Bicistronic LAMP constructs containing immune response enhancing genes and methods of use thereof

Abstract

The present disclosure provides a nucleic acid molecule (e.g., a plasmid or vector) that includes a nucleic acid sequence encoding a bicistronic or multicistronic LAMP construct that includes a specific fragment of the LAMP luminal domain and an antigenic domain heterologous to the LAMP protein to provide at least one antigen for priming an immune response, and the antigen expressed by the construct is optionally processed and presented on an MHC class II molecule, and also provides a nucleic acid sequence encoding an immune response enhancing polypeptide that is optionally secreted from the host cell. The nucleic acid molecule can be used, for example, for the treatment of diseases, particularly allergies, infectious diseases, diabetes, hyperproliferative disorders and / or cancer.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 329,463, filed April 10, 2022, the contents of which are incorporated herein in their entirety.

[0002] Sequence Listing This application contains a sequence listing in ST26 format, which is incorporated by reference in its entirety. (Filename: 2023-04-10_01305-0023-00PCTST26; File size: 1,417,543 bytes.)

[0003] The present disclosure relates to isolated nucleic acid molecules (e.g., plasmids or vectors) encoding bicistronic or multicistronic LAMPs (lysosomal associated membrane proteins). Constructs comprising a LAMP fusion protein and a second, optionally secreted protein such as from an immune response enhancing gene (IREG), and their use in treating subjects suffering from infectious diseases, diabetes, allergies, hyperproliferative disorders and / or cancer, particularly COVID-19. Furthermore, the bicistronic LAMP constructs described herein can be used to generate antibodies in non-human vertebrates, preferably the genome of the non-human vertebrate comprises at least partially human and / or humanized immunoglobulin regions. [Background technology]

[0004] In the following discussion, certain articles and methods are described for purposes of background and introduction. Nothing contained herein should be construed as an "admission" of prior art. Applicants expressly reserve the right, where appropriate, to demonstrate that the articles and methods referenced herein do not constitute prior art under applicable statutory provisions.

[0005] Vaccines are novel and promising candidates for the development of both prophylactic and therapeutic vaccines. They have been proven safe, and the lack of immune response to the vector backbone may be a crucial advantage if repeated cycles of vaccination are required to achieve clinical benefit. However, one recognized drawback of conventional vaccines is their low immunogenicity in humans. A key limiting step in the immunogenicity of epitope-based vaccines may be the access of epitopes to the major histocompatibility (MHC) class II presentation pathway to T cells, which is likely a stochastic process in the case of vaccines without targeted technology.

[0006] Various designs of LAMP-antigen constructs have been previously described, for example, in US Pat. No. 11,203,629 (see FIG. 1 therein). One type of construct described in US Pat. No. 11,203,629, named ILC-4 (shown in FIG. 1 herein), comprises at least one antigen of interest fused between a first homologous domain of a LAMP protein and a second homologous domain of a LAMP protein (or between at least two cysteine-conserved fragments), for example, at least one antigen of interest may be located in the LAMP hinge region. In some embodiments, the construct also comprises a transmembrane domain of a LAMP protein and / or a cytosolic tail of a LAMP protein. The two homologous domains may be derived, for example, from LAMP-1, LAMP-2, LAMP-3, or Endolyn proteins. Alternatively, two homologous domains from two different LAMP proteins may be used. The inventors have unexpectedly found that improved LAMP constructs such as ILC-4, for example, can elicit strong T cell and antibody responses against the antigen(s) of interest, making them viable candidates for use as vaccines.

[0007] Notwithstanding the above, there is a further need to design new and improved LAMP constructs, and the nucleic acid molecules encoding them, which can be used as vaccines and / or in the generation of useful antibodies, for example, to effectively treat allergies, infectious diseases, diabetes, hyperproliferative disorders and / or cancer. Summary of the Invention [Means for solving the problem]

[0008] As further described herein, the inventors have found that isolated nucleic acid molecules can be designed that not only express a LAMP construct, such as those described in FIG. 1 herein, but also express a second polypeptide of a particular type, often a secreted polypeptide, encoding a gene such as CD40L, CD80, OX40, IL-12, IL-21, IL-15, or Flt3L, which have been found to enhance immune responses to tumors or infectious diseases in vivo, and that expressing these two polypeptides from the isolated nucleic acid molecule unexpectedly enhances the immune response, not only compared to previous LAMP constructs, but also compared to bicistronic LAMP constructs that include a particular secreted antigen, such as a second disease antigen.

[0009] This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. Other features, details, utilities, and advantages of the claimed subject matter will become apparent from the Detailed Description set forth below, including the aspects illustrated in the accompanying drawings and defined in the appended claims.

[0010] One objective of the present disclosure is to provide novel nucleic acid molecules encoding constructs comprising specific fragments and / or variants of LAMP domains ("bicistronic LAMP constructs") that effectively present antigen(s) of interest to the immune system to generate enhanced immune responses. These bicistronic LAMP constructs effectively target antigens to lysosomal / endosomal compartments where they are processed and presented to major histocompatibility complex (MHC) class II molecules, resulting in preferential stimulation of helper T cells and / or generation of antibodies with the ability to enhance immune responses.

[0011] The nucleic acid molecule and method encoding the bicistronic LAMP construct described herein can induce an immune response in a subject. The immune response can be an immune response to the epitope of the antigen encoded in the bicistronic LAMP construct (e.g., a vaccine). The vaccine arms the immune system of the subject so that the immune system can detect and destroy the antigen-containing object of the vaccine in the subject. The nucleic acid molecule and method encoding the bicistronic LAMP construct described herein can induce a Thl immune response in a subject. The Thl immune response can include the secretion of inflammatory cytokines (e.g., IFNγ, TNFa) by a subset of immune cells (e.g., antigen-specific T cells). In some cases, the inflammatory cytokine activates another subtype of immune cells (e.g., cytotoxic T cells) that can destroy the antigen-containing object in the subject.

[0012] In some cases, the antigen used in the bicistronic LAMP constructs and methods described herein can be recognized by the immune system of a subject to induce a Thl immune response and release type I cytokines. A Thl response can be initiated by the interaction between an epitope and a T cell, more specifically, a major histocompatibility complex (MHC) expressed by a T cell. For example, high affinity binding of an epitope to an MHC receptor can stimulate a Thl response. The MHC receptor can be at least one of multiple types of MHC receptors. The MHC receptor bound on a T cell can vary between individuals in a population.

[0013] In some cases, the immune response is a type 1 immune response. In some cases, the immune response is characterized by a ratio of type I cytokine production to type II cytokine production that is greater than 1. In some cases, the immune response is characterized by a ratio of type I cytokine production to type II cytokine production that is less than 1. In some cases, the immune response is characterized by a ratio of IFNγ production to IL-10 production that is greater than 1. In some cases, the immune response is characterized by a ratio of IFNγ production to IL-10 production that is less than 1.

[0014] The nucleic acid molecule encoding the bicistronic LAMP construct described herein can also be used to provide expression of an immune regulatory element (IRE) or immune response enhancing gene (IREG) to induce an enhanced immune response in a subject (e.g., an immune response including a significantly higher antibody titer). For example, the nucleic acid molecule (e.g., a plasmid or vector) can provide expression of a bicistronic LAMP construct that includes a LAMP-antigen polypeptide that is processed and presented on MHC class II molecules so that helper T cells are preferentially stimulated, memory cells are initiated, and / or antibodies are generated, and can provide expression of an additional IREG or IRE polypeptide that can be secreted into the circulation of a subject, for example, to further enhance both humoral and cellular immune responses to the LAMP antigen.

[0015] In one aspect, the nucleic acid molecule encoding the bicistronic LAMP construct is a suitable vaccine vector for vaccinating a subject.In another aspect, the present disclosure provides a delivery vehicle for facilitating the introduction of the nucleic acid molecule encoding the bicistronic LAMP construct, which comprises the polynucleotide encoding an epitope and / or antigen, into cells.The delivery vehicle can be lipid-based (e.g., liposomal formulation), viral-based (e.g., comprising viral protein that encapsulates the nucleic acid molecule), or cell-based.

[0016] In some embodiments, the present disclosure provides an injectable composition comprising a nucleic acid molecule as described herein that encodes a bicistronic LAMP construct for inducing an immune response (e.g., antibody production) in a subject against an antigen. In some embodiments, the vaccine generates a preferential Th1 response over a Th2 response.

[0017] The present disclosure also provides a cell comprising the nucleic acid molecule described herein that encodes a bicistronic LAMP construct that can be used to generate an immune response. In one aspect, the cell is an antigen-presenting cell. The antigen-presenting cell may be a professional antigen-presenting cell (e.g., dendritic cell, macrophage, B cell, etc.) or an engineered antigen-presenting cell (e.g., a non-professional antigen-presenting cell that is engineered to express a molecule required for antigen presentation, such as an MHC class II molecule). The molecule required for antigen presentation may be derived from other cells, e.g., naturally occurring, or may itself be engineered (e.g., mutated or modified to express a desired property, such as a higher or lower affinity for an antigen epitope).

[0018] The present disclosure further provides a kit comprising a plurality of cells comprising the nucleic acid molecule described herein that encodes a bicistronic LAMP construct.At least two of the cells may express different MHC class II molecules, and each cell may comprise the same LAMP construct.In one aspect, a kit is provided that comprises a viral vector that encodes a bicistronic LAMP construct.

[0019] The present disclosure also provides a transgenic animal comprising at least one of the cells and / or at least one of the nucleic acid molecules encoding the bicistronic LAMP construct as described herein.The present disclosure also provides a transgenic animal comprising at least one of the cells described herein.

[0020] The present disclosure further provides a method for generating an enhanced immune response to an antigen in a subject (e.g., a human or non-human vertebrate), comprising administering to the subject the above-mentioned cells, wherein the cells express or can be induced to express the bicistronic LAMP construct in the subject. In one aspect, the cells comprise MHC class II molecules that are compatible with the MHC protein of the subject, such that the subject does not generate an immune response to the MHC class II molecule.

[0021] In a further aspect, the present disclosure provides a method for inducing an enhanced immune response to an antigen, comprising administering to a subject, for example, a human or non-human vertebrate, a nucleic acid molecule encoding the bicistronic LAMP construct described herein.Preferably, the nucleic acid molecule is infectious to the cells of the subject.For example, the nucleic acid molecule encoding the bicistronic LAMP construct can be a viral vector, such as a vaccinia vector.

[0022] The present disclosure also includes a method for producing antibodies in a non-human vertebrate, which is to inject the non-human vertebrate with a nucleic acid molecule encoding the bicistronic LAMP construct described herein. The produced antibodies can be isolated from the blood of the vertebrate (as polyclonals) and then further isolated to produce monoclonal antibodies using standard techniques.

[0023] The methods described herein can be used in the production and / or optimization of antibodies, including fully human, humanized, chimeric antibodies, for diagnostic and therapeutic applications. Hybridomas producing such antibodies are also a further object of the present disclosure.

[0024] Particular embodiments of the present disclosure include: 1. An isolated nucleic acid molecule comprising: a. a first polynucleotide sequence encoding a polypeptide comprising two homologous domains of the luminal domain of a LAMP protein and an antigenic domain heterologous to said LAMP protein (collectively a "LAMP-antigen construct"), said antigenic domain being disposed between said two homologous domains; b. a second polypeptide sequence encoding at least one second polypeptide comprising an immune response enhancing gene polypeptide (IREG) or an extracellular domain of an IREG comprising a secretory signal sequence; 2. The isolated nucleic acid molecule of embodiment 1, wherein the LAMP protein is selected from LAMP-1, LAMP2, LAMP-3, lysosomal integral membrane protein-2 ("LIMP 2"), Macrosialin, Endolyn, LAMP5, or Limbic-associated membrane protein ("LIMBIC"). 3. The isolated nucleic acid molecule of embodiment 2, wherein the LAMP protein is selected from any one of SEQ ID NOs: 1-113. 4. The isolated nucleic acid molecule of embodiment 1 or 2, wherein the LAMP protein is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NOs: 1-113. 5. The isolated nucleic acid molecule of embodiment 2, wherein the LAMP protein is LAMP-1 and the two homologous domains of the LAMP-antigen construct comprise LAMP-1 homology domain 1 and LAMP-1 homology domain 2. 6. The isolated nucleic acid molecule of embodiment 5, wherein the human LAMP-1 homology domain 1 comprises the amino acid sequence of residues 29-194 of SEQ ID NO:1, or the amino acid sequence of residues 29-195 of SEQ ID NO:198, or an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of residues 29-194 of SEQ ID NO:1 or the amino acid sequence of residues 29-195 of SEQ ID NO:198, or the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:199, or a nucleotide sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO:199 (wherein if the nucleotide sequence is RNA, T is replaced by U). 7. The isolated nucleic acid molecule according to embodiment 5 or 6, wherein the human LAMP-1 homology domain 2 comprises the amino acid sequence of residues 228-381 of SEQ ID NO:1 or residues 228-382 of SEQ ID NO:1, or comprises the amino acid sequence of SEQ ID NO:202, or comprises an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of residues 228-381 of SEQ ID NO:1 or the amino acid sequence of SEQ ID NO:202, or the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:203 or a nucleotide sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO:203 (wherein, if the nucleotide sequence is RNA, T is replaced by U). 8. The isolated nucleic acid molecule according to any one of the preceding embodiments, wherein the LAMP-antigen construct comprises a linker between at least one of the two homologous domains and the antigenic domain. 9. The isolated nucleic acid molecule of embodiment 8, wherein the linker comprises the amino acid sequence of GPGPG or PMGLP. 10. The isolated nucleic acid molecule according to any one of embodiments 1 to 9, wherein the LAMP-antigen construct further comprises a transmembrane domain of a LAMP protein. 11. The isolated nucleic acid molecule of embodiment 10, wherein the transmembrane domain comprises residues 383-405 of SEQ ID NO:1. 12. The isolated nucleic acid molecule according to any one of embodiments 1 to 11, wherein the LAMP-antigen construct further comprises a signal sequence. 13. The isolated nucleic acid molecule of embodiment 12, wherein the signal sequence is derived from a LAMP protein. 14. The isolated nucleic acid molecule of any one of embodiments 1 to 13, wherein the LAMP-antigen construct further comprises the cytoplasmic domain of a LAMP protein. 15. The isolated nucleic acid molecule of embodiment 14, wherein the cytoplasmic domain comprises residues 406-417 of SEQ ID NO:1. 16. The IREG comprises one or more of CD40L, CD80, OX40, Flt3L, GM-CSF, IL-12, IL-21, IL-23, IL-15, CD70, CD86, IL-7, IL-18, or IL-33 (or SEQ ID NOs: 133, 145, 147, 149, 151, 155, 159, 165, 169, 173, 177, 181, 189, 191, 204, 238, 242, 246, 250, 252, 254, 256, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 3 43, 252, or 253), or an extracellular domain thereof, and optionally comprising an amino acid sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, or 100% identical to the amino acid sequence of any one of CD40L, CD80, OX40, Flt3L, GM-CSF, IL-12, IL-21, IL-23, or 16. The isolated nucleic acid molecule of any one of embodiments 1-15, wherein IL-15 is fused to an Fc domain of an immunoglobulin, or wherein the isolated nucleic acid molecule comprises a nucleotide sequence encoding CD40L, CD80, OX40, Flt3L, GM-CSF, IL-12, IL-21, IL-23, IL-15, CD70, CD86, IL-7, IL-18, or IL-33, optionally fused to an Fc domain, and wherein the nucleotide sequence is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, or 100% identical to any one of SEQ ID NOs: 134, 146, 148, 150, 152, 160, 166, 170, 174, 178, 182, 190, 192, 205, 239, 244, or 881. 17. The isolated nucleic acid molecule of any one of embodiments 1-16, wherein the secretory signal sequence is heterologous to the IREG. 18. The isolated polypeptide of embodiment 17, wherein the secretory signal sequence is derived from IgKVIII (e.g., SEQ ID NO: 122), Ig-kappa (e.g., SEQ ID NO: 120), tetranectin, or IL-2, and / or the second polypeptide further comprises pulmonary surfactant associated protein D (SPD) (e.g., SEQ ID NO: 131). 19. The isolated nucleic acid molecule of embodiment 18, wherein the second polypeptide is expressed under the control of an EF-1α core promoter, such as the promoter of SEQ ID NO: 124. 20. A composition comprising an isolated nucleic acid molecule according to any one of embodiments 1 to 18. 21. A host cell comprising an isolated nucleic acid according to any one of embodiments 1 to 18. 22. A composition comprising a host cell according to embodiment 20. 23. A method of treating a subject having a disease or disorder, or inducing an immune response in a subject having a disease or disorder, or inducing an immune response in a subject at risk of developing a disease or disorder, comprising administering to the subject an isolated nucleic acid molecule according to any one of embodiments 1-18, a composition according to embodiment 19, or a host cell according to embodiment 20, in an amount sufficient to treat the disease or disorder or induce an immune response in the subject. 24. The method of embodiment 23, further comprising administering at least one second therapeutic agent to the subject. 25. An isolated nucleic acid molecule comprising: a. a first polynucleotide sequence encoding a polypeptide comprising two homologous domains of the luminal domain of a LAMP protein and an antigenic domain comprising a HER2 extracellular domain (collectively "HER2-LAMP"), wherein the antigenic domain is disposed between the two LAMP homologous domains; b. A second polypeptide sequence encoding at least a second polypeptide comprising an immune response enhancing gene polypeptide (IREG) or the extracellular domain of an IREG comprising a secretory signal sequence. 26. The isolated nucleic acid molecule of embodiment 25, wherein the LAMP protein is selected from LAMP-1, LAMP2, LAMP-3, lysosomal integral membrane protein-2 ("LIMP 2"), Macrosialin, Endolyn, LAMP5, or Limbic-associated membrane protein ("LIMBIC"). 27. The isolated nucleic acid molecule according to embodiment 26, wherein the LAMP protein is selected from any one of SEQ ID NOs: 1-113. 28. The isolated nucleic acid molecule of embodiment 25 or 26, wherein the LAMP protein is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NOs: 1-113. 29. The isolated nucleic acid molecule of embodiment 26, wherein the LAMP protein is LAMP-1 and the two homologous domains of HER2-LAMP include LAMP-1 homology domain 1 and LAMP-1 homology domain 2. 30. The isolated nucleic acid molecule according to embodiment 29, wherein the human LAMP-1 homology domain 1 comprises the amino acid sequence of residues 29-194 of SEQ ID NO: 1, or the amino acid sequence of residues 29-195 of SEQ ID NO: 198, or an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of residues 29-194 of SEQ ID NO: 1 or the amino acid sequence of residues 29-195 of SEQ ID NO: 198, or the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 199 or a nucleotide sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 199 (wherein, if the nucleotide sequence is RNA, T is replaced by U). 31. The isolated nucleic acid molecule according to embodiment 29 or 30, wherein the human LAMP-1 homology domain 2 comprises the amino acid sequence of residues 228-381 or 228-382 of SEQ ID NO: 1, or the amino acid sequence of residues 228-382 of SEQ ID NO: 1, or the amino acid sequence of residues 228-382 of SEQ ID NO: 1, or the amino acid sequence of SEQ ID NO: 202, the amino acid sequence of residues 228-381 of SEQ ID NO: 1, or the amino acid sequence of SEQ ID NO: 202 is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, or the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 203, or the nucleotide sequence of SEQ ID NO: 203 is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, (wherein if the nucleotide sequence is RNA, T is replaced by U). 32. The isolated nucleic acid molecule according to any one of embodiments 25 to 31, wherein HER2-LAMP comprises a linker between at least one of the two homology domains and the antigenic domain. 33. The isolated nucleic acid molecule of embodiment 32, wherein the linker comprises the amino acid sequence of GPGPG or PMGLP. 34. The isolated nucleic acid molecule according to any one of embodiments 25 to 33, wherein HER2-LAMP further comprises the transmembrane domain of the LAMP protein. 35. The isolated nucleic acid molecule of embodiment 34, wherein the transmembrane domain comprises residues 383 to 405 of SEQ ID NO:1. 36. The isolated nucleic acid molecule according to any one of embodiments 25 to 35, wherein HER2-LAMP further comprises a signal sequence. 37. The isolated nucleic acid molecule of embodiment 36, wherein the signal sequence is derived from a LAMP protein. 38. The isolated nucleic acid molecule according to any one of embodiments 25 to 37, wherein HER2-LAMP further comprises the cytoplasmic domain of the LAMP protein. 39. The isolated nucleic acid molecule of embodiment 38, wherein the cytoplasmic domain comprises residues 406 to 417 of SEQ ID NO:1. 40. The isolated nucleic acid molecule according to any one of embodiments 25 to 39, wherein the antigen domain comprises or consists of the amino acid sequence of SEQ ID NO: 200. 41. An isolated nucleic acid molecule according to any one of embodiments 25 to 40, wherein HER2-LAMP comprises or consists of the amino acid sequence of residues 1 to 194 of SEQ ID NO: 1 or SEQ ID NO: 202, followed by the amino acid sequence of SEQ ID NO: 200, followed by the amino acid sequence of residues 228 to 381 of SEQ ID NO: 1 or SEQ ID NO: 198. 42. The IREG comprises one or more of CD40L, CD80, OX40, Flt3L, GM-CSF, IL-12, IL-21, IL-23, IL-15, CD70, CD86, IL-7, IL-18, or IL-33 (or SEQ ID NOs: 133, 145, 147, 149, 151, 155, 159, 165, 169, 173, 177, 181, 189, 191, 193, 204, 238, 242, 243, 252, or 253), or an extracellular domain thereof, and optionally comprising an amino acid sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, or 100% identical, to the amino acid sequence of any one of CD40L, CD80, OX40, Flt3L, GM-CSF, IL-12, IL-21, IL-23, or I 42. The isolated nucleic acid molecule of any one of embodiments 25-41, wherein CD40L, CD80, OX40, Flt3L, GM-CSF, IL-12, IL-21, IL-23, IL-15, CD70, CD86, IL-7, IL-18, or IL-33 is fused to an Fc domain of an immunoglobulin, or wherein the isolated nucleic acid molecule comprises a nucleotide sequence encoding CD40L, CD80, OX40, Flt3L, GM-CSF, IL-12, IL-21, IL-23, IL-15, CD70, CD86, IL-7, IL-18, or IL-33, optionally fused to an Fc domain, and wherein the nucleotide sequence is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, or 100% identical to any one of SEQ ID NOs: 134, 146, 148, 150, 152, 160, 166, 170, 174, 178, 182, 190, 192, 194, 205, 239, 244, or 881. 43. The isolated nucleic acid molecule of any one of embodiments 25 to 42, wherein the secretory signal sequence is heterologous to the IREG. 44. The isolated nucleic acid molecule of embodiment 43, wherein the secretory signal sequence is derived from IgKVIII (e.g., SEQ ID NO: 122), Ig-kappa (e.g., SEQ ID NO: 120), tetranectin, or IL-2. 45. The second polypeptide is a fusion of SPD and soluble CD40L (sCD40L), a fusion of SPD and Flt3L, IL-12, IL-21, OX40L fused to an Fc domain, CD80 fused to an Fc domain, or IL-15 (e.g., comprises an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, or 100% identical, to SEQ ID NO: 233, 238, 242, or 252, or is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, or 100% identical, to the amino acid sequence of SEQ ID NO: 131 followed by one of the amino acid sequences of SEQ ID NO: 204, 151, 145, 147, 149, 193, 181, 155, 159, 169, 252, or 253). 45. The isolated nucleic acid molecule of any one of embodiments 25-44, wherein the nucleic acid comprises a nucleotide sequence encoding a fusion of the SPD and soluble CD40L (sCD40L), a fusion of the SPD and Flt3L, IL-12, IL-21, OX40L fused to an Fc domain, CD80 fused to an Fc domain, or IL-15 (e.g., comprising the sequence of SEQ ID NO: 132 followed by an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical or 100% identical to the amino acid sequence of one of SEQ ID NOs: 205, 152, 146, 148, 150, 194, 182, 156, 170, or 881). 46. ​​The isolated nucleic acid molecule according to embodiment 45, wherein the second polypeptide is expressed under the control of an EF-1α core promoter, such as the promoter of SEQ ID NO: 124. 47. A composition comprising an isolated nucleic acid molecule according to any one of embodiments 25 to 46. 48. A host cell comprising an isolated nucleic acid according to any one of embodiments 25 to 46. 49. A composition comprising a host cell according to embodiment 48. 50. A method for treating a subject having cancer, comprising administering to the subject an isolated nucleic acid molecule described in any one of embodiments 25 to 46, a composition described in embodiment 47, or a host cell described in embodiment 48, in an amount sufficient to treat the cancer or to induce an immune response against the cancer in the subject. 51. The method of embodiment 50, further comprising administering at least one second therapeutic agent to the subject. 52. An isolated nucleic acid molecule comprising: a. a first polynucleotide sequence encoding a polypeptide comprising two homologous domains of the luminal domain of a LAMP protein and an antigenic domain comprising a coronavirus spike protein antigen (collectively "Spike-LAMP"), wherein the antigenic domain is disposed between the two LAMP homologous domains; b. A second polypeptide sequence encoding at least a second polypeptide comprising an immune response enhancing gene polypeptide (IREG) or the extracellular domain of an IREG comprising a secretory signal sequence. 53. The isolated nucleic acid molecule of embodiment 52, wherein the LAMP protein is selected from LAMP-1, LAMP2, LAMP-3, lysosomal integral membrane protein-2 ("LIMP2"), Macrosialin, Endolyn, LAMP5, or Limbic-associated membrane protein ("LIMBIC"). 54. The isolated nucleic acid molecule according to embodiment 53, wherein the LAMP protein is selected from any one of SEQ ID NOs: 1-113. 55. The isolated nucleic acid molecule of embodiment 52 or 53, wherein the LAMP protein is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NOs: 1-113. 56. The isolated nucleic acid molecule according to embodiment 53, wherein the LAMP protein is LAMP-1 and the two homologous domains of Spike-LAMP comprise LAMP-1 homologous domain 1 and LAMP-1 homologous domain 2. 57. The isolated nucleic acid molecule according to embodiment 56, wherein the human LAMP-1 homology domain 1 comprises the amino acid sequence of residues 29-194 of SEQ ID NO: 1, or the amino acid sequence of residues 29-195 of SEQ ID NO: 198, or an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of residues 29-194 of SEQ ID NO: 1 or the amino acid sequence of residues 29-195 of SEQ ID NO: 198, or the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 199 or a nucleotide sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 199 (wherein, if the nucleotide sequence is RNA, T is replaced by U). 58. The isolated nucleic acid molecule according to embodiment 56 or 57, wherein the human LAMP-1 homology domain 2 comprises residues 228-381 or 228-382 of SEQ ID NO: 1, or the amino acid sequence of residues ... SEQ ID NO: 202, or the amino acid sequence of residues 228-381 of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 202 is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of residues 228-381 of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 202, or wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 203, or a nucleotide sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 203 (wherein, if the nucleotide sequence is RNA, T is replaced by U). 59. The isolated nucleic acid molecule according to any one of embodiments 52 to 58, wherein Spike-LAMP comprises a linker between at least one of the two homologous domains and the antigenic domain. 60. The isolated nucleic acid molecule of embodiment 59, wherein the linker comprises the amino acid sequence of GPGPG or PMGLP. 61. The isolated nucleic acid molecule according to any one of embodiments 52 to 60, wherein Spike-LAMP further comprises a transmembrane domain of a LAMP protein. 62. The isolated nucleic acid molecule of embodiment 61, wherein the transmembrane domain comprises residues 383 to 405 of SEQ ID NO:1. 63. The isolated nucleic acid molecule according to any one of embodiments 52 to 62, wherein Spike-LAMP further comprises a signal sequence. 64. The isolated nucleic acid molecule of embodiment 63, wherein the signal sequence is derived from a LAMP protein. 65. The isolated nucleic acid molecule according to any one of embodiments 52 to 64, wherein the Spike-LAMP further comprises the cytoplasmic domain of a LAMP protein. 66. The isolated nucleic acid molecule of embodiment 65, wherein the cytoplasmic domain comprises residues 406 to 417 of SEQ ID NO:1. 67. The isolated nucleic acid molecule according to any one of embodiments 52 to 66, wherein the antigenic domain comprises an amino acid sequence comprising Spike S1 and / or S2, or the sequence of SEQ ID NO: 118 or 119. 68. An isolated nucleic acid molecule according to any one of embodiments 52 to 67, wherein Spike-LAMP comprises or consists of the amino acid sequence of residues 1 to 194 of SEQ ID NO: 1 or SEQ ID NO: 202, followed by the amino acid sequence of SEQ ID NO: 231, followed by the amino acid sequence of residues 228 to 381 of SEQ ID NO: 1 or SEQ ID NO: 198. 69. The IREG comprises one or more of CD40L, CD80, OX40, Flt3L, GM-CSF, IL-12, IL-21, IL-23, IL-15, CD70, CD86, IL-7, IL-18, or IL-33 (or SEQ ID NOs: 133, 145, 147, 149, 151, 155, 159, 165, 169, 173, 177, 181, 189, 191, 204, 238, 242, 246, 250, 252, 254, 256, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 3 43, 252, or 253), or an extracellular domain thereof, and optionally, CD40L, CD80, OX40, Flt3L, GM-CSF, IL-12, IL-21, IL-23, or an amino acid sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, or 100% identical, to the amino acid sequence of any one of CD40L, CD80, OX40, Flt3L, GM-CSF, IL-12, IL-21, IL-23, or an extracellular domain thereof. or IL-15 is fused to an Fc domain of an immunoglobulin; or the isolated nucleic acid molecule comprises a nucleotide sequence encoding CD40L, CD80, OX40, Flt3L, GM-CSF, IL-12, IL-21, IL-23, IL-15, CD70, CD86, IL-7, IL-18, or IL-33, optionally fused to an Fc domain, and the nucleotide sequence is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, or 100% identical to any one of SEQ ID NOs: 134, 146, 148, 150, 152, 160, 166, 170, 174, 178, 182, 190, 192, 205, 239, 244, or 881. 70. The isolated nucleic acid molecule according to any one of embodiments 52-68, wherein the second polypeptide comprises an SPD-sCD40L fusion polypeptide. 71. The isolated nucleic acid molecule of any one of embodiments 52-70, wherein the secretory signal sequence is heterologous to the IREG. 72. The isolated nucleic acid molecule according to embodiment 71, wherein the secretory signal sequence is derived from the SPD. 73. The isolated nucleic acid molecule according to any one of embodiments 52 to 72, wherein the second polypeptide is expressed under the control of an EF-1α core promoter, such as the promoter of SEQ ID NO: 124. 74. A composition comprising an isolated nucleic acid molecule according to any one of embodiments 52 to 73. 75. A host cell comprising an isolated nucleic acid according to any one of embodiments 52 to 73. 76. A composition comprising a host cell according to embodiment 75. 77. A method for treating a subject having or at risk of developing a coronavirus, such as COVID-19, infection, comprising administering to the subject an isolated nucleic acid molecule described in any one of embodiments 52-73, a composition described in embodiment 74, or a host cell described in embodiment 75, in an amount sufficient to treat or prevent the onset of or reduce the severity of symptoms of a coronavirus, such as COVID-19, infection. 78. A method for inducing an immune response against a coronavirus, such as SARS Co-V2, in a subject, comprising administering to the subject an isolated nucleic acid molecule described in any one of embodiments 52-73, a composition described in embodiment 74, or a host cell described in embodiment 75, in an amount sufficient to induce an immune response against a coronavirus, such as SARS Co-V2, in the subject. 79. An isolated nucleic acid molecule comprising: a. a first polynucleotide sequence encoding a polypeptide comprising two homologous domains of the luminal domain of a LAMP protein and an antigenic domain comprising a NY-ESO1 or CD161 protein antigen ("NY-ESO1-LAMP" or "CD161-LAMP" LAMP-antigen construct), wherein the antigenic domain is located between the two LAMP homologous domains; b. A second polypeptide sequence encoding at least a second polypeptide comprising an immune response enhancing gene polypeptide (IREG) or the extracellular domain of an IREG comprising a secretory signal sequence. 80. The isolated nucleic acid molecule of embodiment 79, wherein the LAMP protein is selected from LAMP-1, LAMP2, LAMP-3, lysosomal integral membrane protein-2 ("LIMP2"), Macrosialin, Endolyn, LAMP5, or Limbic system-associated membrane protein ("LIMBIC"). 81. The isolated nucleic acid molecule according to embodiment 80, wherein the LAMP protein is selected from any one of SEQ ID NOs: 1-113. 82. The isolated nucleic acid molecule according to embodiment 79 or 80, wherein the LAMP protein is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NOs: 1-113. 83. The isolated nucleic acid molecule of embodiment 79, wherein the LAMP protein is LAMP-1 and the two homologous domains of NY-ESO1-LAMP or CD161-LAMP include LAMP-1 homology domain 1 and LAMP-1 homology domain 2. 84. The isolated nucleic acid molecule according to embodiment 83, wherein the human LAMP-1 homology domain 1 comprises the amino acid sequence of residues 29-194 of SEQ ID NO: 1, or the amino acid sequence of residues 29-195 of SEQ ID NO: 198, or an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of residues 29-194 of SEQ ID NO: 1 or the amino acid sequence of residues 29-195 of SEQ ID NO: 198, or the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 199 or a nucleotide sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 199 (wherein, if the nucleotide sequence is RNA, T is replaced by U). 85. The isolated nucleic acid molecule according to embodiment 83 or 84, wherein the human LAMP-1 homology domain 2 comprises the amino acid sequence of residues 228-381 or 228-382 of SEQ ID NO: 1, or the amino acid sequence of residues ... SEQ ID NO: 202, or the amino acid sequence of residues 228-381 of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 202 is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of residues 228-381 of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 202, or the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 203, or the nucleotide sequence of SEQ ID NO: 203 is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 203 (wherein, if the nucleotide sequence is RNA, T is replaced by U). 86. The isolated nucleic acid molecule according to any one of embodiments 79 to 85, wherein the LAMP-antigen construct comprises a linker between at least one of the two homologous domains and the antigenic domain. 87. The isolated nucleic acid molecule of embodiment 86, wherein the linker comprises the amino acid sequence of GPGPG or PMGLP. 88. The isolated nucleic acid molecule according to any one of embodiments 79 to 87, wherein the LAMP-antigen construct further comprises a transmembrane domain of a LAMP protein. 89. The isolated nucleic acid molecule of embodiment 88, wherein the transmembrane domain comprises residues 383 to 405 of SEQ ID NO:1. 90. The isolated nucleic acid molecule according to any one of embodiments 79 to 89, wherein the LAMP-antigen construct further comprises a signal sequence. 91. The isolated nucleic acid molecule of embodiment 90, wherein the signal sequence is derived from a LAMP protein. 92. The isolated nucleic acid molecule according to any one of embodiments 79 to 91, wherein the LAMP-antigen construct further comprises the cytoplasmic domain of a LAMP protein. 93. The isolated nucleic acid molecule of embodiment 92, wherein the cytoplasmic domain comprises residues 406 to 417 of SEQ ID NO:1. 94. The isolated nucleic acid molecule according to any one of embodiments 79 to 93, wherein the LAMP-antigen construct comprises or consists of the amino acid sequence of residues 1 to 194 of SEQ ID NO:1 or SEQ ID NO:202, followed by the amino acid sequence of SEQ ID NO:223, followed by the amino acid sequence of residues 228 to 381 of SEQ ID NO:1 or SEQ ID NO:198, or the amino acid sequence of residues 1 to 194 of SEQ ID NO:1 or SEQ ID NO:202, followed by the amino acid sequence of SEQ ID NO:236, followed by the amino acid sequence of residues 228 to 381 of SEQ ID NO:1 or SEQ ID NO:198. 95. The IREG comprises one or more of CD40L, CD80, OX40, Flt3L, GM-CSF, IL-12, IL-21, IL-23, IL-15, CD70, CD86, IL-7, IL-18, or IL-33 (or SEQ ID NOs: 133, 145, 147, 149, 151, 155, 159, 165, 169, 173, 177, 181, 189, 191, 204, 238, 242, 246, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 3 3, 252, or 253), or an extracellular domain thereof, and optionally comprising an amino acid sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, or 100% identical to the amino acid sequence of any one of CD40L, CD80, OX40, Flt3L, GM-CSF, IL-12, IL-21, IL-23, or 95. The isolated nucleic acid molecule of any one of embodiments 79-94, wherein IL-15 is fused to an Fc domain of an immunoglobulin, or wherein the isolated nucleic acid molecule comprises a nucleotide sequence encoding CD40L, CD80, OX40, Flt3L, GM-CSF, IL-12, IL-21, IL-23, IL-15, CD70, CD86, IL-7, IL-18, or IL-33, optionally fused to an Fc domain, and wherein the nucleotide sequence is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, or 100% identical to any one of SEQ ID NOs: 134, 146, 148, 150, 152, 160, 166, 170, 174, 178, 182, 190, 192, 205, 239, 244, or 881. 96. The isolated nucleic acid molecule of any one of embodiments 79 to 95, wherein the second polypeptide comprises an SPD-sCD40L fusion polypeptide or an IL-15 polypeptide (e.g., SEQ ID NO: 233 or 169 or 225). 97. The isolated nucleic acid molecule of any one of embodiments 79-96, wherein the secretory signal sequence is heterologous to the IREG. 98. The isolated nucleic acid molecule of embodiment 97, wherein the secretory signal sequence is derived from SPD or comprises SEQ ID NO: 120 or 122. 99. The isolated nucleic acid molecule according to any one of embodiments 79 to 98, wherein the second polypeptide is expressed under the control of an EF-1α core promoter, such as the promoter of SEQ ID NO: 124. 100. A composition comprising an isolated nucleic acid molecule according to any one of embodiments 79 to 99. 101. A host cell comprising an isolated nucleic acid according to any one of embodiments 79 to 99. 102. A composition comprising a host cell according to embodiment 101. 103. A method for inducing an immune response in a subject in need thereof, comprising administering to the subject an amount of an isolated nucleic acid molecule described in any one of embodiments 79 to 99, a composition described in embodiment 100, or a host cell described in embodiment 101, in an amount sufficient to induce an immune response in the subject. 104. An isolated nucleic acid molecule comprising: a. a first polynucleotide sequence encoding a polypeptide comprising two homologous domains of the luminal domain of a LAMP protein and a pp65 antigen, such as comprising SEQ ID NO: 291, 292, or 293, optionally further comprising a gB antigen, such as comprising SEQ ID NO: 294, 295, 296, or 297, and one or both 1E1 antigens, such as comprising SEQ ID NO: 298, 299, or 300, and optionally an antigenic domain comprising a linker peptide between the pp65 and the gB and / or 1E1 antigen sequences (collectively "pp65-LAMP"), wherein the antigenic domain is disposed between the two LAMP homologous domains; b. A second polypeptide sequence encoding at least a second polypeptide comprising an immune response enhancing gene polypeptide (IREG) or the extracellular domain of an IREG comprising a secretory signal sequence. 105. The isolated nucleic acid molecule of embodiment 104, wherein the LAMP protein is selected from LAMP-1, LAMP2, LAMP-3, lysosomal integral membrane protein-2 ("LIMP2"), Macrosialin, Endolyn, LAMP5, or Limbic system-associated membrane protein ("LIMBIC"). 106. The isolated nucleic acid molecule according to embodiment 105, wherein the LAMP protein is selected from any one of SEQ ID NOs: 1-113. 107. The isolated nucleic acid molecule according to embodiment 104 or 105, wherein the LAMP protein is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NOs: 1-113. 108. The isolated nucleic acid molecule according to embodiment 105, wherein the LAMP protein is LAMP-1 and the two homologous domains of HER2-LAMP include LAMP-1 homology domain 1 and LAMP-1 homology domain 2. 109. The isolated nucleic acid molecule according to embodiment 108, wherein the human LAMP-1 homology domain 1 comprises the amino acid sequence of residues 29-194 of SEQ ID NO: 1, or comprises the amino acid sequence of residues 29-194 of SEQ ID NO: 199, or comprises an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of residues 29-195 of SEQ ID NO: 198, or to the amino acid sequence of residues 29-195 of SEQ ID NO: 198, or the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 199, or a nucleotide sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 199, or to the nucleotide sequence of SEQ ID NO: (wherein, if the nucleotide sequence is RNA, T is replaced by U). 110. The isolated nucleic acid molecule according to embodiment 108 or 109, wherein the human LAMP-1 homology domain 2 comprises the amino acid sequence of residues 228-381 or 228-382 of SEQ ID NO: 1, or the amino acid sequence of residues ... SEQ ID NO: 202, or the amino acid sequence of residues 228-381 of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 202 is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of residues 228-381 of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 202, or the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 203, or the nucleotide sequence of SEQ ID NO: 203 is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 203 (wherein, if the nucleotide sequence is RNA, T is replaced by U). 111. An isolated nucleic acid molecule according to any one of embodiments 104 to 110, wherein pp65-LAMP comprises a linker between at least one of the two homologous domains and the antigenic domain. 112. The isolated nucleic acid molecule according to embodiment 111, wherein the linker comprises the amino acid sequence of GPGPG or PMGLP. 113. The isolated nucleic acid molecule according to any one of embodiments 104 to 112, wherein pp65-LAMP further comprises a transmembrane domain of the LAMP protein. 114. The isolated nucleic acid molecule of embodiment 113, wherein the transmembrane domain comprises residues 383 to 405 of SEQ ID NO:1. 115. The isolated nucleic acid molecule according to any one of embodiments 104 to 114, wherein pp65-LAMP further comprises a signal sequence. 116. The isolated nucleic acid molecule according to embodiment 115, wherein the signal sequence is derived from a LAMP protein. 117. The isolated nucleic acid molecule according to any one of embodiments 104 to 116, wherein pp65-LAMP further comprises the cytoplasmic domain of the LAMP protein. 118. The isolated nucleic acid molecule of embodiment 117, wherein the cytoplasmic domain comprises residues 406 to 417 of SEQ ID NO:1. 119. The IREG comprises one or more of CD40L, CD80, OX40, Flt3L, GM-CSF, IL-12, IL-21, IL-23, IL-15, CD70, CD86, IL-7, IL-18, or IL-33 (or SEQ ID NOs: 133, 145, 147, 149, 151, 155, 159, 165, 169, 173, 177, 181, 189, 191, 204, 238, 242, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 269, 270, 272, 274, 276, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 43, 252, or 253), or an extracellular domain thereof, and optionally comprising an amino acid sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, or 100% identical, to the amino acid sequence of any one of CD40L, CD80, OX40, Flt3L, GM-CSF, IL-12, IL-21, IL-23, or I 119. The isolated nucleic acid molecule of any one of embodiments 104-118, wherein L-15 is fused to an Fc domain of an immunoglobulin, or wherein the isolated nucleic acid molecule comprises a nucleotide sequence encoding CD40L, CD80, OX40, Flt3L, GM-CSF, IL-12, IL-21, IL-23, IL-15, CD70, CD86, IL-7, IL-18, or IL-33, optionally fused to an Fc domain, and wherein the nucleotide sequence is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, or 100% identical to any one of SEQ ID NOs: 134, 146, 148, 150, 152, 160, 166, 170, 174, 178, 182, 190, 192, 205, 239, 244, or 881. 120. The isolated nucleic acid molecule of any one of embodiments 104 to 119, wherein the secretory signal sequence is heterologous to the IREG. 121. The isolated nucleic acid molecule of embodiment 120, wherein the secretory signal sequence is derived from IgKVIII (e.g., SEQ ID NO: 122), Ig-kappa (e.g., SEQ ID NO: 120), tetranectin, or IL-2. 122. The second polypeptide comprises a fusion of SPD with soluble CD40L (sCD40L), a fusion of SPD with Flt3L, IL-12, IL-21, OX40L fused to an Fc domain, CD80 fused to an Fc domain, or IL-15 (e.g., comprising an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, or 100% identical, to the amino acid sequence of one of SEQ ID NOs: 204, 151, 145, 147, 149, 193, 181, 155, 159, 169, 252, or 253), or 122. The isolated nucleic acid molecule of any one of embodiments 104-121, comprising a fusion of SPD with soluble CD40L (sCD40L), a fusion of SPD with Flt3L, IL-12, IL-21, OX40L fused to an Fc domain, CD80 fused to an Fc domain, or IL-15 (e.g., comprising an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, or 100% identical, to SEQ ID NO: 132 followed by one of SEQ ID NOs: 205, 152, 146, 148, 150, 194, 182, 156, 170, or 881). 123. The isolated nucleic acid molecule according to embodiment 122, wherein the second polypeptide is expressed under the control of an EF-1α core promoter, such as the promoter of SEQ ID NO: 124. 124. A composition comprising an isolated nucleic acid molecule according to any one of embodiments 104 to 123. 125. A host cell comprising an isolated nucleic acid according to any one of embodiments 104 to 123. 126. A composition comprising a host cell according to embodiment 125. 127. A method for treating a subject having cancer, comprising administering to the subject an isolated nucleic acid molecule described in any one of embodiments 104 to 123, a composition described in embodiment 124, or a host cell described in embodiment 125, in an amount sufficient to treat the cancer or to induce an immune response against the cancer in the subject. 128. The method of embodiment 127, further comprising administering at least one second therapeutic agent to the subject. 129. The method of embodiment 127 or 128, wherein the cancer is selected from glioblastoma, breast cancer, prostate cancer, colorectal cancer, and head and neck cancer. 130. An isolated nucleic acid molecule comprising: a. a first polynucleotide sequence encoding a polypeptide comprising two homologous domains of the luminal domain of a LAMP protein and an antigenic domain comprising a large T antigen, such as one comprising the amino acid sequence of SEQ ID NO: 254, 255, or 256 ("large T-LAMP"), wherein the antigenic domain is disposed between the two LAMP homologous domains; b. A second polypeptide sequence encoding at least a second polypeptide comprising an immune response enhancing gene polypeptide (IREG) or the extracellular domain of an IREG comprising a secretory signal sequence. 131. The isolated nucleic acid molecule of embodiment 130, wherein the LAMP protein is selected from LAMP-1, LAMP2, LAMP-3, lysosomal integral membrane protein-2 ("LIMP2"), Macrosialin, Endolyn, LAMP5, or Limbic system-associated membrane protein ("LIMBIC"). 132. The isolated nucleic acid molecule according to embodiment 131, wherein the LAMP protein is selected from any one of SEQ ID NOs: 1-113. 133. The isolated nucleic acid molecule according to embodiment 130 or 131, wherein the LAMP protein is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NOs: 1-113. 134. The isolated nucleic acid molecule of embodiment 131, wherein the LAMP protein is LAMP-1 and the two homologous domains of HER2-LAMP include LAMP-1 homology domain 1 and LAMP-1 homology domain 2. 135. The isolated nucleic acid molecule according to embodiment 134, wherein the human LAMP-1 homology domain 1 comprises the amino acid sequence of residues 29-194 of SEQ ID NO: 1, or the amino acid sequence of residues 29-195 of SEQ ID NO: 198, or an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of residues 29-194 of SEQ ID NO: 1 or the amino acid sequence of residues 29-195 of SEQ ID NO: 198, or the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 199, or a nucleotide sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 199 (wherein if the nucleotide sequence is RNA, T is replaced by U). 136. The isolated nucleic acid molecule according to embodiment 134 or 135, wherein the human LAMP-1 homology domain 2 comprises the amino acid sequence of residues 228-381 or 228-382 of SEQ ID NO: 1, or the amino acid sequence of residues ... SEQ ID NO: 202, or the amino acid sequence of residues 228-381 of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 202 is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of residues 228-381 of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 202, or the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 203, or the nucleotide sequence of SEQ ID NO: 203 is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence of SEQ ID NO: 203 (wherein if the nucleotide sequence is RNA, T is replaced by U). 137. The isolated nucleic acid molecule of any one of embodiments 130 to 136, wherein the large T-LAMP comprises a linker between at least one of the two homologous domains and the antigenic domain. 138. The isolated nucleic acid molecule according to embodiment 137, wherein the linker comprises the amino acid sequence of GPGPG or PMGLP. 139. The isolated nucleic acid molecule according to any one of embodiments 130 to 138, wherein large T-LAMP further comprises a transmembrane domain of a LAMP protein. 140. The isolated nucleic acid molecule of embodiment 140, wherein the transmembrane domain comprises residues 383 to 405 of SEQ ID NO:1. 141. The isolated nucleic acid molecule according to any one of embodiments 130 to 140, wherein pp65-LAMP further comprises a signal sequence. 142. The isolated nucleic acid molecule of embodiment 141, wherein the signal sequence is derived from a LAMP protein. 143. The isolated nucleic acid molecule according to any one of embodiments 130 to 142, wherein large T-LAMP further comprises the cytoplasmic domain of the LAMP protein. 144. The isolated nucleic acid molecule of embodiment 143, wherein the cytoplasmic domain comprises residues 406 to 417 of SEQ ID NO:1. 145. The IREG comprises one or more of CD40L, CD80, OX40, Flt3L, GM-CSF, IL-12, IL-21, IL-23, IL-15, CD70, CD86, IL-7, IL-18, or IL-33 (or SEQ ID NOs: 133, 145, 147, 149, 151, 155, 159, 165, 169, 173, 177, 181, 189, 191, 204, 238, 242, 246, 250, 252, 254, 256, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 43, 252, or 253), or an extracellular domain thereof, and optionally comprising an amino acid sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, or 100% identical to the amino acid sequence of any one of CD40L, CD80, OX40, Flt3L, GM-CSF, IL-12, IL-21, IL-23, or 145. The isolated nucleic acid molecule of any one of embodiments 130-144, wherein IL-15 is fused to an Fc domain of an immunoglobulin, or wherein the isolated nucleic acid molecule comprises a nucleotide sequence encoding CD40L, CD80, OX40, Flt3L, GM-CSF, IL-12, IL-21, IL-23, IL-15, CD70, CD86, IL-7, IL-18, or IL-33, optionally fused to an Fc domain, and wherein the nucleotide sequence is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, or 100% identical to any one of SEQ ID NOs: 134, 146, 148, 150, 152, 160, 166, 170, 174, 178, 182, 190, 192, 205, 239, 244, or 881. 146. The isolated nucleic acid molecule of any one of embodiments 130 to 145, wherein the secretory signal sequence is heterologous to the IREG. 147. The isolated nucleic acid molecule of embodiment 146, wherein the secretory signal sequence is derived from IgKVIII (e.g., SEQ ID NO: 122), Ig-kappa (e.g., SEQ ID NO: 120), tetranectin, or IL-2. 148. The second polypeptide comprises a fusion of SPD and soluble CD40L (sCD40L), a fusion of SPD and Flt3L, IL-12, IL-21, OX40L fused to an Fc domain, CD80 fused to an Fc domain, or IL-15 (e.g., an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, or 100% identical, to the amino acid sequence of SEQ ID NO: 233, 238, 242, or 252, or an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, or 100% identical, to the amino acid sequence of SEQ ID NO: 131 followed by any of the amino acid sequences of SEQ ID NOs: 204, 151, 145, 147, 149, 193, 181, 155, 159, 169, 252, or 253). 148. The isolated nucleic acid molecule of any one of embodiments 130-147, comprising a nucleotide sequence encoding a fusion of the SPD with soluble CD40L (sCD40L), a fusion of the SPD with Flt3L, IL-12, IL-21, OX40L fused to an Fc domain, CD80 fused to an Fc domain, or IL-15 (e.g., comprising an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical or 100% identical to the sequence of one of SEQ ID NOs: 205, 152, 146, 148, 150, 194, 182, 156, 170, or 881). 149. The isolated nucleic acid molecule according to embodiment 148, wherein the second polypeptide is expressed under the control of an EF-1α core promoter, such as the promoter of SEQ ID NO: 124. 150. A composition comprising an isolated nucleic acid molecule according to any one of embodiments 130 to 149. 151. A host cell comprising an isolated nucleic acid according to any one of embodiments 130 to 149. 152. A composition comprising a host cell according to embodiment 151. 153. A method for treating a subject having cancer, comprising administering to the subject an isolated nucleic acid molecule according to any one of embodiments 130 to 149, a composition according to embodiment 150, or a host cell according to embodiment 151, in an amount sufficient to treat the cancer or to induce an immune response against the cancer in the subject. 154. The method of embodiment 153, further comprising administering at least one second therapeutic agent to the subject. 155. The method of embodiment 153 or 154, wherein the cancer is a skin cancer, such as Merkel cell carcinoma. 156. The isolated nucleic acid molecule of any one of embodiments 1-19, 25-46, 52-73, 79-99, 104-123, or 130-149, comprising DNA, mRNA, or self-amplifying RNA. 157. A set of polypeptides encoded by the isolated nucleic acid of any one of embodiments 1-19, 25-46, 52-73, or 79-99, 104-123, or 130-149.

[0025] Additional objects and advantages will be set forth in part in the description which follows and in part will be understood from the description or may be learned by practice. The objects and advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.

[0026] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the scope of the appended claims.

[0027] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one or more embodiments and, together with the description, serve to explain the principles described herein.

[0028] The objects and features of the present disclosure may be better understood with reference to the following detailed description and the accompanying drawings. [Brief description of the drawings]

[0029] [Figure 1] Figure 1 illustrates a general scheme of the different types of improved LAMP-antigen constructs (identified as ILC-1, ILC-2, ILC-3, ILC-4, ILC-5, and ILC-6) that can be used as described herein. Specific scaffold constructs are further described in U.S. Patent No. 11,203,629, the disclosure of which is incorporated by reference in its entirety.

[0030] [Diagram 2] Figure 2B shows the domains of the LAMP proteins as defined herein, and Figure 2A defines the specific amino acid boundaries of these domains for human LAMP-1 (SEQ ID NO: 1), human LAMP-2 (SEQ ID NO: 2), human LAMP-3 (SEQ ID NO: 3), human LIMP-2 (SEQ ID NO: 4), human Endolyn (SEQ ID NO: 5), human Macrosialin (SEQ ID NO: 80), human LAMP-5 (SEQ ID NO: 93) and human LIMBIC (SEQ ID NO: 67). As described herein, the LAMP luminal domain, homology domain, transmembrane domain, cytoplasmic tail and signal sequence can be used to generate bicistronic LAMP constructs as described herein.

[0031] [Diagram 3] Figure 3 provides an alignment of LAMP-1 proteins found in other species compared to human LAMP-1 (SEQ ID NO: 1). Equivalent domains from these other species can be used to generate the bicistronic LAMP constructs described herein and are readily identifiable by comparing the domains identified for human LAMP-1 in Figures 2 and 3 with the alignment shown in Figure 3.

[0032] [Figure 4]Figure 4 provides an alignment of LAMP-2 proteins found in other species compared to human LAMP-2 (SEQ ID NO: 2). Equivalent domains from these other species can be used to generate the bicistronic LAMP constructs described herein and are readily identifiable by comparing the domains identified for human LAMP-2 in Figures 2 and 4 with the alignment shown in Figure 4.

[0033] [Diagram 5] Figure 5 provides an alignment of LAMP-3 proteins found in other species compared to human LAMP-3 (SEQ ID NO: 3). Equivalent domains from these other species can be used to generate the bicistronic LAMP constructs described herein and are readily identifiable by comparing the domains identified for human LAMP-3 in Figures 2 and 5 with the alignment shown in Figure 5.

[0034] [Figure 6] Figure 6 provides an alignment of LIMP-2 proteins found in other species compared to human LIMP-2 (SEQ ID NO: 4). The equivalent domains of these other species can be used to generate the bicistronic LAMP constructs described herein and are readily identifiable by comparing the domains identified for human LIMP-2 in Figures 2 and 6 to the alignment shown in Figure 6.

[0035] [Figure 7] Figure 7 provides an alignment of LIMBIC proteins found in other species compared to human LIMBIC (SEQ ID NO: 67). Equivalent domains from these other species can be used to generate the bicistronic LAMP constructs described herein and are readily identifiable by comparing the domains identified for human LIMBIC in Figures 2 and 7 to the alignment shown in Figure 7.

[0036] [Figure 8] Figure 8 provides an alignment of Endolyn proteins found in other species compared to human Endolyn (SEQ ID NO: 5). Equivalent domains from these other species can be used to generate the bicistronic LAMP constructs described herein and are readily identifiable by comparing the domains identified for human Endolyn in Figures 2 and 8 with the alignment shown in Figure 8.

[0037] [Figure 9] Figure 9 provides an alignment of macrosialin proteins found in other species compared to human macrosialin (SEQ ID NO: 80). The equivalent domains of these other species can be used to generate the bicistronic LAMP constructs described herein and are readily identifiable by comparing the domains identified for human macrosialin in Figures 2 and 9 to the alignment shown in Figure 9.

[0038] [Figure 10] Figure 10 provides an alignment of LAMP-5 proteins found in other species compared to human LAMP-5 (SEQ ID NO: 93). Equivalent domains from these other species can be used to generate the bicistronic LAMP constructs described herein and can be readily identified by comparing the domains identified for human LAMP-5 in Figures 2 and 10 with the alignment shown in Figure 10.

[0039] [Figure 11] FIG. 11 shows the design of an exemplary bicistronic construct HER2-LAMP-sCD40L.

[0040] [Figure 12]FIG. 12 shows the detection of sCD40L in the supernatant of 293T cells transfected with bicistronic HER2-LAMP-sCD40L vaccine. The asterisk indicates the protein band indicating the presence of sCD40L. Immunoprecipitation experiments with biotinylated anti-CD40L antibody were used to detect sCD40L in control 293T cells, 293T cells transfected with bicistronic HER2-LAMP-sCD40L, or 293T cells transfected with a control vector expressing GFP. Cell culture supernatant was used as input (I). Flow-through (FT) indicates proteins that did not bind to biotinylated anti-CD40L, while bound (B) indicates proteins that bound to the biotinylated anti-CD40L antibody.

[0041] [Figure 13] Figures 13A-D show detection of spike-specific T cell responses in mice after one (Figure 13A-B) or two (Figure 13C-D) immunizations with ITI-bicistronic or 2-V COVID vaccines. Figures 13A and 13C: ELISPOT. Figures 13B and 13D: Spot counts. CV = control vector (plasmid alone). SFC = spot forming cells. Significance was determined using Student's T test. *p<0.05, **p<0.01, ***p<0.001.

[0042] [Figure 14] Figure 14 shows the detection of spike-specific CD4 and CD8 T cells by flow cytometry. Both CD4+ and CD8+ T cell responses were enhanced after vaccination with the 2-V vaccine.

[0043] [Figure 15] FIG. 15 shows intracellular cytokine staining (ICS) of IFNγ, TNFα, and IL-2 in CD4+ and CD8+ T cells.

[0044] [Figure 16]Figures 16A-F show measurements of S1-specific antibodies after one immunization (Figures 16A, 16C, and 16E) versus two immunizations (Figures 16B, 16D, and 16F) with either the ITI-bicistronic or 2-V vaccines. Significance was determined using Student's T-test.

[0045] [Figure 17] Figures 17A-B: Detection of HER2-LAMP-sCD40L in the supernatant of transfected 293T cells. Figure 17A shows the sCD40L standard curve for ELISA. Figure 17B shows the detection of sCD40L in the supernatant. Control cells = cells not transfected with the bicistronic construct.

[0046] [Figure 18] Figures 18A-F show that bicistronic vaccines can induce strong T cell and antibody responses. Figure 18A shows an exemplary vaccination schedule, where mice were immunized by intradermal (ID) injection with 20 μg of control vector, HER2-LAMP, or bicistronic HER2-LAMP-sCD40L. Figure 18B shows IFNγ spot forming cells (ELISPOT). Figure 18C shows the mean IFNγ spot forming cells + SEM. Figures 18D-F show HER2-specific total IgG (Figure 18D), IgG1 (Figure 18E), and IgG2a (Figure 18F) by ELISA; N=7. One-way ANOVA was used for statistical analysis. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0047] [Figure 19] Figures 19A-B show intracellular staining of cytokines IFNγ (IFNg), TNFα (TNFa), and IL-2 in CD4 and CD8 T cells. Figure 19A shows % of stained cells as mean + SEM. Figure 19B shows representative FACS plots. *p<0.05, **p<0.01, ***p<0.001.

[0048] [Figure 20] FIG. 20 shows HER2-specific antibody responses determined by ELISA.

[0049] [Figure 21] Figures 21A-B show that HER2-LAMP-sCD40L protects mice from HER2-expressing breast tumors. Figure 21A shows the results by ELISPOT. Figure 21B shows the tumor size measured by caliper.

[0050] [Figure 22] Figures 22A-B show that HER2-LAMP-sCD40L enhances survival in mice. Figure 22A is a schematic of the experimental design. Figure 22B shows mouse survival after tumor challenge in a single experiment. N=10.

[0051] [Figure 23] Figure 23 shows the effect of HER2-LAMP vaccine with immune response enhancing gene (IREG; HER2-LAMP-IREG) on tumor volume. IREG=CD40L, Flt3L, IL-21, IL-12, and OX40L. **p<0.01; ****p<0.0001.

[0052] [Figure 24] Figure 24 shows the effect of HER2-LAMP-IREG on mouse survival.

[0053] [Diagram 25] Figure 25 shows that HER2-LAMP-IL-15 induces strong antigen-specific antibody responses as determined by ELISA. N=5 per group. t-test was used for statistical analysis. *p<0.05, **p<0.01.

[0054] [Figure 26]Figure 26 shows that HER2-LAMP-IL-15 induces strong T cell responses as determined by ELISPOT. Data represent mean IFNγ spot forming cells + SEM. N=5. Statistical analysis was performed using t-test. *p<0.05, **p<0.01.

[0055] [Figure 27] Figures 27A-B show that two doses of HER2-LAMP-IL-15 induce strong T cell responses as determined by ELISPOT. Data represent original spots (Figure 27A) and mean IFNγ spot forming cells + SEM (Figure 27B). N=5. t-test was used for statistical analysis. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0056] [Figure 28] Figures 28A-C relate to a first generation bicistronic LAMP construct comprising SARS CoV-2 S1 spike protein. Figure 28A shows a general scheme of a vector (bicistronic-S1-LAMP-EF1 IgK-S 2P; 7427 bp) encoding a first generation bicistronic LAMP construct (based on ILC-4) that contains a fragment of SARS CoV-2 S1 spike protein as a target antigen and an expression cassette for expressing SARS CoV-2 S2 spike protein operably linked to an Ig-kappa leader (secretion signal) as a second antigen for secretion. Figure 28B is another representation (as a circular plasmid) of the first generation ITI-COVID bicistronic construct, showing a representative but preferred example of the arrangement of the polynucleotide encoding sequences. Figure 28C shows the complete polynucleotide and encoded polypeptide domains of the first generation ITI-COVID bicistronic construct.

[0057] [Figure 29]Figures 29A-D show the suppression of tumor growth by bicistronic HER2-LAMP-sCD40L (soluble CD40 ligand) administered as a DNA vector in a mouse TSA breast cancer model compared to control (CV) and non-bicistronic HER2-LAMP vectors. Figure 29A shows the experimental protocol. Figure 29B shows the changes in tumor growth for each mouse, and Figure 29C shows the changes in tumor growth for 7 mice in each group (control, HER2-LAMP and HER2-LAMP-sCD40L). The control (CV; top curve) is shown by a filled circle, HER2-LAMP (middle curve in Figure 29C) is shown by a filled square, and HER2-LAMP-sCD40L (bottom curve in Figure 29C) is shown by a filled triangle. Figure 29D shows the tumor weight measured at the end of the experiment (* indicates p<0.05, ** indicates p<0.01).

[0058] [Diagram 30] FIG. 30 shows that HER2-LAMP-sCD40L induces the production of CD3+ memory T cells compared to the control vector.

[0059] [Diagram 31] Figures 31A-C show that HER2-LAMP-sCD40L promotes infiltration of T cells into the tumor microenvironment compared to HER2-LAMP and control vector. Figure 31A shows the number of CD3+ T cells in the tumor after tumor harvest, washing, and dissociation. Figure 31B shows the number of CD4+ T cells. Figure 31C shows the number of CD8+ T cells. * indicates p<0.05, ** indicates p<0.01.

[0060] [Diagram 32] Figures 32A-C show that soluble CD40 ligand (sCD40L) produced by the HER2-LAMP-sCD40L vector enhances activation of IL-12-producing type 1 dendritic cells (DC1) in draining lymph nodes. Figure 32A shows the gating protocol used in the experiment, Figure 32B shows CD8-expressing DC1 cells, and Figure 32C shows IL-12-producing DC1 cells. * indicates p<0.05.

[0061] [Diagram 33] Figure 33A-B shows that bicistronic HER2-LAMP-sCD40L activates inflammatory signals in the tumor microenvironment. Specifically, Figure 33A shows the number of CD4+CD69+ cells in the tumor after tumor harvesting, washing, and dissociation, and Figure 33B shows the number of CD8+CD69+ cells in the tumor. * indicates p<0.05.

[0062] [Diagram 34] Figure 34A-B shows that bicistronic HER2-LAMP-sCD40L promotes PD-1 producing T cells in the tumor microenvironment. Specifically, Figure 33A shows the number of CD4+PD-1+ cells in the tumor after tumor harvest, washing and dissociation, and Figure 33B shows the number of CD8+PD-1+ cells in the tumor. * indicates p<0.05, ** indicates p<0.01.

[0063] [Diagram 35] Figure 35 shows the results of ELISPOT analysis of splenocytes from mice following the protocol described in Figure 29A after incubating the splenocytes with several different pooled peptides of the HER2 extracellular domain. The results show that bicistronic HER2-LAMP-sCD40L induced stronger responses to certain pooled HER2 peptides than control or HER2-LAMP constructs.

[0064] [Diagram 36] FIG. 36 shows results from a parallel experiment to that described in FIG. 29A-C, using 5 mice per control, HER2-LAMP and HER2-LAMP-sCD40L group, confirming that the bicistronic construct suppressed tumor growth more than the other two groups (p<0.05).

[0065] [Figure 37]Figures 37A-D show the results of FACS analysis of splenocytes from the experiment shown in Figure 36 (following the protocol of Figure 29A) and show that HER2-LAMP-sCD40L induces polyfunctional CD4 effector memory T cells ("TEM" cells) in the spleen. Figure 37A shows the percentage of CD4 TEM cells. Figure 37B shows the percentage of CD8 TEM cells. The amount of CD4 or CD8 TEM cells expressing IFNg, TNFa, or both IFNg and TNFa is shown in a bar graph. The remaining figure panels show the response to pooled HER2 peptides by CD4 TEM cells (Figure 37C) or CD8 TEM cells (Figure 37D) expressing both IFNg and TNFa. *P<0.05, **p<0.01, ****p<0.0001.

[0066] [Figure 38] Figures 38A-B show that soluble CD40L expressed from the HER2-LAMP-sCD40L construct enhances activation of DC1 dendritic cells in the spleen in the mouse TSA model of Figure 29A. The FACS gating strategy is shown in Figure 38A, and the percentage of DC1 dendritic cells is shown in Figure 38B. * indicates p<0.05, ** indicates p<0.01.

[0067] [Figure 39] Figures 39A-B show results from cell staining experiments demonstrating that soluble CD40L expressed from the HER2-LAMP-sCD40L construct increases the presence of CD4+ (Figure 39A) and CD8+ T cells (Figure 39B) in the tumor in the mouse TSA model of Figure 29A.

[0068] [Diagram 40]Figures 40A-B show results from an experiment in which mice were injected with control vector (CV), Her2-LAMP (i.e., HER2-Hinge-LAMP), Her2-LAMP-sCD40L, Her2-LAMP-mFlt3L, or a combination of both Her2-LAMP-sCD40L and Her2-LAMP-mFlt3L (7 mice per group), followed by ELISPOT analysis of splenocytes. Figure 40A shows the mean IFNg forming cells + / - SEM for each group, and Figure 40B shows the number of cells recognizing various HER2 peptide pools from each of the five groups.

[0069] [Diagram 41] FIG. 41 shows the antibody titers determined by ELISA for each group in FIG. 40A.

[0070] [Diagram 42] Figures 42A-D show the percentages of different CD4 and CD8 TEM cells that recognize different HER2 extracellular domain peptide pools following the experiment of Figure 40A, specifically CD8 IFNg TNFa cells (Figure 42A), CD8 IFNg cells (Figure 42B), CD4 IFNg TNFa cells (Figure 42C), and CD4 IFNg cells (Figure 42D).

[0071] [Diagram 43] FIG. 43 shows the effect of combined administration of two bicistronic constructs, HER2-LAMP-IL-12 and HER2-LAMP-mFlt3L, on serum antibody titers as measured by ELISA. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0072] The present disclosure encompasses nucleic acid molecules encoding bicistronic or multicistronic LAMP constructs, which can be used, for example, to generate vaccines and / or to elicit antibody and / or humoral immune responses. The nucleic acid molecules can be used to induce immune responses. In one aspect, the present disclosure provides a method for treating a subject with an allergy, an infectious disease such as coronavirus or Covid-19, cancer, or a hyperproliferative disorder by providing a nucleic acid (e.g., a plasmid or vector) encoding the bicistronic LAMP construct described herein. The nucleic acid molecules encoding the bicistronic LAMP constructs can also be used to generate antibodies in non-human vertebrates, preferably non-human mammals.

[0073] A.Definition The following definitions are provided for specific terms used in the written description that follows.

[0074] As used in this specification and claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells (including mixtures thereof). The term "nucleic acid molecule" includes a plurality of nucleic acid molecules.

[0075] As used herein, the term "comprising" is intended to mean that the nucleic acid molecule or bicistronic LAMP construct and method includes the recited elements but does not exclude other elements. In the case of amino acid or nucleotide sequences, it is intended to mean that other sequence elements may be added to either end of the sequence. "Consisting essentially of," when used to define nucleic acid molecules, bicistronic LAMP constructs and methods, is intended to mean excluding other elements that are essentially important to the combination or its function. Thus, bicistronic LAMP constructs that consist essentially of the elements defined herein do not exclude trace contaminants from the isolation and purification methods, as well as pharmacy-acceptable carriers such as phosphate-buffered saline, preservatives, etc. "Consisting of" is intended to mean excluding more than trace elements of other components and substantial method steps for administering the nucleic acid molecule encoding the bicistronic LAMP construct described herein. In the case of amino acid or nucleotide sequences, "consisting of" indicates that no additional sequence elements are added to either end of the sequence, but that the recited sequence is capable of incorporating physiologically occurring modifications to the amino acids or nucleotides, such as DNA methylation or glycosylation. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0076] The term "about" or "approximately" means within an acceptable range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" can mean within a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a value. Unless otherwise specified, the term "about" means within an acceptable range of error for a particular value, e.g., ±1-20%, preferably ±1-10%, and more preferably ±1-5%.

[0077] When a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range, and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limits in the stated range. Where a stated range includes one or both of the limits, ranges excluding both of those included limits are also included in the disclosure.

[0078] As used herein, "lysosomal / endosomal compartment" refers to a membrane-bound acidic vacuole that contains LAMP molecules in its membrane, hydrolases that function in antigen processing, and MHC class II molecules for antigen recognition and presentation. This compartment serves as a site for degradation of foreign substances that are internalized from the cell surface by any of a variety of mechanisms, including endocytosis, phagocytosis, and pinocytosis, as well as intracellular substances that are delivered to this compartment by specialized autolysis events (de Duve, Eur. J. Biochem. 137:391, 1983). As used herein, the term "endosome" encompasses lysosomes.

[0079] As used herein, "lysosome-associated organelle" refers to any organelle that contains a lysosome, including, but not limited to, MIIC, CIIV, melanosome, secretory granule, lytic granule, platelet-rich granule, basophil granule, Birbeck granule, phagolysosome, secretory lysosome, etc. Preferably, such organelle lacks mannose 6-phosphate receptor and contains LAMP, but may or may not contain MHC class II molecule. For review, see, e.g., Blot and Griffiths, Nature Reviews, Molecular Cell Biology, 2002; Dell'Angelica, et al., The FASEB Journal 14:1265-1278, 2000.

[0080] As used herein, the terms "polynucleotide" and "nucleic acid molecule" are used interchangeably to refer to polymeric forms of nucleotides of any length. A polynucleotide may contain deoxyribonucleotides, ribonucleotides, and / or their analogs. Thus, in some cases, the bicistronic LAMP construct nucleic acid molecule herein may be a DNA molecule, such as a DNA vector, e.g., a DNA virus vector, and in other cases, it may be an RNA molecule, including a self-amplifying RNA vector (also known as a self-replicating RNA vector).

[0081] Nucleotides may have any three-dimensional structure and may perform any function, known or unknown. Depending on the context, the term "polynucleotide" also includes, for example, single-stranded, double-stranded and triple-helical molecules, genes or gene fragments, exons, introns, mRNA, tRNA, rRNA, ribozymes, antisense molecules, cDNA, recombinant polynucleotides, branched polynucleotides, aptamers, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Nucleic acid molecules may also include modified nucleic acid molecules (e.g., containing modified bases, sugars, and / or internucleotide linkers).

[0082] As used herein, the term "peptide" refers to a compound of two or more subunit amino acids, amino acid analogs, or peptidomimetics. The subunits may be linked by peptide bonds or other bonds (e.g., as esters, ethers, etc.).

[0083] As used herein, the term "amino acid" refers to any natural and / or unnatural or synthetic amino acid, including glycine and both D or L optical isomers, as well as amino acid analogs and peptidomimetics. A peptide of three or more amino acids is generally called an oligopeptide if the peptide chain is short. If the peptide chain is long (e.g., longer than about 10 amino acids), the peptide is generally called a polypeptide or protein. The term "protein" encompasses the term "polypeptide", although a "polypeptide" may be shorter than a full-length protein.

[0084] As used herein, "LAMP protein" or "LAMP polypeptide" refers to any of the mammalian lysosome-associated membrane proteins human LAMP-1, human LAMP-2, human LAMP-3, human LIMP-2, human Endolyn, human LIMBIC, human LAMP-5, or human Macrosialin described herein, as well as orthologs (such as the LAMP proteins depicted in Figures 3-10), and allelic variants thereof. As used herein, LAMP-1, LAMP2, LAMP-3, LIMP2, Macrosialin, Endolyn, LAMP5, or LIMBIC refer to the human proteins as depicted in Figures 3-10, and allelic variants thereof, unless expressly stated otherwise.

[0085] As used herein, a LAMP "homology domain" comprises at least four evenly spaced cysteine ​​residues as shown in Figures 3-10. These cysteine ​​residues are labeled 1, 2, 3, and 4 in each homology domain as shown in Figures 3-10 (and in LIMP-2 and macrosialin, five cysteines are identified, in LIMBIC, six cysteines are identified, and in endrin, eight cysteines are identified), and are defined herein as "cysteine-conserved fragments." Additional amino acids can be included at either the N-terminus and / or C-terminus of the cysteine-conserved fragment to generate up to and including the complete homology domain of a LAMP protein. These additional added amino acids can be derived from the homology domain from which the cysteine-conserved fragment is derived, or from other LAMP protein homology domains. Thus, as used herein, a LAMP homology domain comprises and / or consists of one cysteine-conserved fragment. At least two LAMP homology domains constitute the luminal domain of LAMP-1, LAMP-2, LAMP-3, or Endolyn.

[0086] As used herein, "expression" refers to the process by which a polynucleotide is transcribed into mRNA and / or translated into a peptide, polypeptide, or protein. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA transcribed from the genomic DNA.

[0087] As used herein, "under transcriptional control" or "operably linked" refers to expression (e.g., transcription or translation) of a polynucleotide sequence controlled by appropriate juxtaposition of expression control elements and a coding sequence. In one aspect, a nucleic acid sequence is "operably linked" to an expression control sequence when the expression control sequence controls and regulates the transcription of that sequence. In another context, the term "operably linked" refers to the linkage of a peptide, polypeptide or protein, such as an epitope or antigen, to a signal sequence, such as a secretory signal sequence, to effect secretion of the peptide, polypeptide or protein from a host cell.

[0088] As used herein, "signal sequence" or "signal peptide" refers to an endoplasmic reticulum translocation sequence. This sequence encodes a signal peptide that communicates with the cell to direct the polypeptide to which it is linked (e.g., via chemical bond) to the endoplasmic reticulum vesicular compartment, enter the extracellular / endocytic organelle, deliver to either the cellular vesicular compartment, the cell surface, or secrete the polypeptide. This signal sequence may be cut off by the cell in the maturation of the polypeptide. Signal sequences can be found associated with various proteins native to prokaryotes and eukaryotes. "Secretory signal sequence" refers to a signal sequence that causes the polypeptide to which it is linked to be secreted by the cell.

[0089] As used herein, a "coding sequence" is a sequence that is transcribed and translated into a peptide, polypeptide or protein when placed under the control of appropriate expression control sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. Coding sequences can include, but are not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. Polyadenylation signals and transcription termination sequences are usually located 3' to the coding sequence.

[0090] As used herein, two coding sequences "correspond" to each other if the sequences or their complementary sequences encode the same amino acid sequence.

[0091] As used herein, "transport" refers to the movement or progression of a polypeptide encoded by a bicistronic LAMP construct through an organelle or compartment in the pathway from the rough endoplasmic reticulum to an endosomal / lysosomal compartment or related organelle, where antigen processing and binding to MHC II occurs.

[0092] The term "antigen" or "antigen of interest" as used herein encompasses any polypeptide sequence encoded by a polynucleotide sequence cloned into a nucleic acid molecule encoding a bicistronic LAMP construct used to elicit an innate or adaptive immune response. "Antigen" encompasses both a single antigen as well as multiple antigenic sequences (derived from the same or different proteins). In some cases, the "antigen" provides a specific "epitope" or antibody recognition site. In some cases, the "antigen" is a "target antigen," meaning that it represents a specific protein expressed by a diseased cell, such as a tumor antigen expressed by a tumor cell, or a specific foreign antigen from an infectious disease, such as a spike protein from a coronavirus or other type of virus. In some cases, the antigen is expressed within a LAMP fusion protein, creating a "LAMP-antigen construct." Different arrangements of LAMP-antigen constructs that can be used herein are illustrated in FIG. 1 as ILC-1 to ILC-6.

[0093] As used herein, "bicistronic LAMP construct" and "bicistronic LAMP construct comprising an antigen" and "bicistronic LAMP construct comprising a target antigen" and "bicistronic LAMP construct comprising an antigen of interest" and "bicistronic LAMP-antigen construct" are used interchangeably and refer to a nucleic acid construct that encodes or expresses two polypeptides, i.e., a first polypeptide and a second polypeptide (which may encode an IREG polypeptide or a second antigen in some embodiments) comprising a LAMP-antigen construct. In some cases, the bicistronic LAMP construct nucleic acid molecule herein may be a DNA molecule, such as a DNA vector, e.g., a DNA virus vector, and in other cases, it may be an RNA molecule, including a self-amplifying RNA vector (also known as a self-replicating RNA vector). In other cases, based on the context, "bicistronic LAMP construct" refers to the polypeptides collectively expressed from the nucleic acid construct.

[0094] As used herein, "immune response element" or "immune response enhancing gene" or "IREG" refers broadly to a gene that encodes a protein that can enhance immune response, such as humoral and / or cellular immune response, in a subject.In some cases, the abbreviation IREG can also be used to refer to the encoded polypeptide of such a gene, or the extracellular domain of such a protein, or a fusion molecule that includes such a protein or extracellular domain.Examples of IREG include certain cytokines or immune proteins, such as CD40L, CD80, OX40, Flt3L, GM-CSF, IL-12, IL-21, IL-23, IL-15, CD70, CD86, IL-7, IL-18, or IL-33.

[0095] As used herein, a "bicistronic LAMP construct delivery vehicle" is defined as any molecule or group of molecules or macromolecules that can carry a nucleic acid molecule encoding a bicistronic LAMP construct into a host cell.

[0096] As used herein, "bicistronic LAMP construct delivery" or "bicistronic LAMP construct transfer" refers to the introduction of a nucleic acid molecule encoding a bicistronic LAMP construct into a host cell, regardless of the method used for introduction. The introduced nucleic acid molecule can be stably or transiently maintained in the host cell. Stable maintenance typically requires that the introduced nucleic acid molecule encoding a bicistronic LAMP construct contains a replication origin compatible with the host cell or is integrated into a host cell replicon, such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome.

[0097] As used herein, "viral bicistronic LAMP construct" refers to a virus or virus particle that contains a nucleic acid molecule that contains a bicistronic LAMP construct and is delivered to a host cell in vivo, ex vivo, or in vitro. Examples of viral bicistronic LAMP constructs include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, and the like. In an embodiment in which gene transfer is mediated by an adenovirus vector, the viral bicistronic LAMP construct comprises an adenovirus genome or a portion thereof, and a selected non-adenovirus gene, together with an adenovirus capsid protein.

[0098] As used herein, "adenovirus-mediated gene transfer" or "adenovirus transduction" refers to the process in which a nucleic acid molecule encoding a bicistronic LAMP construct is introduced into a host cell by an adenovirus that enters the cell. Preferably, the nucleic acid molecule is capable of replicating and / or integrating and being transcribed within the cell.

[0099] As used herein, an "adenovirus particle" refers to an individual adenovirus virion that is composed of an outer capsid and a nucleic acid molecule that encodes a bicistronic LAMP construct, and the capsid is further composed of an adenovirus envelope protein. The adenovirus envelope protein can be modified to include a fusion polypeptide that includes a polypeptide ligand covalently linked to a viral protein, for example, to target the adenovirus particle to a specific cell and / or tissue type.

[0100] As used herein, the term "administering" or "immunizing" or "injecting" a nucleic acid molecule encoding a bicistronic LAMP construct refers to transducing, transfecting, microinjecting, electroporating, or bombarding a cell with the nucleic acid molecule. In some embodiments, the nucleic acid molecule encoding a bicistronic LAMP construct is introduced into a target cell by contacting the target cell with a delivery cell (e.g., by cell fusion or by lysing the delivery cell when the delivery cell is in close proximity to the target cell).

[0101] As used herein, the term "treat" or "treatment" refers broadly to the improvement or amelioration of disease or disorder in a subject, for example, in the case of a tumor, the improvement or amelioration of at least one symptom or marker associated with the disease or disorder, such as, for example, the reduction in size of the tumor, or the change in a biochemical marker associated with the tumor, or the reduction in disease symptoms. In the case of a disease such as cancer or an infectious disease, treat or treatment also refers to the alleviation of at least one symptom of the disease or disorder. Treat or treatment also refers to, for example, the prevention or delay of onset of a disease or disorder, or the prevention or alleviation of one or more symptoms upon onset (including, for example, the onset of asymptomatic disease versus symptomatic disease). Treat or treatment also refers to the use in immunization or vaccination to induce an immune response in a subject that can prevent the onset of symptoms of a disease or disorder in a subject, reduce the severity of symptoms, or improve at least one existing symptom.

[0102] As used herein, the phrase "targeted enhancement" or simply "enhancement" of immune response describes the use of a nucleic acid molecule encoding a "LAMP antigen construct" that includes a target antigen associated with the disease or disorder to be treated in a LAMP fusion polypeptide, and (2) an additional secreted polypeptide that encodes a protein (i.e., an IREG protein) intended to enhance the immune response. Generally, such targeted enhancement allows for simultaneous delivery of the target antigen and the secreted IREG. In some embodiments, this approach can improve both humoral and cellular immune responses.

[0103] The term "secreted" as used herein refers to the intracellular process and pathway that results in the transport of a peptide, polypeptide or protein through the cell wall so that the peptide, polypeptide or protein can be released into the extracellular environment, for example, into the circulation of a subject. A peptide, polypeptide or protein that is destined for the extracellular environment (i.e., secreted) typically comprises a secretory signal sequence, generally located at the N-terminus, which directs ribosomes to translate the peptide, polypeptide or protein to the rough endoplasmic reticulum (rough ER), from where the newly made peptide, polypeptide or protein can be incorporated into small transport or secretory vesicles that transport the peptide, polypeptide or protein to the cell surface for release.

[0104] As used herein, "hybridization" refers to a reaction in which one or more polynucleotides react to form a complex stabilized through hydrogen bonds between the bases of nucleotide residues. Hydrogen bonds can occur by Watson-Crick base pairing, Hoogsteen binding, or any other sequence-specific manner. The complex can include two strands forming a duplex structure, three or more strands forming a multistranded complex, a single self-hybridizing strand, or any combination of these. A hybridization reaction can constitute a step in a more extensive process, such as the initiation of a PCR reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme.

[0105] As used herein, a polynucleotide or polynucleotide region (or polypeptide or polypeptide region) having a particular percentage (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%) "sequence identity" to another sequence means that percentage of bases (or amino acids) are the same in comparing the two sequences when maximally aligned using software programs routine in the art.

[0106] Two sequences are "substantially homologous" or "substantially similar" if at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90 or at least 95% of the nucleotides match over a defined length of the DNA sequence. Similarly, two polypeptide sequences are "substantially homologous" or "substantially similar" if at least 50%, at least 60%, at least 66%, at least 70%, at least 75%, at least 80%, at least 90 or at least 95% of the amino acid residues of the polypeptides match over a defined length of the polypeptide sequence. Substantially homologous sequences can be identified by comparing sequences using standard software available in sequence databanks. Substantially homologous nucleic acid sequences can also be identified, for example, in Southern hybridization experiments under stringent conditions as defined for that particular system. It is within the skill of the art to define appropriate hybridization conditions. For example, stringent conditions can be as follows: hybridization at 42°C with 5xSSC and 50% formamide, and washing at 60°C with 0.1xSSC and 0.1% sodium dodecyl sulfate. Further examples of stringent hybridization conditions include an incubation temperature of about 25°C to about 37°C; a hybridization buffer concentration of about 6xSSC to about 10xSSC; a formamide concentration of about 0% to about 25%; and a washing solution of about 6xSSC. Examples of moderate hybridization conditions include an incubation temperature of about 40°C to about 50°C; a buffer concentration of about 9xSSC to about 2xSSC; a formamide concentration of about 30% to about 50%; and a washing solution of about 5xSSC to about 2xSSC. Examples of high stringency conditions include an incubation temperature of about 55°C to about 68°C; a buffer concentration of about 1xSSC to about 0.1xSSC; a formamide concentration of about 55% to about 75%; and a wash solution of about 1xSSC, 0.1xSSC, or deionized water.Generally, hybridization incubation times are between 5 minutes and 24 hours, with one, two, or more washing steps, with washing incubation times of about 1, 2, or 15 minutes. SSC is 0.15 M NaCl and 15 mM citrate buffer. It is understood that equivalents of SSC using other buffer systems can be used. Similarity can be verified by sequencing, but is preferably also or alternatively verified by function (e.g., ability to transport to endosomal compartments, etc.) using assays appropriate for the particular domain in question.

[0107] The terms "percent sequence similarity", "percent sequence identity", and the like generally refer to the degree of identity or correspondence between different nucleotide sequences of nucleic acid molecules or amino acid sequences of polypeptides that may or may not share a common evolutionary origin (see Reeck et al., supra). Sequence identity can be determined using any of a number of publicly available sequence comparison algorithms, such as BLAST, FASTA, DNA Strider, GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin).

[0108] To determine the percent identity between two amino acid sequences or two nucleic acid molecules, the sequences are aligned for optimal comparison purposes.The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity=number of identical positions / total number of positions (e.g., overlapping positions)×100).In one embodiment, the two sequences are the same length or approximately the same length.The percent identity between two sequences can be determined using techniques similar to those described below, with or without gaps.When calculating percent sequence identity, typically exact matches are counted.

[0109] Determining percent identity between two sequences can be accomplished using mathematical algorithms. A non-limiting example of a mathematical algorithm that can be used to compare two sequences is the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 1993, 87:2264, modified as in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 1990, 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al, J. Mol. Biol. 1990; 215:403. BLAST nucleotide searches can be performed using the NBLAST program, score=100, wordlength=12, to obtain nucleotide sequences homologous to the sequences of the present disclosure. BLAST protein searches can be performed using the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to the protein sequences of the present disclosure. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 1997, 25:3389. Alternatively, PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See ncbi.nlm.nih.gov / BLAST / on the World Wide Web.

[0110] Another non-limiting example of a mathematical algorithm utilized for comparing sequences is the algorithm of Myers and Miller, CABIOS 1988;4:11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program to compare amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

[0111] In one embodiment, the percent identity between two amino acid sequences is determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 1970, 48:444-453) as incorporated into the GAP program in the GCG software package (Accelrys, Burlington, MA; available on the World Wide Web at accelrys.com) using either a Blossum 62 matrix or a PAM250 matrix, gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6. In yet another embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package using a NWSgapdna.CMP matrix, gap weights of 40, 50, 60, 70, or 80, and length weights of 1, 2, 3, 4, 5, or 6. A particular set of parameters (and one that can be used if the practitioner is uncertain as to which parameters to apply to determine whether a molecule is at the limit of sequence identity or homology of the present disclosure) uses a Blossum 62 scoring matrix with a gap open penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0112] Another non-limiting example of how percent identity can be determined is by using software programs such as those described in Current Protocols In Molecular Biology (FM Ausubel et al., eds., 1987) Appendix 30, section 7.7.18, table 7.7.1. Preferably, default parameters are used for the alignment. The alignment program is BLAST using default parameters. In particular, the programs are BLASTN and BLASTP using the following default parameters: Genetic code=standard; Filter=none; Strand=both; Cutoff=60; Expectation=10; Matrix=BLOSUM62; Description=50 sequences; Sort=HIGH SCORE; Database=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translation+SwissProtein+SPupdate+PIR. Details of these programs can be found at the following Internet address: http: / / www.ncbi.nlm.nih.gov / cgi-bin / BLAST.

[0113] Statistical analysis of the characteristics described herein can be performed by standard tests, such as t-tests, ANOVA, or chi-square tests. Typically, statistical significance is measured to a level of p=0.05 (5%), more preferably p=0.01, p=0.001, p=0.0001, p=0.000001.

[0114] "Conservatively modified variants" of domain sequences can also be provided. With respect to a particular nucleic acid sequence, conservatively modified variants refer to nucleic acids that code identical or essentially identical amino acid sequences, or, if the nucleic acid does not code for an amino acid sequence, essentially identical sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced with mixed base and / or deoxyinosine residues (Batzer, et al., 1991, Nucleic Acid Res. 19: 5081; Ohtsuka, et al., 1985, J. Biol. Chem. 260: 2605-2608; Rossolini et al., 1994, Mol. Cell. Probes 8: 91-98).

[0115] The terms "biologically active fragment," "biologically active form," "biologically active equivalent," and "functional derivative" of a wild-type protein have a biological activity that is at least substantially equal to (e.g., not significantly different from) the biological activity of the wild-type protein, as measured using an assay suitable for detecting the activity.

[0116] As used herein, "in vivo nucleic acid delivery, nucleic acid transfer, nucleic acid therapy" and the like refer to the direct transfer of a nucleic acid molecule encoding a bicistronic LAMP construct described herein into the body of a subject, such as a human or non-human mammal, whereby the nucleic acid molecule is transferred in vivo into the cells of such an organism.

[0117] As used herein, the term "in situ" refers to a type of in vivo nucleic acid delivery in which the nucleic acid molecule encoding the bicistronic LAMP construct is in close proximity to the target cell (e.g., the nucleic acid is not administered systemically). For example, in situ delivery methods include, but are not limited to, injecting the nucleic acid molecule encoding the bicistronic LAMP construct directly into a site (e.g., into a tissue such as a tumor or myocardium), contacting the nucleic acid molecule with a cell or tissue through an open surgical field, or using a medical access device such as a catheter to deliver the nucleic acid molecule to a site.

[0118] As used herein, the term "isolated" or "purified" means that a polynucleotide, peptide, polypeptide, protein, antibody, or fragment thereof is separated (or substantially free) from cellular and other components with which it is normally naturally associated. For example, with respect to an isolated nucleic acid molecule encoding a bicistronic LAMP construct as described herein, an isolated polynucleotide is one that is separated from the 5' and 3' sequences with which it is normally associated in the chromosome. As will be apparent to one of skill in the art, a non-natural polynucleotide, peptide, polypeptide, protein, antibody, or fragment thereof does not require "isolation" to distinguish it from its natural counterpart. Substantially free or substantially purified means that at least 50%, preferably at least 70%, more preferably at least 80%, and even more preferably at least 90% of the population is free of naturally associated components.

[0119] As used herein, "target cell" or "recipient cell" refers to an individual cell or cells that are desired to be or have been recipients of the nucleic acid molecules encoding the bicistronic LAMP constructs described herein. The term is also intended to include the progeny of a single cell, and the progeny may not necessarily be completely identical (in morphology or in genomic or total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutations. Target cells may be in contact with other cells (e.g., as in tissues) or may be found circulating within the body of an organism.

[0120] As used herein, a "subject" is a human unless otherwise specified. In such other cases, the subject may be a mammal. Mammals include, but are not limited to, mice, monkeys, humans, livestock, sport animals, and pets. In some cases, the "subject" is a rodent (e.g., rat, mouse, or rabbit), llama, camel, cow, guinea pig, hamster, dog, cat, horse, non-human primate, ape (e.g., monkey or ape), monkey (e.g., marmoset, baboon, rhesus), or ape (e.g., gorilla, chimpanzee, orangutan, gibbon). In some embodiments, non-human mammals, particularly mammals that are conventionally used as models to demonstrate therapeutic efficacy in humans (e.g., mice, primates, pigs, dogs, or rabbits) may be employed.

[0121] The terms "cancer," "neoplasm," and "tumor" are used interchangeably and in either the singular or plural form refer to cells that have undergone malignant transformation that renders them pathological to the host organism. Transformation of primary cancer cells that renders them pathological to the host organism. Primary cancer cells (i.e., cells obtained from near the site of malignant transformation) can be readily distinguished from noncancerous cells by well-established techniques, particularly histological examination. The definition of cancer cells as used herein includes not only primary cancer cells, but any cells derived from a cancer cell ancestor. This includes metastasized cancer cells, as well as in vitro cultures and cell lines derived from cancer cells. When referring to types of cancer that usually manifest as solid tumors, a "clinically detectable" tumor is one that is detectable based on the tumor mass, for example, by procedures such as CAT scan, MR imaging, X-ray, ultrasound, or palpation, and / or is detectable due to the expression of one or more cancer-specific antigens in a sample that can be obtained from the patient.

[0122] In some embodiments, the cancer (including all stages of progression including hyperplasia) is an adenocarcinoma, a sarcoma, a skin cancer, a melanoma, a bladder cancer, a brain cancer, a breast cancer, a uterine cancer, an ovarian cancer, a prostate cancer, a lung cancer (including but not limited to NSCLC, SCLC, squamous cell carcinoma), a colorectal cancer, anal cancer, a rectal cancer, a cervical cancer, a liver cancer, a head and neck cancer, an oral cancer, a salivary gland cancer, an esophageal cancer, a pancreatic cancer, a pancreatic ductal adenocarcinoma (PDA), a renal cancer, a gastric cancer, a renal cancer, a multiple myeloma or a brain cancer.

[0123] Nucleic acid molecules encoding the bicistronic LAMP constructs described herein can also be used to treat allergies, such as, for example, food allergies (e.g., peanut allergens such as Ara H1, Ara H2 and / or Ara H3), or environmental allergens, such as, for example, pollen (e.g., tree pollen such as CRY J1 or CRY J2), dog dander, cat saliva, or dust mites.

[0124] In some cases, the bicistronic LAMP construct may contain an antigen useful in the treatment of infectious diseases, such as viral or bacterial diseases. In one example, the bicistronic LAMP construct can be used to treat coronavirus infections, such as from Covid-19.

[0125] Other diseases and / or disorders that can be treated with the bicistronic LAMP constructs described herein include, for example, infectious diseases and diabetes.

[0126] As used herein, the term "pharmaceutical acceptable carrier" includes any of the standard pharmaceutical carriers, such as phosphate buffered saline, water, and emulsions such as oil / water or water / oil emulsions, as well as various types of wetting agents. The composition containing the nucleic acid molecule encoding the bicistronic LAMP construct can also contain stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see Martin Remington's Pharm.Sci., 15th Ed. (Mack Publ.Co., Easton (1975)).

[0127] A cell has been "transformed," "transduced," or "transfected" by a nucleic acid molecule encoding a bicistronic LAMP construct when such a nucleic acid molecule is introduced into the cell. The transforming DNA may or may not be integrated (covalently linked) with the chromosomal DNA that constitutes the genome of the cell. In prokaryotes, yeast, and mammalian cells, for example, the nucleic acid molecule encoding the bicistronic LAMP construct may be maintained on an episomal element, such as a plasmid. In eukaryotic cells, a stably transformed cell is one in which the nucleic acid molecule encoding the bicistronic LAMP construct has been integrated into a chromosome so that it is inherited by daughter cells via chromosomal replication. This stability is demonstrated by the ability of the eukaryotic cell to establish a cell line or clone consisting of a population of daughter cells that contain the nucleic acid molecule. A "clone" is a population of cells derived from a single cell or a common ancestor by mitosis. A "cell line" is a clone of a primary cell that can grow stably in vitro for many generations (e.g., at least 10).

[0128] As used herein, an "effective amount" or "therapeutically effective amount" is an amount sufficient to affect a beneficial or desired result, e.g., to treat a subject or to induce an immune response in a subject.

[0129] An "antibody" is any immunoglobulin, including antibodies and fragments thereof, that binds to a specific antigen. The term encompasses polyclonal, monoclonal, and chimeric antibodies (e.g., bispecific antibodies). An "antibody combining site" is the structural portion of an antibody molecule consisting of the variable and hypervariable regions of the heavy and light chains that specifically binds to an antigen. Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules, and portions of immunoglobulin molecules that contain the paratope, including the Fab, Fab', F(ab')2, and F(v) portions. Thus, the term antibody encompasses whole antibody molecules as well as antibody fragments and variants of antibodies and antibody fragments, including derivatives such as fusion proteins. Examples of molecules described by the term "antibody" in this application include, but are not limited to, single chain Fv (scFv), Fab fragments, Fab' fragments, F(ab')2, disulfide-linked Fv (sdFv), Fv, and fragments comprising or consisting of either the VL or VH domains. The term "single-chain Fv" or "scFv" as used herein refers to a polypeptide comprising the VL domain of an antibody linked to the VH domain of an antibody. See Carter (2006) Nature Rev. Immunol. 6:243.

[0130] Furthermore, antibodies that can be generated using nucleic acid molecules encoding the bicistronic LAMP constructs described herein include, but are not limited to, monoclonal, multispecific, bispecific, human, humanized, murine, or chimeric antibodies, single chain antibodies, camelid antibodies, Fab fragments, F(ab') fragments, anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies to the antibodies of the present disclosure), domain antibodies, and epitope-binding fragments of any of the above. The immunoglobulin molecule can be any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass of immunoglobulin molecule.

[0131] As used herein, a "human" antibody includes antibodies having the amino acid sequence of a human immunoglobulin, including antibodies isolated from human immunoglobulin libraries and from xenogeneic mice or other organisms that have been genetically engineered to produce human antibodies. Nucleic acid molecules encoding the bicistronic LAMP constructs described herein can be used in combination with known techniques for generating human antibodies and human monoclonal antibodies, as described in the exemplified protocols, see, e.g., PCT Publications WO98 / 24893; WO92 / 01047; WO96 / 34096; WO96 / 33735; European Patent No. 0598877; U.S. Patent Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598; and Lonberg and Huszar, Int. Rev. Immunol. 13:65-93 (1995).

[0132] Human antibodies or "humanized" chimeric monoclonal antibodies can be produced using nucleic acid molecules encoding bicistronic LAMP constructs in combination with techniques described herein or otherwise known in the art. For example, standard methods for producing chimeric antibodies are known in the art. For review, see the following references: Morrison, Science 229:1202(1985);Oi et al., BioTechniques 4:214(1986);Cabilly et al., US Pat. No. 4,816,567;Taniguchi et al., EP171496;Morrison et al., EP173494;Neuberger et al., WO8601533;Robinson et al., WO8702671; Boulianne et al., Nature 312:643(1984); Neuberger et al., Nature 314:268(1985).

[0133] The antibodies that can be generated using the nucleic acid molecules encoding the bicistronic LAMP constructs described herein can be monovalent, bivalent, trivalent or multivalent. For example, monovalent scFvs can be multimerized either chemically or by association with another protein or substance. scFvs fused to hexahistidine or flag tags can be multimerized using Ni-NTA agarose (Qiagen) or using anti-flag antibodies (Stratagene, Inc.). Furthermore, the nucleic acid molecules encoding the bicistronic LAMP constructs can be used to generate monospecific, bispecific, trispecific or higher multispecific antibodies against the encoded antigen(s) contained in the bicistronic LAMP construct. For example, PCT Publication WO93 / 17715;WO92 / 08802;WO91 / 00360;WO92 / 05793;Tutt, et al., J. Immunol. 147:60-69 (1991); U.S. Patent No. 4,474,893; U.S. Patent No. 4,714,681; U.S. Pat. et.al., J. Immunol. 148:1547-1553 (1992).

[0134] An "epitope" is a structure that is usually composed of a short peptide sequence or oligosaccharide that is specifically recognized or specifically bound by components of the immune system. T cell epitopes are generally shown to be linear oligopeptides. Two epitopes correspond to each other if they can be specifically bound by the same antibody. Two epitopes correspond to each other if both can bind to the same B cell receptor or the same T cell receptor, and the binding of one antibody to its epitope substantially prevents the binding by the other epitope (e.g., less than about 30%, preferably less than about 20%, more preferably less than about 10%, 5%, 1%, or about 0.1% of the other epitope is bound). It is understood by those skilled in the art that multiple epitopes can constitute an antigen.

[0135] The term "antigen-presenting cell" as used herein includes any cell that presents an antigen on its surface associated with a major histocompatibility complex molecule or a portion thereof, or one or more non-classical MHC molecules or portions thereof. Examples of suitable APCs are discussed in detail below and include, but are not limited to, whole cells such as macrophages, dendritic cells, B cells, hybrid APCs, and foster antigen-presenting cells.

[0136] As used herein, "engineered antigen-presenting cell" refers to an antigen-presenting cell that has a non-natural molecular moiety on its surface.For example, such a cell may not naturally have a costimulatory factor on its surface, or may have an artificial costimulatory factor on its surface in addition to the natural costimulatory factor, or may express a non-natural class II molecule on its surface.In some embodiments, engineered antigen-presenting cell has an antigen expressed from a bicistronic LAMP construct on its surface.

[0137] As used herein, "immune effector cell" refers to a cell that can bind to an antigen and mediate an immune response. These cells include, but are not limited to, T cells, B cells, monocytes, macrophages, NK cells, and cytotoxic T lymphocytes (CTLs), such as CTL lines, CTL clones, and CTLs derived from tumors, inflammation, or other infiltrates.

[0138] As used herein, "partially human" refers to a nucleic acid having sequences derived from both humans and non-human vertebrates. In the context of a partially human sequence, the partially human nucleic acid has sequences based on human immunoglobulin coding regions and non-coding sequences of endogenous immunoglobulin regions of a non-human vertebrate. When used in reference to endogenous non-coding sequences from a non-human vertebrate, the term "based on" refers to sequences that correspond to non-coding sequences and share a relatively high degree of homology with non-coding sequences of the endogenous locus of the host vertebrate, e.g., the non-human vertebrate from which the ES cells are derived. Preferably, the non-coding sequences share at least 80%, more preferably 90%, homology with the corresponding non-coding sequences found in the endogenous locus of the non-human vertebrate host cell into which the partially human molecule comprising the non-coding sequence has been introduced.

[0139] The term "immunoglobulin variable region" as used herein refers to a nucleotide sequence that encodes all or part of the variable region of an antibody molecule, or all or part of the regulatory nucleotide sequence that controls the expression of an antibody molecule. The immunoglobulin region of a heavy chain may include, but is not limited to, all or part of the V, D, J, and switch regions, including introns. The immunoglobulin region of a light chain may include, but is not limited to, the V and J regions, their upstream adjacent sequences, and introns associated with or adjacent to the light chain constant region gene.

[0140] "Transgenic animal" refers to a non-human animal, usually a mammal, that has an exogenous nucleic acid sequence present in some of its cells as an extrachromosomal element or stably integrated into its germline DNA (i.e., most or all of the genomic sequence of its cells). In generating transgenic animals containing human sequences, partially human nucleic acid is introduced into the germline of such a transgenic animal, for example, by genetic manipulation of the host animal's embryos or embryonic stem cells, according to methods well known in the art.

[0141] "Vector" includes plasmids and viruses, as well as any DNA or RNA molecule, whether autonomously replicating or not, that can be used to transform or transfect cells.

[0142] As used herein, "genetic modification" refers to any addition, deletion or disruption of the normal nucleotide of cells.Artificially recognized methods include virus-mediated gene transfer, liposome-mediated transfer, transformation, transfection and transduction, for example, the nucleic acid molecule encoding the bicistronic LAMP construct based on DNA viruses such as adenovirus, adeno-associated virus and herpes virus, and virus-mediated gene transfer such as retrovirus-based vectors.

[0143] The practice of the present disclosure, in certain embodiments, unless otherwise indicated, employs conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are fully described in the literature. See, for example, Maniatis, Fritsch & Sambrook, In Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, Volumes I and II (DN Glover, ed., 1985); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Nucleic Acid Hybridization (BD Hames & SJ Higgins, eds., 1985); Transcription and Translation (BD Hames & SI Higgins, eds., 1984); Animal Cell Culture (RI Freshney, ed., 1986); Immobilized Cells and Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide to Molecular Cloning (1984).

[0144] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. All publications mentioned herein are incorporated by reference for the purpose of describing and disclosing devices, formulations, and methodologies that can be used in connection with the disclosure described herein.

[0145] B. Exemplary LAMP-Antigen Fusion Polypeptides The following are representative embodiments:

[0146] An isolated nucleic acid molecule that encodes a bicistronic LAMP construct, i.e., encodes a LAMP-antigen fusion protein (LAMP construct) and an immune enhancing protein, both of which are expressed in a host cell or subject. In some cases, the nucleic acid herein may encode a multicistronic construct, for example, when a third polypeptide is also expressed.

[0147] In some cases, the antigen is a target antigen of a particular disease or disorder. In some cases, the immune enhancing protein is a polypeptide or polypeptide domain, e.g., an extracellular domain, derived from an IREG, and is optionally fused to an additional molecule, such as the Fc domain of an immunoglobulin. In some cases, the immune enhancing protein can be secreted, and is therefore operably linked to a secretory signal sequence. In some cases, the LAMP-antigen fusion protein can have the backbone structure of any one of ILC-1, ILC-2, ILC-3, ILC-4, ILC-5 or ILC-6 in Figure 1 herein. In some cases, it has the backbone structure of ILC-4. In some cases, the LAMP-antigen construct comprises an antigen located between two homologous domains of the luminal domain of the LAMP protein. In other cases, the antigen is located before the N-terminus of the LAMP homologous domain or after the C-terminus of the LAMP homologous domain, but before the LAMP transmembrane domain. As used herein, the LAMP protein may be selected from LAMP-1, LAMP2, LAMP-3, LIMP2, macrosialin, endrin, LAMP5 or LIMBIC. In additional embodiments, the LAMP protein is selected from an amino acid sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of amino acid SEQ ID NOs: 1-113. In some cases, the LAMP protein is LAMP-1.

[0148] In some cases, the LAMP component comprises at least two homologous domains of the LAMP, as well as a transmembrane domain of the LAMP or a heterologous protein, and optionally a signal sequence of the LAMP or a heterologous protein, and optionally a cytoplasmic domain of the LAMP. In some such cases, the LAMP protein is LAMP-1.

[0149] In particular, LAMP-1 (Chen et al., J. Biol. Chem. 263:8754, 1988), as deduced from a cDNA clone, consists of a polypeptide core of approximately 382 amino acids with a large (346 residue) luminal amino-terminal domain followed by a 24-residue hydrophobic transmembrane region and a short (12 residue) carboxyl-terminal cytoplasmic tail. See Figures 2A and 2B. The luminal domain is heavily glycosylated, substituted with approximately 20 asparagine-linked complex-type oligosaccharides, and consists of two approximately 160-residue "homologous domains" separated by a proline / serine-rich 22-residue "hinge" region. Each of these "homology domains" contains four evenly spaced cysteine ​​residues that are disulfide bonded to form four 36-38 residue loops symmetrically arranged within the two halves of the luminal domain (Arterburn et al., J. Biol. Chem. 265:7419, 1990; see also Chen et al., J. Biol. Chem. 25:263(18):8754-8, 1988). Figure 2A shows a schematic of the conserved domains among LAMP-1, LAMP-2, LAMP-3, Endolyn, LIMBIC, LAMP5, or Macrosialin.

[0150] Previously reported LAMP constructs contained the following elements in this particular arrangement: (a) the complete luminal domain of the LAMP-1 protein, the antigen, then the complete transmembrane / cytoplasmic tail of the LAMP-1 protein; or (b) the antigen and the complete transmembrane / cytoplasmic tail of the LAMP-1 protein.

[0151] In example (a), the antigenic sequence is inserted between the complete luminal domain of the LAMP-1 protein and the complete transmembrane domain / cytoplasmic tail of LAMP-1. Both constructs have been shown to successfully target the antigen sequence to lysosomes / endosomes, and are referred to as "complete LAMP constructs" as shown in FIG. 1, in comparison with the improved LAMP constructs ILC-1 to ILC-6 described herein. The bicistronic LAMP constructs described herein do not include complete LAMP constructs. Instead, the bicistronic LAMP constructs described herein may include at least one antigen fused to the N-terminus of the luminal domain of the LAMP protein, the N- or C-terminus of at least one homologous domain of the LAMP protein, or the N- or C-terminus of at least one cysteine-conserved fragment of the LAMP protein (see, for example, ILC-1 to ILC-6 in FIG. 1). However, some bicistronic LAMP constructs include at least one antigen fused between a first homologous domain of a LAMP protein and a second homologous domain of a LAMP protein (or between at least two cysteine-conserved fragments) (see, e.g., ILC-4 in FIG. 1). For example, the at least one antigen may be located in or replace the LAMP hinge region. In some embodiments, the construct also includes a transmembrane domain of a LAMP protein and / or a cytosolic tail of a LAMP protein.

[0152] Specifically, in some embodiments, the bicistronic LAMP construct comprises two homologous domains (e.g., ILC-4 in FIG. 1). In some embodiments, these constructs also comprise the transmembrane domain of the LAMP protein and / or the cytosolic tail of the LAMP protein. In other embodiments, if the antigen contains a transmembrane domain, the transmembrane domain of the LAMP protein and / or the cytosolic tail of the LAMP protein are not required. In yet other embodiments, the two homologous domains are derived from LAMP-1, LAMP-2, LAMP-3, or Endolyn proteins. Alternatively, the two homologous domains are derived from different LAMP proteins. In these constructs that comprise two homologous domains, a LAMP hinge domain may also be included.

[0153] When the LAMP-antigen portion of the bicistronic LAMP construct comprises an ILC-4 structure (shown in FIG. 1), for example, when the first polynucleotide sequence of the LAMP construct comprises or encodes a polypeptide comprising two homologous domains of the luminal domain of the LAMP protein and an antigenic domain heterologous to the LAMP protein, with the antigenic domain being disposed between the two homologous domains, the two LAMP protein homologous domains may be selected, for example, from the homologous domain 1 and homologous domain 2 amino acid sequences shown in FIG. 3, which are derived from SEQ ID NOs: 1-113, or amino acid sequences that are at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto. Thus, for example, when the LAMP in such a construct is LAMP-1, the native human LAMP-1 homologous domain 1 and homologous domain 2 may surround the antigenic domain of the LAMP-antigen construct. In some cases, the homology domain may comprise amino acid residues 29-194 and 228-381 of SEQ ID NO: 1, or an amino acid sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto, or one of the sequences of such domains shown in Figures 2A and 3, or, for example, residues 29-195 of SEQ ID NO: 198 and 202. If the construct is a polynucleotide, the construct may further encode a signal sequence prior to the start of the LAMP homology domain 1 coding sequence, for example, residues 1-28 of SEQ ID NO: 1, or a signal sequence otherwise shown in Figures 2A and 3, or, for example, residues 1-28 of SEQ ID NO: 198. In some cases, a heterologous (i.e., non-LAMP) signal sequence may also be used. Thus, in some embodiments, the first homology domain of a LAMP comprises a polypeptide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity of residue 29 to the C-terminus of SEQ ID NO: 198 or residues 29-194 of SEQ ID NO: 1. In some cases, a LAMP signal sequence, such as residues 1-28 of SEQ ID NO: 198 or SEQ ID NO: 1, may also be included.In some cases, the second homologous domain of the LAMP comprises a polypeptide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:202 or residues 228-381 of SEQ ID NO:1. In some cases, a LAMP transmembrane domain may be included, such as that shown in Figures 2A and 3, i.e., comprising residues 383-405 of SEQ ID NO:1, or comprising another native LAMP transmembrane domain sequence. In some cases, a cytoplasmic tail of the LAMP may also be included, such as that comprising residues 406-417 of SEQ ID NO:1, or as shown in Figures 2A and 3, or comprising an otherwise native LAMP cytoplasmic tail sequence.

[0154] In some other embodiments, the bicistronic LAMP construct comprises at least one antigen fused to the C-terminus of a single homologous domain of a LAMP protein or a single cysteine-conserved fragment of a LAMP protein. See, for example, ILC-3 and ILC-5 in FIG. 1. In some embodiments, these constructs also comprise a transmembrane domain of a LAMP protein and / or a cytosolic tail of a LAMP protein. In other embodiments, if the antigen contains a transmembrane domain, the transmembrane domain of a LAMP protein and / or the cytosolic tail of a LAMP protein are not required.

[0155] The bicistronic LAMP constructs described above can be generated using the domains defined in the figures. For example, it is specifically contemplated that the domains included in the bicistronic LAMP constructs shown in Figure 1 can be derived, for example, from sequences derived from orthologous sequences. See, for example, Figures 3-10. It is specifically contemplated to generate bicistronic LAMP constructs using the vector backbone shown in Figure 1 for orthologous sequences using the equivalent domains defined in Figures 2A and 2B. Furthermore, the orthologous sequences shown in Figures 3-10 are representative of sequences that can be used to generate domains. It is well within the skill of the art to identify other orthologous sequences and / or isotypes and compare them to the alignments shown in Figures 3-10. Thus, the alignments shown in Figures 3-10 can be used to generate the bicistronic LAMP constructs described herein by identifying the equivalent boundaries defined in Figures 2A and 2B for human LAMP proteins.

[0156] As will be appreciated by those skilled in the art, the boundaries of each domain are approximate and may be adjusted by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids based on cloning considerations and restriction enzyme placement. Thus, when a particular domain (e.g., a LAMP homology domain) is included in a bicistronic LAMP construct, the amino acid start and end of the domain may be adjusted by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids as the boundaries defined in Figure 2A.

[0157] Each of the bicistronic LAMP constructs described herein can further include a signal sequence and / or additional amino acids between each domain for cloning purposes, as is well known in the art. Furthermore, the LAMP homology domain, LAMP luminal domain, LAMP transmembrane domain, and / or LAMP cytoplasmic tail domain can be derived from the same LAMP protein (e.g., human LAMP-1) or different LAMP proteins (e.g., the luminal domain from human LAMP-1 and the transmembrane domain from human LAMP-2, and / or a mixture of orthologous domains in the same gene family (e.g., LAMP-1) or different gene families (LAMP-1 and LAMP-2).

[0158] Polypeptide variants of the described LAMP constructs are contemplated. For example, polypeptides at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to any of the bicistronic LAMP constructs described herein, as well as polynucleotides encoding these variants. Variants of bicistronic LAMP constructs retain the ability to function by targeting antigenic sequences to lysosomes. For example, modified luminal sequences must retain the ability to transport both membrane and non-membrane antigenic materials to endosomal compartments with at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% efficiency compared to original domain sequences, i.e., efficiency that results in sufficient antigen presentation by cells that contain chimeric sequences to initiate immune responses. In one embodiment, sequences containing suitable transport signals can be identified by constructing bicistronic LAMP constructs that contain the well-characterized antigen domain of ovalbumin, the transmembrane domain, and the cytoplasmic domain of a protein that contains a putative lysosomal / endosomal targeting signal. Targeting efficiency can be measured by determining the ability of antigen-presenting cells expressing bicistronic LAMP constructs to stimulate HA epitope-specific MHC class II-restricted T cells (see, for example, Example 5 of U.S. Patent No. 5,633,234).

[0159] Polynucleotides encoding any of the bicistronic LAMP constructs described herein are some embodiments of the present disclosure, along with polynucleotides with at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 96%, 97%, 98% or 99% identity to any of the bicistronic LAMP construct polynucleotides described herein. Variants of bicistronic LAMP constructs retain the ability to function by targeting antigenic sequences to lysosomes. For example, modified luminal sequences must retain the ability to transport both membrane and non-membrane antigenic materials to endosomal compartments with at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% efficiency compared to the original domain sequence, i.e., efficiency that results in sufficient antigen presentation by cells containing chimeric sequences to initiate an immune response. In one embodiment, sequences containing suitable transport signals can be identified by constructing bicistronic LAMP constructs that contain the well-characterized antigen domain of ovalbumin, the transmembrane domain, and the cytoplasmic domain of a protein that contains a putative lysosomal / endosomal targeting signal. Targeting efficiency can be measured by determining the ability of antigen-presenting cells expressing bicistronic LAMP constructs to stimulate HA epitope-specific MHC class II-restricted T cells (see, for example, Example 5 of U.S. Patent No. 5,633,234).

[0160] C. Construction of an exemplary bicistronic LAMP construct The bicistronic LAMP construct herein can be constructed from an isolated nucleotide sequence, for example, in which a second promoter / enhancer and a coding sequence for a second polypeptide are present in the nucleic acid, either before or after the promoter / enhancer and coding sequence of the LAMP-antigen construct. In some cases, the second polypeptide can be a polypeptide intended to enhance immune response, i.e., a protein or an extracellular domain of a protein expressed from an immune response enhancing gene (IREG). In some cases, the second polypeptide can be secreted, and thus the nucleotide can include an appropriate secretion signal sequence for the second polypeptide, which can be already included in the polypeptide coding sequence or can be from a different protein. In some cases, the second polypeptide is a fusion protein (e.g., an extracellular domain or a complete IREG polypeptide fused to another polypeptide sequence (e.g., an Fc domain of an immunoglobulin, an additional antigen, etc.)).

[0161] As described below, the antigen in the LAMP-antigen construct used in the bicistronic LAMP construct may be a target antigen of an infectious disease, such as a viral spike protein, or alternatively, a cancer antigen, such as a polypeptide that is overexpressed in a particular tumor or tumor cell.

[0162] For example, as described in the following examples, certain bicistronic LAMP proteins were made using spike proteins or spike protein subunits from the SARS Co-V2 virus, as well as cancer antigens such as the HER2 extracellular domain, or using NY-ESO1 or CD161. Thus, in some embodiments, the LAMP-antigen construct comprises a LAMP fused to a cancer antigen or a viral spike protein antigen. The coding region of such an antigen may be combined with an IREG coding region, in some cases. Examples of IREGs include certain cytokines or immune proteins, such as CD40L, CD80, OX40, Flt3L, GM-CSF, IL-12, IL-21, IL-23, IL-15, CD70, CD86, IL-7, IL-18, or IL-33. In some embodiments, the IREG comprises a homologous or heterologous secretory signal sequence coding region, such that the second polypeptide is secreted when expressed.

[0163] As described in the Examples below, the following bicistronic LAMP constructs were constructed: (1) HER2-LAMP-sCD40L (FIG. 11; SEQ ID NO: 197), (2) HER2-LAMP-mFLT3L (SEQ ID NO: 208), (3) HER2-LAMP-IL-12 (SEQ ID NO: 212), (4) HER2-LAMP-IL-21 (SEQ ID NO: 216), (5) HER2-LAMP-OX40L (SEQ ID NO: 241), (6) HER2-LAMP-CD80 (SEQ ID NO: 251), (7) NY-ESO1-LAMP-IL-15 (SEQ ID NO: 222), (8) CD161-LAMP-sCD40L (SEQ ID NO: 235), (9) Spike-LAMP-sCD40L (2-V (10) ITI-COVID-19 bicistronic vaccine (ITI-bicistronic-S1-LAMP-RBG pA-EF2-S2P BHG pA; 1st generation COVID-19 vaccine; SEQ ID NO: 228). The sequences of these constructs and their components are provided in the table below.

[0164] For example, a viral vector can be constructed that includes a first polynucleotide sequence to generate the different structures ILC-1-ILC-6 shown in FIG. 1, including the structure of ILC-4 present in some embodiments, or a similar structure that includes a first antigen of interest (priming antigen) in or replacing the LAMP hinge region between the first and second homologous domains (or between at least two cysteine-conserved fragments) of the LAMP protein. The LAMP domains shown in FIG. 1 were derived from the amino acid sequences shown in FIG. 3-FIG. 10. Corresponding domains can also be cloned from orthologous sequences by identifying equivalent domains when compared to human sequences. Antigens of interest (including one or more antigens of interest) can be cloned individually or in combination into the described LAMP constructs. Viral vectors can also be constructed to encode an expression cassette that includes a second polynucleotide sequence encoding an IREG or a second antigen, which can be operably linked to a secretion signal sequence.

[0165] The relatively "compact" size of the ILC-4 LAMP construct is advantageous in some embodiments, since it may reduce the size constraints associated with including a second polynucleotide sequence. Furthermore, as described in the above-mentioned U.S. Patent No. 11,203,629, the ILC-4 LAMP construct has been found to provide a stronger immune response (e.g., a stronger T cell and / or antibody response) than other LAMP constructs tested. Thus, in some embodiments, the bicistronic LAMP construct uses the ILC-4 design, i.e., includes the general structure of the ILC-4 LAMP-antigen construct.

[0166] In some embodiments, the isolated nucleic acid, e.g., vector or vaccine, comprising an expression cassette encoding a bicistronic LAMP construct is a DNA vector, but in other cases, it is an RNA vector, including a self-amplifying RNA vector.

[0167] D. Antigens for Use in Bicistronic LAMP Constructs 1. SARS-CoV-2 viral antigens and bicistronic constructs In some embodiments, the antigen in the LAMP-antigen construct may include an antigen from another viral infectious agent, such as SARS CoV2 or a viral spike protein. In some cases, the bicistronic construct may also express a second antigen from the same infectious agent for secretion. In other cases, the bicistronic construct may express an IREG polypeptide as a second polypeptide.

[0168] In one particular example, a first generation vector was constructed for use as a vaccine against severe acute respiratory syndrome coronavirus (SARS-CoV-2 virus, otherwise known as COVID-19). Apart from the generation of neutralizing antibodies by effective vaccines, T cells are a key component of naturally acquired protective immunity against many infectious diseases, and many vaccines and vaccines in development against viral infections often induce virus-specific T cell responses that have the capacity to activate innate immunity, have direct effector functions, and aid antibody responses, which can be used for prophylactic and therapeutic purposes.

[0169] To induce both T cell and antibody responses to prevent SARS-Cov-2 infection or to reduce symptoms associated with infection, we designed vectors in which two viral proteins were expressed separately. The first fusion protein, composed of LAMP and viral spike S1 subunit protein, aimed to induce a rapid and strong S1-specific CD4+ T cell response. The second protein was a full-length spike protein with two proline substitutions, which was driven by an independent promoter, human elongation factor-1α (EF1), and also had a sequence peptide (IgK SP) at its N-terminus. The nub design allowed the generation of pre-fusion stabilized spike protein and its secretion to present antigen to B cells. The robust S1-specific CD4 T cells induced by the first promoter not only supported the function of CD8 T cells but also boosted neutralizing antibodies against SARS-Cov-2 (see Figure 28A-C for vector description).

[0170] The first generation ITI-COVID-19 bicistronic vaccine encodes for the expression of the S1 and S2 subunits of the spike glycoprotein anchored on the viral surface. The S1 and S2 subunits of the spike mediate the entry of the SARS-CoV-2 virus into host cells. Using the nucleic acid molecule of the ILC-4 LAMP construct, the S1 coding sequence (GenBank MN908974) was placed between polynucleotide sequences encoding two LAMP homology domains (N-LAMP and luminal domain 2). The S1 coding sequence was operably linked to the CMV promoter under the influence of the CMV enhancer sequence, such that expression in the host cell resulted in an ILC-4 LAMP construct containing the S1 antigen for processing and presentation to MHC class II molecules (i.e., providing a "priming antigen"). The S2 coding sequence is provided elsewhere on the vector and operably linked to a polynucleotide sequence encoding an Ig-kappa secretion signal (leader sequence) and an EF1 promoter sequence, such that expression in a host cell results in the S2 antigen for secretion (i.e., providing a "booster antigen"). The vector thereby provided a single nucleic acid molecule for introduction into a suitable host or target cell that can provide both priming and boosting antigens to elicit an enhanced immune response. This may therefore confer a significant advantage over the use of vectors encoding only bicistronic LAMP constructs, since any desire or requirement to enhance an immune response elicited by a LAMP construct would require administration of a booster vaccine (e.g., containing an antigen) administered separately at one or more time intervals.

[0171] A second generation COVID-19 vector, Spike-LAMP-sCD40L, was later designed and tested as described in Examples 1 and 2 below, and was found to have an unexpectedly even higher immune response to the first generation vector. This second generation vector encodes the LAMP-antigen sequence provided in SEQ ID NO: 229 in an ILC-4 format, with the specific spike derived from the B1.351 variant. It should be understood that as COVID-19 further evolves, other variant spike antigens may be used to replace the antigen used for this vector. And instead of a second COVID-19 antibody as the second polypeptide, this second generation vector encodes a fusion protein of pulmonary surfactant binding protein D (SPD) and CD40L extracellular domain, as provided in SEQ ID NO: 233.

[0172] It is understood that variants of such isolated nucleic acids are encompassed herein, where the LAMP-antibody construct comprises a COVID-19 spike protein or a similar viral antigen protein and an IREG polypeptide, such as SPD-CD40L or the extracellular domain or full protein sequence of another IREG, such as CD80, OX40, Flt3L, GM-CSF, IL-12, IL-21, IL-23, IL-15, CD70, CD86, IL-7, IL-18, or IL-33, optionally further fused to a domain such as an immunoglobulin Fc domain. A linker may also be present in some embodiments between the domains of the first or second polypeptide. And, in some embodiments, the second polypeptide may be secreted and thus operably linked to a secretory signal sequence.

[0173] a) Exemplary Spike Protein Sequences In some embodiments, the LAMP-antigen construct may include an infectious disease antigen, such as a bacterial or viral antigen. In some embodiments, the viral antigen is a spike protein or a domain of a spike protein. In some cases, the viral antigen is derived from a coronavirus, such as a SARS virus, such as the SARS-CoV-2 (COVID-19) virus. In some embodiments, the antigen used in the bicistronic construct herein is selected from antigens encoded by the SARS-CoV-2 virus (e.g., the S1 spike subunit or the S2 spike subunit). Examples of such S1 spike subunits include, but are not limited to, an amino acid sequence with at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:118, and / or an amino acid sequence with at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:119 or SEQ ID NO:231. In some embodiments, the bicistronic construct comprises a polynucleotide encoding an amino acid sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 229. In some embodiments, the bicistronic construct comprises a polynucleotide having at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 232.

[0174] In some cases, the corresponding second polypeptide expressed by the bicistronic construct is an IREG polypeptide, e.g., SPD-CD40L, or the extracellular domain or complete protein sequence of another IREG, e.g., CD80, OX40, Flt3L, GM-CSF, IL-12, IL-21, IL-23, IL-15, CD70, CD86, IL-7, IL-18, or IL-33, which may optionally be further fused to a domain such as an immunoglobulin Fc domain.

[0175] 2. Cancer-associated antigens In other embodiments, the LAMP-antigen construct may comprise a cancer antigen. Candidates for cancer immunotherapy using vaccines comprising the bicistronic LAMP constructs described herein are any patient with cancer, such as, for example, patients with documented Epstein-Barr virus-associated lymphoma, patients with HPV-associated cervical cancer, patients with chronic HCV, or patients with defined rearrangements or mutations in oncogenes or tumor suppressor genes.

[0176] In some embodiments, cancers that can be treated using vaccines comprising the bicistronic LAMP constructs described herein include, but are not limited to, all advanced stages of adenocarcinoma, sarcoma, skin cancer, melanoma, Merkel cell carcinoma, bladder cancer, brain cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, lung cancer (including but not limited to NSCLC, SCLC, squamous cell carcinoma), colorectal cancer, anal cancer, rectal cancer, cervical cancer, liver cancer, head and neck cancer, oral cancer, salivary gland cancer, esophageal cancer, pancreatic (pancreatic) cancer, pancreatic ductal adenocarcinoma (PDA), renal cancer, gastric cancer, renal cancer, multiple myeloma, or hyperplasia of brain cancer.

[0177] It is envisioned that once identified, treatments using vaccine compositions comprising the nucleic acid molecules described herein can be utilized at any time during an individual's cancer course. It is also possible to vaccinate high-risk patients to prevent subsequent cancer development.

[0178] Examples of such cancer antigens include HER2, CD161, and NY-ESO1, or their extracellular domains.

[0179] a) HER2 In some embodiments, the LAMP-antigen construct comprises a HER2 antigen. In some cases, the HER2 antigen comprises an extracellular domain (ECD) portion of HER2. In some cases, the antigen is selected from an amino acid sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 200. In some embodiments, the nucleotide sequence encoding the HER2 antigen has at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 201.

[0180] In some embodiments, the HER2-LAMP-antigen construct encodes an amino acid sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 195. In some embodiments, the LAMP-antigen construct comprises a nucleotide sequence encoding a HER2-LAMP and having at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 196. In some embodiments, the HER2-LAMP antigen construct has a nucleotide coding sequence with at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 199 followed by SEQ ID NO: 201 followed by SEQ ID NO: 203. In some embodiments, the HER2-LAMP antigen construct has a polypeptide sequence with at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 198 followed by SEQ ID NO: 200 followed by SEQ ID NO: 202, optionally with one or two linker sequences between these segments.

[0181] In some cases, the corresponding second polypeptide expressed by the bicistronic construct is an IREG polypeptide, e.g., SPD-CD40L, or the extracellular domain or complete protein sequence of another IREG, e.g., CD80, OX40, Flt3L, GM-CSF, IL-12, IL-21, IL-23, IL-15, CD70, CD86, IL-7, IL-18, or IL-33, which may optionally be further fused to a domain such as an immunoglobulin Fc domain.

[0182] b) NY-ESO1 In some embodiments, the LAMP-antigen construct comprises an antigen of NY-ESO1. Examples of such NY-ESO1 antigen sequences include, but are not limited to, amino acid sequences with at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:223. In some embodiments, the NY-ESO1 nucleotide coding sequence has at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:224.

[0183] In some embodiments, the LAMP-antigen construct comprises (a) a polynucleotide encoding an amino acid sequence of at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 221. In some embodiments, the LAMP-antigen construct coding sequence has at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 199 followed by SEQ ID NO: 224 followed by SEQ ID NO: 203.

[0184] In some cases, the corresponding second polypeptide expressed by the bicistronic construct is an IREG polypeptide, e.g., the extracellular domain or the complete protein sequence of IL-15 or another IREG, e.g., CD40, CD80, OX40, Flt3L, GM-CSF, IL-12, IL-21, IL-23, CD70, CD86, IL-7, IL-18, or IL-33, which may optionally be further fused to a domain such as an immunoglobulin Fc domain.

[0185] c) CD161 In some embodiments, the LAMP-antigen construct comprises an antigen of CD161, such as the ECD of CD161. Examples of such CD161 sequences include, but are not limited to, amino acid sequences with at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 236. In some embodiments, the CD161 ECD nucleotide coding sequence has at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 237.

[0186] In some embodiments, the LAMP-antigen construct comprises (a) a polynucleotide encoding an amino acid sequence of at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 234. In some embodiments, the LAMP-antigen construct coding sequence has at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 199 followed by SEQ ID NO: 237 followed by SEQ ID NO: 203.

[0187] In some cases, the corresponding second polypeptide expressed by the bicistronic construct is an IREG polypeptide, e.g., SPD-CD40L, or the extracellular domain or complete protein sequence of another IREG, e.g., CD80, OX40, Flt3L, GM-CSF, IL-12, IL-21, IL-23, IL-15, CD70, CD86, IL-7, IL-18, or IL-33, which may optionally be further fused to a domain such as an immunoglobulin Fc domain.

[0188] d) Further cancer antigens The following antigens shown in Table A can also be cloned into each of the bicistronic LAMP-antigen constructs described herein using techniques known to those skilled in the art. For example, the sequences / fragments / epitopes listed in the fourth column can also be cloned into the LAMP-antigen constructs described herein. Furthermore, any one of the cancer antigens listed in Table A can be combined with any other antigen listed in Table A that contains the sequences / fragments / epitopes listed in the fourth column and inserted into the LAMP-antigen constructs as described herein. Alternatively, any one of the cancer antigens in Table A can be combined with any other cancer antigens described in this disclosure and inserted into the LAMP-antigen constructs herein.

[0189] In some cases, the corresponding second polypeptide expressed by a bicistronic construct comprising a cancer antigen from Table A is an IREG polypeptide, e.g., SPD-CD40L, or the extracellular domain or complete protein sequence of another IREG, e.g., CD80, OX40, Flt3L, GM-CSF, IL-12, IL-21, IL-23, IL-15, CD70, CD86, IL-7, IL-18, or IL-33, which may optionally be further fused to a domain, such as an immunoglobulin Fc domain.

[0190] [Table 1]

[0191]

Table 2

[0192]

Table 3

[0193]

Table 4

[0194]

Table 5

[0195]

Table 6

[0196]

Table 7

[0197]

Table 8

[0198]

Table 9

[0199]

Table 10

[0200]

Table 11

[0201]

Table 12

[0202]

Table 13

[0203]

Table 14

[0204]

Table 15

[0205]

Table 16

[0206]

Table 17

[0207]

Table 18

[0208]

Table 19

[0209]

Table 20

[0210]

Table 21

[0211]

Table 22

[0212]

Table 23

[0213]

Table 24

[0214]

Table 25

[0215]

Table 26

[0216]

Table 27

[0217]

Table 28

[0218]

Table 29

[0219]

Table 30

[0220]

Table 31

[0221]

Table 32

[0222] Additionally, the antigens listed in Table A (including sequences / fragments / epitopes listed in column 4) can be cloned individually or in combination with each other into the LAMP-antigen constructs described herein. Thus, each one of the sequences listed in column 1 of Table A, including the epitopes / fragments listed in column 4 of Table 1, can be used in combination with another sequence also selected from column 1 or column 4 of Table A to generate a LAMP-antigen construct. In the case of a nucleic acid construct or a cell carrying such a nucleic acid construct, the construct can, for example, encode one or more of the antigens listed in Table A or elsewhere herein. Additionally, an IREG sequence can also be included as a second polypeptide (or, in the case of a nucleic acid vector or a cell carrying such a vector, the coding sequence for the IREG) to generate a bicistronic LAMP construct.

[0223] The order of antigen combinations in a particular LAMP-antigen construct may also vary, for example, the names pp65, gB, IE1, MTRII, US28, IGFBP2, IL10, UL144, UL141, US11, HOS, IE2, TERT, survivin, tetanus, NY-ESO-1, HER2, HER3, HVEM, HPV16E6, HPV18E6, HPV16E7, HPV18E7, EBNA1, EBNA trunc, gp350, LMP2, GCP3, Middle S, X Protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, MUCIN-1, MUCIN-1 iso, CD40-L, WT-1, WT-1 trunc, PRAME, LAGE-1, and / or MAGE. A3 refers to the proteins set out in Table 1, and is expressly intended to refer generally to not only the full length sequences set out in column 1 of Table A, but also to sequences / fragments / epitopes as set out in column 4 of Table A. Thus, each one of the sequences set out in column 1 of Table A, including the epitopes / fragments set out in column 4 of Table 1, can be used to generate a LAMP construct, which can then be incorporated, for example, with an IREG sequence to create a bicistronic LAMP construct.

[0224]

[0043] Without limiting the disclosure, to illustrate different, additional possible antibody combinations, antibody combinations (comprising sequences shown in column 1 of Table A and / or sequences / fragments / epitopes set forth in column 4 of Table A) can be cloned into LAMP constructs as follows: (a) pp65 with gB, IE1, MTRII, US28, IGFBP2, IL10, UL144, UL141, US11, HOS, IE2, TERT, survivin, tetanus, NY-ESO-1, HER2, HER3, HVEM, HPV16E6, HPV18E6, HPV16E7, HPV18E7, EBNA1, EBNA trunc, gp350, LMP2, GCP3, Middle S,X Protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, MUCIN-1, MUCIN-1 iso, CD40-L, WT-1, WT-1 trunc, PRAME, LAGE-1, and / or MAGE A3; (b) gB and at least one of pp65, IE1, MTRII, US28, IGFBP2, IL10, UL144, UL141, US11, HOS, IE2, TERT, survivin, tetanus, NY-ESO-1, HER2, HER3, HVEM, HPV16E6, HPV18E6, HPV16E7, HPV18E7, EBNA1, EBNA trunc, gp350, LMP2, GCP3, Middle S,X Protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, MUCIN-1, MUCIN-1 iso, CD40-L, WT-1, WT-1 with at least one of trunc, PRAME, LAGE-1, and / or MAGE A3;(c) IE1 and pp65, gB, MTRII, US28, IGFBP2, IL10, UL144, UL141, US11, HOS, IE2, TERT, survivin, tetanus, NY-ESO-1, HER2, HER3, HVEM, HPV16E6, HPV18E6, HPV16E7, HPV18E7, EBNA1, EBNA trunc, gp350, LMP2, GCP3, Middle S, A3; (d) MTRII and at least one of pp65, gB, IE1, US28, IGFBP2, IL10, UL144, UL141, US11, HOS, IE2, TERT, survivin, tetanus, NY-ESO-1, HER2, HER3, HVEM, HPV16E6, HPV18E6, HPV16E7, HPV18E7, EBNA1, EBNA trunc, gp350, LMP2, GCP3, Middle S,X Protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, MUCIN-1, MUCIN-1 iso, CD40-L, WT-1, WT-1 trunc, PRAME, LAGE-1, and / or MAGE. (e) US28 and at least one of pp65, gB, IE1, MTRII, IGFBP2, IL10, UL144, UL141, US11, HOS, IE2, TERT, survivin, tetanus, NY-ESO-1, HER2, HER3, HVEM, HPV16E6, HPV18E6, HPV16E7, HPV18E7, EBNA1, EBNA trunc, gp350, LMP2, GCP3, Middle S,X Protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, MUCIN-1, MUCIN-1 iso, CD40-L, WT-1, WT-1 trunc, PRAME, LAGE-1, and / or MAGE A3;(f) IGFBP2 and pp65, gB, IE1, MTRII, US28, IL10, UL144, UL141, US11, HOS, IE2, TERT, survivin, tetanus, NY-ESO-1, HER2, HER3, HVEM, HPV16E6, HPV18E6, HPV16E7, HPV18E7, EBNA1, EBNA trunc, gp350, LMP2, GCP3, Middle S, A3; (g) IL10 and at least one of pp65, gB, IE1, MTRII, US28, IGFBP2, UL144, UL141, US11, HOS, IE2, TERT, survivin, tetanus, NY-ESO-1, HER2, HER3, HVEM, HPV16E6, HPV18E6, HPV16E7, HPV18E7, EBNA1, EBNA trunc, gp350, LMP2, GCP3, Middle S,X Protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, MUCIN-1, MUCIN-1 iso, CD40-L, WT-1, WT-1 trunc, PRAME, LAGE-1, and / or MAGE. (h) UL144 and at least one of pp65, gB, IE1, MTRII, US28, IGFBP2, IL10, UL141, US11, HOS, IE2, TERT, survivin, tetanus, NY-ESO-1, HER2, HER3, HVEM, HPV16E6, HPV18E6, HPV16E7, HPV18E7, EBNA1, EBNA trunc, gp350, LMP2, GCP3, Middle S,X Protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, MUCIN-1, MUCIN-1 iso, CD40-L, WT-1, WT-1 trunc, PRAME, LAGE-1, and / or MAGE A3;(i) UL141, pp65, gB, IE1, MTRII, US28, IGFBP2, IL10, UL144, US11, HOS, IE2, TERT, survivin, tetanus, NY-ESO-1, HER2, HER3, HVEM, HPV16E6, HPV18E6, HPV16E7, HPV18E7, EBNA1, EBNA trunc, gp350, LMP2, GCP3, Middle S, A3; (j) US11 and at least one of pp65, gB, IE1, MTRII, US28, IGFBP2, IL10, UL144, UL141, HOS, IE2, TERT, survivin, tetanus, NY-ESO-1, HER2, HER3, HVEM, HPV16E6, HPV18E6, HPV16E7, HPV18E7, EBNA1, EBNA trunc, gp350, LMP2, GCP3, Middle S,X Protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, MUCIN-1, MUCIN-1 iso, CD40-L, WT-1, WT-1 trunc, PRAME, LAGE-1, and / or MAGE. (k) IE2 and at least one of pp65, gB, IE1, MTRII, US28, IGFBP2, IL10, UL144, UL141, US11, HOS, TERT, survivin, tetanus, NY-ESO-1, HER2, HER3, HVEM, HPV16E6, HPV18E6, HPV16E7, HPV18E7, EBNA1, EBNA trunc, gp350, LMP2, GCP3, Middle S,X Protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, MUCIN-1, MUCIN-1 iso, CD40-L, WT-1, WT-1 trunc, PRAME, LAGE-1, and / or MAGE A3;(l) TERT, pp65, gB, IE1, MTRII, US28, IGFBP2, IL10, UL144, UL141, US11, HOS, IE2, Survivin, Tetanus, NY-ESO-1, HER2, HER3, HVEM, HPV16E6, HPV18E6, HPV16E7, HPV18E7, EBNA1, EBNA trunc, gp350, LMP2, GCP3, Middle S, A3; (m) survivin and at least one of pp65, gB, IE1, MTRII, US28, IGFBP2, IL10, UL144, UL141, US11, HOS, IE2, TERT, tetanus, NY-ESO-1, HER2, HER3, HVEM, HPV16E6, HPV18E6, HPV16E7, HPV18E7, EBNA1, EBNA trunc, gp350, LMP2, GCP3, Middle S,X Protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, MUCIN-1, MUCIN-1 iso, CD40-L, WT-1, WT-1 trunc, PRAME, LAGE-1, and / or MAGE. (n) tetanus and at least one of pp65, gB, IE1, MTRII, US28, IGFBP2, IL10, UL144, UL141, US11, HOS, IE2, TERT, survivin, NY-ESO-1, HER2, HER3, HVEM, HPV16E6, HPV18E6, HPV16E7, HPV18E7, EBNA1, EBNA trunc, gp350, LMP2, GCP3, Middle S,X Protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, MUCIN-1, MUCIN-1 iso, CD40-L, WT-1, WT-1 trunc, PRAME, LAGE-1, and / or MAGE A3;(o)NY-ESO-1, pp65, gB, IE1, MTRII, US28, IGFBP2, IL10, UL144, UL141, US11, HOS, IE2, TERT, Survivin, Tetanus, HER2, HER3, HVEM, HPV16E6, HPV18E6, HPV16E7, HPV18E7, EBNA1, EBNA trunc, gp350, LMP2, GCP3, Middle S, A3 and at least one of;(p)HER2 and pp65, gB, IE1, MTRII, US28, IGFBP2, IL10, UL144, UL141, US11, HOS, IE2, TERT, survivin; Tetanus, NY-ESO-1, HER3, HVEM, HPV16E6, HPV18E6, HPV16E7, HPV18E7, EBNA1, EBNA trunc, gp350, LMP2, GCP3, Middle S, X Protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, MUCIN-1, MUCIN-1 iso, CD40-L, WT-1, WT-1 trunc, PRAME, LAGE-1, and / or MAGE A3; (q) HER3 and at least one of pp65, gB, IE1, MTRII, US28, IGFBP2, IL10, UL144, UL141, US11, HOS, IE2, TERT, survivin, tetanus, NY-ESO-1, HER2, HVEM, HPV16E6, HPV18E6, HPV16E7, HPV18E7, EBNA1, EBNA trunc, gp350, LMP2, GCP3, Middle S,X Protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, MUCIN-1, MUCIN-1 iso, CD40-L, WT-1, WT-1 trunc, PRAME, LAGE-1, and / or MAGE. (r) HVEM and at least one of pp65, gB, IE1, MTRII, US28, IGFBP2, IL10, UL144, UL141, US11, HOS, IE2, TERT, survivin, tetanus, NY-ESO-1, HER2, HER3, HPV16E6, HPV18E6, HPV16E7, HPV18E7, EBNA1, EBNA trunc, gp350, LMP2, GCP3, Middle S,X Protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, MUCIN-1, MUCIN-1 iso, CD40-L, WT-1, WT-1 trunc, PRAME, LAGE-1, and / or MAGE A3;(s)HOS, pp65, gB, IE1, MTRII, US28, IGFBP2, IL10, UL144, UL141, US11, IE2, TERT, Survivin, Tetanus, NY-ESO-1, HER2, HER3, HVEM, HPV16E6, HPV18E6, HPV16E7, HPV18E7, EBNA1, EBNA trunc, gp350, LMP2, GCP3, Middle S, and at least one of: (t) HPV16E6 and pp65, gB, IE1, MTRII, US28, IGFBP2, IL10, UL144, UL141, US11, HOS, IE2, TERT, survivin, tetanus, NY-ESO-1, HER2, HER3, HVEM, HPV18E6, HPV16E7, HPV18E7, EBNA1, EBNA trunc, gp350, LMP2, GCP3, Middle S,X Protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, MUCIN-1, MUCIN-1 iso, CD40-L, WT-1, WT-1 trunc, PRAME, LAGE-1, and / or MAGE. (u) HPV18E6 and at least one of pp65, gB, IE1, MTRII, US28, IGFBP2, IL10, UL144, UL141, US11, HOS, IE2, TERT, survivin, tetanus, NY-ESO-1, HER2, HER3, HVEM, HPV16E6, HPV16E7, HPV18E7, EBNA1, EBNA trunc, gp350, LMP2, GCP3, Middle S,X Protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, MUCIN-1, MUCIN-1 iso, CD40-L, WT-1, WT-1 trunc, PRAME, LAGE-1, and / or MAGE A3;(v) HPV16E7, pp65, gB, IE1, MTRII, US28, IGFBP2, IL10, UL144, UL141, US11, HOS, IE2, TERT, Survivin, Tetanus, NY-ESO-1, HER2, HER3, HVEM, HPV16E6, HPV18E6, HPV18E7, EBNA1, EBNA trunc, gp350, LMP2, GCP3, Middle S, and at least one of: (w) HPV18E7 and pp65, gB, IE1, MTRII, US28, IGFBP2, IL10, UL144, UL141, US11, HOS, IE2, TERT, survivin, tetanus, NY-ESO-1, HER2, HER3, HVEM, HPV16E6, HPV18E6, HPV16E7, EBNA1, EBNA trunc, gp350, LMP2, GCP3, Middle S,X Protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, MUCIN-1, MUCIN-1 iso, CD40-L, WT-1, WT-1 trunc, PRAME, LAGE-1, and / or MAGE (x) EBNA1 and at least one of pp65, gB, IE1, MTRII, US28, IGFBP2, IL10, UL144, UL141, US11, HOS, IE2, TERT, survivin, tetanus, NY-ESO-1, HER2, HER3, HVEM, HPV16E6, HPV18E6, HPV16E7, HPV18E7, EBNA trunc, gp350, LMP2, GCP3, Middle S, X Protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, MUCIN-1, MUCIN-1 iso, CD40-L, WT-1, WT-1 trunc, PRAME, LAGE-1, and / or MAGE A3;(y)EBNA1 trunc, pp65, gB, IE1, MTRII, US28, IGFBP2, IL10, UL144, UL141, US11, HOS, IE2, TERT, survivin, tetanus, N Y-ESO-1, HER2, HER3, HVEM, HPV16E6, HPV18E6, HPV16E7, HPV18E7, EBNA1, gp350, LMP2, GCP3, Middle S, and at least one of: (z) gp350 and pp65, gB, IE1, MTRII, US28, IGFBP2, IL10, UL144, UL141, US11, HOS, IE2, TERT, survivin, tetanus, NY-ESO-1, HER2, HER3, HVEM, HPV16E6, HPV18E6, HPV16E7, HPV18E7, EBNA1, EBNA trunc, LMP2, GCP3, Middle S,X Protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, MUCIN-1, MUCIN-1 iso, CD40-L, WT-1, WT-1 trunc, PRAME, LAGE-1, and / or MAGE. (aa) LMP2 and at least one of pp65, gB, IE1, MTRII, US28, IGFBP2, IL10, UL144, UL141, US11, HOS, IE2, TERT, survivin, tetanus, NY-ESO-1, HER2, HER3, HVEM, HPV16E6, HPV18E6, HPV16E7, HPV18E7, EBNA1, EBNA trunc, gp350, GCP3, Middle S,X Protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, MUCIN-1, MUCIN-1 iso, CD40-L, WT-1, WT-1 trunc, PRAME, LAGE-1, and / or MAGE A3;(ab) GCP3 and at least one of pp65, gB, IE1, MTRII, US28, IGFBP2, IL10, UL144, UL141, US11, HOS, IE2, TERT, survivin, tetanus, NY-ESO-1, HER2, HER3, HVEM, HPV16E6, HPV18E6, HPV16E7, HPV18E7, EBNA1, EBNA trunc, gp350, LMP2, Middle S,X Protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, MUCIN-1, MUCIN-1 iso, CD40-L, WT-1, WT-1 trunc, PRAME, LAGE-1, and / or MAGE A3; (ac) Middle and at least one of pp65, gB, IE1, MTRII, US28, IGFBP2, IL10, UL144, UL141, US11, HOS, IE2, TERT, survivin, tetanus, NY-ESO-1, HER2, HER3, HVEM, HPV16E6, HPV18E6, HPV16E7, HPV18E7, EBNA1, EBNA trunc, gp350, LMP2, GCP3, X Protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, MUCIN-1, MUCIN-1 iso, CD40-L, WT-1, WT-1 trunc, PRAME, LAGE-1, and / or MAGE A3; (ad)X Protein and at least one of pp65, gB, IE1, MTRII, US28, IGFBP2, IL10, UL144, UL141, US11, HOS, IE2, TERT, survivin, tetanus, NY-ESO-1, HER2, HER3, HVEM, HPV16E6, HPV18E6, HPV16E7, HPV18E7, EBNA1, EBNA trunc, gp350, LMP2, GCP3, Middle S, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, MUCIN-1, MUCIN-1 iso, CD40-L, WT-1, WT-1 trunc, PRAME, LAGE-1, and / or MAGE A3;(ae)TIGIT, IE1, MTRII, US28, IGFBP2, IL10, UL144, UL141, US11, HOS, IE2, TERT. NY-ESO-1, HER2, HER3, HVEM, HPV16E6, HPV18E6, HPV16E7, HPV18E7, EBNA1, EBNA1 trunc, gp350, LMP2, GCP3, Middle S, X Protein, TEM8, TEM1, HER2 ECD+™, CEA, TARP, PROSTEIN, PSMA, BIRC4, MUCIN-1, MUCIN-1 iso、CD40-L、WT-1、WT-1 trunc、PR; and at least one of AME, LAGE-1, and / or MAGE A3; (af) TEM8 and at least one of pp65, gB, IE1, MTRII, US28, IGFBP2, IL10, UL144, UL141, US11, HOS, IE2, TERT, survivin, tetanus, NY-ESO-1, HER2, HER3, HVEM, HPV16E6, HPV18E6, HPV16E7, HPV18E7, EBNA1, EBNA trunc, gp350, LMP2, GCP3, Middle S,X Protein, TIGIT, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, MUCIN-1, MUCIN-1 iso, CD40-L, WT-1, WT-1 trunc, PRAME, LAGE-1, and / or MAGE (ag)TEM1 and at least one of pp65, gB, IE1, MTRII, US28, IGFBP2, IL10, UL144, UL141, US11, HOS, IE2, TERT, survivin, tetanus, NY-ESO-1, HER2, HER3, HVEM, HPV16E6, HPV18E6, HPV16E7, HPV18E7, EBNA1, EBNA trunc, gp350, LMP2, GCP3, Middle S,X Protein, TIGIT, TEM8, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, MUCIN-1, MUCIN-1 iso, CD40-L, WT-1, WT-1 trunc, PRAME, LAGE-1, and / or MAGE A3;(ah)HER2 ECD+TM and pp65, gB, IE1, MTRII, US28, IGFBP2, IL10, UL144, UL141, US11, HOS, IE2, TERT, Survivin, Tetanus, NY-ESO-1, HER2, HER3, HVEM, HPV16E6, HPV18E6, HPV16E7, HPV18E7, EBNA1, EBNA trunc, gp350, LMP2, GCP3, Middle S, (ai) CEA and at least one of pp65, gB, IE1, MTRII, US28, IGFBP2, IL10, UL144, UL141, US11, HOS, IE2, TERT, survivin, tetanus, NY-ESO-1, HER2, HER3, HVEM, HPV16E6, HPV18E6, HPV16E7, HPV18E7, EBNA1, EBNA trunc, gp350, LMP2, GCP3, Middle S,X Protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, TARP, PROSTEIN, PSMA, BIRC4, MUCIN-1, MUCIN-1 iso, CD40-L, WT-1, WT-1 trunc, PRAME, LAGE-1, and / or MAGE. (aj) TARP and at least one of pp65, gB, IE1, MTRII, US28, IGFBP2, IL10, UL144, UL141, US11, HOS, IE2, TERT, survivin, tetanus, NY-ESO-1, HER2, HER3, HVEM, HPV16E6, HPV18E6, HPV16E7, HPV18E7, EBNA1, EBNA trunc, gp350, LMP2, GCP3, Middle S,X Protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, PROSTEIN, PSMA, BIRC4, MUCIN-1, MUCIN-1 iso, CD40-L, WT-1, WT-1 trunc, PRAME, LAGE-1, and / or MAGE A3;(ak)PROSTEIN, pp65, gB, IE1, MTRII, US28, IGFBP2, IL10, UL144, UL141, US11, HOS, IE2, TERT, Survivin, Tetanus, NY-ESO-1, HER2, HER3, HVEM, HPV16E6, HPV18E6, HPV16E7, HPV18E7, EBNA1, EBNA trunc, gp350, LMP2, GCP3, Middle S, A3; (a1) PSMA and at least one of pp65, gB, IE1, MTRII, US28, IGFBP2, IL10, UL144, UL141, US11, HOS, IE2, TERT, survivin, tetanus, NY-ESO-1, HER2, HER3, HVEM, HPV16E6, HPV18E6, HPV16E7, HPV18E7, EBNA1, EBNA trunc, gp350, LMP2, GCP3, Middle S,X Protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, BIRC4, MUCIN-1, MUCIN-1 iso, CD40-L, WT-1, WT-1 trunc, PRAME, LAGE-1, and / or MAGE. (am) BIRC4 and at least one of pp65, gB, IE1, MTRII, US28, IGFBP2, IL10, UL144, UL141, US11, HOS, IE2, TERT, survivin, tetanus, NY-ESO-1, HER2, HER3, HVEM, HPV16E6, HPV18E6, HPV16E7, HPV18E7, EBNA1, EBNA trunc, gp350, LMP2, GCP3, Middle S,X Protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, MUCIN-1, MUCIN-1 iso, CD40-L, WT-1, WT-1 trunc, PRAME, LAGE-1, and / or MAGE A3;(an) MUCIN-1 and at least one of pp65, gB, IE1, MTRII, US28, IGFBP2, IL10, UL144, UL141, US11, HOS, IE2, TERT, survivin, tetanus, NY-ESO-1, HER2, HER3, HVEM, HPV16E6, HPV18E6, HPV16E7, HPV18E7, EBNA1, EBNA trunc, gp350, LMP2, GCP3, Middle S,X Protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, MUCIN-1 iso, CD40-L, WT-1, WT-1 trunc, PRAME, LAGE-1, and / or MAGE A3; (ao) MUCIN-1 iso, pp65, gB, IE1, MTRII, US28, IGFBP2, IL10, UL144, UL141, US11, HOS, IE2, TERT, survivin, tetanus, NY-ESO-1, HER2, HER3, HVEM, HPV16E6, HPV18E6, HPV16E7, HPV18E7, EBNA1, EBNA trunc, gp350, LMP2, GCP3, Middle S, (ap)CD40-L and at least one of pp65, gB, IE1, MTRII, US28, IGFBP2, IL10, UL144, UL141, US11, HOS, IE2, TERT, survivin, tetanus, NY-ESO-1, HER2, HER3, HVEM, HPV16E6, HPV18E6, HPV16E7, HPV18E7, EBNA1, EBNA trunc, gp350, LMP2, GCP3, Middle S,X Protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, MUCIN-1, MUCIN-1 iso, WT-1, WT-1 trunc, PRAME, LAGE-1, and / or MAGE A3;(aq) WT-1 and at least one of pp65, gB, IE1, MTRII, US28, IGFBP2, IL10, UL144, UL141, US11, HOS, IE2, TERT, survivin, tetanus, NY-ESO-1, HER2, HER3, HVEM, HPV16E6, HPV18E6, HPV16E7, HPV18E7, EBNA1, EBNA trunc, gp350, LMP2, GCP3, Middle S,X Protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, MUCIN-1, MUCIN-1 iso, CD40-L, WT-1 trunc, PRAME, LAGE-1, and / or MAGE A3; (ar) WT-1 trunc, pp65, gB, IE1, MTRII, US28, IGFBP2, IL10, UL144, UL141, US11, HOS, IE2, TERT, survivin, tetanus, NY-ESO-1, HER2, HER3, HVEM, HPV16E6, HPV18E6, HPV16E7, HPV18E7, EBNA1, EBNA trunc, gp350, LMP2, GCP3, Middle S, and (as) PRAME and at least one of pp65, gB, IE1, MTRII, US28, IGFBP2, IL10, UL144, UL141, US11, HOS, IE2, TERT, survivin, tetanus, NY-ESO-1, HER2, HER3, HVEM, HPV16E6, HPV18E6, HPV16E7, HPV18E7, EBNA1, EBNA trunc, gp350, LMP2, GCP3, Middle S,X Protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, MUCIN-1, MUCIN-1 iso, CD40-L, WT-1, WT-1 trunc, LAGE-1, and / or MAGE A3;(at) LAGE-1 and at least one of pp65, gB, IE1, MTRII, US28, IGFBP2, IL10, UL144, UL141, US11, HOS, IE2, TERT, survivin, tetanus, NY-ESO-1, HER2, HER3, HVEM, HPV16E6, HPV18E6, HPV16E7, HPV18E7, EBNA1, EBNA trunc, gp350, LMP2, GCP3, Middle S,X Protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, MUCIN-1, MUCIN-1 iso, CD40-L, WT-1, WT-1 trunc, PRAME, and / or MAGE A3; and / or (au) MAGE A3 and pp65, gB, IE1, MTRII, US28, IGFBP2, IL10, UL144, UL141, US11, HOS, IE2, TERT, survivin, tetanus, NY-ESO-1, HER2, HER3, HVEM, HPV16E6, HPV18E6, HPV16E7, HPV18E7, EBNA1, EBNA; At least one of the following antigens may be combined in a particular LAMP construct: trunc, gp350, LMP2, GCP3, Middle S, X Protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, MUCIN-1, MUCIN-1 iso, CD40-L, WT-1, WT-1 trunc, PRAME, and / or LAGE-1. The order of the combination of the above antigens in a particular LAMP construct may vary, as this list describes what the LAMP construct contains, and does not necessarily describe the arrangement of the antigens in a particular construct. Furthermore, it is specifically envisioned that these antigens can be combined in a single LAMP construct or delivered in a composition that includes multiple LAMP constructs.

[0225] Further examples of antigens that can be used in the bicistronic LAMP constructs of the present specification include those disclosed in, for example, International Publication No. WO2018 / 204534, Table 1 and Figures 19-20 of that publication, or International Publication No. WO2021 / 077051, Table 1 and Figure 11A of that publication. Both of these publications are incorporated by reference in their entirety herein.

[0226] In some cases, the antigen used in the bicistronic LAMP construct comprises a pp65 antigen, such as one or more of SEQ ID NO:291, 292, or 293, or a portion of SEQ ID NO:291 shown in column 4 of Table A. In some cases, the antigen comprises SEQ ID NO:292 or 293. In some cases, the antigen used in the bicistronic LAMP construct comprises a gB antigen, such as one or more of SEQ ID NO:294, 295, 296, or 297, or an antigen fragment from SEQ ID NO:294 shown in column 4 of Table A. In some cases, the antigen comprises SEQ ID NO:296 or 297. In some cases, the antigen used in the bicistronic LAMP construct comprises a 1E1 antigen, such as one or more of SEQ ID NO:298, 299, or 300, or a 1E1 polypeptide sequence shown in column 4 of Table A. In some cases, the antigen comprises SEQ ID NO:299 or 300. In some cases, the antigen comprises two or more of the pp65, gB, and 1E1 antigen sequences linked by one or more linker peptide sequences, such as, for example, those shown in column 4 of Table A. In some cases, the antigen comprises each of the pp65, gB, and 1E1 antigen sequences, for example, sequences selected from the set of antigen sequences comprising (a) SEQ ID NO: 292 or 293, (b) SEQ ID NO: 296 or 297, and (c) SEQ ID NO: 299 or 300. For example, such polynucleotide coding sequences of pp65, gB, and 1E1 antigens can be used in a bicistronic LAMP construct together with an IREG protein coding sequence. For example, such constructs in the form of a polypeptide, polynucleotide (i.e., a DNA vector or a self-replicating RNA vector), or cell may be used in the treatment of various cancers such as those listed above, including, for example, where the cancer (including all stages of progression including hyperplasia) is an adenocarcinoma, a sarcoma, a skin cancer, a melanoma, a bladder cancer, a brain tumor, a breast cancer, a uterine cancer, an ovarian cancer, a prostate cancer, a lung cancer (including but not limited to NSCLC, SCLC, squamous cell carcinoma), a colorectal cancer, anal cancer, a rectal cancer, a cervical cancer, a liver cancer, a head and neck cancer, an oral cancer, a salivary gland cancer, an esophageal cancer, a pancreatic cancer, a pancreatic ductal adenocarcinoma (PDA), a renal cancer, a gastric cancer, a renal cancer, a multiple myeloma, or a brain tumor.In some cases, the cancer is glioblastoma multiforme. In some cases, the cancer is breast cancer. In some cases, the cancer is prostate cancer. In some cases, the cancer is head and neck cancer. In some cases, the cancer is colorectal cancer.

[0227] In other cases, the antigen comprises a large T antigen, such as one comprising the amino acid sequence of SEQ ID NO: 254, 255, or 256. In some cases, the antigen comprises the amino acid sequence of SEQ ID NO: 255 or SEQ ID NO: 256. In some cases, the LAMP-antigen construct within the bicistronic LAMP construct comprises the amino acid sequence of SEQ ID NO: 879 or SEQ ID NO: 880, both of which comprise the amino acid sequence of SEQ ID NO: 256, flanked by the homology domains of LAMP1 and including a signal sequence. SEQ ID NO: 879 further comprises the LAMP transmembrane domain and cytoplasmic region. In the bicistronic LAMP constructs herein, the construct may further comprise or encode an IREG protein. Constructs in which the antigen is a large T antigen may be used, for example, in the treatment of cancer (e.g., skin cancer (e.g., Merkel cell carcinoma)). Such antigens may be combined with IREG as a second polypeptide, examples of which are provided below and elsewhere in this disclosure.

[0228] E. Exemplary Immune Response Enhancement Genes (IREGs) for Use in Bicistronic LAMP Constructs In some embodiments, the second polypeptide may comprise a domain or antigen encoded by an immune response enhancing gene (IREG) that may increase T cell and / or antibody responses to the bicistronic LAMP-antigen construct. Examples of IREG polypeptides include, for example, CD40L, CD80, OX40, Flt3L, GM-CSF, IL-12, IL-21, IL-23, IL-15, CD70, CD86, IL-7, IL-18, IL-33, GM-CSF, 4-1BB, 4-1BBL, IL-27, or CCL20.

[0229] 1. CD40 Ligand (CD40L) In some embodiments, the IREG is CD40L. CD40L is a transmembrane protein expressed on the surface of activated T cells, particularly CD4 T cells. CD40L stimulates the CD40-dependent activation of antigen-presenting cells (APCs), such as dendritic cells (DCs) and macrophages, as well as B cells to enhance T cell and antibody responses. In some embodiments, CD40L is a soluble version of CD40L (sCD40L). In some embodiments, sCD40L is a protein complex that includes a tetramer, i.e., a tetramer of trimers of CD40L. In some embodiments, sCD40L is more soluble and / or has better secretion than native CD40L. Examples of such CD40L sequences include, but are not limited to, amino acid sequences that have at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:204.

[0230] In some embodiments, the sCD40L is fused to another polypeptide, such as an SPD. In some embodiments, the bicistronic construct comprises a second polynucleotide encoding an amino acid sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or 100% sequence identity to SEQ ID NO: 196, 233, or 238, or a combination of SEQ ID NO: 131 or 133 followed by SEQ ID NO: 204. In some embodiments, the bicistronic construct comprises a second polynucleotide that encodes or has 100% sequence identity to at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence of SEQ ID NO: 196, 233, or 238, or a combination of SEQ ID NO: 131 or 133 followed by SEQ ID NO: 204. In some cases, the coding sequence for the second polypeptide comprises or has 100% sequence identity to at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% nucleotide sequence of SEQ ID NO: 239 or 237 or 205.

[0231] 2. FLT3L In some embodiments, the IREG is FLT3L. Examples of such FLT3L sequences include, but are not limited to, amino acid sequences with at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 151 or 209.

[0232] In some embodiments, the bicistronic construct encodes a second polypeptide comprising a human Flt3L polypeptide preceded by an SPD polypeptide, thus creating a fusion protein. In some such cases, the bicistronic construct encodes a second polypeptide having at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence of SEQ ID NO:207, or having 100% sequence identity.

[0233] 3.IL-12 In some embodiments, the IREG is IL-12. Examples of such IL-12 sequences include, but are not limited to, amino acid sequences with at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NOs: 137, 139, 143, 145, 147, 149, 187, 189, 193, or 213.

[0234] In some embodiments, the nucleotide coding sequence of IL-12 comprises a polynucleotide sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or 100% sequence identity to SEQ ID NO: 138, 140, 144, 146, 148, 150, 188, 190, 194, or 214.

[0235] 4.IL-21 In some embodiments, the IREG is IL-21. Examples of such IL-21 sequences include, but are not limited to, amino acid sequences with at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 179, 181, or 217.

[0236] In some embodiments, the nucleotide coding sequence of IL-21 comprises a polynucleotide sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or 100% sequence identity to SEQ ID NO: 180, 182, or 218.

[0237] 5. OX40 Ligand (OX40L) In some embodiments, IREG is OX40L.Examples of such OX40L sequences include, but are not limited to, the amino acid sequence of at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity of SEQ ID NO: 153, 155, or 243.In some cases, OX40L is fused to a heterologous signal peptide, such as that derived from IL-2.In some cases, the OX40L sequence is an extracellular domain sequence.In some cases, OX40L extracellular domain is also fused to the Fc domain of immunoglobulin. In some cases, the bicistronic construct comprises a second polynucleotide that encodes at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the amino acid sequence of, or has 100% sequence identity to, SEQ ID NO:242, or a combination of SEQ ID NO:246 or 248 followed by SEQ ID NO:243.

[0238] 6.IL-15 In some embodiments, the IREG is IL-15. Examples of such IL-15 sequences include, but are not limited to, amino acid sequences with at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 167 or 169. In some embodiments, the nucleotide coding sequence of IL-15 comprises a polynucleotide sequence with at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or 100% sequence identity to SEQ ID NO: 168 or 170.

[0239] In some cases, IL-15 is expressed behind a heterologous signal sequence, such as IgKVIII or Ig-kappa. Examples of such IL-15 sequences include, but are not limited to, amino acid sequences with at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 225. In some embodiments, the nucleotide coding sequence of IL-15 comprises a polynucleotide sequence with at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or 100% sequence identity to SEQ ID NO: 226.

[0240] 7.CD80 In some embodiments, the IREG is CD80. Examples of such CD80 sequences include, but are not limited to, amino acid sequences with at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 157, 159, or 253. In some embodiments, the nucleotide coding sequence of CD80 comprises a polynucleotide sequence with at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or 100% sequence identity to SEQ ID NO: 158 or 160 or 254.

[0241] In some cases, CD80 is expressed behind a heterologous signal sequence, such as an IL-2 signal sequence. In some cases, CD80 is the extracellular domain of CD80. In some cases, the extracellular domain of CD80 is further fused to the Fc domain of an immunoglobulin. Examples of such sequences include, but are not limited to, amino acid sequences with at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 252 or 253.

[0242] F. Exemplary Bicistronic LAMP Construct Sequences In some embodiments, the bicistronic construct comprises a polynucleotide sequence encoding a LAMP-antigen polypeptide comprising two homologous domains of the luminal domain of the LAMP protein and an antigenic domain heterologous to the LAMP protein, the antigenic domain being disposed between the two homologous domains. In some cases, the first homologous domain of the LAMP comprises a polypeptide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the C-terminus of SEQ ID NO:198 or residues 29-194 of SEQ ID NO:1. In some cases, the second homologous domain of the LAMP comprises a polypeptide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:202 or residues 228-381 of SEQ ID NO:1.

[0243] In some embodiments, the bicistronic construct comprises a polynucleotide sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:230.

[0244] In some embodiments, the bicistronic construct comprises a polynucleotide sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:228.

[0245] In some embodiments, the bicistronic construct comprises a polynucleotide sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:197.

[0246] In some embodiments, the bicistronic construct comprises a polynucleotide sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:208.

[0247] In some embodiments, the bicistronic construct comprises a polynucleotide sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:212.

[0248] In some embodiments, the bicistronic construct comprises a polynucleotide sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:216.

[0249] In some embodiments, the bicistronic construct comprises a polynucleotide sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:222.

[0250] In some embodiments, the bicistronic construct comprises a polynucleotide sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:241.

[0251] In some embodiments, the bicistronic construct comprises a polynucleotide sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:251.

[0252] In some embodiments, the bicistronic construct comprises a polynucleotide sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:235.

[0253] G. Assembly of sequences encoding bicistronic LAMP constructs The procedure for constructing a bicistronic LAMP construct containing an antigen of interest is well known in the art (see, for example, Williams, et al., J. Cell Biol. 111:955, 1990). The DNA sequence encoding the desired segment can be obtained from readily available recombinant DNA materials (e.g., those available from the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852, USA) or from a DNA library containing the desired DNA.

[0254] For example, a DNA segment corresponding to a desired domain sequence can be assembled with appropriate control and signal sequences using conventional procedures of recombinant DNA methodology (see, e.g., U.S. Patent No. 4,593,002, and Langford, et al., Molec. Cell. Biol. 6: 3191, 1986).

[0255] A DNA sequence encoding a protein or polypeptide can be chemically synthesized or isolated by one of several approaches. The DNA sequence to be synthesized can be designed with the appropriate codons for the desired amino acid sequence. In general, the codons that are preferred for the intended host in which the sequence will be used for expression are selected. The complete sequence can be assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, for example, Edge, Nature 292:756, 1981; Nambair, et al. Science 223:1299, 1984; Jay, et al., J.Biol.Chem.259:6311, 1984.

[0256] In one embodiment, one or more of the polynucleotides encoding the domain sequences of the bicistronic LAMP construct are individually isolated using polymerase chain reaction (MA Innis, et al., In PCR Protocols: A Guide to Methods and Applications, Academic Press, 1990). The domains are preferably isolated from publicly available clones known to contain them, but they can also be isolated from genomic DNA or cDNA libraries. Preferably, the isolated fragments are bounded by compatible restriction endonuclease sites that allow the construction of the bicistronic LAMP construct encoding the antigen sequence. This technique is well known to those skilled in the art. The domain sequences may be directly fused to each other (e.g., without intervening sequences), or inserted into each other (e.g., when the domain sequences are discontinuous), or separated by intervening sequences (e.g., linker sequences, etc.).

[0257] The basic strategies for preparing oligonucleotide primers, probes and DNA libraries, and their screening by nucleic acid hybridization, are well known to those skilled in the art. See, e.g., Sambrook, et al., 1989, supra; Perbal, 1984, supra. The construction of suitable genomic DNA or cDNA libraries is within the skill of the art. See, e.g., Perbal, 1984, supra. Alternatively, suitable DNA libraries or publicly available clones are available from suppliers of biological research materials, such as Clonetech and Stratagene, and from public depositories, such as the American Type Culture Collection.

[0258] Selection can be accomplished by expressing sequences from an expression library of DNA and immunologically detecting the expressed peptides. Clones expressing peptides that bind to MHC II molecules and the desired antibody / T cell receptor are selected. These selection procedures are well known to those of skill in the art (see, e.g., Sambrook, et al., 1989, supra).

[0259] Once a clone containing the coding sequence for the desired polypeptide sequence has been prepared or isolated, the sequence can be cloned into any appropriate vector, preferably containing an origin of replication for maintaining the sequence in a host cell.

[0260] H. Nucleic Acid Delivery Vehicles In one aspect, the present disclosure provides a nucleic acid molecule (e.g., a plasmid or vector) comprising: (i) a first polynucleotide sequence encoding an antigen described herein fused between a first homologous domain of a LAMP protein and a second homologous domain of a LAMP protein (or between at least two cysteine-conserved fragments) (e.g., at least one antigen of interest may be located in or replace the LAMP hinge region); and (ii) a second polynucleotide sequence encoding at least one IREG or additional antigen operably linked to a secretion signal sequence, wherein the IREG or additional antigen is secreted into the circulation of a subject. Further exemplary nucleic acid embodiments are described in the Summary and Claims sections, as well as throughout the disclosure herein.

[0261] The nucleic acid molecule can be provided as a vaccine composition and introduced into a cell. The cell can be a host cell for replicating the nucleic acid molecule or for expressing the bicistronic LAMP construct (providing a LAMP-antigen construct) and the IREG or second antigen operably linked to a secretion signal sequence (so that the second polypeptide comprising the IREG or second antigen is secreted from the cell). Preferably, the host cell is an antigen-presenting cell (described further below). In some embodiments, the vaccine comprises DNA, mRNA, or self-amplifying RNA.

[0262] In some embodiments, the first polynucleotide sequence encoding the LAMP-antigen construct further comprises a polynucleotide sequence for insertion into a target cell and an expression control sequence operably linked thereto for controlling the expression (e.g., transcription and / or translation) of the first polynucleotide sequence in the cell. Similarly, in some embodiments, the second polynucleotide sequence encoding a second polypeptide comprising an IREG or an additional antigen further comprises a polynucleotide sequence for insertion into a target cell and an expression control sequence operably linked thereto for controlling the expression (e.g., transcription and / or translation) of the second polynucleotide sequence in the cell. The nucleic acid molecule comprising the first and second polynucleotide sequences can be provided, for example, as a plasmid, phage, autonomously replicating sequence (ARS), centromere, and other sequences that can replicate or replicate in vitro or in a host cell (e.g., bacteria, yeast, or insect cell, etc.) and / or in a target cell (e.g., a mammalian cell, preferably an antigen-presenting cell, etc.) and / or can transport the expressed sequence to a desired location within the target cell.

[0263] Recombinant expression vectors can be derived from microorganisms that easily infect animals, including humans, horses, cows, pigs, llamas, giraffes, dogs, cats, or chickens. Specific vectors herein include those already used as live vaccines, such as vaccinia. These recombinants can be directly inoculated into the host, conferring immunity not only to the microbial vector, but also to express the foreign antigen. Some vectors contemplated herein as live recombinant vaccines include RNA viruses, adenoviruses, herpes viruses, polio viruses, and vaccinia and other pox viruses. See, for example, Flexner, Adv. Pharmacol. 21: 51, 1990.

[0264] Expression control sequences include, but are not limited to, promoter sequences that bind RNA polymerase, enhancer sequences or negative regulatory elements that bind transcriptional activators and repressors, respectively, and / or translation initiation sequences for ribosome binding. For example, bacterial expression vectors may contain a promoter, such as the lac promoter, and a Shine-Dalgarno sequence and the start codon AUG for transcription initiation (Sambrook et al., 1989, supra). Similarly, eukaryotic expression vectors preferably contain a heterologous, homologous, or chimeric promoter for RNA polymerase II, a downstream polyadenylation signal, the start codon AUG, and a termination codon for detachment of the ribosome.

[0265] Expression control sequences can be obtained from naturally occurring genes or can be designed. Designed expression control sequences include, but are not limited to, mutated and / or chimeric expression control sequences, or synthetic or cloned consensus sequences. Vectors that contain both a promoter and a cloning site into which a polynucleotide can be operably linked are well known in the art. Such vectors can transcribe RNA in vitro or in vivo and are commercially available from sources such as Stratagene (La Jolla, Calif.) and Promega Biotech (Madison, Wis.).

[0266] To optimize expression and / or transcription, it may be necessary to remove, add, or modify the 5' and / or 3' untranslated portions of the vector to eliminate extra or alternative translation initiation codons or other sequences that may disrupt or reduce expression at either the transcriptional or translational level. Alternatively, a consensus ribosome binding site can be inserted directly 5' of the initiation codon to enhance expression. A wide variety of expression control sequences (sequences that control the expression of a polynucleotide sequence operably linked thereto) can be used in these vectors to express the polynucleotide sequences of the present disclosure. Such useful expression control sequences include, for example, early or late promoters of SV40, CMV, vaccinia, polyoma, adenovirus, herpes virus, and other sequences known to control the expression of genes in mammalian cells, as well as various combinations thereof.

[0267] The first and second polynucleotide sequences (encoding the LAMP-antigen construct and the second polypeptide) can be expressed from the same or different expression control sequences. For example, a single promoter may be used for transcription of the bicistronic mRNA molecule encoding both polypeptides, or different promoters may be used to control the expression of two different polypeptides. Those skilled in the art will recognize that the "second" translation of the encoded protein can be achieved by including a translation enhancing element such as an internal ribosome entry site (IRES) (Plank et al., Wiley Interdiscip. Rev. RNA 3:195-212, 2012) or an unstructured junction sequence to achieve translational reinitiation after termination (Onishi et al., G3 (Bethesda) 6(12):4115-4125, 2016). However, in some embodiments, the polynucleotide sequences encoding the two polypeptides are expressed from different expression control sequences (e.g., different promoters).

[0268] To achieve secretion of the second polynucleotide, the coding sequence may include a polynucleotide sequence encoding a secretory signal sequence (also known as a leader sequence), typically 16-30 amino acids in length, such that expression of the second polynucleotide sequence is operably linked to the secretory signal sequence. Those skilled in the art are well aware of suitable secretory signal sequences, including, for example, the signal sequences of interleukin-2, CD5, immunoglobulin kappa light chain (hereinafter referred to as Ig-kappa leader), trypsinogen, serum albumin, and prolactin (Stern et al., Trends Cell Mol. Biol. 2:1-17, 2007; Kober et al., Biotechnol. Bioengin. 110:1164-1173, 2013). The secretory signal sequence may be a secretory signal sequence that is "native" to the IREG or the second polypeptide antigen, as the case may be.

[0269] In one embodiment, the nucleic acid molecule comprises an origin of replication for replication. Preferably, the origin functions in at least one type of host cell that can be used to generate a sufficient number of copies of the sequence for use in delivery to the target cell. Thus, suitable origins include, but are not limited to, origins that function in bacterial cells (e.g., Escherichia sp., Salmonella sp., Proteus sp., Clostridium sp., Klebsiella sp., Bacillus sp., Streptomyces sp., and Pseudomonas sp.), yeast (e.g., Saccharomyces sp. or Pichia sp.), insect cells, and mammalian cells. In one embodiment, an origin of replication is provided that functions in the target cell (e.g., a mammalian cell such as a human cell) into which the nucleic acid delivery vehicle is introduced. In another embodiment, at least two origins of replication are provided, one that functions in the host cell and one that functions in the target cell.

[0270] Alternatively or additionally, the nucleic acid molecule may comprise a polynucleotide sequence(s) for facilitating integration of at least a portion of the nucleic acid molecule (e.g., a delivery vector) into a target cell chromosome. For example, the nucleic acid molecule may comprise a region of homology to the target cell chromosomal DNA. In one embodiment, the nucleic acid molecule is provided as a delivery vector that comprises two or more recombination sites flanking the nucleic acid sequence encoding the LAMP-antigen construct and the second polypeptide, and / or the bicistronic LAMP construct itself.

[0271] Vectors may further include detectable and / or selectable markers to verify that the vector has been successfully introduced into and / or can be expressed by the target cell. These markers may code for an activity such as the production of RNA, peptides, or proteins, or may provide binding sites for RNA, peptides, proteins, inorganic and organic compounds or compositions, and the like.

[0272] Examples of detectable / selectable marker genes include, but are not limited to, DNA segments that encode products that provide resistance to other toxic compounds (e.g., antibiotics); DNA segments that encode products that are otherwise deficient in the recipient cell (e.g., tRNA genes, auxotrophic markers); DNA segments that encode products that suppress the activity of a gene product; DNA segments that encode products that can be easily identified (e.g., phenotypic markers such as β-galactosidase, fluorescent proteins (GFP, CFP, YFG, BFP, RFP, EGFP, EYFP, EBFP, dsRed, mutant, modified, or enhanced forms thereof, etc.), and cell surface proteins); DNA segments that bind products that are deleterious to cell survival and / or function; DNA segments that inhibit the activity of other nucleic acid segments (e.g., antisense oligonucleotides); DNA segments that bind products that modify substrates (e.g., restriction endonucleases); DNA segments that can be used to isolate or identify desired molecules (e.g., segments that encode specific protein binding sites); primer sequences; DNA segments that, when absent, directly or indirectly confer resistance or sensitivity to a particular compound; and / or DNA segments that encode products that are toxic in the recipient cell.

[0273] The marker gene can be used as a conformational marker of successful gene transfer and / or to isolate cells expressing the transferred gene and / or to recover the transferred gene from the cell. For example, in one embodiment, the marker gene is used to isolate and purify antigen-presenting cells expressing the bicistronic LAMP construct described herein.

[0274] Substantially similar genes can be provided, for example, genes with identity of more than about 50%, more than about 70%, more than 80%, more than about 90%, preferably more than about 95% to known genes.Substantially similar domain sequences can be identified by first selecting sequences that specifically hybridize to the domain sequence of interest under stringent hybridization conditions.To carry out an assay to determine the suitability of homologous, mutant or modified domain sequences, it is simply a matter of screening sequences that express appropriate activity.Such screening is routine in the art.

[0275] The bicistronic LAMP construct encoding the LAMP-antigen construct and the second polypeptide can be provided as a naked nucleic acid molecule or in a delivery vehicle associated with one or more molecules to facilitate the entry of the nucleic acid into cells. Suitable delivery vehicles include, but are not limited to, liposome formulations, polypeptides, polysaccharides, lipopolysaccharides, viral formulations (including, for example, viruses, viral particles, artificial viral envelopes, etc.), cell delivery vehicles, etc.

[0276] I. Lipid-Based Formulations A delivery vehicle designed to facilitate intracellular delivery of a nucleic acid molecule encoding a bicistronic LAMP construct must interact with both non-polar and polar environments (e.g., in or on cell membranes, tissue fluids, intracellular compartments, etc.). Thus, preferably, the delivery vehicle is designed to contain both polar and non-polar domains or translocation sequences to translocate the nucleic acid molecule encoding the bicistronic LAMP construct into the cell.

[0277] Compounds with polar and non-polar domains are called amphiphiles. Cationic amphiphiles have polar groups that can become positively charged at or near physiological pH in order to interact with negatively charged polynucleotides such as DNA.

[0278] The nucleic acid molecule comprising the bicistronic LAMP construct can be provided in a formulation comprising a lipid monolayer or bilayer to facilitate the movement of the vector across the cell membrane. Any form of liposome or lipid membrane can be used, such as planar lipid membrane or the cell membrane of an intact cell (e.g., red blood cell). The liposome formulation can be administered by any means, including intravenous or oral administration.

[0279] Liposomes and liposomal formulations can be prepared according to standard methods and are well known in the art, see, for example, Remington's; Akimaru, 1995, Cytokines Mol. Ther. 1: 197-210; Alving, 1995, Immunol. Rev. 145: 5-31; Szoka, 1980, Ann. Rev. Biophys. Bioeng. 9: 467; U.S. Patent No. 4,235,871; U.S. Patent No. 4,501,728; and U.S. Patent No. 4,837,028. In one embodiment, the liposome comprises a targeting molecule for targeting the liposome:nucleic acid molecule (the bicistronic LAMP construct herein) complex to a specific cell type. In certain embodiments, the targeting molecule comprises a binding partner (e.g., a ligand or receptor) for a biomolecule (e.g., a receptor or ligand) on the surface of blood vessels or cells found in the target tissue.

[0280] The charge of the liposome is an important determinant in liposome clearance from the blood, with negatively charged liposomes being taken up more rapidly by the reticuloendothelial system (Juliano, 1975, Biochem.Biophys.Res.Commun.63:651) and therefore having a shorter half-life in the bloodstream. Incorporation of phosphatidylethanolamine derivatives enhances circulation time by preventing liposome aggregation. For example, incorporation of N-(omega-carboxy)acylamidophosphatidylethanolamines into large unilamellar vesicles of L-α-distearoylphosphatidylcholine dramatically increases the circulation lifetime of liposomes in vivo (e.g., Ahl, 1997, Biochim.Biophys.Acta 1329:370-382). Liposomes with extended circulation half-lives are typically desirable for therapeutic and diagnostic applications. For a general discussion of pharmacokinetics, see, e.g., Remington's, Chapters 37-39, Lee, et al., In Pharmacokinetic Analysis: A Practical Approach (Technomic Publishing AG, Basel, Switzerland 1996).

[0281] Typically, liposomes are prepared with about 5-15 mole percent of negatively charged phospholipids (e.g., phosphatidylglycerol, phosphatidylserine, or phosphatidyl-inositol). Addition of negatively charged phospholipids such as phosphatidylglycerol also helps prevent spontaneous liposome aggregation, thus minimizing the risk of forming small-sized liposome aggregates. Membrane stiffening agents such as sphingomyelin or saturated neutral phospholipids at a concentration of at least 50 mole percent, and 5-15 mole percent of monosialylganglioside, can also desirably impart liposomal properties such as stiffness (see, e.g., U.S. Pat. No. 4,837,028).

[0282] Additionally, liposomal suspensions can contain lipid-protective agents that protect lipids from free radical and lipid peroxidative damage upon storage. Lipophilic free radical quenchers such as α-tocopherol and water-soluble iron-specific chelators such as ferrioxanine can be used.

[0283] The bicistronic LAMP constructs described herein can also be incorporated into heterogeneously sized multilamellar vesicles. For example, the vesicle-forming lipids can be dissolved in a suitable organic solvent or solvent system and dried under vacuum or inert gas to form a thin lipid film. If desired, the film can be redissolved in a suitable solvent such as tertiary butanol and then lyophilized to form a more homogenous lipid mixture in a powder-like form that is more easily hydrated. This film is covered with an aqueous solution of the peptide or polypeptide complex and allowed to hydrate, typically with stirring, for 15 to 60 minutes. The size distribution of the resulting multilamellar vesicles can be shifted to smaller sizes by hydrating the lipids under more vigorous stirring conditions or by adding a solubilizing detergent such as deoxycholate. The hydration medium preferably contains nucleic acid at the concentration desired in the interior volume of the liposomes in the final liposome suspension.

[0284] After liposome preparation, the liposomes can be sized to achieve a desired size range and a relatively narrow distribution of liposome sizes. One exemplary size range is about 0.2-0.4 microns, which allows the liposome suspension to be sterilized by filtration through a conventional filter, typically a 0.22 micron filter. When the liposomes are reduced to about 0.2-0.4 microns, sterilization by filtration can be performed on a high-throughput basis. Several techniques are available for sizing liposomes to the desired size (see, for example, U.S. Patent No. 4,737,323).

[0285] Suitable lipids include DOTMA (Felgner, et al., 1987, Proc. Natl. Acad. Sci. USA 84: 7413-7417), DOGS or Transfectain™ (Behr, et al., 1989, Proc. Natl. Acad. Sci. USA 86: 6982-6986), DNERIE or DORIE (Felgner, et al., Methods 5: 67-75), DCCHOL (Gao and Huang, 1991, BBRC 179: 280-285), DOTAP™ (McLachlan, et al., 1995, Gene Therapy 2: 674-622), Lipofectamine™, and glycerolipid compounds (see, for example, EP901463 and WO98 / 37916).

[0286] Other molecules suitable for conjugation with bicistronic LAMP constructs may include cationic molecules such as polyamidoamines (Haensler and Szoka, 1993, Bioconjugate Chem. 4: 372-379), dendritic polylysines (WO 95 / 24221), polyethylene irinine or polypropylene h-nin (WO 96 / 02655), polylysine (U.S. Pat. No. 5,595,897; FR 2 719 316), chitosan (U.S. Pat. No. 5,744,166), DNA-gelatin coacervates (see, e.g., U.S. Pat. Nos. 6,207,195; 6,025,337; and 5,972,707) or DEAE dextran (Lopata, et al., 1984, Nucleic Acid Res. 12: 5707-5717).

[0287] J. Viral-Based Gene Delivery Vehicles In one embodiment, the nucleic acid molecule comprising the bicistronic LAMP construct is provided as a delivery vehicle comprising a virus or a viral particle.In this embodiment, preferably, the nucleic acid molecule comprises a viral vector.Viral vectors such as retroviruses, adenoviruses, adeno-associated viruses, and herpes viruses are often composed of two components, a modified viral genome and a coat structure surrounding it (see, for example, Smith et al., 1995, Ann. Rev. Microbiol. 49: 807-838), but sometimes viral vectors are introduced in naked form or coated with proteins other than viral proteins.Most of the current vectors have coat structures similar to wild-type viruses.This structure provides a means for packaging and protecting viral nucleic acid and binding to and entering target cells.

[0288] Preferably, the viral vector comprising the bicistronic LAMP construct described herein is modified from the wild-type viral genome so as to disable viral propagation in target cells while allowing viral propagation in host cells (such as packaging cells or helper cells) used to prepare infectious particles.Vector nucleic acid generally contains the essential cis-acting viral sequences for replication and packaging in helper cells, as well as expression control sequences for regulating the expression of polynucleotides delivered to target cells.Other viral functions are expressed in trans in certain packaging or helper cell lines, as known in the art.

[0289] The viral vector may be derived from a virus selected from the group consisting of herpesvirus, cytomegalovirus, foamy virus, lentivirus, Semliki Forest virus, AAV (adeno-associated virus), poxvirus, adenovirus and retrovirus. Such viral vectors are well known in the art.

[0290] In one embodiment, the viral vector used is an adenoviral vector. The adenoviral genome consists of a linear double-stranded DNA molecule of about 36 kb with more than about 30 genes necessary to complete the viral replication cycle. The early genes are divided into four regions (E1-E4) that are essential for viral replication, except for the E3 region, which is thought to regulate the antiviral host immune response. The E1 region (EIA and EIB) encodes proteins responsible for regulating the transcription of the viral genome. Expression of the E2 region genes (E2A and E2B) results in the synthesis of polypeptides necessary for viral replication. The proteins encoded by the E3 region prevent cell lysis by cytotoxic T cells and tumor necrosis factor (Wold and Gooding, 1991, Virology 184:1-8). The proteins encoded by the E4 region are involved in DNA replication, late gene expression and splicing, and host cell shutoff (Halbert, et al., 1985, J.Virol.56:250-257). Late genes generally code for structural proteins that contribute to viral capsid. In addition, adenovirus genomes possess cis-acting 5' and 3' ITRs (inverted terminal repeats) and packaging sequences essential for DNA replication. ITRs carry the origin of DNA replication, while encapsidation regions are necessary for packaging adenovirus DNA into infectious particles.

[0291] Adenoviral vectors can be engineered to be conditionally replicable (CRAd vectors) to selectively replicate in certain cells (such as proliferating cells) as described in Heise and Kim (2000, J. Clin. Invest. 105: 847-85 1). In another embodiment, the adenoviral vector is replication-deficient for E1 function (e.g., by total or partial deletion or mutagenesis of E1). The adenoviral backbone of the vector can include further modifications (deletions, insertions or mutations in one or more viral genes). An example of E2 modification is illustrated by a temperature-sensitive mutation located on the gene encoding DBP (DNA-binding protein) (Ensinger et al., 1972, J. Virol. 10: 328-339). Adenoviral sequences may also be deleted of all or part of the E4 region (e.g., EP 974 668; Christ, et al., 2000, Human Gene Ther. 11: 415-427; Lusky, et al., 1999, J. Virol. 73: 8308-8319). Further deletions in the non-essential E3 region may allow the size of the delivered polynucleotide to be increased (Yeh, et al., 1997, FASEB Journal 11: 615 623). However, it may be advantageous to retain all or part of the E3 sequence that encodes a polypeptide (such as gp19k) that allows the virus to evade the immune system (Gooding, et al., 1990, Critical Review of Immunology 10: 53-71) or inflammatory responses (EP 00440267.3).

[0292] To improve long-term expression of expressed genes in transduced cells, second generation vectors may also be used that retain the ITRs and packaging sequences and contain substantial genetic modifications to abolish residual synthesis of viral antigens (see, e.g., WO 94 / 28152; Lusky, et al., 1998, J. Virol 72:2022-2032).

[0293] The nucleic acid molecule of the present disclosure introduced into the cell can be inserted at any position in the viral genome, except for cis-acting sequences. Preferably, it is inserted in place of the deleted regions (E1, E3 and / or E4), preferably within the deleted E1 region.

[0294] The adenovirus may be derived from any human or animal source, in particular canine (e.g., Genbank reference numbers for CAV-1 or CAV-2 are CAVIGENOM and CAV77082, respectively), avian (Genbank reference number, AAVEDSDNA), bovine (e.g., BAV3; Reddy, et al., 1998, J. Virol. 72: 1394 1402), murine (Genbank reference number, ADRMUSMAVI), ovine, feline, porcine or simian sources, or may be a hybrid virus. Any serotype may be used. In some cases, human adenoviruses of the C subgroup are used, in particular adenoviruses 2 (Ad2) and 5 (Ad5). Such viruses are available, for example, from the ATCC.

[0295] Adenovirus particles or empty adenovirus capsids can also be used to transfer nucleic acid molecules encoding bicistronic LAMP constructs by a virus-mediated co-internalization process as described in U.S. Patent No. 5,928,944. This process can be achieved in the presence of cationic agents such as polycarbenes or lipid vesicles containing one or more lipid layers.

[0296] Adenoviral particles can be prepared and propagated according to any conventional technique in the art (e.g., WO 96 / 17070) by using complementation cell line or helper virus that provides the defective viral gene required for viral replication in trans.Cell line 293 (Graham et al., 1977, J.Gen.Virol.36:59-72) and PERC6 (Fallaux et al., 1998, Human Gene Therapy 9:1909-1917) are commonly used to complement E1 deletion. Other cell lines have been engineered to complement defective vectors (Yeh, et al., 1996, J. Virol. 70: 559-565; Kroughak and Graham, 1995, Human Gene Ther. 6: 1575-1586; Wang, et al., 1995, Gene Ther. 2: 775-783; Lusky, et al., 1998, J. Virol. 72: 2022-203; EP 919627 and WO97 / 04119). Adenoviral particles can be recovered from the culture supernatant, but also from the cells after lysis, and, if necessary, can be further purified according to standard techniques (e.g., chromatography, ultracentrifugation, as described in WO96 / 27677, WO98 / 00524, WO98 / 26048 and WO00 / 50573).

[0297] Cell type specific targeting can be achieved by using vectors derived from adenoviruses with a wide host range, by modifying viral surface proteins.For example, the specificity of adenovirus infection is determined by attachment to cell receptors present on the surface of permissive cells.In this regard, the fiber and penton present on the surface of adenovirus capsid play an important role in cell adhesion (Defer, et al.,1990, J.Virol.64:3661-3673).Therefore, adenovirus cell targeting can be carried out by genetic modification of viral genes that code for fiber and / or penton, producing modified fiber and / or penton that can specifically interact with unique cell surface receptors. Examples of such modifications are described in Wickarn, et al., 1997, J. Virol. 71: 8221-8229; Arriberg, et al., 1997, Virol. Chem 268: 6866-6869; Roux, et al., 1989, Proc. Natl. Acad. Sci. USA 86: 9079-9083; Miller and Vile, 1995, FASEB J. 9: 190-199; WO 93 / 09221, and WO 95 / 28494.

[0298] In certain embodiments, adeno-associated virus sequences are used as vectors. Vectors derived from the human parvovirus AAV-2 (adeno-associated virus type 2) are one of the most promising gene delivery vehicles currently being developed. Some of the features of this system for packaging single-stranded DNA suggest it as a possible alternative to naked DNA for delivery. In contrast to other viral vectors such as vaccinia or adenovirus, a major attractive feature is that AAV vectors do not express any viral genes. The only viral DNA sequence included in the vaccine construct is the 145 bp inverted terminal repeat (ITR). Thus, as in the case of naked DNA immunization, the only gene expressed is that of the antigen or antigen chimera. Furthermore, AAV vectors are known to transduce both dividing and non-dividing cells (e.g., human peripheral blood monocyte-derived dendritic cells) with sustained transgene expression and with the possibility of oral and intranasal delivery for the generation of mucosal immunity. Moreover, the amount of DNA required is as little as 50 μg or about 10 15 As opposed to a naked DNA dose of 10 copies 10 ~10 11 The maximum response at doses of particles or DNA copies appears to be several orders of magnitude less.

[0299] In one embodiment, AAV vectors are packaged by co-transfection of a suitable cell line (e.g., human 293 cells) with DNA contained in a construct encoding an AAV ITR chimeric protein and the AAV helper plasmid ACG2, which contains the AAV coding region without the ITRs (AAV rep and cap genes). The cells are then infected with adenovirus Ad5. The vectors can be purified from cell lysates using methods known in the art (e.g., cesium chloride density gradient ultracentrifugation) and confirmed (e.g., by cytopathic effect bioassay) to ensure that they do not contain detectable replication-competent AAV or adenovirus. AAV titers can be determined by quantitative PCR using viral DNA samples prepared after digestion with proteinase K. Preferably, vector titers produced by such methods are about 5×10 per ml. 12 ~1×10 13 It is a DNase-resistant particle.

[0300] In other embodiments, retroviral vectors are used. Retroviruses are a class of integrative viruses that replicate using virally encoded reverse transcriptase to replicate viral RNA genomes into double-stranded DNA that is integrated into the chromosomal DNA of infected cells (e.g., target cells). Such vectors include those derived from murine leukemia viruses, particularly Moloney (Gilboa et al., 1988, Adv. Exp. Med. Biol. 241:29) or Friend's FB29 strain (WO 95 / 01447). In general, retroviral vectors are deleted of all or part of the viral genes gag, pol and env, and retain the 5' and 3' LTRs and encapsidation sequences. These elements can be modified to increase the expression level or stability of the retroviral vector. Such modifications include the replacement of retroviral encapsidation sequences with one of the retrotransposons (e.g., VL30) (see, e.g., U.S. Patent No. 5,747,323). Preferably, the nucleic acid molecule of the present disclosure is inserted downstream of the encapsidation sequence, preferably in the opposite orientation relative to the retroviral genome. Cell-specific targeting can be achieved by conjugating antibodies or antibody fragments to retroviral envelope proteins, as known in the art.

[0301] Retroviral particles are prepared in the presence of helper virus or in a suitable complementing (packaging) cell line that contains retroviral genes (e.g., gag / pol and env) that retroviral vector is missing integrated into its genome. Such cell lines are described in the prior art (Miller and Rosman, 1989, BioTechniques 7:980; Danos and Mulligan, 1988, Proc.Natl.Acad.Sci.USA 85:6460; Markowitz, et al., 1988, Virol.167:400). The product of the env gene is responsible for binding of viral particles to viral receptors present on the surface of target cells, thus determining the host range of retroviral particles. In the context of the present disclosure, it is advantageous to use packaging cell lines such as PA317 cells (ATCC CRL 9078) or 293EI6 (WO97 / 35996) that contain amphotropic envelope proteins to allow infection of human and other species target cells. Retroviral particles are preferably recovered from the culture supernatant and, if necessary, can be further purified according to standard techniques (e.g., chromatography, ultracentrifugation).

[0302] Other suitable viruses include poxviruses. The genomes of several members of the Poxviridae family have been mapped and sequenced. Poxvirus vectors can be obtained from any member of the Poxviridae family, in particular canarypox, fowlpox and vaccinia viruses. Suitable vaccinia viruses include the Copenhagen strain (Goebel et al., 1990, Virol. 179:247-266; Johnson et al., 1993, Virol. 196:381-401), the Wyeth strain and the modified Ankara (MVA) strain (Antoine, et al., 1998, Virol. 244:365-396). Suitable vaccinia viruses include, but are not limited to, the Copenhagen strain (Goebel, et al., 1990, Virol. 179: 247-266; Johnson, et al., 1993, Virol. 196: 381-401), the Wyeth strain and the modified Ankara (MVA) strain (Antoine, et al., 1998, Virol. 244: 365-396). The general conditions for constructing vaccinia virus vectors are known in the art (see, for example, EP 83 286 and EP 206 920; Mayr et al., 1975, Infection 3: 6-14; Sutter and Moss, 1992, Proc. Natl. Acad. Sci. USA 89: 10847-10851). Preferably, the polynucleotide of interest is inserted within a non-essential locus, such as a non-coding intergenic region, or any gene whose inactivation or deletion does not significantly impair viral growth and replication.

[0303] Poxvirus particles are prepared as described in the art (Piccini et al., 1987, Methods of Enzymology 153:545-563; U.S. Patent No. 4,769,330; U.S. Patent No. 4,772,848; U.S. Patent No. 4,603,112; U.S. Patent No. 5,100,587 and U.S. Patent No. 5,179,993). In general, a donor plasmid is constructed, amplified by growth in E. coli, and isolated by conventional procedures. It is then introduced into a suitable cell culture (e.g., chicken embryo fibroblasts) together with the poxvirus genome to produce poxvirus particles by homologous recombination. These can be recovered from the culture supernatant or from the cultured cells after a lysis step (e.g., chemical lysis, freeze / thaw, osmotic shock, sonication, etc.). Successive rounds of plaque purification can be used to remove contaminating wild-type virus. The viral particles can then be purified using techniques known in the art, such as chromatographic methods or ultracentrifugation on cesium chloride or sucrose gradients.

[0304] The use of vaccinia as a live viral vaccine in the worldwide campaign to eradicate smallpox made it an obvious choice for development as a live recombinant vaccine vector. Live recombinant vaccinia viruses expressing nearly 100 different foreign proteins have been reported, many of which are effective experimental vaccines (reviewed by Moss and Flexner, 1987). Vaccinia is particularly versatile as an expression vector because of its large genome size, its ability to accept foreign DNA of at least 25,000 base pairs, and its ability to infect most eukaryotic cell types, including insect cells (ibid.). Unlike other DNA viruses, poxviruses replicate exclusively in the cytoplasm of infected cells, reducing the possibility of genetic exchange of recombinant viral DNA with the host chromosome. Recombinant vaccinia vectors have been shown to properly process and express proteins from a variety of sources, including humans, other mammals, parasites, RNA and DNA viruses, bacteria and bacteriophages.

[0305] Expression of the DNA encoding the foreign protein is controlled by host virus regulatory elements, including upstream promoter sequences and, where necessary, RNA processing signals. Insertion of foreign DNA into non-essential regions of the vaccinia virus genome has been achieved by homologous recombination (Panicali et al., Proc. Nat'l. Acad. Sci. USA, 79:4927, 1982; Mackett et al., Proc. Nat'l. Acad. Sci. USA, 79:7415, 1982).

[0306] The expression of polypeptides by the nucleic acid molecules of the present disclosure can occur due to transcriptional regulatory elements at or near the insertion site, or by more precise genetic engineering. Plasmid vectors have been constructed that greatly facilitate the insertion and expression of foreign genes (Mackett, et al., J. Virol, 49: 857, 1982). These vectors contain an expression site that is composed of a vaccinia transcription promoter and one or more unique restriction endonuclease sites for the insertion of foreign coding sequences flanked by DNA from non-essential regions of the vaccinia genome. The choice of promoter determines both the time (e.g., early or late) and level of expression, while the flanking DNA sequences determine the site of homologous recombination.

[0307] Only about one in 1000 virus particles produced by this procedure is a recombinant. Recombinant virus plaques can be identified by DNA hybridization, but efficient selection procedures have been developed. By using segments of the non-essential vaccinia virus thymidine kinase (TK) gene as flanking sequences, the foreign gene is recombined into the TK locus, inactivating the TK gene by insertion. Selection of TK viruses is achieved by performing a virus plaque assay in TK cells in the presence of 5-bromodeoxyuridine. Phosphorylation of the nucleoside analogue and the resulting lethal incorporation into viral DNA occurs only in cells infected with the TK+ parent virus. Depending on the efficiency of transfection and recombination, up to 80 plaques are the desired recombinants, and the remainder are spontaneous TK mutants.

[0308] A plasmid vector containing the E. coli β-galactosidase gene as well as an expression site for a second gene allows an alternative method of distinguishing recombinant viruses from the parent virus (Chakrabarti, et al., Mol. Cell. Biol., 5: 3403, 1985). Plaques formed by such recombinants can be positively identified by the blue color they form upon addition of an appropriate indicator. By combining both TK selection and β-galactosidase expression, recombinant viruses are easily and quickly isolated. Recombinants are then amplified by growth in an appropriate cell line, and expression of the inserted gene is checked by appropriate enzymatic, immunological or physical procedures.

[0309] The upper limit of the amount of genetic information that can be added to the vaccinia virus genome is not yet known. However, the addition of approximately 25,000 base pairs of foreign DNA had no apparent detrimental effect on virus yield (Smith, et al., Gene, 25:21, 1983). If necessary, large segments of the vaccinia virus genome can be deleted to provide additional capacity (Moss, et al., J. Virol. 40: 387, 1981).

[0310] The viral capsid molecule may include a targeting moiety to facilitate targeting and / or entry into cells. Suitable targeting molecules include, but are not limited to, chemical conjugates, lipids, glycolipids, hormones, sugars, polymers (e.g., PEG, polylysine, PEI, etc.), peptides, polypeptides (e.g., see WO94 / 40958), vitamins, antigens, lectins, antibodies, and fragments thereof. Preferably, such targeting molecules recognize and bind to cell-specific markers, tissue-specific markers, cell receptors, viral antigens, antigenic epitopes, or tumor-associated markers.

[0311] Compositions containing viral particle-based bicistronic LAMP constructs are 10-10 14 iu (infectious unit), preferably 10 to 10 11 iu doses. The potency can be determined by conventional techniques. The dose of the bicistronic LAMP construct is preferably 0.01-10 mg / kg, more specifically 0.1-2 mg / kg.

[0312] K. Self-replicating RNA Self-replicating RNA virus vectors (also called self-amplifying RNA virus vectors) can also be constructed using the bicistronic LAMP constructs described herein.For example, alphavirus, flavivirus, measles virus and rhabdovirus can be used to make self-replicating RNA virus vaccines.Exemplary strains of self-replicating RNA virus include but are not limited to rabies virus (RABV), vesicular stomatisitis virus (VSV), West Nile virus, Kunjin virus, Semliki Forest virus (SFV), Sindbis virus (SIN) and / or Venezuelan equine encephalitis virus (VEE).

[0313] Self-replicating RNA viruses express native antigens upon delivery to tissues, thus mimicking live attenuated vaccines without the risk of reversion to pathogenicity. They also stimulate the innate immune system, thus enhancing responses. See, for example, Ljungberg, K. "Self-replicating alphavirus RNA vaccines," Expert Rev Vaccines (2):177-94 (2015); Lundstrom, K., "Oncolytic Alphaviruses in Cancer Immunotherapy," Vaccines 5:9 (2017); Lundstrom, K. "Replicon RNA Viral Vectors as Vaccines," Vaccines 4:39 (2016). (Incorporated herein by reference in its entirety). The use of self-replicating vaccines comprising the bicistronic LAMP constructs described herein can also be used in prime-boost protocols.

[0314] In addition, self-replicating RNA viruses can also be encapsulated by liposomes as described herein to improve delivery and targeting. Immunization with self-replicating RNA viruses containing the nucleic acid molecules described herein can provide higher transient expression levels of antigens, leading to the generation of neutralizing antibody responses and protection against lethal challenge under safe conditions.

[0315] L. Cell-Based Delivery Vehicles A nucleic acid molecule according to the present disclosure can be delivered to a target cell by another cell (a "delivery cell") that contains a construct. Methods for introducing nucleic acid molecules into cells are known in the art and include microinjection of DNA into the nucleus of a cell (Capechi, et al., 1980, Cell 22:479-488); transfection with CaP04 (Chen and Okayama, 1987, Mol. Cell Biol. 7:2745 2752), electroporation (Chu, et al., 1987, Nucleic Acid Res. 15:1311-1326); lipofection / liposome fusion (Feigner, et al., 1987, Proc. Natl. Acad. Sci. USA 84:7413-7417), and particle bombardment (Yang, et al., 1990, Proc. Natl. Acad. Sci. USA 87:9568-9572). Suitable cells include autologous and non-autologous cells, and may include xenogeneic cells. The delivery cells can be induced to deliver their contents to the target cells by inducing their death (e.g., by providing these cells with an inducible suicide gene).

[0316] M. Accessory molecules Compositions comprising nucleic acid molecules according to the present disclosure may include one or more accessory molecules to facilitate introduction of the nucleic acid molecule into a cell, and / or to enhance a particular therapeutic effect, and / or to enhance antibody production.

[0317] Additionally, the compositions may contain one or more stabilizing substances, such as lipids, nuclease inhibitors, hydrogels, hyaluronidase (WO98 / 53853), collagenase, polymers, chelating agents (EP 890362), to inhibit degradation in the animal / human body and / or to improve transfection / infection of the vector into target cells. Such substances may be used alone or in combination (e.g., cationic lipids and neutral lipids).

[0318] It has also been shown that adenovirus proteins can destabilize endosomes and enhance DNA uptake into cells.Mixing adenovirus into the solution containing lipid-complexed DNA vectors, or binding DNA to polylysine covalently linked to adenovirus using protein crosslinking agent, can substantially improve the uptake and expression of bicistronic LAMP constructs containing nucleic acid molecules (e.g., Curiel, et al., 1992, Am.I.Respir.Cell.Mol.Biol.6:247-252).

[0319] N. host cell Nucleic acid molecules according to the present disclosure can be expressed in a variety of host cells, including, but not limited to, prokaryotic cells (e.g., E. coli, Staphylococcus spp., Bacillus spp.); yeast cells (e.g., Saccharomyces spp.); insect cells; nematode cells; plant cells; amphibian cells (e.g., Xenopus laevis); avian cells; and mammalian cells (e.g., human cells, murine cells, mammalian cell lines, primary cultured mammalian cells from dissected tissues, etc.).

[0320] The molecule can be expressed in a host cell isolated from an organism, a host cell that is part of an organism, or a host cell introduced into an organism. In one embodiment, the nucleic acid molecule is expressed in a host cell in vitro, e.g., in culture. In another embodiment, the nucleic acid molecule is expressed in a transgenic organism (e.g., a transgenic mouse, rat, rabbit, pig, primate, etc.) that contains somatic and / or germ cells that contain a nucleic acid encoding the bicistronic LAMP construct of the present invention. Methods for constructing transgenic animals are well known and routine in the art.

[0321] The nucleic acid molecules described herein can also be introduced into cells in vitro, and the cells (e.g., stem cells, hematopoietic cells, lymphocytes, etc.) can be introduced into a host organism. The cells can be xenogeneic or autologous to the host organism. For example, cells can be obtained from a host organism, the nucleic acid molecules can be introduced into the cells in vitro, and then reintroduced into the host organism.

[0322] O. Antigen presenting cells In one aspect of the disclosure, the nucleic acid molecules described herein are introduced into natural or engineered antigen-presenting cells.

[0323] The term "antigen-presenting cell" (APC) as used herein refers to any cell that presents an antigen associated with a major histocompatibility complex molecule, preferably an MHC class II molecule, or a portion thereof, on its surface. Examples of suitable APCs are discussed in detail below and include, but are not limited to, whole cells such as macrophages, dendritic cells, B cells, hybrid APCs, and foster antigen-presenting cells. Methods for making hybrid APCs are described and known in the art.

[0324] Dendritic cells (DCs) are potent antigen-presenting cells. DCs have been shown to provide all the signals necessary for T cell activation and proliferation. These signals can be classified into two types. The first type, which confers specificity to the immune response, is mediated through the interaction between the T cell receptor / CD3 ("TCR / CD3") complex and antigenic peptides presented by major histocompatibility complex ("MHC" defined above) class I or II proteins on the surface of APCs. This interaction is necessary but not sufficient for T cell activation to occur. In fact, in the absence of the second type of signal, the first type of signal can lead to T cell anergy. The second type of signal, called costimulatory signals, is neither antigen-specific nor MHC-restricted and can lead to a full proliferative response of T cells and induction of T cell effector functions in the presence of the first type of signal.

[0325] Several molecules have been shown to enhance costimulatory activity, including, but not limited to, heat-stable antigen (HSA), chondroitin sulfate-modified MHC invariant chain (Ii-CS), intracellular adhesion molecule I (ICAM-1), and the B7 costimulatory molecule on the surface of APCs, as well as its counter-receptors CD28 or CTLA-4 on T cells.

[0326] Other important costimulatory molecules are CD40, CD54, CD80, CD86. As used herein, the term "costimulatory molecule" includes any single molecule or combination of molecules that, when acting together with the peptide / MHC complex bound by the TCR on the surface of the T cell, provide a costimulatory effect that achieves activation of the T cell that binds to the peptide. Thus, the term includes B7, or other costimulatory molecules on APCs, fragments thereof (alone, complexed with another molecule, or as part of a fusion protein), which, together with the peptide / MHC complex, bind to the cognate ligand and result in activation of the T cell when the TCR on the surface of the T cell specifically binds to the peptide. Costimulatory molecules are commercially available from a variety of sources, including, for example, Beckman Coulter.

[0327] In one embodiment of the present disclosure, Romani et al., J.Immunol.Methods 196:135-151, 1996, and Bender et al.Methods 196:121-135, 1996 are used to generate both immature and mature dendritic cells from peripheral blood mononuclear cells (PBMCs) of mammals such as mice, monkeys, or humans. Briefly, isolated PBMCs are pretreated by immunomagnetic techniques to deplete T cells and B cells. The lymphocyte-depleted PBMCs are then cultured (e.g., for about 7 days) in RPMI medium supplemented with human plasma (preferably autologous plasma) and GM-CSF / IL-4 to generate dendritic cells. Dendritic cells are non-adherent when compared to their monocyte precursors. Thus, on about day 7, non-adherent cells are collected for further processing.

[0328] Dendritic cells derived from PBMCs in the presence of GM-CSF and IL-4 are immature in that they lose their non-adhesive properties and can revert to a macrophage cell fate when cytokine stimulation is removed from the culture. Dendritic cells in the immature state are highly efficient at processing natural protein antigens for the MHC class II restricted pathway (Romani, et al., J. Exp. Med. 169:1169, 1989). Further maturation of cultured dendritic cells is achieved by culturing for 3 days in macrophage conditioned medium (CM) containing the necessary maturation factors. Mature dendritic cells are less capable of capturing new proteins for presentation, but are much more capable of stimulating resting T cells (both CD4 and CD8) to proliferate and differentiate.

[0329] Mature dendritic cells can be identified by changes in morphology, such as the formation of more motile cytoplasmic processes; by non-adherence; by the presence of at least one of the following markers: CD83, CD68, HLA-DR, or CD86; or by the loss of Fc receptors, such as CD115 (Steinman, Annu.Rev.Immunol.9:271, 1991). Mature dendritic cells can be collected and analyzed using typical cytofluorimetric and cell sorting techniques and devices, such as FACScan and FACStar. Primary antibodies used for flow cytometry are specific for cell surface antigens of mature dendritic cells and are commercially available. Secondary antibodies can be biotinylated Ig followed by FITC or PE-conjugated streptavidin.

[0330] Alternatively, other investigators have reported methods to upregulate (activate) dendritic cells and convert monocytes to an activated dendritic cell phenotype. The method involves the addition of calcium ionophore to the medium to convert monocytes to activated dendritic cells. For example, addition of calcium 21 ionophore A23187 at the beginning of a 24-48 h culture period resulted in uniform activation and dendritic cell phenotype conversion of the pooled "monocyte+DC" fraction: characteristically, the activated population becomes uniformly CD14(Leu M3) negative, and upregulates HLA-DR, HLA-DQ, ICAM-1, 137.1, and 137.2. Furthermore, this activated bulk population also functions, on a minority basis, as a further purified version. Certain combinations of cytokines have been used successfully to amplify (or partially replace) the activation / transduction achieved with calcium ionophores: these cytokines include, but are not limited to, G-CSF, GM-CSF, IL-2, and IL-4. Each cytokine, when given alone, is insufficient for optimal upregulation.

[0331] The second approach to isolate APCs is to collect the relatively large number of pre-committed APCs already circulating in the blood. Conventional techniques for isolating committed APCs from human peripheral blood have included a combination of physical procedures such as metrizamide gradient and adherence / non-adherence steps (Freudenthal et al. PNAS 87: 7698-7702, 1990); Percoll gradient separation (Mehta-Damani, et al., J. Immunol. 153: 996-1003, 1994); and fluorescence-activated cell sorting techniques (Thomas et al., J. Immunol. 151: 6840-52, 1993).

[0332] Many other methods exist that are routine in the art for isolating professional antigen-presenting cells (or their precursors), and such methods and others that may be developed are encompassed within the scope of this disclosure without limitation.

[0333] In one embodiment, the APCs, and thus the cells presenting one or more antigens, are autologous, hi another embodiment, the antigen-presenting APCs are allogeneic, i.e., derived from a different subject.

[0334] As discussed herein, the nucleic acid molecules described herein can be introduced into APCs using the above methods or other methods known in the art, including but not limited to transfection, electroporation, fusion, microinjection, viral-based delivery, or cell-based delivery. Arthur et al., Cancer Gene Therapy 4(1): 17-25, 1997, reports a comparison of gene transfer methods in human dendritic cells.

[0335] The known partial and predicted human leukocyte antigens (HLA), gene names, amino acid and nucleotide sequences, including consensus sequences, of human MHC have been published (see, e.g., Zemmour and Parham, Immunogenetics 33:310-320, 1991), and cell lines expressing HLA variants are also known and publicly available, many available from the American Type Culture Collection ("ATCC"). Thus, using PCR, nucleotide sequences encoding MHC class II are readily operably linked to the expression vectors of the present disclosure and then used to transform appropriate cells for expression therein.

[0336] Specialized APCs such as macrophages, B cells, monocytes, dendritic cells, and Langerhans cells can be used. They are collected from the blood or tissue of 1) an autologous donor; 2) a xenogeneic donor with a different HLA specificity than the host being treated; or 3) a xenogeneic donor of a different species using standard procedures (Coligan et al., Current Protocols in Immunology, Sections 3 and 14, 1994). Cells can be isolated from normal hosts or from patients with infectious diseases, cancer, autoimmune diseases, or allergies.

[0337] Professional APCs can be obtained from peripheral blood using leukapheresis and "FICOLL / HYPAQUE" density gradient centrifugation (stepwise centrifugation through Ficoll and discontinuous Percoll density gradients). A procedure is utilized that avoids exposure of APCs to antigens that may be internalized by the APCs and leads to activation of T cells that are not specific for the antigen of interest.

[0338] Cells that do not naturally present antigens can be engineered to present antigens by introducing sequences encoding the appropriate molecules. For example, nucleic acid sequences encoding MHC class II molecules, accessory molecules, costimulatory molecules, and antigen processing auxiliary molecules can be introduced after direct synthesis, cloning, purification of DNA from cells containing such genes, and the like. One convenient means for obtaining genes for encoding molecules used in the bicistronic LAMP constructs and methods described herein is by polymerase chain reaction (PCR) amplification on selected nucleic acid templates with selected oligonucleotide primer pairs. For example, epithelial cells, endothelial cells, tumor cells, fibroblasts, activated T cells, eosinophils, keratinocytes, astrocytes, microglia, thymic cortical epithelial cells, Schwann cells, retinal pigment epithelial cells, myoblasts, vascular smooth muscle cells, chondrocytes, intestinal cells, thyroid cells, and renal tubule cells can be used. These can be primary cells that have been recently explanted from the host and have not been passaged extensively in cell culture to form an established cell line, or established cell lines that are relatively homogenous and can be propagated for many generations or indefinitely.

[0339] Cells that are not professional APCs are isolated from autologous donors; xenogeneic donors or any tissues of xenogeneic donors where they are present using various known separation methods (Darling, Animal Cells: Culture and Media. J. Wiley, New York, 1994; Freshney, Culture of Animal Cells. Alan R. Liss, Inc., New York, 1987). Non-autologous cells, such as xenogeneic or xenogeneic cells, can be engineered ex vivo to express HLA class I and class II molecules that match known human HLA specificities. These cells can then be introduced into a human subject that matches the HLA specificity of the engineered cells. The cells are further engineered ex vivo to express one or more LAMP constructs according to the present disclosure.

[0340] The engineered cells are maintained in cell culture by standard cell culture methods (Darling, Animal Cells: Culture and Media, J. Wiley, New York, 1994; Freshney, Culture of Animal Cells, Alan R. Liss, Inc., New York, 1987). Cell lines for use in the present disclosure can be obtained from a variety of sources (e.g., ATCC Catalogue of Cell Lines & Hybidomas, American Type Culture Collection, 8th edition, 1995) or produced using standard methods (Freshney, Culture of Immortalized Cells, Wiley-Liss, New York, 1996). Non-transformed cell lines are preferred for use in human subjects.

[0341] In one embodiment, CD34+ precursors differentiated into dendritic cells under the influence of GM-CSF are obtained from the body of a subject, and the nucleic acid molecule encoding the bicistronic LAMP construct is introduced into the cells, which are then injected into the subject.The use of the nucleic acid molecule described herein enhances the association of peptides derived from specific antigens with MHC class II molecules on transduced antigen-presenting cells, resulting in significantly stronger systemic T cell-dependent immune responses and / or antibody production.The antigen-presenting cells transfected in this strategy are preferably autologous cells, but any MHC class II cells that effectively present antigens in the host can be used as described above.

[0342] P. Administration Vaccine material according to the present disclosure may contain a nucleic acid molecule encoding the immunostimulatory bicistronic LAMP construct described herein, or may be a recombinant microorganism or antigen-presenting cell expressing the immunostimulatory bicistronic LAMP construct. Preparation and administration of such nucleic acid molecules for immunization of an individual is accomplished according to principles of immunization well known to those skilled in the art.

[0343] Large quantities of these substances can be obtained by culturing recombinant or transformed cells that contain nucleic acid molecules. Cultivation methods are well known to those skilled in the art and are taught in one or more of the documents cited above. Vaccines that contain the nucleic acid molecules described herein are generally produced by culturing recombinant or transformed cells and formulated into pharmacologically acceptable solutions or suspensions, which are usually physiologically compatible aqueous solutions, or into coated tablets, tablets, capsules, suppositories, or ampoules, as described in the art (e.g., U.S. Pat. No. 4,446,128, incorporated herein by reference). Administration can be by any suitable route, including oral, rectal, intranasal, or injection (injection can be, for example, transdermal, subcutaneous, intramuscular, or intravenous).

[0344] The nucleic acid molecules described herein can be administered to a mammal in an amount sufficient to induce an immune response in the mammal. In some embodiments, the minimum amount for administration is the amount required to induce antibody formation to a concentration at least 4-fold higher than that present prior to administration. A typical initial dose for administration is 10 μg of recombinant vector. 5 ~10 11 Although the amount of plaque forming units can be adjusted by the administering clinician, as typically occurs in the administration of vaccines and other agents to induce an immune response, a single dose may usually be sufficient to induce immunity.

[0345] Vaccines comprising the nucleic acid molecules described herein can be first tested in non-human mammals (e.g., mice or primates). For example, assays of immune responses of inoculated mice can be used to demonstrate greater antibody, T cell proliferation, and cytotoxic T cell responses to the bicistronic LAMP construct than to the wild-type antigen. The vaccine can be evaluated in rhesus monkeys to determine whether the highly effective vaccine formulation in mice also induces an adequate monkey immune response. In one embodiment, each monkey receives a total of 5 mg of nucleic acid molecule per immunization, delivered IM, split into two sites, immunized on day 0 and at weeks 4, 8, and 20, with additional doses being optional. Antibody responses, ADCC, CD4+ and CD8+ T cell cytokine production, CD4+ and CD8+ T cell antigen-specific cytokine staining can be measured to monitor the immune response to the vaccine.

[0346] Further description of suitable methods of formulation and administration according to the present disclosure can be found in U.S. Pat. No. 4,454,116 (constructs), U.S. Pat. No. 4,681,762 (recombinant bacteria), and U.S. Pat. Nos. 4,592,002 and 4,920,209 (recombinant viruses).

[0347] Q. Treatment Procedures In one embodiment, the nucleic acid molecule encoding the bicistronic LAMP construct described herein can be injected into the patient at any suitable time during the course of the patient's malignancy.For example, the nucleic acid molecule described herein is injected at a stage where the tumor burden is low.In an alternative embodiment in which the nucleic acid molecule is introduced into the antigen-presenting cells of an individual, the antigen-presenting cells or precursors of mature antigen-presenting cells are drawn from either the bone marrow or peripheral blood of an individual by venipuncture.These cells are established in culture and then transduced with the nucleic acid molecule.After transduction has occurred, these antigen-presenting cells are injected back into the patient.

[0348] In certain embodiments, the present disclosure provides a method of treatment for cancer patients with low tumor burden, such as at the beginning of disease, after resection of neoplastic tumor, or when tumor cell burden is otherwise reduced.In this method, a cell population is obtained from the patient, including autologous stem cells that can differentiate into antigen-presenting cells that express MHC class II molecules.These cells are cultured and transformed by introducing the bicistronic LAMP construct described herein, and deliver antigen that associates with MHC class II molecules either in the compartment / organelle or in another compartment / organelle where the antigen is delivered, and secrete a second antigen or IREG into circulation.

[0349] The transfected stem cell population is then reintroduced into the patient, where the stem cells are capable of transducing antigen-driven T h Differentiate into antigen-presenting cells that express MHC class II molecules complexed with epitopes.The immune response to antigen is enhanced by enhancing the stimulation of helper T cell population.The secreted antigen or IREG enhances immune response, for example, by expanding memory response.

[0350] More generally, in one embodiment, the present disclosure provides a vaccine composition comprising a nucleic acid molecule encoding a bicistronic LAMP construct for modulating an immune response in a mammal to an antigen (i.e., stimulating, enhancing, or reducing such a response).

[0351] R.Kit The present disclosure further includes a kit for facilitating the implementation of the methods described herein. In one embodiment, the kit includes a nucleic acid molecule encoding the bicistronic LAMP construct described herein and a cell for receiving the nucleic acid molecule. In one embodiment, the cell is a professional APC. The cell may or may not express a costimulatory molecule. In an embodiment, if the cell does not express a costimulatory molecule, the antigen encoded by the bicistronic LAMP construct is an autoantigen. In another embodiment, a panel of cells expressing different MHC molecules (e.g., known to be expressed in humans) is provided. In a further embodiment, the kit includes a reagent (e.g., lipid-based formulation, viral packaging material, cells, etc.) for facilitating the entry of the nucleic acid molecule into cells. In yet another embodiment, one or more T cell lines specific for the antigen encoded by the nucleic acid molecule are provided to verify the ability of the bicistronic LAMP construct to induce, modulate, or enhance an immune response.

[0352] S. Additional Embodiments Further embodiments herein include the following: 1. A nucleic acid molecule comprising: a. a first polynucleotide sequence encoding a LAMP-antigen construct comprising an antigen and a cysteine-conserved fragment of a LAMP protein; b. a second polynucleotide sequence encoding at least one IREG polypeptide operably linked to a secretory signal sequence; c. wherein said first and second polynucleotide sequences are operably linked to an expression control sequence(s) for expression of the LAMP-antigen construct and an IREG in a host or target cell. 2. The nucleic acid molecule of embodiment 1, wherein: a. The antigen is located at the N-terminus of the cysteine ​​conserved fragment; b. the antigen is located at the C-terminus of the cysteine-conserved fragment; or c. The antigen is located between two cysteine-conserved fragments. 3. A nucleic acid molecule comprising: a. a first polynucleotide sequence encoding a LAMP-antigen construct comprising an antigen disposed between two cysteine ​​conserved fragments; b. a second polynucleotide sequence encoding at least one IREG polypeptide operably linked to a secretory signal sequence; c. wherein the first and second polynucleotide sequences are operably linked to an expression control sequence(s) for expression of the LAMP-antigen construct and the IREG polypeptide in a host or target cell. 4. The nucleic acid molecule of any one of the preceding embodiments, wherein the improved LAMP construct comprises the structure shown in FIG. 1 for ILC-1, ILC-2, ILC-3, ILC-4, ILC-5, or ILC-6 (i.e., the first polynucleotide sequence encodes a polypeptide comprising the structure shown in FIG. 1 for ILC-1, ILC-2, ILC-3, ILC-4, ILC-5, or ILC-6). 5. The nucleic acid molecule of any one of the preceding embodiments, wherein the cysteine ​​conserved fragment comprises a homology domain of a LAMP protein. 6. The nucleic acid molecule of any one of the preceding embodiments, wherein the improved LAMP construct further comprises a transmembrane domain of a LAMP protein. 7. The nucleic acid molecule of any one of the preceding embodiments, wherein the improved LAMP construct further comprises a signal sequence. 8. The nucleic acid molecule of embodiment 7, wherein the signal sequence is derived from a LAMP protein. 9. The nucleic acid molecule of any one of the preceding embodiments, wherein the antigen comprising the LAMP-antigen construct is located at or replaces the LAMP hinge region. 10. The method of any one of the preceding embodiments, wherein the LAMP protein is selected from LAMP-1, LAMP2, LAMP-3, LIMP2, Macrosialin, Endolyn, LAMP5, or LIMBIC. 11. The nucleic acid molecule of embodiment 10, wherein the LAMP protein is selected from any one of SEQ ID NOs: 1 to 113. 12. The nucleic acid molecule of embodiment 10, wherein the LAMP protein is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NOs: 1-113. 13. The nucleic acid molecule of any one of the preceding embodiments, wherein expression of the LAMP-antigen construct in a host or target cell results in antigen processing and presentation to the MHC class II pathway to elicit an immune response. 14. The nucleic acid molecule of any one of the preceding embodiments, wherein expression of the IREG in a host or target cell results in secretion of the IREG. 15. The nucleic acid molecule of embodiment 14, wherein secretion of the IREG can enhance an immune response elicited by an antigen. 16. The method of any one of embodiments 1 to 15, wherein the antigen is an antigen associated with Covid-19, a cancer, the SARS-CoV-2 virus S1 spike subunit, HER2, NY-ESO1, or CD161, or a domain of the SARS-CoV-2 virus S1 spike subunit, HER2, NY-ESO1, or CD161. 17. The nucleic acid molecule of any one of the preceding embodiments, wherein the nucleic acid molecule is a plasmid or vector. 18. The nucleic acid molecule of embodiment 17, wherein the nucleic acid molecule is a viral vector. 19. A host cell comprising a nucleic acid molecule according to any one of embodiments 1 to 18. 20. A composition comprising a nucleic acid molecule according to any one of embodiments 1 to 18, or a host cell according to embodiment 19. 21. A method for treating a subject having a disease or disorder, comprising administering to a subject in need thereof a nucleic acid molecule according to any one of embodiments 1 to 18, or a host cell according to embodiment 19, or a composition according to embodiment 20, in an amount sufficient to alleviate or treat said disease or disorder. EXAMPLES

[0353] The present disclosure will now be further illustrated with reference to the following examples. It will be understood that the following description is merely exemplary and that changes can be made in detail while still falling within the scope of the present disclosure.

[0354] Example 1. Construction of a vector encoding a bicistronic LAMP construct We designed a first generation COVID-19 vaccine candidate that encodes one COVID-19 antibody as a LAMP fusion protein and a second COVID-19 antibody as a secreted protein. This construct, named ITI-COVID-19 bicistronic construct (ITI-bicistronic-S1-LAMP-RBG pA-EF1-S2P BGH pA; SEQ ID NO: 228, Figure 28C), was demonstrated to induce both SARS-Cov-2 specific antibody and T cell responses. To obtain optimal antibody and T cell responses, the vaccine dose (20 μg) and the time period between prime and boost (e.g., 14 days) were studied. After two immunizations with 20 μg of ITI-COVID-19 bicistronic vaccine, robust SARS-Cov-2 spike-specific T cells and antibodies were induced. Importantly, the ITI-COVID-19 bicistronic vaccine elicited immune responses that neutralized SARS-Cov-2.

[0355] The S1 and S2 subunits of the spike mediate SARS-CoV-2 viral entry into host cells. Using the nucleic acid molecule of the ILC-4 LAMP construct, an S1 coding sequence (GenBank MN908974) was placed between polynucleotide sequences encoding two LAMP homology domains (N-LAMP and luminal domain 2). The S1 coding sequence was operably linked to a CMV promoter under the influence of a CMV enhancer sequence, such that expression in a host cell produced an ILC-4 LAMP construct containing the S1 antigen for processing and presentation to MHC class II molecules (i.e., providing a "priming antigen"). The S2 coding sequence was provided elsewhere on the vector and operably linked to a polynucleotide sequence encoding an Ig-κ secretion signal (leader sequence) and an EF1 promoter sequence, such that expression in a host cell produced the S2 antigen for secretion (i.e., providing a "boosting antigen"). Thus, the vector provides a single nucleic acid molecule for introduction into suitable host or target cells that can provide both antigen priming and boosting to induce enhanced immune response.This can therefore confer a significant advantage over the use of vectors that only code for bicistronic LAMP constructs, since any desire or requirement to boost the immune response induced by the LAMP construct requires administration of a booster vaccine (e.g., containing an antigen) that is administered separately at one or more time intervals.

[0356] The first generation ITI-COVID-19 bicistronic vaccine expresses the S1 and S2 subunits of the spike glycoprotein anchored on the viral surface. The S1 and S2 subunits of the spike mediate the entry of the SARS-CoV-2 virus into host cells. Using the nucleic acid molecule of the ILC-4 LAMP construct, the S1 coding sequence (GenBank MN908974) was placed between polynucleotide sequences encoding two LAMP homology domains (N-LAMP and luminal domain 2). The S1 coding sequence is operably linked to the CMV promoter under the influence of the CMV enhancer sequence, such that expression in the host cell results in an ILC-4 LAMP construct containing the S1 antigen for processing and presentation to MHC class II molecules (i.e., providing a "priming antigen"). The S2 coding sequence is provided elsewhere on the vector and is operably linked to a polynucleotide sequence encoding an Ig-κ secretion signal (leader sequence) and an EF1 promoter sequence, such that expression in a host cell results in the S2 antigen for secretion (i.e., providing a "booster antigen"). The vector thereby provides a single nucleic acid molecule for introduction into a suitable host or target cell that can provide a target antigen and a boosting antigen to elicit an enhanced immune response. This may therefore confer a significant advantage over the use of a vector encoding only a bicistronic LAMP construct, since any desire or requirement to boost an immune response elicited by the LAMP construct would require administration of a booster vaccine (e.g., containing an antigen) administered separately at one or more time intervals.

[0357] The polynucleotide sequence of this first generation COVID-19 bicistronic construct is shown in Figure 28C. The DNA sequences of S1 and S2 were obtained, for example, from Genbank (ncbi.nlm.nih.gov / genbank / sars-cov-2-seqs / #nucleotide-sequences). In the vector shown in Figure 28A, the S2 polynucleotide sequence encodes an S2 variant containing two proline (P) substitutions. In particular, the ITI-COVID-19 bicistronic vaccine (first generation vaccine) expresses a spike protein (residues 16-1273) with an S1-LAMP sequence (residues 950-2004 of SEQ ID NO:227 shown in Figure 28C) and an Ig-kappa leader sequence (see Figure 28C; residues 2145-3423 of SEQ ID NO:227), as described in more detail below.

[0358] Table 1 below provides exemplary DNA and protein sequences of the bicistronic LAMP constructs described above, as well as various exemplary promoter / enhancer sequences and polypeptide sequences that can be used in constructing the bicistronic LAMP constructs described herein.

[0359] The following additional bicistronic LAMP constructs were constructed using standard molecular biology techniques well known to those of skill in the art: (1) HER2-LAMP-sCD40L (Figure 11; SEQ ID NO: 197), (2) HER2-LAMP-mFLT3L (SEQ ID NO: 208), (3) HER2-LAMP-IL-12 (SEQ ID NO: 212), (4) HER2-LAMP-IL-21 (SEQ ID NO: 216), (5) HER2-LAMP-OX40L (SEQ ID NO: 241), (6) HER2-LAMP-CD80 (SEQ ID NO: 251), (7) NY-ESO1-LAMP-IL-15 (SEQ ID NO: 222), (8) CD161-LAMP-sCD40L (SEQ ID NO: 235), and the second generation Covid-19 construct (9) Spike-LAMP-sCD40L (2-V Covid vaccine; SEQ ID NO: 230). The sequences of these constructs and their components are provided in the table below.

[0360] HER2-LAMP-sCD40L expresses HER2-LAMP polypeptide SEQ ID NO: 195 and mSPD-sCD40L fusion protein SEQ ID NO: 196. HER2-LAMP-mFLT3L expresses HER2-LAMP polypeptide (SEQ ID NO: 195) and SPD-mFLT3L polypeptide SEQ ID NO: 207. HER2-LAMP-IL-12 expresses HER2-LAMP polypeptide (SEQ ID NO: 195) and mouse IL-12 p36-P2A-IL-12p40 polypeptide SEQ ID NO: 213. HER2-LAMP-IL-21 expresses HER2-LAMP polypeptide (SEQ ID NO: 195) and mouse IL-21 polypeptide SEQ ID NO: 217. HER2-LAMP-OX40L expresses HER2-LAMP polypeptide (SEQ ID NO: 195) and OX40L extracellular domain (ECD). Fc fusion protein with mouse IL-2 signal peptide (SP) SEQ ID NO: 242. HER2-LAMP-CD80 expresses HER2-LAMP polypeptide (SEQ ID NO: 195) and CD80 ECD with mouse IL-2 SP SEQ ID NO: 252. Spike-LAMP-sCD40L expresses Spike-LAMP polypeptide SEQ ID NO: 229 and SPD-sCD40L polypeptide SEQ ID NO: 233. A table showing these and other sequences is provided below. Compared to the US provisional patent application, SEQ ID NO: 195 has been updated to reflect that the HER2-LAMP polypeptide sequence includes SEQ ID NO: 198, followed by SEQ ID NO: 200, followed by SEQ ID NO: 202, and the duplicate HER2-LAMP sequence included in the provisional patent application has been deleted without changing the overall numbering of the surrounding sequences. SEQ ID NOs: 221 and 229 are similarly updated to reflect that the NY-ESO1-LAMP and Spike-LAMP polypeptide sequences include SEQ ID NO: 198, followed by the NY-ESO1 antigen or spike antigen, followed by SEQ ID NO: 202.

[0361] [Table 33]

[0362] [Table 34]

[0363]

Table 35

[0364]

Table 36

[0365]

Table 37

[0366]

Table 38

[0367]

Table 39

[0368]

Table 40

[0369]

Table 41

[0370]

Table 42

[0371]

Table 43

[0372]

Table 44

[0373]

Table 45

[0374]

Table 46

[0375]

Table 47

[0376]

Table 48

[0377]

Table 49

[0378]

Table 50

[0379]

Table 51

[0380]

Table 52

[0381]

Table 53

[0382]

Table 54

[0383]

Table 55

[0384]

Table 56

[0385]

Table 57

[0386]

Table 58

[0387]

Table 59

[0388]

Table 60

[0389]

Table 61

[0390]

Table 62

[0391]

Table 63

[0392]

Table 64

[0393]

Table 65

[0394]

Table 66

[0395]

Table 67

[0396]

Table 68

[0397]

Table 69

[0398]

Table 70

[0399]

Table 71

[0400]

Table 72

[0401]

Table 73

[0402]

Table 74

[0403]

Table 75

[0404] [Table 76]

[0405] Example 2. Second generation bicistronic COVID vaccine (2-V-COVID) induces T cell-mediated and immune responses in mice. A major challenge to generate sufficient antigen-specific T cell and antibody responses is the low immunogenicity of DNA vaccines. To increase T cell responses to DNA vaccines, a novel bicistronic DNA vaccine was designed. This example considers a second generation COVID-19 DNA vaccine, Spike-LAMP-sCD40L (2-V-Covid vaccine; SARS-CoV-2 B.1.351 spike (South African variant, SP and TM free) + LAMP + EF-1α + SPD + sCD40L; SEQ ID NOs: 242-243), which contains (1) full-length SARS-CoV-2 spike protein fused with LAMP, and (2) soluble CD40L (sCD40L), both of which were expressed separately. This bicistronic DNA vaccine was designed to induce local expression of sCD40L at relatively low levels, thereby providing a safe approach to using systemic recombinant CD40L or agonistic anti-CD40 antibodies (van Mierlo et al., 2002).

[0406] CD40 ligand (CD40L; CD154) enhances adaptive immune responses by stimulating dendritic cells and B cells. DNA vaccines containing the CD40L gene have been shown to enhance T cell and antibody responses in vivo. (Not shown.) To produce a COVID vaccine with improved immunogenicity, a second generation vaccine was designed to express SARS-CoV-2 full-length spike protein and soluble CD40L in two separate cassettes. The immunogenicity of this 2-V COVID vaccine was evaluated in BALB / c mice. After two immunizations, the new vaccine induced stronger spike-specific T cell responses and higher levels of spike-specific immune responses compared to the first generation COVID-19 DNA vaccine described in Example 1. The data discussed in the following examples suggest that the new vaccine is more immunogenic and has the potential to improve protection against COVID19 and future emerging infectious diseases.

[0407] CD40L is a transmembrane protein expressed on the surface of activated T cells, especially CD4 T cells. CD40L stimulates the CD40-dependent activation of antigen-presenting cells (APCs) such as dendritic cells (DCs) and macrophages, as well as B cells to enhance T cell and antibody responses (Grewal & Flavell, 1998; Schoenberger et al., 1998). Recombinant soluble CD40L or agonistic antibodies have been used in the clinic and have shown promising results in various cancers (Beatty et al., 2011, 2017; Vonderheide et al., 2001). As a result, these immune-stimulating functions of CD40L have made it a promising vaccine adjuvant against infectious diseases and cancer. As discussed herein and in the Examples below, sCD40L enhanced spike-specific T cell and antibody responses. The data discussed herein support the use of bicistronic DNA vaccines against infectious diseases.

[0408] A. Materials and Methods 1. Vaccine Constructs An ITI-COVID-19 bicistronic vaccine (ITI-bicistronic-S1-LAMP-RBG pA-EF2-S2P BHG pA; first generation COVID-19 DNA vaccine, "ITI-bicistronic vaccine"; shown in Figure 28C, SEQ ID NO: 228) was previously constructed as described in Example 1 above.

[0409] A second generation Spike-LAMP-sCD40L construct ("2-V COVID vaccine", SEQ ID NO:228) was also constructed according to the methods described in Example 1 above. The 2-V COVID vaccine had two separate cassettes independently driven by the CMV and EF1 promoters. The first cassette expressed the full-length spike gene of the SARS-CoV-2 South African variant (B.1.351) infectious clone (SEQ ID NO:227). The second cassette expressed the soluble mouse CD40L extracellular domain fused to the mouse pulmonary surfactant associated protein D (SPD) protein (SEQ ID NO:233).

[0410] The control vector (CV) used is a vector that does not contain any gene insert.

[0411] 2. Reagents Antibodies for flow cytometry and enzyme-linked immunospot (ELISPOT) were purchased from Biolegend, and SA-HRP and AEC kits were purchased from BD. Antibodies against spike protein, as well as recombinant spike (S1) (cat. no. 40591-V08H) and RBD (cat. no. 40592-V08B) proteins, were purchased from Sino Biologics. Rabbit polyclonal anti-S1 antibody was purchased from Sino Biologics. Antibody titers were assessed using HRP anti-mouse antibody from Southern Biotech. Renilla luciferase reporter assay was purchased from Promega (cat. no. E2710). Epivax peptides were synthesized by GenScript.

[0412] Premade lentiviral particles for overexpression of human ACE2 were ordered from GenTarget Inc (catalog number LvP1310).

[0413] JPT PepMix™ SARS-CoV-2 overlapping peptide pool was purchased from JPT (catalog number PM-WCPV-S). The peptide pool contained a total of 315 peptides (delivered in two subpools of 158 and 157 peptides) derived from peptide scans (15mer segments with 11 amino acid overlaps) via Spike (UniProt:P0DTC2).

[0414] Pseudotyped luciferase rSARS-Cov-2 spike virus was purchased from Creative Diagnostics (No: Cov-PS01, Lot. No.: CL-114A), which is based on SARS-Cov-2 Wuhan-Hu-1 with luciferase as a reporter.

[0415] 3. Transient Transfection of Constructs 293T cells were transiently transfected with control vector (CV) or bicistronic vaccine constructs using Lipofectamine 2000 (Invitrogen). 48 hours after transfection, the supernatants were collected, centrifuged, and filtered. The supernatants were analyzed for the expression of soluble CD40L.

[0416] In some embodiments, 293T cells can be transduced with human ACE2. The 293T-ACE2 cell line was generated by transducing 293T with lentivirus expressing human ACE2 (angiotensin I converting enzyme 2, NM_021804), which was generated by GeneTarget and contains RFP and blasticidin dual selection markers. After infection, 15ug / ml blasticidin is used to isolate single cell clones expressing both RFP and ACEs. As shown in the figure, the expression of human ACE2 was detected by staining with anti-human ACE2 antibody (Sino Biological, Rabbit Pab, Catalog No.: 10108-T60). 293T-ACE3 clone 2 and clone 5 have higher expression of hACE2, and clone 5 was used for neutralization test in this study.

[0417] 4. Western Blot and Immunoprecipitation Biotinylated anti-CD40L antibody (Clone MR1 BioLegend Cat#106503) was bound to streptavidin-coated magnetic beads and used to isolate CD40L. The beads were loaded onto gels for Western blot imaging. The following antibodies were also used for Western blot: (1) primary anti-CD40L / CD154 antibody (Invitrogen PA5-78983) and secondary antibody of goat anti-rabbit IgG-HRP (Southern Biotech 4030-05).

[0418] 5. Immunization and Serum Collection Female BALB / c mice aged 6 to 8 weeks were bred and maintained in an approved breeding facility.

[0419] The immunization schedule for ITI-bicistronic and 2-V COVID vaccines is shown in Table 2. Mice were immunized with DNA vaccines by ID injection in the ear followed by electroporation. Blood samples were collected before immunization and 14 days after the second immunization. On day 28, mice were sacrificed as scheduled and spleens and serum were harvested for measurement of T cell and antibody responses. [Table 77]

[0420] 6. Assessment of antigen-specific T cell responses by ELISPOT. To assess antigen-specific T cell responses in vaccinated mice, splenocytes from vaccinated mice were assessed for antigen-specific IFNγ by enzyme-linked immunospot (ELISPOT). ELISPOT assays were performed as described herein. Briefly, splenocytes were cultured at 3×10 5 Cells / well were plated and co-cultured with 1 μg / ml JPT overlapping spike peptide, 0.25 μg / mL concanavalin A, or medium alone in a total volume of 200 μl / well of T cell medium for 48 h at 37 °C in 5% CO2. Plates were developed with 50 μl / well of AEC development solution for up to 30 min. Development was stopped by washing under running tap water. After air drying, colored spots were counted using an AID ELISPOT High-Resolution Reader System and AID ELISPOT software version 3.5 (Autoimmun Diagnostika GmbH). Student T-test was used to determine significant differences between cells transfected with ITI-bicistronic and 2-V COVID vaccines.

[0421] 7.ELISA Mouse antibody responses to the vaccine were assessed by indirect ELISA. ELISA plates (MaxiSorp) were coated overnight with 1 μg / ml recombinant SARS-Cov-2 spike, S1, or RBD protein, then blocked with 2% BSA in PBS. Serum samples were diluted (1:2) in PBS-T. Samples were detected with 1:6000 goat anti-mouse IgG-HRP (Southern Biotech, Birmingham, AL). Reactions were developed with SureBlue TMB substrate and stopped with TMB stop solution from KPL (Gaithersburg, MD). Plates were read (OD450) using an Epoch ELISA reader (BioTek, Winooski, VT).

[0422] 8. SARS-COV-2 Pseudovirus Neutralization Test (PVNT) In some embodiments, a SARS-CoV-2 pseudovirus neutralization test was used. Pseudotyped luciferase rSARS-CoV-2 Spike was purchased from Creative Diagnostics Inc., and this lentivirus-based SARS-CoV-2 S pseudotyped virus is a replication-restricted recombinant pseudotyped lentivirus particle containing the SARS-CoV-2 spike protein (based on the Wuhan-Hu-1 isolate). The infectivity of pseudotyped luciferase rSARS-CoV-2 is limited to one round of replication, so it encodes Renilla luciferase in the lentivirus vector genome. When the genome is integrated after entering cells, luciferase expression and activity are proportional to the number of transduced cells.

[0423] To determine pVNT, 20 μL of SARS-CoV-2 spike-pseudotyped virus (105 RLU) can be pre-incubated with two-fold serially diluted test serum samples (starting with a dilution of 1:10) in a final volume of 50 μL for 1 h at 37 °C, followed by the addition of 5 × 104 HEK293T-ACE2 cells in a volume of 50 μL. At 48–72 h post-infection, an equal amount of Renilla luciferase substrate (Promega, catalog no. 2710) is added and the luminescence signal is measured using a microplate reader (BioTek) with Gen5 software. Measurements can be performed in duplicate for CV pooled serum samples and in triplicate for group B pooled serum samples. % neutralization and IC50 of pVNT are calculated as previously described (Le Bert et al., 2020).

[0424] 9. Flow Cytometry and Intracellular Cytokine Staining (ICS) Splenocytes were stimulated with spike peptides at a concentration of 2ug / mL for 6 hours. After 6 hours of incubation, cells were stained with Zombie aqua followed by surface staining, fixing with Perm / fix solution (BD Biosciences) and staining with intracellular antibodies in 1x perm / wash buffer. Samples were analyzed on a CytoFlex flow cytometer (Beckman Coulter) and analyzed using Cauja software (Beckman Coulter).

[0425] Cells were stained for intracellular cytokines for IFNγ, TNFα, and IL-2. CD4 T cells (effector memory CD4) were gated on CD3+CD4+CD8-CD44+CD62L- lymphocytes. CD8 T cells (effector memory CD4) were gated on CD3+CD4-CD8+CD44+CD62L- lymphocytes.

[0426] B. Results 1. Soluble CD40 ligand (SCD40L) expressed by 2-V COVID vaccine 293T cells were transfected with the 2-V COVID vaccine and analyzed for expression of sCD40L. Immunoprecipitation and Western blotting were used to confirm the expression of sCD40L from the 2-V COVID vaccine (Figure 12; boxed band indicates sCD40L).

[0427] 2.2-V COVID vaccine elicited T cell responses The immunogenicity of the 2-V COVID vaccine was determined 14 days after the first immunization and 14 days after the second immunization (Table 2). The data show that the 2-V COVID vaccine induced significantly enhanced T cell-mediated responses compared to the first generation COVID-19 vaccine (Figure 13A-D). The data show that the presence of sCD40L improved the 2-V COVID vaccine by inducing a greater T cell-mediated response.

[0428] 3.2-V COVID vaccine stimulated spike-specific CD4 and CD8 T cells in vivo A higher number of CD4+ and CD8+ T cells were observed in splenocytes from mice vaccinated with the 2-V vaccine compared to the ITI-bicistronic vaccine (Figure 14). The data show that the presence of sCD40L provided enhancement. CD4+ and CD8+ T cells were also stained for intracellular cytokines, with a higher percentage of IFNγ, TNFα, and IL-2 staining in mice vaccinated with the 2-V vaccine compared to the ITI-bicistronic vaccine (Figure 15).

[0429] 4.2-V COVID vaccine elicited spike-specific antigen responses in vivo Sera from immunized mice were analyzed by ELISA for S1-specific antibodies after one or two immunizations. Figures 16A-B show total IgG S1-binding antibodies. Figures 16C-D show IgG2a antibodies. Figures 16E-F show IgG1 antibodies. The data demonstrate that the 2-V COVID vaccine elicited superior S1-specific antigen responses compared to the ITI-bicistronic vaccine. The IgG1 response was particularly prominent after a single dose of the 2-V COVID vaccine (Figure 16E).

[0430] 5. Overview The 2-V vaccine significantly enhanced spike-specific T cell responses, including both CD4+ and CD8+ T cell responses, compared with the first generation vaccine. The 2-V vaccine also enhanced S1-specific antibody responses, especially IgG1 levels, after a single immunization dose.

[0431] Example 3. Soluble CD40L (SCD40L) Expressed from HER2-LAMP-SCD40L Bicistronic Vaccine In this example, we consider HER2-LAMP-sCD40L (Figure 11; SEQ ID NO: 197), a bicistronic DNA vaccine encoding HER2-LAMP and the 4-trimeric version of sCD40L. The data discussed herein support the use of bicistronic DNA vaccines against cancer.

[0432] CD40 ligand (CD40L) is a transmembrane protein expressed on the surface of activated T cells, particularly CD4 T cells, and stimulates CD40-dependent activation of antigen-presenting cells (APCs), resulting in enhanced T cell and antibody responses. The soluble multimeric form of CD40L (sCD40L) can act as an adjuvant to enhance vaccine immunogenicity. As described in this example, a HER2-LAMP-sCD40L bicistronic construct was tested against a construct expressing HER2-LAMP but not any second polypeptide such as sCD40L. HER2-LAMP-sCD40L elicited significantly enhanced HER2-specific T cell and antibody responses in mice compared to mice immunized with control HER2-LAMP DNA. Intracellular staining revealed that the inclusion of sCD40L in the vaccine induced strong antigen-specific T cell (IFNγ) production, primarily in CD4 T cells. Furthermore, in murine TSA breast cancer mocels, HER2-LAMP-sCD40L significantly inhibited tumor growth and prolonged survival in a therapeutic vaccine setting, suggesting that HER2-LAMP-sCD40L vaccine is an effective strategy to promote antitumor efficacy in vivo.

[0433] A. Materials and Methods Generally, the materials and methods used in this example were as described in Examples 1-2 above, with the following modifications.

[0434] 1. Vaccine Constructs The HER2-LAMP-sCD40L bicistronic construct discussed in these examples (Figure 11; SEQ ID NO: 197) contains two expression cassettes. The first cassette is driven by the CMV promoter to express the LAMP-HER2 / ErBB2 fusion protein (SEQ ID NO: 195; or SEQ ID NO: 198 followed by SEQ ID NO: 200 followed by SEQ ID NO: 202). The second cassette is driven by the EF1 promoter to express the soluble mouse CD40 ligand (sCD40L; GenBank accession number X65453.2) encoding the 4-trimeric soluble CD40L (Gomez et al., 2009; Stone et al., 2006) fused to the body of surfactant protein D (SPD) (SEQ ID NO: 196). A three amino acid HRR is present between the SPD and the sCD40L. The constructs are designed to deliver the HER2 antigen to the MHC II compartment, which may enhance both antibody production and CD4 T cell responses, whereas the sCD40L polypeptide constructs are secreted.

[0435] The HER2-LAMP construct without sCD40L was used as a control vaccine.

[0436] A bicistronic DNA vaccine encoding Spike (Spike-LAMP) was used as a control vector (i.e., negative control).

[0437] 2. Immunization and Serum Collection Female C57BL / 6 mice aged 6-8 weeks were bred and maintained in an approved breeding facility. The immunization schedule for the HER2-LAMP-sCD40L vaccine is shown in Figure 13A and Table 3. Mice were immunized with 20 μg of control vector or vaccine by intradermal (ID) injection into the ear. The experiment was terminated 1 week after the second dose, i.e., on day 22. Splenocytes were treated with 1 μg / mL of HER2 pool peptide for 48 h. [Table 78]

[0438] 3. Transient Transfection of Constructs and ELISA HER2-LAMP-sCD40L transfected 293T cells were analyzed for sCD40L expression. Spike-encoding bicistronic DNA vaccine (Spike-LAMP) served as a negative control. Five days after transfection, supernatants from these cells were harvested and sCD40L was detected using ELISA.

[0439] 4. Evaluation of antigen-specific T cell responses by ELISPOT Materials and methods were as described in Example 2 above, except that overlapping HER2 peptides (purchased from Genscript or JPT) were used instead of overlapping spike peptides.

[0440] 5. Flow Cytometry and Intracellular Cytokine Staining (ICS) Splenocytes from immunized mice (FIG. 18A) were incubated with brefeldin A and monensin with or without the HER2 peptide pool for 5 hours.

[0441] 6. ELISA Mouse antibody responses to HER2 were assessed by indirect ELISA: ELISA plates (MaxiSorp) were coated overnight with 1 μg / ml HER2 protein.

[0442] 7. Statistics Statistical analysis was performed using Prism software. Groups were compared using one-way ANOVA.

[0443] 8. Mouse Breast Tumor Model Mice were immunized with two doses of HER2-LAMP-sCD40L or HER2-LAMP, followed by 2 × 10 5 HER2 expressing TSA (mouse breast cancer) cells were injected. Blood cells were stimulated with 1 μg / mL of the HER2 peptide pool and analyzed by ELISPOT....

Claims

1. Isolated nucleic acid molecules, comprising the following: a. A first polynucleotide sequence encoding a polypeptide (collectively, a "LAMP-antigen construct") comprising two homologous domains of the luminal domain of a LAMP protein and a heterologous antigen domain relative to the LAMP protein, wherein the antigen domain is positioned between the two homologous domains; and b. A second polynucleotide sequence encoding an immune response-enhancing gene polypeptide (IREG) or at least one second polypeptide comprising an extracellular domain of an IREG containing a secretory signaling sequence.

2. The isolated nucleic acid molecule according to Claim 1, wherein the LAMP protein is selected from LAMP-1, LAMP2, LAMP-3, lysosomal membrane endogenous protein-2 ("LIMP 2"), macrosailin, endolyn, LAMP5, or limbic system-associated membrane protein ("LIMBIC").

3. The isolated nucleic acid molecule according to claim 2, wherein the LAMP protein comprises an amino acid sequence selected from any one of SEQ ID NOs: 1 to 113, or comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NOs: 1 to 113.

4. The isolated nucleic acid molecule according to claim 2, wherein the LAMP protein is LAMP-1, and the two homologous domains of the LAMP-antigen construct comprise LAMP-1 homologous domain 1 and LAMP-1 homologous domain 2.

5. The aforementioned human LAMP-1 homologous domain 1, (a) containing the amino acid sequence of residues 29-194 of SEQ ID NO: 1, or containing the amino acid sequence of residues 29-195 of SEQ ID NO: 198, (b) A variant of (a) wherein the variant contains at least 95% or at least 95% identical amino acids to the amino acid sequence of (a); and / or the human LAMP-1 homologous domain 2 contains the amino acid sequence of residues 228-381 of SEQ ID NO: 1 The isolated nucleic acid molecule according to claim 4.

6. The isolated nucleic acid molecule according to any one of claims 1 to 5, wherein the LAMP-antigen construct includes a linker between at least one of the two homologous domains and the antigenic domain.

7. The isolated nucleic acid molecule according to claim 6, wherein the linker comprises the amino acid sequence GPPGG or PMGLP.

8. The isolated nucleic acid molecule according to any one of claims 1 to 5, wherein the LAMP-antigen construct further comprises the transmembrane domain and / or cytoplasmic domain of the LAMP protein.

9. The isolated nucleic acid molecule according to claim 8, wherein the transmembrane domain comprises residues 383 to 405 of SEQ ID NO: 1, and / or the cytoplasmic domain comprises residues 406 to 417 of SEQ ID NO:

1.

10. The isolated nucleic acid molecule according to any one of claims 1 to 5, wherein the LAMP-antigen construct further comprises a signal sequence.

11. The isolated nucleic acid molecule according to claim 10, wherein the signal sequence is derived from a LAMP protein such as a signal sequence containing residues 1 to 28 of SEQ ID NO: 1 or residues 1 to 28 of SEQ ID NO:

198.

12. The isolated nucleic acid molecule according to any one of claims 1 to 5, wherein the IREG comprises one or more of the following: CD40L, CD80, OX40, Flt3L, GM-CSF, IL-12, IL-21, IL-23, IL-15, CD70, CD86, IL-7, IL-18, or IL-33, or their extracellular domains, and optionally the CD40L, CD80, OX40, Flt3L, GM-CSF, IL-12, IL-21, IL-23, or IL-15 is fused to the Fc domain of the immunoglobulin.

13. The isolated nucleic acid molecule according to any one of claims 1 to 5, wherein the secretory signal sequence is heterogeneous to IREG and / or derived from IgKVIII, Ig-kappa, tetranectin, or IL-2, and / or the second polypeptide further comprises pulmonary surfactant-related protein D (SPD).

14. The isolated nucleic acid molecule according to claim 13, wherein the second polypeptide is expressed under the control of an EF-1α core promoter such as the promoter of SEQ ID NO:

124.

15. The isolated nucleic acid molecule according to any one of claims 1 to 5, wherein the first polynucleotide sequence encodes a polypeptide comprising two homologous domains of the luminal domain of a LAMP protein and an antigenic domain comprising the HER2 extracellular domain (collectively, "HER2-LAMP"), and the antigenic domain is positioned between the two LAMP homologous domains.

16. An isolated nucleic acid molecule according to any one of claims 1 to 5, comprising DNA, mRNA, or self-amplified RNA.

17. A composition comprising an isolated nucleic acid molecule according to any one of claims 1 to 5.

18. A host cell containing an isolated nucleic acid molecule according to any one of claims 1 to 5.

19. A composition comprising the host cells described in claim 18.

20. A pharmaceutical composition for treating a disease or disorder in a subject, or for inducing an immune response in a subject having a disease or disorder, or a subject at risk of developing a disease or disorder, comprising an isolated nucleic acid molecule as described in any one of claims 1 to 5.

21. The pharmaceutical composition according to claim 20, wherein the treatment or induction further comprises administering at least one second therapeutic agent to the subject.