Compositions comprising nucleic acid molecules encoding t cell receptors and methods of treating cancer using the same

Abstract

The disclosure relates to compositions, pharmaceutical compositions, methods of making, and methods of treating related to DNA vaccines against TCR and neoantigens.

Description

DOCKET NO: WIST-020-PCT PCT APPLICATIONCOMPOSITIONS COMPRISING NUCLEIC ACID MOLECULES ENCODING T CELL RECEPTORS AND METHODS OF TREATING CANCER USING THE SAMECROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. provisional application No. 63 / 765,486, which was filed February 28, 2025, entitled “Compositions Comprising Nucleic Acid Molecules Encoding T Cell Receptors and Methods of Treating Cancer f and is incorporated herein by reference in its entirety.SEQUENCE LISTING

[0002] The sequence listing filed herewith, titled “WIST-020-PCT_SL.xml,” prepared on January 23, 2026, and having a file size of 40,626 bytes is incorporated herein by reference in its entirety.FIELD

[0003] The disclosure relates to compositions and pharmaceutical compositions comprising DNA vaccines; and methods of making, and of treating hyperproliferative disease in a subject by administering DNA vaccines against TCR and neoantigens.BACKGROUND

[0004] T cell lymphomas (TCLs) represent about 10% of all non-Hodgkin’s Lymphomas (NHL) and can be divided into several categories based on maturation stage of the T cell at time of tumorigenesis1’2. TCLs are a relatively rare tumor with fewer than 8000 new diagnosis annually3. TCLs are extremely heterogenous and can be subdivided into more than 30 different subcategories according to the World Health Organization4. The diversity and heterogeneity of TCLs account for significant differences in patient prognosis and results in very few therapeutic options which are specific just for TCLs3. Peripheral T cell lymphomas (PTCLs) are the most common form of TCL and have very few therapeutic options, especially in the relapsed / refractory setting2. For CTCL, which is considered a less aggressive tumor, the median survival for patients diagnosed with stage III / IV disease is 5 years3. On the other hand, the median survival for PTCL patients is 5 months for refractory patients and 11 months for relapsed patients6. There are very few treatment options for patients whose tumors do not respond to therapy and there is an urgent need for newer therapies for these patients.DOCKET NO: WIST-020-PCT PCT APPLICATION

[0005] Over the last few years, immunotherapies such as monoclonal antibodies, immune checkpoint inhibitors (ICI), bispecific antibodies and CART cells have rapidly changed the treatment landscape for cancer. Immunotherapy has demonstrated promising results in patients with relapsed and refractory cancers in solid tumors as well as B cell cancers7'10. Slow growing TCLs such as CTCL are characterized by an infiltration of CD8+ T cells which are mainly exhausted and express several immune inhibitory markers such as PD1, Tim3 and Lag3n. Exhausted T cells are also found in more aggressive T cell tumors and are associated with disease progression2. These results highlight that TCLs could potentially benefit from reinvigoration exhausted T cells and that immunotherapy could be successful in treatment of TCLs. Given the efficacy of immunotherapies in the clinic, their efficacy for treatment of TCLs should also be explored.

[0006] Over the last few years there has been a resurgence of interest in cancer vaccines. The targets for cancer vaccines can be derived from several distinct sources such as tumor associated antigens (TAAs), lineage markers, viral oncogenes and neoantigens (shared or personal)12. Additionally, for B and T cell cancers, the immune cell receptor (BCR or TCR) can also serve as an antigenic target. The hypervariable regions of the receptors are not encoded by a single gene, but are in fact produced via random joining of the V,D,J segments with further mutations and insertions / deletions of amino acids13. This suggests that the immune cell receptor can potentially be immunogenic as the T cells which would recognize them would not have been subject to central T cell tolerance. The BCR of a B cell cancer was shown to be the driver mutation for the disease highlighting that it would be difficult for B cell leukemias or lymphomas to lose their BCR in response to targeted therapy14. It has been shown that in patients suffering from B and T cell cancers, peptides derived from cancerous BCR / TCR can specifically bind to the patient’s Class I MHC and be expressed as peptide-MHC complexes for recognition by CD8+ T cells15'17. Clinically, an idiotype vaccine targeting the B cell receptor was safe, produced idiotype specific T cell responses in 87.5% of the patients and reduced tumorigenic B cell (but not plasma cell) burden in 66.7% of the patients in a Phase 1 trial18.

[0007] Neoantigens are antigenic peptides derived from somatic mutations in tumor cells. Rapid cell division, a feature of tumor cells, leads to several errors in replication. This process can be further enhanced due to either inherited or de novo mutations in enzymes which ensure fidelity of DNA replication, increasing the tumor mutational burden19. These mutations can be recognized byDOCKET NO: WIST-020-PCT PCT APPLICATIONthe adaptive immune system because they are not subject to central immune tolerance. Further, neoantigen expression is limited to tumor cells only making them ideal targets for therapeutic cancer vaccines20. While the majority of neoantigens are derived from non-synonymous mutations, they can be also be derived from in / del mutations which modify the open reading frame and gene / RNA fusions21'22. Every tumor evolves differently and hence each patient needs a highly personalized vaccine for their tumor therapy.SUMMARY

[0008] The disclosure relates to a composition comprising at least a first nucleic acid molecule comprising expressible nucleic acid sequence. The disclosure also relates to a composition comprising at least a first nucleic acid molecule comprising one or a plurality of nucleic acid sequences, wherein the one or plurality of nucleic acid sequences comprise: (i) one or a plurality of nucleic acid sequences encoding one or a plurality of T Cell Receptor (TCR) alpha subunits; (ii) one or a plurality of nucleic acid sequences encoding one or a plurality of TCR beta subunits; (iii) one or a plurality of nucleic acid sequences encoding one or a plurality of TCR gamma subunits; and (iv) one or a plurality of T cell antigens. In some embodiments, the composition comprises a first nucleic acid molecule and a second nucleic acid molecule, wherein the first nucleic acid molecule comprises: (i) one or a plurality of nucleic acid sequences encoding one or a plurality of T Cell Receptor (TCR) alpha amino acid sequences; (ii) one or a plurality of nucleic acid sequences encoding one or a plurality of TCR beta chain amino acid sequences; and (iii) one or a plurality of nucleic acid sequences encoding one or a plurality of TCR gamma chain amino acid sequences; and wherein the second nucleic acid molecule comprises one or a plurality of T cell antigens. In some embodiments, the first or second nucleic acid molecule comprise one or a plurality of T cell neoantigens, a regulatory sequence and an expressible nucleic acid sequence comprising Formula I or Formula II, wherein each antigen expression domain comprises a T cell neoantigen.

[0009] The disclosure also relates to compositions disclosed herein comprising a first nucleic acid molecule and a second nucleic acid molecule, wherein the first nucleic acid molecule is SEQ ID NO:25 or a functional variant thereof comprising at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:25; and wherein second nucleic acidDOCKET NO: WIST-020-PCT PCT APPLICATIONmolecule is SEQ ID NO:26 or a functional variant thereof comprising at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:26.

[0010] Some aspects of the disclosure relate to pharmaceutical compositions. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the composition of any of the one or combination of nucleic acid molecules disclosed herein and a pharmaceutically acceptable carrier.

[0011] In some embodiments, the therapeutically effective amount comprises from about 0.1 mg to about 100 mg of the first nucleic acid molecule. In some embodiments, the therapeutically effective amount comprises from about 0.1 mg to about 100 mg of the first nucleic acid molecule; and from about 0.1 mg to about 100 mg of a second nucleic acid molecule. The disclosure also relates to a method of treating or preventing a hyperproliferative disorder in a subject in need thereof comprising administering to the subject the pharmaceutical composition disclosed herein. In some embodiments, the hyperproliferative disorder is a T cell lymphoma. In some embodiments, the T cell lymphoma is a peripheral T cell lymphoma. In some embodiments, the step of administering is repeated at least two times over the course of 3, 4, 5, 6, 7, or 8 weeks. In some embodiments, the method comprises a composition comprising at least one nucleic acid molecule comprising a nucleic acid sequence encoding one or more subunits of IL- 12 or a functional variant thereof. Some embodiments relate to methods of treating or preventing cancer in a subject in need thereof further comprising administering one or more chemotherapeutic agents in and one or more disclosed nucleic acid molecules. In some embodiments, the step of administering is performed orally, intravenously, sub-cutaneously, intramuscularly, intravaginally, intradermally, or intraperitoneally. In some embodiments, at least a first nucleic acid molecule (an optionally a second) comprise one or more sequences identified in Table 3 or functional variants thereof that comprise at least about 75% sequence identity to the sequences identified in Table 3.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The following detailed description of embodiments of the present invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings certain embodiments. It is understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown. In the drawings:DOCKET NO: WIST-020-PCT PCT APPLICATION

[0013] FIGS. 1A through II illustrate TCRfullvax vaccine design and characterization. A) TCRfullvax design representing order in which each TCR chain was included. B) Schematic representation of immunization schedule for TCRfullvax immunogenicity characterization. C)Mean IFNy spots generated against TCRa, TCRP and TCRy chains by the TCRfullvax. D) IFNy spots generated against each peptide pool derived from the TCRa chain. E) IFNy spots generated against each peptide pool derived from the TCRP chain. F) IFNy spots generated against each peptide pool derived from the TCRy chain. G) Representative images showing IFNy spots against the most immunogenic peptide pools derived from the TCRa chain. H) Representative images showing IFNy spots against the most immunogenic peptide pools derived from the TCR chain. I) Representative images showing IFNy spots against the most immunogenic peptide pools derived from the TCRy chain.

[0014] FIGS. 2A through 21 illustrate TCRfullvax controls EL4 tumor growth. A) Schematic of tumor challenge testing efficacy of TCRfullvax with EL4 cells in vivo. B) Mean EL4 tumor sizes in mice treated with TCRfullvax or empty vector control pVax. C) Tumor volume v. Days PTI. D) Survival of EL4 tumor bearing mice demonstrating statistically significant improvement in survival of mice treated with TCRfullvax. E) %TCRp+ TCRV 12+ (TCR expressing EL4 cells) in tumors of mice immunized with either pVax or TCRfullvax. F) Flow cytometry plots showing TCRP+ TCRV 12+ double positive cells in tumors of mice immunized with either pVax or TCRfiillvax.

[0015] FIGS. 2G through K illustrate TCRfullvax treated tumors have greater CD8+ T cell infiltration. G) CD8+ T cell infiltration in tumors of mice immunized with either pVax or TCRfullvax. H) %PD1 expression on CD8+ T cells in tumors of mice immunized with either pVax or TCRfullvax. I) %KLRG1 expression on CD8+ T cells in tumors of mice immunized with either pVax or TCRfullvax. J) PD 1 MFI on CD8+ T cells in tumors of mice immunized with either pVax or TCRfullvax. K) KLRG 1 MFI on CD8+ T cells in tumors of mice immunized with either pVax or TCRfullvax.

[0016] FIGS. 3 A through 3H illustrate EL4neovax design and characterization. A) Bar graphs representing number of single nucleotide variants (SNVs) and Frameshift (FS) mutations which were encoded into EL4neovax. B) Bar graphs representing breakdown of neoantigens based on predicted binding affinity to Class I MHC. C) Schematic of EL4neo vaccine design. D)Mean IFNy spots generated against 5 immunogenic neoantigens by EL4neovax. E) Breakdown of percentage of neoantigens that were immunogenic vs non-immunogenic. F) Representative images showing IFNy spots generated against the 5 immunogenic neoantigens in EL4neovax. G) Bar graphs indicating binding affinity of the 5 immunogenic neoantigens in EL4neovax. H) Bar graphs indicating whether each individual epitope elicited CD4+, CD8+ T cell response or both.DOCKET NO: WIST-020-PCT PCT APPLICATION

[0017] FIGS. 4A through 4D illustrate that EL4neo vaccine controls EL4 tumors in mice. A) Schematic of tumor challenge testing efficacy of EL4neovax with EL4 cells in vivo. B) Mean EL4 tumor sizes in mice treated with EL4neovax or empty vector control pVax. C) Tumor size of each individual mouse treated with either EL4neovax or pVax. D) Survival of EL4 tumor bearing mice demonstrating improvement in survival of mice treated with EL4neovax.

[0018] FIGS. 5 A through 5D illustrate a synergistic effect of TCRfull and EL4neovax. A) Schematic of tumor challenge testing synergy of TCRfullvax and EL4neovax for controlling EL4 cells in vivo. B) Mean EL4 tumor sizes in mice treated with TCRfullvax, EL4neovax, both vaccines co-delivered or empty vector control pVax. C) Tumor size of each individual mouse treated with either TCRfullvax, EL4neovax, both vaccines co-delivered or pVax. D) Survival of EL4 tumor bearing mice demonstrating improved survival of mice treated with combination therapy compared to either vaccine alone.

[0019] FIGS 6A through 6D illustrate IFNy Responses elicited by TCRfull vaccine against the A)TCRa B) TCRp and C)TCRy chains. Bar graphs represent mean+ SEM of total of all pools added up.

[0020] FIGS. 6A through 6C illustrate IFNy SFU / million spleenocytes for naive versus TCFfull for A) TCRa, B) TCR , and C) TCRy.

[0021] FIGS. 7A and 7B illustrate safety of targeting TCR using the TCRfull vaccine. A) Mean number of splenocytes in naive, mice vs mice vaccinated with TCRfull. B) Percentage of Live CD3+ T cells amongst splenocytes in naive mice vs mice vaainated with TCRfull (n=5 mice / group).

[0022] FIGS. 8A through 8C illsutrate flow cytometry analysis demonstrating CD4+ and CD8+ T cell responses elicited by TCRfullvax.

[0023] FIGS. 9A through 9H illustrate A) EL4 tumor size of each mouse treated with either pvax or TCRfullvax. B) Proportion of intratumoralCD8+ T cells that are KLRG1-PD1+, KLRG1-PD1-, KLRG1+ PD1+ or KLRG1+ PD1- in pvax vs TCRfullvax treatedmice. C) Comparison of KLRG1+ PD1+, KLRG1-PD1+, KLRG1-PD1- and KLRG1+ PD1- intratumoral CD8+ T cells in pvax vsTCRfullvax treated mice. D) Ratio KLRG1+ PD1+, KLRG1-PD1+, KLRG1-PD1- and KLRG1+ PD1- intratumoral CD8+ T cells inpvax vs TCRfullvax treated mice of E)PD1 MFI on intra-tumoral CD8+ Tcm cells. F) PD1 MFI on intra-tumoral CD8+ Teffcells. G) KLRG1 MFI on intra-tumoral CD8+ Tcm cells. H) KLRG1 MFI on intra-tumoral CD8+ Teff cells.

[0024] FIG. 10 illustrates a gating strategy for TCR Vpi2 expression in EL4 tumors.

[0025] FIG. 11 illustrates decreased expression of TCRVP12 in response to TCRfullvax.DOCKET NO: WIST-020-PCT PCT APPLICATION

[0026] FIGS. 12A through 12C illustrate expression of A) IFNy, B) TNFa and C) IL2 from CD8+ and CD4+ T cells generated by EL4neos vaccine.

[0027] FIG 13 illustrates EL4 sequencing and vaccine design.

[0028] FIG 13 illustrates TCRfull generates robust immune response against TCRa and TCRp chains.

[0029] FIG 13 illustrates TCRfull generates robust immune response against TCRy chain.

[0030] FIG 13 illustrates Immune response against TCRa is predominantly CD4+ T cell driven.

[0031] FIG 13 illustrates Immune response against TCRy is predominantly CD4+ T cell.

[0032] FIG 13 illustrates 5 / 15 Neoantigens in EL4neos generate strong immune responseNaiveE14.

[0033] FIG 13 illustrates TCRfull and EL4 neos control growth of EL4 tumors, codelivery of both vaccines is better than single treatment.

[0034] FIG. 13: EL4 sequencing and vaccine design.

[0035] FIGS. 14A and 14B: TCRfull generates robust immune response against TCRa and TCRP chains. FIG. 14A discloses SEQ ID NO: 1. FIG. 14B discloses SEQ ID NOS 2-7, respectively, in order of appearance.

[0036] FIG. 15: TCRfull generates robust immune response against TCRy chain. FIG. 15 discloses SEQ ID NOS 8 and 9, respectively, in order of appearance.

[0037] FIGS. 16A-16F: Immune response against TCRa is predominantly CD4+ T cell driven.

[0038] FIGS. 17A-17F: Immune response against TCRy is predominantly CD4+ T cell.

[0039] FIGS. 18A-18F: 5 / 15 Neoantigens in EL4neos generate strong immune responseNaiveE14.

[0040] FIGS. 19A-19F: TCRfull and EL4 neos control growth of EL4 tumors, codelivery of both vaccines is better than single treatment.DETAILED DESCRIPTION

[0041] The present disclosure relates to strategies for the treatment of cancer, by administering a therapeutically effective amount of a pharmaceutical composition (e g., a TCR vaccine and / or a cancer vaccine) comprising one or more TCR antigens and / or a plurality of tumor specific neoantigens to a subject.DOCKET NO: WIST-020-PCT PCT APPLICATIONDefinitions

[0042] It must be noted that as used herein and in the appended claims, the singular forms “a”, “an,” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to “a nucleic acid sequence” includes a plurality of nucleotides that are formed, reference to “the nucleic acid sequence” is a reference to one or more nucleic acid sequences and equivalents thereof known to those skilled in the art, and so forth.

[0043] Ranges may be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, also specifically contemplated and considered disclosed is the range from the one particular value and / or to the other particular value unless the context specifically indicates otherwise. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another, specifically contemplated embodiment that should be considered disclosed unless the context specifically indicates otherwise. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint unless the context specifically indicates otherwise. The term “about” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0044] As used herein, the terms “activate,” “stimulate,” “enhance” “increase” and / or “induce” (and like terms) are used interchangeably to generally refer to the act of improving or increasing, either directly or indirectly, a concentration, level, function, activity, or behavior relative to the natural, expected, or average, or relative to a control condition. “Activate” in context of an immunotherapy refers to a primary response induced by ligation of a cell surface moiety. For example, in the context of receptors, such stimulation entails the ligation of a receptor and a subsequent signal transduction event. Further, the stimulation event may activate a cell and upregulate or downregulate expression or secretion of a molecule. Thus, indirect or direct ligation of cell surface moieties, even in the absence of a direct signal transduction event, may result in the reorganization of cytoskeletal structures, or in the coalescing of cell surface moieties, each of which could serve to enhance, modify, or alter subsequent cellular responses. As used herein, the terms “activating CD8± T cells” or “CD8± T cell activation” refer to a process (e.g., a signaling event) causing or resulting in one or more cellular responses of a CD8± T cell (CTL), selectedDOCKET NO: WIST-020-PCT PCT APPLICATIONfrom: proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers. As used herein, an “activated CD8+ T cell” refers to a CD8+ T cell that has received an activating signal, and thus demonstrates one or more cellular responses, selected from proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers. Suitable assays to measure CD8+ T cell activation are known in the art and are described herein.

[0045] The term “combination therapy” as used herein is meant to refer to administration of one or more therapeutic agents in a sequential manner, that is, wherein each therapeutic agent is administered at a different time, as well as administration of these therapeutic agents, or at least two of the therapeutic agents, in a substantially simultaneous manner. Substantially simultaneous administration can be accomplished, for example, by administering to the subject a single dose having a fixed ratio of each therapeutic agent or in multiple, individual doses for each of the therapeutic agents. For example, one combination of the present disclosure may comprise a pooled sample of one or more nucleic acid molecules comprising one or a plurality of expressible nucleic acid sequences and optionally an adjuvant and / or an anti-viral agent administered at the same or different times. In some embodiments, the pharmaceutical composition of the disclosure can be formulated as a single, co-formulated pharmaceutical composition comprising one or more nucleic acid molecules comprising one or a plurality of expressible nucleic acid sequences and optionally one or more adjuvants and / or one or more anti-viral agents. As another example, a combination of the present disclosure (e.g., DNA vaccines and anti-viral agent) may be formulated as separate pharmaceutical compositions that can be administered at the same or different time. As used herein, the term “simultaneously” is meant to refer to administration of one or more agents at the same time. For example, in certain embodiments, antiviral vaccine or immunogenic composition and antiviral agents are administered simultaneously). Simultaneously includes administration contemporaneously or immediately sequentially, that is during the same period of time. In certain embodiments, the one or more agents are administered simultaneously in the same hour, or simultaneously in the same day. Sequential or substantially simultaneous administration of each therapeutic agent can be effected by any appropriate route including, but not limited to, oral routes, intravenous routes, sub-cutaneous routes, intramuscular routes, direct absorption through mucous membrane tissues (e.g., nasal, mouth, vaginal, and rectal), and ocular routes (e.g., intravitreal, intraocular, etc.). The therapeutic agents can be administered by the same route or by differentDOCKET NO: WIST-020-PCT PCT APPLICATIONroutes. For example, one component of a particular combination may be administered by intravenous injection while the other component(s) of the combination may be administered intramuscularly only. The components may be administered in any therapeutically effective sequence. A “combination” embraces groups of compounds or non-small chemical compound therapies useful as part of a combination therapy.

[0046] As used herein, “expression” refers to the process by which a polynucleotide is transcribed from a DNA template (such as into and mRNA or other RNA transcript) and / or the process by which a transcribed mRNA (or administered mRNA) is translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides may be collectively referred to as “gene product.” If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. In some embodiments, the at least first expressible nucleic acid sequence comprises only DNA nucleotides, RNA nucleotides or comprises both RNA and DNA nucleotides. In some embodiments, the at least first expressible nucleic acid consist of RNA. In some embodiments, wherein the at least first expressible nucleic acid consists of DNA. In some embodiments, the composition comprises a nucleic acid molecule comprising a first expressible nucleic acid sequence, a second nucleic acid sequence, and a third nucleic acid sequence, each of the first, second and third nucleic acid sequences encoding at least one amino acid sequence that is from about 8 to about 50 amino acids from a T cell receptor sequence.

[0047] The terms “functional fragment” means any portion of a polypeptide or nucleic acid sequence from which the respective full-length polypeptide or nucleic acid relates that is of a sufficient length and has a sufficient structure to confer a biological affect that is at least similar or substantially similar to the full-length polypeptide or nucleic acid upon which the fragment is based. In some embodiments, a functional fragment is a portion of a full-length or wild-type nucleic acid sequence that encodes any one of the nucleic acid sequences disclosed herein, and said portion encodes a polypeptide of a certain length and / or structure that is less than full-length but encodes a domain that still biologically functional as compared to the full-length or wild-type protein. In some embodiments, the functional fragment may have a reduced biological activity, about equivalent biological activity, or an enhanced biological activity as compared to the wildtype or full-length polypeptide sequence upon which the fragment is based (such wild-type or full length sequences “reference sequences” or each individually a “reference sequence”). In some embodiments, the functional fragment is derived from the sequence of an organism, such as aDOCKET NO: WIST-020-PCT PCT APPLICATIONhuman. In such embodiments, the functional fragment may retain about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% sequence identity to the wild-type human sequence upon which the sequence is derived. In some embodiments, the functional fragment may retain about 85%, 80%, 75%, 70%, 65%, or 60% sequence identity to the wild-type sequence upon which the sequence is derived. In some embodiments, the functional fragment may retain about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% sequence identity to the wild-type human sequence upon which the sequence is derived and retains no less than about 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 50% biological function of the full-length sequence upon which it is based.

[0048] By “fragment” is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or about 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more nucleotides or amino acids.

[0049] “Optional” or “optionally” means that the subsequently described event, circumstance, or material may or may not occur or be present, and that the description includes instances where the event, circumstance, or material occurs or is present and instances where it does not occur or is not present.

[0050] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to “A and / or B,” when used in conjunction with open-ended language such as “comprising” can refer, in some embodiments, to A without B (optionally including elements other than B); in another embodiments, to B without A (optionally including elements other than A); in yet another embodiments, to both A and B (optionally including other elements); etc.

[0051] As used herein in the specification and in the claims, “or” should he understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items.DOCKET NO: WIST-020-PCT PCT APPLICATIONOnly terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0052] As used herein an “antigen” is meant to refer to any substance that elicits an immune response.

[0053] As used herein, the term “protein of interest” refers to any protein. In some embodiments, the term “protein of interest” refers to any protein that can be expressed in any of the constructs disclosed herein. In some embodiments, the “protein of interest” is a viral antigen. In some embodiments, the “protein of interest” is any of the viral antigens disclosed herein. In some embodiments, the “protein of interest” is a cancer antigen. In some embodiments, the “protein of interest” is a cancer antigen disclosed herein. In some embodiments, the “protein of interest” is any of the protein antigens disclosed herein.

[0054] As used herein, the term “electroporation,” “electro-permeabilization,” or “electro-kinetic enhancement” (“EP”), are used interchangeably and are meant to refer to the use of a transmembrane electric field pulse to induce microscopic pathways (pores) in a bio-membrane; their presence allows biomolecules such as plasmids, oligonucleotides, siRNA, drugs, ions, and / or water to pass from one side of the cellular membrane to the other. In some of the disclosed methods of treatment or prevention, the method comprises a step of electroporation of a subject’s tissue for a sufficient time and with a sufficient electrical field capable of inducing uptake of the pharmaceutical compositions disclosed herein into the antigen-presenting cells. In some embodiments, the cells are antigen presenting cells.

[0055] The term “pharmaceutically acceptable excipient,” “pharmaceutically acceptable carrier” or “pharmaceutically acceptable diluent” as used herein is meant to refer to an excipient, carrier or diluent that can be administered to a subject, together with an agent or the pharmaceutical compositions disclosed herein, and which is inert or fails to eliminate the pharmacological activity of the active agent of the pharmaceutical composition. In some embodiments, the pharmaceutically acceptable carrier does fails to destroy or is incapable of eliminating the pharmacological activity of an active agent / vaccine and is nontoxic when administered in doses sufficient to deliver aDOCKET NO: WIST-020-PCT PCT APPLICATIONtherapeutic amount of the active agent. The term “pharmaceutically acceptable salt” of nucleic acids as used herein may be an acid or base salt that is generally considered in the art to be suitable for use in contact with the tissues of human beings or animals without excessive toxicity, irritation, allergic response, or other problem or complication. Such salts include mineral and organic acid salts of basic residues such as amines, as well as alkali or organic salts of acidic residues such as carboxylic acids. Specific pharmaceutical salts include, but are not limited to, salts of acids such as hydrochloric, phosphoric, hydrobromic, malic, glycolic, fumaric, sulfuric, sulfamic, suifanilic, formic, toluenesulfonie, methanesulfonic, benzene sulfonic, ethane disulfonic, 2- hydroxyethyl sulfonic, nitric, benzoic, 2-acetoxybenzoic, citric, tartaric, lactic, stearic, salicylic, glutamic, ascorbic, pamoic, succinic, fumaric, maleic, propionic, hydroxymaleic, hydroiodic, phenyiacetic, alkanoic such as acetic, H00C-(CH2)n-C00H where n is 0-4, and the like. Similarly, pharmaceutically acceptable cations include, but are not limited to sodium, potassium, calcium, aluminum, lithium and ammonium. Those of ordinary skill in the art will recognize from this disclosure and the knowledge in the art that further pharmaceutically acceptable salts for the pooled viral specific antigens or polynucleotides provided herein, including those listed by Remington’s Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, p. 1418 ( 1985). In general, a pharmaceutically acceptable acid or base salt can be synthesized from a parent compound that contains a basic or acidic moiety by any conventional chemical method. Briefly, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in an appropriate solvent.

[0056] As used herein, the terms “prevent,” “preventing,” “prevention,” “prophylactic treatment,” and the like, are meant to refer to reducing the probability of developing a disease or condition in a subject, who does not have, but is at risk of or susceptible to developing a disease or condition.

[0057] As used herein, the term “purified” means that the polynucleotide or polypeptide or fragment, variant, or derivative thereof is substantially free of other biological material with which it is naturally associated, or free from other biological materials derived, e.g., from a recombinant host cell that has been genetically engineered to express the polypeptide of the present disclosure. That is, e.g., a purified polypeptide of the present disclosure is a polypeptide that is at least from about 70 to 100% pure, i.e., the polypeptide is present in a composition wherein the polypeptide constitutes from about 70 to about 100% by weight of the total composition. In some embodiments, the purified polypeptide of the present disclosure is from about 75% to about 99%DOCKET NO: WIST-020-PCT PCT APPLICATIONby weight pure, from about 80% to about 99% by weight pure, from about 90 to about 99% by weight pure, or from about 95% to about 99% by weight pure.

[0058] As used herein, the terms “subject,” “individual,” “host,” and “patient,” are used interchangeably herein and refer to a vertebrate individual, including but not limited to a mammal or human, for whom diagnosis, treatment or therapy is desired, particularly humans. Mammals include, but are not limited to, murines, simians, humans, farm animals, cows, pigs, goats, sheep, horses, dogs, sport animals, and domesticated animals. Tissues, cells and their progeny obtained in vivo or cultured in vitro are also encompassed by the definition of the term “subject.” The methods described herein are applicable to both human therapy and veterinary applications. In some instances in the description of the present disclosure, the term “patient” refers to human patients suffering from a particular disease or disorder. In some embodiments, the subject may be a human suspected of having or being identified as at risk to develop a viral infection or having or being at risk to have a hyperproliferative disorder, such as cancer. In some embodiments, the subject may be diagnosed as having human immunodeficiency virus-1 (HIV-1) and of having or being identified as at risk to develop autoimmune deficiency syndrome or AIDS. In some embodiments, the subject is a mammal, and, in other embodiments, the subject is a human.

[0059] The term “hyperproliferative disorder” refers to a disease or disorder characterized by abnormal proliferation, abnormal growth, abnormal senescence, abnormal quiescence, or abnormal removal (or apoptosis) of cells in an organism, and includes all forms of hyperplasias, neoplasias, and cancer, malignant or benign. In some embodiments, the hyperproliferative disease is a cancer derived from the gastrointestinal tract or urinary system. In some embodiments, a hyperproliferative disease is a cancer of the adrenal gland, bladder, blood, bone, bone marrow, brain, spine, breast, cervix, gall bladder, ganglia, gastrointestinal tract, stomach, colon, heart, kidney, liver, lung, lymphatic tissue, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, or uterus. In some embodiments, the term hyperproliferative disease is a cancer chosen from: lung cancer, bone cancer, CMML, pancreatic cancer, skin cancer, cancer of the head and neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, colon cancer, breast cancer, testicular, gynecologic tumors (e.g., uterine sarcomas, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina or carcinoma of the vulva), Hodgkin's disease, cancer of the esophagus, cancer of the small intestine,DOCKET NO: WIST-020-PCT PCT APPLICATIONcancer of the endocrine system (e.g., cancer of the thyroid, parathyroid or adrenal glands), sarcomas of soft tissues, cancer of the urethra, cancer of the penis, prostate cancer, chronic or acute leukemia, solid tumors of childhood, lymphocytic lymphomas, cancer of the bladder, cancer of the kidney or ureter (e.g., renal cell carcinoma, carcinoma of the renal pelvis), or neoplasms of the central nervous system (e g., primary CNS lymphoma, spinal axis tumors, brain stem gliomas or pituitary adenomas). In some embodiments, the hyperproliferative disorder is T cell lymphoma or a solid tumor.

[0060] The term “therapeutic effect” as used herein is meant to refer to some extent of relief of one or more of the symptoms of a disorder or its associated pathology. A “therapeutically effective amount” as used herein is meant to refer to an amount of an agent which is effective, upon single or multiple dose administration (such as a first, second and / or third booster) to the cell or subject, in prolonging the survivability of the patient with such a disorder, reducing one or more signs or symptoms of the disorder, preventing or delaying, and the like beyond that expected in the absence of such treatment. A “therapeutically effective amount” is intended to qualify the amount required to achieve a therapeutic effect. A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the “therapeutically effective amount” (e.g., ED50) of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the present disclosure employed in a pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0061] The terms “treat,” “treated,” “treating,” “treatment,” and the like as used herein are meant to refer to reducing or ameliorating a disorder and / or symptoms associated therewith (e.g., a viral infection). “Treating” can refer to administration of the DNA vaccines described herein to a subject after the onset, or suspected onset, of a viral infection. “Treating” includes the concepts of “alleviating,” which refers to lessening the frequency of occurrence or recurrence, or the severity, of any symptoms or other ill effects related to a virus and / or the side effects associated with viral therapy. The term “treating” also encompasses the concept of “managing” which refers to reducing the severity of a particular disease or disorder in a patient or delaying its recurrence, e.g., lengthening the period of remission in a patient who had suffered from the disease. It is appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition, or symptoms associated therewith be completely eliminated.DOCKET NO: WIST-020-PCT PCT APPLICATION

[0062] The term “prophylactically effective amount” refers to the amount of the subject compound or composition that will elicit the biological or medical response of a tissue, system, or subject that is being sought by the researcher, veterinarian, medical doctor or other clinician in preventing a disease or disordered state. The term “prophylactically effective amount” includes that amount of a compound or composition that, when administered, is sufficient to prevent development of to some extent, one or more of the signs or symptoms of a disease or disordered state. The prophylactically effective amount will vary depending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated.

[0063] The term “therapeutically effective amount” refers to the amount of the subject compound or composition that will elicit the biological or medical response of a tissue, system, or subject that is being sought by the researcher, veterinarian, medical doctor or other clinician. The term “therapeutically effective amount” includes that amount of a compound or composition that, when administered, is sufficient to alleviate to some extent, one or more of the signs or symptoms of the disease or disordered state being treated. The therapeutically effective amount will vary depending on the compound or composition, the disease and its severity and the age, weight, etc., of the subject to be treated.

[0064] For any therapeutic agent described herein the therapeutically effective amount may be initially determined from preliminary in vitro studies and / or animal models. A therapeutically effective dose may also be determined from human data. The applied dose can be adjusted based on the relative bioavailability and potency of the administered agent. Adjusting the dose to achieve maximal efficacy based on the methods described above and other well-known methods is within the capabilities of the ordinarily skilled artisan. General principles for determining therapeutic effectiveness, which may be found in Chapter 1 of Goodman and Gilman’s The Pharmacological Basis of Therapeutics, 10th Edition, McGraw-Hill (New York) (2001), incorporated herein by reference, are summarized below. Pharmacokinetic principles provide a basis for modifying a dosage regimen to obtain a desired degree of therapeutic efficacy with a minimum of unacceptable adverse effects. In situations where the drug’s plasma concentration can be measured and related to the therapeutic window, additional guidance for dosage modification can be obtained. Drug products are considered to be pharmaceutical equivalents if they contain the same active ingredients and are identical in strength or concentration, dosage form, and route of administration. Two pharmaceutically equivalent drug products are considered to be bioequivalent when the ratesDOCKET NO: WIST-020-PCT PCT APPLICATIONand extents of bioavailability of the active ingredient in the two products are not significantly different under suitable test conditions.

[0065] The terms “polynucleotide,” “oligonucleotide” and “nucleic acid” are used interchangeably throughout and include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs (e.g., peptide nucleic acids and non-naturally occurring nucleotide analogs), and hybrids thereof. Thus, ther term “expressible nucleic acid” or “expressible nucleic acid sequence” as used herein refers to expressible DNA or RNA molecules or expressible DNA or RNA sequences.

[0066] The nucleic acid molecule and / or sequences of each embodiment can be single-stranded or double-stranded. In some embodiments, the nucleic acid molecules of the disclosure comprise a contiguous open reading frame encoding an antibody, or a fragment thereof, as described herein. “Nucleic acid” or “oligonucleotide” or “polynucleotide” as used herein may mean at least two nucleotides covalently linked together. The depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of a depicted single strand. Many variants of a nucleic acid may he used for the same purpose as a given nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and complements thereof. A single strand provides a probe that may hybridize to a target sequence under stringent hybridization conditions. Thus, a nucleic acid also encompasses a probe that hybridizes under stringent hybridization conditions. Nucleic acids may be single stranded or double stranded, or may contain portions of both double stranded and single stranded sequence. The nucleic acid may be DNA, both genomic and cDNA, RNA, or a hybrid, where the nucleic acid may contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine. Nucleic acids may be obtained by chemical synthesis methods or by recombinant methods. A nucleic acid will generally contain phosphodiester bonds, although nucleic acid analogs maybe included that may have at least one different linkage, e.g., phosphoramidate, phosphorothioate, phosphorodi thioate, or o-methylphosphoroamidite linkages and peptide nucleic acid backbones and linkages. Other analog nucleic acids include those with positive backbones; non-ionic backbones, and non-ribose backbones, including those described in U.S. Pat. Nos.5,235,033 and 5,034,506, which are incorporated by reference in their entireties. Nucleic acids containing one or more non-naturally occurring or modified nucleotides are also included withinDOCKET NO: WIST-020-PCT PCT APPLICATIONone definition of nucleic acids. The modified nucleotide analog may he located for example at the 5 ’-end and / or the 3 ’-end of the nucleic acid molecule. Representative examples of nucleotide analogs may be selected from sugar- or backbone-modified ribonucleotides. It should be noted, however, that also nucleobase-modified ribonucleotides, i.e. ribonucleotides, containing a non-naturally occurring nucleobase instead of a naturally occurring nucleobase such as uridines or cytidines modified at the 5-position, e.g. 5-(2-amino)propyl uridine, 5-bromo uridine; adenosines and guanosines modified at the 8-position, e.g. 8-bromo guanosine; deaza nucleotides, e.g. 7-deaza-adenosine; 0- and N-alkylated nucleotides, e g. N6-methyl adenosine are suitable. The 2’-OH-group may be replaced by a group selected from H, OR, R, halo, SH, SR, NH2, NHR, N2 or CN, wherein R is Ci-Ce alkyl, alkenyl or alkynyl and halo is F, Cl, Br or I. Modified nucleotides also include nucleotides conjugated with cholesterol through, e.g., a hydroxyprolinol linkage as described in Krutzfeldt et al., Nature (Oct. 30, 2005), Soutschek et al., Nature 432: 173-178 (2004), and U.S. Patent Publication No. 20050107325, which are incorporated herein by reference in their entireties. Modified nucleotides and nucleic acids may also include locked nucleic acids (LNA), as described in U.S. Patent No. 20020115080, which is incorporated herein by reference. Additional modified nucleotides and nucleic acids are described in U.S. Patent Publication No.20050182005, which is incorporated herein by reference in its entirety. Modifications of the ribose-phosphate backbone may be done for a variety of reasons, e.g., to increase the stability and half-life of such molecules in physiological environments, to enhance diffusion across cell membranes, or as probes on a biochip. Mixtures of naturally occurring nucleic acids and analogs may be made; alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made. In some embodiments, the expressible nucleic acid sequence is in the form of DNA. In some embodiments, the expressible nucleic acid is in the form of RNA with a sequence that encodes the polypeptide sequences disclosed herein and, in some embodiments, the expressible nucleic acid sequence is an RNA / DNA hybrid molecule that encodes any one or plurality of polypeptide sequences disclosed herein.

[0067] As used herein, the term “nucleic acid molecule” is a molecule that comprises one or more nucleotide sequences that encode one or more proteins. In some embodiments, a nucleic acid molecule comprises initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of the individual to whom the nucleic acid molecule is administered. In some embodiments, the nucleicDOCKET NO: WIST-020-PCT PCT APPLICATIONacid molecule also includes a plasmid containing one or more nucleotide sequences that encode one or a plurality of tumor antigens, neoantigens, or malignant cancer antigens, of the blood or solid tumor. In some embodiments, the disclosure relates to a pharmaceutical composition comprising a first, second, third or more nucleic acid molecule, each of which encoding one or a plurality of antigens from a tissue exhibiting a hyperproliferative disorder and at least one of each plasmid comprising one or more of the compositions disclosed herein. In some embodiments, the compositions can comprise a nucleic acid molecule that comprises a first, second, third or more expressible nucleic acid sequences, wherein at least one of the first, second or third expressible nucleic acid sequences comprise the domains disclosed herein. In some embodiments, one, two or each of the first, second or third expressible nucleic acid sequences comprises one or a plurality of neoantigens. In some embodiments, the neoantigens are from a T cell lymphoma, B cell lymphoma or solid tumor. In some embodiments, methods of the disclosure comprise a step of identifying one or a plurality of neoantigens from a subject and then administering a therapeutically effective amount of a vaccine encoding one or the plurality of neoantigens from the subject to the subject.

[0068] The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-natural amino acids or chemical groups that are not amino acids. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. As used herein the term “amino acid” includes natural and / or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics.

[0069] The “percent identity” or “percent homology” of two polynucleotide or two polypeptide sequences is determined by comparing the sequences using the GAP computer program (a part of the GCG Wisconsin Package, version 10.3 (Accelrys, San Diego, Calif.)) using its default parameters. “Identical” or “identity” as used herein in the context of two or more nucleic acids or amino acid sequences, may mean that the sequences have a specified percentage of residues that are the same over a specified region. The percentage may be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs in both sequences to yield the number of matchedDOCKET NO: WIST-020-PCT PCT APPLICATIONpositions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity. In cases where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of single sequence are included in the denominator but not the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) may be considered equivalent. Identity may he performed manually or by using a computer sequence algorithm such as BLAST or BLAST 2.0. Briefly, the BLAST algorithm, which stands for Basic Local Alignment Search Tool is suitable for determining sequence similarity. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (ncbi.nlm.nih.gov). This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length Win the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension for the word hits in each direction are halted when: 1) the cumulative alignment score falls off by the quantity X from its maximum achieved value; 2) the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or 3) the end of either sequence is reached. The Blast algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The Blast program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 10915-10919, which is incorporated herein by reference in its entirety) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands. The BLAST algorithm (Karlin et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 5873-5787, which is incorporated herein by reference in its entirety) and Gapped BLAST perform a statistical analysis of the similarity between two sequences. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide sequences would occur by chance. For example, a nucleic acid is considered similar to another if the smallest sum probability in comparison of the test nucleic acid to the other nucleic acid is less than about 1, less than about 0.1, less than about 0.01, and less than about 0.001. TwoDOCKET NO: WIST-020-PCT PCT APPLICATIONsingle-stranded polynucleotides are “the complement” of each other if their sequences can be aligned in an anti-parallel orientation such that every nucleotide in one polynucleotide is opposite its complementary nucleotide in the other polynucleotide, without the introduction of gaps, and without unpaired nucleotides at the 5’ or the 3’ end of either sequence. A polynucleotide is “complementary” to another polynucleotide if the two polynucleotides can hybridize to one another under moderately stringent conditions. Thus, a polynucleotide can be complementary to another polynucleotide without being its complement.

[0070] The term “hybridization” or “hybridizes” as used herein refers to the formation of a duplex between nucleotide sequences that are sufficiently complementary to form duplexes via Watson-Crick base pairing. Two nucleotide sequences are “complementary” to one another when those molecules share base pair organization homology. “Complementary” nucleotide sequences will combine with specificity to form a stable duplex under appropriate hybridization conditions. For instance, two sequences are complementary when a section of a first sequence can bind to a section of a second sequence in an anti-parallel sense wherein the 3 ’-end of each sequence binds to the 5’-end of the other sequence and each A, T(U), G and C of one sequence is then aligned with a T(U), A, C and G, respectively, of the other sequence. RNA sequences can also include complementary G=U or U=G base pairs. Thus, two sequences need not have perfect homology to be “complementary.” Usually two sequences are sufficiently complementary when at least about 90% (preferably at least about 95%) of the nucleotides share base pair organization over a defined length of the molecule.

[0071] By “substantially identical” is meant nucleic acid molecule (or polypeptide) exhibiting at least about 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least about 60%, more preferably about 80% or 85%, and more preferably about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even 99% identical at the amino acid level or nucleic acid level to the sequence used for comparison.

[0072] A nucleotide sequence is “operably linked” to a regulatory sequence if the regulatory sequence affects the expression (e.g., the level, timing, or location of expression) of the nucleotide sequence. A “regulatory sequence” is a nucleic acid that affects the expression (e.g., the level, timing, or location of expression) of a nucleic acid to which it is operably linked. The regulatoryDOCKET NO: WIST-020-PCT PCT APPLICATIONsequence can, for example, exert its effects directly on the regulated nucleic acid, or through the action of one or more other molecules (e.g., polypeptides that bind to the regulatory sequence and / or the nucleic acid). Examples of regulatory sequences include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Further examples of regulatory sequences are described in, for example, Goeddel, 1990, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif, and Baron et al., 1995, Nucleic Acids Res.23:3605-06.

[0073] A “vector” is a nucleic acid that can be used to introduce another nucleic acid linked to it into a cell. One type of vector is a “plasmid,” which refers to a linear or circular double stranded DNA molecule into which additional nucleic acid segments can be ligated. Another type of vector is a viral vector (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), comprising additional, exogenous DNA, RNA or hybrid DNA or RNA molecules that can be introduced into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors comprising a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. An “expression vector” is a type of vector that can direct the expression of a chosen polynucleotide. The disclosure relates to any one or plurality of vectors that comprise nucleic acid sequences encoding any one or plurality of amino acid sequence disclosed herein. In some embodiments, the expression vector includes from about 30 to about 100,000 nucleotides (e.g., from about 30 to about 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 1,000, from 30 to 1,500, from 30 to 3,000, from 30 to 5,000, from 30 to 7,000, from 30 to 10,000, from 30 to 25,000, from 30 to 50,000, from 30 to 70,000, from 100 to 250, from 100 to 500, from 100 to 1,000, from 100 to 1,500, from 100 to 3,000, from 100 to 5,000, from 100 to 7,000, from 100 to 10,000, from 100 to 25,000, from 100 to 50,000, from 100 to 70,000, from 100 to 100,000, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 3,000, from 500 to 5,000, from 500 to 7,000, from 500 to 10,000, from 500 to 25,000, from 500 to 50,000, from 500 to 70,000, from 500 to 100,000, from 1,000 to 1,500, from 1,000 to 2,000, from about 1,000 to about 3,000, from 1,000 to 5,000, from 1,000 to 7,000, from 1,000 to 10,000, from 1,000 to 25,000, from 1,000 to 50,000, from 1,000 to 70,000, from 1,000 to 100,000, from 1,500 to 3,000, from 1,500 to 5,000, from 1,500 to 7,000, from 1,500 to 10,000, from 1,500 toDOCKET NO: WIST-020-PCT PCT APPLICATION25,000, from 1,500 to 50,000, from 1,500 to 70,000, from 1,500 to 100,000, from 2,000 to 3,000, from 2,000 to 5,000, from 2,000 to 7,000, from 2,000 to 10,000, from 2,000 to 25,000, from 2,000 to 50,000, from about 2,000 to about 70,000, and from 2,000 to 100,000).

[0074] The term “vaccine” as used herein is meant to refer to a composition for generating immunity for the prophylaxis and / or treatment of diseases (e.g., viral infections). Accordingly, vaccines are medicaments which comprise antigens in protien and / or nucleic acid forms and are in animals for generating specific defense and protective substance by vaccination. A “vaccine composition” or a “DNA vaccine composition” can include a pharmaceutically acceptable excipient, carrier or diluent. A “DNA vaccine composition” as used herein can comprise a DNA vaccine, a RNA vaccine or a combination thereof.

[0075] “Variants” are intended to mean substantially similar sequences. For nucleic acid molecules, a variant comprises a nucleic acid molecule having deletions (i.e., truncations) at the 5’ and / or 3’ end; deletion and / or addition of one or more nucleotides at one or more internal sites in the native polynucleotide; and / or substitution of one or more nucleotides at one or more sites in the native polynucleotide. As used herein, a “native” nucleic acid molecule or polypeptide comprises a naturally occurring or endogenous nucleotide sequence or amino acid sequence, respectively. For nucleic acid molecules, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of one of the polypeptides of the disclosure. Variant nucleic acid molecules also include synthetically derived nucleic acid molecules, such as those generated, for example, by using site-directed mutagenesis but which still encode a protein of the disclosure. Generally, variants of a particular nucleic acid molecule of the disclosure will have at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to that particular polynucleotide as determined by sequence alignment programs and parameters as described elsewhere herein. Variants of a particular nucleic acid molecule of the disclosure (i.e., the reference DNA sequence) can also be evaluated by comparison of the percent sequence identity between the polypeptide encoded by a variant nucleic acid molecule and the polypeptide encoded by the reference nucleic acid molecule. Percent sequence identity between any two polypeptides can be calculated using sequence alignment programs and parameters described elsewhere herein. Where any given pair of nucleic acid molecule of the disclosure is evaluated by comparison of the percent sequence identity shared by the two polypeptides that they encode, the percent sequence identity betweenDOCKET NO: WIST-020-PCT PCT APPLICATIONthe two encoded polypeptides is at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity. In some embodiments, the term “variant” protein is intended to mean a protein derived from the native protein by deletion (so-called truncation) of one or more amino acids at the N-terminal and / or C-terminal end of the native protein; deletion and / or addition of one or more amino acids at one or more internal sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein. Variant proteins encompassed by the present disclosure are biologically active, that is they continue to possess the desired biological activity of the native protein as described herein. Such variants may result from, for example, genetic polymorphism or from human manipulation. Biologically active variants of a protein of the disclosure will have at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence for the native protein as determined by sequence alignment programs and parameters described elsewhere herein. A biologically active variant of a protein of the disclosure may differ from that protein by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue. The proteins or polypeptides of the disclosure may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants and fragments of the proteins can be prepared by mutations in the nucleic acid sequence that encode the amino acid sequence recombinantly.

[0076] Finally, it should be understood that all of the individual values and sub-ranges of values contained within an explicitly disclosed range are also specifically contemplated and should be considered disclosed unless the context specifically indicates otherwise. The foregoing applies regardless of whether in particular cases some or all of these embodiments are explicitly disclosed.

[0077] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed method and compositions belong. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present method and compositions, the particularly useful methods, devices, and materials are as described. Publications cited herein and the material for which they are cited are hereby specifically incorporated by reference. Nothing herein is to be construed as an admission that the present disclosure is not entitled to antedate such disclosure by virtue of prior disclosure. No admission is made that any reference constitutes priorDOCKET NO: WIST-020-PCT PCT APPLICATIONart. The discussion of references states what their authors assert, and applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of publications are referred to herein, such reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

[0078] Throughout the description and claims of this specification, the word “comprise” and variations of the word, such as “comprising” and “comprises,” means “including but not limited to,” and is not intended to exclude, for example, other additives, components, integers or steps. In particular, in methods stated as comprising one or more steps or operations it is specifically contemplated that each step comprises what is listed (unless that step includes a limiting term such as “consisting of’), meaning that each step is not intended to exclude, for example, other additives, components, integers or steps that are not listed in the step.

[0079] The term “neoantigen” as used herein is meant to refer to a class of cancer antigens which arises from cancer cell-specific mutations in expressed protein of a subject. In some embodiments, the neoantigen is derived directly from a cancer cell of a subject. In some embodiments, the neoantigen is a known cancer cell associated antigen which may be a consensus sequence known to elicit an immune response against a cell expressing the cancer cell antigen but not necessarily expressed by a cancer cell of the subject.Nucleic Acid CompositionsTCR

[0080] In some embodiments, the disclosure relates to one or a plurality of first nucleic acid sequences, each encoding a TCRP antigen. In some embodiments, the TCRP antigen amino acid sequence encoded by each first nucleic acid sequence is the identical. In some embodiments, the TCRP antigen amino acid sequence encoded by each first nucleic acid sequence is independently selected for each first nucleic acid sequence.

[0081] In some embodiments, the disclosure relates to one or a plurality of second nucleic acid sequences, each encoding a TCRa antigen. In some embodiments, the TCRa antigen amino acid sequence encoded by each second nucleic acid sequence is the identical. In some embodiments, the TCRa antigen amino acid sequence encoded by each second nucleic acid sequence is independently selected for each first nucleic acid sequence.DOCKET NO: WIST-020-PCT PCT APPLICATION

[0082] In some embodiments, the disclosure relates to one or a plurality of third nucleic acid sequences, each encoding a TCRy antigen. In some embodiments, the TCRy antigen amino acid sequence encoded by each third nucleic acid sequence is the identical. In some embodiments, the TCRy antigen amino acid sequence encoded by each third nucleic acid sequence is independently selected for each first nucleic acid sequence.

[0083] In some embodiments, the disclosure relates to one or a plurality of fourth nucleic acid sequences, each encoding a linker sequence. In some embodiments, the linker amino acid sequence encoded by each fourth nucleic acid sequence is the identical. In some embodiments, the linker amino acid sequence encoded by each fourth nucleic acid sequence is independently selected for each fourth nucleic acid sequence. In some embodiments, the linker is a cleavable linker. In some embodiments, the cleavable linker is a furin linker.

[0084] In some embodiments, the disclosure relates to fifth nucleic acid sequence encoding a leader sequence. In some embodiments, the leader sequence is an IgE leader sequence.

[0085] In some embodiments, the disclosure relates to a nucleic acid molecule comprising at least one of the one or a plurality of first nucleic acid sequences, the one or a plurality of second nucleic acid sequences, one or a plurality of third nucleic acid sequences, the one or a plurality of fourth nucleic acid sequences, and the fifth nucleic acid sequence. In some embodiments, the one or a plurality of first nucleic acid sequences, the one or a plurality of second nucleic acid sequences, one or a plurality of third nucleic acid sequences, the one or a plurality of fourth nucleic acid sequences are arranged contiguously with one of the fourth nucleic acid sequences separating each of the first, second, and third nucleic acid sequences. The first, second, and third nucleic acid sequences, in some embodiments, appear in any order despite the use of “first,” “second,” and “third.” For example, the arrangement of first, second, third, and fourth nucleic sequences, in some embodiments, may be third, fourth, second, fourth, third, fourth, first, fourth, first, fourth, second fourth, and third. In some embodiments, the arrangement is first, fourth, second, fourth, third. These examples of arrangement are non-limiting. In some embodiments, the fifth nucleic acid sequence may precede all of the first, second, third, and fourth nucleic acid sequence.

[0086] In some embodiments, at least a portion of the one or more first nucleic acid sequences encode an amino acid sequence comprising an amino acid sequence independently selected from SEQ ID NOS: 2 through 7, or a functional variant thereof having at least about 70%, 80%, 85%, 90% , 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 2 through 7. In someDOCKET NO: WIST-020-PCT PCT APPLICATIONembodiments, at least a portion of the one or more second nucleic acid sequences each encode an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 1 or a functional variant thereof having at least about 70%, 80%, 85%, 90% , 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1. In some embodiments, at least a portion of the one or more third nucleic acid sequences encode an amino acid sequence comprising an amino acid sequence independently selected from SEQ ID NOS: 8 and 9 or a functional variant thereof having at least 70%, 80%, 85%, 90% , 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 8 or 9.

[0087] In some embodiments, at least a portion of the one or more first nucleic acid sequences encode an amino acid sequence comprising an independently selected antigenic fragment of TCR0. In some embodiments, the antigenic fragment is about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 amino acids in length. In some embodiments, the antigenic fragment comprises an amino acid sequence comprising at least a portion of or all of one of SEQ ID NOS: 21 or 22. In some embodiments, the portion has a length selected from any of the foregoing lengths. In some embodiments, the antigenic fragment comprises an amino acid sequence comprising at least about 70%, 80%, 85%, 90% , 95%, 96%, 97%, 98% or 99% sequence identity to at least a portion of or all of SEQ ID NOS: 21 or 22. In some embodiments, the antigenic fragment comprises an amino acid sequence comprising at least about 70%, 80%, 85%, 90% , 95%, 96%, 97%, 98% or 99% sequence identity to one of the foregoing lengths of amino acid residues within one SEQ ID NOS: 21 or 22.

[0088] In some embodiments, at least a portion of the one or more second nucleic acid sequences each encode an amino acid sequence comprising an independently selected antigenic fragment of TCRa. In some embodiments, the antigenic fragment is about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 amino acids in length. In some embodiments, the antigenic fragment comprises an amino acid sequence comprising at least a portion of or all of one of SEQ ID NOS: 19 or 20. Tin some embodiments, the portion has a length selected from any of the foregoing lengths. In some embodiments, the antigenic fragment comprises an amino acid sequence comprising at least about 70%, 80%, 85%, 90% , 95%, 96%, 97%, 98% or 99% sequence identity to at least a portion of orDOCKET NO: WIST-020-PCT PCT APPLICATIONall of SEQ ID NOS: 19 or 20. In some embodiments, the antigenic fragment comprises an amino acid sequence comprising at least about 70%, 80%, 85%, 90% , 95%, 96%, 97%, 98% or 99% sequence identity to one of the foregoing lengths of amino acid residues within one SEQ ID NOS: 19 or 20.

[0089] In some embodiments, at least a portion of the one or more third nucleic acid sequences each encode any amino acid sequence comprising an independently selected antigenic fragment of TCRy. In some embodiments, the antigenic fragment is about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 amino acids in length. In some embodiments, the antigenic fragment comprises an amino acid sequence comprising at least a portion of or all of one of SEQ ID NOS: 23 or 24. In some embodiments, the portion has a length selected from any of the foregoing lengths. In some embodiments, the antigenic fragment comprises an amino acid sequence comprising at least about 70%, 80%, 85%, 90% , 95%, 96%, 97%, 98% or 99% sequence identity to at least a portion of or all of SEQ ID NOS: 23 or 24. In some embodiments, the antigenic fragment comprises an amino acid sequence comprising at least about 70%, 80%, 85%, 90% , 95%, 96%, 97%, 98% or 99% sequence identity to one of the foregoing lengths of amino acid residues within one SEQ ID NOS: 23 or 24.

[0090] In some embodiments, the disclosure relates to a nucleic acid sequence comprising SEQ ID NO: 25 or a variant thereof comprising at least about 70%, 80%, 85%, 90% , 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 25. In some embodiments a nucleic acid sequence comprising SEQ ID NO: 25 or a variant thereof comprising at least about 70%, 80%, 85%, 90% , 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 25 comprises the first, second, third, fourth, and fifth nucleic acid sequences. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence comprising SEQ ID NO: 25 or a variant thereof comprising at least about 70%, 80%, 85%, 90% , 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 25.EL4TCRFULL GCTGCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATT AATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATDOCKET NO: WIST-020-PCT PCT APPLICATIONAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGT CAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGG GTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAA GTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTA CATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTAC CATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGG GGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATC AACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTA GGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCA CTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTA GCGTTTAAACTTAAGCTTGGTACCGAGCTCGGATCCACTAGTCCAGTGTGGTGGAATT CGCCACCATGGATTGGACTTGGATTCTCTTTCTCGTTGCAGCTGCTACACGCGTTCAC AGTATGGGAATCCAGACCCTGTGTTGTGTGATCTTTTATGTGCTGATCGCAAATCACA CCGATGCCGGCGTGACACAGACACCCAGACACGAGGTGGCCGAGAAGGGACAGAC AATCATCCTGAAGTGCGAGCCCGTGTCCGGCCACAACGACCTGTTCTGGTATAGGCA GACAAAGATCCAGGGCCTGGAGCTGCTGAGCTATTTCAGAAGCAAGAGCCTGATGGA GGACGGCGGCGCCTTCAAGGATAGGTTTAAGGCCGAGATGCTGAATTCTAGCTTTTCC ACACTGAAGATCCAGCCAACAGAGCCTAAGGATAGCGCCGTGTACCTGTGCGCCTCT TCCACCGGCACCGAGACACTGTACTTTGGAAGCGGGACCAGACTGACCGTCCTGCG GGGACGCAAAAGGAGGTCCATGAAGAGACTGCTGTGCAGCCTGCTGGGCCTGCTGT GTACCCAGGTGTGCTGGCTGAAGGAGCAGCAGGTGCAGCAGTCCCCAGCATCCCTG GTGCTGCAGGAGGCCGAGAACGCCGAGCTGCAGTGCAGCTTCTCTATCTTCACCAAC CAGGTGCAGTGGTTTTACCAGCGCCCCGGCGGACGCCTGGTGTCTCTGCTGTACAAT CCAAGCGGCACCAAGCAGTCTGGCAGGCTGACATCCACCACCGTGATCAAGGAGAG ACGGTCTAGCCTGCACATCAGCTCCAGCCAGATCACCGATTCCGGGACATACCTGTGT GCAATGGACCTGCCTCTGATGAACACAGAGGGCGCCGATAGACTGACCTTTGGAAAA GGCACCCAGCTGATCATCCAGCCCAGGGGCCGCAAGAGAAGATCCATGCTGCTGCTG AGGATGTTCACCAGCTGTTGTTGCCTGTGGGTGTGGCTGGGCCAGCTGGAGCAGACC GAGCTGTCCGTGACACGGGCCACCGACGAGTCTGCCCAGATCTCTTGCATCGTGTCC CTGCCTTGCTTCAGCAACACAGCCATCCACTGGTATCGGCAGAAGCCTAACCAGCAG TTCGAGTATCTGATCTATGTGGAGACAAACTATAATCAGCAGCCACTGGGCGGCAAAADOCKET NO: WIST-020-PCT PCT APPLICATIONATAAGAAGATCGAGGCCTCTAAGGACTTCCAGACCAGCACATCCACCCTGAAGATCA ATTACCTGAAGAAGGAGGACGAGGCCACCTACTACTGCGCAGTGTGGATCGAGTACT CTTCCGGATTCCACAAGGTGTTTGCAGAGGGCACCAAGCTGATCGTGATCCCAAGCT GATAAGCGGCCGCTCGAGTCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACT GTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCT GGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGT CTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGA GGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTACT GGGCGGTTTTATGGACAGCAAGCGAACCGGAATTGCCAGCTGGGGCGCCCTCTGGTA AGGTTGGGAAGCCCTGCAAAGTAAACTGGATGGCTTTCTTGCCGCCAAGGATCTGAT GGCGCAGGGGATCAAGCTCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTG AACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCT ATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAG CGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAAC TGCAAGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCA GCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTG CCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGG CTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCA AGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCA GGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCT CAAGGCGAGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTT GCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTG GGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGC TTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTC GCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAATTATTAACGCTTACA ATTTCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATCAGG TGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATT CAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATAGCACGTG CTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCAT GACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAA GATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACADOCKET NO: WIST-020-PCT PCT APPLICATIONAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTT TTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGT AGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCT GCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTG GACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTC GTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCG TGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGG TAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGC CTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGT GATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTAC GGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTT (SEQ ID NO: 25)

[0091] In some embodiments, each fourth nucleic acid sequence encoding a linker is independently selectable from about 0 to about 25, about 1 to about 25, about 2 to about 25, about 3 to about 25, about 4 to about 25, about 5 to about 25, about 6 to about 25, about 7 to about 25, about 8 to about 25, about 9 to about 25, about 10 to about 25, about 11 to about 25, about 12 to about 25, about 13 to about 25, about 14 to about 25, about 15 to about 25, about 16 to about 25, about 17 to about 25, about 18 to about 25, about 19 to about 25, about 20 to about 25, about 21 to about 25, about 22 to about 25, about 23 to about 25, about 24 to about 25 natural or non-natural nucleic acids in length. In some embodiments, the fourth nucleic acid sequence encoding each linker is about 0, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25 natural or non-natural nucleic acids in length. In some embodiments, the fourth nucleic acid sequence encoding each linker is independently selectable from about 0, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25 natural or non-natural nucleic acids in length. In some embodiments, each fourth nucleic acid sequence encoding a linker is about 21 natural or non-natural nucleic acids in length.

[0092] In some embodiments, each fourth nucleic acid sequence encoding a linker is of a different length. For example, in some embodiments, one the fourth nucleic acid sequences is about 0, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, aboutDOCKET NO: WIST-020-PCT PCT APPLICATION12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about21, about 22, about 23, about 24, about 25 natural or non-natural nucleic acids in length, and the length of a second fourth nucleic acid sequence is about 0, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25 natural or non-natural nucleic acids in length, where the length of the first fourth nucleic acid sequence is different from the length of the second fourth nucleic acid sequence. Various configurations can be envisioned by the present disclosure, comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more fourth nucleic acid sequences encoding linkers wherein the linkers are of similar or different lengths.

[0093] In some embodiments, each of the first nucleic acid sequences, each of the second nucleic acid sequences, and each of the third nucleic acid sequences are independently selectable from about 20 to about 2,000 nucleotides in length. In some embodiments, each of the first nucleic acid sequences, each of the second nucleic acid sequences, and each of the third nucleic acid sequences are independently about 20 to about 2,000 nucleotides in length, about 50 to about 2,000 nucleotides in length, about 100 to about 2,000 nucleotides in length, about 500 to about 2,000 nucleotides in length, about 1500 to about 2,000 nucleotides in length. In some embodiments, each of the first nucleic acid sequences, each of the second nucleic acid sequences, and each of the third nucleic acid sequences are independently about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 1,100, about 1,200, about 1,300, about 1,400, about 1,500, about 1,600, about 1,700, about 1,800, about 1900, about 2000 nucleotides in length. In some embodiments, each of the first nucleic acid sequences, each of the second nucleic acid sequences, and each of the third nucleic acid sequences are independently selectable from about 15 to about 150 nucleotides in length, for example about 15 to about 150 nucleotides in length, about 15 to about 125 nucleotides in length, about 15 to about 100, about 15 to about 90 nucleotides in length, about 15 to about 90 nucleotides in length, about 15 to about 80 nucleotides in length, about 15 to about 70 nucleotides in length, about 15 to about 60 nucleotides in length, about 15 to about 50 nucleotides in length, about 15 to about 40 nucleotides in length, about 15 to about 30 nucleotides in length, about 15 to about 20 nucleotides in length.

[0094] In some embodiments, each of the first nucleic acid sequences, each of the second nucleic acid sequences, and each of the third nucleic acid sequences are independently about 15 to aboutDOCKET NO: WIST-020-PCT PCT APPLICATION100 nucleotides in length, for example about 3 to about 120 nucleotides in length, from about 15 to about 100, from about 15 to about 90 nucleotides in length, about 15 to about 90 nucleotides in length, about 15 to about 80 nucleotides in length, about 15 to about 70 nucleotides in length, about 15 to about 60 nucleotides in length, about 15 to about 50 nucleotides in length, about 15 to about 40 nucleotides in length, about 15 to about 30 nucleotides in length, about 15 to about 20 nucleotides in length.

[0095] In some embodiments, each of the first nucleic acid sequences, each of the second nucleic acid sequences, and each of the third nucleic acid sequences are independently about 15 to about 50 nucleotides in length, for example about 15 to about 50 nucleotides in length, about 15 to about 40 nucleotides in length, about 15 to about 30 nucleotides in length, about 15 to about 20 nucleotides in length.

[0096] In some embodiments, at least one linker comprises a furin protease cleavage site.

[0097] Furin is a protease which resides in the trans-Golgi network of eukaryotic cells. Its function is to cleave proteins at a step just prior to their delivery to their final cellular destination. Furin recognizes a consensus amino acid sequence, RXRR, RXRK or KXKR (where X is any amino acid, Moehring et al., 1993, incorporated by reference in its entirety herein) and cuts proteins which contain these sequences when they reach the trans-Golgi network. Furin is a Ca2+-dependent serine endoprotease that cleaves protein precursors with a high specificity after the multiple basic motifs shown in Table 1 below.<"

[0098] In some embodiments, the furin- sensitive cleavage site selected from the sequence R-X-[R / K] -R, where R denotes arginine, X is any amino acid, and K is lysine. The "R / K" indicates that this amino acid may be either arginine or lysine.

[0099] In some embodiments, the leader sequence is an IgE leader sequence: Met Asp Trp Thr Trp He Leu Phe Leu Vai Ala Ala Ala Thr Arg Vai (SEQ ID NO: 10) or a leader sequence that is aDOCKET NO: WIST-020-PCT PCT APPLICATIONfunctional fragment thereof comprising at least about 70%, 80%, 85%, 90% , 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 10.Neoantigens

[0100] In some embodiments, the disclosure relates to a sixth nucleic acid sequence comprising Formula I: (AED)i-(NA linker)i-(AED)2-(NA linker)2 . . . (AED)n-(NA linker )n, wherein each AED is an independently selectable antigen expression domain comprising an expressible nucleic acid sequence, and each NA linker is independently selectable from about 0 to about 300 natural or non-natural nucleic acids in length. In some embodiments, each antigen expression domain from about 12 to about 15,000 nucleotides in length and encodes an epitope. In some embodiments, n is any positive integer from about 1 to about 500. The term NA linker is used to refer to the nucleic acid sequence linking one AED to another in Formula 1. An NA linker, in some embodiments, encodes a linker, as referred to in the above section describing TCR vaccines. An NA linker, in some embodiments, encodes a cleavable linker. In some embodiments, the disclosure relates to a sixth nucleic acid sequence comprising Formula IE (AED) i-(NA linker) I-(AED)2-(NA linker)2 . . . (AED)n, wherein each AED is an independently selectable antigen expression domain comprising an expressible nucleic acid sequence, and each NA linker is independently selectable from about 0 to about 300 natural or non-natural nucleic acids in length. In some embodiments, each antigen expression domain from about 12 to about 15,000 nucleotides in length and encodes an epitope. In some embodiments, n is any positive integer from about 1 to about 500. The term NA linker is used to refer to the nucleic acid sequence linking one AED to another in Formula 1. An NA linker, in some embodiments, encodes a linker, as referred to in the above section describing TCR vaccines. An NA linker, in some embodiments, encodes a cleavable linker or amino acid sequence recognized by cellular enzymes. In some embodiments, the NA linker is a nucleic acid sequence encoding a furin linker. In some embodiments, an AED herein encodes any one or more epitope herein.

[0101] In some embodiments, the disclosure relates to a composition comprising at least a first nucleic acid molecule, wherein the first nucleic acid molecule comprises:(i) a DNA backbone region, and(ii) an expressible nucleic acid sequence operably linked to a regulatory sequence. In some embodiments, the expressible nucleic acid sequence is a DNA comprising Formula I that encodesDOCKET NO: WIST-020-PCT PCT APPLICATIONa chimeric polypeptide comprising a concatemer of a linked series of neoantigens or antigenic determinants thereof from the cancer, Formula I comprising independently selected, non-repetitive antigen expression domains (AED):(AEDi)-(linker)i-(AED2)-(linker)2-(AED3)-(linker)3- . . . (AEDn)-(linker)n - [AEDn+1] Formula I,In some embodiments, at least one of, a plurality of, or each AED encodes one of the neoantigens or antigenic determinants thereof. In some embodiments, at least one of, a plurality of, or each linker has a length independently selectable from about 15 to about 300 nucleotides and encodes a furin cleavage site. In some embodiments, the length at least one, a plurality of, or each AED is independently selectable from about 90 to about 250 nucleotides. In some embodiments, n is any positive integer from about 10 to about 100. In some embodiments, the DNA backbone region comprises a backbone of a pVAX vector. In some embodiments, n is any positive integer from 19 to about 60. In some embodiments, n is any positive integer from 19 to about 55. In some embodiments, n is any positive integer from 19 to about 58. In some embodiments, n is any positive integer from 19 to about 70. In some embodiments, n is any positive integer from 19 to about 80. In some embodiments, n is any positive integer from 19 to about 90. In some embodiments, n is any positive integer from 19 to about 95. In some embodiments, n is any positive integer from 19 to about 110. In some embodiments, at least one, a plurality of, or each of the AED encodes one or more epitope, antigen, or neoantigen herein or functional variant thereof comprising at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity thereto. In some embodiments, methods of the disclosure comprises a method of treating cancer in a subject having cancer, the method comprising administering to the subject at least the first nucleic acid molecule of this paragraph.

[0102] In some embodiments, methods of the disclosure comprises a method of treating cancer in a subject having cancer, the method comprising administering to the subject at least a first nucleic acid molecule, wherein the first nucleic acid molecule comprises:(i) a DNA backbone region, and(ii) an expressible nucleic acid sequence operably linked to a regulatory sequence; wherein the expressible nucleic acid sequence is a DNA comprising Formula I that encodes a chimeric polypeptide comprising a concatemer of a linked series of neoantigens or antigenicDOCKET NO: WIST-020-PCT PCT APPLICATIONdeterminants thereof from the cancer, Formula T comprising independently selected, non-repetitive antigen expression domains (AED):(AEDi)-(linker)i-(AED2)-(linker)2-(AED3)-(linker)3- . . . (AEDn)-(linker)n- [AEDn+l] Formula I,wherein each AED encodes one of the neoantigens or antigenic determinants thereof; wherein each linker has a length independently selectable from about 15 to about 300 nucleotides and encodes a furin cleavage site;wherein the length of each AED is independently selectable from about 90 to about 250 nucleotides;wherein n is any positive integer from about 10 to about 100, and wherein the DNA backbone region comprises a backbone of a pVAX vector. In some embodiments, n is any positive integer from 19 to about 60. In some embodiments, n is any positive integer from 19 to about 55.

[0103] In some embodiments, n is any positive integer from 19 to about 58.

[0104] In some embodiments, n is any positive integer from 19 to about 70.

[0105] In some embodiments, n is any positive integer from 19 to about 80.

[0106] In some embodiments, n is any positive integer from 19 to about 90.

[0107] In some embodiments, n is any positive integer from 19 to about 95.

[0108] In some embodiments, n is any positive integer from 19 to about 110.

[0109] In some embodiments, n is any positive integer from 19 to about 120.

[0110] In some embodiments, the AED encodes one or more epitope, antigen, or neoantigen herein or functional variant thereof comprising at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity thereto.

[0111] In some embodiments, a vaccine comprising one or more nucleic acid herein targets an immune cell receptor (BCR or TCR). In some embodiments, a BCR or TCR, fragment thereof, or functional variants thereof can serve as an antigenic target. In some embodiments, one or more AED herein encodes a BCR or TCR, fragment thereof, or functional variants thereof. In some embodiments, the TCR is one disclosed in: Willemsen RA et al, Gene Therapy 2000; 7: 1369-1377; Zhang T et al, Cancer Gene Ther 2004; 11: 487-496; or Aggen et al, Gene Ther. 2012 Apr;19(4):365-74 (each of which is incorporated herein by reference in its entirety) or functional variants thereof comprising at least about 70%, about 75%, about 80%, about 85%, about 90%,DOCKET NO: WIST-020-PCT PCT APPLICATIONabout 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity thereto. In some embodiments, the BCR is one disclosed in Kim, D. and Park, D. “Deep sequencing of B cell receptor repertoire” (2019) BMB Rep. 52(9): 540-547, which is incorporated herein by reference in its entirety, functional variants thereof comprising at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity thereto. In some embodiments, the BCR is MPGGPGVLQALPATIFLLFLLSAVYLGPGCQALWMHKVPASLMVSLGEDAHFQCPHNSS NNANVTWWRVLHGNYTWPPEFLGPGEDPNGTLIIQNVNKSHGGIYVCRVQEGNESYQQ SCGTYLRVRQPPPRPFLDMGEGTKNRIITAEGIILLFCAVVPGTLLLFRKRWQNEKLGLDA GDEYEDENLYEGLNLDDCSMYEDISRGLQGTYQDVGSLNIGDVQLEKP (Pl 1912 CD79A_HUMAN) or a functional variant thereof comprising at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity thereto. In some embodiments, the BCR is MARLALSPVPSHWMVALLLLLSAEPVPAARSEDRYRNPKGSACSRIWQSPRFIARKRGFT VKMHCYMNSASGNVSWLWKQEMDENPQQLKLEKGRMEESQNESLATLTIQGIRFEDN GIYFCQQKCNNTSEVYQGCGTELRVMGFSTLAQLKQRNTLKDGIIMIQTLLIILFIIVPIFL LLDKDDSKAGMEEDHTYEGLDIDQTATYEDIVTLRTGEVKWSVGEHPGQE (P40259 CD79B_HUMAN) or a functional variant thereof comprising at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity thereto. In some embodiments, the BCR is DLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQ PLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQI VSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF(P01850 ■ TRBC1_HUMAN) or a functional variant thereof comprising at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity thereto.

[0112] In some embodiments, the disclosure relates to a nucleic acid molecule comprising one or more nucleic acid sequences encoding antigen or antigen determinants. In some embodiments, theDOCKET NO: WIST-020-PCT PCT APPLICATIONnucleic acid molecule comprises SEQ ID NO: 26 or a variant thereof comprising at least about 70%, 80%, 85%, 90% , 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 26. In some embodiments, the disclosure relate to a first nucleic acid molecule comprising one or more nucleic acid sequences encoding antigen or antigen determinants and a second nucleic acid molecule encoding one or more nucleic acid sequences encoding one or more antigens or antigen determinants.

[0113] In some embodiments a nucleic acid sequence comprising SEQ ID NO: 25 or a variant thereof comprising at least about 70%, 80%, 85%, 90% , 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 25 comprises the sixth nucleic acid sequence. In some embodiments, the nucleic acid molecule or the second nucleic acid molecule comprises a nucleic acid sequence comprising SEQ ID NO: 25 or a variant thereof comprising at least about 70%, 80%, 85%, 90% , 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 25. In some embodiments, compositions of the disclosure comprise a first nucleic acid molecule comprising SEQ ID NO:25 or a variant thereof comprising at least about 70%, 80%, 85%, 90% , 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 25; and a second nucleic acid molecule comprising SEQ ID NO: 26 or a variant thereof comprising at least about 70%, 80%, 85%, 90% , 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 26.EL4neo GCTGCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATT AATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACAT AACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGT CAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGG GTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAA GTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTA CATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTAC CATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGG GGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATC AACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTA GGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCA CTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTADOCKET NO: WIST-020-PCT PCT APPLICATIONGCGTTTAAACTTAAGCTTGGTACCGAGCTCGGATCCACTAGTCCAGTGTGGTGGAATT CGCCACCATGGATTGGACTTGGATTCTTTTTCTCGTTGCTGCAGCTACACGCGTTCAC AGTGATAGGCCCTCTCTGTCCAAGAAGTTTTGTGAGGCCTGCTGGAGATTCCTGTTTT ACCTGTGTTCCTTCGTGGGAGGCACCAGCATCCTGTATCACGAGCGCGGCCGGAAGA GAAGGTCCCCAGTGCAGCCTTTTCTGCCCGGCCAGAGCCACCAGGCCCCTGGCGTGA GCGATGGCCTGAGACAGCAGCTGGGGCCACGCCTGGCCTGGACCCTGACCAGAGGC AGGAAGCGGCGGTCTGCCGGCTTCCTGTGCGGAGAGAATGAGGACGACAGGGGAAG GTTTGCCCTGGTGCTGGGACTGCTGAAGCCACACCTGAACAAGGAGACATGGAAAA AGAGAGGCAGGAAGAGGCGGTCCGCAGCCCCTTCTGCCCTGAGCCCAGAGAGCCCT GTGCTGGCCACATCTACCGAGCCATGGGGCCCCTCCCTGTCTGCAAGCCCTGAGAGC CGGAAGCCTCGGGGCCGGAAGAGGAGAAGCCCAGAGCCTAGAAAGCGGGCCCTGTT CCCAGAGTCCCGCAAGCACGTGCTGCTGCCCGAGCTGCCTAAGTCCGCAGTGTTCTC TGACGCCCAGAAGGCCAGAGGCAGAAAGAGACGGTCTGAGCCACAGGGAGCCGGC CCTCTGATCAGCCCCGAGCCACAGGCACCTAGCCTGCCTGCCGAGGCCAGCAAGGCC GCACCAGTGCCATGTCCCGAAAGGAGGGGCAGAAAGCGCCGCTCTGCCAACATCAA TAAGCCTGACTGCGAGGGCGAGACACCTATCCACAAGGTGGCAAGGAGCGGAAGCC TGGAGTGTATCACAGCACTGGTGGGCTCCGGCAGAGGCCGCAAGAGGCGGTCTAAG AACCAGGAGCGCACATACTCTACAAAGGAGAGATCCGAGAGGAGGACAGTGTTCAG AGCCCCACAGGAGGCCTCTTTTGCAGGCAGCTTCGAGGAGCGGGGCAGAAAGCGCA GGTCCCTGGAGGGCAATGCCGTGGTGAGCTATAGCAGGCCAACCGGCATCCCAGCAG GCTGTCCCATGCGCGGCAGGAAAAGACGCTCCACACCTAATAACCCCTCCCCATGGA CATATTACTGCCCAGACGCCACCCTGTATGAGAGCTCTTCCTCCAGAACCCCCTCTAC CTCTGCCTGGTGGAGCAGGGGCAGGAAGAGGAGAAGCTCCCAGAAAAGGGAGTGC GGAGTGGGGTGCCACAGGTGGGGCCCACAGCACCTGCTGCCCGACCCCGAGGACGT GCTGCACGCCCAGTGGACCCACGATAGGGGCAGAAAGAGACGCAGCCCCACACTGT CCCCAACCCTGTGGCTGCCCGGACAGTCTCAGGTGTGTCACACCGGCCAGGAGGGA CCTCAGGCCCAGCACCAGAGAGCCGGACTGTGGCGCGGCAGAAAGAGAAGATCCTC TACCAAAAAGACCGAGCAGATCATCATCAAGCAGGATCTGTGCTGTCTGCTGAAGAA GCTGGATTGGTCCTGGAAGCGCGGACGGAAGCGCCGGAGCACACCCCACCTGGTGT ACTCCCAGGCAGCCACCTCCACCAATGATCTGTGCAGCACACCCAACAACCCTAGCC CATGGACCTACTACTGCCCAGACGCACGGGGCAGAAAGAGAAGGTCCATCGGCCTGDOCKET NO: WIST-020-PCT PCT APPLICATIONGTGGGCAATCACATCGACGTGCTGACCGGGAAGTGGGTGGCACAGGATGCAGGAAT CGGACTGGTGTGGATCCCCATCCTGAGCACCTGGTGATAAGCGGCCGCTCGAGTCTA GAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATC TGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTC CTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCT GGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGG CATGCTGGGGATGCGGTGGGCTCTATGGCTTCTACTGGGCGGTTTTATGGACAGCAAG CGAACCGGAATTGCCAGCTGGGGCGCCCTCTGGTAAGGTTGGGAAGCCCTGCAAAG TAAACTGGATGGCTTTCTTGCCGCCAAGGATCTGATGGCGCAGGGGATCAAGCTCTG ATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAG GTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAA TCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTT TGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAAGACGAGGCAGCGCGGC TATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTG AAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCAT CTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCA TACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCG AGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCA TCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGAGCATGCCCGACG GCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAA TGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAG GACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGAC CGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCG CCTTCTTGACGAGTTCTTCTGAATTATTAACGCTTACAATTTCCTGATGCGGTATTTTCT CCTTACGCATCTGTGCGGTATTTCACACCGCATCAGGTGGCACTTTTCGGGGAAATGT GCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAG ACAATAACCCTGATAAATGCTTCAATAATAGCACGTGCTAAAACTTCATTTTTAATTTA AAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGA GTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGAT CCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGG TGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGDOCKET NO: WIST-020-PCT PCT APPLICATIONCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTC AAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTG CTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGG ATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAG CGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACG CTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGG AGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGG GTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGC CTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTT TTGCTCACATGTTCTT (SEQ ID NO: 26)

[0114] In some embodiments, each NA linker is independently selectable from about 0 to about 25, about 1 to about 25, about 2 to about 25, about 3 to about 25, about 4 to about 25, about 5 to about 25, about 6 to about 25, about 7 to about 25, about 8 to about 25, about 9 to about 25, about 10 to about 25, about 11 to about 25, about 12 to about 25, about 13 to about 25, about 14 to about 25, about 15 to about 25, about 16 to about 25, about 17 to about 25, about 18 to about 25, about 19 to about 25, about 20 to about 25, about 21 to about 25, about 22 to about 25, about 23 to about 25, about 24 to about 25 natural or non-natural nucleic acids in length. In some embodiments, each NA linker is independently about 0, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25 natural or nonnatural nucleic acids in length. In some embodiments, each NA linker is independently selectable from about 0, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25 natural or non-natural nucleic acids in length. In some embodiments, each NA linker is about 21 natural or non-natural nucleic acids in length.

[0115] In some embodiments, each NA linker is of a different length. For example, in some embodiments, one NA linker is about 0, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25 natural or non-natural nucleic acids in length, and the length of a second NA linker is about 0, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, aboutDOCKET NO: WIST-020-PCT PCT APPLICATION13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21 , about 22, about 23, about 24, about 25 natural or non-natural nucleic acids in length, where the length of the first fourth nucleic acid sequence is different from the length of the second fourth nucleic acid sequence. Various configurations can be envisioned by the present disclosure, comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NA linkers wherein the NA linkers are of similar or different lengths.

[0116] In some embodiments, each of the AEDs are independently selectable from about 20 to about 2,000 nucleotides in length. In some embodiments, each of the AEDs are independently about 20 to about 2,000 nucleotides in length, about 50 to about 2,000 nucleotides in length, about 100 to about 2,000 nucleotides in length, about 500 to about 2,000 nucleotides in length, about 1500 to about 2,000 nucleotides in length. In some embodiments, each of the AEDs are independently about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 1,100, about 1,200, about 1,300, about 1,400, about 1,500, about 1,600, about 1,700, about 1,800, about 1900, about 2000 nucleotides in length. In some embodiments, each of the AEDs are independently selectable from about 15 to about 150 nucleotides in length, for example about 15 to about 150 nucleotides in length, about 15 to about 125 nucleotides in length, about 15 to about 100, about 15 to about 90 nucleotides in length, about 15 to about 90 nucleotides in length, about 15 to about 80 nucleotides in length, about 15 to about 70 nucleotides in length, about 15 to about 60 nucleotides in length, about 15 to about 50 nucleotides in length, about 15 to about 40 nucleotides in length, about 15 to about 30 nucleotides in length, about 15 to about 20 nucleotides in length.

[0117] In some embodiments, each of the AEDs are independently about 15 to about 100 nucleotides in length, for example about 3 to about 120 nucleotides in length, from about 15 to about 100, from about 15 to about 90 nucleotides in length, about 15 to about 90 nucleotides in length, about 15 to about 80 nucleotides in length, about 15 to about 70 nucleotides in length, about 15 to about 60 nucleotides in length, about 15 to about 50 nucleotides in length, about 15 to about 40 nucleotides in length, about 15 to about 30 nucleotides in length, about 15 to about 20 nucleotides in length. In some embodiments, each of the AEDs are independently about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, or 015 nucleotides in length.DOCKET NO: WIST-020-PCT PCT APPLICATION

[0118] In some embodiments, each of the AEDs are independently about 15 to about 50 nucleotides in length, for example about 15 to about 50 nucleotides in length, about 15 to about 40 nucleotides in length, about 15 to about 30 nucleotides in length, about 15 to about 20 nucleotides in length.

[0119] In some embodiments, at least one NA linker comprises a furin protease cleavage site.

[0120] Furin recognizes a consensus amino acid sequence, RXRR, RXRK or KXKR (where X is any amino acid, Moehring et al., 1993, incorporated by reference in its entirety herein) and cuts proteins which contain these sequences when they reach the trans-Golgi network. Furin is a Ca2+-dependent serine endoprotease that cleaves protein precursors with a high specificity after the multiple basic motifs shown in Table 1 below.

[0121] In some embodiments, the furin-sensitive cleavage site selected from the sequence R-X-[R / K] -R, where R denotes arginine, X is any amino acid, and K is lysine. The "R / K" indicates that this amino acid may be either arginine or lysine. In some embodiments, the furin cleavage site comprises no more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length and comprises any of the sequences of Table 1 below.

[0122] In some embodiments, the sixth nucleic acid further encodes a leader sequence. The leader sequence, in some embodiments, is an IgE leader sequence: Met Asp Trp Thr Trp He Leu Phe Leu Vai Ala Ala Ala Thr Arg Vai (SEQ ID NO: 10) or a leader sequence that is a functional fragment thereof comprising at least about 70%, 80%, 85%, 90% , 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 10.

[0123] In some embodiments, the nucleic acid molecule further comprises the sixth nucleic acid.

[0124] In some embodiments, the disclosure relates to second nucleic acid molecule comprising the sixth nucleic acid.

[0125] In some embodiments, each AED encodes a neoantigen of a functional variant thereof.DOCKET NO: WIST-020-PCT PCT APPLICATION

[0126] In some embodiments, the nucleic acid molecule and / or the second nucleic acid molecule comprise one or more regulatory sequences operably linked to the nucleic acid sequences thereon. In some embodiment, the one or more regulatory sequences comprise one or more promoters capable of driving expression of the nucleic acid sequences. In some embodiments, the nucleic acid sequences are DNA and free of RNA. In some embodiments, one of the nucleic acid sequences comprises DNA and is free of RNA. In some embodiments, any and all nucleic acid molecules are DNA and are free of RNA.

[0127] In some embodiments, the nucleic acid sequences and / or nucleic acid molecules are cloned into a plasmid. In some embodiments, one or more nucleic acid molecule is a plasmid comprising one or more nucleic acid sequence herein. In some embodiments, the plasmid into which a nucleic acid sequence or nucleic acid molecule is cloned is pVAX:

[0128] Full-length pVAX sequence is as follows: gctgcttcgcgatgtacgggccagatatacgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagccc atatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgt atgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtat catatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctactt ggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacgggg atttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccat tgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaactagagaacccactgcttactggcttatc gaaattaatacgactcactatagggagacccaagctggctagcgtttaaacttaagcttggtaccgagctcggatccactagtccagtgtggt ggaattctgcagatatccagcacagtggcggccgctcgagtctagagggcccgtttaaacccgctgatcagcctcgactgtgccttctagtt gccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgca tcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcat gctggggatgcggtgggctctatggcttctactgggcggttttatggacagcaagcgaaccggaattgccagctggggcgccctctggtaa ggttgggaagccctgcaaagtaaactggatggctttcttgccgccaaggatctgatggcgcaggggatcaagctctgatcaagagacagg atgaggatcgtttcgcatgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctatgactgggcac aacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgcc ctgaatgaactgcaagacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaa gcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatgg ctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactc ggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctcaaggcgDOCKET NO: WIST-020-PCT PCT APPLICATIONagcatgcccgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatc gactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggct gaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgaattattaacgctt acaatttcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatcaggtggcacttttcggggaaatgtgcgcggaacccc tatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatagcacgtgctaaaacttcattttt aatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtag aaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgc cggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttagg ccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtctt accgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagc gaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatcc ggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgcc acctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctgg ccttttgctggccttttgctcacatgttctt (SEQ ID NO: 18)

[0129] In some embodiments, the nucleic acid molecule comprises a first nucleic acid sequence encoding a TCR chain alpha and a TCR chain beta. In some embodiments, the first nucleic acid molecule encodes a TCR chain alpha, a TCR chain beta, and a TCR chain gamma amino acid sequence. In some embodiments, at least one nucleic acid molecule comprises a a nucleic acid sequence encoding one or a combination of any of the sequences in Tables 1 and 2 or functional variants thereof In some embodiments, at least one nucleic acid molecule comprises a nucleic acid sequence encoding one or a combination of any of the sequences in Table 3 or functional variants thereof. In some embodiments, compositions of the disclosure comprise a first nucleic acid molecule and a second nucleic acid molecule, wherein the first nucleic acid molecule comprises a nucleic acid sequence that encodes TCR chain alpha or a functional variant thereof, TCR chain beta or a functional variant thereof, and, optionally, TCR chain gamma or a functional variant thereof; and wherein the second nucleic acid molecule comprises a nucleic acid sequence encoding one or a plurality of neoantigens from one or a plurality of cancer cells. In some embodiments, the cancer cells are tumor tissue or a plurality of cells from a biopsy. In some embodiments, the cancer cells or tumor is a T cell lymphoma or peripheral T cell lymphoma. In some embodiments, the TCR is one disclosed in: Willemsen RA et al, Gene Therapy 2000; 7: 1369-1377; Zhang T et al, Cancer Gene Ther 2004; 11: 487-496; or Aggen et al, Gene Ther. 2012 Apr;19(4):365-74, whichDOCKET NO: WIST-020-PCT PCT APPLICATIONdisclose T cell receptor alpha (TCR-aplha), TCR-beta, and TCR-gamma subunits, or functional variants thereof comprising at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity thereto. Any mutations disclosed or endogenous TCR subunit sequences disclosed in the above-identified references are incorporated herein by reference in their entireties. In some embodiments, the composition encoding TCR subunit or subunits is a mutation identified in the above-identified references or a variant thereof comprising at least about 80%, 90% or 99% sequence identity to the amino acid sequences disclosed in the above-identified references.

[0130] In some embodiments, the nucleic acid molecule comprises a first nucleic acid sequence encoding a BCR or BCR fragment, or functional variant thereof. In some embodiments, compositions of the disclosure comprise a first nucleic acid molecule and a second nucleic acid molecule, wherein the first nucleic acid molecule comprises a nucleic acid sequence that encodes a BCR or BCR fragment, or functional variant thereof; and wherein the second nucleic acid molecule comprises a nucleic acid sequence encoding one or a plurality of neoantigens from one or a plurality of cancer cells. In some embodiments, the cancer cells are tumor tissue or a plurality of cells from a biopsy. In some embodiments, the BCR is one disclosed in Kim, D. and Park, D. “Deep sequencing of B cell receptor repertoire” (2019) BMB Rep. 52(9): 540-547, which is incorporated herein by reference in its entirety, functional variants thereof comprising at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity thereto. In some embodiments, the BCR is MPGGPGVLQALPATIFLLFLLSAVYLGPGCQALWMHKVPASLMVSLGEDAHFQCPHNSS NNANVTWWRVLHGNYTWPPEFLGPGEDPNGTLIIQNVNKSHGGIYVCRVQEGNESYQQ SCGTYLRVRQPPPRPFLDMGEGTKNRIITAEGIILLFCAVVPGTLLLFRKRWQNEKLGLDA GDEYEDENLYEGLNLDDC SMYEDISRGLQGTYQDVGSLNIGDVQLEKP (P 11912 CD79A_HUMAN) or a functional variant thereof comprising at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity thereto. In some embodiments, the BCR is MARLALSPVPSHWMVALLLLLSAEPVPAARSEDRYRNPKGSACSRIWQSPRFIARKRGFTDOCKET NO: WIST-020-PCT PCT APPLICATIONVKMHCYMNSASGNVSWLWKQEMDENPQQLKLEKGRMEESQNESLATLTIQGIRFEDN GIYFCQQKCNNTSEVYQGCGTELRVMGFSTLAQLKQRNTLKDGIIMIQTLLIILFIIVPIFL LLDKDDSKAGMEEDHTYEGLDIDQTATYEDIVTLRTGEVKWSVGEHPGQE (P40259 CD79B HUMAN) or a functional variant thereof comprising at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity thereto. In some embodiments, the BCR is DLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQ PLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQI VSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF(P01850 ■ TRBC1 HUMAN) or a functional variant thereof comprising at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity thereto.Pharmaceutical Compositions

[0131] In some embodiments, a pharmaceutical composition comprises (i) one or a plurality of any of the nucleic acid molecules described herein, or a pharmaceutically acceptable salt thereof and (ii) a pharmaceutically acceptable carrier. Optionally, the pharmaceutical composition may further comprise one or more therapeutic agents, such as a biologic therapeutic or a small molecule. In some embodiment, one of the therapeutic agents is (i) a checkpoint inhibitor or functional fragment thereof, or (ii) a nucleic acid sequence that encodes a checkpoint inhibitor or functional fragment thereof. In some embodiments, the checkpoint inhibitor or functional fragment thereof associates or inhibits a checkpoint protein selected from the group consisting of CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN- 15049, CHK 1, CHK2, A2aR, and B-7 family ligands or a combination thereof. In some embodiments, the checkpoint inhibitor is an inhibitor of the programmed death- 1 (PD-1) pathway, or a nucleic acid encoding the same. In some embodiments, the checkpoint inhibitor is an anti -cytotoxic T- lymphocyte-associated antigen 4 (CTLA4) antibody. In some embodiments, a pharmaceutical composition comprises a pharmaceutically effective amount of: (i) one or a plurality of any of the nucleic acid molecules described herein or nucleic acid sequences that have about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceDOCKET NO: WIST-020-PCT PCT APPLICATIONidentity to any nucleic acid sequence listed herein, or a pharmaceutically acceptable salt thereof; and (ii) a pharmaceutically acceptable carrier.

[0132] In some embodiments, the therapeutic agent is an adjuvant or functional fragment thereof. In some embodiments, the adjuvant or functional fragment thereof is (i) selected from the group consisting of: poly-ICLC, 1018 ISS, aluminum salts, Amplivax. AS15, BCG, CP- 870,893, CpG7909, CyaA, GM-CSF, IC30, IC31, Imiquimod, ImuFact 1MP321, IS Patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, monophosphoryf lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PEPTEL, vector system, PLGA micropartieles, resiquimod, S LI 72, Virosomes and other Viruslike particles, YF-17D, VEGF trap, R848, beta-gluean, Pam3Cys, acrylic or methacrylic polymers, copolymers of maleic anhydride and Aquila's QS21 stimulon, and a functional fragment of any thereof; or (ii) a nucleic acid molecule encoding an adjuvant selected from the group consisting of: poly-ICLC, 1018 ISS, Amplivax AS15, BCG, CP- 870,893, CpG7909, CyaA, GM-CSF, IC30, IC31 , Imiquimod, ImuFact 1MP321 , IS Patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, monophosphoryf lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PEPTEL, vector system, resiquimod, S L172, Virosomes and other Virus-like particles, YF-17D, VEGF trap, R848, beta-gluean, Pam3Cys, and Aquila's QS21 stimulon, or functional fragment thereof. IL-12, IL-15 ( protein or plasmid or NA).

[0133] In some embodiments, the adjuvant can be selected from the group consisting of: a-interferon (IFN-a), (3-interferon (IFN-0), ^-interferon, platelet derived growth factor (PDGF), TNFa, TNFP, GM-CSF, epidermal growth factor (EGF), cutaneous T cell-attracting chemokine (CTACK), epithelial thymus-expressed chemokine (TECK), mucosae-associated epithelial chemokine (MEC), IL-12, IL-15, MHC, CD80, CD86 including IL-15 having the signal sequence deleted and optionally including the signal peptide from IgE. The adjuvant can be IL-12, IL-15, IL-28, CTACK, TECK, platelet derived growth factor (PDGF), TNF.alpha., TNF.beta., GM-CSF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18, or a combination thereof.

[0134] Other genes which can be useful adjuvants include those encoding: MCP-1, MIP-la, MIP-Ip, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-DOCKET NO: WIST-020-PCT PCT APPLICATION4, mutant forms of IL-18, CD40, CD40L, vascular growth factor, fibroblast growth factor, TL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Fit, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, Caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, Inactive NIK, SAP K, SAP-1, JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, 0x40, 0x40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAPI, TAP2 and functional fragments thereof.

[0135] Human IL-12 alpha subunit is set forth in GenBank Accession Nos. NP_000873.2, NM_000882.3, incorporated by reference in their entireties herein. An exemplary human IL-12 alpha subunit amino acid sequence is shown below:MCPARSLLLV ATLVLLDHLS LARNLPVATP DPGMFPCLHH SQNLLRAVSN MLQKARQTLE FYPCTSEEID HEDITKDKTS TVEACLPLEL TKNESCLNSRETSFITNGSC LASRKTSFMM ALCLSSIYED LKMYQVEFKT MNAKLLMDPK RQIFLDQNML AVIDELMQAL NFNSETVPQK SSLEEPDFYK TKIKLCILLH AFRIRAVTID RVMSYLNAS(SEQ ID NO: 11)

[0136] Human IL-12 beta subunit is set forth in GenBank Accession No. NP_002178.2, incorporated by reference in its entirety herein. An exemplary human IL-12 beta subunit amino acid sequence is shown below:MCHQQLVISW FSLVFLASPL VAIWELKKDV YVVELDWYPD APGEMVVLTC DTPEEDGITW TLDQSSEVLG SGKTLTIQVK EFGDAGQYTC HKGGEVLSHS LLLLHKKEDG IWSTDILKDQ KEPKNKTFLR CEAKNYSGRF TCWWLTTIST DLTFSVKSSR GSSDPQGVTC GAATLSAERV RGDNKEYEYS VECQEDSACP AAEESLPIEV MVDAVHKLKY ENYTSSFFIR DIIKPDPPKN LQLKPLKNSR QVEVSWEYPD TWSTPHSYFS LTFCVQVQGK SKREKKDRVF TDKTSATVIC RKNASISVRA QDRYYSSSWS EWASVPCS (SEQ ID NO: 12)

[0137] Human IL-15 is set forth in GenBank Accession Nos. NP_000576.1, NP_751915.1, AAI00962.1 incorporated by reference in their entireties herein. An exemplary human IL- 15 amino acid sequence is shown below:DOCKET NO: WIST-020-PCT PCT APPLICATIONMRISKPHLRS ISIQCYLCLL LNSHFLTEAG IHVFILGCFS AGLPKTEANW VNVISDLKKI EDLIQSMHID ATLYTESDVH PSCKVTAMKC FLLELQVISL ESGDASIHDT VENLIILANN SLSSNGNVTE SGCKECEELE EKNIKEFLQS FVHIVQMFIN TS (SEQ ID NO: 13)

[0138] Human IL-17 is set forth in GenBank Accession Nos. NP 002181.1, NM_002190.2, incorporated by reference in their entireties herein. An exemplary human IL- 17 amino acid sequence is shown below:MTPGKTSLVS LLLLLSLEAI VKAGITIPRN PGCPNSEDKN FPRTVMVNLN IHNRNTNTNP KRSSDYYNRS TSPWNLHRNE DPERYPSVIW EAKCRHLGCI NADGNVDYHM NSVPIQQEIL VLRREPPHCP NSFRLEKILV SVGCTCVTPI VHHVA(SEQ ID NO: 14)

[0139] Human IL-8 is set forth in GenBank Accession Nos. NP 000575.1, NM_000584.3, incorporated by reference in their entireties herein. An exemplary human IL-8 amino acid sequence is shown below:MTSKLAVALL AAFLISAALC EGAVLPRSAK ELRCQCIKTY SKPFHPKFIK ELRVIESGPH CANTEIIVKL SDGRELCLDP KENWVQRVVE KFLKRAENS (SEQ ID NO: 15)

[0140] Human C-C motif chemokine 5 (processed form RANTES(3-68) ) is set forth in GenBank Accession Nos. NP_002976.2, NM_002985.2, incorporated by reference in their entireties herein. An exemplary human C-C motif chemokine 5 amino acid sequence is shown below:MKVSAAALAV ILIATALCAP ASASPYSSDT TPCCFAYIAR PLPRAHIKEY FYTSGKCSNP AVVFVTRKNR QVCANPEKKW VREYINSLEM S (SEQ ID NO: 16)

[0141] Human Macrophage inflammatory protein 1-alpha (MIP-la) is set forth in GenBank Accession Nos. NP 002974.1, NM 002983.2, incorporated by reference in their entireties herein. An exemplary human C-C motif chemokine 5 amino acid sequence is shown below:MQVSTAALAV LLCTMALCNQ FSASLAADTP TACCFSYTSR QIPQNFIADY FETSSQCSKP GVIFLTKRSRQVCADPSEEWVQKYVSDLEL SA (SEQ ID NO: 17)

[0142] Other exemplary adjuvants include, but are not limited to, poly-ICLC (see Pharmacol Ther.2015 Feb;146:120-31, incorporated by reference in its entirety herein), 1018 ISS (see Vaccine.2003 lun 2;21(19-20): 2461-7, incorporated by reference in its entirety herein), aluminum salts, Amplivax AS 15, Bacillus Colmette-Guerin (BCG) (see Clin Immunol. 2000 Jan;94(l):64-72, incorporated by reference in its entirety herein) , CP- 870,893, CpG7909 (GenBank Accession No.DOCKET NO: WIST-020-PCT PCT APPLICATIONCS576603.1), CyaA (GenBank Accession No. KP670536.1), GM-CSF (GenBank Accession No. Ml 1220.1), IC30 (see Expert Rev Vaccines. 2007 Oct;6(5):741-6, incorporated by reference in its entirety herein), IC31 (see Expert Rev Vaccines. 2007 Oct;6(5):741-6, incorporated by reference in its entirety herein), Imiquimod (see Vaccine. 2006 Mar 10;24(ll): 1958-6, incorporated by reference in its entirety herein), ImuFact 1MP321 , IS Patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PEPTEL, vector system, PLGA microparticles, resiquimod, S LI 72, Virosomes and other Virus-like particles, YF-17D, VEGF trap, R848, beta-glucan, Pam3Cys, acrylic or methacrylic polymers, copolymers of maleic anhydride and Aquila's QS21 stimulon, and a functional fragment of any thereof; or (ii) a nucleic acid molecule encoding an adjuvant selected from the group consisting of (i) poly-ICLC, 1018 ISS, aluminum salts, Amplivax AS 15, BCG, CP- 870,893, CpG7909, CyaA, GM-CSF, IC30, IC31 , Imiquimod, ImuFact 1MP321, IS Patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM- 197-MP-EC, ONTAK, PEPTEL, vector system, PLGA microparticles, resiquimod, S LI 72, Virosomes and other Virus-like particles, YF-17D, VEGF trap, R848, beta-glucan, Pam3Cys, acrylic or methacrylic polymers, copolymers of maleic anhydride and Aquila's QS21 stimulon, or functional fragment thereof.

[0143] In another embodiment, the therapeutic agent is an immunostimulatory agent or functional fragment thereof. For example, in some embodiments, the imunostimulatory agent is an interleukin or functional fragment thereof.

[0144] In another embodiment, the therapeutic agent is a chemotherapeutic agent. Examples of chemotherapeutic agents include, but are not limited to, aldesleukin, altretamine, amifostine, asparaginase, bleomycin, capecitabine, carboplatin, carmustine, cladribine, cisapride, cisplatin, cyclophosphamide, cytarabine, dacarbazine (DTIC), dactinomycin, docetaxel, doxorubicin, dronabinol, epoetin alpha, etoposide, filgrastim, fludarabine, fluorouracil, gemcitabine, granisetron, hydroxyurea, idarubicin, ifosfamide, interferon alpha, irinotecan, lansoprazole, levamisole, leucovorin, megestrol, mesna, methotrexate, metoclopramide, mitomycin, mitotane, mitoxantrone, omeprazole, ondansetron, paclitaxel (Taxol®), pilocarpine, prochloroperazine, rituximab, tamoxifen, taxol, topotecan hydrochloride, trastuzumab, vinblastine, vincristine andDOCKET NO: WIST-020-PCT PCT APPLICATIONvinorelbine tartrate. For prostate cancer treatment, a preferred chemotherapeutic agent with which anti-CTLA-4 can be combined is paclitaxel (Taxol®).

[0145] In some embodiments, the adjuvant can include a nucleic acid plasmid that encodes any cytokine or functional fragment thereof that is administered sequentially with a pharmaceutical composition comprising a plasmid encoding a plurality of neoantigens, optionally with one or a plurality of tumor associated antigens not derived from a subject. In some embodiments, the cytokine is IL-12 or a subunit of IL-12. In some embodiments, adjuvant is a nucleic acid sequence that encodes an amino acid sequence that comprises at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ NO: 54 or a functional fragment thereof. In some embodiments, adjuvant is a nucleic acid sequence that encodes an amino acid sequence that comprises at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ NO: 55 or a functional fragment thereof. In some embodiments, adjuvant is a first nucleic acid sequence that encodes an amino acid sequence that comprises at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ NO: 54 and a second amino acid sequence that comprises at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ NO: 55 or a functional fragment thereof. In some embodiments, if the nucleic acid sequence encoding a cytokine or functional fragment thereof comprise two subunits, the disclosure relates to nucleic acid molecule comprises a first nucleic acid sequence encoding the first subunit and a second nucleic acid encoding the second subunit, each of the first or second nucleic acid sequences operably linked to at least a first promoter, such as a CMV promoter. In some embodiments, if the nucleic acid sequence encoding a cytokine or functional fragment thereof comprise two subunits, the disclosure relates to nucleic acid molecule comprises a first nucleic acid sequence encoding the first subunit and a second nucleic acid encoding the second subunit, the first nucleic acid sequence is operably linked to at least a first promoter and the second nucleic acid sequence is operably linked to at least a second promoter.

[0146] In some embodiments, the IL- 12 sequences and nucleic acids sequences encoding the same can be found in US Pat. Nos. 9,981,036 and 9,272,024, each of which is incorporated by reference in its entirety. In some embodiments, the therapeutic agent is: (i) an immunostimulatory agent or functional fragment thereof; or (ii) a nucleic acid sequence encoding an immunostimulatory agent or a functional fragment thereof. In some embodiments, the immunostimulatory agent is anDOCKET NO: WIST-020-PCT PCT APPLICATIONinterleukin or a functional fragment thereof. Tn some embodiments, the therapeutic agent is: (i) an chemotherapeutic agent or functional fragment thereof; or (ii) a nucleic acid sequence encoding an chemotherapeutic agent or a functional fragment thereof.

[0147] In embodiments, the pharmaceutical compositions contain a pharmaceutically acceptable carrier, excipient, or diluent, which includes any pharmaceutical agent that does not itself induce the production of an immune response harmful to a subject receiving the composition, and which may be administered without undue toxicity. As used herein, the term “pharmaceutically acceptable” means being approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopia, European Pharmacopia or other generally recognized pharmacopia for use in mammals, and more particularly in humans. These compositions can be useful for treating and / or preventing viral infection and / or autoimmune disease.

[0148] A thorough discussion of pharmaceutically acceptable carriers, diluents, and other excipients is presented in Remington's Pharmaceutical Sciences (17th ed., Mack Publishing Company) and Remington: The Science and Practice of Pharmacy (21 st ed., Lippincott Williams & Wilkins), which are hereby incorporated by reference. The formulation of the pharmaceutical composition should suit the mode of administration. In embodiments, the pharmaceutical composition is suitable for administration to humans, and can be sterile, non-particulate and / or non-pyrogenic.

[0149] In some embodiments, the pharmaceutical composition comprises a pharmaceutically effective amount of: (i) one or a plurality of any of the nucleic acid molecules described herein comprising one or a combination of any component of a plasmid disclosed herein or nucleic acid sequences that are about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to any nucleic acid sequence that is a component of the plasmid listed herein. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence that encodes one or more nucleic acids that encode neoantigens and one or more linkers. In some embodiments, the disclosure relates to a pharmaceutical composition comprising a nucleic acid molecule that is pGX4505 or a nucleic acid sequence comprising at least about 70% sequence identity to the sequence of pGX4505, wherein its multiple cloning site is replaced by any of the Formulae disclosed herein.VaccinesDOCKET NO: WIST-020-PCT PCT APPLICATION

[0150] In an exemplary embodiment, the present disclosure is directed to an immunogenic composition, e.g., a vaccine, composition comprising the nucleic acid sequences disclosed herein, capable of eliciting an antigen-specific immune response, and, in some embodiments, an antigenspecific T-cell response comprising both active CD4+ and therapeutically effective CD8+ T cell response. In some embodiments, the present disclosure is directed to an immunogenic composition, e.g., a vaccine, composition comprising the nucleic acid sequences disclosed herein, capable of eliciting a neoantigen-specific immune response, and, in some embodiments, an antigen-specific T-cell response comprising a therapeutically effective number of active CD4+ and / or therapeutically effective amount CD8+ T cells responsive to one or a plurality of neoantigens from a subject.

[0151] DNA vaccines are described in U.S. Patent Nos. 5,593,972, 5,739,118, 5,817,637,

[0152] 5,830,876, 5,962,428, 5,981,505, 5,580,859, 5,703,055, 5,676,594, and the priority applications cited therein, which are each incorporated herein by reference. In addition to the delivery protocols described in those applications, alternative methods of delivering DNA are described in US. Patent Nos. 4,945,050 and 5,036,006, which are both incorporated herein by reference.

[0153] In certain embodiments, the vaccine composition comprises mutant neo-antigenic nucleic acid molecules as described herein (e.g. comprising a nucleic acid sequence comprising the formula: [(antigen expression domain 1) - (linker) - (antigen expression domain 2) - (linker)] n), corresponding to tumor specific neo-antigens identified by the methods described herein. A suitable vaccine will preferably contain a plurality of tumor specific neo-antigenic nucleic acid molecules. In some embodiments, the vaccine comprises from about 1 to about 200 nucleic acid sequences that encode neoantigens or neoantigenic epitopes. In some embodiments, the neoantigenic epitopes are from about 8 to about 15 amino acids in length encoded by a nucleic acid sequence.

[0154] In some embodiments, the vaccine comprises from about 2 to about 100, from about 2 to about 58, from about 2 to about 29, or over 20 nucleic acid sequences that encode neoantigens or neoantigenic epitopes. In certain embodiments, the vaccine will include about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleic acid sequences that encode neoantigens or neoantigenic epitopes.DOCKET NO: WIST-020-PCT PCT APPLICATION

[0155] In certain embodiments, the vaccine will include about 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleic acid sequences that encode neoantigens or neoantigenic epitopes.

[0156] In certain embodiments, the vaccine will include about 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 nucleic acid sequences that encode neoantigens or neoantigenic epitopes.

[0157] In certain embodiments, the vaccine will include about 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 or 90 nucleic acid sequences that encode neoantigens or neoantigenic epitopes.

[0158] In certain embodiments, the vaccine will include about 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 nucleic acid sequences that encode neoantigens or neoantigenic epitopes.

[0159] In some embodiments, the vaccines comprising nucleic acid sequence encoding tumor antigens or antigens from tissue exhibiting a hyperproliferative disorder encode from about 30 to about 35 amino acids in length for one or a plurality of the antigen sequences. In some embodiments, each antigen sequence encoding a neoantigen encodes an antigen or antigenic fragment thereof that has a length from about 30 to about 35 amino acids.

[0160] In certain embodiments, the vaccine composition is capable of enhancing a CD8+ T cell immune response in a subject. In some embodiments, enhancing the CD8+ T cell immune response comprises activating CD8+ T cells. In another embodiment, enhancing the CD8+ T cell immune response comprises expanding CD8+ T cells. In other embodiments, the vaccine composition is capable of raising a specific cytotoxic T-cells response and / or a specific helper T-cell response.

[0161] The vaccine composition can further comprise an adjuvant and / or a carrier.

[0162] Adjuvants are described herein and are any substance whose admixture into the vaccine composition increases or otherwise modifies the immune response to the mutant peptide. Carriers are scaffold structures, for example a polypeptide or a polysaccharide, to which the neo-antigenic peptides, is capable of being associated. Optionally, adjuvants are conjugated covalently or non-covalently to the peptides or polypeptides of the disclosure.

[0163] The ability of an adjuvant to increase the immune response to an antigen is typically manifested by a significant increase in immune-mediated reaction, or reduction in diseaseDOCKET NO: WIST-020-PCT PCT APPLICATIONsymptoms. For example, an increase in humoral immunity is typically manifested by a significant increase in the titer of antibodies raised to the antigen, and an increase in T-cell activity is typically manifested in increased cell proliferation, or cellular cytotoxicity, or cytokine secretion. An adjuvant may also alter an immune response, for example, by changing a primarily humoral or Th2 response into a primarily cellular, or Thl response. Suitable adjuvants are described herein.

[0164] A vaccine composition according to the present disclosure may comprise more than one different adjuvant. Furthermore, the disclosure encompasses a therapeutic composition comprising any adjuvant substance including any of the above or combinations thereof. It is also contemplated that the nucleic acid molecule, and the adjuvant can be administered separately in any appropriate sequence.

[0165] Cytotoxic T-cells (CTLs) recognize an antigen in the form of a peptide bound to an MHC molecule rather than the intact foreign antigen itself. The MHC molecule itself is located at the cell surface of an antigen presenting cell. Thus, an activation of CTLs is only possible if a trimeric complex of peptide antigen, MHC molecule, and APC is present. Therefore, in some embodiments the vaccine composition according to the present disclosure additionally contains at least one antigen-presenting cell.

[0166] The antigen-presenting cell (or stimulator cell) typically has an MHC class I or II molecule on its surface, and in some embodiments is substantially incapable of itself loading the MHC class I or II molecule with the selected antigen.

[0167] In some embodiments, the antigen-presenting cells are dendritic cells. In some embodiments, the dendritic cells are autologous to a subject. In some embodiments of the present disclosure the antigen presenting cell comprises an expression construct comprising the nucleic acid molecules of the present disclosure. The nucleic acid molecules are capable of transducing the dendritic cell, thus resulting in the presentation of a peptide and induction of immunity.

[0168] The disclosure features a method of making an individualized cancer vaccine for a subject suspected of having or diagnosed with a cancer, comprising identifying a plurality of mutations in a sample from the subject; analyzing the plurality of mutations to identify one or more neoantigen mutations; and producing, based on the identified subset, a personalized cancer vaccine.

[0169] In some embodiments, identifying comprises sequencing the cancer. Methods for carrying out sequencing are described herein.DOCKET NO: WIST-020-PCT PCT APPLICATION

[0170] In some embodiments, identifying comprises sequencing the tumor associated antigens from one or a plurality of cells exhibiting a hyperproliferative disorder.

[0171] In some embodiments, analyzing further comprises determining one or more binding characteristics associated with the neoantigen mutation, the binding characteristics selected from the group consisting of binding of the subject-specific peptides to T-cell receptor, binding of the subject-specific peptides to a HLA protein of the subject and binding of the subject-specific peptides to transporter associated with antigen processing (TAP); and ranking, based on the determined characteristics, each of the neo-antigenic mutations.

[0172] In some embodiments, the method further comprises cloning nucleic acid sequences encoding the one or plurality of neoantigen mutations into a nucleic acid molecule.

[0173] In some embodiments, the nucleic acid molecule is a plasmid. In another embodiment, the nucleic acid molecule comprises a nucleic acid sequence of Formula I that is positioned within the multiple cloning site of a plasmid selected from the group consisting of selected from the group consisting of pGXOOOl, pGX4501, pGX4503, pGX4504, pGX4505, pGX4506 and pGX6001.

[0174] In some embodiments, the nucleic acid sequence of Formula I is positioned with the multiple cloning site of pGXOOOl . In some embodiments, the nucleic acid sequence of Formula I is positioned with the multiple cloning site of pGX4501. In some embodiments, the nucleic acid sequence of Formula I is positioned with the multiple cloning site of pGX4503.

[0175] In some embodiments, the nucleic acid sequence of Formula I is positioned with the multiple cloning site of pGX4504. In some embodiments, the nucleic acid sequence of Formula I is positioned with the multiple cloning site of pGX4505. In some embodiments, the nucleic acid sequence of Formula I is positioned with the multiple cloning site of pGX4506. In some embodiments, the nucleic acid sequence of Formula I is positioned with the multiple cloning site ofpGX6001.

[0176] In some embodiments, the plasmid is pGXOOOl. In some embodiments, the plasmid comprises the backbone and linker sequence of pGXOOOl comprising a nucleic acid sequence that is an expressible nucleic acid sequence, the expressible nucleic acid sequence comprising at least two or more AEDs that encoding one or more neoantigens from a subject; and wherein at least one neoantigen is an amino acid associated with a T cell lymphoma or B cell lymphoma.DOCKET NO: WIST-020-PCT PCT APPLICATION

[0177] In some embodiments, the plasmid is pGX4505. In some embodiments, the plasmid comprises the backbone and linker sequence of pGX4505 with at least two or more AED nucleotide sequence encoding one or more neoantigens from a subject.Methods

[0178] In some embodiments, a method of treating and / or preventing cancer in a subject comprises administering to the subject in need thereof a pharmaceutically effective amount of any of the nucleic acid molecules, or any of the pharmaceutical compositions described in this disclosure. In some embodiments, treatment is determined by a clinical outcome, an increase, enhancement or prolongation of anti-cancer activity by T cells, an increase in the number of anti-cancer T cells or activated T cells as compared with the number prior to treatment, or a combination thereof. In some embodiments, the outcome is one or more of retard cancer growth, induce cancer cell death, induce cancer regression, prevent or delay cancer recurrence, prevent cancer growth, prevent cancer spread and / or induce cancer elimination.

[0179] One of skill in the art can determine which therapeutic regimen is appropriate on a subject by subject basis, depending, for example, on the state of the cancer and their immune status (e.g., T-cell, B cell or NK cell activity and / or numbers).

[0180] According to the present disclosure, a host cell can be transfected in vivo (i.e., in an animal) or ex vivo (i.e., outside of an animal). Transfection of a nucleic acid molecule into a host cell can be accomplished by any method by which a nucleic acid molecule can be inserted into the cell. Transfection techniques include, but are not limited to, transfection, electroporation, microinjection, lipofection, adsorption, and protoplast fusion.

[0181] In some embodiments, the disclosure relates to a composition comprising one, two, three or more nucleic acid molecules, each nucleic acid molecule comprising at least one coding sequence comprising one or more of the first nucleic acid sequence, the second nucleic acid sequence, the third nucleic acid sequence, the fourth nucleic acid sequence, the fifth nucleic acid sequence, or the sixth nucleic acid sequence. In some embodiments, the sixth nucleic acid sequence comprises at least one AED that is a neoantigen and at least AED that is a tumor associated antigen that is not derived from a subject. In some embodiments, the the sixth nucleic acid sequence comprises at least one coding sequence comprising at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 AEDs derived from a subject.DOCKET NO: WIST-020-PCT PCT APPLICATIONIn some embodiments, the sixth nucleic acid sequence comprises at least one coding sequence comprising at least about 20 AEDs derived from a subject. In some embodiments, the sixth nucleic acid sequence comprises at least one coding sequence comprising at least about 30 AEDs derived from a subject, the sixth nucleic acid sequence comprises at least one coding sequence comprising at least about 40 AEDs derived from a subject. In some embodiments, the sixth nucleic acid sequence comprises at least one coding sequence comprising at least about 50 AEDs derived from a subject. In some embodiments, the sixth nucleic acid sequence comprises at least one coding sequence comprising at least about 60 AEDs derived from a subject.

[0182] In some embodiments, the sixth nucleic acid sequence comprises at least one coding sequence comprising at least about 10 AEDs that are each independently a cancer associated antigen that is not derived from a subject. In some embodiments, the sixth nucleic acid sequence comprises at least about 20 AEDs that are each independently a cancer associated antigen that is not derived from a subject. In some embodiments, the sixth nucleic acid sequence comprises at least one coding sequence comprising at least about 30 AEDs that are each independently a cancer associated antigen that is not derived from a subject, n some embodiments, the sixth nucleic acid sequence comprises at least one coding sequence comprising at least about 40 AEDs that are each independently a cancer associated antigen that is not derived from a subject. In some embodiments, the sixth nucleic acid sequence comprises at least one coding sequence comprising at least about 50 AEDs that are each independently a cancer associated antigen that is not derived from a subject. In some embodiments, the sixth nucleic acid sequence comprises at least one coding sequence comprising at least about 60 AEDs that are each independently a cancer associated antigen that is not derived from a subject. Any ratio of nucleic acid seqeunce encoding a neoantigemnucleic acid seqeunce encoding a tumor associated antigen not derived from the subject may be included in the embodiments, such as 1:1, 2:1:, 1:2, 1:4, 4:1, 5:1, 1:5, 1:3, 3:1, etc.

[0183] When the agents described herein are administered as pharmaceuticals to humans or animals, they can be given per se or as a pharmaceutical composition containing active ingredient in combination with a pharmaceutically acceptable carrier, excipient, or diluent.

[0184] Actual dosage levels and time course of administration of the active ingredients in the pharmaceutical compositions of the invention can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. Generally, agents orDOCKET NO: WIST-020-PCT PCT APPLICATIONpharmaceutical compositions of the invention are administered in an amount sufficient to reduce or eliminate symptoms associated with viral infection and / or autoimmune disease.

[0185] The composition comprising one or a plurality of nucleic acid molecules described herein preferably comprise DNA quantities of from about 1 nanogram to 10 milligrams; about 1 microgram to about 10 milligrams; or preferably about 0.1 microgram to about 10 milligrams; or more preferably about 100 microgram to about 1 milligram. In some preferred embodiments, DNA plasmid vaccines according to the present invention comprise about 5 nanograms to about 1000 micrograms of DNA. In some preferred embodiments, the DNA plasmid vaccines contain about 10 nanograms to about 800 micrograms of DNA. In some preferred embodiments, the DNA plasmid vaccines contain about 0.1 to about 500 micrograms of DNA. In some preferred embodiments, the DNA plasmid vaccines contain about 1 to about 350 micrograms of DNA. In some preferred embodiments, the DNA plasmid vaccines contain about 25 to about 250 micrograms of DNA. In some preferred embodiments, the DNA plasmid vaccines contain about 100 microgram to about 1 milligram DNA.

[0186] The pharmaceutical compositions according to the present invention are formulated according to the mode of administration to be used. In cases where pharmaceutical compositions are injectable pharmaceutical compositions, they are sterile, pyrogen free and particulate free. An isotonic formulation is preferably used. Generally, additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol and lactose. In some cases, isotonic solutions such as phosphate buffered saline are preferred. Stabilizers include gelatin and albumin. In some embodiments, a vasoconstriction agent is added to the formulation.

[0187] Preferably the DNA formulations for use with a muscle or skin EP device described herein have high DNA concentrations, preferably concentrations that include microgram to tens of milligram quantities, and preferably milligram quantities, of DNA in small volumes that are optimal for delivery to the skin, preferably small injection volume, ideally 25-200 microliters (pL). In some embodiments, the DNA formulations have high DNA concentrations, such as 1 mg / mL or greater (mg DNA / volume of formulation). More preferably, the DNA formulation has a DNA concentration that provides for gram quantities of DNA in 200 pL of formula, and more preferably gram quantities of DNA in 100 pL of formula.

[0188] The DNA plasmids for use with the electroporation devices of the present invention can be formulated or manufactured using a combination of known devices and techniques, but preferablyDOCKET NO: WIST-020-PCT PCT APPLICATIONthey are manufactured using an optimized plasmid manufacturing technique that is described in U.S. Patent Application Publication No. 20090004716, incorporated by reference in its entirety herein. In some examples, the DNA plasmids used in these studies can be formulated at concentrations greater than or equal to 10 mg / mL. The manufacturing techniques also include or incorporate various devices and protocols that are commonly known to those of ordinary skill in the art, in addition to those described in U.S. Patent Application Publication No. 20090004716 and those described in U.S. Patent No. 7,238,522, incorporated by reference in their entireties herein. The high concentrations of plasmids used with the skin electroporation devices and delivery techniques described herein allow for administration of plasmids into the ID / SC space in a reasonably low volume and aids in enhancing expression and immunization effects.

[0189] Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose, as hereinabove recited, or an appropriate fraction thereof, of the administered ingredient.

[0190] The dosage regimen for treating a disorder or a disease with the tumor specific neo-antigenic peptides of this invention and / or compositions of this invention is based on a variety of factors, including the type of disease, the age, weight, sex, medical condition of the patient, the severity of the condition, the route of administration, and the particular compound employed. Thus, the dosage regimen may vary widely, but can be determined routinely using standard methods.

[0191] The amounts and dosage regimens administered to a subject will depend on a number of factors, such as the mode of administration, the nature of the condition being treated, the body weight of the subject being treated and the judgment of the prescribing physician.

[0192] The quantity of DNA included within therapeutically active formulations according to the present embodiments is an effective amount for treating the disease or condition. Determination of an effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. Generally, an efficacious or effective amount of an agent is determined by first administering a low dose of the agent(s) and then incrementally increasing the administered dose or dosages until a desired effect (e.g., reduce or eliminate symptoms associated with viral infection or autoimmune disease) is observed in the treated subject, with minimal or acceptable toxic side effects. Applicable methods for determining an appropriate dose and dosing schedule for administration of a pharmaceutical composition of the present invention are described, for example, in Goodman and Gilman's The Pharmacological Basis of Therapeutics, Goodman et al., eds., 11th Edition, McGraw-Hill 2005, and Remington: The Science and PracticeDOCKET NO: WIST-020-PCT PCT APPLICATIONof Pharmacy, 20th and 21 st Editions, Gennaro and University of the Sciences in Philadelphia, Eds., Lippencott Williams & Wilkins (2003 and 2005), each of which is hereby incorporated by reference.

[0193] In some embodiments, the pharmaceutical composition is administered once daily; in other embodiments, the pharmaceutical composition is administered twice daily; in yet other embodiments, the pharmaceutical composition is administered once every two days, once every three days, once every four days, once every five days, once every six days, once every seven days, once every two weeks, once every three weeks, once every four weeks, once every two months, once every six months, or once per year. The dosing interval can be adjusted according to the needs of individual patients. For longer intervals of administration, extended release or depot formulations can be used.

[0194] In some embodiments, several divided dosages, as well as staggered dosages, can be administered daily or sequentially, or the dose can be continuously infused, or can be a bolus injection. Further, the dosages of the compound(s) of the disclosure can be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.

[0195] In some embodiments, the present disclosure also relates to methods for administration of the pharmaceutical compositions described herein using a prime-boost regimen. The term of "prime- boost" refers to the successive administrations of two different immunogenic or immunological composition types having at least one immunogen in common. The priming administration (priming) is the administration of a first immunogenic or immunological composition type and may comprise one, two or more administrations. The boost administration is the administration of a second immunogenic or immunological composition type and may comprise one, two or more administrations, and, for instance, may comprise or consist essentially of annual administrations. The "boost" may be administered from about 2 weeks to about 32 weeks after the "priming", or from about 4 to about 30 weeks after the priming, or from about 8 to about 28 weeks after the priming, advantageously from about 16 to about 24 weeks after the priming, and more advantageously, about 24 weeks after the priming.

[0196] The pharmaceutical compositions described herein can be used to treat diseases and disease conditions that are acute, and may also be used for treatment of chronic conditions. In certain embodiments, the pharmaceutical composition of the invention are administered for time periods exceeding two weeks, three weeks, one month, two months, three months, four months, fiveDOCKET NO: WIST-020-PCT PCT APPLICATIONmonths, six months, one year, two years, three years, four years, or five years, ten years, or fifteen years; or for example, any time period range in days, months or years in which the low end of the range is any time period between 14 days and 15 years and the upper end of the range is between 15 days and 20 years (e.g., 4 weeks and 15 years, 6 months and 20 years). In some cases, it may be advantageous for the pharmaceutical composition of the invention to be administered for the remainder of the patient's life. In preferred embodiments, the patient is monitored to check the progression of the disease or disorder, and the dose is adjusted accordingly. In preferred embodiments, treatment according to the invention is effective for at least two weeks, three weeks, one month, two months, three months, four months, five months, six months, one year, two years, three years, four years, or five years, ten years, fifteen years, twenty years, or for the remainder of the subject's life.

[0197] In some embodiments, the disclosure relates to a method treating T cell lymphoma in a subject in need thereof comprises administering to the a pharmaceutically effective amount of any of one or more nucleic acid molecules, a pharmaceutical composition comprising the one or more nucleic acid molecules, or any of the pharmaceutical compositions as described in this disclosure. In some embodiments, the treatment is of sufficient magnitude or efficacy to inhibit or retard cancer growth, induce cancer cell death, induce cancer regression, prevent or delay cancer recurrence, prevent cancer growth, prevent cancer spread and / or induce cancer elimination. In some embodiments, the T cell lymphoma is a peripheral T cell lymphoma. In some embodiments, the step of administering is repeated at least two times over the course of 3, 4, 5, 6, 7, or 8 weeks. In some embodiments, the pharmaceutical composition comprises at least one nucleic acid molecule comprising a nucleic acid sequence encoding one or more subunits of IL-12 or a functional variant thereof. In some embodiments, the method further comprises administering one or more chemotherapeutic agents. In some embodiments, the step of administering is performed orally, intravenously, sub-cutaneously, intramuscularly, intravaginally, intradermally, or intraperitoneally. In some embodiments, at least one of, a plurality of, or each of the one or more nucleic acid molecules comprise:(i) a DNA backbone region, and(ii) an expressible nucleic acid sequence operably linked to a regulatory sequence. In some embodiments, the expressible nucleic acid sequence is a DNA comprising Formula I that encodes a chimeric polypeptide comprising a concatemer of a linked series of neoantigens or antigenicDOCKET NO: WIST-020-PCT PCT APPLICATIONdeterminants thereof from the cancer, Formula T comprising independently selected, non-repetitive antigen expression domains (AED):(AEDi)-(linker)i-(AED2)-(linker)2-(AED3)-(linker)3- . . . (AEDn)-(linker)n- [AEDn+l] Formula I,In some embodiments, at least one of, a plurality of, or each AED encodes one of the neoantigens or antigenic determinants thereof. In some embodiments, at least one of, a plurality of, or each linker has a length independently selectable from about 15 to about 300 nucleotides and encodes a furin cleavage site. In some embodiments, the length at least one, a plurality of, or each AED is independently selectable from about 90 to about 250 nucleotides. In some embodiments, n is any positive integer from about 10 to about 100. In some embodiments, the DNA backbone region comprises a backbone of a pVAX vector. In some embodiments, n is any positive integer from 19 to about 60. In some embodiments, n is any positive integer from 19 to about 55. In some embodiments, n is any positive integer from 19 to about 58. In some embodiments, n is any positive integer from 19 to about 70. In some embodiments, n is any positive integer from 19 to about 80. In some embodiments, n is any positive integer from 19 to about 90. In some embodiments, n is any positive integer from 19 to about 95. In some embodiments, n is any positive integer from 19 to about 110. In some embodiments, at least one, a plurality of, or each of the AED encodes one or more epitope, antigen, or neoantigen herein or functional variant thereof comprising at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity thereto.

[0198] In some embodiments, the disclosure relates to a method treating B cell lymphoma in a subject in need thereof comprises administering to the a pharmaceutically effective amount of any of one or more nucleic acid molecules, a pharmaceutical composition comprising the one or more nucleic acid molecules, or any of the pharmaceutical compositions as described in this disclosure. In some embodiments, the treatment is of sufficient magnitude or efficacy to inhibit or retard cancer growth, induce cancer cell death, induce cancer regression, prevent or delay cancer recurrence, prevent cancer growth, prevent cancer spread and / or induce cancer elimination. In some embodiments, the step of administering is repeated at least two times over the course of 3, 4, 5, 6, 7, or 8 weeks. In some embodiments, the pharmaceutical composition comprises at least one nucleic acid molecule comprising a nucleic acid sequence encoding one or more subunits of IL- 12 or a functional variant thereof. In some embodiments, the method further comprisesDOCKET NO: WIST-020-PCT PCT APPLICATIONadministering one or more chemotherapeutic agents. In some embodiments, the step of administering is performed orally, intravenously, sub-cutaneously, intramuscularly, intravaginally, intradermally, or intraperitoneally. In some embodiments, at least one of, a plurality of, or each of the one or more nucleic acid molecules comprise:(i) a DNA backbone region, and(ii) an expressible nucleic acid sequence operably linked to a regulatory sequence. In some embodiments, the expressible nucleic acid sequence is a DNA comprising Formula I that encodes a chimeric polypeptide comprising a concatemer of a linked series of neoantigens or antigenic determinants thereof from the cancer, Formula I comprising independently selected, non-repetitive antigen expression domains (AED):(AEDi)-(linker)i-(AED2)-(linker)2-(AED3)-(linker)3- . . . (AEDn)-(linker)n- [AEDn+l] Formula I,In some embodiments, at least one of, a plurality of, or each AED encodes one of the neoantigens or antigenic determinants thereof. In some embodiments, at least one of, a plurality of, or each linker has a length independently selectable from about 15 to about 300 nucleotides and encodes a furin cleavage site. In some embodiments, the length at least one, a plurality of, or each AED is independently selectable from about 90 to about 250 nucleotides. In some embodiments, n is any positive integer from about 10 to about 100. In some embodiments, the DNA backbone region comprises a backbone of a pVAX vector. In some embodiments, n is any positive integer from 19 to about 60. In some embodiments, n is any positive integer from 19 to about 55. In some embodiments, n is any positive integer from 19 to about 58. In some embodiments, n is any positive integer from 19 to about 70. In some embodiments, n is any positive integer from 19 to about 80. In some embodiments, n is any positive integer from 19 to about 90. In some embodiments, n is any positive integer from 19 to about 95. In some embodiments, n is any positive integer from 19 to about 110. In some embodiments, at least one, a plurality of, or each of the AED encodes one or more epitope, antigen, or neoantigen herein or functional variant thereof comprising at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity thereto.

[0199] In some embodiments, a method of enhancing an immune response against a plurality of heterogeneous hyperproliferative cells in a subject comprises administering to the subject in need thereof a pharmaceutically effective amount of any of the nucleic acid molecules or any of theDOCKET NO: WIST-020-PCT PCT APPLICATIONpharmaceutical compositions as described in this disclosure. In some embodiments, the immune response is of a sufficient magnitude or efficacy to inhibit or retard cancer growth, induce cancer cell death, induce cancer regression, prevent or delay cancer recurrence, prevent cancer growth, prevent cancer spread and / or induce cancer elimination.

[0200] In some embodiments, a method of inducing an immune response in a subject comprises administering to the subject in need thereof a pharmaceutically effective amount of any of the nucleic acid molecules or any of the pharmaceutical composition of the present disclosure. In some embodiments, the immune response is a CD8+ T cell immune response. In some embodiments, inducing the CD8+ T cell immune response comprises activating 0.01% to about 50% CD8+ T cells. In some embodiments, inducing the CD8+ T cell immune response comprises expanding CD8+ T cells.

[0201] In some embodiments, a method of enhancing an immune response in a subject comprises administering to the subject in need thereof a pharmaceutically effective amount of any of the nucleic acid molecules or any of the pharmaceutical compositions disclosed herein.

[0202] In some embodiments, the immune response is a CD8+ T cell immune response. In some embodiments, enhancing the CD8+ T cell immune response comprises activating 0.01% to about 50% CD8+ T cells. In some embodiments, enhancing the CD8+ T cell immune response comprises expanding CD8+ T cells.

[0203] In some embodiments, a method of identifying one or more subject-specific DNA neoantigen mutations in a subject, wherein the subject has a cancer characterized by the presence or quantity of a plurality of neoantigen mutations comprises:sequencing a nucleic acid sample from a tumor of the subject and of a non-tumor sample of the subject;analyzing the sequence to determine coding and non-coding regions;identifying sequences comprising tumor-specific non-synonymous or non-silent mutations not present in the non-tumor sample;identifying single nucleotide variations and single nucleotide insertions and deletions; producing subject-specific peptides encoded by the sequences comprising tumor-specific non-synonymous or non-silent mutations not present in the non-tumor sample; and measuring the binding characteristics of the of the subject-specific peptides,DOCKET NO: WIST-020-PCT PCT APPLICATIONwherein each subject-specific peptide is an expression product of subject-specific DNA neoantigen not present in the non-tumor sample,thereby identifying one or more subject-specific DNA neoantigens in a subject.

[0204] In some embodiments, the step of measuring the binding characteristics of the of the subject-specific peptides is carried out by one or more of:measuring the binding of the subject-specific peptides to T-cell receptor; measuring the binding of the subject-specific peptides to a HLA protein of the subject; or measuring the binding of the subject-specific peptides to transporter associated with antigen processing (TAP).

[0205] In some embodiments, the subject-specific peptides bind to HLA proteins of the subject with an IC50 of less than about 500 nM. In some embodiments, the step of ranking the subjectspecific peptides based on the binding characteristics. In some embodiments, the method further comprises the step of measuring the CD8+ T cell immune response generated by the subjectspecific peptides. In some embodiments, the method further comprises formulating the subjectspecific DNA neoantigens into a immunogenic composition for administration to the subject. In some embodiments, about 200 ranked neoantigen mutations are included in the immunogenic composition.

[0206] In some embodiments, the method further comprises steps of providing a culture comprising dendritic cells obtained from the subject, and contacting the dendritic cells with the immunogenic composition. In some embodiments, the method further comprises steps of: administering to the subject the dendritic cells, obtaining a population of CD8+ T cells from a peripheral blood sample from the subject, wherein the CD8+ cells recognize the at least one neoantigen, and expanding the population of CD8+ T cells that recognizes the neoantigen. In some embodiments, the method further comprises administering to the subject the expanded population ofCD8+ T cells.

[0207] In some embodiments, a method of making an individualized cancer vaccine for a subject suspected of having or diagnosed with a cancer comprises:identifying a plurality of mutations in a sample from the subject;analyzing the plurality of mutations to identify one or more neoantigen mutations; and producing, based on the identified subset, a personalized cancer vaccine.DOCKET NO: WIST-020-PCT PCT APPLICATION

[0208] In some embodiments, the step of identifying comprises sequencing the cancer. In some embodiments, the step of analyzing further comprises determining one or more binding characteristics associated with the neoantigen mutation, the binding characteristics selected from the group consisting of binding of the subject-specific peptides to T-cell receptor, binding of the subject-specific peptides to a HLA protein of the subject and binding of the subject-specific peptides to transporter associated with antigen processing (TAP), and ranking, based on the determined characteristics, each of the neo-antigenic mutations.

[0209] In some embodiments, the method comprises cloning nucleic acid sequences encoding the one or plurality of TCRa, TCR0, TCRy, and / or neoantigen mutations into a nucleic acid molecule, such as a plasmid. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence of Formula I that is positioned within the multiple cloning site of a plasmid selected from the group consisting of pGX4501, pGX4503, pGX 4504, pGX4505, and pGX4506.

[0210] In some embodiments, the disclosure relates to a method of manufacturing a synthetic DNA vaccine. In some embodiments, the method comprises synthesizing a DNA comprising:(i) a DNA backbone region, and(ii) an expressible nucleic acid sequence operably linked to a regulatory sequence. In some embodiments, the expressible nucleic acid sequence is a DNA comprising Formula I that encodes a chimeric polypeptide comprising a concatemer of a linked series of neoantigens or antigenic determinants thereof from the cancer, Formula I comprising independently selected, non-repetitive antigen expression domains (AED):(AEDi)-(linker)i-(AED2)-(linker)2-(AED3)-(linker)3- . . . (AEDn)-(linker)n- [AEDn+l] Formula I,In some embodiments, at least one of, a plurality of, or each AED encodes one of the neoantigens or antigenic determinants thereof. In some embodiments, at least one of, a plurality of, or each linker has a length independently selectable from about 15 to about 300 nucleotides and encodes a furin cleavage site. In some embodiments, the length at least one, a plurality of, or each AED is independently selectable from about 90 to about 250 nucleotides. In some embodiments, n is any positive integer from about 10 to about 100. In some embodiments, the DNA backbone region comprises a backbone of a pVAX vector. In some embodiments, n is any positive integer from 19 to about 60. In some embodiments, n is any positive integer from 19 to about 55. In some embodiments, n is any positive integer from 19 to about 58. In some embodiments, n is any positiveDOCKET NO: WIST-020-PCT PCT APPLICATIONinteger from 19 to about 70. In some embodiments, n is any positive integer from 19 to about 80. In some embodiments, n is any positive integer from 19 to about 90. In some embodiments, n is any positive integer from 19 to about 95. In some embodiments, n is any positive integer from 19 to about 110. In some embodiments, at least one, a plurality of, or each of the AED encodes one or more epitope, antigen, or neoantigen herein or functional variant thereof comprising at least about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity thereto.Combination Therapy

[0211] According to embodiments of the disclosure, the pharmaceutical compositions described herein may be administered with one or more additional therapeutic agents. Various combination therapies contemplated by the present invention are described throughout.

[0212] In certain embodiments, any of the additional therapeutic agents is administered chronologically after or simultaneously with the DNA vaccine. In certain embodiments, the additional therapeutic agent is administered 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9, days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 1 month, or any combination thereof, before the DNA vaccine or immunogenic compositions is administered. In certain embodiments, the additional therapeutic agent is administered 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9, days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 1 month, or any combination thereof, after the DNA vaccine or immunogenic compositions is administered.

[0213] In an exemplary embodiment, the present invention is directed to an immunogenic composition, e.g., a vaccine, composition comprising the nucleic acid molecules described herein, capable of raising an immune response, and in particular a specific T-cell response.

[0214] DNA vaccines are described in US. Patent Nos. 5,593,972, 5,739,118, 5,817,637,

[0215] 5,830,876, 5,962,428, 5,981,505, 5,580,859, 5,703,055, 5,676,594, and the priority applications cited therein, which are each incorporated herein by reference. In addition to theDOCKET NO: WIST-020-PCT PCT APPLICATIONdelivery protocols described in those applications, alternative methods of delivering DNA are described in US. Patent Nos. 4,945,050 and 5,036,006, which are both incorporated herein by reference.

[0216] In certain embodiments, the vaccine composition comprises mutant neo-antigenic nucleic acid molecules as described herein (e.g. comprising a nucleic acid sequence comprising the formula: [ (antigen expression domain 1) - (linker) - (antigen expression domain 2) - (linker)] n), corresponding to tumor specific neo-antigens identified by the methods described herein. A suitable vaccine will preferably contain a plurality of tumor specific neo-antigenic nucleic acid molecules. In an embodiment, the vaccine will include between about 1 to about 200 nucleic acid molecules, between about 2 to about 100 nucleic acid molecules, between about 2 to about 58 nucleic acid molecules, between about 2 to about 29 nucleic acid molecules. In certain embodiments, the vaccine will include about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleic acid molecules. In certain embodiments, the vaccine will include about 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleic acid molecules. In certain embodiments, the vaccine will include about 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 nucleic acid molecules. In certain embodiments, the vaccine will include about 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 or 90 nucleic acid molecules. In certain embodiments, the vaccine will include about 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 nucleic acid molecules.

[0217] In certain embodiments, the vaccine composition is capable of enhancing a CD8+ T cell immune response in a subject. In some embodiments, enhancing the CD8+ T cell immune response comprises activating CD8+ T cells. In another embodiment, enhancing the CD8+ T cell immune response comprises expanding CD8+ T cells. In other embodiments, the vaccine composition is capable of raising a specific cytotoxic T-cells response and / or a specific helper T-cell response.

[0218] The vaccine composition can further comprise an adjuvant and / or a carrier.

[0219] Adjuvants are described herein, and are any substance whose admixture into the vaccine composition increases or otherwise modifies the immune response to the mutant peptide. Carriers are scaffold structures, for example a polypeptide or a polysaccharide, to which the neo-antigenicDOCKET NO: WIST-020-PCT PCT APPLICATIONpeptides, is capable of being associated. Optionally, adjuvants are conjugated covalently or non-covalently to the peptides or polypeptides of the invention.

[0220] The ability of an adjuvant to increase the immune response to an antigen is typically manifested by a significant increase in immune-mediated reaction, or reduction in disease symptoms. For example, an increase in humoral immunity is typically manifested by a significant increase in the titer of antibodies raised to the antigen, and an increase in T-cell activity is typically manifested in increased cell proliferation, or cellular cytotoxicity, or cytokine secretion. An adjuvant may also alter an immune response, for example, by changing a primarily humoral or Th2 response into a primarily cellular, or Thl response. Suitable adjuvants are described herein.

[0221] vaccine composition according to the present invention may comprise more than one different adjuvant. Furthermore, the invention encompasses a therapeutic composition comprising any adjuvant substance including any of the above or combinations thereof. It is also contemplated that the nucleic acid molecule, and the adjuvant can be administered separately in any appropriate sequence.

[0222] Cytotoxic T-cells (CTLs) recognize an antigen in the form of a peptide bound to an MHC molecule rather than the intact foreign antigen itself. The MHC molecule itself is located at the cell surface of an antigen presenting cell. Thus, an activation of CTLs is only possible if a trimeric complex of peptide antigen, MHC molecule, and APC is present. Therefore, in some embodiments the vaccine composition according to the present invention additionally contains at least one antigen presenting cell.

[0223] The antigen-presenting cell (or stimulator cell) typically has an MHC class I or II molecule on its surface, and In some embodiments is substantially incapable of itself loading the MHC class I or II molecule with the selected antigen.

[0224] Preferably, the antigen presenting cells are dendritic cells. In some embodiments, the dendritic cells are autologous to a subject. In some embodiments of the present invention the antigen presenting cell comprises an expression construct comprising the nucleic acid molecules of the present invention. The nucleic acid molecules are capable of transducing the dendritic cell, thus resulting in the presentation of a peptide and induction of immunity.

[0225] In one aspect, the disclosure features a method of making an individualized cancer vaccine for a subject suspected of having or diagnosed with a cancer, comprising identifying a plurality of mutations in a sample from the subject; analyzing the plurality of mutations to identify one or moreDOCKET NO: WIST-020-PCT PCT APPLICATIONneoantigen mutations; and producing, based on the identified subset, a personalized cancer vaccine.

[0226] In some embodiments, identifying comprises sequencing the cancer. Methods for carrying out sequencing are described herein.

[0227] In some embodiments, identifying comprises sequencing the cancer.

[0228] In another embodiment, analyzing further comprises determining one or more binding characteristics associated with the neoantigen mutation, the binding characteristics selected from the group consisting of binding of the subject-specific peptides to T-cell receptor, binding of the subject-specific peptides to a HLA protein of the subject and binding of the subject-specific peptides to transporter associated with antigen processing (TAP); and ranking, based on the determined characteristics, each of the neo-antigenic mutations.

[0229] In some embodiments, the method further comprises cloning nucleic acid sequences encoding the one or plurality of neoantigen mutations into a nucleic acid molecule.

[0230] In some embodiments, the nucleic acid molecule is a plasmid. In another embodiment, the nucleic acid molecule comprises a nucleic acid sequence of Formula I that is positioned within the multiple cloning site of a plasmid selected from the group consisting of selected from the group consisting of pGX4501, pGX4503, pGX 4504, pGX4505, and pGX4506. In some embodiments, the nucleic acid sequence of Formula I is positioned with the multiple cloning site of pGX4501. In some embodiments, the nucleic acid sequence of Formula I is positioned with the multiple cloning site of a plasmid selected from the group consisting of pGX4503. In some embodiments, the nucleic acid sequence of Formula I is positioned with the multiple cloning site of a plasmid selected from the group consisting of ppGX 4504. In some embodiments, the nucleic acid sequence of Formula I is positioned with the multiple cloning site of a plasmid selected from the group consisting of pGX4505. In some embodiments, the nucleic acid sequence of Formula I is positioned with the multiple cloning site of a plasmid selected from the group consisting of pGX4506. In some embodiments, the plasmid is pGX4505. In some embodiments, the plasmid comprises the backbone and linker sequence of pGX4505 with at least two or more AED nucleotide sdequence encoding one or more neoantigens from a subject.KitsDOCKET NO: WIST-020-PCT PCT APPLICATION

[0231] The present disclosure provides a kit comprising a pharmaceutical composition comprising one or a plurality of nucleic acid molecules as described herein. The components of the kit are preferably formulated in pharmaceutically acceptable carriers.

[0232] Also included in the kit are instructions for use in methods of treating cancer in a subject or enhancing a CD8+ T cell immune response in a subject.

[0233] The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture” (Freshney, 1987); “Methods in Enzymology” “Handbook of Experimental Immunology” (Wei, 1996); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Current Protocols in Molecular Biology” (Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994); “Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

[0234] Other embodiments are described in the following non-limiting Examples.References

[0235] The references cited throughout this application, are incorporated by reference in their entireties and as if each reference was fully set forth. A citation of a reference at a particular location indicates a manner(s) in which the teachings of the reference are incorporated. However, a citation of a reference at a particular location does not limit the manner in which all of the teachings of the cited reference are incorporated for all purposes.

[0236] It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but is intended to cover all modifications which are within the spirit and scope of the invention as defined by the appended claims; the above description; and / or shown in the attached drawings.DOCKET NO: WIST-020-PCT PCT APPLICATION1. A clinical evaluation of the International Lymphoma Study Group classification of nonHodgkin’s lymphoma. The Non-Hodgkin’s Lymphoma Classification Project. Blood 89(1997).2. Paola Ghione, Alison J. Moskowitz, Nadia E.K. De Paola, Steven M. Horwitz and Marco Ruella. Novel immunotherapies for T-cell lymphoma and leukemia. Current Hematologic malignancy reports 13(2018).3. Enrica Marchi, Owen A. O’Connor The rapidly changing landscape in mature T-cell lymphoma (MTCL) biology and management. CA: A Cancer Journal for Clinicians (2019).4. Swerdlow S, Campo E, Harris N et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues, in International Agency for Research on Cancer (Lyon, 2017). 5. Youn H Kim, Howard L Liu, Serena Mraz-Gernhard, Anna Varghese, Richard T Hoppe.Long-term outcome of 525 patients with mycosis fungoides and Sezary syndrome: clinical prognostic factors and risk for disease progression. Archives of Dermatology 139(2003).6. Bellei M, Foss FM, Shustov AR, Horwitz SM, Marcheselli L, Kim WS, et al. The outcome of peripheral T-cell lymphoma patients failing first line therapy: a report from the prospective, international T-Cell project. Haematologica (2018).7. DG., Maloney. Anti-CD20 antibody therapy for B-cell lymphomas. The New England Journal of Medicine 66, 2008-2016 (2012).8. June CH, O’Connor RS, Kawalekar OU, Ghassemi S, Milone MC. CAR T cell immunotherapy for human cancer. Science (New York, NY) 359, 1361-1365 (2018).9. Brody J, Kohrt H, Marabelle A, Levy R. Active and passive immunotherapy for lymphoma:proving principles and improving results. Journal of clinical oncology 29, 1864-1875 (2011).10. Xu-Monette ZY, Zhou J, Young KH. PD-1 expression and clinical PD-1 blockade in B-cell lymphomas. Blood 131, 68-83 (2018).11. Murray D, Eldershaw SA, Pearce H, Davies N, McMurray J, Scarisbrick JJ, et al. T Cell Versus T Cell; A Study of the Immune Checkpoint Landscape in Cutaneous T Cell Lymphoma. Blood 130(2017).DOCKET NO: WIST-020-PCT PCT APPLICATION12. Chang-Gon Kim, Yun-Beom Sang, Ji-Hyun Lee and Hong-Jae Chon. Combining Cancer Vaccines with Immunotherapy: Establishing a New Immunological Approach. International Journal of Molecular Sciences 22(2021).13. David Jung, Frederick W Alt. Unraveling V(D)J Recombination. Cell 116, 299-311 (2004).14. Schleiss, C.; Ilias, W.; Tahar, O.; Giiler, Y; Miguet, L.; Mayeur-Rousse, C.; Mauvieux, L.;Fomecker, L.M.; Toussaint, E.; Herbrecht, R.; et al. BCR-associated factors driving chronic lymphocytic leukemia cells proliferation ex vivo. Scientific Reports 9(2019).15. Carole L. BERGER, B. Jack LONGLEY, Suguru IMAEDA, Inger CHRISTENSEN, Peter HEALD and Richard L. EDELSON. TUMOR- SPECIFIC PEPTIDES IN CUTANEOUS T-CELL LYMPHOMA: ASSOCIATION WITH CLASS I MAJOR HISTOCOMPATIBILITY COMPLEX AND POSSIBLE DERIVATION FROM THE CLONOTYPIC T-CELL RECEPTOR. INternational Journal of Cancer 76, 304-311 (1998).16. Michael S Khodadoust, Niclas Olsson, Binbin Chen, Brian Sworder, Tanaya Shree, Chih Long Liu, Lichao Zhang, Debra K Czerwinski, Mark M Davis, Ronald Levy, Joshua E Elias, Ash AAlizadeh. B-cell lymphomas present immunoglobulin neoantigens. Blood 133, 878-881 (2019).17. Michael S Khodadoust, Niclas Olsson, Lisa E Wagar, Ole A W Haabeth, Binbin Chen, Kavya Swaminathan, Keith Rawson, Chih Long Liu, David Steiner, Peder Lund et al. Antigen presentation profiling reveals recognition of lymphoma immunoglobulin neoantigens. Nature 543, 723-727 (2017).18. Szymon J. Szymura, Lin Wang, Tiantian Zhang, Soung-chul Cha, Joo Song, Zhenyuan Dong, Aaron Anderson, Elizabeth Oh, Vincent Lee, Zhe Wang, et al. Personalized neoantigen vaccines as early intervention in untreated patients with lymphoplasmacytic lymphoma: a non-randomized phase 1 trial. Nature Communications 15(2024).19. Mardis, Elaine R. Neoantigens and genome instability: impact on immunogenomic phenotypes and immunotherapy response. Genome Medicine 11(2019).20. Elizabeth K. Duperret, Alfredo Perales-Puchalt, Regina Stoltz, Hiranjith G.H., Nitin Mandloi, James Barlow, Amitabha Chaudhuri, Niranjan Y. Sardesai and David B.Weiner. A Synthetic DNA, Multi-Neoantigen Vaccine Drives Predominately MHC Class I CD8+ T-cell Responses, Impacting Tumor Challenge. Cancer Immunology Research 7(2019).DOCKET NO: WIST-020-PCT PCT APPLICATION21. Vabret, Matthew Brown & Nicolas. Alternative RNA splicing generates shared clonal neoantigens across different types of cancer. Nature reviews immunology 24(2024).22. Na Xie, Guobo Shen, Wei Gao, Zhao Huang, Canhua Huang and Li Fu. Neoantigens:promising targets for cancer therapy. Signal Transduction and targeted therapy 8(2023).23. Weiner, Ebony N. Gary and David B. DNA vaccines: prime time is now. Current opinion in Immunology 65, 21-27 (2020).24. Pratik S Bhojnagarwala, Alfredo Peral es-Puchalt, Neil Cooch, Niranjan Y Sardesai, David B Weiner. A synDNA vaccine delivering neoAg collections controls heterogenous, multifocal murine lung and ovarian tumors via robust T cell generation. Molecular Therapy-Oncolytis 21(2021).25. Kimberly A Kraynyak, Michele A Kutzl er, Neil J Cisper, Amir S Khan, Ruxandra Draghia- Akli, Niranjan Y Sardesai, Mark G Lewis, Jian Yan, David B Weiner. Systemic immunization with CCL27 / CTACK modulates immune responses at mucosal sites in mice and macaques. Vaccine 28(2010).26. J I Sin, J J Kim, R L Arnold, K E Shroff, D McCallus, C Pachuk, S P McElhiney, M W Wolf, S J Pompa-de Bruin, T J Higgins, R B Ciccarelli, D B Weiner. IL- 12 gene as a DNA vaccine adjuvant in a herpes mouse model: IL-12 enhances Thl-type CD4+ T cell- mediated protective immunity against herpes simplex virus-2 challenge. Journal of Immunology 162(1999).27. Lauren A Hirao, Ling Wu, Amir S Khan, David A Hokey, lian Yan, Anlan Dai, Michael R Betts, Ruxandra Draghia-Akli, David B Weiner. Combined effects of IL-12 and electroporation enhances the potency of DNA vaccination in macaques. Vaccine 26(2008).28. Spyros A Kalams, Scott Parker, Xia lin, Mamie Elizaga, Barbara Metch, Maggie Wang, lohn Hural, Michael Lubeck, John Eldridge, et al. Safety and immunogenicity of an HIV- 1 gag DNA vaccine with or without IL-12 and / or IL-15 plasmid cytokine adjuvant in healthy, HIV-1 uninfected adults. PloS One 7(2012).29. Mark Yarchoan, Edward J. Gane, Thomas U. Marron, Renzo Perales-Linares, Jian Yan, Neil Cooch, Daniel H. Shu, Elana J. Fertig, Luciane T. Kagohara, Gabor Bartha et al. Personalized neoantigen vaccine and pembrolizumab in advanced hepatocellular carcinoma: a phase 1 / 2 trial. Nature Medicine 30, 1044-1053 (2024).DOCKET NO: WIST-020-PCT PCT APPLICATION30. Weiner, Elizabeth K. Duperret; Alfredo Perales-Puchalt; Regina Stoltz; Hiranjith G.H.;Nitin Mandloi; James Barlow; Amitabha Chaudhuri; Niranjan Y. Sardesai and David B. A Synthetic DNA, Multi-Neoantigen Vaccine Drives Predominately MHC Class I CD8+ T- cell Responses, Impacting Tumor Challenge. Cancer Immunology Research 7(2019). 31. Lundin J, Hagberg H, Repp R, Cavallin-Stahl E, Freden S, Juliusson G, et al. Phase 2 study of alemtuzumab (anti-CD52 monoclonal antibody) in patients with advanced mycosis fungoides / Sezary syndrome. Blood 101, 4267-4272 (2003).32. Karlin L, Coiffier B. The changing landscape of peripheral T-cell lymphoma in the era of novel therapies. Seminars in hematology 51(2014).33. Ramos CA, Ballard B, Zhang H, Dakhova O, Gee AP, Mei Z, et al. Clinical and immunological responses after CD30-specific chimeric antigen receptor-redirected lymphocytes. The Journal of Clinical Investigation 127, 3462-3471 (2017).34. Khodadoust M, Rook AH, Porcu P, Foss FM, Moskowitz AJ, Shustov AR, et al.Pembrolizumab for Treatment of Relapsed / Refractory Mycosis Fungoides and Sezary Syndrome: Clinical Efficacy in a Citn Multicenter Phase 2 Study. Blood 128(2016).35. Lesokhin AM, Ansell SM, Armand P, Scott EC, Halwani A, Gutierrez M, et al. Nivolumab in Patients With Relapsed or Refractory Hematologic Malignancy: Preliminary Results of a Phase lb Study. Journal of cliniccd oncology : official journal of the American Society of Clinical Oncology 34, 2698-2704 (2016).36. Chan TSY, Li J, Loong F, Khong PL, Tse E, Kwong YL. PD1 blockade with low-dose nivolumab in NK / T cell lymphoma failing L-asparaginase: efficacy and safety. Annals of hematology 97(2018).37. Kwong YL, Chan TSY, Tan D, Kim SJ, Poon LM, Mow B, et al. PD1 blockade with pembrolizumab is highly effective in relapsed or refractory NK / T-cell lymphoma failing 1- asparaginase. Blood 129, 2437-2442 (2017).38. Li X, Cheng Y, Zhang M, Yan J, Li L, Fu X, et al. Activity of pembrolizumab in relapsed / refractory NK / T-cell lymphoma. Journal of Hematology and oncology 11(2018).39. Ratner L, Waldmann TA, Janakiram M, Brammer JE. Rapid Progression of Adult T-Cell Leukemia-Lymphoma after PD-1 Inhibitor Therapy. The New England Journal of Medicine 378, 1947-1948 (2018).DOCKET NO: WIST-020-PCT PCT APPLICATION40. Kim YH, Gratzinger D, Harrison C, Brody ID, Czerwinski DK, Ai WZ, et al. In situ vaccination against mycosis fungoides by intratumoral injection of a TLR9 agonist combined with radiation: a phase 1 / 2 study. Blood 119, 355-363 (2012).41. Tanja Maier, Adrian Tun-Kyi, Anatoli Tassis, Karl -Peter Jungius, Gu' nter Burg, Reinhard Dummer, and Frank O. Nestle. Vaccination of patients with cutaneous T-cell lymphoma using intranodal injection of autologous tumor-lysate-pulsed dendritic cells. Blood 102, 2338-2344 (2003).42. Amy R. Rappaport, Chrisann Kyi, Monica Lane, Meghan G. Hart, Melissa L. Johnson, Brian S. Henick, Chih-Yi Liao, Amit Mahipal, Ardaman Shergill, Alexander I. Spira et al. A shared neoantigen vaccine combined with immune checkpoint blockade for advanced metastatic solid tumors: phase 1 trial interim results. Nature Medicine 30, 1013-1022 (2024).43. Luis A. Rojas, Zachary Sethna, Kevin C. Soares, Cristina Olcese, Nan Pang, Erin Patterson, Jay on Lihm, Nicholas Ceglia, Pablo Guasp, Alexander Chu, et al. Personalized RNA neoantigen vaccines stimulate T cells in pancreatic cancer. Nature 168(2023).44. Jeffrey S Weber, Matteo S Carlino, Adnan Khattak, Tarek Meniawy, George Ansstas, Matthew H Taylor, Kevin B Kim, Meredith McKean, Georgina V Long, Ryan J Sullivan et al. Individualised neoantigen therapy mRNA-4157 (V940) plus pembrolizumab versus pembrolizumab monotherapy in resected melanoma (KEYNOTE-942): a randomised, phase 2b study. Lancet 403, 632-644 (2024).45. Xiaofei Song, Shiun Chang, Lucia Seminario- Vidal, Alvaro de Mingo Pulido, Leticia Tordesillas, Xingzhi Song, Rhianna A. Reed, Andrea Harkins, Shannen Whiddon, Jonathan V. Nguyen. Genomic and single-cell landscape reveals novel drivers and therapeutic vulnerabilities of transformed cutaneous T-cell lymphoma. Cancer Discovery 12, 1294- 1313 (2022).46. Marina Tusup, Severin Lauchli, Natalia Teresa Jarzebska, Lars E. French, Yun-Tsan Chang, Maya Vonow-Ei senring, Andreas Su, Thomas M. Kundig, Emmanuella Guenova and Steve Pascolo. mRNA-Based Anti-TCR CDR3 Tumour Vaccine for T-Cell Lymphoma. MDPI Pharmaceutics 13(2021).DOCKET NO: WIST-020-PCT PCT APPLICATION47. Marie Gonthier, Re'gine Llobera, Jacques Arnaud and Bent Rubin. Self-Reactive T Cell Receptor-Reactive CD8+ T Cells Inhibit T Cell Lymphoma Growth In Vivo. The Journal of Immunology 173, 7062-7069 (2004).48. Karosiene E, Lundegaard C, Lund O, Nielsen M. NetMHCcons: a consensus method for the major histocompatibility complex class I predictions. Immunogenetics 64, 177-186 (2012).EXAMPLES

[0237] T cell lymphomas constitute approximately 10% of all non-Hodgkin lymphomas and are associated with poor prognosis. Patients experiencing early relapse exhibit a 5-year overall survival rate of 11%, underscoring the urgent need for novel therapeutic strategies. T cell cancers are predominantly clonal, making the T cell receptor (TCR) an appealing target for immunotherapy. In this study, we developed and evaluated a synDNA vaccine against the TCRa, P, and y chains (TCRfullvax) of the EL4 murine T cell lymphoma model. Immunogenicity studies revealed robust immune responses against all three TCR chains, with identification of immunodominant epitopes for each chain. Notably, no significant differences in the number of live T cells were observed between TCRfullvax-vaccinated mice and control groups, indicating the vaccine’s ability to selectively break tolerance against vaccinated TCR without broadly depleting T cells. In a minimal residual disease model, TCRfullvax delayed EL4 tumor progression. Analysis of tumors from TCRfullvax -treated mice revealed downregulation of TCR expression, suggesting a potential immune escape mechanism. Neoantigens, derived from somatic mutations within tumor cells, present another promising target for anti-cancer vaccine development and we developed a second vaccine targeting 15 neoantigens identified through sequencing of the EL4 cells (EL4neovax). The EL4neo vaccine elicited strong immune responses against 5 / 15 encoded neoantigens and was also able to control growth of EL4 tumors. Co-administration of the TCRfullvax and EL4neovax demonstrated superior tumor control compared to either vaccine alone, demonstrating that neoantigen targeting can partially mitigate TCR loss. These findings highlight the potential of combining TCR-targeted and neoantigen-based immunotherapies for the treatment of T-cell lymphomas. Further investigation of this dual-vaccine approach is warranted to optimize its therapeutic efficacy.DOCKET NO: WIST-020-PCT PCT APPLICATION

[0238] In this study, we tested for the first time whether a synDNA vaccine targeting the TCR can control the growth of EL4 tumors (a mouse syngeneic T cell lymphoma cell line). We observed robust T cell immune responses against the TCRa, TCRP and TCRy chains of the EL4 cell line. We identified the most immunogenic epitopes from each chain and found that most of them were part of the CDR regions of the TCR which highlights the potential safety of this approach as the CDR regions of each TCR are unique and there are minimal chances of cross reactivity against non-tumorigenic T cells. The TCRfull vaccine was able to control growth of EL4 tumors in vivo and enhanced survival of tumor bearing mice. However, we observed that tumors downregulate TCR expression suggesting a potential means that tumors develop in response to immune pressure to evade the immune system. We hypothesized that a second vaccine targeting neoantigens derived from EL4 tumors would further improve tumor control. We performed whole exome sequencing and RNA sequencing of EL4 tumors and identified 27 mutations. We selected 15 mutations to be included in the vaccine based on mRNA expression and predicted binding affinity to class I MHC. The EL4neos vaccine generated T cell immune responses against 33.3% of the neoantigens. These were both CD4+ and CD8+ T cell derived and led to control of EL4 tumors in vivo. Finally, we demonstrate that mice simultaneously treated with TCRfull and EL4neos vaccines had improved tumor control compared to those treated with either vaccine alone and this combination therapy significantly improved animal survival compared to those treated with empty vector controls. These results highlight that synDNA vaccines could be useful in treating T cell cancers and further development of synDNA vaccines for this difficult to treat cancer is warranted.Results

[0239] TCRfull vaccine design and characterization

[0240] For TCRfullvax, the sequences of the TCRa, TCR0 and TCRy chains were obtained from direct RNA sequencing data. The sequences were optimized for codon usage and mRNA expression. A codon optimized human IgE leader sequence was added at the beginning of the vaccine insert to improve the expression and induced immune responses as previously reported32. Each individual chain was separated by furin cleavage sites to allow for individual expression of single chains and minimize formation of junctional epitopes (FIG. 1A). For immunogenicity assessment, each mouse was immunized with 25ug DNA in a prime / boost schedule (FIG. IB). Spleens were harvested one week post the final vaccination and immunogenicity was measured via ZFNy ELISpot and intracellular cytokine staining by flow cytometry.DOCKET NO: WIST-020-PCT PCT APPLICATION

[0241] We observed the strongest immune response against the TCRy chain with a mean of 3900 IFNy spots per million splenocytes. This was followed by the TCRa chain (mean 633 IFNy spots), and the TCRP chain which was the least immunogenic with a mean of 362 IFNy spots (FIG. 1C). We performed epitope mapping using mixed matrix peptide pools to identify the most immunogenic epitopes for each TCR chain, the results of which are listed in Table 1. For the TCRa chain, the strongest immune response was against pools 2 and 7 (FIG. ID and FIG. 6A) which contain the epitope or SIFTNQ which is predicted to be in the top 1.5% of binders to HLA H2-Kb. This epitope is derived from the CDR1 region of the TCRa chain highlighting that the strongest immune response is being made against the variable regions of the TCR chain. The epitope SF SIFTNQ (SEQ ID NO: 27) was detected with a strong immune response. For TCRp chain, the immune responses were more diverse and pools 3, 4, 6, 8 and 9 elicited similar immune responses (FIG. IE and FIG. 1H). There are several epitopes contained in each pool that are predicted to be within the top 2% of binders to either HLA H2-Kb or H2-Db. Of these the epitope ASSTGTETL (SEQ ID NO: 28) (derived from the CDR3 region) is predicted to be within the top 1% of binders to HLAH2-Db and is represented in pools 4 and 9 as well as partially in pool 3 which are amongst the most immunogenic pools. For the TCRy chain, the strongest immune responses were against Pools 4 and 9 and pools 2 and 7 had the second highest immune response (FIG. IF and FIG. II). Pools 4 and 9 contain the epitope VWIEYSSGF (SEQ ID NO: 29) (derived from CDR3 region) and pools 2 and 7 contain the epitope VSLPCFSNT (SEQ ID NO: 30) (derived from CDR1 region) both of which are predicted to be within the top 1% of binders to HLA H2-Kb. These results highlight that a synDNA vaccine against the TCR chains can break tolerance which results in generation of strong immune responses against TCRa, TCRP and TCRy chains. This is further supported by the fact that we did not observe any differences in the number of total splenocytes or percentage of live CD3+ T cells amongst the naive mice and TCRfullvax mice (FIG. 7A and 7B) suggesting that the immune responses generated are specific for the EL4 TCR chains only and that we do not see any off target immune responses.

[0242] To further characterize the immune response via flow cytometry, we cultured splenocytes from vaccinated mice with peptide pools comprised of overlapping peptides derived from TCRa, TCRp or TCRy chains for 5 hrs. For the TCRa and TCRp chains, we detected IFNy, TNFa and IL2 expression on CD8+ T cells, and TNFa and IL2 expression on CD4+ T cells (FIGS. 8A-8C). For the TCRy chain, we observed robust secretion of all three cytokines by both CD4+ and CD8+DOCKET NO: WIST-020-PCT PCT APPLICATIONT cells (FIG. 8A-8C). This highlights that TCRfullvax can elicit functionally active T cells that can secrete multiple cytokines.

[0243] To test whether the immune responses generated are sufficient to control tumor growth, we vaccinated C57B16 mice three times, and one week post the final dose, we injected 2e5 EL4 tumor cells subcutaneously on the right flank (FIG. 2A). There was a significant delay in tumor growth in the vaccinated mice compared to mice immunized with empty vector pVax controls (FIG. 2Band FIG. 9A). The delay in tumor growth led to a statistically significant improvement in survival of TCRfullvax treated mice (Median survival 18 days vs 16 days) (FIG. 2D).

[0244] TCRfullvax treated tumors have greater CD8+ T cell infiltration

[0245] To further characterize the impact of vaccine generated immunity on the tumors, we collected tumors at the terminal timepoint, dissociated them into single cells and performed flow cytometry. We observed greater CD8+ T cell infiltration in the tumors from TCRfullvax treated mice compared to the pVax treated mice (FIG. 2G). These CD8+ T cells in tumors isolated from TCRfullvax mice were less exhausted and less anergic as evidenced by decreased expression of PD1 (FIG. 2H and 2J) and KLRG1 (FIG. 21 and 2K). Boolean gating for PD1 and KLRG1 revealed that 94.6% of the CD8+ T cells from TCRfullvax mice did not express either PD1 or KLRG1 and only 0.17% were PD1+ KLRG1+ double positive T cells (FIG. 9B and 9C). In contrast, only 59.25% of CD8+ T cells from pVax treated tumors were PD1- KLRG1- double negative and 2% of all CD8+ T cells were PD1+ KLRG1+ double positive (FIG. 9B and 9C). Intratumoral CD8+ T cells from pvax mice were 11.7 times more KLRG1+PD1+, 8.7 times more KLRG1- PD1+, 2.5 times more KLRG1+ PD1- and only 0.6 times KLRG1-PD1- compared to those from TCRfullvax treated mice (FIG. 9D). These data indicate that CD8+ T cells from pVax treated mice were more exhausted compared to those from TCRfullvax treated mice. We extended our analysis to different memory subsets of the CD8+ T cell population and observed lower expression of PD1 and KLRG1 on both central memory (Tan) and effector memory (Tetr) populations (FIG. 9E-9H).

[0246] To elucidate mechanisms of resistance to the TCRfullvax, we isolated tumors and digested them into single cell suspensions for flow cytometry analysis (FIG. 10). We observed a significant decrease in expression of TCR V 12 in the vaccinated mice vs control mice (FIG. 2E, 2F and FIG.11). This highlights a potential immune escape mechanism as the tumor has downregulated expression of target antigen in response to targeted immune pressure. It would be desirable to have longer term control even in this highly aggressive mouse model.DOCKET NO: WIST-020-PCT PCT APPLICATION

[0247] EL4neovax controls EL4 tumors in mice

[0248] To test whether the immune responses against neoantigens can impact EL4 tumor growth, we vaccinated mice 3 times and one week post final dose, we injected EL4 tumors on the right flank (FIG. 4A). We observed a statistically significant delay in the growth of EL4 tumors in mice vaccinated with EL4neovax compared to those immunized with empty vector controls (FIG. 4B and 4C). While there was a trend in improvement of survival in vaccinated animals (median survival 25 days vs 22 days), this did not reach statistical significance (p value=0.08) (FIG. 4D).

[0249] EL4neo vaccine design and characterization

[0250] Neoantigens are derived from mutations in the tumor cells and serve as important and unique targets for therapeutic cancer vaccines24,3°. We hypothesized that an immunogen design targeting neoantigens derived from the EL4 cells would be immunogenic and should also provide T cell control the growth of EL4 tumors. We identified a total of 27 nonsynonymous mutations that could potentially serve as neoantigen vaccine targets. We down selected 15 mutations to encode in our vaccine based on the selection criteria described previously30. The designed immunogen contained 8 single nucleotide variants, and 7 mutations derived from frameshift mutations (FIG. 3 A). Of the 1 , 2 had a predicted binding affinity of <500nM (high affinity), 7 had a binding affinity 500nM-2000nM (medium affinity) and 6 had a binding affinity lower than 2000nM (low affinity) based on NetMHC4.0 predictions (FIG. 3B). Each neoantigen was encoded as a 33 amino acid sequence and separated by furin cleavage sites (FIG. 3C). We immunized C57B16 mice as in FIG. IB and analyzed immune responses one week after final dose. Based on ELISPOT data, 5 / 15 mice elicited significantly higher numbers of IFNy SFUs compared to naive controls (FIG. 3D-3F). Of the neoantigens that elicited significant immunity, 4 / 5 were predicted to be medium affinity binders and 1 / 5 was predicted to be a high affinity binder to class I MHC (FIG. 3G). We next performed flow cytometry to further characterize immune responses in the mice (12A-12C). Of the 5 neoantigens that were immunogenic, 2 epitopes generated both CD4+ and CD8+ T cell responses, 2 epitopes generated CD4+ T cell response only and 1 epitope generated CD8+ T cell response only (FIG. 3G).

[0251] EL4neo vaccine controls EL4 tumors in mice

[0252] To test whether the immune responses against neoantigens can control E14 tumor growth, we vaccinated mice 3 times and one week post final dose, we injected EL4 tumors on the right flank (FIG. 4A). We observed a significant delay in the growth of EL4 tumors in mice vaccinated with E14neovax compared to those immunized with empty vector controls (FIG. 4B and 4C). WhileDOCKET NO: WIST-020-PCT PCT APPLICATIONthere was a small improvement in survival of vaccinated animals (median survival 25 days vs 22 days), this did not reach statistical significance (p value 0.08) (FIG. 4D).

[0253] Synergistic effect of TCRfull and EL4neovax

[0254] Given the impact of both individual vaccines in slowing EL4 tumor growth, we next tested whether the combination of both vaccines would be further synergistic in controlling this tumor challenge. We vaccinated C57BL63 times with both vaccines and injected EL4 tumors on the right flank 7 days post the third dose (FIG. 5A). Mice that received single vaccination had significantly delayed tumor growth compared to pVax controls. Mice that received dual vaccination, had smaller tumors than the mice that received either TCRfullvax or EL4neovax (FIG. 5B and 5C). All groups that received treatment significantly improved animal survival compared to pVax controls (FIG.5D and Table 2). These data highlight that the loss of antigen expression driven by the TCRfullvax can at least partially be overcome by combining with DNA vaccine targeting neoantigens.Discussion

[0255] Here, we demonstrate that a synDNA vaccine against the TCR of a T cell lymphoma is highly immunogenic and can impact the growth of TCR expressing tumors in vivo. TCRfullvax elicited strong CD4+ and CD8+ T cell responses and majority of the T cell responses were against the CDR regions suggesting that healthy T cells would be spared by the vaccine generated immune response. We observed CD8+ IFNy responses against all three chains (TCRa, TCRp and TCRy) included in the vaccine whereas CD4+ IFNy responses were only observed against the TCRy chain, highlighting the value of our vaccine in driving stronger CD8+ T cell responses. We observed a decrease in expression of TCR in mice vaccinated with TCRfullvax indicating a potential immune evasion strategy employed by the tumor cells. Several studies have demonstrated that tumor cells downregulate or modulate antigen expression in response to immunotherapies such as CART cells, Bispecific T cell engagers and vaccine induced immune pressure33'36. To counteract the loss of antigen expression, we sequenced the EL4 tumors and identified 27 potential neoantigens. We down selected 15 neoantigens based on their predicted binding affinity to Class I MHC and RNA expression data and designed a new EL4neovax construct. The EL4neovax elicited potent CD4+ and CD8+ T cell responses against 5 / 15 neoantigens, including against frameshift mutations, and controlled growth of EL4 tumors in mice. Historically, neoantigen based cancer vaccine development has focused predominantly on the targeting of neoantigens predicted to have high binding affinity to class I MHC (kd < 500 nm). TCLs and other hematological malignancies areDOCKET NO: WIST-020-PCT PCT APPLICATIONoften categorized as tumors with a low tumor mutational burden (TMB), with small numbers of targetable neoantigens and therefore unlikely to benefit from targeting of small numbers of private neoantigens37. Our data demonstrates that the immune system has the ability to drive strong T cell responses to more neoantigens including against those that are not predicted to be the highest affinity candidates. Indeed, we observed T cell responses to approximately similar proportions of predicted high and medium affinity neoantigens - i.e. 1 of 2 (50%) high affinity antigens and 4 of 7 (57.1%); cumulatively 5 / 9 (55.5%) medium and high affinity antigens - thereby expanding the tumor directed T cell repertoire via vaccination with a broader range of targetable neoantigens21’26’31. Finally, we show that animals treated with combination of TCRfullvax and EL4neovax had better tumor control compared to mice treated with either vaccine alone highlighting that downregulation of TCR expression to evade the immune response can at least partially be overcome by vaccinating against tumor derived neoantigens.

[0256] Immunotherapies such as monoclonal and bispecific antibodies, CART cells and ICI are playing an essential and growing role in cancer care over the last few years which has improved clinical outcomes for solid tumors and B cell cancers7'10. The role of immunotherapy in treatment of TCLs has been limited so far. Three monoclonal antibodies targeting CD52, CD30 and CCR4 have been approved for different subtypes of TCLs depending on antigen expression and clinical subtype2. Of these alemtuzumab, an anti-CD52 antibody had an overall response rate of -35% for the treatment of peripheral or cutaneous T cell lymphoma (PTCL or CTCL). Suppression of T cell activity is one of the side effects of anti-CD52 antibodies and treatment with alemtuzumab led to a high rate of opportunistic infections including some that were fatal in about 30% of the patients highlighting the risks associated with this therapy38’39. CART cells are another type of immunotherapy that are being tried for TCL therapy. Most targets for CART cell therapy are also expressed on healthy T cells, which creates the risk of fratricide which leads to manufacturing issues. It can also lead to T cell aplasia which can significantly increase the risk of infections and compromise the quality of life2. CART cells against CD30 have been tried in 2 patients with Anaplastic Large Cell Lymphoma (ALCL), one of which experienced a complete response without compromising anti-viral immunity40. This highlights the potential utility of CART cell therapy against TCLs and several clinical trials against different subtypes targeting different antigens are ongoing, the results of which will be exciting to see2. Amongst immune checkpoint inhibitors, in early-stage trials with small numbers of patients, anti-PDl inhibitors have demonstrated an ORRDOCKET NO: WIST-020-PCT PCT APPLICATIONof 15-100% in different TCL subtypes41'45. The highest response rates were observed in patients with NKTCLs which is linked with EBV infection which most likely provides additional antigens for T cell targeting and results in higher response rates. These results need to be confirmed in larger trials with more patients. Additionally, in cases of TCL, PD1 can also act as a tumor suppressor and PD1 inhibition can lead to accelerated tumor growth. Ratner et al reported this phenomenon in a clinical trial of adult T cell lymphoma-leukemia which was terminated after the first 3 patients experienced rapid progression of tumors after anti-PDl therapy46. This serves as a cautionary tale for the use of anti-PDl therapy for TCLs.

[0257] Anti-cancer vaccine therapy for TCL has been limited to using dendritic cells pulsed with whole tumor lysates and intratumoral injection of TLR9 agonist to stimulate CD8+ T cell activity in CTCL patients with limited efficacy47’48. In recent times, personalized therapeutic vaccines targeting neoantigens have demonstrated impressive results across different tumor types using different vaccine platforms such as DNA, RNA and adenoviral vectors31 49'51. This includes shrinking of large, established HCC tumors which is traditionally considered an immunologically cold tumor31. The neoantigen approach led to induction of CD8+ T cell responses in 50% of the patients (although with limited numbers of T cell clones driven) in PDAC patients, another type of cancer that is considered a low TMB tumor and is immunologically cold50. CTCL which is a relatively less aggressive form of TCL, is perhaps an optimal candidate for personalized anticancer vaccines targeting TCR and / or neoantigens. Recently Song et al described CTCL as having a high tumor mutation burden52which suggests that CTCL tumors could harbor a high number of neoantigens and it would be critical to target as many as possible to get optimal clinical response. The synDNA platform is optimally placed to address this need as preclinical and clinical studies have shown the feasibility of targeting up to 40 neoantigens simultaneously without compromising immune response or tumor control2531. In preclinical studies, a 40-mer vaccine was able to control growth of murine lung and ovarian tumors in the same mouse highlighting the value of including as many neoantigens as possible in controlling multifocal tumors with different mutational profiles25. This suggests that a synDNA vaccine targeting the TCR and neoantigens derived from CTCL could be effective in the potential treatment of CTCL52.

[0258] Targeting the TCR region of T cell lymphoma has previously been reported to provide some minimal protection in different mouse models36,53. Gonthier et al used a peptide-based vaccine against the TCR V012 variable region and observed protection in 40-60% of the miceDOCKET NO: WIST-020-PCT PCT APPLICATIONchallenged with L12R4 (a mouse TCL cell line)53. The study did not specifically analyze vaccine induced immune responses or reasons for why the vaccine was only effective in 40-60% of the mice. This strategy can potentially lead to immune responses against other TCRV 12 T cells which can also deplete healthy T cells. Tusup et al describe a mRNA-based vaccine encoding just the CDR3 region of a and P chains of the EL4 cell line which demonstrated some control of the EL4 tumor growth in mice. The authors also observed a decrease in TCR expression indicative of a potential escape mechanism. The authors do not report vaccine induced immune responses and while they did observe a delay in tumor growth, they did not observe an improvement in animal survival36. With our vaccine TCRfullvax designs, we did not see any changes in numbers of splenocytes or live T cells. Additionally, majority of the T cell response was against the CDR regions highlighting the safety of the vaccine construct and suggesting that healthy T cells would not be affected by the anti-vaccine immune responses.

[0259] Tumors from TCRfullvax treated mice exhibited greater CD8+ T cells compared to those from control mice. These CD8+ T cells also expressed lower levels of PD1 and KLRG1 compared to intra-tumoral CD8+ T cells from the control mice. High PD1 expression on T cells is associated with a more exhausted phenotype leading to decreased cytokine secretion, proliferation and cytotoxic potential54. KLRG1+ T cells are also poor at proliferation and cytokine productions suggesting diminished ability to kill the tumors53. Co-expression of PD1 and KLRG1 on T cells is further associated with an inhibitory phenotype and presence of PD1 KLRG1 double positive CD8+ T cells in the tumors would indicate a reduced ability of these T cells to fight the tumors36. Detailed flow cytometry analysis of the tumor infiltrating lymphocytes revealed that tumors from TCRfullvax treated mice had almost no CD8+ PD1+ KLRG1+ T cells whereas those from pVax treated mice had almost 2% of their CD8+ T cells co-express PD1 and KLRG1 (11.7-fold higher). Thus, TCRfullvax not only drives greater CD8+ T cells to the tumor, but these tumors are better equipped to control the tumor compared to controls suggesting a mechanistic insight into vaccine’s efficacy in slowing down tumor growth.

[0260] In conclusion, the combination of TCR and neoantigen targeting vaccine can be effective in controlling TCLs. This approach would most likely have synergistic effects with other immunotherapy approaches such as ICI which are also being developed for TCLs and IFNa which is used for some TCL therapy. Clinically, the combination of gene delivered pIL12 as a cytokine adjuvant has demonstrated improved T cell responses to vaccine antigens in the cancer andDOCKET NO: WIST-020-PCT PCT APPLICATIONinfectious disease settings31’?7'61. Such an approach has not been tested in settings of hematological cancers, and it would be interesting to study if cytokine adjuvants can further boost immune responses for enhanced treatment of TCLs. Further development of this approach for treatment of T cell lymphomas is warranted.>>>DOCKET NO: WIST-020-PCT PCT APPLICATIONMaterials and Methods

[0261] Animals and cell lines

[0262] 6- to 8-week-old female C57BL / 6 mice were purchased from The Jackson Laboratory. All animal experiments were approved by the Institutional Animal Care and Use Committee at The Wistar Institute. The EL4 cell line was purchased from ATCC. EL4 tumors were generated by injecting 200,000 EL4 cells into the right flank. All cell lines were maintained at low passage (<10 passages) and thawed directly from a master stock generated upon receipt of the cells for all experiments. The cell lines were not genetically authenticated but were examined for morphologic authenticity in cell culture.

[0263] Mice were vaccinated by injecting indicated amounts of DNA resuspended in 50 pL of water into the tibialis anterior muscle followed by electroporation with the CELLECTRA-3PDOCKET NO: WIST-020-PCT PCT APPLICATIONdevice (Inovio Pharmaceuticals). For each vaccination, mice were delivered two 0.1 -amp electric constant current square-wave pulses.

[0264] DNA and RNA sequencing

[0265] We sequenced EL4 cell lines from in vitro cultures. As a control, we used tails from C57B1 / 6 mice. The mouse exome and RNA sequencing were performed on the Illumina HiSeq-2500 platform. The SureSelect Mouse All Exon Kit (Agilent Technologies; cat #5190-4642) was used. All samples generated greater than 13 Gb of data, with greater than 98% of the exomes covered at >150*. Overall, 99% of the reads aligned to the mouse reference genome. Mapping quality for 80% of the aligned reads was >Q60. Duplicate % was low: 4%-6%. Somatic variant calling was performed using Strelka program vl.0.14 (Illumina Inc.). The identified somatic variants were further filtered (using Strelka parameters such as read filtering, indel calling, SNV calling, and other parameters described in https: / / github.com / Illumina / strelka.), and only passed and on-target variants were considered for further analysis.

[0266] The RNA sequencing was done using TrueSeq RNA library prep kit v2 (Illumina, cat. # G9641B). All samples generated >100 million reads. Reads mapping to the ribosomal and mitochondrial genome were removed before performing alignment. The reads were aligned using STAR (2.4.1) aligner (open source software distributed under GPLv3). Overall 96% to 98% of the total preprocessed reads were mapped to the reference gene model / genome (Mus musculus GRCm38 DNA). The gene expression was estimated using Cufflinks v2.2.1 (Trapnell and colleagues, Broad Institute of MIT and Harvard).

[0267] Vaccine design

[0268] For TCR full vaccine (or TCRfullvax), the sequences for the TCRa, TCRP and TCRy chains were obtained from the RNA sequencing data. Each chain was separated by furin cleavage sites to allow for individual expression of each chain and minimize formation of junctional epitopes.

[0269] For EL4_neovax (or EL4neovax), neoepitopes were prioritized from either nonsynonymous coding missense mutants or frameshift mutations, where the mutant allele expression was >1 FPKM. MHC class I binding analysis was performed for all coding mutations. The 9-mer epitopes were analyzed using NetMHCons v4.0the C57B1 / 6 MHC alleles (H-2-Kb and H-2-Db). We included a total of 15 neoantigens with a wide range of predicted binding affinitiesDOCKET NO: WIST-020-PCT PCT APPLICATIONfor class 1 MHC. For all mutations, we kept the 9-mer epitope including the mutation in the central position and included 12 wildtype amino acids on each side to create a 33-mer epitope. Each epitope was separated by furin cleavage sites.

[0270] The human IgE leader sequence was added to each construct to improve expression. All constructs were codon and RNA optimized for expression and encoded into pVaxl expression vector.

[0271] Flow cytometry

[0272] Antibodies conjugated to murine CD4 (RM4-5), CD8b (YTS 156.7.7), IFN-y (XMG1.2), TNF-a (MP6-XT22), and IL-2 (JES6-5H4) were acquired from BioLegend. A Live / Dead Violet viability kit from Invitrogen was utilized to exclude dead cells from the analysis. Intracellular cytokine production was analyzed by culturing splenocytes (1 million) from vaccinated mice with peptides (5 pg / mL) derived from corresponding mutated neoantigens (or pooled peptides derived from the TCRa, TCR0 and TCRy chains) along with a protein transport inhibitor (eBioscience) for 5 hours. Following surface staining, we permeabilized the splenocytes using the eBioscience FoxP3 staining kit. Data was acquired on a BD FAC Symphony (BD Biosciences) and analyzed with FlowJo. The neoantigen peptides were designed as 15-mer sequences overlapping by 9 amino acids, covering the entire 33-mer used in vaccination. We vaccinated mice three times at 2-week intervals euthanized mice one week after the final vaccination. We collected spleens, and splenocyte suspensions were prepared using a Stomacher 80 Biomaster (Thomas Scientific), followed by red blood cell lysis (Thermo Fisher Scientific).

[0273] ELISPOTs

[0274] Mice were vaccinated three times at 2-week intervals. 1 week following the final vaccination, splenocytes were harvested and co-incubated with each neoantigen-derived peptide pool comprising 15-mer overlapping by 9 aa (5 pg / mL). To determine immunogenic epitopes in TCR chains, peptides (15-mer overlapping by 9 aa) derived from TCRa and TCRy chains were pooled into 10 pools and TCRfl was pooled into 9 distinct pools. Splenocytes were cultured at 37°C. 24 h later, we performed the murine IFN-y ELISPOT according to the manufacturer’s instructions (Mabtech, no. 3321-4 APT- 10). Spots were quantified using MabTech IRIS Fluorospot / ELIspot reader, normalized to an unstimulated control (R10). Concanavalin A was used as a positive control. The threshold for immunogenicity was set at 30 SFU per million splenocytes.

[0275] Tumor challengeDOCKET NO: WIST-020-PCT PCT APPLICATION

[0276] Mice were vaccinated with indicated construct 3x at 2-week intervals. One week after the final dose, 200,000 EL4 cells (in PBS) were injected on the right flank subcutaneously. Tumor size was monitored via caliper measurements. Mice were euthanized when the length of tumor reached 20 mm or tumor volume reached greater than 2,000 mm3. Tumor volume was calculated using the formula V= [(length x width2) x 3.14] / 2, where width is considered the side with smaller measurement. Graph represents data from two independent experiments (n=5 mice / group). For TCR expression analysis, the tumors from a separate experiment were isolated at day 10 and broken down into a single cell suspension using the mouse tumor dissociation kit (Miltenyi Biotec). The cells were cultured for 7 days before staining for anti-mouse TCR and anti-mouse TCR VBeta 12 for FACS analysis.

[0277] Statistical analysis

[0278] The difference between the means of experimental groups was calculated using a Tukey’s multiple comparison test. Comparisons between two or more groups with multiple subjects was done using ordinary one-way ANOVA. Comparisons between tumor size at each time point were done using two-way ANOVA with Fisher’s least significant difference (LSD) test. Error bars represent standard error of the mean. For mouse survival analysis, significance was determined using a log-rank (Mantel-Cox) test. All statistical analyses were done using GraphPad Prism 10.0. p < 0.05 was considered statistically significant. Error bars represent SEM unless otherwise stated.

Claims

DOCKET NO: WIST-020-PCT PCT APPLICATIONCLAIMS1. A composition comprising at least a first nucleic acid molecule comprising expressible nucleic acid sequence:(i) one or a plurality of nucleic acid sequences encoding one or a plurality of T Cell Receptor (TCR) alpha subunits;(ii) one or a plurality of nucleic acid sequences encoding one or a plurality of TCR beta subunits; (iii) one or a plurality of nucleic acid sequences encoding one or a plurality of TCR gamma subunits; and(iv) one or a plurality of T cell antigens.

2. The composition of claim 1 comprising a first nucleic acid molecule and a second nucleic acid molecule, wherein the first nucleic acid molecule comprises:(i) one or a plurality of nucleic acid sequences encoding one or a plurality of T Cell Receptor (TCR) alpha amino acid sequences;(ii) one or a plurality of nucleic acid sequences encoding one or a plurality of TCR beta chain amino acid sequences; and(iii) one or a plurality of nucleic acid sequences encoding one or a plurality of TCR gamma chain amino acid sequences; andwherein the second nucleic acid molecule comprises one or a plurality of T cell antigens.

3. The composition of either of claims 1 or 2, wherein the first or second nucleic acid molecule comprising one or a plurality of T cell neoantigens comprises a regulatory sequence and an expressible nucleic acid sequence comprising Formula I or Formula II, wherein each antigen expression domain comprises a T cell neoantigen.

4. The composition of any of claims 1 through 3 comprising a first nucleic acid molecule and a second nucleic acid molecule, wherein the first nucleic acid molecule is SEQ ID NO:25 or a functional variant thereof comprising at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:25; and wherein second nucleic acid molecule isDOCKET NO: WIST-020-PCT PCT APPLICATIONSEQ ID NO:26 or a functional variant thereof comprising at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:26.

5. A pharmaceutical composition comprising a therapeutically effective amount of the composition of any of claims 1 through 4; and a pharmaceutically acceptable carrier.

6. The pharmaceutical composition of claim 5, wherein the therapeutically effective amount comprises from about 0.1 mg to about 100 mg of the first nucleic acid molecule.

7. The pharmaceutical composition of claim 5, wherein the therapeutically effective amount comprises from about 0.1 mg to about 100 mg of the first nucleic acid molecule; and from about 0.1 mg to about 100 mg of a second nucleic acid molecule.

8. A method of treating or preventing a hyperproliferative disorder in a subject in need thereof comprising administering to the subject the pharmaceutical composition of any of claims 5 through 7.

9. The method of claim 8, wherein the hyperproliferative disorder is a T cell lymphoma.

10. The method of claim 9, wherein the T cell lymphoma is a peripheral T cell lymphoma.

11. The method of any of claims 8 through 10, wherein the step of administering is repeated at least two times over the course of 3, 4, 5, 6, 7, or 8 weeks.

12. The method of any of claims 8 through 11, wherein the composition comprises at least one nucleic acid molecule comprising a nucleic acid sequence encoding one or more subunits of IL-12 or a functional variant thereof.

13. The method of any of claims 8 through 12 further comprising administering one or more chemotherapeutic agents.DOCKET NO: WIST-020-PCT PCT APPLICATION14. The method of any of claims 8 through 13, wherein the step of administering is performed orally, intravenously, sub-cutaneously, intramuscularly, intravaginally, intradermally, or intraperitoneally.

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