morboteinib, icotinib, dacomitinib, poziotinib, cetuximab, ervantumab, morboteinib, vormetinib, DZD9008, CLN-081, STX-721, YK-029A, HS-10376, zipalletinib, TA K-788, JMT101, ABT-101, BEBT-109, DZG9008, PLB1004, EMB-01, HS-20117, MCLA-129, BLU-945; BDTX-1535; NX-019; JIN-A02; BBT-207; BLU-525; THE-349; STX-241; ABK3376; BI-732; BLU-701; BBT-176, BLU-451, ametinib, and talotinib.

21. The nucleic acid construct according to claim 19, wherein the one or more acquired drug resistance mutations are selected from C797S, C797G, T790M, L858R, L858M, L718V, L718Q, C796S, L798I, L792X, SV768IL, L692V, G719A, G719S, G719C, G719D, S786I, L861Q, L861R, V834X, V843X, G724S, E709K, E709H, E709A, E709G, E709V, D761Y, D761N, R776C, R776H, and T854A.

22. The nucleic acid construct according to claim 1, wherein the nucleic acid sequence encoding EGFR 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 with a nucleic acid sequence selected from SEQ ID NO: 13-32.

23. The nucleic acid construct according to claim 1, wherein the coding sequence of the polypeptide construct comprises a coding sequence of EGFR containing one or more acquired drug resistance mutations selected from the following: S768_D770dup, A767_V769dup, H773dup, T790M, L858R, and C797S.

24. The nucleic acid construct according to claim 23, wherein the coding sequence is in the 5' to 3' orientation.

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

26. The recombinant cell of claim 25, wherein the recombinant cell is a mammalian cell or an insect cell.

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

28. The pharmaceutical composition of claim 27, wherein the composition is formulated together with a delivery medium into a delivery system, wherein the delivery system comprises liposomes, viral replicon particles (VRPs), lipid-based nanoparticles (LNPs), polymer nanoparticles, physiological buffers, microspheres, immunostimulatory complexes (ISCOMs), conjugates of bioactive ligands, or combinations thereof.

29. The pharmaceutical composition of claim 28, wherein the LNP delivery system comprises cationic lipids, ionizable cationic lipids, anionic lipids, or neutral lipids.

30. The pharmaceutical composition of claim 29, wherein the lipids are present in a mass ratio of lipids to RNA of about 100:1 to about 4:

1.

31. The pharmaceutical composition of claim 27, wherein the lipid-based nanoparticles have an average diameter of about 25 nm to about 1000 nm.

32. The pharmaceutical composition of claim 27, wherein the composition is formulated as a vaccine or immunotherapy agent.

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

34. The method of claim 33, wherein the method is a method for inducing an immune response.

35. The method of claim 33, wherein the method is a method for treating cancer.

36. The method of claim 33, wherein the cancer is non-small cell lung cancer (NSCLC).

37. The method of claim 33, wherein the composition is administered to the subject alone as a monotherapy (monotherapy) or as a first therapy in combination with at least one other therapy.

38. A method for inducing at least one pharmacodynamic effect in a subject, the method comprising administering to the subject a composition comprising the nucleic acid construct according to claim 1.

39. The method of claim 38, wherein the applied composition results in the induction of one or more of the following: an immune response and a mediator selected from TNF, IL-1b, IL-12, IL-2, IFNa, IFNb, IL-6 and IFNγ.

40. The method of claim 38, wherein the at least one pharmacodynamic effect comprises one or more of the following: immunogenic effect, biomarker response, therapeutic effect, preventive effect, desired effect, undesirable effect, adverse effect, and effect in a disease model.

41. The method of claim 38, wherein the applied composition enhances antitumor immunity in the tumor microenvironment.

42. The method of claim 41, wherein the subject has cancer.

43. The method of claim 42, wherein the cancer is lung cancer.

44. The method of claim 43, wherein the lung cancer is NSCLC.

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