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

Abstract

The present disclosure provides a nucleic acid molecule (e.g., a plasmid or vector) comprising: a nucleic acid sequence encoding a bicistronic or polycistronic LAMP construct comprising a specific fragment of an LAMP endodomain and an antigen domain heterologous to the LAMP protein to provide at least one antigen for eliciting an immune response, wherein the antigen expressed by the construct is optionally processed and presented to a class II MHC molecule; and a nucleic acid sequence encoding an immune response enhancing polypeptide, optionally secreted from a host cell. The nucleic acid molecules can be used, for example, for the treatment of diseases, in particular allergy, infectious diseases, diabetes, hyperproliferative disorders and / or cancer.

Description

[0001] CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 329,463 filed on April 10, 2022, the contents of which are incorporated herein in their entirety.

[0003] Sequence Listing

[0004] This application includes a sequence listing in ST26 format, which is incorporated herein by reference in its entirety (file name: 2023-04-10_01305-0023-00PCTST26; file size: 1,417,543 bytes). Technical Field

[0005] The present disclosure relates to isolated nucleic acid molecules (e.g., plasmids or vectors) encoding bicistronic or polycistronic LAMP (lysosomal associated membrane protein) constructs comprising a LAMP fusion protein, and a second optionally secreted protein such as a protein from an immune response enhancing gene (IREG), and their use in treating subjects with infectious diseases, diabetes, allergies, hyperproliferative disorders and / or cancer, particularly COVID-19. In addition, the bicistronic LAMP constructs described herein can be used to produce antibodies in non-human vertebrates, preferably wherein the genome of the non-human vertebrate comprises at least a portion of a human immunoglobulin region and / or a humanized immunoglobulin region. Background Art

[0006] In the following discussion, certain articles and methods are described for background and introduction purposes. Nothing contained herein should be construed as an "admission" of prior art. Applicants expressly reserve the right to demonstrate, where appropriate, that the articles and methods cited herein do not constitute prior art under applicable law.

[0007] Vaccines are new and promising candidates for the development of preventive and therapeutic vaccines. They have been shown to be safe, and the lack of immune response to the vector backbone may be a decisive advantage if repeated vaccination cycles are required to achieve clinical benefit. However, a significant disadvantage of conventional vaccines is their low immunogenicity in humans. A key limiting step in the immunogenicity of epitope-based vaccines may be the process of epitope entry into the class II major histocompatibility (MHC) presentation pathway of T cells, which may be a stochastic process in the case of vaccines without targeted technology.

[0008] Various designs of LAMP-antigen constructs have been previously described, for example, in U.S. Pat. No. 11,203,629 (see Figure 1). A construct designated ILC-4 described in U.S. Pat. No. 11,203,629 (depicted in the text) Figure 1 In some embodiments, the construct further comprises a transmembrane domain of a LAMP protein and / or a cytoplasmic tail region of a LAMP protein. The two homologous domains may be derived from, for example, LAMP-1, LAMP-2, LAMP-3 or Endolyn proteins. Alternatively, two homologous domains from two different LAMP proteins may be used. The inventors unexpectedly discovered that improved LAMP constructs such as ILC-4 can, for example, elicit strong T cell and antibody responses against the antigen of interest, making them viable candidates for use as vaccines.

[0009] Nevertheless, there remains a need to design new and further improved LAMP constructs and nucleic acid molecules encoding them, which can be used as vaccines to effectively treat, for example, allergies, infectious diseases, diabetes, hyperproliferative disorders and / or cancer, and / or for generating useful antibodies. Summary of the invention

[0010] As further described herein, the inventors have now discovered that isolated nucleic acid molecules can be designed that not only express a LAMP construct (e.g., Figure 1 ), and expresses a second polypeptide of a particular type, typically a secreted polypeptide, encoding a gene that has been found to enhance the immune response to tumors or infectious diseases in vivo, such as CD40L, CD80, OX40, IL-12, IL-21, IL-15 or Flt3L, etc.; and expressing these two polypeptides from the isolated nucleic acid molecule unexpectedly enhances the immune response not only compared to earlier LAMP constructs, but also compared to bicistronic LAMP constructs containing certain secreted antigens, such as second disease antigens.

[0011] This summary is provided to introduce a series of concepts in a simplified form, which are further described in the following detailed description. This summary is not intended to identify key 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 be apparent from the following written detailed description, including those aspects illustrated in the drawings and defined in the claims.

[0012] An object of the present disclosure is to provide novel nucleic acid molecules encoding constructs ("bicistronic LAMP constructs") comprising specific fragments and / or variants of LAMP domains that effectively present antigens of interest to the immune system to generate an enhanced immune response. These bicistronic LAMP constructs effectively direct the antigens to the lysosomal / endosomal compartments where they are processed and presented to class II major histocompatibility complex (MHC) molecules to preferentially stimulate helper T cells and / or antibody production while having the ability to enhance the immune response.

[0013] The nucleic acid molecules and methods of the encoding bicistronic LAMP constructs described herein can trigger an immune response in a subject. The immune response can be an immune response to an epitope of an 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 substance containing the antigen of the vaccine in the subject. The nucleic acid molecules and methods of the encoding bicistronic LAMP constructs described herein can trigger a Th1 immune response in the subject. The Th1 immune response can include secretion of inflammatory cytokines (e.g., IFNγ, TNFa) by a subgroup of immune cells (e.g., antigen-specific T cells). In some cases, the inflammatory cytokines activate another subtype of immune cells (e.g., cytotoxic T cells), which can destroy the substance containing the antigen in the subject.

[0014] In some cases, the antigen used in the bicistronic LAMP constructs and methods described herein can be recognized by the subject's immune system, thereby initiating a Th1 immune response and releasing type I cytokines. The Th1 response can be initiated by the interaction between the epitope and the T cell, more specifically the major histocompatibility complex (MHC) expressed by the T cell. For example, the high affinity binding of the epitope to the MHC receptor can stimulate the Th1 response. The MHC receptor can be at least one of multiple types of MHC receptors. The MHC receptor engaged on the T cell may vary from individual to individual in the population.

[0015] 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.

[0016] The nucleic acid molecules encoding the bicistronic LAMP constructs described herein can also be used in a manner to provide for the expression of an immunoregulatory element (IRE) or an immune response enhancing gene (IREG), thereby eliciting an enhanced immune response (e.g., an immune response comprising significantly higher antibody titers) in a subject. For example, a nucleic acid molecule (e.g., a plasmid or vector) can provide for the expression of a bicistronic LAMP construct comprising a LAMP-antigen polypeptide that is processed and presented to a class II MHC molecule, thereby preferentially stimulating helper T cells, activating memory cells and / or producing antibodies; and providing for the expression of another IREG or IRE polypeptide that can be secreted into the circulation of the subject and can, for example, further enhance the humoral and cellular immune response to the LAMP antigen.

[0017] In one aspect, the nucleic acid molecule of the coding bicistronic LAMP construct is a vaccine vector suitable for vaccination of a subject. In another aspect, the disclosure provides a delivery vehicle for facilitating the introduction of the nucleic acid molecule (which comprises a polynucleotide encoding an epitope and / or an antigen) of the coding bicistronic LAMP construct into a cell. The delivery vehicle can be lipid-based (e.g., liposome preparation), virus-based (e.g., comprising a viral protein encapsulating the nucleic acid molecule) or cell-based.

[0018] In some embodiments, the present disclosure provides an injectable composition comprising a nucleic acid molecule encoding a bicistronic LAMP construct as described herein for eliciting an immune response (e.g., production of antibodies) to an antigen in a subject. In some embodiments, the vaccine produces a Th1 response that is preferential to a Th2 response.

[0019] The disclosure also provides a kind of cell, it comprises the nucleic acid molecule of encoding bicistronic LAMP construct described herein, can be used for producing immune response.In one aspect, the cell is antigen presenting cell.The antigen presenting cell can be professional antigen presenting cell (for example dendritic cell, macrophage, B cell etc.) or engineered antigen presenting cell (for example, engineered to express the non-professional antigen presenting cell of molecule such as class II MHC molecule required for antigen presentation).The molecule required for antigen presentation can be derived from other cells, for example, naturally occurring, or they themselves can be engineered (for example, mutated or modified to show the desired characteristic, for example, higher or lower affinity to antigen epitope).

[0020] The disclosure also provides a kind of test kit, it comprises multiple cells, and the cell comprises the nucleic acid molecule of the encoding bicistronic LAMP construct described herein.At least two of the cells can express different class II MHC molecules, and each cell can comprise the same LAMP construct.In one aspect, a kind of test kit is provided, it comprises the viral vector of the encoding bicistronic LAMP construct.

[0021] The present disclosure also provides a transgenic animal comprising at least one of the cells and / or at least one nucleic acid molecule encoding a bicistronic LAMP construct as described herein. The present disclosure also provides a transgenic animal comprising at least one of the cells as described herein.

[0022] The present disclosure also provides a method for producing an enhanced immune response to an antigen in a subject (e.g., a human or non-human vertebrate), the method comprising administering a cell as described above to the subject, wherein the cell expresses or can be induced to express the bicistronic LAMP construct in the subject. In one aspect, the cell comprises a class II MHC molecule compatible with the subject's MHC protein, such that the subject does not produce an immune response to the class II MHC molecule.

[0023] In another aspect, the present disclosure provides a method of eliciting an enhanced immune response to an antigen, the method comprising administering to a subject, such as a human or non-human vertebrate, a nucleic acid molecule encoding a bicistronic LAMP construct as 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 virus vector.

[0024] The disclosure also includes methods for producing antibodies in non-human vertebrates, wherein the non-human vertebrates are injected with nucleic acid molecules encoding bicistronic LAMP constructs as described herein. The antibodies produced can be isolated from the blood of the vertebrates (as polyclones) and then further isolated using standard techniques to produce monoclonal antibodies.

[0025] The methods described herein can be used to produce and / or optimize antibodies, including fully human antibodies, humanized antibodies, chimeric antibodies, for diagnostic and therapeutic uses. Hybridomas producing such antibodies are also another object of the present disclosure.

[0026] Specific embodiments of the present disclosure include the following embodiments:

[0027] 1. An isolated nucleic acid molecule comprising:

[0028] 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 the LAMP protein (collectively referred to as a "LAMP-antigen construct"), wherein the antigenic domain is placed between the two homologous domains; and

[0029] b. A second polypeptide sequence encoding at least one second polypeptide comprising an immune response enhancing gene polypeptide (IREG) or an extracellular domain of IREG comprising a secretion signal sequence.

[0030] 2. An isolated nucleic acid molecule according to embodiment 1, wherein the LAMP protein is selected from LAMP-1, LAMP2, LAMP-3, lysosomal integral membrane protein-2 ("LIMP 2"), Macrosailin, Endolyn, LAMP5 or limbic system-associated membrane protein ("LIMBIC").

[0031] 3. The isolated nucleic acid molecule according to embodiment 2, wherein the LAMP protein is selected from any one of SEQ ID NOs: 1-113.

[0032] 4. An isolated nucleic acid molecule according to 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 NO: 1-113.

[0033] 5. The isolated nucleic acid molecule of embodiment 2, wherein the LAMP protein is LAMP-1, and wherein the two homology domains of the LAMP-antigen construct include LAMP-1 homology domain 1 and LAMP-1 homology domain 2.

[0034] 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 comprises the amino acid sequence of residues 29-195 of SEQ ID NO: 198, 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 29-194 of SEQ ID NO: 1 or the amino acid sequence of residues 29-195 of SEQ ID NO: 198, or wherein 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).

[0035] 7. The isolated nucleic acid molecule of 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 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).

[0036] 8. The isolated nucleic acid molecule of any one of embodiments 1-7, wherein the LAMP-antigen construct comprises a linker between at least one of the two homologous domains and the antigenic domain.

[0037] 9. An isolated nucleic acid molecule according to embodiment 8, wherein the linker comprises the amino acid sequence of GPGPG or PMGLP.

[0038] 10. The isolated nucleic acid molecule of any one of embodiments 1-9, wherein the LAMP-antigen construct further comprises a transmembrane domain of a LAMP protein.

[0039] 11. The isolated nucleic acid molecule of embodiment 10, wherein the transmembrane domain comprises residues 383-405 of SEQ ID NO: 1.

[0040] 12. The isolated nucleic acid molecule of any one of embodiments 1-11, wherein the LAMP-antigen construct further comprises a signal sequence.

[0041] 13. An isolated nucleic acid molecule according to embodiment 12, wherein the signal sequence is derived from a LAMP protein.

[0042] 14. The isolated nucleic acid molecule of any one of embodiments 1-13, wherein the LAMP-antigen construct further comprises a cytoplasmic domain of a LAMP protein.

[0043] 15. The isolated nucleic acid molecule of embodiment 14, wherein the cytoplasmic domain comprises residues 406-417 of SEQ ID NO: 1.

[0044] 16. The isolated nucleic acid molecule of any one of embodiments 1-15, wherein 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 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 any one of SEQ ID NOs: 133, 145, 147, 149, 151, 155, 159, 165, 169, 173, 177, 181, 189, 191, 204, 238, 242, 243, 252, or 253), or an extracellular domain thereof, optionally wherein the CD40L, CD80, OX40, Flt3L, GM-CSF, IL-12, IL-21, IL-23, IL-15, CD70, CD86, IL-7, IL-18, or IL-33 X40, Flt3L, GM-CSF, IL-12, IL-21, IL-23 or IL-15 fused to the 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, wherein the nucleotide sequence is the same as SEQ The nucleotide sequence of any one of ID NOs: 134, 146, 148, 150, 152, 160, 166, 170, 174, 178, 182, 190, 192, 205, 239, 244, or 881 is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, or 100% identical.

[0045] 17. The isolated nucleic acid molecule of any one of embodiments 1-16, wherein the secretion signal sequence is heterologous to the IREG.

[0046] 18. An isolated nucleic acid molecule according to embodiment 17, wherein the secretory signal sequence is derived from IgKVIII (e.g., SEQ ID NO: 122), Ig-κ (e.g., SEQ ID NO: 120), tetranectin or IL-2, and / or wherein the second polypeptide further comprises lung surfactant-associated protein D (SPD) (e.g., SEQ ID NO: 131).

[0047] 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.

[0048] 20. A composition comprising the isolated nucleic acid molecule according to any one of embodiments 1-18.

[0049] 21. A host cell comprising the isolated nucleic acid according to any one of embodiments 1-18.

[0050] 22. A composition comprising the host cell according to embodiment 20.

[0051] 23. A method of treating a subject having a disease or condition or inducing an immune response in a subject having a disease or condition or at risk of developing a disease or condition, wherein the method comprises 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 condition or induce an immune response in the subject.

[0052] 24. The method of embodiment 23, wherein the method further comprises administering at least one second therapeutic agent to the subject.

[0053] 25. An isolated nucleic acid molecule comprising:

[0054] 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 the extracellular domain of HER2 (collectively referred to as "HER2-LAMP"), wherein the antigenic domain is placed between the two LAMP homologous domains; and

[0055] b. A second polypeptide sequence encoding at least one second polypeptide comprising an immune response enhancing gene polypeptide (IREG) or an extracellular domain of IREG comprising a secretion signal sequence.

[0056] 26. An isolated nucleic acid molecule according to embodiment 25, wherein the LAMP protein is selected from LAMP-1, LAMP2, LAMP-3, lysosomal integral membrane protein-2 ("LIMP 2"), Macrosailin, Endolyn, LAMP5 or limbic system-associated membrane protein ("LIMBIC").

[0057] 27. The isolated nucleic acid molecule of embodiment 26, wherein the LAMP protein is selected from any one of SEQ ID NOs: 1-113.

[0058] 28. An isolated nucleic acid molecule according to embodiment 25 or 26, wherein the LAMP protein has 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% identity with SEQ ID NO: 1-113.

[0059] 29. An isolated nucleic acid molecule according to embodiment 26, wherein the LAMP protein is LAMP-1, and wherein the two homology domains of HER2-LAMP include LAMP-1 homology domain 1 and LAMP-1 homology domain 2.

[0060] 30. The isolated nucleic acid molecule of 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 comprises the amino acid sequence of residues 29-195 of SEQ ID NO: 198, 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 29-194 of SEQ ID NO: 1 or the amino acid sequence of residues 29-195 of SEQ ID NO: 198, or wherein 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).

[0061] 31. The isolated nucleic acid molecule of embodiment 29 or 30, wherein the human LAMP-1 homology domain 2 comprises the amino acid sequence of residues 228-381 or residues 228-382 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 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).

[0062] 32. An isolated nucleic acid molecule according to any one of embodiments 25-31, wherein the HER2-LAMP comprises a linker between at least one of the two homologous domains and the antigenic domain.

[0063] 33. An isolated nucleic acid molecule according to embodiment 32, wherein the linker comprises the amino acid sequence of GPGPG or PMGLP.

[0064] 34. An isolated nucleic acid molecule according to any one of embodiments 25-33, wherein the HER2-LAMP further comprises a transmembrane domain of a LAMP protein.

[0065] 35. An isolated nucleic acid molecule according to embodiment 34, wherein the transmembrane domain comprises residues 383-405 of SEQ ID NO: 1.

[0066] 36. An isolated nucleic acid molecule according to any one of embodiments 25-35, wherein the HER2-LAMP further comprises a signal sequence.

[0067] 37. An isolated nucleic acid molecule according to embodiment 36, wherein the signal sequence is derived from a LAMP protein.

[0068] 38. An isolated nucleic acid molecule according to any one of embodiments 25-37, wherein the HER2-LAMP further comprises a cytoplasmic domain of a LAMP protein.

[0069] 39. An isolated nucleic acid molecule according to embodiment 38, wherein the cytoplasmic domain comprises residues 406-417 of SEQ ID NO: 1.

[0070] 40. The isolated nucleic acid molecule of any one of embodiments 25-39, wherein the antigenic domain comprises or consists of the amino acid sequence of SEQ ID NO: 200.

[0071] 41. An isolated nucleic acid molecule according to any one of embodiments 25-40, wherein the HER2-LAMP comprises residues 1-194 of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 198, followed by the amino acid sequence of SEQ ID NO: 200, followed by residues 228-381 of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 202, or consists of said amino acid sequence.

[0072] 42. The isolated nucleic acid molecule of any one of embodiments 25-41, wherein the IREG comprises CD40L, CD80, OX40, Flt3L, GM-CSF, IL-12, IL-21, IL-23, IL-15, CD70, CD86, IL-7, IL-18, or IL-33 (or comprises a nucleic acid molecule corresponding to SEQ ID NO: 133, 145, 147, 149, 151, 155, 159, 165, 169, 173, 177, 181, 189, 191, 193, 204, 238, 242, 243, 252 or 253) or one or more of its extracellular domains, optionally wherein the CD40L, CD80 , OX40, Flt3L, GM-CSF, IL-12, IL-21, IL-23 or IL-15 fused to the 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, wherein the nucleotide sequence is the same as SEQ The nucleotide sequence of any one of ID NOs: 134, 146, 148, 150, 152, 160, 166, 170, 174, 178, 182, 190, 192, 194, 205, 239, 244, or 881 is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, or 100% identical.

[0073] 43. An isolated nucleic acid molecule according to any one of embodiments 25-42, wherein the secretion signal sequence is heterologous to the IREG.

[0074] 44. An isolated nucleic acid molecule according to embodiment 43, wherein the secretory signal sequence is derived from IgKVIII (e.g., SEQ ID NO: 122), Ig-κ (e.g., SEQ ID NO: 120), tetranectin or IL-2.

[0075] 45. The isolated nucleic acid molecule of any one of embodiments 25-44, wherein 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., 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: 233, 238, 242, or 252, 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, or 100% identical to the amino acid sequence of SEQ ID NO: 131 followed by the amino acid sequence of SEQ ID NO: NO:204, 151, 145, 147, 149, 193, 181, 155, 159, 169, 252 or 253), or wherein the nucleic acid comprises a nucleotide sequence encoding 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., comprising 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, or 100% identical to the following sequence: SEQ ID NO:132 followed by one of SEQ ID NO:205, 152, 146, 148, 150, 194, 182, 156, 170 or 881).

[0076] 46. ​​The isolated nucleic acid molecule of 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.

[0077] 47. A composition comprising the isolated nucleic acid molecule according to any one of embodiments 25-46.

[0078] 48. A host cell comprising the isolated nucleic acid according to any one of embodiments 25-46.

[0079] 49. A composition comprising the host cell according to embodiment 48.

[0080] 50. A method of treating a subject having cancer, wherein the method comprises administering to the subject an isolated nucleic acid molecule according to any one of embodiments 25-46, a composition according to embodiment 47, or a host cell according to embodiment 48 in an amount sufficient to treat the cancer or induce an immune response against the cancer in the subject.

[0081] 51. The method of embodiment 50, wherein the method further comprises administering at least one second therapeutic agent to the subject.

[0082] 52. An isolated nucleic acid molecule comprising:

[0083] a. a first polynucleotide sequence encoding a polypeptide comprising two homologous domains of the luminal domain of a LAMP protein and an antigenic domain of a coronavirus spike protein antigen (collectively referred to as "Spike-LAMP"), wherein the antigenic domain is placed between the two LAMP homologous domains; and

[0084] b. A second polypeptide sequence encoding at least one second polypeptide comprising an immune response enhancing gene polypeptide (IREG) or an extracellular domain of IREG comprising a secretion signal sequence.

[0085] 53. An isolated nucleic acid molecule according to embodiment 52, wherein the LAMP protein is selected from LAMP-1, LAMP2, LAMP-3, lysosomal integral membrane protein-2 ("LIMP 2"), Macrosailin, Endolyn, LAMP5 or limbic system-associated membrane protein ("LIMBIC").

[0086] 54. The isolated nucleic acid molecule of embodiment 53, wherein the LAMP protein is selected from any one of SEQ ID NOs: 1-113.

[0087] 55. An isolated nucleic acid molecule according to embodiment 52 or 53, wherein the LAMP protein has 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% identity with SEQ ID NO: 1-113.

[0088] 56. An isolated nucleic acid molecule according to embodiment 53, wherein the LAMP protein is LAMP-1, and wherein the two homologous domains of Spike-LAMP include LAMP-1 homologous domain 1 and LAMP-1 homologous domain 2.

[0089] 57. An 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 comprises the amino acid sequence of residues 29-195 of SEQ ID NO: 198, 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 29-194 of SEQ ID NO: 1 or the amino acid sequence of residues 29-195 of SEQ ID NO: 198, or wherein 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).

[0090] 58. An isolated nucleic acid molecule according to embodiment 56 or 57, wherein the human LAMP-1 homology domain 2 comprises the amino acid sequence of residues 228-381 or residues 228-382 of SEQ ID NO: 1, or residues 228-382 of SEQ ID NO: 1, or residues 228-382 of SEQ ID NO: 1, or the amino acid sequence of SEQ ID NO: 202, 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 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).

[0091] 59. An isolated nucleic acid molecule according to any one of embodiments 52-58, wherein the Spike-LAMP comprises a linker between at least one of the two homologous domains and the antigenic domain.

[0092] 60. An isolated nucleic acid molecule according to embodiment 59, wherein the linker comprises an amino acid sequence of GPGPG or PMGLP.

[0093] 61. An isolated nucleic acid molecule according to any one of embodiments 52-60, wherein the Spike-LAMP further comprises a transmembrane domain of a LAMP protein.

[0094] 62. An isolated nucleic acid molecule according to embodiment 61, wherein the transmembrane domain comprises residues 383-405 of SEQ ID NO: 1.

[0095] 63. An isolated nucleic acid molecule according to any one of embodiments 52-62, wherein the Spike-LAMP further comprises a signal sequence.

[0096] 64. An isolated nucleic acid molecule according to embodiment 63, wherein the signal sequence is derived from a LAMP protein.

[0097] 65. An isolated nucleic acid molecule according to any one of embodiments 52-64, wherein the Spike-LAMP further comprises a cytoplasmic domain of a LAMP protein.

[0098] 66. An isolated nucleic acid molecule according to embodiment 65, wherein the cytoplasmic domain comprises residues 406-417 of SEQ ID NO: 1.

[0099] 67. An isolated nucleic acid molecule according to any one of embodiments 52-66, wherein the antigenic domain comprises spike S1 and / or S2 or an amino acid sequence comprising the sequence of SEQ ID NO: 118 or 119.

[0100] 68. An isolated nucleic acid molecule according to any one of embodiments 52-67, wherein the Spike-LAMP comprises residues 1-194 of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 198, followed by the amino acid sequence of SEQ ID NO: 231, followed by residues 228-381 of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 202, or consists of said amino acid sequence.

[0101] 69. The isolated nucleic acid molecule of any one of embodiments 52-68, wherein the IREG comprises CD40L, CD80, OX40, Flt3L, GM-CSF, IL-12, IL-21, IL-23, IL-15, CD70, CD86, IL-7, IL-18, or IL-33 (or comprises a nucleic acid molecule corresponding to SEQ ID NO: 133, 145, 147, 149, 151, 155, 159, 165, 169, 173, 177, 181, 189, 191, 204, 238, 242, 243, 252 or 253 having 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) or one or more of its extracellular domains, optionally wherein said CD40L, CD80, O X40, Flt3L, GM-CSF, IL-12, IL-21, IL-23 or IL-15 fused to the 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, wherein the nucleotide sequence is the same as SEQ The nucleotide sequence of any one of ID NOs: 134, 146, 148, 150, 152, 160, 166, 170, 174, 178, 182, 190, 192, 205, 239, 244, or 881 is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, or 100% identical.

[0102] 70. The isolated nucleic acid molecule of any one of embodiments 52-68, wherein the second polypeptide comprises a SPD-sCD40L fusion polypeptide.

[0103] 71. An isolated nucleic acid molecule according to any one of embodiments 52-70, wherein the secretion signal sequence is heterologous to the IREG.

[0104] 72. An isolated nucleic acid molecule according to embodiment 71, wherein the secretion signal sequence is derived from SPD.

[0105] 73. The isolated nucleic acid molecule of any one of embodiments 52-72, wherein the second polypeptide is expressed under the control of an EF-1α core promoter, such as the promoter of SEQ ID NO: 124.

[0106] 74. A composition comprising the isolated nucleic acid molecule according to any one of embodiments 52-73.

[0107] 75. A host cell comprising the isolated nucleic acid according to any one of embodiments 52-73.

[0108] 76. A composition comprising the host cell according to embodiment 75.

[0109] 77. A method for treating a subject having a coronavirus infection (e.g., from COVID-19) or at risk of developing a coronavirus infection (e.g., from COVID-19), wherein the method comprises administering to the subject an isolated nucleic acid molecule according to any one of embodiments 52-73, a composition according to embodiment 74, or a host cell according to embodiment 75 in an amount sufficient to treat the coronavirus infection (e.g., COVID-19) or prevent its onset or reduce the severity of its symptoms.

[0110] 78. A method for inducing an immune response against a coronavirus, such as SARS Co-V2, in a subject, wherein the method comprises administering to the subject an isolated nucleic acid molecule according to any one of embodiments 52-73, a composition according to embodiment 74, or a host cell according to embodiment 75 in an amount sufficient to induce an immune response against the coronavirus, such as SARS Co-V2, in the subject.

[0111] 79. An isolated nucleic acid molecule comprising:

[0112] a. a first polynucleotide sequence encoding a polypeptide comprising two homologous domains of the luminal domain of a LAMP protein and an antigenic domain of an NY-ESO1 or CD161 protein antigen ("NY-ESO1-LAMP" or "CD161-LAMP" LAMP-antigen construct), wherein the antigenic domain is placed between the two LAMP homologous domains; and

[0113] b. A second polypeptide sequence encoding at least one second polypeptide comprising an immune response enhancing gene polypeptide (IREG) or an extracellular domain of IREG comprising a secretion signal sequence.

[0114] 80. An isolated nucleic acid molecule according to embodiment 79, wherein the LAMP protein is selected from LAMP-1, LAMP2, LAMP-3, lysosomal integral membrane protein-2 ("LIMP 2"), Macrosailin, Endolyn, LAMP5 or limbic system-associated membrane protein ("LIMBIC").

[0115] 81. The isolated nucleic acid molecule of embodiment 80, wherein the LAMP protein is selected from any one of SEQ ID NOs: 1-113.

[0116] 82. An isolated nucleic acid molecule according to embodiment 79 or 80, wherein the LAMP protein has 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% identity with SEQ ID NO: 1-113.

[0117] 83. An isolated nucleic acid molecule according to embodiment 79, wherein the LAMP protein is LAMP-1, and wherein the two homology domains of NY-ESO1-LAMP or CD161-LAMP include LAMP-1 homology domain 1 and LAMP-1 homology domain 2.

[0118] 84. An 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 comprises the amino acid sequence of residues 29-195 of SEQ ID NO: 198, 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 29-194 of SEQ ID NO: 1 or the amino acid sequence of residues 29-195 of SEQ ID NO: 198, or wherein 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).

[0119] 85. An 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 residues 228-382 of SEQ ID NO: 1, or residues 228-382 of SEQ ID NO: 1, or residues 228-382 of SEQ ID NO: 1, or the amino acid sequence of SEQ ID NO: 202, 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 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).

[0120] 86. An isolated nucleic acid molecule according to any one of embodiments 79-85, wherein the LAMP-antigen construct comprises a linker between at least one of the two homologous domains and the antigenic domain.

[0121] 87. An isolated nucleic acid molecule according to embodiment 86, wherein the linker comprises an amino acid sequence of GPGPG or PMGLP.

[0122] 88. An isolated nucleic acid molecule according to any one of embodiments 79-87, wherein the LAMP-antigen construct further comprises a transmembrane domain of a LAMP protein.

[0123] 89. An isolated nucleic acid molecule according to embodiment 88, wherein the transmembrane domain comprises residues 383-405 of SEQ ID NO: 1.

[0124] 90. An isolated nucleic acid molecule according to any one of embodiments 79-89, wherein the LAMP-antigen construct further comprises a signal sequence.

[0125] 91. An isolated nucleic acid molecule according to embodiment 90, wherein the signal sequence is derived from a LAMP protein.

[0126] 92. An isolated nucleic acid molecule according to any one of embodiments 79-91, wherein the LAMP-antigen construct further comprises a cytoplasmic domain of a LAMP protein.

[0127] 93. An isolated nucleic acid molecule according to embodiment 92, wherein the cytoplasmic domain comprises residues 406-417 of SEQ ID NO:1.

[0128] 94. An isolated nucleic acid molecule according to any one of embodiments 79-93, wherein the LAMP-antigen construct comprises or consists of the following amino acid sequence: residues 1-194 of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 198, followed by the amino acid sequence of SEQ ID NO: 223, followed by residues 228-381 of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 202, or residues 1-194 of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 198, followed by the amino acid sequence of SEQ ID NO: 236, followed by residues 228-381 of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 202.

[0129] 95. The isolated nucleic acid molecule of any one of embodiments 79-94, wherein the IREG comprises CD40L, CD80, OX40, Flt3L, GM-CSF, IL-12, IL-21, IL-23, IL-15, CD70, CD86, IL-7, IL-18, or IL-33 (or comprises a nucleic acid molecule corresponding to SEQ ID NO: 133, 145, 147, 149, 151, 155, 159, 165, 169, 173, 177, 181, 189, 191, 204, 238, 242, 243, 252 or 253 having 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) or one or more of its extracellular domains, optionally wherein said CD40L, CD80, O X40, Flt3L, GM-CSF, IL-12, IL-21, IL-23 or IL-15 fused to the 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, wherein the nucleotide sequence is the same as SEQ The nucleotide sequence of any one of ID NOs: 134, 146, 148, 150, 152, 160, 166, 170, 174, 178, 182, 190, 192, 205, 239, 244, or 881 is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, or 100% identical.

[0130] 96. The isolated nucleic acid molecule of any one of embodiments 79-95, wherein the second polypeptide comprises a sPD-sCD40L fusion polypeptide or an IL-15 polypeptide (eg, SEQ ID NO: 233 or 169 or 225).

[0131] 97. An isolated nucleic acid molecule according to any one of embodiments 79-96, wherein the secretion signal sequence is heterologous to the IREG.

[0132] 98. An isolated nucleic acid molecule according to embodiment 97, wherein the secretion signal sequence is derived from SPD or comprises SEQ ID NO: 120 or 122.

[0133] 99. The isolated nucleic acid molecule of any one of embodiments 79-98, wherein the second polypeptide is expressed under the control of an EF-1α core promoter, such as the promoter of SEQ ID NO: 124.

[0134] 100. A composition comprising the isolated nucleic acid molecule according to any one of embodiments 79-99.

[0135] 101. A host cell comprising the isolated nucleic acid according to any one of embodiments 79-99.

[0136] 102. A composition comprising the host cell according to embodiment 101.

[0137] 103. A method of inducing an immune response in a subject in need thereof, the method comprising administering to the subject an isolated nucleic acid molecule according to any one of embodiments 79-99, a composition according to embodiment 100, or a host cell according to embodiment 101 in an amount sufficient to induce an immune response in the subject.

[0138] 104. An isolated nucleic acid molecule comprising:

[0139] a. a first polynucleotide sequence encoding a polypeptide comprising two homologous domains of the luminal domain of a LAMP protein and an antigenic domain, wherein the antigenic domain comprises a pp65 antigen (e.g., comprising SEQ ID NO: 291, 292 or 293), and optionally further comprises one or both of a gB antigen (e.g., comprising SEQ ID NO: 294, 295, 296 or 297) and a 1E1 antigen (e.g., comprising SEQ ID NO: 298, 299 or 300), optionally comprising a linker peptide between the pp65 and gB and / or 1E1 antigen sequences (collectively referred to as "pp65-LAMP"), wherein the antigenic domain is placed between the two LAMP homologous domains; and

[0140] b. A second polypeptide sequence encoding at least one second polypeptide comprising an immune response enhancing gene polypeptide (IREG) or an extracellular domain of IREG comprising a secretion signal sequence.

[0141] 105. An isolated nucleic acid molecule according to embodiment 104, wherein the LAMP protein is selected from LAMP-1, LAMP2, LAMP-3, lysosomal integral membrane protein-2 ("LIMP 2"), Macrosailin, Endolyn, LAMP5 or limbic system-associated membrane protein ("LIMBIC").

[0142] 106. The isolated nucleic acid molecule of embodiment 105, wherein the LAMP protein is selected from any one of SEQ ID NOs: 1-113.

[0143] 107. An isolated nucleic acid molecule according to embodiment 104 or 105, wherein the LAMP protein has 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% identity with SEQ ID NO: 1-113.

[0144] 108. An isolated nucleic acid molecule according to embodiment 105, wherein the LAMP protein is LAMP-1, and wherein the two homology domains of HER2-LAMP include LAMP-1 homology domain 1 and LAMP-1 homology domain 2.

[0145] 109. An 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 29-195 of SEQ ID NO: 198, 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 29-194 of SEQ ID NO: 1 or the amino acid sequence of residues 29-195 of SEQ ID NO: 198, or wherein 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).

[0146] 110. The isolated nucleic acid molecule of embodiment 108 or 109, wherein the human LAMP-1 homology domain 2 comprises the amino acid sequence of residues 228-381 or residues 228-382 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 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).

[0147] 111. An isolated nucleic acid molecule according to any one of embodiments 104-110, wherein the pp65-LAMP comprises a linker between at least one of the two homologous domains and the antigenic domain.

[0148] 112. An isolated nucleic acid molecule according to embodiment 111, wherein the linker comprises an amino acid sequence of GPGPG or PMGLP.

[0149] 113. An isolated nucleic acid molecule according to any one of embodiments 104-112, wherein the pp65-LAMP further comprises a transmembrane domain of a LAMP protein.

[0150] 114. An isolated nucleic acid molecule according to embodiment 113, wherein the transmembrane domain comprises residues 383-405 of SEQ ID NO:1.

[0151] 115. The isolated nucleic acid molecule of any one of embodiments 104-114, wherein the pp65-LAMP further comprises a signal sequence.

[0152] 116. An isolated nucleic acid molecule according to embodiment 115, wherein the signal sequence is derived from a LAMP protein.

[0153] 117. An isolated nucleic acid molecule according to any one of embodiments 104-116, wherein the pp65-LAMP further comprises a cytoplasmic domain of a LAMP protein.

[0154] 118. An isolated nucleic acid molecule according to embodiment 117, wherein the cytoplasmic domain comprises residues 406-417 of SEQ ID NO:1.

[0155] 119. The isolated nucleic acid molecule of any one of embodiments 104-118, wherein the IREG comprises CD40L, CD80, OX40, Flt3L, GM-CSF, IL-12, IL-21, IL-23, IL-15, CD70, CD86, IL-7, IL-18, or IL-33 (or comprises a nucleic acid molecule corresponding to SEQ ID NO: 133, 145, 147, 149, 151, 155, 159, 165, 169, 173, 177, 181, 189, 191, 204, 238, 242, 243, 252 or 253 having 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) or one or more of its extracellular domains, optionally wherein said CD40L, CD80, O X40, Flt3L, GM-CSF, IL-12, IL-21, IL-23 or IL-15 fused to the 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, wherein the nucleotide sequence is the same as SEQ The nucleotide sequence of any one of ID NOs: 134, 146, 148, 150, 152, 160, 166, 170, 174, 178, 182, 190, 192, 205, 239, 244, or 881 is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, or 100% identical.

[0156] 120. An isolated nucleic acid molecule according to any one of embodiments 104-119, wherein the secretion signal sequence is heterologous to the IREG.

[0157] 121. An isolated nucleic acid molecule according to embodiment 120, wherein the secretory signal sequence is derived from IgKVIII (e.g., SEQ ID NO: 122), Ig-κ (e.g., SEQ ID NO: 120), tetranectin or IL-2.

[0158] 122. The isolated nucleic acid molecule of any one of embodiments 104-121, wherein 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., 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: 233, 238, 242, or 252, 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, or 100% identical to the amino acid sequence of SEQ ID NO: 131 followed by the amino acid sequence of SEQ ID NO: NO:204, 151, 145, 147, 149, 193, 181, 155, 159, 169, 252 or 253), or wherein the nucleic acid comprises a nucleotide sequence encoding 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., comprising 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, or 100% identical to the following sequence: SEQ ID NO:132 followed by one of SEQ ID NO:205, 152, 146, 148, 150, 194, 182, 156, 170 or 881).

[0159] 123. An 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.

[0160] 124. A composition comprising the isolated nucleic acid molecule according to any one of embodiments 104-123.

[0161] 125. A host cell comprising the isolated nucleic acid according to any one of embodiments 104-123.

[0162] 126. A composition comprising the host cell according to embodiment 125.

[0163] 127. A method for treating a subject having cancer, wherein the method comprises administering to the subject an isolated nucleic acid molecule according to any one of embodiments 104-123, a composition according to embodiment 124, or a host cell according to embodiment 125 in an amount sufficient to treat the cancer or induce an immune response against the cancer in the subject.

[0164] 128. The method of embodiment 127, wherein the method further comprises administering at least one second therapeutic agent to the subject.

[0165] 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.

[0166] 130. An isolated nucleic acid molecule comprising:

[0167] 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 (e.g., an amino acid sequence comprising SEQ ID NO: 254, 255 or 256) ("large T-LAMP"), wherein the antigenic domain is placed between the two LAMP homologous domains; and

[0168] b. A second polypeptide sequence encoding at least one second polypeptide comprising an immune response enhancing gene polypeptide (IREG) or an extracellular domain of IREG comprising a secretion signal sequence.

[0169] 131. An isolated nucleic acid molecule according to embodiment 130, wherein the LAMP protein is selected from LAMP-1, LAMP2, LAMP-3, lysosomal integral membrane protein-2 ("LIMP 2"), Macrosailin, Endolyn, LAMP5 or limbic system-associated membrane protein ("LIMBIC").

[0170] 132. An isolated nucleic acid molecule according to embodiment 131, wherein the LAMP protein is selected from any one of SEQ ID NOs: 1-113.

[0171] 133. An 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 NO: 1-113.

[0172] 134. An isolated nucleic acid molecule according to embodiment 131, wherein the LAMP protein is LAMP-1, and wherein the two homology domains of HER2-LAMP include LAMP-1 homology domain 1 and LAMP-1 homology domain 2.

[0173] 135. An 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 comprises the amino acid sequence of residues 29-195 of SEQ ID NO: 198, 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 29-194 of SEQ ID NO: 1 or the amino acid sequence of residues 29-195 of SEQ ID NO: 198, or wherein 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).

[0174] 136. An 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 residues 228-382 of SEQ ID NO: 1, or residues 228-382 of SEQ ID NO: 1, or residues 228-382 of SEQ ID NO: 1, or the amino acid sequence of SEQ ID NO: 202, 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 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).

[0175] 137. An isolated nucleic acid molecule according to any one of embodiments 130-136, wherein the large T-LAMP comprises a linker between at least one of the two homologous domains and the antigenic domain.

[0176] 138. An isolated nucleic acid molecule according to embodiment 137, wherein the linker comprises an amino acid sequence of GPGPG or PMGLP.

[0177] 139. An isolated nucleic acid molecule according to any one of embodiments 130-138, wherein the large T-LAMP further comprises a transmembrane domain of a LAMP protein.

[0178] 140. An isolated nucleic acid molecule according to embodiment 140, wherein the transmembrane domain comprises residues 383-405 of SEQ ID NO: 1.

[0179] 141. An isolated nucleic acid molecule according to any one of embodiments 130-140, wherein the pp65-LAMP further comprises a signal sequence.

[0180] 142. An isolated nucleic acid molecule according to embodiment 141, wherein the signal sequence is derived from a LAMP protein.

[0181] 143. An isolated nucleic acid molecule according to any one of embodiments 130-142, wherein the large T-LAMP further comprises a cytoplasmic domain of a LAMP protein.

[0182] 144. An isolated nucleic acid molecule according to embodiment 143, wherein the cytoplasmic domain comprises residues 406-417 of SEQ ID NO:1.

[0183] 145. The isolated nucleic acid molecule of any one of embodiments 130-144, wherein the IREG comprises CD40L, CD80, OX40, Flt3L, GM-CSF, IL-12, IL-21, IL-23, IL-15, CD70, CD86, IL-7, IL-18, or IL-33 (or comprises a nucleic acid molecule corresponding to SEQ ID NO: 133, 145, 147, 149, 151, 155, 159, 165, 169, 173, 177, 181, 189, 191, 204, 238, 242, 243, 252 or 253 having 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) or one or more of its extracellular domains, optionally wherein said CD40L, CD80, O X40, Flt3L, GM-CSF, IL-12, IL-21, IL-23 or IL-15 fused to the 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, wherein the nucleotide sequence is the same as SEQ The nucleotide sequence of any one of ID NOs: 134, 146, 148, 150, 152, 160, 166, 170, 174, 178, 182, 190, 192, 205, 239, 244, or 881 is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical, or 100% identical.

[0184] 146. An isolated nucleic acid molecule according to any one of embodiments 130-145, wherein the secretion signal sequence is heterologous to the IREG.

[0185] 147. An isolated nucleic acid molecule according to embodiment 146, wherein the secretory signal sequence is derived from IgKVIII (e.g., SEQ ID NO: 122), Ig-κ (e.g., SEQ ID NO: 120), tetranectin or IL-2.

[0186] 148. The isolated nucleic acid molecule of any one of embodiments 130-147, wherein 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., 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: 233, 238, 242, or 252, 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, or 100% identical to the amino acid sequence of SEQ ID NO: 131 followed by the amino acid sequence of SEQ ID NO: NO:204, 151, 145, 147, 149, 193, 181, 155, 159, 169, 252 or 253), or wherein the nucleic acid comprises a nucleotide sequence encoding 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., comprising 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, or 100% identical to the following sequence: SEQ ID NO:132 followed by one of SEQ ID NO:205, 152, 146, 148, 150, 194, 182, 156, 170 or 881).

[0187] 149. The isolated nucleic acid molecule of 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.

[0188] 150. A composition comprising the isolated nucleic acid molecule according to any one of embodiments 130-149.

[0189] 151. A host cell comprising the isolated nucleic acid according to any one of embodiments 130-149.

[0190] 152. A composition comprising the host cell according to embodiment 151.

[0191] 153. A method of treating a subject having cancer, wherein the method comprises administering to the subject an isolated nucleic acid molecule according to any one of embodiments 130-149, a composition according to embodiment 150, or a host cell according to embodiment 151 in an amount sufficient to treat the cancer or induce an immune response against the cancer in the subject.

[0192] 154. The method of embodiment 153, wherein the method further comprises administering at least one second therapeutic agent to the subject.

[0193] 155. The method of embodiment 153 or 154, wherein the cancer is skin cancer, such as Merkel cell carcinoma.

[0194] 156. An isolated nucleic acid according to any one of embodiments 1-19, 25-46, 52-73, 79-99, 104-123 or 130-149, wherein the isolated nucleic acid comprises DNA, mRNA or self-amplifying RNA.

[0195] 157. A set of polypeptides encoded by the isolated nucleic acid according to any one of embodiments 1-19, 25-46, 52-73 or 79-99, 104-123 or 130-149.

[0196] Other purposes and advantages will be set forth in part in the following description and will be understood in part from the description or may be learned through practice. Said purposes and advantages will be realized and obtained by means of the elements and combinations particularly pointed out in the claims.

[0197] 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 claims.

[0198] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one or more implementations and, together with the description, serve to explain the principles described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0199] The objects and features of the present disclosure may be better understood with reference to the following detailed description and accompanying drawings.

[0200] Figure 1 The overall scheme of 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 is shown. Certain backbone constructs are further described in U.S. Patent No. 11,203,629, which is incorporated herein by reference in its entirety.

[0201] Figure 2B The domain structure of the LAMP proteins defined herein is shown, and Figure 2A Specific amino acid boundaries of these domains are defined 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, homeodomain, transmembrane domain, cytoplasmic tail, and signal sequence can be used to generate the bicistronic LAMP constructs described herein.

[0202] Figure 3 An alignment of LAMP-1 proteins found in other species compared to human LAMP-1 (SEQ ID NO: 1) is provided. The equivalent domains of these other species can be used to generate the bicistronic LAMP constructs described herein and can be obtained by combining FIG. 2 and Figure 3 The structural domains identified in human LAMP-1 Figure 3 They can be easily identified by comparison with the alignment shown in .

[0203] Figure 4 An alignment of LAMP-2 proteins found in other species compared to human LAMP-2 (SEQ ID NO: 2) is provided. The equivalent domains of these other species can be used to generate the bicistronic LAMP constructs described herein and can be obtained by combining FIG. Figure 4 The structural domains identified in human LAMP-2 Figure 4 They can be easily identified by comparison with the alignment shown in .

[0204] Figure 5 An alignment of LAMP-3 proteins found in other species compared to human LAMP-3 (SEQ ID NO: 3) is provided. The equivalent domains of these other species can be used to generate the bicistronic LAMP constructs described herein and can be obtained by combining FIG. Figure 5 The structural domains identified in human LAMP-3 Figure 5 They can be easily identified by comparison with the alignment shown in .

[0205] Figure 6An alignment of LIMP-2 proteins found in other species compared to human LIMP-2 (SEQ ID NO: 4) is provided. The equivalent domains of these other species can be used to generate the bicistronic LAMP constructs described herein and can be obtained by combining FIG. Figure 6 The structural domains identified in human LIMP-2 Figure 6 They can be easily identified by comparison with the alignment shown in .

[0206] Figure 7 An alignment of LIMBIC proteins found in other species compared to human LIMBIC (SEQ ID NO: 67) is provided. The equivalent domains of these other species can be used to generate the bicistronic LAMP constructs described herein and can be obtained by combining FIG2 and Figure 7 The structural domains identified in human LIMBIC Figure 7 They can be easily identified by comparison with the alignment shown in .

[0207] Figure 8 An alignment of Endolyn proteins found in other species compared to human Endolyn (SEQ ID NO: 5) is provided. The equivalent domains of these other species can be used to generate the bicistronic LAMP constructs described herein and can be obtained by combining FIG. Figure 8 The structural domains identified in human Endolyn Figure 8 They can be easily identified by comparison with the alignment shown in .

[0208] Fig. 9 An alignment of Macrosialin proteins found in other species compared to human Macrosialin (SEQ ID NO: 80) is provided. The equivalent domains of these other species can be used to generate the bicistronic LAMP constructs described herein and can be obtained by combining FIG. Fig. 9 Domains identified in human Macrosialin Fig. 9 They can be easily identified by comparison with the alignment shown in .

[0209] Fig.10 An alignment of LAMP-5 proteins found in other species compared to human LAMP-5 (SEQ ID NO: 93) is provided. The equivalent domains of these other species can be used to generate the bicistronic LAMP constructs described herein and can be obtained by combining FIG. Fig.10 The structural domains identified in human LAMP-5 Fig.10 They can be easily identified by comparison with the alignment shown in .

[0210] Fig.11The design of an exemplary bicistronic construct HER2-LAMP-sCD40L is shown.

[0211] Fig.12 Detection of sCD40L in the supernatant of 293T cells transfected with the bicistronic HER2-LAMP-sCD40L vaccine is shown. The asterisk indicates the protein band indicating the presence of sCD40L. Immunoprecipitation experiments using biotinylated anti-CD40L antibodies were used to detect sCD40L in control 293T cells, 293T cells transfected with the bicistronic HER2-LAMP-sCD40L, or 293T cells transfected with a control vector expressing GFP. Cell culture supernatant was used as input (I). Flowthrough (FT) represents proteins not bound to biotinylated anti-CD40L antibodies, while bound (B) represents proteins bound to biotinylated anti-CD40L antibodies.

[0212] Fig.13A -D shows the results of single immunization with ITI-bicistronic vaccine or 2-V COVID vaccine ( Fig.13A -B) or two immunizations ( Fig. 13C -D) Detection of spike-specific T cell responses in mice. Fig.13A and 13C :ELISPOT. Fig. 13B and 13D : Spot counts. CV = control vector (plasmid alone). SFC = spot forming cells. Student's T test was used to determine significance. *p<0.05, **p<0.01, ***p<0.001.

[0213] Fig.14 Detection of spike-specific CD4 and CD8 T cells by flow cytometry is shown. Both CD4+ and CD8+ T cell responses were enhanced after vaccination with the 2-V vaccine.

[0214] Fig.15 Intracellular cytokine staining (ICS) for IFNγ, TNFα, and IL-2 in CD4+ and CD8+ T cells is shown.

[0215] Fig.16A -F shows the results of one immunization with ITI-bicistronic vaccine or 2-V vaccine ( Fig.16A , 16C and 16E) compared to two immunizations ( Fig. 16B , 16D The Student's T test was used to determine significance.

[0216] Fig.17A-B shows the detection of HER2-LAMP-sCD40L in the supernatant of transfected 293T cells. Fig.17A The sCD40L standard curve used for ELISA is shown. Fig. 17B Detection of sCD40L in supernatants is shown. Control cells = cells not transfected with the bicistronic construct.

[0217] Fig.18A -F shows that the bicistronic vaccine can elicit strong T cell and antibody responses. Fig.18A An exemplary vaccination schedule is shown, in which mice were immunized by intradermal (ID) injection of 20 μg of control vector, HER2-LAMP, or bicistronic HER2-LAMP-sCD40L. Fig.18B IFNγ spot forming cells (ELISPOT) are shown. Fig. 18C Mean ± SEM of IFNγ spot-forming cells are shown. Fig.18D -F shows HER2-specific total IgG detected by ELISA ( Fig.18D )、IgG1( Fig.18E ) and IgG2a( Fig.18F ); N = 7. One-way ANOVA was used for statistical analysis. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0218] Fig.19A -B shows intracellular staining of the cytokines IFNγ (IFNg), TNFα (TNFa), and IL-2 in CD4 and CD8 T cells. Fig.19A Mean ± SEM of % of stained cells are shown. Fig.19B Representative FACS plots are shown. *p<0.05, **p<0.01, ***p<0.001.

[0219] Fig. 20 Shown are HER2-specific antibody responses determined by ELISA.

[0220] Fig.21A -B shows HER2-LAMP-sCD40L protects mice from HER2-expressing breast tumors. Fig.21A The results of ELISPOT are shown. Fig. 21B Tumor sizes measured using calipers are shown.

[0221] Fig.22A -B shows that HER2-LAMP-sCD40L improves survival in mice. Fig.22A A schematic diagram of the experimental design is shown. Fig. 22BShown are mouse survival rates after tumor challenge in a single experiment. N=10.

[0222] Fig.23 Shown is the effect of HER2-LAMP vaccine with immune response enhancing genes (IREGs) (HER2-LAMP-IREG) on tumor volume. IREG = CD40L, Flt3L, IL-21, IL-12, and OX40L. **p<0.01; ****p<0.0001.

[0223] Fig.24 Shown is the effect of HER2-LAMP-IREG on mouse survival.

[0224] Fig.25 It is shown that HER2-LAMP-IL-15 induces a strong antigen-specific antibody response as determined by ELISA. N=5 per group. T-test was used for statistical analysis. *p<0.05, **p<0.01.

[0225] Fig.26 HER2-LAMP-IL-15 is shown to elicit a robust T cell response as determined by ELISPOT. Data represent mean ± SEM of IFNγ spot-forming cells. N=5. T-test was used for statistical analysis. *p<0.05, **p<0.01.

[0226] Fig.27A -B shows that two doses of HER2-LAMP-IL-15 elicited a robust T cell response as determined by ELISPOT. Data represent raw spots ( Fig.27A ) and IFNγ spot-forming cells mean ± SEM ( Fig.27B ). N = 5. T-test was used for statistical analysis. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0227] Fig.28A -C involves the first generation of bicistronic LAMP constructs containing the SARS CoV-2 S1 spike protein. Fig.28A The overall scheme of the vector (Bicistronic-S1-LAMP-EF1 IgK-S2P; 7427 bp) encoding the first generation bicistronic LAMP construct (ILC-4 based) comprising a fragment of the SARS CoV-2S1 spike protein as the target antigen and an expression cassette for the expression of the SARS CoV-2S2 spike protein (as the second antigen for secretion) operably linked to an Ig-κ leader sequence (secretion signal) is shown. Fig.28Bis another representation of a first generation ITI-COVID bicistronic construct (as a circular plasmid), showing a representative but preferred example of the arrangement of the polynucleotide encoding sequence. Fig.28C Shown are the complete polynucleotides and encoded polypeptide domains of the first generation ITI-COVID bicistronic construct.

[0228] Fig.29A -D shows inhibition of tumor growth by bicistronic HER2-LAMP-sCD40L (soluble CD40 ligand) administered as a DNA vector compared to control (CV) and non-bicistronic HER2-LAMP vectors in the murine TSA breast cancer model. Fig.29A The experimental scheme is shown. Fig.29B The changes in tumor growth for each mouse are shown. Fig.29C Tumor growth changes of 7 mice in each group (control, HER2-LAMP and HER2-LAMP-sCD40L) are shown. Control (CV; top curve) is depicted by solid circles; HER2-LAMP ( Fig.29C The middle curve in Figure ) is depicted by solid squares; HER2-LAMP-sCD40L ( Fig.29C The bottom curve in ) is depicted by a solid triangle. Fig.29D Tumor weights measured at the termination of the experiment are shown (* indicates p<0.05 and ** indicates p<0.01).

[0229] Fig.30 It is shown that HER2-LAMP-sCD40L induced the generation of CD3+ memory T cells compared to control vector.

[0230] Fig.31A -C shows that HER2-LAMP-sCD40L promoted T cell infiltration in the tumor microenvironment compared with HER2-LAMP and control vector. Fig.31A Shown are the numbers of CD3+ T cells in tumors after harvesting, cleaning, and dissociation of the tumors. Fig.31B The number of CD4+ T cells is shown. FIG31C shows the number of CD8+ T cells. * indicates p<0.05, and ** indicates p<0.01.

[0231] Fig.32A -C shows that soluble CD40 ligand (sCD40L) produced by HER2-LAMP-sCD40L vector enhances the activation of IL-12-producing type 1 dendritic cells (DC1) in draining lymph nodes. Fig.32A shows the gating scheme used in the experiments, and Fig.32B DC1 cells expressing CD8 are shown. Fig.32CIL-12 producing DC1 cells are shown. * indicates p<0.05.

[0232] Fig.33A -B shows that the bicistronic HER2-LAMP-sCD40L activates inflammatory signals in the tumor microenvironment. Specifically, Fig.33A shows the number of CD4+CD69+ cells in the tumor after harvesting, cleaning and dissociation of the tumor, and Fig.33B The number of CD8+CD69+ cells in tumors is shown. * indicates p<0.05.

[0233] Fig.34A -B shows that the bicistronic HER2-LAMP-sCD40L promotes PD-1-producing T cells in the tumor microenvironment. Specifically, Fig.33A shows the number of CD4+PD-1+ cells in the tumor after harvesting, cleaning and dissociation of the tumor, and Fig.33B The number of CD8+PD-1+ cells in tumors is shown. * indicates p<0.05, and ** indicates p<0.01.

[0234] Fig.35 Shown is the incubation of splenocytes with several different peptide combinations of the HER2 extracellular domain. Fig.29A Results of ELISPOT analysis performed on splenocytes from mice according to the protocol described in. The results show that the bicistronic HER2-LAMP-sCD40L induced stronger responses against certain combined HER2 peptides compared to the control or HER2-LAMP constructs.

[0235] Fig.36 Shown from Fig.29A Results of parallel experiments to those described in -C, using 5 mice each for control, HER2-LAMP, and HER2-LAMP-sCD40L groups, demonstrated that the bicistronic construct inhibited tumor growth more than the other two groups, p<0.05.

[0236] Fig.37A -D shows the Fig.36 The results of FACS analysis of spleen cells from the experiment shown in Fig.29A ), and showed that HER2-LAMP-sCD40L induced multifunctional CD4 effector memory T cells ("TEM" cells) in the spleen. Fig.37A The percentage of CD4 TEM cells is shown; Fig.37BThe percentage of CD8 TEM cells is shown. The amount of CD4 or CD8 TEM cells expressing IFNg, TNFa, or both IFNg and TNFa is indicated in the bar graph. The remaining figures show the CD4 TEM cells expressing both IFNg and TNFa ( Fig.37C ) or CD8 TEM cells ( Fig.37D ) Response to combined HER2 peptides. *P<0.05, **p<0.01, ****p<0.0001.

[0237] Fig.38A -B shows the Fig.29A In the murine TSA model of HER2, soluble CD40L expressed from the HER2-LAMP-sCD40L construct enhanced activation of DC1 dendritic cells in the spleen. The FACS gating strategy is shown in Fig.38A The percentage of DC1 dendritic cells is shown in Fig.38B * indicates p < 0.05, and ** indicates p < 0.01.

[0238] Fig.39A -B shows the results of cell staining experiments, indicating that Fig.29A In a murine TSA model, soluble CD40L expressed from the HER2-LAMP-sCD40L construct increased CD4+ ( Fig.39A ) and CD8+ T cells ( Fig.39B )’s existence.

[0239] Fig.40A -B shows the results of 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 Her2-LAMP-sCD40L and Her2-LAMP-mFlt3L (7 mice per group), and then spleen cells were subjected to ELISPOT analysis. Fig.40A The mean + / - SEM of IFNg-producing cells in each group is shown. Fig.40B The number of cells from each of the five groups that recognized various HER2 peptide combinations is shown.

[0240] Fig.41 Shows Fig.40A The antibody titer of each group was determined by ELISA.

[0241] Fig.42A -D shows the Fig.40AAfter the experiment, the percentage of various CD4 and CD8 TEM cells recognizing various HER2 extracellular domain peptide combinations, specifically CD8IFNgTNFa cells ( Fig.42A )、CD8 IFNg cells ( Fig.42B )、CD4 IFNg TNFa cells ( Fig.42C ) and CD4 IFNg cells ( Fig.42D ).

[0242] Fig.43 Shown is the effect of combined administration of two bicistronic constructs, HER2-LAMP-IL-12 and HER2-LAMP-mFlt3L, on serum antibody titers measured by ELISA. DETAILED DESCRIPTION

[0243] The present disclosure encompasses, for example, nucleic acid molecules encoding bicistronic or polycistronic LAMP constructs, which can be used to produce vaccines and / or for producing antibodies and / or humoral immune responses. The nucleic acid molecules can be used to induce an immune response. In one aspect, the present disclosure provides for treating subjects suffering from allergies, infectious diseases such as coronavirus or Covid-19, diabetes, cancer, or hyperproliferative disorders by providing nucleic acid molecules (e.g., plasmids or vectors) encoding bicistronic LAMP constructs described herein. The nucleic acid molecules encoding bicistronic LAMP constructs can also be used to produce antibodies in non-human vertebrates, preferably non-human mammals.

[0244] A. Definition

[0245] The following definitions are provided for certain terms used in the following written description.

[0246] Unless the context clearly dictates otherwise, references without a specific number as used in this specification and claims include plural referents. For example, the term "cell" includes a plurality of cells, including mixtures thereof. The term "nucleic acid molecule" includes a plurality of nucleic acid molecules.

[0247] As used herein, the term "comprising" means that the nucleic acid molecule or bicistronic LAMP construct and method include the described elements, but do not exclude other elements. In the case of amino acid or nucleotide sequences, this means that other sequence elements can be added to either end of the sequence. When used to define nucleic acid molecules, bicistronic LAMP constructs and methods, "essentially consisting of..." means excluding other elements that are of any essential importance to the combination or its function. Therefore, the bicistronic LAMP constructs consisting essentially of the elements defined herein do not exclude trace contaminants from separation and purification methods and pharmaceutically acceptable carriers such as phosphate buffered saline, preservatives, etc. "Consisting of..." means excluding the elements exceeding trace amounts of other ingredients and the substantial method steps for applying the nucleic acid molecules encoding the bicistronic LAMP constructs described herein. In the case of amino acid or nucleotide sequences, "consisting of..." means that no other sequence elements are added to either end of the sequence, but the described sequence allows the incorporation of physiologically occurring amino acid or nucleotide modifications, such as DNA methylation or glycosylation, etc. The embodiments defined by each of these transitional terms are within the scope of the present disclosure.

[0248] 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 range will depend in part on how the value is measured or determined, such as limitations of the measurement system. For example, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, more preferably up to 1% of a given value. Alternatively, particularly for biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, more preferably within 2-fold of a value. Unless otherwise indicated, the term "about" means within an acceptable error range for a particular value, such as within ±1-20%, preferably ±1-10%, more preferably ±1-5%.

[0249] Where a range of values ​​is provided, it is to be understood that each intervening value between the upper and lower limits of the range and any other stated or intervening values ​​within the stated range are encompassed within the disclosure. The upper and lower limits of these smaller ranges may be independently included within the smaller ranges and are also encompassed within the disclosure, provided that any specifically excluded limits within the stated range are excluded. Where a stated range includes one or two limits, ranges excluding these two included limits are also encompassed within the disclosure.

[0250] "Lysosome / endosome compartment" as used herein refers to a membrane-bound acidic vacuole containing LAMP molecules in the membrane, hydrolases that play a role in antigen processing, and class II MHC molecules for antigen recognition and presentation. The compartment serves as a degradation site (de Duve, Eur. J. Biochem. 137: 391, 1983) for foreign substances internalized from the cell surface by any of various mechanisms (including endocytosis, engulfment, and pinocytosis) and for intracellular substances delivered to the compartment by a special autolysis phenomenon. The term "endosome" as used herein encompasses lysosomes.

[0251] As used herein, "lysosome-associated organelles" refer to any organelles containing lysosomes, including but not limited to MIIC, CIIV, melanosomes, secretory granules, lytic granules, platelet dense granules, basophil granules, Birbeck granules, phagolysosomes, secretory lysosomes, etc. Preferably, such organelles lack mannose 6-phosphate receptors and contain LAMPs, but may or may not contain class II MHC molecules. For review, see, e.g., Blott and Griffiths, Nature Reviews, Molecular Cell Biology, 2002; Dell'Angelica et al., The FASEB Journal 14: 1265-1278, 2000.

[0252] The terms "polynucleotide" and "nucleic acid molecule" used herein are interchangeably used to refer to polymeric forms of nucleotides of any length. A polynucleotide can contain deoxyribonucleotides, ribonucleotides and / or their analogs. Therefore, in some cases, the bicistronic LAMP construct nucleic acid molecule herein can be a DNA molecule such as a DNA vector, such as a DNA viral vector, and in other cases, it can be an RNA molecule, including a self-amplifying RNA vector (also referred to as a self-replicating RNA vector).

[0253] Nucleotide can have any three-dimensional structure, and can perform any known or unknown function.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, DNA separated from any sequence, RNA separated from any sequence, nucleic acid probes and primers.Nucleic acid molecules can also include modified nucleic acid molecules (e.g., comprising modified bases, sugars and / or internucleotide linkers).

[0254] The term "peptide" as used herein refers to a compound of two or more subunit amino acids, amino acid analogs or peptide mimetics. The subunits may be linked by peptide bonds or other bonds (eg, esters, ethers, etc.).

[0255] The term "amino acid" as used herein refers to natural and / or non-natural or synthetic amino acids, including glycine and D or L optical isomers, as well as amino acid analogs and peptide mimetics. Peptides of three or more amino acids are generally referred to as oligopeptides (if the peptide chain is short). If the peptide chain is long (e.g., greater than about 10 amino acids), the peptide is generally referred to as a polypeptide or protein. Although the term "protein" encompasses the term "polypeptide", a "polypeptide" can be a protein less than full length.

[0256] As used herein, "LAMP protein" or "LAMP polypeptide" refers to any of the mammalian lysosomal-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 (e.g., Figure 3-10 Unless otherwise expressly stated, LAMP-1, LAMP2, LAMP-3, LIMP 2, Macrosialin, Endolyn, LAMP5 or LIMBIC as used herein refers to Figure 3-10 The human proteins and their allelic variants indicated in .

[0257] The LAMP "homologous domain" used in this article contains at least Figure 3-10 4 evenly spaced cysteine ​​residues as shown in FIG. Figure 3-10 As shown in, these cysteine ​​residues are labeled 1, 2, 3 and 4 in each homology domain (and five cysteines are identified in LIMP-2 and Macrosialin, six cysteines are identified in LIMBIC, and eight cysteines are identified in Endolyn), and are defined herein as "cysteine ​​conservative fragments". Additional amino acids may be included at the N-terminal end and / or C-terminal end of the cysteine ​​conservative fragment to produce a complete homology domain up to and including the LAMP protein. These additionally added amino acids may be derived from the homology domain from which the cysteine ​​conservative fragment is derived or other LAMP protein homology domains. Therefore, the LAMP homology domain used herein comprises a cysteine ​​conservative fragment and / or is composed of a cysteine ​​conservative fragment. At least two LAMP homology domains constitute the luminal domain of LAMP-1, LAMP-2, LAMP-3 or Endolyn.

[0258] "Expression" as used herein refers to the process in 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 mRNA transcribed from the genomic DNA.

[0259] As used herein, "under transcriptional control" or "operably linked" refers to the expression (e.g., transcription or translation) of a polynucleotide sequence controlled by the appropriate juxtaposition of expression control elements and coding sequences. In one aspect, a sequence is "operably linked" to an expression control sequence when the expression control sequence controls and regulates the transcription of a nucleic acid sequence. In another instance, the term "operably linked" refers to the connection of a peptide, polypeptide, or protein (e.g., an epitope or antigen) to a signal sequence (e.g., a secretory signal sequence) to allow the peptide, polypeptide, or protein to be secreted from a host cell.

[0260] As used herein, "signal sequence" or "signal peptide" means an endoplasmic reticulum translocation sequence. This sequence encodes a signal peptide that communicates with the cell to direct a polypeptide attached thereto (e.g., by a chemical bond) to the endoplasmic reticulum vesicular compartment, into an exocytic / endocytic organelle, delivered to a cellular vesicular compartment, to the cell surface, or to secrete the polypeptide. During maturation of the polypeptide, this signal sequence is sometimes sheared off by the cell. Signal sequences can be found accompanying various proteins native to prokaryotes and eukaryotes. A "secretion signal sequence" refers to a signal sequence that causes a polypeptide attached thereto to be secreted by a cell.

[0261] As used herein, "coding sequence" refers to a sequence that is transcribed and translated into a peptide, polypeptide or protein when placed under the control of a suitable expression control sequence. The boundaries of the coding sequence are determined by the start codon at the 5' (amino) end and the translation stop codon at the 3' (carboxyl) end. The coding sequence may include, but is 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' of the coding sequence.

[0262] As used herein, two coding sequences "correspond to" each other if they, or their complements, encode the same amino acid sequence.

[0263] As used herein, "translocation" refers to the movement or progression of a polypeptide encoded by a bicistronic LAMP construct through an organelle or compartment by way of the rough endoplasmic reticulum to an endosomal / lysosomal compartment or related organelle where antigen processing and binding to MHC II occurs.

[0264] The terms "antigen" or "antigen of interest" as used herein encompass any polypeptide sequence encoded by a polynucleotide sequence cloned into a nucleic acid molecule encoding a bicistronic LAMP construct, which is used to elicit an innate or adaptive immune response. "Antigen" encompasses both a single antigen and multiple antigenic sequences (derived from the same or different proteins). In some cases, an "antigen" provides a specific "epitope" or antibody recognition site. In some cases, an "antigen" is a "target antigen," meaning that it represents a specific protein expressed by diseased cells, 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 to produce a "LAMP-antigen construct." Different arrangements of LAMP-antigen constructs that can be used herein are described in. Figure 1 Described as ILC-1-ILC-6.

[0265] As used herein, "bicistronic LAMP construct", "bicistronic LAMP construct comprising an antigen", "bicistronic LAMP construct comprising a target antigen", "bicistronic LAMP construct comprising an antigen of interest" and "bicistronic LAMP-antigen construct" can be used interchangeably and refer to a nucleic acid construct encoding or expressing two polypeptides (i.e., a first polypeptide and a second polypeptide comprising a LAMP-antigen construct), wherein the second polypeptide may encode an IREG polypeptide or a second antigen in certain embodiments. In some cases, the bicistronic LAMP construct nucleic acid molecule herein may be a DNA molecule such as a DNA vector such as a DNA viral vector, and in other cases it may be an RNA molecule, including a self-amplifying RNA vector (also referred to as a self-replicating RNA vector). In other cases, depending on the context, a "bicistronic LAMP construct" refers to a polypeptide co-expressed from a nucleic acid construct.

[0266] As used herein, "immune response element" or "immune response enhancing gene" or "IREG" refers broadly to a gene encoding a protein that can enhance an immune response, such as a humoral and / or cellular immune response, in a subject. In some cases, the abbreviation IREG may also be used to refer to a polypeptide encoded by such a gene or an extracellular domain of such a protein, or a fusion molecule comprising such a protein or extracellular domain. 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.

[0267] As used herein, "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.

[0268] 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 to introduce it. The introduced nucleic acid molecule can be stably or transiently maintained in the host cell. Stable maintenance generally requires that the introduced nucleic acid molecule encoding the bicistronic LAMP construct either contain an origin of replication compatible with the host cell, or is integrated into a replicon of the host cell, such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear chromosome or a mitochondrial chromosome.

[0269] As used herein, "viral bicistronic LAMP construct" refers to a virus or virus particle comprising a nucleic acid molecule comprising a bicistronic LAMP construct to be delivered to a host cell in vivo, ex vivo or in vitro. Examples of viral bicistronic LAMP constructs include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and the like. In aspects where gene transfer is mediated by an adenoviral vector, the viral bicistronic LAMP construct comprises an adenoviral genome or portion thereof, and a selected non-adenoviral gene associated with an adenoviral capsid protein.

[0270] As used herein, "adenovirus-mediated gene transfer" or "adenovirus transduction" refers to the process of transferring a nucleic acid molecule encoding a bicistronic LAMP construct into a host cell using an adenovirus that enters the host cell. Preferably, the nucleic acid molecule is capable of replicating and / or integrating and transcribed in the cell.

[0271] As used herein, an "adenoviral particle" is a single adenoviral particle consisting of an outer capsid and a nucleic acid molecule encoding a bicistronic LAMP construct, wherein the capsid is further composed of an adenoviral envelope protein. The adenoviral envelope protein can be modified to include a fusion polypeptide containing a polypeptide ligand covalently attached to a viral protein, for example, to target the adenoviral particle to a specific cell and / or tissue type.

[0272] The term "administering" or "immunization" or "injecting" a nucleic acid molecule encoding a bicistronic LAMP construct as used herein refers to transducing, transfecting, microinjecting, electroporating or shooting cells with the nucleic acid molecule. In some aspects, the nucleic acid molecule encoding the bicistronic LAMP construct is introduced into the target cell by contacting the target cell with the delivery cell (e.g., by cell fusion or by cleaving the delivery cell when the delivery cell is close to the target cell).

[0273] As used herein, the terms "treatment" and the like broadly refer to the improvement or amelioration of a disease or condition in a subject, such as the improvement or amelioration of at least one symptom or marker associated with the disease or condition, such as in the case of a tumor, such as a reduction in tumor size or a change in a biochemical marker associated with the tumor or a alleviation of disease symptoms. In the case of a disease such as cancer or an infectious disease, treatment also refers to the alleviation of at least one symptom of the disease or condition. For example, treatment also refers to preventing the onset of a disease or condition or slowing its onset, or preventing or alleviating one or more symptoms after onset (e.g., including the development of asymptomatic disease compared to symptomatic disease). Treatment also refers to use in immunization or vaccination, such as inducing an immune response in a subject, which can, for example, prevent the onset of symptoms, reduce the severity of symptoms, or improve at least one existing symptom of a disease or condition in a subject.

[0274] As used herein, the phrase "target enhancement" or simply "enhancement" of an immune response describes the use of a nucleic acid molecule encoding a "LAMP-antigen construct" comprising: (1) a target antigen associated with a disease or condition to be treated in a LAMP fusion polypeptide, and (2) another secreted polypeptide encoding a protein intended to enhance the immune response (i.e., an IREG protein). In general, such target enhancement allows for the simultaneous delivery of a target antigen and secreted IREG. In some embodiments, this approach can improve both humoral and cellular immune responses.

[0275] As used herein, the term "secretion" refers to processes and pathways within a cell that result in the transport of a peptide, polypeptide, or protein through the cell wall, thereby releasing the peptide, polypeptide, or protein into the extracellular environment and possibly, for example, into the circulation of a subject. Peptides, polypeptides, or proteins destined for the extracellular environment (i.e., to be secreted) are typically provided with a secretory signal sequence, usually at the N-terminus, which directs the ribosomes translating the peptide, polypeptide, or protein to the rough endoplasmic reticulum (rough ER), from where the newly manufactured peptide, polypeptide, or protein can be incorporated into small transport or secretory vesicles, which transport the peptide, polypeptide, and protein to the cell surface for release.

[0276] As used herein, "hybridization" refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized by hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding can occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner. The complex can include two chains that form a duplex structure, three or more chains that form a multi-chain complex, a single self-hybridizing chain, or any combination of these situations. The hybridization reaction can constitute a step in the initiation of a more extensive process such as a PCR reaction or the digestion of a polynucleotide by a ribozyme.

[0277] As used herein, a polynucleotide or polynucleotide region (or polypeptide or polypeptide region) having a certain 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%) of "sequence identity" to another sequence means that when the two sequences are compared for maximum alignment using software programs routine in the art, that percentage of bases (or amino acids) are the same.

[0278] When 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 within the specified length of the DNA sequence, the two sequences are "substantially homologous" or "substantially similar". Similarly, when 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 polypeptide match within the specified length of the polypeptide sequence, the two polypeptide sequences are "substantially homologous" or "substantially similar". Substantially homologous sequences can be identified by comparing sequences using standard software available in sequence databases. In Southern hybridization experiments, substantially homologous nucleic acid sequences can also be identified under stringent conditions, such as those defined for the particular system. It is within the ability of those skilled in the art to define suitable hybridization conditions. For example, stringent conditions can be: hybridization at 42°C in 5xSSC and 50% formamide, and washing at 60°C in 0.1xSSC and 0.1% sodium dodecyl sulfate. Other 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 washing solution of about 1xSSC, 0.1xSSC or deionized water. In general, the hybridization incubation time is 5 minutes to 24 hours, 1, 2 or more washing steps are used, and the washing incubation time is about 1, 2 or 15 minutes. SSC is 0.15M NaCl and 15mM citrate buffer. It will be appreciated that the equivalent of SSC using other buffer systems may be employed. Similarity may be verified by sequencing, but preferably also by function (e.g., ability to transport to endosomal compartments, etc.) using assays appropriate for the particular domain in question.

[0279] 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, which sequences may or may not have a common evolutionary origin (see Reeck et al., supra). Sequence identity can be determined using any of a variety of publicly available sequence comparison algorithms, such as BLAST, FASTA, DNA Strider, GCG (Genetics Computer Group, GCG Software Package Program Manual, 7th Edition, Madison, Wisconsin), and the like.

[0280] In order to determine the percent identity between two amino acid sequences or two nucleic acid molecules, the sequences are compared for the purpose of optimal comparison. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., the total number x 100 of the number of percent identity=identical positions / positions (e.g., overlapping positions)). In one embodiment, the two sequences have or have approximately the same length. The percent identity between the two sequences can be determined using similar techniques as described below, allowing or not allowing spaces. When calculating the percent identity of the sequences, exact matches are usually counted.

[0281] The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. A non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 1990, 87:2264, and modified as described in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 1993, 90:5873-5877. This 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. In order to obtain gapped alignments for comparison purposes, gapped BLAST as described in Altschul et al., Nucleic Acids Res. 1997, 25:3389 can be used. Alternatively, PSI-Blast can be used to perform an iterative search to detect distance relationships between molecules. See Altschul et al., (1997), supra. When using BLAST, gapped BLAST, and PSI-Blast programs, the default parameters of the corresponding programs (e.g., XBLAST and NBLAST) can be used. See ncbi.nlm.nih.gov / BLAST / on the World Wide Web.

[0282] Another non-limiting example of a mathematical algorithm used for the comparison of sequences is the algorithm of Myers and Miller, CABIOS 1988; 4: 11-17. This algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When using 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.

[0283] 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), which has been incorporated into the GAP program in the GCG software package (Accelrys, Burlington, MA; available on the World Wide Web at accelrys.com), using a Blossum 62 matrix or a PAM250 matrix, a gap weight of 16, 14, 12, 10, 8, 6, or 4, and a length weight 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 the NWSgapdna.CMP matrix, a gap weight of 40, 50, 60, 70, or 80, and a length weight of 1, 2, 3, 4, 5, or 6. A particular set of parameters (and a set of parameters that can be used if the practitioner is unsure which parameters should be applied to determine whether a molecule is a sequence identity or homology restriction of the present disclosure) is to use the Blossum 62 scoring matrix, with a gap open penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0284] Another non-limiting example of how the percent identity can be determined is by using a software program, such as the program described in Current Protocols In Molecular Biology (FM Ausubel et al., ed., 1987), Supplement 30, Section 7.7.18, Table 7.7.1. Preferably, the alignment is performed using default parameters. The alignment program is BLAST, using default parameters. Specifically, the programs are BLASTN and BLASTP, using the following default parameters: genetic code = standard; filter = none; chain = two; cutoff = 60; expectation = 10; matrix = BLOSUM62; description = 50 sequences; sorting mode = high score; database = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translation + SwissProtein + SPupdate + PIR. Detailed information on these programs can be found at the following Internet addresses: http: / / www.ncbi.nlm.nih.gov / cgi-bin / BLAST .

[0285] Statistical analysis of the properties described herein can be performed by standard tests such as t-test, ANOVA or chi-square test. Typically, statistical significance will be measured to a level of p=0.05 (5%), more preferably p=0.01, p=0.001, p=0.0001, p=0.000001.

[0286] "Conservatively modified variants" of domain sequences can also be provided. For a particular nucleic acid sequence, conservatively modified variants refer to those nucleic acids that encode the same or substantially the same amino acid sequence, or, where the nucleic acid does not encode an amino acid sequence, to substantially the same sequence. 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 by mixed bases 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).

[0287] 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 equivalent to (e.g., not significantly different from) the biological activity of the wild-type protein, as measured using an assay suitable for detecting said activity.

[0288] As used herein, "in vivo nucleic acid delivery, nucleic acid transfer, nucleic acid therapy" and the like refer to the direct introduction of a nucleic acid molecule encoding a bicistronic LAMP construct described herein into the body of a subject (e.g., a human or non-human mammal), thereby introducing the nucleic acid molecule into the cells of such an organism in vivo.

[0289] The term "in situ" as used herein refers to a type of in vivo nucleic acid delivery in which the nucleic acid molecule encoding the bicistronic LAMP construct is brought into proximity with the target cell (e.g., the nucleic acid is not systemically administered). 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., a tissue such as a tumor or myocardium), contacting the nucleic acid molecule with cells or tissues through an open surgical area, or delivering the nucleic acid molecule to a site using a medical access device such as a catheter.

[0290] As used herein, the term "isolated" or "purified" refers to separation from cells and other components with which the polynucleotide, peptide, polypeptide, protein, antibody or fragment thereof is normally associated in nature (or substantially free of said components). For example, for the isolated nucleic acid molecules encoding the bicistronic LAMP constructs described herein, the isolated polynucleotide is a polynucleotide separated from the 5' and 3' sequences that are normally associated with it in the chromosome. As will be apparent to those skilled in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody or fragment thereof does not require "isolation" to distinguish it from its naturally occurring 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 does not contain the components with which they are associated in nature.

[0291] As used herein, "target cell" or "receptor cell" refers to an individual cell or cell that is intended to be or has become a receptor for a nucleic acid molecule encoding a bicistronic LAMP construct as described herein. The term is also intended to include the progeny of a single cell, and the progeny may not necessarily be identical (in morphology or in genome 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., in a tissue), or may circulate in the body of an organism.

[0292] Unless otherwise specifically stated, "subject" as used herein is a human. In such other cases, the subject may be a mammal. Mammals include, but are not limited to, rodents, apes, humans, farm animals, sports 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 monkey) or ape (e.g., gorilla, chimpanzee, orangutan, gibbon). In some embodiments, non-human mammals may be used, particularly mammals (e.g., rodents, primates, pigs, dogs, or rabbits) that are commonly used as models to demonstrate therapeutic efficacy in humans.

[0293] The terms "cancer", "neoplasm" and "tumor" are interchangeable and used in the singular or plural form to refer to cells that have undergone malignant transformation and are pathological to the host organism. Primary cancer cell transformation makes it pathological to the host organism. Primary cancer cells (i.e., cells obtained from the vicinity of the site of malignant transformation) can be easily distinguished from non-cancerous cells by recognized techniques, particularly histological examination. The definition of cancer cells used herein includes not only primary cancer cells, but also any cells derived from cancer cell ancestors. This includes metastatic cancer cells, as well as in vitro cultures and cell lines derived from cancer cells. When referring to cancer types that usually manifest as solid tumors, "clinically detectable" tumors are tumors that can be detected, for example, by procedures such as CAT scans, MR imaging, X-rays, ultrasound or palpation on the basis of tumor masses, and / or tumors that can be detected due to the expression of one or more cancer-specific antigens in samples that can be obtained from patients.

[0294] In some embodiments, the cancer (including all stages of advancement including hyperplasia) is adenocarcinoma, sarcoma, skin cancer, melanoma, 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 cancer, pancreatic ductal adenocarcinoma (PDA), kidney cancer, stomach cancer, renal cancer, multiple myeloma, or brain cancer.

[0295] The nucleic acid molecules encoding the bicistronic LAMP constructs described herein can also be used to treat allergies, such as food allergies (e.g., peanut allergens, such as Ara H1, H2 and / or H3) or environmental allergens such as pollen (tree pollen, such as CRYJ1 or CRY J2), dog dander, cat saliva or dust mites.

[0296] In some cases, the bicistronic LAMP construct can include an antigen that can be used to treat an infectious disease, such as a viral or bacterial disease. In one example, the bicistronic LAMP construct can be used to treat a coronavirus infection, such as an infection from Covid-19.

[0297] Other diseases and / or conditions that may be treated with the bicistronic LAMP constructs described herein include, for example, infectious diseases and diabetes.

[0298] The term "pharmaceutically acceptable carrier" as used herein encompasses any standard pharmaceutical carrier, such as phosphate buffered saline solution, water and emulsions such as oil / water or water / oil emulsions, and various types of wetting agents. Compositions comprising nucleic acid molecules encoding bicistronic LAMP constructs may also include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see Martin Remington's Pharm. Sci., 15th edition (Mack Publ. Co., Easton (1975)).

[0299] When the nucleic acid molecule encoding the bicistronic LAMP construct has been introduced into a cell, the cell has been "transformed", "transduced" or "transfected" by this nucleic acid molecule. The transforming DNA may be integrated (covalently linked) with the chromosomal DNA constituting the cell genome, or it may not be integrated. For example, in prokaryotes, yeast and mammalian cells, the nucleic acid molecule encoding the bicistronic LAMP construct may be maintained on an episomal element such as a plasmid. In eukaryotic cells, stably transformed cells are cells in which the nucleic acid molecule encoding the bicistronic LAMP construct has been integrated into the chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish a cell line or clone consisting of a daughter cell colony containing the nucleic acid molecule. A "clone" is a colony 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 stably grow for many generations (e.g., at least 10 generations) in vitro.

[0300] As used herein, an "effective amount" or "therapeutically effective amount" is an amount sufficient to affect beneficial or desired results, such as treating a subject or inducing an immune response in a subject.

[0301] "Antibody" is any immunoglobulin that binds to a specific antigen, including antibodies and fragments thereof. The term encompasses polyclonal, monoclonal and chimeric antibodies (e.g., bispecific antibodies). "Antibody binding site" is the structural portion of an antibody molecule, consisting of heavy and light chain variable regions and hypervariable regions that specifically bind to an antigen. Exemplary antibody molecules are complete immunoglobulin molecules, substantially complete immunoglobulin molecules, and those portions of immunoglobulin molecules that contain a fill, including Fab, Fab', F(ab')2, and F(v) portions. Therefore, the term antibody includes not only whole antibody molecules, but also 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 (scF), Fab fragments, Fab' fragments, F(ab')2, disulfide-linked Fv (sdFv), Fv, and fragments comprising or consisting of 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 connected to the VH domain of an antibody. See Carter (2006) Nature Rev. Immunol. 6:243.

[0302] In addition, antibodies that can be produced using the nucleic acid molecules encoding the bicistronic LAMP constructs described herein include, but are not limited to, monoclonal, multispecific, bispecific, human, humanized, mouse 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 antibodies of the present disclosure), domain antibodies, and epitope-binding fragments of any of the above. The immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0303] As used herein, "human" antibodies include antibodies having the amino acid sequence of a human immunoglobulin, and include antibodies isolated from human immunoglobulin libraries and xenogeneic mice or other organisms that have been genetically engineered to produce human antibodies. The nucleic acid molecules encoding the bicistronic LAMP constructs described herein can be used in combination with known techniques for producing human antibodies and human monoclonal antibodies as described in the exemplary schemes, see, e.g., PCT Publications WO 98 / 24893, WO 92 / 01047, WO 96 / 34096, WO 96 / 33735, European Patent No. 0 598 877, U.S. Pat. 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).

[0304] Human antibodies or "humanized" chimeric monoclonal antibodies can be produced using a combination of nucleic acid molecules encoding the bicistronic LAMP constructs described herein or techniques 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., U.S. Patent No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671; Boulianne et al., Nature 312: 643 (1984); Neuberger et al., Nature 314: 268 (1985).

[0305] The antibody that can be produced using the nucleic acid molecules of the coding bicistronic LAMP construct described herein can be monovalent, divalent, trivalent or multivalent. For example, monovalent scFv can be polymerized by chemical methods or by being combined with another protein or substance. The scFv fused with hexa-histidine tag or Flag tag can be polymerized using Ni-NTA agarose (Qiagen) or using anti-Flag antibody (Stratagene, Inc.). In addition, the nucleic acid molecules of the coding bicistronic LAMP construct can be used to produce monospecific, bispecific, trispecific or larger multispecific for the coded antigen included in the bicistronic LAMP construct. See, e.g., PCT Publications WO 93 / 17715, WO 92 / 08802, WO 91 / 00360, WO 92 / 05793; Tutt et al., J. Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893, 4,714,681, 4,925,648, 5,573,920, 5,601,819; Kostelny et al., J. Immunol. 148:1547-1553 (1992).

[0306] An "epitope" is a structure that is specifically recognized or specifically bound by a component of the immune system, typically consisting of a short peptide sequence or oligosaccharide. T cell epitopes are typically shown to be linear oligopeptides. If two epitopes can be specifically bound by the same antibody, they correspond to each other. If both epitopes are able to 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 of the other epitope (e.g., the binding of the other epitope is less than about 30%, preferably less than about 20%, more preferably less than about 10%, 5%, 1% or about 0.1%), then the two epitopes correspond to each other. One of ordinary skill in the art will appreciate that multiple epitopes can constitute an antigen.

[0307] As used herein, the term "antigen presenting cell" includes any cell that presents an antigen bound to a major histocompatibility complex molecule or portion thereof or one or more non-classical MHC molecules or portions thereof on its surface. Examples of suitable APCs are discussed in detail below and include, but are not limited to, intact cells such as macrophages, dendritic cells, B cells, mixed APCs, and foster antigen presenting cells.

[0308] As used herein, "engineered antigen presenting cells" refer to antigen presenting cells having non-natural molecular moieties on their surface. For example, such cells may not naturally have costimulators on their surface, or may have additional artificial costimulators in addition to natural costimulators on their surface, or may express non-natural class II molecules on their surface. In some embodiments, the engineered antigen presenting cells have antigens expressed from the bicistronic LAMP construct on their surface.

[0309] "Immune effector cell" as used herein 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 from tumors, inflammatory or other infiltrates.

[0310] As used herein, "partially human" refers to a nucleic acid having sequences from both humans and non-human vertebrates. In the context of partially human sequences, the partially human nucleic acid has the sequence of a human immunoglobulin coding region and a sequence of a non-coding sequence based on an endogenous immunoglobulin region of the non-human vertebrate. When used to refer to an endogenous non-coding sequence from a non-human vertebrate, the term "based on" refers to a sequence that corresponds to the non-coding sequence and has a relatively high degree of homology with a non-coding sequence of an endogenous locus of the host vertebrate (e.g., a non-human vertebrate from which an ES cell is derived). Preferably, the non-coding sequence has at least 80%, more preferably 90% homology with a corresponding non-coding sequence found in an endogenous locus of a non-human vertebrate host cell into which a partially human molecule comprising the non-coding sequence has been introduced.

[0311] The term "immunoglobulin variable region" as used herein refers to a nucleotide sequence encoding all or part of the variable region of an antibody molecule or all or part of a regulatory nucleotide sequence that controls the expression of an antibody molecule. The heavy chain region of an immunoglobulin may include, but is not limited to, all or part of the V, D, J, and switch regions, including introns. The light chain region of an immunoglobulin may include, but is not limited to, V and J regions associated with or adjacent to the light chain constant region gene, their upstream flanking sequences, and introns.

[0312] "Transgenic animals" refer to non-human animals, usually mammals, that have exogenous nucleic acid sequences present as extrachromosomal elements in some of their cells or stably integrated into their germline DNA (i.e., in the genomic sequence of most or all of their cells). In producing transgenic animals containing human sequences, some human nucleic acids are introduced into the germline of such transgenic animals by genetic manipulation of, for example, embryos or embryonic stem cells of the host animal according to methods well known in the art.

[0313] "Vector" includes plasmids and viruses, and any DNA or RNA molecule, whether self-replicating or not, that can be used to transform or transfect cells.

[0314] "Genetic modification" as used herein refers to any addition, deletion or destruction of normal nucleotides of a cell. Methods recognized in the art include viral-mediated gene transfer, liposome-mediated transfer, transformation, transfection and transduction, such as viral-mediated gene transfer, such as using nucleic acid molecules encoding bicistronic LAMP constructs based on DNA viruses (e.g., adenovirus, adeno-associated virus and herpes virus) and retroviral-based vectors.

[0315] In certain aspects, unless otherwise indicated, the practice of the present disclosure employs conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are fully explained in the literature. See, e.g., Maniatis, Fritsch and Sambrook, Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, Vols. I and II (DN Glover, ed., 1985); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Nucleic Acid Hybridization (BD Hames and SJ Higgins, eds., 1985); Transcription and Translation (BD Hames and 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).

[0316] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing devices, formulations and methods that may be used in conjunction with the disclosure described herein.

[0317] B. Exemplary LAMP-Antigen Fusion Polypeptides

[0318] The following are representative implementations:

[0319] A kind of isolated nucleic acid, it encodes a bicistronic LAMP construct, i.e., encodes a LAMP-antigen fusion protein (LAMP construct) and an immunopotentiating protein, so that both are expressed in a host cell or a subject. In some cases, for example, if a third polypeptide is also expressed, the nucleic acid herein can encode a polycistronic construct.

[0320] In some cases, the antigen is a target antigen for a specific disease or condition. In some cases, the immune enhancing protein is a polypeptide or polypeptide domain such as an extracellular domain from IREG, optionally fused to another molecule such as an Fc domain of an immunoglobulin. In some cases, the immune enhancing protein can be secreted and is therefore operably linked to a secretion signal sequence. In some cases, the LAMP-antigen fusion protein can have a Figure 1 In some cases, the LAMP-antigen construct comprises an antigen placed between two homology domains of the luminal domain of the LAMP protein. In other cases, the antigen is placed before the N-terminus of the LAMP homology domain, or after the C-terminus of the LAMP homology domain but before the LAMP transmembrane domain. When used in this article, the LAMP protein can be selected from LAMP-1, LAMP2, LAMP-3, LIMP 2, Macrosialin, Endolyn, LAMP5 or LIMBIC. In other 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 with any one of amino acid SEQ ID NOs: 1-113. In some cases, the LAMP protein is LAMP-1.

[0321] In some cases, in addition to at least two homologous domains of LAMP, the LAMP component also includes a transmembrane domain of LAMP or a heterologous protein, and an optional signal sequence of LAMP or a heterologous protein, and in some cases also includes a cytoplasmic domain of LAMB. In some such cases, the LAMP protein is LAMP-1.

[0322] Specifically, LAMP-1 deduced from a cDNA clone (Chen et al., J. Biol. Chem. 263:8754, 1988) consists of a polypeptide core of approximately 382 amino acids, with a large (346 residues) intraluminal amino-terminal domain, followed by a 24-residue hydrophobic transmembrane region and a short (12 residues) carboxyl-terminal cytoplasmic tail. Figure 2A and Figure 2B The endodomain is heavily glycosylated, substituted with about 20 asparagine-linked complex oligosaccharides, and consists of two "homeodomains" of about 160 residues separated by a 22-residue "hinge" region rich in proline / serine. Each of these "homeodomains" contains four evenly spaced cysteine ​​residues, disulfide-bonded to form four 36-38 residue loops, symmetrically placed in the two halves of the endodomain (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 The conserved domains among LAMP-1, LAMP-2, LAMP-3, Endolyn, LIMBIC, LAMP5 or Macrosialin are schematically shown.

[0323] Previously reported LAMP constructs contained the following elements in this specific arrangement: (a) the entire luminal domain of the LAMP-1 protein, the antigen, and then the entire transmembrane / cytoplasmic tail of the LAMP-1 protein; or (b) the antigen and the entire transmembrane / cytoplasmic tail of the LAMP-1 protein.

[0324] 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 antigenic sequence to lysosomes / endosomes and will be referred to as "complete LAMP constructs" when compared to the improved LAMP constructs ILC-1-ILC-6 described herein, e.g. Figure 1 As shown in . The bicistronic LAMP constructs described herein do not include the complete LAMP construct. Instead, the bicistronic LAMP constructs described herein may comprise 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, e.g. Figure 1However, some bicistronic LAMP constructs contain at least one antigen fused between the first homology domain of the LAMP protein and the second homology domain of the LAMP protein (or at least between two cysteine ​​conserved fragments) (see, e.g. Figure 1 For example, the at least one antigen may be placed in the LAMP hinge region or may replace the LAMP hinge region. In some embodiments, the construct further comprises a transmembrane domain of a LAMP protein and / or a cytoplasmic tail region of a LAMP protein.

[0325] Specifically, in some embodiments, the bicistronic LAMP construct comprises two homologous domains (e.g. Figure 1 In some embodiments, these constructs further comprise a transmembrane domain of a LAMP protein and / or a cytoplasmic tail of a LAMP protein. In other embodiments, when the antigen contains a transmembrane domain, the transmembrane domain of a LAMP protein and / or the cytoplasmic tail of a LAMP protein is optional. In 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 comprising two homologous domains, a LAMP hinge domain may also be included.

[0326] The LAMP-antigen portion of the bicistronic LAMP construct comprises an ILC-4 structure (eg Figure 1 ), for example, wherein the first polynucleotide sequence of the LAMP construct comprises or encodes a polypeptide comprising two homologous domains of the luminal domain of a LAMP protein and an antigenic domain heterologous to the LAMP protein, wherein the antigenic domain is placed between the two homologous domains, the two LAMP protein homologous domains can be selected from Figure 3 The amino acid sequences of homology domain 1 and homology domain 2 shown in , which are derived from SEQ ID NO: 1-113 or amino acid sequences having, for example, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with those sequences. Thus, for example, where the LAMP in such a construct is LAMP-1, native human LAMP-1 homology domain 1 and homology domain 2 may surround the antigenic domain of the LAMP-antigen construct. In some cases, the homology domain may, for example, comprise amino acid residues 29-194 and 228-381 of SEQ ID NO: 1, or amino acid sequences having at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with those sequences, or Figure 2A and3 , or residues 29-195 of SEQ ID NOs: 198 and 202. Where the construct is a polynucleotide, the construct may further encode a signal sequence before the start of the LAMP homology domain 1 coding sequence, e.g., residues 1-28 of SEQ ID NO: 1 or residues 29-195 of SEQ ID NOs: 198 and 202. Figure 2A and Figure 3 In some cases, a heterologous (i.e., non-LAMP) signal sequence may also be used. Thus, in some embodiments, the first homology 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 with residues 29 to the C-terminus of SEQ ID NO: 198 or residues 29 to 194 of SEQ ID NO: 1. In some cases, a LAMP signal sequence may also be included, such as residues 1 to 28 of SEQ ID NO: 198 or SEQ ID NO: 1. In some cases, the second homology 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 with residues 228 to 381 of SEQ ID NO: 202 or SEQ ID NO: 1. In some cases, for example, Figure 2A and 3 The LAMP transmembrane domain depicted in SEQ ID NO: 1, i.e., comprising residues 383-405 of SEQ ID NO: 1 or comprising another natural LAMP transmembrane domain sequence. In some cases, the cytoplasmic tail region of LAMP may also be included, e.g., comprising residues 406-417 of SEQ ID NO: 1, or as Figure 2A and 3 as depicted in, or comprising a native LAMP cytoplasmic tail sequence.

[0327] In some other embodiments, the bicistronic LAMP construct comprises at least one antigen fused to the C-terminus of a single homology domain of a LAMP protein or a single cysteine ​​conserved fragment of a LAMP protein. See, e.g. Figure 1 In some embodiments, these constructs further comprise a transmembrane domain of a LAMP protein and / or a cytoplasmic tail of a LAMP protein. In other embodiments, when the antigen contains a transmembrane domain, the transmembrane domain of the LAMP protein and / or the cytoplasmic tail of the LAMP protein is optional.

[0328] The above bicistronic LAMP constructs can be generated using the domains defined in the figure. For example, specifically contemplated are domains such as those contained in Figure 1 The domains in the bicistronic LAMP constructs shown in can be derived from sequences derived from orthologous sequences. See e.g. Figure 3-10 . Clearly envisioned Figure 2A and Figure 2B The equivalent domains defined in can be used to Figure 1 The vector backbone shown in generates the bicistronic LAMP construct of orthologous sequences. In addition, Figure 3-10 The orthologous sequences shown in are representative of sequences that can be used to generate the domains. It is well within the capabilities of those skilled in the art to identify other orthologous sequences and / or isoforms and to compare them with Figure 3-10 Therefore, by using Figure 3-10 The comparison shown in Figure 2A and Figure 2B Using the equivalent boundaries of human LAMP proteins defined in , one can generate the bicistronic LAMP constructs described herein.

[0329] As will be well understood by the skilled artisan, 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 depending 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 start and end amino acids of the domain may be adjusted by at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, such as Figure 2A Those boundaries defined in .

[0330] As is known in the art, for cloning purposes, each of the bicistronic LAMP constructs described herein may additionally comprise a signal sequence and / or additional amino acids between each domain. In addition, the LAMP homology domain, LAMP endodomain, LAMP transmembrane domain and / or LAMP cytoplasmic tail domain may be derived from the same LAMP protein (e.g., human LAMP-1) or different LAMP proteins (e.g., the endodomain 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).

[0331] The polypeptide variants of the described LAMP constructs are envisioned. For example, polypeptides 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% identity with any bicistronic LAMP construct described herein and the polynucleotides encoding these variants. The variants of the bicistronic LAMP constructs retain the ability to play a role by targeting the antigenic sequence to the lysosome. For example, the modified luminal sequence must retain the ability to transport both membrane and non-membrane antigenic substances to the endosomal compartment, and compared with the original domain sequence, its efficacy is at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100%, and the efficacy promptly causes the cells comprising its chimeric sequence to present antigens fully to produce the efficacy of an immune response. In one aspect, sequences containing suitable transport signals can be identified by constructing a bicistronic LAMP construct containing a well-characterized antigenic domain of ovalbumin, a transmembrane domain, and a cytoplasmic domain of a protein containing a putative lysosomal / endosomal targeting signal. Targeting efficiency can be measured by determining the ability of antigen presenting cells expressing the bicistronic LAMP construct to stimulate HA epitope-specific class II MHC restricted T cells (see, e.g., Example 5 of U.S. Patent No. 5,633,234).

[0332] Some embodiments of the present disclosure include polynucleotides encoding any of the described bicistronic LAMP constructs, and polynucleotides having 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. The variants of the bicistronic LAMP construct retain the ability to act by targeting the antigenic sequence to the lysosome. For example, the modified luminal sequence must retain the ability to transport both membrane and non-membrane antigenic substances to the endosomal compartment, and its efficacy is at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% compared to the original domain sequence, which efficacy results in the cell containing its chimeric sequence fully presenting the antigen to produce an immune response. In one aspect, sequences containing suitable transport signals can be identified by constructing a bicistronic LAMP construct containing a well-characterized antigenic domain of ovalbumin, a transmembrane domain, and a cytoplasmic domain of a protein containing a putative lysosomal / endosomal targeting signal. Targeting efficiency can be measured by determining the ability of antigen presenting cells expressing the bicistronic LAMP construct to stimulate HA epitope-specific class II MHC restricted T cells (see, e.g., Example 5 of U.S. Patent No. 5,633,234).

[0333] C. Construction of exemplary bicistronic LAMP constructs

[0334] The bicistronic LAMP construct herein can be for example constructed from an isolated nucleotide sequence, wherein in the nucleic acid, behind or in front of the promoter / enhancer of the LAMP-antigen construct and the coding sequence is the second promoter / enhancer and the coding sequence of the second polypeptide. In some cases, the second polypeptide can be a polypeptide intended to enhance immune response, i.e., a protein or the extracellular domain of protein expressed from an immune response enhancing gene (IREG). In some cases, the second polypeptide can be secreted, so the nucleotides can include a suitable secretion signal sequence of the second polypeptide, which can be included in the coding sequence of the polypeptide, or can be from different proteins. In some cases, the second polypeptide is a fusion protein, for example, an extracellular domain or a complete IREG polypeptide fused with another polypeptide sequence such as the Fc domain of an immunoglobulin, another antigen, etc.

[0335] As described below, the antigen in the LAMP-antigen construct used in the bicistronic LAMP construct can be a target antigen for infectious diseases, such as a viral spike protein, or it can also be a cancer antigen, such as a polypeptide overexpressed in certain tumors or tumor cells.

[0336] For example, as described in the examples below, some bicistronic LAMP proteins are made using spike proteins or spike protein subunits from SARS-Co-V2 viruses and using cancer antigens such as HER2 extracellular domains or using NY-ESO1 or CD161. Therefore, in some embodiments, the LAMP-antigen construct comprises a LAMP fused to a cancer antigen or a viral spike protein antigen. In some cases, the coding region of such antigens can be combined with an IREG coding region. 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 secretion signal sequence coding region so that the second polypeptide is secreted when expressed.

[0337] 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 Covid vaccine; SEQ ID NO: 230) and (10) ITI-COVID-19 bicistronic vaccine (ITI-bicistronic-S1-LAMP-RBG pA-EF2-S2P BHG pA; first generation COVID-19 vaccine; SEQ ID NO: 228). The sequences of these constructs and their components are provided in the table below.

[0338] For example, a viral vector comprising a first polynucleotide sequence can be constructed to produce Figure 1 The different structures of ILC-1 to ILC-6 shown in , including the structure of ILC-4 present in some embodiments, or a similar structure in which the first antigen of interest (priming antigen) is included in the LAMP hinge region between the first and second homologous domains of the LAMP protein (or at least between two cysteine ​​conserved fragments) or the LAMP hinge region is replaced with the first antigen of interest. Figure 1 The LAMP domain shown in Figure 3-10 The amino acid sequence shown in . By identifying the domains equivalent to the human sequence, the corresponding domains can also be cloned from the orthologous sequences. The antigens of interest (including one or more antigens of interest) can be cloned into the LAMP construct individually or in combination. Viral vectors can also be constructed to encode expression cassettes, which include a second polynucleotide sequence encoding IREG or a second antigen, which can be operably connected to a secretion signal sequence.

[0339] The relatively "compact" size of the ILC-4 LAMP construct is advantageous in some embodiments because it can reduce the size restrictions associated with the inclusion of a second polynucleotide sequence. In addition, as described in the aforementioned U.S. Patent No. 11,203,629, it has been found that the ILC-4 LAMP construct provides a stronger immune response (e.g., a stronger T cell and / or antibody response) than other LAMP constructs tested. Therefore, in some embodiments, the bicistronic LAMP construct uses the ILC-4 design, i.e., the overall structure of the ILC-4 LAMP-antigen construct is included.

[0340] In some embodiments, the isolated nucleic acid, such as a vector or vaccine, comprising an expression cassette encoding a bicistronic LAMP construct is a DNA vector, while in other cases it is an RNA vector, including a self-amplifying RNA vector.

[0341] D. Antigens for Bicistronic LAMP Constructs

[0342] 1. SARS-CoV-2 Viral Antigens and Bicistronic Constructs

[0343] In some embodiments, the antigen in the LAMP-antigen construct can include an antigen from SARS CoV2 or other viral infectious agents, such as a viral spike protein. In some cases, the bicistronic construct can also express a second antigen from the same infectious agent for secretion. In other cases, the bicistronic construct can express an IREG polypeptide as a second polypeptide.

[0344] In one specific example, a first generation vector was constructed for use as a vaccine against severe acute respiratory syndrome coronavirus (SARS-CoV-2 virus, also known as COVID-19). In addition to the production of neutralizing antibodies by effective vaccines, T cells are an important component of naturally acquired protective immunity against many infectious diseases, and many vaccines against viral infections and vaccines under development are generally intended to elicit virus-specific T cell responses that have the potential to activate innate immunity, have direct effector functions, and contribute to antibody responses, which can be used as a recommendation for prevention and treatment.

[0345] In order to induce both T cell and antibody responses to prevent SARS-Cov-2 infection or alleviate symptoms associated with infection, a vector was designed in which two viral proteins were expressed separately. The first fusion protein composed of LAMP and the viral spike S1 subunit protein is intended to elicit a rapid and strong S1-specific CD4+T cell response. The second protein is a full-length spike protein with two proline substitutions, which is driven by an independent promoter, human elongation factor-1α (EF1), and also has a sequence peptide (IgK SP) at its N-terminus. The 54nub design enables the pre-fusion stabilized spike protein to be produced and secreted to present the antigen to B cells. The strong S1-specific CD4 T cells elicited by the first promoter not only contribute to the function of CD8 T cells, but also enhance neutralizing antibodies against SARS-Cov-2. (For a description of the vector, see Fig.28A -C. )

[0346] The first generation ITI-COVID-19 bicistronic vaccine encodes the expression of the S1 and S2 subunits of the spike glycoprotein anchored on the surface of the virus. The S1 and S2 subunits of the spike mediate the entry of the SARS-CoV-2 virus into the host cell. Using the nucleic acid molecule of the ILC-4LAMP construct, the S1 coding sequence (GenBank MN908974) is located between the polynucleotide sequences encoding two LAMP homologous domains (N-Lamp and endodomain 2). The S1 coding sequence is operably connected to the CMV promoter under the influence of the CMV enhancer sequence, so that the expression in the host cell produces an ILC-4LAMP construct containing the S1 antigen, which is used for processing and presentation to class II MHC molecules (i.e., providing "primary antigens"). The S2 coding sequence is provided elsewhere on the vector and is operably connected to the polynucleotide sequence (leader sequence) encoding the Ig-κ secretion signal and the EF1 promoter sequence, so that the expression in the host cell produces the S2 antigen for secretion (i.e., providing "boosting antigens"). Thus, the vector provides a single nucleic acid molecule for introduction into a suitable host or target cell that can provide both a primary immunization and a booster antigen to elicit an enhanced immune response. Thus, this can provide significant advantages over the use of vectors encoding only a bicistronic LAMP construct, since any need or requirement to enhance an immune response elicited by a LAMP construct requires administration of a booster vaccine (e.g., comprising the antigen) administered separately at one or more time intervals.

[0347] As described in Examples 1 and 2 below, a second generation COVID-19 vector: Spike-LAMP-sCD40L was subsequently designed and tested and found to have an unexpectedly even higher immune response than the first generation vector. This second generation vector encodes the LAMP-antigen sequence provided in SEQ ID NO: 229 in an ILC-4 format, wherein the specific spike is 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 spike antigen used for this vector. And, instead of the second COVID-19 antigen as the second polypeptide, the second generation vector encodes a fusion protein of the lung surfactant binding protein D (SPD) and the CD40L extracellular domain provided in SEQ ID NO: 233.

[0348] It should be understood that variants of such isolated nucleic acids are contemplated herein, wherein the LAMP-antigen construct comprises a COVID-19 spike protein or a similar viral antigen protein and an IREG polypeptide, such as an extracellular domain or complete protein sequence of SPD-CD40L or 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 in some cases may optionally be further fused to a domain such as an immunoglobulin Fc domain. In some embodiments, a linker may also be present between the domains of the first or second polypeptide. And in some embodiments, the second polypeptide may be secreted and is therefore operably connected to a secretion signal sequence.

[0349] a) Exemplary spike protein sequences

[0350] In some embodiments, the LAMP-antigen construct may comprise 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 a SARS-CoV-2 (COVID-19) virus. In some embodiments, the antigen used in the bicistronic construct herein is selected from an antigen encoded by a SARS-CoV-2 virus, such as an S1 spike subunit or an S2 spike subunit. Examples of such S1 spike subunits include, but are not limited to, amino acid sequences 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: 118 and / or amino acid sequences 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: 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.

[0351] In some cases, the corresponding second polypeptide expressed by the bicistronic construct is an IREG polypeptide, such as SPD-CD40L or the extracellular domain or the entire 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 in some cases may optionally be further fused to a domain such as an immunoglobulin Fc domain.

[0352] 2. Cancer-associated antigens

[0353] In other embodiments, the LAMP-antigen construct may comprise a cancer antigen.Candidates for cancer immunotherapy using a vaccine comprising a bicistronic LAMP construct as described herein would be any cancer patient, such as a documented Epstein-Barr virus-associated lymphoma patient, an HPV-associated cervical cancer patient, a chronic HCV patient, or a patient with a defined rearrangement or mutation in an oncogene or tumor suppressor gene.

[0354] In some embodiments, cancers that can be treated using a vaccine comprising a bicistronic LAMP construct described herein include, but are not limited to, all stages of progression of the following cancers, including hyperplasia: 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 cancer, pancreatic ductal adenocarcinoma (PDA), kidney cancer, gastric cancer, renal cancer, multiple myeloma, or brain cancer.

[0355] It is contemplated that once an individual's cancer is identified, therapy with a vaccine composition comprising the nucleic acid molecules described herein may be used at any stage during its course. Vaccination may also be performed in high-risk patients to prevent subsequent cancer.

[0356] Examples of such cancer antigens include HER2, CD161 and NY-ESO1 or their extracellular domains.

[0357] a) HER2

[0358] 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 with SEQ ID NO: 200. In some embodiments, the encoding nucleotide sequence of 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%, at least 99% or 100% sequence identity with SEQ ID NO: 201.

[0359] 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 encodes HER2-LAMP and comprises a nucleotide 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: 196. In some embodiments, the HER2-LAMP antigen construct has a nucleotide coding 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 the following sequence: 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 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 the following sequence: 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.

[0360] In some cases, the corresponding second polypeptide expressed by the bicistronic construct is an IREG polypeptide, such as SPD-CD40L or the extracellular domain or the 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 in some cases may optionally be further fused to a domain such as an immunoglobulin Fc domain.

[0361] b) NY-ESO1

[0362] 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 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: 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.

[0363] In some embodiments, the LAMP-antigen construct comprises: (a) 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: 221. In some embodiments, the coding sequence of the LAMP-antigen construct 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 the following sequences: SEQ ID NO: 199, followed by SEQ ID NO: 224, followed by SEQ ID NO: 203.

[0364] In some cases, the corresponding second polypeptide expressed by the bicistronic construct is an IREG polypeptide, such as 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 in some cases may optionally be further fused to a domain such as an immunoglobulin Fc domain.

[0365] c) CD161

[0366] In some embodiments, the LAMP-antigen construct includes an antigen of CD161, such as the ECD of CD161. Examples of such CD161 sequences include, but are not limited to, 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 with 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 with SEQ ID NO: 237.

[0367] In some embodiments, the LAMP-antigen construct comprises: (a) 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: 234. In some embodiments, the LAMP-antigen construct encoding 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 the following sequence: SEQ ID NO: 199, followed by SEQ ID NO: 237, followed by SEQ ID NO: 203.

[0368] In some cases, the corresponding second polypeptide expressed by the bicistronic construct is an IREG polypeptide, such as SPD-CD40L or the extracellular domain or the 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 in some cases may optionally be further fused to a domain such as an immunoglobulin Fc domain.

[0369] d) Other cancer antigens

[0370] The antigens shown in Table A below can also be cloned into each bicistronic LAMP-antigen construct described herein using techniques known to the skilled person. For example, the sequence / fragment / epitope described in the fourth column can also be cloned into the LAMP-antigen construct described herein. In addition, any one of the cancer antigens listed in Table A can be combined with any other antigen listed in Table A (including the sequence / fragment / epitope described in the fourth column) and inserted into the LAMP-antigen construct described herein. Alternatively, any one of the cancer antigens of Table A can be combined with any other cancer antigen described in the present disclosure and inserted into the LAMP-antigen construct herein.

[0371] In some cases, the corresponding second polypeptide expressed by the bicistronic construct including a cancer antigen from Table A is an IREG polypeptide, such as SPD-CD40L or the extracellular domain or the entire 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 in some cases may optionally be further fused to a domain such as an immunoglobulin Fc domain.

[0372]

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[0399]

[0400]

[0401]

[0402]

[0403]

[0404]

[0405] In addition, the antigens described in Table A (including the sequences / fragments / epitopes shown in column 4) can be cloned into the LAMP-antigen constructs described herein, either alone or in combination with each other. Thus, each sequence shown in column 1 of Table A, including the epitopes / fragments described 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 produce a LAMP-antigen construct. For example, in the case of a nucleic acid construct or a cell carrying such a nucleic acid construct, the construct can encode one or more antigens listed in Table A or elsewhere herein. In addition, the 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 of IREG) to produce a bicistronic LAMP construct.

[0406] The order of the antigen combination in a particular LAMP-antigen construct can also be different. 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, intermediate S, X protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, mucin-1, mucin-1iso, CD40-L, WT-1, WT-1trunc., PRAME, LAGE-1, and / or MAGE A3 refers to the proteins described in Table 1 and is expressly intended to refer not only to the full-length sequences shown in column 1 of Table A, but also to the sequences / fragments / epitopes described in column 4 of Table A. Thus, each of the sequences shown in column 1 of Table A, including the epitopes / fragments described in column 4 of Table 1, can be used to generate a LAMP construct, which can then be combined with, for example, an IREG sequence to generate a bicistronic LAMP construct.

[0407] To illustrate different, additional possible antigen combinations, but in no way to limit the present disclosure, combinations of the following antigens (including the sequences shown in column 1 of Table A and / or the sequences / fragments / epitopes described in column 4 of Table A) can be cloned into the LAMP construct: (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, intermediate S, X protein, TIGIT, TEM1, HER2, TEM8, TEM9, TEM10, TEM11, TEM12, TEM13, TEM14, TEM15, TEM16, TEM17, TEM18, TEM19, TEM20, TEM21, TEM22, TEM23, TEM24, TEM25 at least one of ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, mucin-1, mucin-1iso, CD40-L, WT-1, WT-1trunc., PRAME, LAGE-1 and / or MAGE A3; (b) gB and 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, intermediate S, X protein, TIGIT, TEM8, TEM1, HER2 ECD + TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, mucin-1, mucin-1iso, CD40-L, WT-1, WT-1trunc., 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, intermediate S, X protein, TIGIT, TEM8, TEM1, HER2ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, mucin-1, mucin-1iso, CD40-L, WT-1, WT-1trunc., PRAME, LAGE-1 and / or MAGEA3; (d) MTRII and 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, intermediate S, X protein, TIGIT, TEM8, TEM1, HER2ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, mucin-1, mucin-1iso, CD40-L, WT-1, WT-1trunc., PRAME, LAGE-1 and / or MAGE A3; (e) US28 and 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, intermediate S, X protein, TIGIT, TEM8, TEM1, HER2ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, mucin-1, mucin-1iso, CD40-L, WT-1, WT-1trunc., PRAME, LAGE-1 and / or MAGE (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, intermediate S, X protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, mucin-1, mucin-1iso, CD40-L, WT-1, WT-1trunc., PRAME, LAGE-1 and / or MAGE 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, intermediate S, X protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, mucin-1, mucin-1iso, CD40-L, WT-1, WT-1trunc., PRAME, LAGE-1 and / or MAGE at least one of A3; (h) UL144 and 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, intermediate S, X protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, mucin-1, mucin-1iso, CD40-L, WT-1, WT-1trunc., PRAME, LAGE-1 and / or MAGE A3; (i) UL141 and 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, intermediate S, X protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, mucin-1, mucin-1iso, CD40-L, WT-1, WT-1trunc., PRAME, LAGE-1 and / or MAGE A3; (j) US11 and 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, intermediate S, X protein, TIGIT, TEM8, TEM1, HER2 at least one of ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, mucin-1, mucin-1iso, CD40-L, WT-1, WT-1trunc., PRAME, LAGE-1 and / or MAGEA3; (k) IE2 and 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, intermediate S, X protein, TIGIT, TEM8, TEM1, HER2 At least one of ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, mucin-1, mucin-1iso, CD40-L, WT-1, WT-1trunc., PRAME, LAGE-1 and / or MAGE A3; (l) TERT and 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, intermediate S, X protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, mucin-1, mucin-1iso, CD40-L, WT-1, WT-1trunc., PRAME, LAGE-1 and / or MAGE A3; (m) survivin and 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, intermediate S, X protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, mucin-1, mucin-1iso, CD40-L, WT-1, WT-1trunc., PRAME, LAGE-1 and / or MAGE A3; (n) tetanus and 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, intermediate S, X protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, mucin-1, mucin-1iso, CD40-L, WT-1, WT-1trunc., PRAME, LAGE-1 and / or MAGE A3; (o) NY-ESO-1 and 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, intermediate S, X protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, mucin-1, mucin-1iso, CD40-L, WT-1, WT-1trunc., PRAME, LAGE-1 and / or MAGE A3; (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, intermediate S, X protein, TIGIT, TEM8, TEM1, HER2 at least one of ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, mucin-1, mucin-1iso, CD40-L, WT-1, WT-1trunc., PRAME, LAGE-1 and / or MAGEA3; (q) HER3 and 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, intermediate S, X protein, TIGIT, TEM8, TEM1, HER2 at least one of ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, mucin-1, mucin-1iso, CD40-L, WT-1, WT-1trunc., PRAME, LAGE-1 and / or MAGE A3; (r) HVEM and 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, intermediate S, X protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, mucin-1, mucin-1iso, CD40-L, WT-1, WT-1trunc., PRAME, LAGE-1 and / or MAGE A3; (s) HOS and 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, intermediate S, X protein, TIGIT, TEM8, TEM1, HER2ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, mucin-1, mucin-1iso, CD40-L, WT-1, WT-1trunc., PRAME, LAGE-1 and / or MAGEA3; (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, intermediate S, X protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, mucin-1, mucin-1iso, CD40-L, WT-1, WT-1trunc., PRAME, LAGE-1 and / or MAGE A3; (u) HPV18E6 and 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, intermediate S, X protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, mucin-1, mucin-1iso, CD40-L, WT-1, WT-1trunc., PRAME, LAGE-1 and / or MAGE A3; (v) HPV16E7 and 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, intermediate S, X protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, mucin-1, mucin-1iso, CD40-L, WT-1, WT-1trunc., PRAME, LAGE-1 and / or MAGE A3; (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, intermediate S, X protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, mucin-1, mucin-1iso, CD40-L, WT-1, WT-1trunc., PRAME, LAGE-1 and / or MAGE A3; (x) EBNA1 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, EBNA trunc, gp350, LMP2, GCP3, intermediate S, X protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, mucin-1, mucin-1iso, CD40-L, WT-1, WT-1trunc., PRAME, LAGE-1 and / or MAGE A3; (y) EBNA1 trunc 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, gp350, LMP2, GCP3, intermediate S, X protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, mucin-1, mucin-1iso, CD40-L, WT-1, WT-1trunc., PRAME, LAGE-1 and / or MAGE (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, intermediate S, X protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, mucin-1, mucin-1iso, CD40-L, WT-1, WT-1trunc., PRAME, LAGE-1 and / or MAGE A3 at least one; (aa) LMP2 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, GCP3, intermediate S, X protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, mucin-1, mucin-1iso, CD40-L, WT-1, WT-1trunc., PRAME, LAGE-1 and / or MAGE A3; (ab) GCP3 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, intermediate S, X protein, TIGIT, TEM8, TEM1, HER2ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, mucin-1, mucin-1iso, CD40-L, WT-1, WT-1trunc., PRAME, LAGE-1 and / or MAGE at least one of A3; (ac) intermediate S 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, X protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, mucin-1, mucin-1iso, CD40-L, WT-1, WT-1trunc., PRAME, LAGE-1 and / or MAGE At least one of A3; (ad)X protein 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, intermediate S, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, mucin-1, mucin-1iso, CD40-L, WT-1, WT-1trunc., PRAME, LAGE-1 and / or MAGE A3; (ae) TIGIT 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, intermediate S, X protein, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, mucin-1, mucin-1iso, CD40-L, WT-1, WT-1trunc., PRAME, LAGE-1 and / or MAGE A3; (af) TEM8 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, EBNAtrunc, gp350, LMP2, GCP3, intermediate S, X protein, TIGIT, TEM1, HER2 at least one of ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, mucin-1, mucin-1iso, CD40-L, WT-1, WT-1trunc., PRAME, LAGE-1 and / or MAGEA3; (ag)TEM1 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, intermediate S, X protein, TIGIT, TEM8, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, mucin-1, mucin-1iso, CD40-L, WT-1, WT-1trunc., 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, intermediate S, X protein, TIGIT, TEM8, TEM1, CEA, TARP, PROSTEIN, PSMA, BIRC4, mucin-1, mucin-1iso, CD40-L, WT-1, WT-1trunc., PRAME, LAGE-1 and / or MAGE at least one of A3; (ai) CEA 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, intermediate S, X protein, TIGIT, TEM8, TEM1, HER2ECD+TM, TARP, PROSTEIN, PSMA, BIRC4, mucin-1, mucin-1iso, CD40-L, WT-1, WT-1trunc., PRAME, LAGE-1 and / or MAGE A3; (aj) TARP 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, intermediate S, X protein, TIGIT, TEM8, TEM1, HER2ECD+TM, CEA, PROSTEIN, PSMA, BIRC4, mucin-1, mucin-1iso, CD40-L, WT-1, WT-1trunc., PRAME, LAGE-1 and / or MAGE A3; (ak) PROSTEIN 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, intermediate S, X protein, TIGIT, TEM8, TEM1, HER2ECD+TM, CEA, TARP, PSMA, BIRC4, mucin-1, mucin-1iso, CD40-L, WT-1, WT-1trunc., PRAME, LAGE-1 and / or MAGE at least one of A3; (al) PSMA 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, intermediate S, X protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, BIRC4, mucin-1, mucin-1iso, CD40-L, WT-1, WT-1trunc., PRAME, LAGE-1 and / or MAGE At least one of A3; (am) BIRC4 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, intermediate S, X protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, mucin-1, mucin-1iso, CD40-L, WT-1, WT-1trunc., PRAME, LAGE-1 and / or MAGE A3; (an) mucin-1 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, intermediate S, X protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, mucin-1iso, CD40-L, WT-1, WT-1trunc., PRAME, LAGE-1 and / or MAGE at least one of A3; (ao) mucin-1 iso 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, intermediate S, X protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, mucin-1, CD40-L, WT-1, WT-1trunc., PRAME, LAGE-1 and / or MAGE A3; (ap) CD40-L 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, intermediate S, X protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, mucin-1, mucin-1iso, WT-1, WT-1trunc., PRAME, LAGE-1 and / or MAGE A3; (aq) WT-1 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, intermediate S, X protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, mucin-1, mucin-1iso, CD40-L, WT-1trunc., PRAME, LAGE-1 and / or MAGE A3; (ar) WT-1trunc 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, intermediate S, X protein, TIGIT, TEM8, TEM1, HER2 ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, mucin-1, mucin-1iso, CD40-L, WT-1, PRAME, LAGE-1 and / or MAGE At least one of A3; (as) PRAME 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, EBNAtrunc, gp350, LMP2, GCP3, intermediate S, X protein, TIGIT, TEM8, TEM1, HER2ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, mucin-1, mucin-1iso, CD40-L, WT-1, WT-1trunc., 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, intermediate S, X protein, TIGIT, TEM8, TEM1, HER2ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, mucin-1, mucin-1iso, CD40-L, WT-1, WT-1trunc., PRAME and / or MAGE A3; and / or (au) MAGE A3 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, intermediate S, X protein, TIGIT, TEM8, TEM1, HER2ECD+TM, CEA, TARP, PROSTEIN, PSMA, BIRC4, mucin-1, mucin-1iso, CD40-L, WT-1, WT-1trunc., PRAME and / or LAGE-1. The order of the combination of antigens described above in a particular LAMP construct may vary, as this list describes the antigens contained in the LAMP construct and does not necessarily describe the arrangement of the antigens within a particular construct. Furthermore, it is specifically contemplated that these antigens may be combined within a single LAMP construct or may be delivered in a composition comprising multiple LAMP constructs.

[0408] Other examples of antigens that can be used in the bicistronic LAMP constructs herein include, for example, those described in International Publication WO 2018 / 204534 (e.g., Table 1 and Figures 19-20 of the disclosure) or International Publication WO 2021 / 077051 (e.g., Table 1 and Figures 20-21 of the disclosure). Fig.11 Both publications are incorporated herein by reference in their entirety.

[0409] In some cases, the antigen used in the bicistronic LAMP construct comprises a pp65 antigen, e.g., comprising SEQ ID NO: 291, 292, or 293, or one or more portions of SEQ ID NO: 291 as 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, e.g., comprising SEQ ID NO: 294, 295, 296, or 297, or one or more antigenic fragments of SEQ ID NO: 294 as 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 an 1E1 antigen, e.g., comprising SEQ ID NO: 298, 299, or 300, or one or more of the 1E1 polypeptide sequences 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 more than one of the pp65, gB and 1E1 antigen sequences, e.g., linked by one or more linker peptide sequences, as shown in column 4 of Table A. In some cases, the antigen comprises each of the pp65, gB and 1E1 antigen sequences, e.g., selected from the group of antigen sequences (a) comprising SEQ ID NO: 292 or 293, (b) comprising SEQ ID NO: 296 or 297, and (c) comprising SEQ ID NO: 299 or 300. For example, such polynucleotide coding sequences for 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 polypeptides, polynucleotides (i.e., DNA vectors or self-replicating RNA vectors), or cells can be used to treat various cancers, such as those listed above, including, for example, where the cancer (including all stages of progression, including hyperplasia) is adenocarcinoma, sarcoma, skin cancer, melanoma, 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 cancer, pancreatic ductal adenocarcinoma (PDA), kidney cancer, gastric cancer, renal cancer, multiple myeloma, or brain cancer. 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.

[0410] In other cases, the antigen comprises a large T antigen, e.g., comprising an amino acid sequence of SEQ ID NO: 254, 255, or 256. In some cases, the antigen comprises an 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 an amino acid sequence of SEQ ID NO: 879 or SEQ ID NO: 880, both of which comprise an amino acid sequence of SEQ ID NO: 256 flanked by homology domains of LAMP1, and comprise a signal sequence. SEQ ID NO: 879 further comprises a LAMP transmembrane domain and a cytoplasmic region. In the bicistronic LAMP constructs herein, the construct may further comprise or encode an IREG protein. For example, constructs in which the antigen is a large T antigen may be used to treat 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 the disclosure.

[0411] E. Exemplary Immune Response Enhancer Genes (IREGs) for Bicistronic LAMP Constructs

[0412] In some embodiments, the second polypeptide may include a domain or antigen encoded by an immune response enhancing gene (IREG), which may increase T cell responses 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.

[0413] 1.CD40 ligand (CD40L)

[0414] 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 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, the CD40L is a soluble version of CD40L (sCD40L). In some embodiments, the sCD40L is a 4-trimer, i.e., a protein complex comprising a tetramer of a CD40L trimer. In some embodiments, the 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 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:204.

[0415] In some cases, the sCD40L is fused to another polypeptide, such as 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 the following sequence: 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 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 cases, the coding sequence for the second polypeptide comprises a nucleotide 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: 239, or 237, or 205.

[0416] 2. FLT3L

[0417] In some embodiments, the IREG is FLT3L. Examples of such FLT3L sequences include, but are not limited to, amino acid sequences 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: 151 or 209.

[0418] In some embodiments, the bicistronic construct encodes a second polypeptide comprising a human Flt3L polypeptide preceded by an SPD polypeptide, thereby producing a fusion protein. In certain such cases, the bicistronic construct encodes a second polypeptide having an amino acid sequence that is 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% identical to SEQ ID NO: 207.

[0419] 3. IL-12

[0420] In some embodiments, the IREG is IL- 12. Examples of such IL-12 sequences include, but are not limited to, amino acid sequences 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: 137, 139, 143, 145, 147, 149, 187, 189, 193, or 213.

[0421] In some embodiments, the nucleotide encoding 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%, at least 99%, or 100% sequence identity to SEQ ID NO: 138, 140, 144, 146, 148, 150, 188, 190, 194, or 214.

[0422] 4. IL-21

[0423] In some embodiments, the IREG is IL-21. Examples of such IL-21 sequences include, but are not limited to, amino acid sequences 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: 179, 181 or 217.

[0424] In some embodiments, the nucleotide encoding 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.

[0425] 5.OX40 ligand (OX40L)

[0426] In some embodiments, the IREG is OX40L. Examples of such OX40L sequences include, but are not limited to, amino acid sequences 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: 153, 155, or 243. In some cases, the OX40L is fused to a heterologous signal peptide, such as a signal peptide from IL-2. In some cases, the OX40L sequence is an extracellular domain sequence. In some cases, the OX40L extracellular domain is also fused to the Fc domain of an immunoglobulin. In some cases, 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: 242, or a combination of SEQ ID NO: 246 or 248 followed by SEQ ID NO: 243.

[0427] 6.IL-15

[0428] In some embodiments, the IREG is IL-15. Examples of such IL-15 sequences include, but are not limited to, amino acid sequences 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: 167 or 169. In some embodiments, the nucleotide encoding sequence of IL-15 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: 168 or 170.

[0429] In some cases, the IL-15 is expressed behind a heterologous signal sequence, such as IgKVIII or Ig-κ. Examples of such IL-15 sequences include, but are not limited to, amino acid sequences 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: 225. In some embodiments, the nucleotide encoding sequence of IL-15 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: 226.

[0430] 7.CD80

[0431] In some embodiments, the IREG is CD80. Examples of such CD80 sequences include, but are not limited to, amino acid sequences 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: 157, 159 or 253. In some embodiments, the nucleotide encoding sequence of CD80 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: 158 or 160 or 254.

[0432] In some cases, the CD80 is expressed behind a heterologous signal sequence, such as an IL-2 signal sequence. In some cases, the 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 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 with SEQ ID NO: 252 or 253.

[0433] F. Exemplary bicistronic LAMP construct sequences

[0434] In some embodiments, the bicistronic construct comprises a polynucleotide sequence encoding a LAMP-antigenic polypeptide, the polypeptide comprising two homologous domains of the luminal domain of a LAMP protein and an antigenic domain heterologous to the LAMP protein, wherein the antigenic domain is placed 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 with residues 29 to 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 with residues 228-381 of SEQ ID NO: 202 or SEQ ID NO: 1.

[0435] 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.

[0436] 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.

[0437] 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.

[0438] 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.

[0439] 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.

[0440] 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.

[0441] 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.

[0442] 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.

[0443] 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.

[0444] 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.

[0445] G. Assembly of sequences encoding bicistronic LAMP constructs

[0446] Methods for constructing bicistronic LAMP constructs comprising an antigen of interest are well known in the art (see, e.g., Williams et al., J. Cell Biol. 111: 955, 1990). DNA sequences encoding the desired segments can be obtained from readily available recombinant DNA materials, such as those available from the American Type Culture Collection (12301 Parklawn Drive, Rockville, Md. 20852, USA) or from DNA libraries containing the desired DNA.

[0447] For example, DNA fragments corresponding to the desired domain sequence can be assembled with appropriate control and signal sequences using conventional procedures of recombinant DNA methods, as described in, for example, U.S. Pat. No. 4,593,002 and Langford et al., Molec. Cell. Biol. 6:3191, 1986.

[0448] The DNA sequence encoding the protein or polypeptide can be synthesized by chemical methods or isolated by one of several methods. The DNA sequence to be synthesized can be designed to have codons suitable for the desired amino acid sequence. In general, people will choose preferred codons for the intended host in which the sequence will be used for expression. 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; Nambaier et al., Science 223:1299, 1984; Jay et al., J.Biol.Chem.259:6311, 1984.

[0449] In one aspect, one or more polynucleotides encoding the domain sequences of the bicistronic LAMP construct are individually isolated using polymerase chain reaction (MAInnis 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 a bicistronic LAMP construct encoding the antigen sequence. This technology is well known to those skilled in the art. The domain sequences can be directly fused to each other (e.g., without intervening sequences), or inserted into each other (e.g., wherein the domain sequences are discontinuous), or can be separated by intervening sequences (e.g., linker sequences).

[0450] The basic strategies for preparing oligonucleotide primers, probes, and DNA libraries, and for screening them by nucleic acid hybridization, are well known to those of ordinary skill in the art. See 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 can be obtained from suppliers of biological research materials such as Clonetech and Stratagene, or from public depositories such as the American Type Culture Collection.

[0451] Selection can be achieved by expressing sequences from a DNA expression library and immunodetecting 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 ordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).

[0452] Once a clone containing the coding sequence for the desired polypeptide sequence is prepared or isolated, the sequence may be cloned into any suitable vector, which preferably contains an origin of replication for maintaining the sequence in the host cell.

[0453] H. Nucleic Acid Delivery Vehicles

[0454] 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 homology domain of a LAMP protein and a second homology domain of a LAMP protein (or at least between two cysteine ​​conserved fragments), for example, the at least one antigen of interest can be placed in the LAMP hinge region, or can replace the LAMP hinge region; and (ii) a second polynucleotide sequence encoding at least one IREG or other antigen operably linked to a secretion signal sequence, wherein the IREG or other antigen is secreted into the circulation of the subject. Other exemplary nucleic acid embodiments are described in the Summary of the Invention and Claims sections and throughout the disclosure herein.

[0455] 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 the LAMP-antigen construct) and the IREG or the second antigen operably connected to the secretion signal sequence (so that the second polypeptide comprising the IREG or the 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-amplification RNA.

[0456] 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 connected thereto to control 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 the second polypeptide comprising IREG or other antigens further comprises a polynucleotide sequence for insertion into a target cell and an expression control sequence operably connected thereto to control 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 as, for example, a plasmid, a bacteriophage, an autonomously replicating sequence (ARS), a centromere, and other sequences capable of replicating or being replicated in vitro or in a host cell (e.g., a bacterium, a yeast, or an insect cell) and / or a target cell (e.g., a mammalian cell, preferably an antigen presenting cell) and / or transporting the expressed sequence to a desired location in the target cell.

[0457] Recombinant expression vectors can be derived from microorganisms that are susceptible to infecting animals, including humans, horses, cows, pigs, llamas, giraffes, dogs, cats, or chickens. Some of the vectors herein include vectors that have been used as live vaccines, such as vaccinia. These recombinants can be directly inoculated into the host, not only to provide immunity to the microbial vector, but also to express foreign antigens. As taught in, for example, Flexner, Adv. Pharmacol. 21: 51, 1990, some of the vectors contemplated herein as live recombinant vaccines include RNA viruses, adenoviruses, herpes viruses, polioviruses, and vaccinia and other poxviruses.

[0458] Expression control sequences include, but are not limited to, promoter sequences that bind RNA polymerase, enhancer sequences or negative regulatory elements that bind to transcriptional activators and repressors, respectively, and / or translation initiation sequences for ribosome binding. For example, bacterial expression vectors can include promoters such as the lac promoter, and Shine-Dalgarno sequences and start codon AUG (Sambrook et al., 1989, supra) for transcription initiation. Similarly, eukaryotic expression vectors preferably include heterologous, homologous or chimeric promoters for RNA polymerase II, downstream polyadenylation signals, start codon AUG and stop codons for ribosome separation.

[0459] Expression control sequences can be obtained from naturally occurring genes or can be designed. Designed expression control sequences include, but are not limited to, mutant and / or chimeric expression control sequences or synthetic or cloned consensus sequences. Vectors containing cloning sites to which promoters and polynucleotides can be operably linked are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo and are commercially available from sources such as Stratagene (La Jolla, Calif.) and Promega Biotech (Madison, Wis.).

[0460] In order to optimize expression and / or transcription, it may be necessary to remove, add or change the 5' and / or 3' non-translated portion of the carrier to eliminate additional or optional translation initiation codons or other sequences that may interfere or reduce expression at the transcription or translation level. Alternatively, a consensus ribosome binding site may be inserted close to the 5' of the start codon to enhance expression. A variety of expression control sequences (sequences controlling the expression of the polynucleotide sequence operably connected thereto) can be used in these carriers to express the polynucleotide sequence of the present disclosure. This type of useful expression control sequence includes, for example, early or late promoters of SV40, CMV, vaccinia, polyoma virus, adenovirus, herpes virus and other sequences known to control mammalian cell gene expression, and their various combinations.

[0461] 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 can be used for the transcription of a bicistronic mRNA molecule encoding two polypeptides, or different promoters can be used to control the expression of the two different polypeptides. Those skilled in the art will appreciate that the translation of the "second" encoded protein can be achieved by including a translation enhancing element such as an internal ribosome entry site (IRES) (Wiley Interdiscip. Rev. RNA 3: 195-212, 2012) or an unstructured junction sequence to achieve the termination of translation and then restarting (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).

[0462] In order to achieve the secretion of the second polynucleotide, its coding sequence can include a polynucleotide sequence encoding a secretion signal sequence (also referred to as a leader sequence) of generally 16-30 amino acids in length, so as to express the second polynucleotide sequence operably connected to the secretion signal sequence. Suitable secretion signal sequences are well known to those skilled in the art, including, for example, signal sequences of interleukin-2, CD5, immunoglobulin kappa light chain (hereinafter referred to as Ig-kappa leader sequence), 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). In some cases, the secretion signal sequence can be a "native" secretion signal sequence for the IREG or the second polypeptide antigen.

[0463] In one aspect, the nucleic acid molecule comprises a replication origin for replication. Preferably, the origin works in at least one type of host cell, and the host cell can be used to produce a sufficient number of sequence copies for delivery to a target cell. Therefore, suitable origins include but are not limited to those origins that work in bacterial cells (e.g., Escherichia species (Escherichia sp.), Salmonella species (Salmonella sp.), Proteus species (Proteus sp.), Clostridium species (Clostridium sp.), Klebsiella species (Klebsiella sp.), Bacillus species (Bacillus sp.), Streptomyces species (Streptomyces sp.) and Pseudomonas species (Pseudomonas sp.)), yeast (e.g., Saccharomyces species (Saccharamyces sp.) or Pichia species (Pichia sp.)), insect cells and mammalian cells. In one aspect, there is provided a replication origin that works in a target cell (e.g., mammalian cell, such as a human cell) introducing a nucleic acid delivery medium. In another aspect, at least two origins of replication are provided, one functional in the host cell and another functional in the target cell.

[0464] The nucleic acid molecule may alternatively or additionally comprise a polynucleotide sequence to promote at least a portion of the nucleic acid molecule (e.g., delivery vector) to be integrated into the chromosome of the target cell. For example, the nucleic acid molecule may comprise a region homologous to the target cell chromosome DNA. In one aspect, the nucleic acid molecule is provided as a delivery vector comprising two or more recombination sites, the recombination sites being flanking the nucleic acid sequence encoding the LAMP-antigen construct and the second polypeptide and / or the bicistronic LAMP construct itself.

[0465] The vector may additionally contain a detectable and / or selectable marker to verify that the vector has been successfully introduced into the target cell and / or can be expressed by the target cell. These markers may encode an activity, such as but not limited to the production of RNA, peptides or proteins, or may provide binding sites for RNA, peptides, proteins, inorganic and organic compounds or compositions, etc.

[0466] Examples of detectable / selectable marker genes include, but are not limited to, DNA segments encoding products that provide resistance to otherwise toxic compounds (e.g., antibiotics); DNA segments encoding products that are otherwise lacking in recipient cells (e.g., tRNA genes, auxotrophic markers); DNA segments encoding products that inhibit the activity of a gene product; DNA segments encoding products that are 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). DNA segments that bind to products that are otherwise detrimental to cell survival and / or function; DNA segments that otherwise inhibit the activity of other nucleic acid segments (e.g., antisense oligonucleotides); DNA segments that bind to products that modify substrates (e.g., restriction endonucleases); DNA segments that can be used to isolate or identify desired molecules (e.g., fragments encoding specific protein binding sites); primer sequences; DNA segments that, when absent, directly or indirectly confer resistance or sensitivity to specific compounds; and / or DNA segments that encode products that are toxic in recipient cells.

[0467] The marker gene can be used as a marker of the conformation of successful gene transfer, and / or for isolating cells expressing the transferred gene and / or recovering the transferred gene from the cell. For example, in one aspect, the marker gene is used to separate and purify antigen presenting cells expressing the bicistronic LAMP construct described herein.

[0468] Substantially similar genes can be provided, for example, genes having greater than about 50%, greater than about 70%, greater than 80%, greater than about 90%, preferably greater than about 95% identity to known genes. Substantially similar domain sequences can be initially identified by selecting sequences that specifically hybridize to the domain sequence of interest under stringent hybridization conditions. Making assays to determine the suitability of homologous, variant, or modified domain sequences is simply a matter of screening for sequences that express suitable activity. Such screening is routine in the art.

[0469] The bicistronic LAMP construct encoding the LAMP-antigen construct and the second polypeptide can be provided as a naked nucleic acid molecule, or combined with one or more molecules and provided in a delivery vehicle to promote the entry of nucleic acids into cells. Suitable delivery vehicles include, but are not limited to, liposome preparations, polypeptides, polysaccharides, lipopolysaccharides, viral preparations (e.g., including viruses, virions, artificial viral envelopes, etc.), cell delivery vehicles, etc.

[0470] I. Lipid-based formulations

[0471] Delivery vehicles designed to facilitate intracellular delivery of nucleic acid molecules encoding bicistronic LAMP constructs must interact with both non-polar and polar environments (e.g., in or on the plasma membrane, tissue fluid, intracellular compartments, etc.). Therefore, preferably, the delivery vehicle is designed to contain both polar and non-polar domains or a translocation sequence for translocating the nucleic acid molecule encoding the bicistronic LAMP construct into the cell.

[0472] Compounds with polar and nonpolar domains are called amphiphiles. Cationic amphiphiles have polar groups that are positively charged at or near physiological pH and interact with negatively charged polynucleotides such as DNA.

[0473] The nucleic acid molecule comprising the bicistronic LAMP construct can be provided in the form of a preparation comprising a lipid monolayer or a bilayer to facilitate the transfer of the vector across the cell membrane. Liposomes or any form of lipid membrane, such as a planar lipid membrane or a cell membrane of a complete cell (such as an erythrocyte) can be used. Liposomal preparations can be administered by any means, including intravenous or oral administration.

[0474] Liposomes and liposome 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. Pat. No. 4,235,871; U.S. Pat. No. 4,501,728; and U.S. Pat. No. 4,837,028. In one aspect, the liposomes include targeting molecules for targeting the liposome: nucleic acid molecule (bicistronic LAMP construct herein) complex to a specific cell type. In a specific aspect, the targeting molecule includes a binding partner (e.g., a ligand or receptor) of a biological molecule (e.g., a receptor or ligand) on the surface of a blood vessel or cell found in a target tissue.

[0475] Liposome charge is an important determinant of liposome clearance from the blood, and negatively charged liposomes are absorbed more quickly by the reticuloendothelial system (Juliano, 1975, Biochem.Biophys.Res.Commun.63:651), and therefore have a shorter half-life in the bloodstream. Phosphatidylethanolamine derivatives are incorporated to prolong circulation time by preventing liposome aggregation. For example, N-(ω-carboxyl)amidophosphatidylethanolamine is incorporated into large unilamellar vesicles of L-α-distearoylphosphatidylcholine, significantly extending the liposome circulation life in vivo (see, e.g., Ahl, 1997, Biochim.Biophys.Acta 1329:370-382). Liposomes with extended circulation half-life are generally suitable for therapeutic and diagnostic purposes. For a general discussion of pharmacokinetics, see, eg, Remington's, Chapters 37-39; Lee et al., in Pharmacokinetic Analysis: A Practical Approach (Technomic Publishing AG, Basel, Switzerland 1996).

[0476] Typically, liposomes are prepared using about 5 to 15 mol% of negatively charged phospholipids such as phosphatidylglycerol, phosphatidylserine or phosphatidylinositol. The addition of negatively charged phospholipids such as phosphatidylglycerol is also used to prevent spontaneous aggregation of liposomes, thereby minimizing the risk of unwanted liposome aggregate formation. Membrane hardeners such as sphingomyelin or saturated neutral phospholipids at a concentration of at least 50 mol%, and 5 to 15 mol% of monosialogangliosides, can also impart desirable liposome properties such as rigidity (see, e.g., U.S. Pat. No. 4,837,028).

[0477] In addition, the liposomal suspension may include a lipid protectant that protects the lipids from free radical and lipid peroxidation damage during storage. Lipophilic free radical quenchers such as alpha-tocopherol and water-soluble iron-specific chelators such as ferrioxianine may be used.

[0478] Bicistronic LAMP constructs as described herein can also be incorporated into multilamellar vesicles of varying sizes. For example, the lipids forming the vesicles 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 necessary, the film can be redissolved in a suitable solvent such as tert-butyl alcohol, and then lyophilized to form a more uniform lipid mixture, which takes a powdered form that is more easily hydrated. This film is covered with an aqueous solution of peptide or polypeptide complex, and is allowed to be hydrated under stirring for a period of 15 to 60 minutes, usually. By hydrating the lipids under more intense stirring conditions or by adding a solubilizing detergent such as deoxycholate, the size distribution of the multilamellar vesicles obtained can be migrated to a smaller size direction. The hydration medium is preferably included in the nucleic acid of the desired concentration in the liposome internal volume in the final liposome suspension.

[0479] After liposome preparation, the size of the liposome can be adjusted to achieve a desired size range and a relatively narrow liposome size distribution. An exemplary size range is about 0.2 to 0.4 microns, which allows the liposome suspension to be sterilized by filtering through a conventional filter (usually a 0.22 micron filter). If the size of the liposome has been reduced to about 0.2 to 0.4 microns, filter sterilization can be performed on a high-throughput basis. Several techniques are available for adjusting liposomes to a desired size (see, e.g., U.S. Patent No. 4,737,323).

[0480] Suitable lipids include, but are not limited to, DOTMA (Felgner et al., 1987, Proc. Natl. Acad. Sci. USA 84:7413-7417), DOGS or Transfectain TM (Behr et al., 1989, Proc. Natl. Acad. Sci. USA 86:6982-6986), DNERIE or DORIE (Felgner et al., Methods 5:67-75), DC-CHOL (Gao and Huang, 1991, BBRC 179:280-285), DOTAP TM (McLachlan et al., 1995, Gene Therapy 2:674-622), Lipofectamine TM and glycerolipid compounds (see, for example, EP901463 and WO98 / 37916).

[0481] Other molecules suitable for complexing with the bicistronic LAMP construct may include cationic molecules such as polyamidoamine (Haensler and Szoka, 1993, Bioconjugate Chem. 4:372-379), dendritic polylysine (WO 95 / 24221), polyethyleneimine or polypropyleneimine (WO 96 / 02655), polylysine (U.S. Pat. No. 5,595,897; FR2 719316), chitosan (U.S. Pat. No. 5,744,166), DNA-gelatin aggregates (see, e.g., U.S. Pat. No. 6,207,195; U.S. Pat. No. 6,025,337; U.S. Pat. No. 5,972,707), or DEAE dextran (Lopata et al., 1984, Nucleic Acid Res. 12:5707-5717).

[0482] J. Virus-based gene delivery vectors

[0483] In one aspect, the nucleic acid molecule comprising the bicistronic LAMP construct is provided as a delivery vehicle comprising a virus or virion. In this respect, preferably, the nucleic acid molecule comprises a viral vector. Viral vectors such as retrovirus, adenovirus, adeno-associated virus and herpes virus are usually composed of two components, i.e., modified viral genome and a capsid structure around it (see, for example, Smith et al., 1995, Ann.Rev.Microbiol.49:807-838), although sometimes viral vectors are introduced in naked form or coated with proteins other than viral proteins. At present, the capsid structure of most vectors is similar to wild-type virus. This structure packages and protects viral nucleic acid, and provides means for combining and entering target cells.

[0484] Preferably, the viral vector comprising the bicistronic LAMP construct described herein is modified from a wild-type viral genome so that the virus cannot grow in a target cell, while enabling the virus to grow in a host cell (e.g., packaging or helper cell) for preparing infectious particles. The vector nucleic acid typically comprises essential cis-acting viral sequences for replication and packaging in a helper cell line, and expression control sequences for regulating the expression of the polynucleotides delivered to the target cell. As known in the art, other viral functions are expressed in trans in a specific packaging or helper cell line.

[0485] The viral vector may be derived from a virus selected from the group consisting of herpes virus, cytomegalovirus, foamy virus, lentivirus, Semliki Forest virus, AAV (adeno-associated virus), poxvirus, adenovirus and retrovirus. Such viral vectors are well known in the art.

[0486] In one aspect, the viral vector used is an adenovirus vector. The adenovirus genome consists of a linear double-stranded DNA molecule of about 36kb, carrying more than about 30 genes required to complete the viral replication cycle. Early genes are divided into 4 regions (E1 to E4), except for the E3 region, the remaining regions are crucial for viral replication, and the E3 region is considered to regulate antiviral host immune responses. The E1 region (EIA and EIB) encodes proteins responsible for regulating viral genome transcription. The expression of E2 region genes (E2A and E2B) leads to the synthesis of polypeptides required for viral replication. The proteins encoded by the E3 region prevent cell lysis of cytotoxic T cells and tumor necrosis factor (Wold and Gooding, 1991, Virology 184: 1-8). The proteins encoded by the E4 region participate in DNA replication, late gene expression and splicing, and host cell closure (Halbert et al., 1985, J.Virol.56: 250-257). The late genes usually encode structural proteins that contribute to the viral capsid. In addition, the adenoviral genome carries cis-acting 5' and 3' ITRs (inverted terminal repeats) and packaging sequences essential for DNA replication. The ITRs carry the DNA replication origin, while the encapsidation region is required for the packaging of adenoviral DNA into infectious particles.

[0487] As described in Heise and Kim (2000, J. Clin. Invest. 105: 847-851), adenoviral vectors can be engineered to be conditionally replicative (CRAd vectors) so as to selectively replicate in specific cells (e.g., proliferative cells). In another aspect, the adenoviral vector is replication-deficient for E1 function (e.g., by complete or partial deletion or mutation of E1). The adenoviral backbone of the vector may contain additional modifications (deletions, insertions, or mutations in one or more viral genes). Examples of E2 modifications are illustrated by thermosensitive mutations located on the gene encoding DBP (DNA binding protein) (Ensinger et al., 1972, J. Virol. 10: 328-339). Adenoviral sequences may also be deleted in whole or in part from the E4 region (see, e.g., EP974 668; Christ et al., 2000, Human Gene Ther. 11: 415-427; Lusky et al., 1999, J. Virol. 73: 8308-8319). Additional deletions within the non-essential E3 region can allow the size of the polynucleotide to be delivered 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 encoding a polypeptide (e.g., gp19k), which 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).

[0488] Second generation vectors that retain the ITRs and packaging sequences and contain extensive genetic modifications to abolish residual synthesis of viral antigens can also be used to improve long-term expression of expressed genes in transduced cells (see, e.g., WO 94 / 28152; Lusky et al., 1998, J. Virol 72:2022-2032).

[0489] The nucleic acid molecule of the present disclosure introduced into the cell can be inserted into any position of the viral genome, except for the cis-acting sequence. Preferably, it is inserted to replace the deleted region (E1, E3 and / or E4), preferably within the deleted E1 region.

[0490] Adenoviruses may be derived from any human or animal origin, in particular canine (e.g., CAV-1 or CAV-2, Genbank reference numbers 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 origin, or may be hybrid viruses. Any serotype may be used. In some cases, human adenoviruses of subgroup C are used, in particular adenoviruses 2 (Ad2) and 5 (Ad5). Such viruses may be obtained, for example, from ATCC.

[0491] As described in U.S. Pat. No. 5,928,944, adenoviral particles or empty adenoviral capsids can also be used to transfer nucleic acid molecules encoding bicistronic LAMP constructs by a virus-mediated co-internalization process. This process can be accomplished in the presence of a cationic agent such as polycarbene or a lipid vesicle comprising one or more lipid layers.

[0492] Adenoviral particles can be prepared and propagated according to any conventional technique in the art (e.g., WO 96 / 17070) using a complementing cell line or helper virus that provides the deleted viral genes required for viral replication in trans. Cell lines 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 the 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; EP919627 and WO 97 / 04119). Adenoviral particles can be recovered from the culture supernatant or from the cells after lysis and optionally further purified according to standard techniques (e.g., chromatography, ultracentrifugation, as described in WO 96 / 27677, WO 98 / 00524, WO 98 / 26048 and WO 00 / 50573).

[0493] Using vectors derived from adenoviruses with a broad host range, cell type specific targeting can be achieved by modifying viral surface proteins. For example, the infection specificity of adenoviruses is determined by attachment to cell receptors present at the surface of permissive cells. In this regard, the fibers and pentons present at the surface of adenovirus capsids play a key role in cell attachment (Defer et al., 1990, J. Virol. 64: 3661-3673). Therefore, cell targeting of adenoviruses can be performed by genetically modifying viral genes encoding fibers and / or pentons to produce modified fibers and / or pentons 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.

[0494] In one particular aspect, adeno-associated virus sequences are used as vectors. Vectors derived from human parvovirus AAV-2 (adeno-associated virus type 2) are one of the most promising gene delivery vectors currently under development. Several features of this system packaging single-stranded DNA indicate that it is a possible alternative to naked DNA delivery. Compared with 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 a 145bp inverted terminal repeat (ITR). Therefore, as in immunization using naked DNA, the only gene expressed is the gene of the antigen or antigen chimera. In addition, it is known that AAV vectors transduce both dividing and non-dividing cells, such as human peripheral blood mononuclear cell-derived dendritic cells, with sustained expression of the transferred genes, and it is possible to deliver them orally and intranasally to produce mucosal immunity. In addition, the amount of DNA required seems to be much less, reduced by several orders of magnitude, with the maximum response occurring at 10 10 Up to 10 11 DNA particles or copies, compared to 50 ug or about 10 15 copies.

[0495] In one aspect, the AAV vector is packaged by the following steps: a suitable cell line (e.g., human 293 cells) is co-transfected with DNA contained in a construct encoding an AAV ITR chimeric protein and an AAV helper plasmid ACG2 containing an AAV coding region (AAV rep and cap genes) but without the ITR. 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 verified to ensure that they do not contain detectable replication-competent AAV or adenovirus (e.g., by cytopathic effect bioassay). The AAV titer can be determined by quantitative PCR using a viral DNA sample prepared after digestion with proteinase K. Preferably, the vector titer produced by this method is about 5x10 12 Up to 1x10 13 DNase-resistant particles / ml.

[0496] In other aspects, retroviral vectors are used. Retrovirus is a class of integrative viruses that replicate using a virally encoded reverse transcriptase to replicate the viral RNA genome into double-stranded DNA, which is integrated into the chromosomal DNA of infected cells (e.g., target cells). Such vectors include vectors derived from murine leukemia viruses, particularly Moloney (Gilboa et al., 1988, Adv. Exp. Med. Biol. 241: 29) or Friend's FB29 strains (WO 95 / 01447). Typically, retroviral vectors will delete all or part of the viral genes gag, pol, and env, and retain 5' and 3' LTR and encapsidation sequences. These elements can be modified to improve the expression level or stability of retroviral vectors. Such modifications include replacing the retroviral encapsidation sequence with one of the retrotransposons, such as VL30 (see, e.g., U.S. Patent No. 5,747,323). Preferably, the nucleic acid molecules of the present disclosure are inserted downstream of the encapsidation sequence, preferably in the opposite orientation relative to the retroviral genome. As known in the art, cell-specific targeting can be achieved by coupling antibodies or antibody fragments to retroviral envelope proteins.

[0497] Retroviral particles are prepared in the presence of a helper virus or in a suitable complementing (packaging) cell line containing retroviral genes (e.g., gag / pol and env) that are defective for the retroviral vector integrated into its genome. Such cell lines have been 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 the binding of the virion to the viral receptors present on the surface of the target cell, thus determining the host range of the 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) containing amphotropic envelope proteins, to allow infection of target cells of humans and other species. The retroviral particles are preferably recovered from the culture supernatant and may optionally be further purified according to standard techniques (eg chromatography, ultracentrifugation).

[0498] Other suitable viruses include poxviruses. The genomes of several members of the Poxviridae have been mapped and sequenced. Poxvirus vectors can be obtained from any member of the Poxviridae, in particular canarypox, fowlpox and vaccinia viruses. Suitable vaccinia viruses include, but are not limited to, Copenhagen strains (Goebel et al., 1990, Virol. 179: 247-266; Johnson et al., 1993, Virol. 196: 381-401), Wyeth strains and modified Ankara (MVA) strains (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 into a non-essential locus, such as a non-coding intergenic region, or into any gene whose inactivation or deletion does not significantly impair viral growth and replication.

[0499] Poxvirus particles are prepared as described in the art (Piccini et al., 1987, Methods of Enzymology 153: 545-563; U.S. Pat. No. 4,769,330; U.S. Pat. No. 4,772,848; U.S. Pat. No. 4,603,112; U.S. Pat. No. 5,100,587 and U.S. Pat. No. 5,179,993). Typically, a donor plasmid is constructed, amplified by growth in Escherichia coli (E. coli), and isolated by conventional procedures. It is then introduced into a suitable cell culture (e.g., chicken embryo fibroblasts) along with the poxvirus genome to produce poxvirus particles by homologous recombination. These particles can be recovered from culture supernatants or 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 viruses. The viral particles can then be purified using techniques known in the art (eg, chromatography or ultracentrifugation on cesium chloride or sucrose gradients).

[0500] The use of vaccinia virus as a live viral vaccine in the global smallpox eradication campaign made vaccinia virus an obvious choice for the development of live recombinant vaccine vectors. 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 virus is particularly versatile as an expression vector because of its large genome size, its ability to accept at least 25,000 base pairs of foreign DNA, and its ability to infect most eukaryotic cell types, including insect cells (supra). Unlike other DNA viruses, poxviruses replicate only in the cytoplasm of infected cells, reducing the likelihood of genetic exchange of recombinant viral DNA with host chromosomes. Recombinant vaccinia vectors have been shown to correctly process and express proteins from a variety of sources, including humans, other mammals, parasites, RNA and DNA viruses, bacteria, and bacteriophages.

[0501] The expression of DNA encoding foreign proteins is controlled by host viral regulatory elements (including upstream promoter sequences and, if necessary, RNA processing signals). Insertion of foreign DNA into non-essential regions of the vaccinia virus genome is performed 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).

[0502] The nucleic acid molecules of the present disclosure can express polypeptides due to transcriptional regulatory elements at or near the insertion site or by more precise genetic engineering. Plasmid vectors that greatly facilitate the insertion and expression of foreign genes have been constructed (Mackett et al., J. Virol, 49: 857, 1982). These vectors contain an expression site consisting of a vaccinia virus transcriptional promoter and one or more unique restriction endonuclease sites for inserting foreign coding sequences, flanked by DNA from non-essential regions of the vaccinia virus genome. The choice of promoter determines the time (e.g., early or late) and level of expression, while the flanking DNA sequence determines the site of homologous recombination.

[0503] Only about one thousandth of the virus particles produced by this procedure are recombinant. Although recombinant virus plaques can be identified by DNA hybridization, efficient selection procedures have been developed. By using a segment of the non-essential vaccinia virus thymidine kinase (TK) gene as a flanking sequence, the foreign gene is recombined into the TK locus and the TK gene is inactivated by insertion. Selection of TK virus is achieved by performing a viral plaque assay in TK cells in the presence of 5-bromodeoxyuridine. Phosphorylation of the nucleoside analog and subsequent lethal incorporation into viral DNA only occurs in cells infected with the TK+ parental virus. Depending on the efficiency of transfection and recombination, up to 80 plaques are the desired recombinants and the rest are spontaneous TK mutants.

[0504] Plasmid vectors containing the Escherichia coli β-galactosidase gene and an expression site for a second gene allow an alternative method to distinguish recombinants from parental viruses (Chakrabarti et al., Mol. Cell. Biol., 5:3403, 1985). Plaques formed by such recombinants can be positively identified by the blue color formed after adding a suitable indicator. By combining TK selection and β-galactosidase expression, recombinant viruses are easily and quickly isolated. The recombinants are then amplified by propagation in a suitable cell line and the expression of the inserted gene is checked by appropriate enzymatic, immunological or physical procedures.

[0505] The upper limit of the amount of genetic information that can be added to the vaccinia virus genome is not known. However, the addition of nearly 25,000 base pairs of foreign DNA had no apparent deleterious 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).

[0506] Virus capsid molecules can include targeting moieties 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 (see, e.g., WO 94 / 40958), vitamins, antigens, lectins, antibodies, and fragments thereof. Preferably, such targeting molecules recognize and bind cell-specific markers, tissue-specific markers, cell receptors, viral antigens, antigenic epitopes, or tumor-associated markers.

[0507] Virus-based compositions containing bicistronic LAMP constructs can be prepared in the form of 10 to 10 14 iu (infectious unit), preferably 10 to 10 11 iu. The titer can be determined by conventional techniques. The dosage of the bicistronic LAMP construct is preferably between 0.01 and 10 mg / kg, more particularly between 0.1 and 2 mg / kg.

[0508] K. Self-replicating RNA

[0509] Self-replicating RNA viral vectors (also referred to as self-amplifying RNA viral vectors) can also be constructed using the bicistronic LAMP constructs described herein. For example, alphavirus, flavivirus, measles virus and rhabdovirus can be used to produce self-replicating RNA viral vaccines. Exemplary strains of self-replicating RNA viruses include, but are not limited to, rabies virus (RABV), vesicular stomatitis virus (VSV), West Nile virus, Kunjin virus, Semliki Forest virus (SFV), Sindbis virus (SIN) and / or Venezuelan equine encephalitis virus (VEE).

[0510] Self-replicating RNA viruses express natural antigens after delivery to tissues, thereby simulating live attenuated vaccines without the risk of restoring pathogenicity. They also stimulate the innate immune system, thereby 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) (entirely incorporated herein by reference). The use of self-replicating vaccines comprising bicistronic LAMP constructs described herein can also be used in primary immunity-boosting regimens.

[0511] In addition, as described herein, self-replicating RNA viruses can also be encapsulated by liposomes to improve delivery and targeting. Immunization with self-replicating RNA viruses comprising nucleic acid molecules described herein can provide higher levels of transient expression of antigens, leading to the generation of neutralizing antibody responses and providing protection against lethal challenges under safe conditions.

[0512] L. Cell-based delivery vehicles

[0513] According to the nucleic acid molecules disclosed herein, they can be delivered to target cells by other cells ("delivery cells") comprising the construct. Methods for introducing nucleic acid molecules into cells are known in the art and include microinjection of DNA into the nucleus (Capechi et al., 1980, Cell 22: 479-488), transfection with CaPO4 (Chen and Okayama, 1987, Mol. Cell Biol. 7: 2745 2752), electroporation (Chu et al., 1987, Nucleic Acid Res. 15: 1311-1326), lipid staining / 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 may be induced to deliver their contents to the target cells by inducing their death, for example by providing these cells with an inducible suicide gene.

[0514] M. Auxiliary molecules

[0515] Compositions comprising nucleic acid molecules according to the present disclosure may comprise one or more accessory molecules for facilitating the introduction of the nucleic acid molecules into cells and / or enhancing a particular therapeutic effect and / or increasing antibody production.

[0516] In addition, the composition may include one or more stabilizing substances, such as lipids, nuclease inhibitors, hydrogels, hyaluronidase (WO 98 / 53853), collagenase, polymers, chelating agents (EP 890362), so as to inhibit degradation in animals / humans and / or improve transfection / infection of the vector in target cells. Such substances may be used alone or in combination (e.g., cationic and neutral lipids).

[0517] It has also been shown that adenovirus proteins can destabilize endosomes and enhance the uptake of DNA in cells. A mixture of adenovirus with a solution containing a lipid-complexed DNA vector, or the combination of DNA with polylysine covalently attached to adenovirus using a protein cross-linking agent, can significantly increase the uptake and expression of bicistronic LAMP constructs comprising nucleic acid molecules (see, e.g., Curiel et al., 1992, Am. I. Respir. Cell. Mol. Biol. 6: 247-252).

[0518] N. Host cells

[0519] 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., Escherichia coli, Staphylococcus sp., Bacillus sp.); yeast cells (e.g., Saccharomyces sp.); insect cells; nematode cells; plant cells; amphibian cells (e.g., Xenopus); avian cells; and mammalian cells (e.g., human cells, mouse cells, mammalian cell lines, primary cultured mammalian cells, such as cells from dissected tissue).

[0520] The molecule can be expressed in a host cell separated from an organism, a host cell as a part of an organism, or a host cell introduced into an organism. In one aspect, the nucleic acid molecule is expressed in vitro, for example, in a host cell in culture. In another aspect, the nucleic acid molecule is expressed in a transgenic organism (for example, a transgenic mouse, rat, rabbit, pig, primate, etc.), the organism comprising somatic cells and / or germline cells, the cell comprising a nucleic acid encoding a bicistronic LAMP construct herein. The method for constructing a transgenic animal is well known in the art and is conventional.

[0521] 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 heterologous or autologous relative to the host organism. For example, cells can be obtained from the host organism, nucleic acid molecules can be introduced into the cells in vitro, and then reintroduced into the host organism.

[0522] O. Antigen presenting cells

[0523] In one aspect of the invention, the nucleic acid molecules described herein are introduced into natural or engineered antigen presenting cells.

[0524] As used herein, the term "antigen presenting cell" (APC) means any cell that presents an antigen bound to a major histocompatibility complex molecule (preferably a class II MHC molecule) or a portion thereof on its surface. Examples of suitable APCs are discussed in detail below and include, but are not limited to, intact 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.

[0525] Dendritic cells (DC) are potent antigen presenting cells. Shown, DC provides all signals required for T cell activation and proliferation. These signals can be divided into two types. The first type provides specificity for immune response, mediated by the interaction between the antigenic peptide presented by T cell receptor / CD3 ("TCR / CD3") complex and the major histocompatibility complex ("MHC", defined as above) Class I or Class II protein on the APC surface. This interaction is necessary for T cell activation to occur, but is not sufficient. In fact, in the absence of the second type of signal, the first type of signal can cause T cell anergy. The second type of signal is referred to as costimulatory signal, which is neither antigen-specific nor MHC-restricted, and in the presence of the first type of signal, can cause the complete proliferation response of T cells and the induction of T cell effector function.

[0526] Several molecules have been shown to enhance co-stimulatory activity. These molecules include, but are not limited to, heat stable antigen (HSA), chondroitin sulfate-modified MHC invariant chain (Ii-CS), intracellular adhesion molecule I (ICAM-1), and B7 co-stimulatory molecules on the surface of APCs and their counter-receptors CD28 or CTLA-4 on T cells.

[0527] Other important co-stimulatory molecules are CD40, CD54, CD80, CD86. The term "co-stimulatory molecule" as used herein covers any single molecule or combination of molecules, which, when acting together with the peptide / MHC complex bound by the TCR on the T cell surface, provides a co-stimulatory effect, and achieves the activation of the T cell bound to the peptide. Therefore, the term covers other co-stimulatory molecules on B7 or APC, fragments thereof (alone, in combination with another molecule or as part of a fusion protein), which are bound to cognate ligands together with the peptide / MHC complex, and cause T cell activation when the TCR on the T cell surface specifically binds to the peptide. Co-stimulatory molecules are commercially available from various sources, including, for example, Beckman Coulter.

[0528] In one aspect of the present disclosure, immature and mature dendritic cells are produced from peripheral blood mononuclear cells (PBMCs) of mammals (e.g., mice, monkeys, or humans) using methods described in Romani et al., J. Immunol. Methods 196: 135-151, 1996 and Bender et al., J. Immunol. Methods 196: 121-135, 1996. In brief, isolated PBMCs are pretreated using 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 produce dendritic cells. Dendritic cells are non-adherent compared to their monocyte ancestors. Therefore, at about the 7th day, non-adherent cells are harvested for further processing.

[0529] Dendritic cells derived from PBMC in the presence of GM-CSF and IL-4 are immature because they can lose their non-adhesive properties and revert to the cell fate of macrophages if cytokine stimulation is removed from the culture. Dendritic cells in an immature state are very effective in processing natural protein antigens of MHC class II restricted pathways (Romani et al., J. Exp. Med. 169: 1169, 1989). The further maturation of cultured dendritic cells is achieved by culturing for 3 days in macrophage conditioned medium (CM) containing necessary maturation factors. Mature dendritic cells have a lower ability to capture new proteins for presentation, but are much better in stimulating resting T cells (both CD4 and CD8) growth and differentiation.

[0530] Mature dendritic cells can be identified by changes in their morphology, such as the formation of more motile cytoplasmic processes; their non-adhesiveness; the presence of at least one of the following markers: CD83, CD68, HLA-DR or CD86; or the loss of Fc receptors such as CD 115 (reviewed in Steinman, Annu. Rev. Immunol. 9: 271, 1991). Mature dendritic cells can be collected and analyzed using typical cell fluorescence imaging and cell sorting techniques and devices (e.g., FACScan and FACStar). The first antibodies used for flow cytometry are those that are specific for cell surface antigens of mature dendritic cells and are commercially available. The second antibody can be a biotinylated Ig, followed by FITC or PE-coupled streptavidin.

[0531] Alternatively, others have reported a method of raising (activating) dendritic cells and converting monocytes into activated dendritic cell phenotypes. This method includes adding calcium ionophores to the culture medium to convert monocytes into activated dendritic cells. For example, calcium 21 ionophore A23187 is added at the beginning of the culture period of 24-48 hours, resulting in uniform activation and dendritic cell phenotype conversion of the "monocyte plus DC" fraction merged: characteristically, the activated population uniformly becomes CD 14 (Leu M3) negative, and HLA-DR, HLA-DQ, ICAM-1, 137.1 and 137.2 are raised. In addition, this activated mixed population also plays the role of a further purified population on the basis of a small number. The specific combination of cytokines has been successfully used to expand (or partially replace) the activation / conversion achieved using calcium ionophores: these cytokines include but are not limited to G-CSF, GM-CSF, IL-2 and IL-4. Each cytokine given alone is not enough to achieve optimal upregulation.

[0532] A second method for isolating APCs is to collect relatively large numbers of pre-typed APCs that are already circulating in the blood. Previous techniques for isolating typed APCs from human peripheral blood have involved a combination of physical procedures, such as mepanitol gradients and adhesion / non-adhesion 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 (Thomas et al., J. Immunol. 151:6840-52, 1993).

[0533] There are many other routine methods in the art for isolating professional antigen presenting cells (or precursors thereof), and such methods and other methods that may be developed are non-limiting and are encompassed within the scope of the present disclosure.

[0534] In one embodiment, the APCs, and therefore the cells presenting one or more antigens, are autologous. In another embodiment, the antigen presenting APCs are allogeneic, ie, derived from a different subject.

[0535] 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 reported a comparison of gene transfer methods in human dendritic cells.

[0536] The amino acid and nucleotide sequences (including consensus sequences) of known, partial and putative human leukocyte antigens (HLA), the genetic name for human MHC, have been published (see, e.g., Zemmour and Parham, Immunogenetics 33:310-320, 1991), and cell lines expressing HLA variants are known and commonly available, many from the American Type Culture Collection ("ATCC"). Thus, using PCR, a nucleotide sequence encoding class II MHC can be readily operably linked to an expression vector of the present disclosure, which can then be used to transform suitable cells for expression therein.

[0537] Professional APCs, such as macrophages, B cells, monocytes, dendritic cells, and Langerhans cells can be used. These cells are collected using standard procedures from 1) autologous donors, 2) allogeneic donors with HLA specificities different from the host to be treated, or 3) blood or tissues of xenogeneic donors of different species (Coligan et al., Current Protocols in Immunology, Sections 3 and 14, 1994). The cells can be isolated from normal hosts or patients with infectious diseases, cancer, autoimmune diseases, or allergies.

[0538] 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). The procedure employed avoids exposure of APCs to antigens that could be internalized by the APCs, thereby leading to the activation of T cells that are not specific for the antigen of interest.

[0539] By introducing the sequence of coding suitable molecules, non-natural antigen presenting cells can be engineered into antigen presenting cells.For example, the nucleic acid sequence encoding class II MHC molecules, auxiliary molecules, costimulatory molecules and antigen processing auxiliary molecules can be introduced after direct synthesis, cloning, DNA purification from cells containing such genes, etc. A kind of expedient means of obtaining the gene of the molecule used in the bicistronic LAMP construct and method for encoding described in this article is by using selected oligonucleotide primers to carry out polymerase chain reaction (PCR) amplification on selected nucleic acid template.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 epithelial cells, thyroid cells and renal tubular cells can be used. These cells can be primary cells that have been recently explanted from a host and have not been extensively passaged in cell culture to form an established cell line, or they can be established cell lines that are relatively homogeneous and capable of proliferation for many generations or indefinitely.

[0540] Cells that are non-professional APCs are isolated from any tissue of an autologous donor, allogeneic donor, or xenogeneic donor in which they reside using various known isolation 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 allogeneic or xenogeneic cells, can be engineered ex vivo to express class I and class II HLA molecules that match known human HLA specificities. These cells can then be introduced into human subjects that match 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.

[0541] 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 used in the present disclosure are obtained from various 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 preferably used for human subjects.

[0542] In one aspect, CD34+ precursors that differentiate into dendritic cells under the influence of GM-CSF are obtained from the subject's body, and nucleic acid molecules encoding bicistronic LAMP constructs are introduced into the cells, which are then injected into the subject. The use of nucleic acid molecules described herein will enhance the binding of peptides derived from specific antigens to class II MHC molecules on transduced antigen-presenting cells, resulting in significantly stronger systemic T cell-dependent immune responses and / or antibody production. Although the antigen-presenting cells transfected with this strategy are preferably autologous cells, as described above, any MHC class II cells that effectively present antigens in the host can be used.

[0543] P. Application

[0544] Vaccine materials according to the present disclosure may contain nucleic acid molecules encoding immunostimulatory bicistronic LAMP constructs described herein, or may be recombinant microorganisms or antigen presenting cells expressing the immunostimulatory bicistronic LAMP constructs. The preparation and use of such nucleic acid molecules for individual immunization are completed according to immunization principles well known to those skilled in the art.

[0545] By cultivating the recombinant or transformed cells containing the nucleic acid molecules, a large amount of these materials can be obtained. Culture methods are well known to those skilled in the art and are taught in one or more of the files cited above. Vaccines comprising nucleic acid molecules as described herein are usually produced by cultivating recombinant or transformed cells, and are formulated into pharmacologically acceptable solutions or suspensions, typically physiologically compatible aqueous solutions, or are formulated into coated tablets, tablets, capsules, suppositories or ampoules, as in the art, such as described in U.S. Patent No. 4,446,128, incorporated herein by reference. Administration can be any suitable route, including oral, rectal, intranasal or injection, wherein injection can be, for example, transdermal, subcutaneous, intramuscular or intravenous.

[0546] 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 administered is the amount required to elicit antibody formation to a concentration at least 4 times the concentration present before administration. A typical initial dose administered is 10 5 Up to 10 11 The amount of plaque-forming units of the recombinant vector used in the administration may be 100,0 ...

[0547] Vaccines comprising nucleic acid molecules described herein can initially be tested in non-human mammals (e.g., mice or primates). For example, the immune response assay of inoculated mice can be used to prove that the bicistronic LAMP construct has more antibodies, T cell proliferation, and cytotoxic T cell responses compared to wild-type antigens. Vaccines can be evaluated in rhesus monkeys to determine whether highly effective vaccine formulations in mice will also trigger appropriate monkey immune responses. In one aspect, each monkey receives 5 mg of nucleic acid molecules per immunization, IM delivers and is divided into 2 positions, immunization is performed at the 0th day and the 4th, 8th, and 20th week, and optionally additional administration is performed. Antibody response, 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 for the vaccine.

[0548] 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).

[0549] Q. Treatment Procedure

[0550] In one embodiment, the nucleic acid molecules of coding bicistronic LAMP construct described herein can be injected into the patient at any suitable time in the patient's malignant tumor process.For example, nucleic acid molecules described herein are injected at a stage where tumor load is low.In the optional embodiment in which the nucleic acid molecules are introduced into the antigen presenting cells of an individual, precursors or mature antigen presenting cells of antigen presenting cells are extracted from the bone marrow or peripheral blood of the individual by venipuncture.These cells are set up in culture, and then transduced with the nucleic acid molecules.In case transduction occurs, these antigen presenting cells are injected back into the patient.

[0551] In a specific embodiment, the present disclosure provides a method for treating a cancer patient with a low tumor burden, such as in the early stages of the disease, after resection of a neoplastic tumor, or when the tumor cell burden is otherwise reduced. In this method, a cell population containing autologous stem cells capable of differentiating into antigen presenting cells expressing class II MHC molecules is obtained from the patient. These cells are cultured and transformed by introducing a bicistronic LAMP construct described herein to deliver the antigen associated with the class II MHC molecule to the compartment / organelle or another compartment / organelle to which the antigen is delivered, and secrete a second antigen or IREG into the circulation.

[0552] The transfected stem cell population is then reintroduced into the patient, where the stem cells differentiate into antigen presenting cells that express T cells derived from the antigen. h Epitope complexed class II MHC molecules. Through enhanced stimulation of helper T cell populations, the immune response to the antigen will be enhanced. The secreted antigen or IREG enhances the immune response by, for example, expanding the memory response.

[0553] More generally, in one embodiment, the present disclosure provides a vaccine composition comprising a nucleic acid molecule encoding a bicistronic LAMP construct for use in modulating an immune response to an antigen in a mammal (ie, stimulating, enhancing or reducing such a response).

[0554] R. Kit

[0555] The present disclosure also includes a kit for facilitating the execution of the method described herein. In one aspect, the kit comprises a nucleic acid molecule encoding a bicistronic LAMP construct as described herein and a cell for receiving the nucleic acid molecule. In one aspect, the cell is a professional APC. The cell may or may not express a costimulatory molecule. In one aspect, when the cell does not express a costimulatory molecule, the antigen encoded by the bicistronic LAMP construct is an autoantigen. In another aspect, a group of cells expressing different MHC molecules (e.g., MHC molecules known to be expressed in humans) are provided. In another aspect, the kit comprises a reagent (e.g., a lipid-based preparation, a viral packaging material, a cell, etc.) that promotes the entry of the nucleic acid molecule into the cell. In another aspect, one or more T cell lines specific to the antigen encoded by the nucleic acid molecule are provided to verify the ability of the bicistronic LAMP construct to trigger, regulate or enhance an immune response.

[0556] S. Additional Implementations

[0557] Additional embodiments herein include the following:

[0558] 1. A nucleic acid molecule comprising:

[0559] a. a first polynucleotide sequence encoding a LAMP-antigen construct comprising an antigen and a cysteine ​​conserved fragment of a LAMP protein; and

[0560] b. a second polynucleotide sequence encoding at least one IREG polypeptide operably linked to a secretory signal sequence;

[0561] c. wherein the first and second polynucleotide sequences are operably linked to expression control sequences for expressing the LAMP-antigen construct and IREG in a host or target cell.

[0562] 2. The nucleic acid molecule of embodiment 1, wherein:

[0563] a. the antigen is placed at the N-terminus of the cysteine ​​conserved fragment;

[0564] b. the antigen is placed at the C-terminus of the cysteine ​​conserved fragment; or

[0565] c. The antigen is placed between two cysteine ​​conserved segments.

[0566] 3. A nucleic acid molecule comprising:

[0567] a. a first polynucleotide sequence encoding a LAMP-antigen construct comprising an antigen placed between two cysteine ​​conserved segments; and

[0568] b. a second polynucleotide sequence encoding at least one IREG polypeptide operably linked to a secretory signal sequence;

[0569] c. wherein the first and second polynucleotide sequences are operably linked to expression control sequences for expressing the LAMP-antigen construct and the IREG polypeptide in a host or target cell.

[0570] 4. A nucleic acid molecule according to any one of the preceding embodiments, wherein the improved LAMP construct comprises Figure 1 The structure of ILC-1, ILC-2, ILC-3, ILC-4, ILC-5 or ILC-6 shown in (i.e., wherein the polypeptide encoded by the first polynucleotide sequence comprises Figure 1 The structure of ILC-1, ILC-2, ILC-3, ILC-4, ILC-5 or ILC-6 shown in FIG.

[0571] 5. The nucleic acid molecule according to any one of the preceding embodiments, wherein the cysteine ​​conserved fragment comprises a homologous domain of a LAMP protein.

[0572] 6. The nucleic acid molecule according to any one of the preceding embodiments, wherein the improved LAMP construct further comprises a transmembrane domain of a LAMP protein.

[0573] 7. The nucleic acid molecule according to any one of the preceding embodiments, wherein the improved LAMP construct further comprises a signal sequence.

[0574] 8. The nucleic acid molecule of embodiment 7, wherein the signal sequence is derived from a LAMP protein.

[0575] 9. The nucleic acid molecule according to any one of the preceding embodiments, wherein the antigen comprised in the LAMP-antigen construct is placed in or instead of the LAMP hinge region.

[0576] 10. The nucleic acid molecule according to any one of the preceding embodiments, wherein the LAMP protein is selected from LAMP-1, LAMP2, LAMP-3, LIMP 2, Macrosailin, Endolyn, LAMP5 or LIMBIC.

[0577] 11. The nucleic acid molecule according to embodiment 10, wherein the LAMP protein is selected from any one of SEQ ID NOs: 1-113.

[0578] 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.

[0579] 13. The nucleic acid molecule according to any of the preceding embodiments, wherein expression of the LAMP-antigen construct in a host or target cell results in processing and presentation of the antigen to the MHC class II pathway to elicit an immune response.

[0580] 14. The nucleic acid molecule according to any one of the preceding embodiments, wherein expression of the IREG in a host or target cell results in secretion of the IREG.

[0581] 15. The nucleic acid molecule of embodiment 14, wherein the secretion of the IREG can enhance the immune response elicited by the antigen.

[0582] 16. A nucleic acid molecule according to any one of embodiments 1-15, wherein the antigen is an antigen associated with an infectious disease such as Covid-19, an antigen associated with 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.

[0583] 17. The nucleic acid molecule according to any one of the preceding embodiments, wherein the nucleic acid molecule is a plasmid or a vector.

[0584] 18. The nucleic acid molecule of embodiment 17, wherein the nucleic acid molecule is a viral vector.

[0585] 19. A host cell comprising the nucleic acid molecule according to any one of embodiments 1 to 18.

[0586] 20. A composition comprising the nucleic acid molecule according to any one of embodiments 1 to 18 or the host cell according to embodiment 19.

[0587] 21. A method for treating a subject having a disease or condition, wherein the method comprises administering to a subject in need thereof a nucleic acid molecule according to any one of embodiments 1-18, a host cell according to embodiment 19, or a composition according to embodiment 20 in an amount sufficient to alleviate or treat the disease or condition.

[0588] Example

[0589] The present disclosure will now be further illustrated with reference to the following examples. It should be understood that the following is merely exemplary and that modifications may be made to the details while still falling within the scope of the present disclosure.

[0590] Example 1. Construction of a vector encoding a bicistronic LAMP construct

[0591] A first generation COVID-19 vaccine candidate was designed that encodes one COVID-19 antigen as a LAMP fusion protein and a second COVID-19 antigen as a secreted protein. The construct was named ITI-COVID-19 bicistronic construct (ITI-Bicistronic-S1-LAMP-RBG pA-EF1-S2P BGH pA; SEQ ID NO: 228, Fig.28C ), and was shown to induce both SARS-Cov-2 specific antibodies and T cell responses. To obtain the best antibody and T cell responses, the dose of the vaccine (20 μg) and the length of time between the primary immunization and the booster (e.g., 14 days) were studied. After two immunizations with 20 μg of the ITI-COVID-19 bicistronic vaccine, strong SARS-Cov-2 spike-specific T cells and antibodies were induced. Importantly, the ITI-COVID-19 bicistronic vaccine elicited an antibody response that neutralized SARS-Cov-2.

[0592] The S1 and S2 subunits of the spike mediate the entry of SARS-CoV-2 viruses into host cells. Using nucleic acid molecules for ILC-4LAMP constructs, the S1 coding sequence (GenBank MN908974) is located between polynucleotide sequences encoding two LAMP homologous domains (N-LAMP and endodomain 2). The S1 coding sequence is operably connected to the CMV promoter under the influence of the CMV enhancer sequence, so that the expression in the host cell produces an ILC-4LAMP construct comprising the S1 antigen, and the S1 antigen is used for processing and presentation to class II MHC molecules (i.e., providing "primary immunity antigen"). The S2 coding sequence is provided elsewhere on the carrier, and is operably connected to the polynucleotide sequence (leader sequence) and EF1 promoter sequence encoding Ig-κ secretion signal, so that the expression in the host cell produces S2 antigen for secretion (i.e., providing "enhanced antigen"). Therefore, the carrier provides a single nucleic acid molecule for being introduced into a suitable host or target cell, which can provide both primary immunity and enhanced antigens to trigger an enhanced immune response. This may therefore provide significant advantages over the use of a vector encoding only the bicistronic LAMP construct, as any need or requirement to boost the immune response elicited by the LAMP construct requires the administration of a booster vaccine (e.g. comprising the antigen) administered separately at one or more time intervals.

[0593] The first generation ITI-COVID-19 bicistronic vaccine expresses the S1 and S2 subunits of the spike glycoprotein anchored on the surface of the virus. The S1 and S2 subunits of the spike mediate the entry of the SARS-CoV-2 virus into the host cell. Using the nucleic acid molecule for the ILC-4LAMP construct, the S1 coding sequence (GenBank MN908974) is located between the polynucleotide sequences encoding two LAMP homologous domains (N-Lamp and endodomain 2). The S1 coding sequence is operably connected to the CMV promoter under the influence of the CMV enhancer sequence, so that the expression in the host cell produces an ILC-4LAMP construct containing the S1 antigen, which is used for processing and presentation to class II MHC molecules (i.e., providing "primary immunity antigen"). The S2 coding sequence is provided elsewhere on the vector and is operably connected to the polynucleotide sequence (leader sequence) encoding the Ig-κ secretion signal and the EF1 promoter sequence, so that the expression in the host cell produces the S2 antigen for secretion (i.e., providing "boosting antigen"). Thus, the vector provides a single nucleic acid molecule for introduction into a suitable host or target cell that can provide both the target and the booster antigen to elicit an enhanced immune response. Thus, this can provide significant advantages over the use of vectors encoding only the bicistronic LAMP construct, since any need or requirement to boost the immune response elicited by the LAMP construct requires administration of a booster vaccine (e.g., comprising the antigen) administered separately at one or more time intervals.

[0594] The polynucleotide sequence of this first generation COVID-19 bicistronic construct is shown in Fig.28C The DNA sequences of S1 and S2 were obtained from, for example, Genbank (ncbi.nlm.nih.gov / genbank / sars-cov-2-seqs / #nucleotide-sequences). Fig.28A In the vector shown, the S2 polynucleotide sequence encodes an S2 variant including two proline (P) substitutions. Specifically, the ITI-COVID-19 bicistronic vaccine (first generation vaccine) expresses the S1-LAMP sequence described in more detail below ( Fig.28C 950-2004 residues of SEQ ID NO: 227 shown in ) and the spike protein with an Ig-κ leader sequence (residues 16-1273) (see Fig.28C ; residues 2145-3423 of SEQ ID NO:227).

[0595] Table 1 below provides exemplary DNA and protein sequences for the above-described bicistronic LAMP constructs, as well as various exemplary promoter / enhancer sequences and polypeptide sequences that can be used to construct the bicistronic LAMP constructs described herein.

[0596] The following additional bicistronic LAMP constructs were constructed using standard molecular biology techniques known to those skilled in the art: (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), 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.

[0597] HER2-LAMP-sCD40L expresses the HER2-LAMP polypeptide SEQ ID NO: 195 and the mSPD-sCD40L fusion protein SEQ ID NO: 196. HER2-LAMP-mFLT3L expresses the HER2-LAMP polypeptide (SEQ ID NO: 195) and the SPD-mFLT3L polypeptide SEQ ID NO: 207. HER2-LAMP-IL-12 expresses the HER2-LAMP polypeptide (SEQ ID NO: 195) and the murine IL-12p36-P2A-IL-12p40 polypeptide SEQ ID NO: 213. HER2-LAMP-IL-21 expresses the HER2-LAMP polypeptide (SEQ ID NO: 195) and the murine IL-21 polypeptide SEQ ID NO: 217. HER2-LAMP-OX40L expresses the HER2-LAMP polypeptide (SEQ ID NO: 195) and the OX40L extracellular domain (ECD) Fc fusion protein with the murine IL-2 signal peptide (SP) SEQ ID NO: 242. HER2-LAMP-CD80 expresses the HER2-LAMP polypeptide (SEQ ID NO: 195) and the CD80 ECD with the murine IL-2 SP SEQ ID NO: 252. Spike-LAMP-sCD40L expresses the Spike-LAMP polypeptide SEQ ID NO: 229 and the SPD-sCD40L polypeptide SEQ ID NO: 233. Below is a table illustrating these and other sequences. Compared to the U.S. provisional priority application, SEQ ID NO: 195 has been updated to reflect that the HER2-LAMP polypeptide sequence comprises SEQ ID NO: 198, followed by SEQ ID NO: 200, followed by SEQ ID NO: 202, and that the repeated HER2-LAMP sequence included in the provisional priority application has been deleted without changing the overall numbering of the surrounding sequence. SEQ ID NOs: 221 and 229 have been similarly updated to reflect that the NY-ESO1-LAMP and Spike-LAMP polypeptide sequences comprise SEQ ID NO: 198, followed by the NY-ESO1 antigen or the Spike antigen, followed by SEQ ID NO: 202.

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[0661] Example 2. Second-generation bicistronic COVID vaccine (2-V-Covid) elicits T cell and antibody responses in mice

[0662] The main challenge in generating sufficient antigen-specific T cell and antibody responses is the low immunogenicity of DNA vaccines. In order to improve the T cell response to DNA vaccines, a new bicistronic DNA vaccine was designed. This example discusses the second generation COVID-19 DNA vaccine, namely Spike-LAMP-sCD40L (2-V-Covid vaccine; SARS-CoV-2B.1.351 Spike (South African variant, without SP and TM) + LAMP + EF-1α + SPD + sCD40L; SEQ ID NO: 242-243), which contains (1) full-length SARS-CoV-2 spike protein fused to LAMP, and (2) soluble CD40L (sCD40L), both expressed separately. This bicistronic DNA vaccine is designed to induce local expression of sCD40L at relatively low levels, thereby providing a safe method for using systemic recombinant CD40L or agonistic anti-CD40 antibodies (van Mierlo et al., 2002).

[0663] 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). In order to produce a COVID vaccine with improved immunogenicity, a second-generation vaccine was designed to express the SARS-CoV-2 full-length spike protein and soluble CD40L in two separate expression 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 antibody responses compared to the first-generation COVID-19 DNA vaccine described in Example 1. The data discussed in the examples below show that the new vaccine has higher immunogenicity and has the potential to improve protection against COVID 19 and future emerging infectious diseases.

[0664] CD40L is a transmembrane protein expressed on the surface of activated T cells, particularly CD4 T cells. CD40L stimulates CD40-dependent activation of antigen presenting cells (APCs) such as dendritic cells (DCs) and macrophages and B cells to enhance T cell and antibody responses (Grewal and Flavell, 1998; Schoenberger et al., 1998). Recombinant soluble CD40L or agonistic antibodies have been used clinically and have shown promising results in various cancers (Beatty et al., 2011, 2017; Vonderheide et al., 2001). Therefore, these immunostimulatory functions of CD40L make it a promising vaccine adjuvant against infectious diseases and cancer. As discussed herein and in the examples below, sCD40L enhances spike-specific T cells and antibody responses. The data discussed herein support the use of bicistronic DNA vaccines to combat infectious diseases.

[0665] A. Materials and Methods

[0666] 1. Vaccine Constructs

[0667] 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 Fig.28C , SEQ ID NO: 228) was previously constructed as described in Example 1 above.

[0668] The second generation spike-LAMP-sCD40L construct "2-V COVID vaccine" (SEQ ID NO: 228) was also constructed according to the method described in Example 1 above. The 2-V COVID vaccine has two independent expression cassettes, independently driven by the CMV promoter and the EF1 promoter. The first expression cassette expresses the full-length spike gene of the infectious clone of the SARS-CoV-2 South African variant (B.1.351) (SEQ ID NO: 227). The second expression cassette expresses the soluble mouse CD40L extracellular domain fused to the mouse lung surfactant-associated protein D (SPD) protein (SEQ ID NO: 233).

[0669] The control vector (CV) used was a vector without any gene insert.

[0670] 2. Reagents

[0671] Antibodies for flow cytometry and ELISPOT were purchased from Biolegend, and SA-HRP and AEC kits were purchased from BD. Antibodies against the spike protein and recombinant spike (S1) (Catalog No. 40591-V08H) and RBD (Catalog No. 405092-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. The Renilla luciferase reporter assay system was purchased from Promega (Catalog No. E2710). Epivax peptides were synthesized by GenScript.

[0672] Pre-prepared lentiviral particles for human ACE2 overexpression were ordered from GenTarget Inc (Catalog No. LvP1310).

[0673] JPT PepMix TM SARS-CoV-2 overlapping peptide pools were purchased from JPT (Catalog No. PM-WCPV-S). The peptide pool contained a total of 315 peptides (delivered in two sub-pools of 158 and 157 peptides) derived from peptide scanning of the spike (15mer segments with 11 amino acid overlaps) (UniProt: P0DTC2).

[0674] Pseudo-luciferase rSARS-Cov-2 spike virus was purchased from Creative Diagnostics (No. Cov-PS01, Lot No. CL-114A).

[0675] 3. Transient Transfection of Constructs

[0676] 293T cells were transiently transfected with control vector (CV) or bicistronic vaccine constructs using lipofectamine 2000 (Invitrogen). 48 hours after transfection, supernatants were collected, centrifuged and filtered. The supernatants were analyzed for expression of soluble CD40L.

[0677] In some embodiments, 293T cells can be transduced with human ACE2. 293T-ACE2 cell lines are prepared by transducing 293T cells with a lentivirus expressing human ACE2 (angiotensin I converting enzyme 2, NM_021804), which is prepared by GeneTarget Inc, containing RFP and blasticidin dual selection markers. After infection, 15ug / ml of blasticidin is used to separate single cell clones expressing both RFP and ACE. As shown, the expression of human ACE2 is detected by staining with anti-human ACE2 antibody (SinoBiological, rabbit Pab, catalog number 10108-T60). 293T-ACE3 clone 2 and clone 5 have higher hACE2 expression, and clone 5 is used for neutralization experiments in this study.

[0678] 4. Western Blotting and Immunoprecipitation Methods

[0679] Biotinylated anti-CD40L antibody (clone MR1 BioLegend catalog number 106503) was bound to streptavidin-coated magnetic beads for separation of CD40L. The beads were loaded onto gels for Western blot imaging. The following antibodies were also used for Western blots: (1) anti-CD40L / CD154 primary antibody (Invitrogen PA5-78983) and goat anti-rabbit IgG-HRP secondary antibody (Southern Biotech 4030-05).

[0680] 5. Immunization and Serum Collection

[0681] Female BALB / c mice, 6 to 8 weeks old, were bred and maintained in a licensed animal facility.

[0682] The immunization schedule for the ITI-bicistronic vaccine and the 2-V COVID vaccine is shown in Table 2. Mice were immunized with DNA vaccines by ID injection into 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 planned and spleens and serum were collected for measurement of T cell and antibody responses.

[0683]

[0684] 6. Assessment of Antigen-specific T Cell Responses by ELISPOT

[0685] To evaluate antigen-specific T cell responses in vaccinated mice, antigen-specific IFNγ was assessed in splenocytes from vaccinated mice by enzyme-linked immunospot (ELISPOT). The ELISPOT assay was performed as described herein. Briefly, splenocytes were plated at 3×10 5 The cells were plated at a density of 10 cells / well and co-cultured with 1 μg / ml of JPT overlapping spike peptide, 0.25 μg / mL of concanavalin A or culture medium alone in a total volume of 200 μl / well of T cell culture medium at 37°C and 5% CO2 for 48 hours. The plates were developed for up to 30 minutes with 50 μl / well of AEC color development solution. The color development was stopped by washing under running tap water. After air drying, the colored spots were counted using the AID ELISPOT high-resolution plate reader system and AID ELISPOT software version 3.5 (Autoimmun Diagnostika GmbH). The Student T test was used to determine the significant differences between cells transfected with the ITI-bicistronic vaccine and the 2-V COVID vaccine.

[0686] 7.ELISA

[0687] The antibody response of mice to the vaccine was evaluated by indirect ELISA. ELISA plates (MaxiSorp) were coated overnight with 1 μg / ml recombinant SARS-Cov-2 spike S1 or RBD protein and then blocked with 2% BSA in PBS. Serum samples were diluted in PBS-T (1:2). Samples were detected with 1:6000 goat anti-mouse IgG-HRP (Southern Biotech, Birmingham, Al). The reaction was developed with SureBlue TMB substrate and terminated with TMB stop solution from KPL (Gaithersburg, MD). The plate was read (OD450) using an Epoch ELISA reader (BioTek, Winooski, VT).

[0688] 8. SARS-Cov-2 pseudovirus neutralization test (pVNT)

[0689] In some embodiments, a SARS-CoV-2 pseudovirus neutralization assay is used. Pseudoluciferase rSARS-CoV-2 spike was purchased from Creative Diagnostics Inc. This lentiviral-based SARS-CoV-2S pseudovirus is a replication-restricted recombinant pseudotyped lentiviral particle containing the SARS-CoV-2 spike protein. Since the infectivity of pseudoluciferase rSARS-CoV-2 is limited to a single round of replication, it encodes Renilla luciferase in the lentiviral vector genome. When its genome is integrated after entering the cell, the expression and activity of luciferase are proportional to the number of transduced cells.

[0690] To determine pVNT, 20 μL of SARS-CoV-2 spike pseudotyped virus (10 5 RLU) were pre-incubated with two-fold serial dilutions of test serum samples (starting at 1:10) in a final volume of 50 μL at 37°C for 1 hour, followed by the addition of 50 μL of 5 x 10 4 HEK293T-ACE2 cells. 48-72 hours after infection, an equal volume of Renilla luciferase substrate (Promega, catalog number 2710) was added, and the luminescent signal was measured using a microplate reader (BioTek) with Gen5 software. Measurements can be performed in duplicate for CV combined serum samples and in triplicate for B group combined serum samples. Neutralization % and pVNT IC50 were calculated as previously described (Le Bert et al., 2020).

[0691] 9. Flow Cytometry and Intracellular Cytokine Staining (ICS)

[0692] Splenocytes were stimulated with spike peptide at a concentration of 2ug / mL for 6 hours. After 6 hours of incubation, cells were stained with Zombie aqua, followed by surface staining, fixed with Perm / fixation solution (BD Biosciences), and stained with intracellular antibodies in 1x Perm / wash buffer. Samples were analyzed on a CytoFlex flow cytometer (Beckman Coulter) and analyzed using Kaluza software (Beckman Coulter).

[0693] Cells were stained for intracellular cytokines 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.

[0694] B. Results

[0695] 1.2-V COVID vaccine expressing soluble CD40 ligand (sCD40L)

[0696] 293T cells were transfected with 2-V COVID vaccine and analyzed for sCD40L expression. Using immunoprecipitation and Western blotting methods, expression of sCD40L from 2-V COVID vaccine was confirmed. Fig.12 ; boxed band indicates sCD40L).

[0697] 2.2-V COVID vaccines trigger T cell responses

[0698] 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 showed that the 2-V COVID vaccine induced significantly enhanced T cell responses compared to the first generation COVID-19 vaccine ( Fig.13A -D). The data suggest that the presence of sCD40L improves the 2-VCOVID vaccine by eliciting a greater T cell response.

[0699] 3.2-V COVID vaccines stimulate spike-specific CD4 and CD8 T cells in vivo

[0700] A greater number of CD4+ and CD8+ T cells were observed in the splenocytes of mice vaccinated with the 2-V vaccine compared with the ITI-bicistronic vaccine ( Fig.14 The data suggest that the presence of sCD40L provides the enhancement. Intracellular cytokine staining was also performed on CD4+ and CD8+ T cells, and the percentages of IFNγ, TNFα, and IL-2 were greater in mice vaccinated with the 2-V vaccine compared to the ITI-bicistronic vaccine ( Fig.15 ).

[0701] 4.2-V COVID vaccines induce spike-specific antibody responses in vivo

[0702] S1-specific antibodies were analyzed by ELISA in sera from immunized mice after one or two immunizations. Fig.16A -B shows total IgG of S1 binding antibodies. Fig. 16C -D shows IgG2a antibody. Fig.16E -F shows IgG1 antibodies. The data suggest that the 2-V COVID vaccine elicited superior S1-specific antibody responses compared to the ITI-bicistronic vaccine. The IgG1 response was particularly pronounced after a single dose of the 2-V COVID vaccine ( Fig.16E ).

[0703] 5. Summary

[0704] Compared with the first generation vaccine, the 2-V vaccine significantly enhanced spike-specific T cell responses, including both CD4+ and CD8+ T cell responses. After a single immunization, the 2-V vaccine also enhanced S1-specific antibody responses, especially IgG1 levels.

[0705] Example 3. Expression of soluble CD40L (sCD40L) from HER2-LAMP-sCD40L bicistronic vaccine

[0706] This example discusses HER2-LAMP-sCD40L ( Fig.11 ; SEQ ID NO: 197), a bicistronic DNA vaccine encoding HER2-LAMP and a 4-trimeric version of sCD40L. The data discussed herein support the use of bicistronic DNA vaccines against cancer.

[0707] CD40 ligand (CD40L) is a transmembrane protein expressed on the surface of activated T cells, particularly CD4 T cells, which stimulates the CD40-dependent activation of antigen presenting cells (APCs), resulting in the enhancement of T cells and antibody responses. The soluble polymer form of CD40L (sCD40L) can serve as an adjuvant to enhance the immunogenicity of the vaccine. As described in this embodiment, the HER2-LAMP-sCD40L bicistronic construct was tested for constructs expressing HER2-LAMP but not expressing any second polypeptide (e.g., sCD40L). Compared with mice immunized with control HER2-LAMP DNA, HER2-LAMP-sCD40L triggered significantly enhanced HER2-specific T cells and antibody responses in mice. Intracellular staining showed that the inclusion of sCD40L in the vaccine induced the production of potent antigen-specific T cells (IFNγ), mainly in CD4 T cells. Furthermore, in the mouse TSA breast cancer model, HER2-LAMP-sCD40L significantly inhibited tumor growth and prolonged survival in the context of a therapeutic vaccine, indicating that the HER2-LAMP-sCD40L vaccine is an effective strategy to promote anti-tumor efficacy in vivo.

[0708] A. Materials and Methods

[0709] In general, the materials and methods used in this example were as described above in Examples 1-2, with the following modifications.

[0710] 1. Vaccine Constructs

[0711] In the examples discussed herein, the HER2-LAMP-sCD40L bicistronic construct ( Fig.11; SEQ ID NO: 197) contains two expression cassettes. The first expression 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 expression cassette is driven by the EF1 promoter to express the soluble murine CD40 ligand (sCD40L; GenBank Accession No. X65453.2), which encodes a 4-trimeric soluble CD40L fused to the body of surfactant protein D (SPD) (Gómez et al., 2009; Stone et al., 2006) (SEQ ID NO: 196). There are three amino acids HRR between SPD and sCD40L. The construct is designed to deliver HER2 antigen to the MHC II compartment, which can enhance both antibody production and CD4 T cell responses, while the sCD40L polypeptide construct is secreted.

[0712] The HER2-LAMP construct without sCD40L was used as a control vaccine.

[0713] A bicistronic DNA vaccine encoding the spike (Spike-LAMP) was used as a control vector (ie, negative contr...

Claims

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 the LAMP protein (collectively referred to as a "LAMP-antigen construct"), wherein the antigenic domain is placed between the two homologous domains; and b. A second polypeptide sequence encoding at least one second polypeptide comprising an immune response enhancing gene polypeptide (IREG) or an extracellular domain of IREG comprising a secretion signal sequence.

2. The isolated nucleic acid molecule of claim 1, wherein the LAMP protein is selected from LAMP-1, LAMP2, LAMP-3, lysosomal integral membrane protein-2 ("LIMP 2"), Macrosailin, Endolyn, LAMP5, or limbic system-associated membrane protein ("LIMBIC").

3. The isolated nucleic acid molecule of claim 2, wherein the LAMP protein is selected from any one of SEQ ID NOs: 1-113.

4. The isolated nucleic acid molecule of claim 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 claim 2, wherein the LAMP protein is LAMP-1, and wherein the two homology domains of the LAMP-antigen construct include LAMP-1 homology domain 1 and LAMP-1 homology domain 2. 6 . The isolated nucleic acid molecule according to claim 5 , 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 29-195 of SEQ ID NO:

198.

7. The isolated nucleic acid molecule according to claim 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.

8. The isolated nucleic acid molecule according to any one of claims 1-7, 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 claim 8, wherein the linker comprises the amino acid sequence of GPGPG or PMGLP.

10. The isolated nucleic acid molecule according to any one of claims 1-9, wherein the LAMP-antigen construct further comprises a transmembrane domain of a LAMP protein.

11. The isolated nucleic acid molecule of claim 10, wherein the transmembrane domain comprises residues 383-405 of SEQ ID NO:

1.

12. The isolated nucleic acid molecule of any one of claims 1-11, wherein the LAMP-antigen construct further comprises a signal sequence.

13. The isolated nucleic acid molecule of claim 12, wherein the signal sequence is derived from a LAMP protein, such as a signal sequence comprising residues 1-28 of SEQ ID NO: 1 or residues 1-28 of SEQ ID NO:

198.

14. The isolated nucleic acid molecule of any one of claims 1-13, wherein the LAMP-antigen construct further comprises a cytoplasmic domain of a LAMP protein.

15. The isolated nucleic acid molecule of claim 14, wherein the cytoplasmic domain comprises residues 406-417 of SEQ ID NO:

1.

16. The isolated nucleic acid molecule of any one of claims 1-15, wherein 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 an extracellular domain thereof, optionally wherein the CD40L, CD80, OX40, Flt3L, GM-CSF, IL-12, IL-21, IL-23 or IL-15 is fused to an Fc domain of an immunoglobulin.

17. The isolated nucleic acid molecule of any one of claims 1-16, wherein the secretion signal sequence is heterologous to the IREG.

18. The isolated nucleic acid molecule of claim 17, wherein the secretory signal sequence is derived from IgKVIII, Ig-κ, tetranectin or IL-2, and / or wherein the second polypeptide further comprises surfactant-associated protein D (SPD).

19. The isolated nucleic acid molecule of claim 18, wherein the second polypeptide is expressed under the control of an EF-1 alpha core promoter, such as the promoter of SEQ ID NO:

124.

20. A composition comprising the isolated nucleic acid molecule of any one of claims 1-18.

21. A host cell comprising the isolated nucleic acid of any one of claims 1-18.

22. A composition comprising the host cell according to claim 20.

23. A method of treating a subject suffering from a disease or condition or inducing an immune response in a subject suffering from a disease or condition or at risk of developing a disease or condition, wherein the method comprises administering to the subject an isolated nucleic acid molecule according to any one of claims 1-18, a composition according to claim 19, or a host cell according to claim 20 in an amount sufficient to treat the disease or condition or induce an immune response in the subject.

24. The method of claim 22, wherein the method further comprises administering to the subject at least one second therapeutic agent.

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 the extracellular domain of HER2 (collectively referred to as "HER2-LAMP"), wherein the antigenic domain is placed between the two LAMP homologous domains; and b. A second polypeptide sequence encoding at least one second polypeptide comprising an immune response enhancing gene polypeptide (IREG) or an extracellular domain of IREG comprising a secretion signal sequence.

26. The isolated nucleic acid molecule of claim 25, wherein the LAMP protein is selected from LAMP-1, LAMP2, LAMP-3, lysosomal integral membrane protein-2 ("LIMP 2"), Macrosailin, Endolyn, LAMP5, or limbic system-associated membrane protein ("LIMBIC").

27. The isolated nucleic acid molecule of claim 26, wherein the LAMP protein is selected from any one of SEQ ID NOs: 1-113.

28. The isolated nucleic acid molecule of claim 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 claim 26, wherein the LAMP protein is LAMP-1, and wherein the two homology domains of HER2-LAMP include LAMP-1 homology domain 1 and LAMP-1 homology domain 2.

30. The isolated nucleic acid molecule of claim 29, wherein the human LAMP-1 homology domain 1 comprises the amino acid sequence of residues 29-194 of SEQ ID NO: 1 or residues 29-195 of SEQ ID NO:

198.

31. The isolated nucleic acid molecule according to claim 29 or 30, wherein the human LAMP-1 homology domain 2 comprises the amino acid sequence of residues 228-381 of SEQ ID NO: 1, or comprises the amino acid sequence of SEQ ID NO:

202.

32. The isolated nucleic acid molecule of any one of claims 25-31, wherein the HER2-LAMP comprises a linker between at least one of the two homologous domains and the antigenic domain.

33. The isolated nucleic acid molecule of claim 32, wherein the linker comprises the amino acid sequence of GPGPG or PMGLP.

34. The isolated nucleic acid molecule of any one of claims 25-33, wherein the HER2-LAMP further comprises a transmembrane domain of a LAMP protein.

35. The isolated nucleic acid molecule of claim 34, wherein the transmembrane domain comprises residues 383-405 of SEQ ID NO:

1.

36. The isolated nucleic acid molecule of any one of claims 25-35, wherein the HER2-LAMP further comprises a signal sequence.

37. The isolated nucleic acid molecule of claim 36, wherein the signal sequence is derived from a LAMP protein, such as a signal sequence comprising amino acid residues 1-28 of SEQ ID NO: 1 or residues 1-28 of SEQ ID NO:

198.

38. The isolated nucleic acid molecule of any one of claims 25-37, wherein the HER2-LAMP further comprises a cytoplasmic domain of a LAMP protein.

39. The isolated nucleic acid molecule of claim 38, wherein the cytoplasmic domain comprises residues 406-417 of SEQ ID NO:

1.

40. The isolated nucleic acid molecule of any one of claims 25-39, wherein the antigenic domain comprises or consists of the amino acid sequence of SEQ ID NO:

200.

41. The isolated nucleic acid molecule of any one of claims 25-40, wherein the HER2-LAMP comprises or consists of residues 1-194 of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 198, followed by the amino acid sequence of SEQ ID NO: 200, followed by residues 228-381 of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO:

202.

42. The isolated nucleic acid molecule of any one of claims 25-41, wherein 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 an extracellular domain thereof, optionally wherein the CD40L, CD80, OX40, Flt3L, GM-CSF, IL-12, IL-21, IL-23 or IL-15 is fused to an Fc domain of an immunoglobulin.

43. The isolated nucleic acid molecule of any one of claims 25-42, wherein the secretion signal sequence is heterologous to the IREG.

44. The isolated nucleic acid molecule of claim 43, wherein the secretory signal sequence is derived from IgKVIII, Ig-κ, tetranectin or IL-2.

45. The isolated nucleic acid molecule of any one of claims 25-44, wherein 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.

46. ​​The isolated nucleic acid molecule of claim 45, wherein the second polypeptide is expressed under the control of an EF-1 alpha core promoter, such as the promoter of SEQ ID NO:

124.

47. A composition comprising the isolated nucleic acid molecule of any one of claims 25-46.

48. A host cell comprising the isolated nucleic acid of any one of claims 25-46.

49. A composition comprising the host cell according to claim 48.

50. A method of treating a subject having cancer, wherein the method comprises administering to the subject an isolated nucleic acid molecule according to any one of claims 25-46, a composition according to claim 47, or a host cell according to claim 48 in an amount sufficient to treat the cancer or induce an immune response against the cancer in the subject.

51. The method of claim 50, wherein the method further comprises administering to the subject at least one second therapeutic agent.

52. The isolated nucleic acid of any one of claims 1-19 or 25-46, wherein the isolated nucleic acid comprises DNA, mRNA, or self-amplifying RNA.

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