IL-21, IL-15, and IL-12 polypeptides, compositions comprising them, and methods of their use

Abstract

The present application provides a polypeptide comprising an IL-21 polypeptide, an IL-12 polypeptide, and / or an IL-15 polypeptide, and / or an IL-15 receptor polypeptide; an immune checkpoint polypeptide; compositions comprising them; and methods of their use.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 375,469, filed on September 13, 2022, which is incorporated herein by reference in its entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing that is submitted electronically as an XML file named "260034.000702ST.26Sequence Listing XML", created on September 13, 2023, and having a size of 120,860 bytes. The sequence listing is hereby incorporated by reference in its entirety. Technical Field

[0005] The embodiments provided herein relate to polypeptides comprising IL - 21 polypeptide, IL - 15 polypeptide, and IL - 12 polypeptide, compositions comprising them and immune checkpoint inhibitors, and methods of using them. Background Art

[0006] Interleukin 21 (IL - 21), interleukin 12 (IL - 12), and interleukin 15 (IL - 15) are cytokines that regulate and activate the immune response to treat conditions such as cancer and infection. However, there is a need for improved polypeptides that can be used to stimulate the immune system in a local or systemic manner.

[0007] Immune checkpoint molecules are accessory molecules that promote or inhibit T - cell activation. Two inhibitory molecules include cytotoxic T - lymphocyte antigen 4 (CTLA - 4) and programmed cell death protein 1 (PD - 1). The PD - 1 ligands (PD - L1 and PD - L2) expressed on cancer and other cells interact with PD - 1 on T cells to inhibit T - cell killing of tumor cells. Similarly, B7 family proteins (B7.1 and B7.2) expressed on cancer and other cells interact with CTLA - 4 on T cells and inhibit the activity of T cells. Monoclonal antibody therapies against both PD - 1 and CTLA - 4 have been developed and have shown promise in treating tumors in pre - clinical and clinical studies. However, most patients do not have a sustained anti - tumor response with these therapies. Therefore, there is a need for improved therapies that utilize this key immune checkpoint signaling link.

[0008] The embodiments of the present invention meet these needs and others. Summary of the Invention

[0009] Polypeptides comprising IL-21, IL-12, and IL-15 are provided. In some embodiments, polypeptides comprising the following formula are provided: X1-L1-X2-L2-X3-L3-X4-L4-X5, wherein X1, X2, X3, X4, and X5 are each independently an IL-21 polypeptide, an IL-15 polypeptide, an IL-12p40 polypeptide, an IL-12p35 polypeptide, or an IL-15Rα polypeptide, provided that each of X1, X2, X3, X4, and X5 is different; and L1, L2, L3, and L4 are each independently a polypeptide linker comprising the same or different polypeptide sequences. In some embodiments, one or more of L1, L2, L3, and L4 are each independently a cleavable linker or a non-cleavable linker. In some embodiments, X1 is an IL-21 polypeptide; X2 is an IL-15 polypeptide; X3 is an IL-12p40 polypeptide; X4 is an IL-12p35 polypeptide; X5 is an IL-15Rα polypeptide; L2 is a furin-cleavable linker; and L1, L3, and L4 are non-cleavable linkers.

[0010] In some embodiments, polypeptides comprising the following formula are provided: X1-L1-X2-L2-X3-L3-X4-L4-X5-L5-X6, wherein X1, X2, X3, X4, X5, and X6 are each independently an IL-21 polypeptide, an IL-15 polypeptide, an IL-12p40 polypeptide, an IL-12p35 polypeptide, an IL-15Rα polypeptide, a first fragment of an IL-15Rα polypeptide, or a second fragment of an IL-15Rα polypeptide, provided that each of X1, X2, X3, X4, X5, and X6 is different; and L1, L2, L3, L4, and L5 are each independently a polypeptide linker comprising the same or different polypeptide sequences.

[0011] Compositions comprising multiple polypeptides are also provided. In some embodiments, compositions are provided that comprise:

[0012] A first polypeptide comprising the following formula: X1-L1-X2-L2-X3-L3-X4-L4-X5, wherein X1, X2, X3, X4, and X5 are each independently an IL-21 polypeptide, an IL-15 polypeptide, an IL-12p40 polypeptide, an IL-12p35 polypeptide, or an IL-15Rα polypeptide, provided that each of X1, X2, X3, X4, and X5 is different; and L1, L2, L3, and L4 are each independently a polypeptide linker comprising the same or different polypeptide sequences; and

[0013] A second polypeptide comprising at least one immune checkpoint polypeptide or an active fragment thereof, such as but not limited to a CTLA-4 polypeptide, a PD-1 polypeptide, a PD-L1 polypeptide, a PD-L2 polypeptide, a TIM3 polypeptide, a LAG3 polypeptide, a VISTA polypeptide, a SINGLEC7 polypeptide, a SINGLEC9 polypeptide, a TIGIT polypeptide, a CD96 polypeptide, a BTLA polypeptide, a B7H3 polypeptide, a B7H4 polypeptide, a CD155 polypeptide, a HHLA2 polypeptide, a BTN3A1 polypeptide, a B7.1 polypeptide, a B7.2 polypeptide, or an active fragment thereof.

[0014] In some embodiments, one or more of L1, L2, L3, and L4 are each independently a cleavable linker or a non-cleavable linker. In some embodiments, X1 is an IL-21 polypeptide; X2 is an IL-15 polypeptide; X3 is an IL-12p40 polypeptide; X4 is an IL-12p35 polypeptide; X5 is an IL-15Rα polypeptide; L2 is a furin-cleavable linker; and L1, L3, and L4 are non-cleavable linkers.

[0015] In some embodiments, a composition is provided that comprises:

[0016] A first polypeptide comprising the formula: X1-L1-X2-L2-X3-L3-X4-L4-X5-L5-X6, wherein X1, X2, X3, X4, X5, and X6 are each independently an IL-21 polypeptide, an IL-15 polypeptide, an IL-12p40 polypeptide, an IL-12p35 polypeptide, an IL-15Rα polypeptide, a first fragment of an IL-15Rα polypeptide, or a second fragment of an IL-15Rα polypeptide, provided that each of X1, X2, X3, X4, X5, and X6 is different; and L1, L2, L3, L4, and L5 are each independently a polypeptide linker comprising the same or different polypeptide sequences; and

[0017] A second polypeptide comprising at least one immune checkpoint polypeptide or an active fragment thereof, such as but not limited to a CTLA-4 polypeptide, a PD-1 polypeptide, a PD-L1 polypeptide, a PD-L2 polypeptide, a TIM3 polypeptide, a LAG3 polypeptide, a VISTA polypeptide, a SINGLEC7 polypeptide, a SINGLEC9 polypeptide, a TIGIT polypeptide, a CD96 polypeptide, a BTLA polypeptide, a B7H3 polypeptide, a B7H4 polypeptide, a CD155 polypeptide, a HHLA2 polypeptide, a BTN3A1 polypeptide, a B7.1 polypeptide, a B7.2 polypeptide, or an active fragment thereof.

[0018] In some embodiments, the second polypeptide comprises the formula X A -L A -X B or the formula X B -L A-X A wherein X A is an immune checkpoint polypeptide or an active fragment thereof, such as but not limited to a CTLA-4 polypeptide, a PD-1 polypeptide, a PD-L1 polypeptide, a PD-L2 polypeptide, a TIM3 polypeptide, a LAG3 polypeptide, a VISTA polypeptide, a SINGLEC7 polypeptide, a SINGLEC9 polypeptide, a TIGIT polypeptide, a CD96 polypeptide, a BTLA polypeptide, a B7H3 polypeptide, a B7H4 polypeptide, a CD155 polypeptide, a HHLA2 polypeptide, a BTN3A1 polypeptide or an active fragment thereof; X B is a B7.1 polypeptide or a B7.2 polypeptide; and L A is a polypeptide linker. In some embodiments, the second polypeptide further comprises a leader sequence. In some embodiments, the second polypeptide further comprises an Fc domain.

[0019] In some embodiments, a composition is provided that further comprises an antigen. In some embodiments, the antigen is a tumor antigen, a viral antigen, a bacterial antigen or a microbial antigen. In some embodiments, the antigen is ovalbumin (OVA).

[0020] In some embodiments, a nucleic acid molecule encoding any polypeptide provided herein is provided. In some embodiments, a composition is provided that comprises a first nucleic acid molecule encoding a first polypeptide and a second nucleic acid molecule encoding a second polypeptide. In some embodiments, the provided composition further comprises a third nucleic acid molecule.

[0021] In some embodiments, a vector, a vector composition, a plasmid, a plasmid composition, a virus, a liposome and a pharmaceutical composition are provided, each independently comprising any polypeptide or nucleic acid molecule provided herein.

[0022] In some embodiments, cells and methods of generating cells are provided herein. In some embodiments, the method of generating cells comprises contacting the cells with any vector, vector composition, plasmid, plasmid composition, virus, liposome and pharmaceutical composition provided herein. In some embodiments, the cells are generated in vivo.

[0023] In some embodiments, methods of altering the immune response of a subject are provided, the methods comprising administering to the subject a vector, plasmid, virus, liposome or composition provided herein. In some embodiments, methods of treating cancer in a subject are provided, the methods comprising administering to the subject a vector, plasmid, virus, liposome or composition provided herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Depicts IL-21 signaling of IL-21 / IL-15 / IL-12 polypeptides in the presence and absence of dual checkpoint inhibitor polypeptides.

[0025] Figure 2 Depicts the IL-15, IL-12, and CTLA-4 signals of IL-21 / IL-15 / IL-12 polypeptide with and without dual checkpoint inhibitor polypeptide.

[0026] Figure 3 Depicts the levels of antigen-reactive T cells of IL-21 / IL-15 / IL-12 polypeptide with and without dual checkpoint inhibitor polypeptide and / or antigen.

[0027] Figure 4 Depicts the CD8+ percentage levels of IL-21 / IL-15 / IL-12 polypeptide with and without dual checkpoint inhibitor polypeptide and / or antigen. Detailed Description

[0028] Unless otherwise defined, the meanings of all technical and scientific terms are the same as those commonly understood by one of ordinary skill in the art to which the disclosed embodiments belong. Unless the context requires otherwise, singular terms shall include plural, and plural terms shall include singular. Unless otherwise indicated, the use of "or" means "and / or". The use of the term "including" and other forms (such as "includes" and "included") is not restrictive.

[0029] As used herein, the term "a" or "an" means "at least one" or "one or more", unless the context clearly indicates otherwise.

[0030] As used herein, the term "about" means that the numerical value is approximate, and small variations will not significantly affect the practice of the disclosed embodiments. In the case of using numerical limitations, unless the context otherwise indicates, "about" means that the numerical value can vary by ±10% and remain within the scope of the disclosed embodiments.

[0031] As used herein, the terms "individual" or "subject" or "patient" used interchangeably mean any animal, including mammals, such as mice, rats, other rodents, rabbits, dogs, cats, pigs, cows, sheep, horses, or primates, such as humans.

[0032] As used herein, the term “comprising” (and any form of comprising, such as “comprise,” “comprises,” and “comprised”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), or “containing” (and any form of containing, such as “contains” and “contain”) is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. Any step or composition using the transitional phrase “comprise” or “comprising” may also be said to be described by the transitional phrases “consisting of” or “consists of.”

[0033] As used herein, the term “contact” means bringing two elements in an in vitro system or an in vivo system together. For example, “contacting” a vector with a cell or with an individual or patient or cell includes administering the vector to an individual or patient, such as a human, and, for example, introducing a compound into a sample containing cells or a purified preparation containing cells.

[0034] A “disease” is a health state of an animal in which the animal cannot maintain homeostasis and in which the health of the animal continues to deteriorate if the disease is not ameliorated. In contrast, a “disorder” in an animal is a health state in which the animal is able to maintain homeostasis, but in which the health state of the animal is more adverse than it would be in the absence of the disorder. A disorder does not necessarily result in a further decrease in the health state of the animal if left untreated.

[0035] “Effective amount” or “therapeutically effective amount” are used interchangeably herein and refer to the amount of a compound, formulation, material, or composition as described herein that is effective to achieve a particular biological result or provide a therapeutic or prophylactic benefit. Such results can include, but are not limited to, when administered to a mammal, an amount that causes a detectable level of immune cell activation compared to immune cell activation detected in the absence of the composition. The immune response can be readily evaluated by a number of methods well known in the art. Those skilled in the art will understand that the amount of the composition administered herein will vary and can be readily determined based on many factors, such as the disease or condition being treated, the age and health and physical condition of the mammal being treated, the severity of the disease, the particular compound being administered, etc.

[0036] "Encoding" refers to the inherent property of a specific nucleotide sequence in a polynucleotide, such as a gene, cDNA or mRNA, to serve as a template for the synthesis of other polymers and macromolecules with a defined nucleotide sequence (i.e., rRNA, tRNA and mRNA) or a defined amino acid sequence in a biological process, and the resulting biological properties. Thus, if the transcription and translation of mRNA corresponding to a gene produces a protein in a cell or other biological system, the gene encodes that protein. Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence and is usually provided in the sequence listing) and the non-coding strand (which serves as a template for gene or cDNA transcription) can be said to encode the protein or other product of the gene or cDNA.

[0037] "Expression vector" refers to a vector containing a recombinant polynucleotide that contains an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression; other elements for expression can be provided by the host cell or in an in vitro expression system. Expression vectors include all expression vectors known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., Sendai virus, lentivirus, retrovirus, adenovirus and adeno-associated virus) incorporating the recombinant polynucleotide.

[0038] As used herein, the terms "fusion" or "linkage" when used in reference to a protein having different domains or heterologous sequences mean that the protein domains are part of the same peptide chain and are linked to each other by peptide bonds or other covalent bonds. The domains or segments can be directly linked or fused to each other, or another domain or peptide sequence can be between the two domains or sequences, and such sequences will still be considered to be fused or linked to each other. In some embodiments, the various domains or proteins provided herein are directly linked or fused to each other, or two domains are joined together by a linker sequence such as a glycine / serine sequence.

[0039] "Heterologous" as used herein refers to a non-native nucleic acid or amino acid sequence introduced into a cell, organism or system. The nucleic acid sequence can comprise a polynucleotide of any length. The amino acid sequence can comprise a peptide or polypeptide of any length.

[0040] As used herein, "identity" refers to subunit sequence identity between two polymer molecules, such as between two nucleic acid or amino acid molecules, such as between two polynucleotide or polypeptide molecules. When two amino acid sequences have the same residue at the same position, for example, if the position in each of two polypeptide molecules is occupied by arginine, they are identical at that position. The identity or degree to which two amino acids or two nucleic acid sequences have the same residue at the same position in an alignment is typically expressed as a percentage. The identity between two amino acid or two nucleic acid sequences is a direct function of the number of matching or identical positions, for example, if half of the positions in two sequences are the same, the two sequences are 50% identical; if 90% of the positions (e.g., 9 out of 10) match or are the same, the two amino acid sequences are 90% identical.

[0041] As used herein, "positioned" means to give positional clarity in an amino acid or nucleic acid sequence. For example, a sequence X that is said to be positioned between a first portion A and a second portion B will give rise to potential forms A-X-B or B-X-A.

[0042] In a similar manner, the term "upstream" is intended to give further positional clarity in a nucleic acid or polynucleotide sequence. In the context of a polynucleotide sequence, a sequence X that is said to be upstream of a first portion A will indicate that sequence X is located before portion A, such that the form will read 5'-X-A-3'.

[0043] "Substantially identical" means that a polypeptide or nucleic acid molecule exhibits at least 50% identity with a reference amino acid sequence (e.g., any of the amino acid sequences described herein) or nucleic acid sequence (e.g., any of the nucleic acid sequences described herein). Preferably, such sequences are at least 60%, more preferably 80% or 85%, more preferably 90%, 95% or even 99% identical at the amino acid or nucleotide level to the sequences used for comparison.

[0044] Sequence identity can be measured / determined using sequence analysis software (e.g., the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning a degree of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary method of determining the degree of identity, the BLAST program can be used, where a probability score between e3 and e100 indicates closely related sequences. In some embodiments, sequence identity is determined by using BLAST with default settings.

[0045] For the embodiments provided herein, compositions comprising various proteins are included, which in some cases may contain amino acid sequences having sequence identity with the amino acid sequences disclosed herein. Thus, in certain embodiments, depending on the particular sequence, the degree of sequence identity with the SEQ ID NOs disclosed herein is preferably greater than 50% (e.g., 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more). These proteins can include homologs, orthologs, allelic variants, and functional mutants. Generally, 50% or higher identity between two polypeptide sequences is considered an indication of functional equivalence. Identity between polypeptides is preferably determined by the Smith-Waterman homology search algorithm as implemented in the MPSRCH program (Oxford Molecular), using an affine gap search with parameters gap open penalty = -12 and gap extension penalty = 1.

[0046] Compared to the disclosed proteins, these proteins may include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) conservative amino acid substitutions, i.e., one amino acid is replaced by another amino acid with a related side chain. Genetically encoded amino acids are generally divided into four families: (1) acidic, i.e., aspartic acid, glutamic acid; (2) basic, i.e., lysine, arginine, histidine; (3) non-polar, i.e., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar amino acids, i.e., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes grouped together as aromatic amino acids. Generally, substitution of a single amino acid within these families has no significant effect on biological activity. Relative to the disclosed protein sequence, the protein may have one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) single amino acid deletions. Relative to the disclosed protein sequence, the protein may also include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) insertions (e.g., each of 1, 2, 3, 4, or 5 amino acids).

[0047] As used herein, the phrase "in vivo" with respect to cells transduced, transfected, or transformed in vivo refers to cells transduced, transfected, or transformed in a subject without removing the cells from the subject prior to transducing, transfecting, or transforming such cells.

[0048] "Isolated" means altered or removed from its natural state. For example, a nucleic acid or peptide that occurs naturally in a living animal is not "isolated," but the same nucleic acid or peptide that is partially or completely separated from the coexisting materials in its natural state is "isolated." An isolated nucleic acid or protein may exist in a substantially purified form or may exist in a non-natural environment such as, for example, a host cell.

[0049] As used herein, the term "modified" means an altered state or structure of a molecule or cell as provided herein. A molecule can be modified in many ways, including chemically, structurally, and functionally, such as by mutation, substitution, insertion, or deletion (e.g., internal deletion, truncation). A cell can be modified by introducing nucleic acid or expressing a heterologous protein.

[0050] As used herein, the term "modulate" means to mediate an increase or decrease in the level of a response in a subject compared to the level of the response in a subject in the absence of a treatment or compound, and / or compared to the level of the response in an otherwise identical but untreated subject. The term encompasses interfering with and / or affecting a natural signal or response, thereby mediating a beneficial therapeutic response in a subject such as a human.

[0051] As used herein, the following common nucleic acid base abbreviations are used: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0052] Unless otherwise indicated, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate to one another and encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA may also include introns to the extent that the nucleotide sequence encoding the protein may contain introns in some forms.

[0053] The term "oligonucleotide" generally refers to short polynucleotides. It should be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, C, G), this also provides the corresponding RNA sequence in which "U" replaces "T" (i.e., A, U, C, G).

[0054] As used herein, the term "polynucleotide" is defined as a chain of nucleotides. In addition, a nucleic acid is a polymer of nucleotides. Thus, as used herein, the terms "nucleic acid" and "polynucleotide" are interchangeable. As used herein, polynucleotides include, but are not limited to, all nucleic acid sequences obtained by any method available in the art, including but not limited to recombinant methods, i.e., cloning nucleic acid sequences from recombinant libraries or cell genomes using cloning techniques and PCR, etc., and by synthetic means.

[0055] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound composed of a plurality of amino acid residues covalently linked by peptide bonds. As used herein, the term refers to both short chains (which are also commonly referred to in the art as, for example, peptides, oligopeptides and oligomers) and longer chains (which are commonly referred to in the art as proteins, of which there are many types). "Polypeptides" include, for example, bioactive fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, etc. Polypeptides include natural peptides, recombinant peptides, synthetic peptides or combinations thereof.

[0056] The term "subject" includes living organisms, including organisms in which an immune response can be elicited (e.g., mammals). As used herein, the terms "subject" or "patient" or "individual" are used interchangeably. As used herein, a "subject" can be a human or a non-human mammal. Non-human mammals include, for example, livestock and pets such as sheep, cattle, pigs, dogs, non-human primates, felines and murine mammals. In some embodiments, the subject is a human.

[0057] As used herein, the phrase "in need thereof" means that a subject (animal or mammal) has been identified as in need of a particular method or treatment. In some embodiments, the identification can be by any diagnostic means. In any of the methods and treatments described herein, an animal or mammal may be in need thereof. In some embodiments, the animal or mammal is in an environment where a particular disease, disorder, or condition is endemic or will travel to an environment where a particular disease, disorder, or condition is endemic.

[0058] As used herein, the terms "substituted", "substitution", "mutated", or "mutation" refer to the alteration, deletion, or insertion of one or more amino acids or nucleotides in a polypeptide or polynucleotide sequence to generate a variant of that sequence.

[0059] As used herein, the term "therapeutic" means treatment and / or prevention. A therapeutic effect is obtained by inhibiting, alleviating, or eradicating a disease state.

[0060] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process of transferring or introducing exogenous nucleic acid into a cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed, or transduced with exogenous nucleic acid. Cells include primary subject cells and their progeny. In some embodiments, transfection, transformation, or transduction occurs in vivo.

[0061] As used herein, the term "treating" a disease means reducing the frequency or severity of at least one sign or symptom of the disease or disorder experienced by a subject.

[0062] As used herein, the term "variant" when used in connection with an amino acid sequence means a sequence that is at least or about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a reference sequence. In some embodiments, the variant contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions. In some embodiments, the substitutions are conservative substitutions.

[0063] A "vector" is a physical composition that contains a nucleic acid isolated encoding a protein or peptide. A variety of vectors are known in the art, including but not limited to linear polynucleotides, plasmids, DNA, and RNA. Examples of viral vectors include but are not limited to Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, and the like.

[0064] Ranges: Throughout this disclosure, various aspects of embodiments may be presented in range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, a description of a range such as 1 to 6 should be considered to have specifically disclosed sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range. Unless expressly stated to the contrary, the disclosed ranges also include the endpoints of the range.

[0065] IL-12, IL-15, and IL-21 polypeptides

[0066] Polypeptides are described herein that comprise an IL-21 polypeptide, an IL-12 polypeptide, and an IL-15 polypeptide. In some embodiments, the polypeptide comprises an IL-21 polypeptide, an IL-12p40 polypeptide, an IL-12p35 polypeptide, an IL-15 polypeptide, and an IL-15Rα polypeptide. In some embodiments, the polypeptide comprises an IL-21 polypeptide, an IL-12p40 polypeptide, an IL-12p35 polypeptide, an IL-15 polypeptide, an IL-15Rα polypeptide, and / or a first or second fragment of the IL-15Rα polypeptide. In some embodiments, the IL-12p40 polypeptide is from human. In some embodiments, the IL-12p40 polypeptide is from mouse. In some embodiments, the IL-12p35 polypeptide is from human. In some embodiments, the IL-12p35 polypeptide is from mouse. In some embodiments, the IL-21 polypeptide is from mouse. In some embodiments, the polypeptide can be used to stimulate an immune response. In some embodiments, the polypeptide can be used to be activated by NK cells or CD8+ T cells. In some embodiments, the polypeptide can be used to treat cancer (such as those provided herein), viral infections, bacterial infections (such as but not limited to tuberculosis, listeriosis, etc.), and fungal infections.

[0067] In some embodiments, an IL-12 polypeptide refers to a polypeptide that comprises a functional p40 polypeptide and a p35 polypeptide. These polypeptides can be expressed together to form an IL-12 polypeptide, or the p35 and p40 polypeptides can be joined by using a linker to connect them. In some embodiments, the linker is a polypeptide linker, such as those provided herein.

[0068] In some embodiments, the IL-12p40 polypeptide comprises the following amino acid sequence or an active fragment thereof: MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS (SEQ ID NO:1). The IL-12p40 polypeptide can be processed to produce a mature polypeptide, which can be referred to as the active portion of the polypeptide. In some embodiments, the IL-12p40 polypeptide or an active fragment thereof comprises the following amino acid sequence: IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS (SEQ ID NO:2). In some embodiments, 1, 2, 3, 4, or 5 amino acid residues are deleted from the N-terminus of SEQ ID NO:2 to form the IL-12p40 polypeptide. In some embodiments, 1, 2, 3, 4, or 5 amino acid residues are deleted from the C-terminus of SEQ ID NO:2 to form the IL-12p40 polypeptide. In some embodiments, 1, 2, 3, 4, or 5 amino acid residues are deleted from the N-terminus and / or C-terminus of SEQ ID NO:2 to form the IL-12p40 polypeptide. In some embodiments, SEQ ID NOs: 1 and 2 are or are derived from human IL-12p40.

[0069] In some embodiments, the IL-12p40 polypeptide comprises the following amino acid sequence or an active fragment thereof: MCPQKLTISWFAIVLLVSPLMAMWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRS (SEQ ID NO:3). The IL-12p40 polypeptide can be processed to produce a mature polypeptide, which can be referred to as the active portion of the polypeptide. In some embodiments, the IL-12p40 polypeptide or an active fragment thereof comprises the following amino acid sequence: MWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRS (SEQ ID NO:4). In some embodiments, 1, 2, 3, 4, or 5 amino acid residues are deleted from the N-terminus of SEQ ID NO:4 to form the IL-12p40 polypeptide. In some embodiments, 1, 2, 3, 4, or 5 amino acid residues are deleted from the C-terminus of SEQ ID NO:4 to form the IL-12p40 polypeptide. In some embodiments, 1, 2, 3, 4, or 5 amino acid residues are deleted from the N-terminus and / or C-terminus of SEQ ID NO:4 to form the IL-12p40 polypeptide. In some embodiments, SEQ ID NO:3 and 4 are or are derived from murine IL-12p40.

[0070] In some embodiments, the IL-12p35 polypeptide comprises the following amino acid sequence or an active fragment thereof: MCPARSLLLVATLVLLDHLSLARNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS (SEQ ID NO:5). The IL-12p35 polypeptide can be processed to produce a mature polypeptide, which can be referred to as the active portion of the polypeptide. In some embodiments, the IL-12p35 polypeptide or an active fragment thereof comprises the following amino acid sequence: RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS (SEQ ID NO:6). In some embodiments, 1, 2, 3, 4, or 5 amino acid residues are deleted from the N-terminus of SEQ ID NO:6 to form the IL-12p35 polypeptide. In some embodiments, 1, 2, 3, 4, or 5 amino acid residues are deleted from the C-terminus of SEQ ID NO:6 to form the IL-12p35 polypeptide. In some embodiments, 1, 2, 3, 4, or 5 amino acid residues are deleted from the N-terminus and / or C-terminus of SEQ ID NO:6 to form the IL-12p35 polypeptide. In some embodiments, SEQ ID NO:5 and 6 are or are derived from human IL-12p35.

[0071] In some embodiments, the IL-12p35 polypeptide comprises the following amino acid sequence or an active fragment thereof: MCQSRYLLFLATLALLNHLSLARVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSA (SEQ ID NO:7). The IL-12p35 polypeptide can be processed to produce a mature polypeptide, which can be referred to as the active portion of the polypeptide. In some embodiments, the IL-12p35 polypeptide or an active fragment thereof comprises the following amino acid sequence: RVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSA (SEQ ID NO:8). In some embodiments, 1, 2, 3, 4, or 5 amino acid residues are deleted from the N-terminus of SEQ ID NO:8 to form the IL-12p35 polypeptide. In some embodiments, 1, 2, 3, 4, or 5 amino acid residues are deleted from the C-terminus of SEQ ID NO:8 to form the IL-12p35 polypeptide. In some embodiments, 1, 2, 3, 4, or 5 amino acid residues are deleted from the N-terminus and / or C-terminus of SEQ ID NO:8 to form the IL-12p35 polypeptide. In some embodiments, SEQ ID NOs: 7 and 8 are or are derived from murine IL-12p35.

[0072] In some embodiments, the IL-15 polypeptide comprises the following amino acid sequence or an active fragment thereof: MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO:9). The IL-15 polypeptide can be processed to produce a mature polypeptide, which can be referred to as the active portion of the polypeptide. In some embodiments, the IL-15 polypeptide or an active fragment thereof comprises the following amino acid sequence: GIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO:10). In some embodiments, 1, 2, 3, 4, or 5 amino acid residues are deleted from the N-terminus of SEQ ID NO:10 to form the IL-15 polypeptide. In some embodiments, 1, 2, 3, 4, or 5 amino acid residues are deleted from the C-terminus of SEQ ID NO:10 to form the IL-15 polypeptide. In some embodiments, 1, 2, 3, 4, or 5 amino acid residues are deleted from the N-terminus and / or C-terminus of SEQ ID NO:6 to form the IL-15 polypeptide.

[0073] In some embodiments, the IL-15Rα polypeptide comprises the following amino acid sequence or an active fragment thereof: LQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL (SEQ ID NO:11). In some embodiments, the IL-15Rα polypeptide or an active fragment thereof comprises the following amino acid sequence: ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL (SEQ ID NO:12). In some embodiments, the first fragment of the IL-15Rα polypeptide comprises the following amino acid sequence: ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQR (SEQ ID NO:13). In some embodiments, the second fragment of the IL-15Rα polypeptide comprises the following amino acid sequence: PAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL (SEQ ID NO:14).In some embodiments, 1, 2, 3, 4, or 5 amino acid residues are deleted from the N-terminus of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14 to yield an IL-15Rα polypeptide. In some embodiments, 1, 2, 3, 4, or 5 amino acid residues are deleted from the C-terminus of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14 to yield an IL-15Rα. In some embodiments, 1, 2, 3, 4, or 5 amino acid residues are deleted from the N-terminus and / or C-terminus of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14 to yield an IL-15Rα.

[0074] There are other isoforms of IL-15Rα that can be modified to produce an active polypeptide. Sequences of such isoforms can be found, for example, in GenBank accession numbers: NP_002180.1, AEP26933.1, AAP69528.1, AAH74726.1, NP_001243694.1, XP_016871684.1, XP_011517767.1, NP_001230468.1, and AAI07778.1, each of which is hereby incorporated by reference in its entirety. In some embodiments, the IL-15Rα polypeptide or its transmembrane segment can be replaced or substituted with other transmembrane domains known in the art.

[0075] In some embodiments, the IL-21 polypeptide comprises the following amino acid sequence or an active fragment thereof: MERTLVCLVVIFLGTVAHKSSPQGPDRLLIRLRHLIDIVEQLKIYENDLDPELLSAPQDVKGHCEHAAFACFQKAKLKPSNPGNNKTFIIDLVAQLRRRLPARRGGKKQKHIAKCPSCDSYEKRTPKEFLERLKWLLQKMIHQHLS (SEQ ID NO:71). The IL-21 polypeptide can be processed to produce a mature polypeptide, which can be referred to as the active portion of the polypeptide. In some embodiments, 1, 2, 3, 4, or 5 amino acid residues are deleted from the N-terminus of SEQ ID NO:71 to yield an IL-21 polypeptide. In some embodiments, 1, 2, 3, 4, or 5 amino acid residues are deleted from the C-terminus of SEQ ID NO:71 to yield an IL-21 polypeptide. In some embodiments, 1, 2, 3, 4, or 5 amino acid residues are deleted from the N-terminus and / or C-terminus of SEQ ID NO:71 to yield an IL-21 polypeptide.

[0076] Although the sequences provided herein are human and mouse sequences, other orthologs can also be used because certain orthologs of the same protein have the same or similar activities in different species. For example, murine IL-12 is active on human cells, and thus, the p40 and / or p35 human subunits of IL-12 can be replaced with the murine orthologs provided herein. Similarly, the IL-15, IL-15Rα, and IL-21 polypeptides provided herein can be used in other animals due to high similarity (identity), regardless of whether the polypeptide is from human or mouse. Likewise, any polypeptide provided herein can be replaced with an ortholog from another animal. For example, the IL-21 polypeptide disclosed herein can be replaced with an IL-21 polypeptide from another animal, such as human IL-21.

[0077] In some embodiments, polypeptides comprising the following formula are provided: X1-L1-X2-L2-X3-L3-X4-L4-X5, wherein:

[0078] X1 is an interleukin-21 (IL-21) polypeptide, an interleukin-15 (IL-15) polypeptide, an interleukin-12 p40 subunit (IL-12p40) polypeptide, an interleukin-12 p35 subunit (IL-12p35) polypeptide, or an interleukin-15 receptor (IL-15Rα) polypeptide;

[0079] X2 is an IL-21 polypeptide, an IL-15 polypeptide, an IL-12p40 polypeptide, an IL-12p35 polypeptide, or an IL-15Rα polypeptide;

[0080] X3 is an IL-21 polypeptide, an IL-15 polypeptide, an IL-12p40 polypeptide, an IL-12p35 polypeptide, or an IL-15Rα polypeptide;

[0081] X4 is an IL-21 polypeptide, an IL-15 polypeptide, an IL-12p40 polypeptide, an IL-12p35 polypeptide, or an IL-15Rα polypeptide;

[0082] X5 is an IL-21 polypeptide, an IL-15 polypeptide, an IL-12p40 polypeptide, an IL-12p35 polypeptide, or an IL-15Rα polypeptide;

[0083] L1, L2, L3, and L4 are each independently a polypeptide linker comprising the same or different polypeptide sequences, and

[0084] provided that each of X1, X2, X3, X4, and X5 is different.

[0085] In some embodiments, X1 is an interleukin-21 (IL-21) polypeptide. In some embodiments, X1 is an IL-21 polypeptide; X2 is an IL-15 polypeptide; X3 is an IL-12p40 polypeptide; X4 is an IL-12p35 polypeptide; and X5 is an IL-15Rα polypeptide. In some embodiments, X1 is an IL-21 polypeptide; X2 is an IL-15 polypeptide; X3 is an IL-12p35 polypeptide; X4 is an IL-12p40 polypeptide; and X5 is an IL-15Rα polypeptide. In some embodiments, X1 is an IL-21 polypeptide; X2 is an IL-12p40 polypeptide; X3 is an IL-15 polypeptide; X4 is an IL-12p35 polypeptide; and X5 is an IL-15Rα polypeptide. In some embodiments, X1 is an IL-21 polypeptide; X2 is an IL-12p40 polypeptide; X3 is an IL-12p35 polypeptide; X4 is an IL-15 polypeptide; and X5 is an IL-15Rα polypeptide. In some embodiments, X1 is an IL-21 polypeptide; X2 is an IL-12p35 polypeptide; X3 is an IL-12p40 polypeptide; X4 is an IL-15 polypeptide; and X5 is an IL-15Rα polypeptide. In some embodiments, X1 is an IL-21 polypeptide; X2 is an IL-12p35 polypeptide; X3 is an IL-15 polypeptide; X4 is an IL-12p40 polypeptide; and X5 is an IL-15Rα polypeptide. In some embodiments, X1 is an IL-21 polypeptide; X2 is an IL-12p40 polypeptide; X3 is an IL-12p35 polypeptide; X4 is an IL-15 polypeptide; and X5 is an IL-15Rα polypeptide. In some embodiments, X1 is an IL-21 polypeptide; X2 is an IL-12p40 polypeptide; X3 is an IL-15 polypeptide; X4 is an IL-12p35 polypeptide; and X5 is an IL-15Rα polypeptide. In some embodiments, X1 is an IL-21 polypeptide; X2 is an IL-12p35 polypeptide; X3 is an IL-12p40 polypeptide; X4 is an IL-15 polypeptide; and X5 is an IL-15Rα polypeptide. In some embodiments, X1 is an IL-21 polypeptide; X2 is an IL-12p35 polypeptide; X3 is an IL-15 polypeptide; X4 is an IL-12p40 polypeptide; and X5 is an IL-15Rα polypeptide.

[0086] In some embodiments, a polypeptide comprising the following formula is provided: X1-L1-X2-L2-X3-L3-X4-L4-X5-L5-X6, wherein:

[0087] X1 is interleukin-21 (IL-21) polypeptide, interleukin-15 (IL-15) polypeptide, interleukin-12p40 subunit (IL-12p40) polypeptide, interleukin-12p35 subunit (IL-12p35) polypeptide, interleukin-15 receptor (IL-15Rα) polypeptide, the first fragment of IL-15Rα polypeptide or the second fragment of IL-15Rα polypeptide;

[0088] X2 is IL-21 polypeptide, IL-15 polypeptide, IL-12p40 polypeptide, IL-12p35 polypeptide, IL-15Rα polypeptide, the first fragment of IL-15Rα polypeptide or the second fragment of IL-15Rα polypeptide;

[0089] X3 is IL-21 polypeptide, IL-15 polypeptide, IL-12p40 polypeptide, IL-12p35 polypeptide, IL-15Rα polypeptide, the first fragment of IL-15Rα polypeptide or the second fragment of IL-15Rα polypeptide;

[0090] X4 is IL-21 polypeptide, IL-15 polypeptide, IL-12p40 polypeptide, IL-12p35 polypeptide, IL-15Rα polypeptide, the first fragment of IL-15Rα polypeptide or the second fragment of IL-15Rα polypeptide;

[0091] X5 is IL-21 polypeptide, IL-15 polypeptide, IL-12p40 polypeptide, IL-12p35 polypeptide, IL-15Rα polypeptide, the first fragment of IL-15Rα polypeptide or the second fragment of IL-15Rα polypeptide;

[0092] X6 is IL-21 polypeptide, IL-15 polypeptide, IL-12p40 polypeptide, IL-12p35 polypeptide, IL-15Rα polypeptide, the first fragment of IL-15Rα polypeptide or the second fragment of IL-15Rα polypeptide;

[0093] L1, L2, L3, L4 and L5 are each independently a polypeptide linker containing the same or different polypeptide sequences,

[0094] provided that each of X1, X2, X3, X4, X5 and X6 is different.

[0095] In some embodiments, X1 is an interleukin-21 (IL-21) polypeptide. In some embodiments, X1 is an IL-21 polypeptide; X2 is an IL-15 polypeptide; X3 is a first fragment of an IL-15Rα polypeptide; X4 is an IL-12p40 polypeptide; X5 is an IL-12p35 polypeptide; and X6 is a second fragment of an IL-15Rα polypeptide. In some embodiments, X1 is an IL-21 polypeptide; X2 is an IL-15 polypeptide; X3 is a first fragment of an IL-15Rα polypeptide; X4 is an IL-12p35 polypeptide; X5 is an IL-12p40 polypeptide; and X6 is a second fragment of an IL-15Rα polypeptide. In some embodiments, X1 is an IL-21 polypeptide; X2 is an IL-15 polypeptide; X3 is an IL-12p40 polypeptide; X4 is a first fragment of an IL-15Rα polypeptide; X5 is an IL-12p35 polypeptide; and X6 is a second fragment of an IL-15Rα polypeptide. In some embodiments, X1 is an IL-21 polypeptide; X2 is an IL-15 polypeptide; X3 is an IL-12p40 polypeptide; X4 is an IL-12p35 polypeptide; X5 is a first fragment of an IL-15Rα polypeptide; and X6 is a second fragment of an IL-15Rα polypeptide. In some embodiments, X1 is an IL-21 polypeptide; X2 is an IL-15 polypeptide; X3 is an IL-12p35 polypeptide; X4 is an IL-12p40 polypeptide; X5 is a first fragment of an IL-15Rα polypeptide; and X6 is a second fragment of an IL-15Rα polypeptide. In some embodiments, X1 is an IL-21 polypeptide; X2 is an IL-15 polypeptide; X3 is an IL-12p35 polypeptide; X4 is a first fragment of an IL-15Rα polypeptide; X5 is an IL-12p40 polypeptide; and X6 is a second fragment of an IL-15Rα polypeptide. In some embodiments, X1 is an IL-21 polypeptide; X2 is a first fragment of an IL-15Rα polypeptide; X3 is an IL-15 polypeptide; X4 is an IL-12p40 polypeptide; X5 is an IL-12p35 polypeptide; and X6 is a second fragment of an IL-15Rα polypeptide. In some embodiments, X1 is an IL-21 polypeptide; X2 is a first fragment of an IL-15Rα polypeptide; X3 is an IL-15 polypeptide; X4 is an IL-12p35 polypeptide; X5 is an IL-12p40 polypeptide; and X6 is a second fragment of an IL-15Rα polypeptide. In some embodiments, X1 is an IL-21 polypeptide; X2 is a first fragment of an IL-15Rα polypeptide; X3 is an IL-12p40 polypeptide; X4 is an IL-15 polypeptide; X5 is an IL-12p35 polypeptide; and X6 is a second fragment of an IL-15Rα polypeptide. In some embodiments, X1 is an IL-21 polypeptide; X2 is a first fragment of an IL-15Rα polypeptide; X3 is an IL-12p40 polypeptide; X4 is an IL-12p35 polypeptide; X5 is IL-15; and X6 is a second fragment of an IL-15Rα polypeptide.In some embodiments, X1 is an IL-21 polypeptide; X2 is a first fragment of an IL-15Rα polypeptide; X3 is an IL-12p35 polypeptide; X4 is an IL-12p40 polypeptide; X5 is IL-15; and X6 is a second fragment of an IL-15Rα polypeptide. In some embodiments, X1 is an IL-21 polypeptide; X2 is a first fragment of an IL-15Rα polypeptide; X3 is an IL-12p35 polypeptide; X4 is IL-15; X5 is an IL-12p40 polypeptide; and X6 is a second fragment of an IL-15Rα polypeptide. In some embodiments, X1 is an IL-21 polypeptide; X2 is an IL-12p40 polypeptide; X3 is an IL-15 polypeptide; X4 is a first fragment of an IL-15Rα polypeptide; X5 is an IL-12p35 polypeptide; and X6 is a second fragment of an IL-15Rα polypeptide. In some embodiments, X1 is an IL-21 polypeptide; X2 is an IL-12p40 polypeptide; X3 is an IL-15 polypeptide; X4 is an IL-12p35 polypeptide; X5 is a first fragment of an IL-15Rα polypeptide; and X6 is a second fragment of an IL-15Rα polypeptide. In some embodiments, X1 is an IL-21 polypeptide; X2 is an IL-12p40 polypeptide; X3 is a first fragment of an IL-15Rα polypeptide; X4 is an IL-15 polypeptide; X5 is an IL-12p35 polypeptide; and X6 is a second fragment of an IL-15Rα polypeptide. In some embodiments, X1 is an IL-21 polypeptide; X2 is an IL-12p40 polypeptide; X3 is a first fragment of an IL-15Rα polypeptide; X4 is an IL-12p35 polypeptide; X5 is IL-15; and X6 is a second fragment of an IL-15Rα polypeptide. In some embodiments, X1 is an IL-21 polypeptide; X2 is an IL-12p40 polypeptide; X3 is an IL-12p35 polypeptide; X4 is a first fragment of an IL-15Rα polypeptide; X5 is IL-15; and X6 is a second fragment of an IL-15Rα polypeptide. In some embodiments, X1 is an IL-21 polypeptide; X2 is an IL-12p40 polypeptide; X3 is an IL-12p35 polypeptide; X4 is IL-15; X5 is a first fragment of an IL-15Rα polypeptide; and X6 is a second fragment of an IL-15Rα polypeptide. In some embodiments, X1 is an IL-21 polypeptide; X2 is an IL-12p35 polypeptide; X3 is an IL-15 polypeptide; X4 is a first fragment of an IL-15Rα polypeptide; X5 is an IL-12p40 polypeptide; and X6 is a second fragment of an IL-15Rα polypeptide. In some embodiments, X1 is an IL-21 polypeptide; X2 is an IL-12p35 polypeptide; X3 is an IL-15 polypeptide; X4 is an IL-12p40 polypeptide; X5 is a first fragment of an IL-15Rα polypeptide; and X6 is a second fragment of an IL-15Rα polypeptide.In some embodiments, X1 is an IL-21 polypeptide; X2 is an IL-12p35 polypeptide; X3 is a first fragment of an IL-15Rα polypeptide; X4 is an IL-15 polypeptide; X5 is an IL-12p40 polypeptide; and X6 is a second fragment of an IL-15Rα polypeptide. In some embodiments, X1 is an IL-21 polypeptide; X2 is an IL-12p35 polypeptide; X3 is a first fragment of an IL-15Rα polypeptide; X4 is an IL-12p40 polypeptide; X5 is IL-15; and X6 is a second fragment of an IL-15Rα polypeptide. In some embodiments, X1 is an IL-21 polypeptide; X2 is an IL-12p35 polypeptide; X3 is an IL-12p40 polypeptide; X4 is a first fragment of an IL-15Rα polypeptide; X5 is IL-15; and X6 is a second fragment of an IL-15Rα polypeptide. In some embodiments, X1 is an IL-21 polypeptide; X2 is an IL-12p35 polypeptide; X3 is an IL-12p40 polypeptide; X4 is IL-15; X5 is a first fragment of an IL-15Rα polypeptide; and X6 is a second fragment of an IL-15Rα polypeptide.

[0096] In some embodiments, any polypeptide described herein may lack one or more elements, polypeptides, or linkers. For example, in some embodiments, a polypeptide having the formula X1-L1-X2-L2-X3-L3-X4 is provided, wherein:

[0097] X1 is an interleukin 21 (IL-21) polypeptide, an interleukin 15 (IL-15) polypeptide, an interleukin 12p40 subunit (IL-12p40) polypeptide, an interleukin 12p35 subunit (IL-12p35) polypeptide, or an interleukin 15 receptor (IL-15Rα) polypeptide;

[0098] X2 is an IL-21 polypeptide, an IL-15 polypeptide, an IL-12p40 polypeptide, an IL-12p35 polypeptide, or an IL-15Rα polypeptide;

[0099] X3 is an IL-21 polypeptide, an IL-15 polypeptide, an IL-12p40 polypeptide, an IL-12p35 polypeptide, or an IL-15Rα polypeptide;

[0100] X4 is an IL-21 polypeptide, an IL-15 polypeptide, an IL-12p40 polypeptide, an IL-12p35 polypeptide, or an IL-15Rα polypeptide;

[0101] L1, L2, and L3 are each independently a polypeptide linker comprising the same or different polypeptide sequences, and

[0102] provided that each of X1, X2, X3, and X4 is different.

[0103] In some embodiments, a polypeptide having the formula X1-L1-X2-L2-X3 is provided, wherein:

[0104] X1 is an interleukin-21 (IL-21) polypeptide, an interleukin-15 (IL-15) polypeptide, an interleukin-12p40 subunit (IL-12p40) polypeptide, an interleukin-12p35 subunit (IL-12p35) polypeptide, or an interleukin-15 receptor (IL-15Rα) polypeptide;

[0105] X2 is an IL-21 polypeptide, an IL-15 polypeptide, an IL-12p40 polypeptide, an IL-12p35 polypeptide, or an IL-15Rα polypeptide;

[0106] X3 is an IL-21 polypeptide, an IL-15 polypeptide, an IL-12p40 polypeptide, an IL-12p35 polypeptide, or an IL-15Rα polypeptide;

[0107] L1 and L2 are each independently a polypeptide linker comprising the same or different polypeptide sequences, and

[0108] provided that each of X1, X2, and X3 is different.

[0109] In some embodiments, a polypeptide having the formula X1-L1-X2 is provided, wherein:

[0110] X1 is an interleukin-21 (IL-21) polypeptide, an interleukin-15 (IL-15) polypeptide, an interleukin-12p40 subunit (IL-12p40) polypeptide, an interleukin-12p35 subunit (IL-12p35) polypeptide, or an interleukin-15 receptor (IL-15Rα) polypeptide;

[0111] X2 is an IL-21 polypeptide, an IL-15 polypeptide, an IL-12p40 polypeptide, an IL-12p35 polypeptide, or an IL-15Rα polypeptide;

[0112] L1 is a polypeptide linker comprising the same or different polypeptide sequences, and

[0113] provided that each of X1 and X2 is different.

[0114] In some embodiments, the IL-12p40 polypeptide comprises an amino acid sequence having at least or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 1, 2, 3, or 4 or the IL-12p40 polypeptides provided herein. In some embodiments, the IL-12p40 polypeptide comprises the amino acid sequence of SEQ ID NO: 1, 2, 3, or 4 or the IL-12p40 polypeptides provided herein.

[0115] In some embodiments, the IL-12p35 polypeptide comprises an amino acid having at least or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:5, 6, 7 or 8 or the IL-12p35 polypeptides provided herein. In some embodiments, the IL-12p35 polypeptide comprises the amino acid sequence of SEQ ID NO:5, 6, 7 or 8 or the IL-12p35 polypeptides provided herein.

[0116] In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:9 or 10 or the IL-15 polypeptides provided herein. In some embodiments, the IL-15 polypeptide comprises the amino acid sequence of SEQ ID NO:9 or 10 or the IL-15 polypeptides provided herein.

[0117] In some embodiments, the IL-15Rα polypeptide comprises an amino acid sequence having at least or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 or SEQ ID NO:14 or the IL-15Rα polypeptides provided herein. In some embodiments, the IL-15Rα polypeptide comprises the amino acid sequence of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 or SEQ ID NO:14 or the IL-15Rα polypeptides provided herein.

[0118] In some embodiments, the IL-21 polypeptide comprises an amino acid sequence having at least or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:71 or the IL-21 polypeptides provided herein. In some embodiments, the IL-21 polypeptide comprises the amino acid sequence of SEQ ID NO:71 or the IL-21 polypeptides provided herein.

[0119] Linkers designated L1, L2, L3, L4, or L5 are linkers that can be used to connect different polypeptides provided herein. Although the linkers of L1, L2, L3, L4, and L5 can be the same, they can also be different. The structure (e.g., sequence) of each linker can be independent of the structure of the other linkers. Thus, in some embodiments, L1, L2, L3, L4, and L5 are the same, or each is different, or some are the same and some are different. In some embodiments, L1, L2, L3, L4, and L5 are peptide linkers.

[0120] In some embodiments, the peptide linker is a cleavable linker. Without being bound by theory, a cleavable linker contains at least one cleavage site that can be recognized and cleaved by an enzyme (which can be referred to as a protease). For example, in some embodiments, the enzyme furin recognizes a cleavage site having the general amino acid sequence of RXXR, where X is any amino acid. In some embodiments, the furin cleavage site is RAKR (SEQ ID NO:70). Other cleavable linkers are known in the art and can also be used in place of the furin cleavage site. For example, in some embodiments, the linker is a Val-Cit linker, a Val-Gly linker, a Gly-Gly linker, an Ala-Ala-Asn linker, or a linker comprising polyethylene glycol (PEG). Non-limiting examples of cleavable linkers are provided herein and below.

[0121] Thus, in some embodiments, one or more of L1, L2, L3, L4, or L5 are each independently a cleavable linker. In some embodiments, at least one of L1, L2, L3, L4, or L5 is a cleavable linker. In some embodiments, L1 is a cleavable linker and the remaining linkers are non-cleavable linkers or of unknown cleavability. In some embodiments, L2 is a cleavable linker and the remaining linkers are non-cleavable linkers or of unknown cleavability. In some embodiments, L3 is a cleavable linker and the remaining linkers are non-cleavable linkers or of unknown cleavability. In some embodiments, L1 and L2 are cleavable linkers and the remaining linkers are non-cleavable linkers or of unknown cleavability. In some embodiments, none of L1, L2, L3, L4, or L5 are cleavable linkers. In some embodiments, the cleavable linker is a furin-cleavable linker. In some embodiments, the cleavable linker is a Val-Cit linker, a Val-Gly linker, a Gly-Gly linker, or an Ala-Ala-Asn linker. In some embodiments, the cleavable linker is as shown in the following table and can be selected individually from such tables. In some embodiments, one or more of L1, L2, L3, L4, and L5 contain the sequence of (GSGSGG)n (SEQ ID NO:16), (GGGGS)n (SEQ ID NO:17), (GGGGA)n (SEQ ID NO:18), (GGGSE)n (SEQ ID NO:19), (GGGSK)n (SEQ ID NO:20), (AEEEK)n (SEQ ID NO:21) or a combination thereof, where each n is independently from 1 to 5. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, one or more of v contain the sequence of GSGSGGGSGSGGGSGSGG (SEQ ID NO:22). In some embodiments, each of L1, L2, L3, L4, and L5 contains the sequence of GSGSGGGSGSGGGSGSGG (SEQ ID NO:22).

[0122] Other non-limiting examples of linkers include glycine / serine linkers, which may be or comprise the sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:23) or the sequence GGGGSGGGGSGGGGS (SEQ ID NO:24). These are merely non-limiting examples, and the linker may have a different number of GGGGS (SEQ ID NO:17) repeats as provided herein. In some embodiments, the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 GGGGS (SEQ ID NO:17) repeats.

[0123] In some embodiments, the linker is a flexible linker. In some embodiments, the linker is a rigid linker. In some embodiments, the linker may be as described herein or as shown in the table below:

[0124] Type Sequence SEQ ID Flexible GGGGS SEQ ID NO:17 Flexible GGGGSGGGGSGGGGSGGGGS SEQ ID NO:23 Flexible GGGGSGGGGSGGGGS SEQ ID NO:24 Flexible GGGGGGGG SEQ ID NO:28 Flexible GGGGGG SEQ ID NO:29 Rigid <![CDATA[(EAAAK)3]]> SEQ ID NO:30 Rigid <![CDATA[(EAAK) n (n = 1 - 3)]]> SEQ ID NO:31 Rigid <![CDATA[A(EAAAK)4ALEA(EAAAK)4A]]> SEQ ID NO:32 Rigid AEAAAKEAAAKA SEQ ID NO:33 Rigid PAPAP SEQ ID NO:34 Rigid <![CDATA[(APAP) n (10-34aa)]]> SEQ ID NO:35 Cleavable Disulfide Not applicable Cleavable VSQTSKLTRAETVFPDV SEQ ID NO:36 Cleavable PLGLWA SEQ ID NO:37 Cleavable RVLAEA SEQ ID NO:38 Cleavable EDVVCCSMSY SEQ ID NO:39 Cleavable GGIEGRGS SEQ ID NO:40 Cleavable TRHRQPRGWE SEQ ID NO:41 Cleavable AGNRVRRSVG SEQ ID NO:42 Cleavable RRRRRRRRR SEQ ID NO:43 Cleavable GFLG SEQ ID NO:44 Dipeptide LE Not applicable Flexible SGGGSGGGGSGGGGSGGGSGGGSLQ SEQ ID NO:45 Cleavable SGGGPGGGGRAKRGGGGPSGGGSLQ SEQ ID NO:46 Cleavable SGGGPGGGGRARRGGGGPSGGGSLQ SEQ ID NO:47 Cleavable SGGGPGGGGSAGSGGGGPSGGGSLQ SEQ ID NO:48 Flexible GSGGSGSGGSGSGGSGSGGS SEQ ID NO:49 Flexible SGGGG SEQ ID NO:50

[0125] In some embodiments, the polypeptide may comprise a leader peptide at the N-terminus of the polypeptide. Without being bound by any particular theory, the leader peptide may be used to facilitate the expression and transport of the polypeptide, as it is produced by the cell such that, for example, it may be expressed on the cell surface. In some embodiments, the leader peptide comprises the sequence of the vesicular stomatitis virus G protein (VSV-G). In some embodiments, the leader peptide comprises, consists of, or consists essentially of the sequence MRISKPHLRSISIQCYLCLLLNSHFLTEA (SEQ ID NO:15).

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134] Although the sequences of SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, and SEQ ID NO:79 are shown with specific peptide linkers, these linkers can be modified or replaced with other peptide linkers such as, but not limited to, those provided herein.

[0135] The polypeptides described herein also encompass variants of the peptides provided herein. In some embodiments, the polypeptide comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% substantially similar or identical to the sequences provided herein. These variants include those having the various substitutions described herein and above. In some embodiments, the variant has 1, 2, 3, 4, or 5 additional substitutions. In some embodiments, the substitutions are conservative substitutions. In some embodiments, the conservative substitutions are selected based on the following table:

[0136]

[0137]

[0138]

[0139] The percent identity between two amino acid or two nucleic acid sequences can be determined by visual inspection and mathematical calculation, or, for example, by comparing the sequence information using a computer program. Exemplary computer programs are Genetics Computer Group (GCG; Madison, Wis.), the Wisconsin Package version 10.0 program GAP (Devereux et al. (1984), Nucleic Acids Res., Vol. 12: pp. 387-395). Preferred default parameters for the GAP program include: (1) the GCG implementation of the unary comparison matrix for nucleotides (containing a value of 1 for identity and 0 for non-identity) and the weighted amino acid comparison matrix of Gribskov and Burgess ((1986) Nucleic Acids Res., Vol. 14: p. 6745), as described in Atlas of Polypeptide Sequence and Structure, Schwartz and Dayhoff eds., National Biomedical Research Foundation, pp. 353-358 (1979), or other similar comparison matrices; (2) for amino acid sequences, a penalty of 8 for each gap and an additional penalty of 2 for each character in the gap, or for nucleotide sequences, a penalty of 50 for each gap and an additional penalty of 3 for each character in the gap; (3) no penalty for terminal gaps; and (4) no maximum penalty for long gaps. Other programs used by those skilled in the art of sequence comparison can also be used.

[0140]

[0141]

[0142]

[0143]

[0144] The sequences of SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, and SEQ ID NO:83 are merely exemplary sequences encoding the polypeptides described herein. Due to the degenerate nature of the codons, other nucleic acid molecules can be used. In some embodiments, the nucleic acid molecule is codon-optimized for expression in a bacterial system. In some embodiments, the nucleic acid molecule is codon-optimized for expression in a eukaryotic system or cell. In some embodiments, the nucleic acid molecule is a DNA or RNA molecule encoding the polypeptides provided herein. In some embodiments, the RNA molecule is an mRNA molecule.

[0145] Once the sequence is provided, the nucleic acid molecule can be prepared by synthesis or other conventional techniques known to those skilled in the art, which can be the sequence of the polypeptide to be encoded by the nucleic acid molecule or the nucleic acid sequence itself.

[0146] Another method of constructing a DNA sequence encoding the polypeptides provided herein is chemical synthesis. This includes, for example, directly synthesizing peptides encoding the protein sequences of the polypeptides provided herein by chemical means. This method can incorporate natural and unnatural amino acids at various positions. Alternatively, the nucleic acid molecule encoding the desired protein can be synthesized by chemical means using an oligonucleotide synthesizer. The oligonucleotides are designed based on the amino acid sequence of the desired protein, which can also be selected by using codons that are favorable in the cells that will produce the recombinant variant. It is recognized that the genetic code is degenerate, that is, an amino acid can be encoded by more than one codon. Therefore, it should be understood that for a given DNA sequence encoding a specific polypeptide provided herein, there will be many DNA degenerate sequences that will encode that polypeptide. Thus, in some embodiments, nucleic acid molecules encoding the polypeptides provided herein are provided. The nucleic acid molecule can be DNA or RNA.

[0147] In some embodiments, the nucleic acid molecule will encode a signal sequence or a leader peptide sequence, such as those provided herein. The signal sequence can be selected based on the cell in which it will be expressed. In some embodiments, if the host cell is prokaryotic, the nucleic acid molecule does not contain a signal sequence. In some embodiments, if the host cell is a eukaryotic cell, a signal sequence can be used. In some embodiments, the signal sequence or leader sequence is as provided herein. The signal or leader sequence of a protein can also be from an immature protein.

[0148] "Recombinant" when applied to a polypeptide or protein means that the production of the protein relies on at least one step in which a nucleic acid, which may or may not encode the protein, is introduced into a cell in which it is not naturally present. When a nucleic acid molecule is added exogenously to a cell or system, it can also be referred to as a heterologous molecule.

[0149] A variety of hosts (animal or cell systems) can be used to produce the proteins described herein. Examples of suitable host cells include, but are not limited to, bacteria, fungi (including yeast), plants, insects, mammals, or other suitable animal cells or cell lines, as well as transgenic animals or plants. In some embodiments, these hosts can include well-known eukaryotic and prokaryotic hosts such as Escherichia coli, Pseudomonas, Bacillus, Streptomyces, fungi, yeast, insect cells such as Spodoptera frugiperda (Sf9), animal cells such as Chinese hamster ovary (CHO) and mouse cells such as NS / O, African green monkey cells such as COS1, COS 7, BSC 1, BSC 40 and BNT 10, and human cells, as well as plant cells in tissue culture. For animal cell expression, CHO cells and COS 7 cells in culture can be used, particularly the CHO cell line CHO(DHFR-) or the HKB line. Proteins can also be expressed in vivo by delivering a nucleic acid molecule to cells in the body and having the cells express the protein, whereby the provided polypeptide will be transported to the cell surface. In some embodiments, the nucleic acid molecule is delivered with a vector such as a viral vector, including but not limited to an adenovirus vector. In some embodiments, the nucleic acid molecule is encapsulated in nanoparticles such as lipid nanoparticles to deliver the nucleic acid molecule to cells. In some embodiments, the encapsulated nucleic acid molecule is an RNA molecule. In some embodiments, the encapsulated nucleic acid molecule is a DNA molecule.

[0150] One of ordinary skill in the art can make a selection among various vectors, expression control sequences, and hosts without undue experimentation. For example, when selecting a vector, the host can be considered because the vector must be able to replicate therein, or the polypeptide must be able to be transcribed and / or translated from the vector in a particular host. The vector copy number, the ability to control that copy number, and the expression of any other proteins encoded by the vector such as antibiotic markers can also be considered. Such amplifiable vectors are well known in the art.

[0151] Checkpoint inhibitor polypeptide

[0152] Without being bound by any particular theory, it has been found that the embodiments provided herein use a single polypeptide molecule to simultaneously inhibit multiple immune checkpoint proteins. Current immune checkpoint treatment strategies require the administration of multiple immune checkpoint targeting molecules to target more than one immune checkpoint protein. While the use of combination therapies using these single immune checkpoint targeting protein molecules has demonstrated increased clinical benefit compared to monotherapy treatment, the risk of serious or dose-limiting adverse or long-term systemic off-target immune events also increases. By utilizing a single molecule, adverse and off-target events can be controlled. In addition, incorporating binding domains into the polypeptides of the embodiments provided herein will provide targeted delivery of the immune checkpoint inhibitor polypeptides, further increasing efficacy and reducing unwanted events.

[0153] Thus, the present disclosure describes polypeptide molecules comprising one or more immune checkpoint inhibitors. In some embodiments, a polypeptide is provided that comprises, from the N-terminus to the C-terminus, Formula X A -L A -X B or Formula X B -L A -X A wherein X A and X B are each independently an immune checkpoint inhibitory polypeptide, and L1 is a polypeptide linker. In some embodiments, X A and X B comprise the same immune checkpoint inhibitory polypeptide. In some embodiments, X A and X B comprise different immune checkpoint inhibitory polypeptides. In some embodiments, X A and X B can be any polypeptide or fragment thereof that inhibits an immune checkpoint protein. In some embodiments, X A and X B target the same immune checkpoint protein. In some embodiments, X A and X B target different immune checkpoint proteins. In some embodiments, X A and X B target immune checkpoint proteins selected from the group consisting of, but not limited to, CTLA-4, PD-1, PD-L1, TIM3, LAG3, VISTA, SIGLEC7, SIGLEC9, TIGIT, CD96, BTLA, B7H3, B7H4, CD155, HHLA2, and BTN3A1.

[0154] In some embodiments, the polypeptide comprises, from the N-terminus to the C-terminus, Formula X A -L A -X B . In some embodiments, the polypeptide comprises, from the N-terminus to the C-terminus, Formula X B -L A -X A . In some embodiments, the polypeptide comprises, from the N-terminus to the C-terminus, Formula X A- L A -X A . In some embodiments, the polypeptide comprises, from the N-terminus to the C-terminus, Formula X B -L A -X B .

[0155] In some embodiments, X Ais a peptide selected from the group consisting of: a CTLA-4 polypeptide, a PD-1 polypeptide, a PD-L1 polypeptide, a PD-L2 polypeptide, a B7.1 polypeptide or a B7.2 polypeptide, a TIM3 polypeptide, a LAG3 polypeptide, a VISTA polypeptide, a SINGLEC7 polypeptide, a SINGLEC9 polypeptide, a TIGIT polypeptide, a CD96 polypeptide, a BTLA polypeptide, a B7H3 polypeptide, a B7H4 polypeptide, a CD155 polypeptide, a HHLA2 polypeptide, a BTN3A1 polypeptide, or an active fragment thereof. In some embodiments, X B is a peptide selected from the group consisting of: a CTLA-4 polypeptide, a PD-1 polypeptide, a PD-L1 polypeptide, a PD-L2 polypeptide, a B7.1 polypeptide or a B7.2 polypeptide, a TIM3 polypeptide, a LAG3 polypeptide, a VISTA polypeptide, a SINGLEC7 polypeptide, a SINGLEC9 polypeptide, a TIGIT polypeptide, a CD96 polypeptide, a BTLA polypeptide, a B7H3 polypeptide, a B7H4 polypeptide, a CD155 polypeptide, a HHLA2 polypeptide, a BTN3A1 polypeptide, or an active fragment thereof. In some embodiments, X A and X B are each independently a peptide selected from the group consisting of: a PD-L1 polypeptide, a PD-L2 polypeptide, a B7.1 polypeptide or a B7.2 polypeptide. In some embodiments, X A and X B comprise the same polypeptide. In some embodiments, X A and X B comprise different polypeptides.

[0156] In some embodiments, X A is a PD-L1 polypeptide and X B is a B7.1 polypeptide. In some embodiments, X A is a PD-L1 polypeptide and X B is a B7.2 polypeptide. In some embodiments, X A is a PD-L2 polypeptide and X B is a B7.1 polypeptide. In some embodiments, X A is a PD-L2 polypeptide and X B is a B7.2 polypeptide.

[0157] In some embodiments, the PD-L1 polypeptide comprises the amino acid sequence of SEQ ID NO: 101: MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET (SEQ ID NO: 101) or an active fragment thereof. In any of the embodiments herein, the PD-L1 polypeptide may comprise an amino acid sequence corresponding to the entire PD-L1 protein or any fragment thereof. Additionally, it should be understood that the PD-L1 polypeptide is not limited to a particular isotype, but rather the PD-L1 polypeptides as described herein encompass all PD-L1 isotypes. The PD-L1 polypeptides presented herein may comprise the wild-type PD-L1 protein, or they may comprise any PD-L1 mutant protein known in the art. Furthermore, it should be understood that the PD-L1 polypeptide is not limited to human PD-L1 polypeptides, but rather encompasses PD-L1 polypeptide sequences from other related species, including but not limited to mice, rats, non-human primates, cattle, horses, etc.

[0158] In some embodiments, the PD-L1 polypeptide comprises the extracellular domain of PD-L1. In some embodiments, the PD-L1 polypeptide comprises an amino acid sequence substantially similar to SEQ ID NO: 102: FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNER (SEQ ID NO: 102). In some embodiments, the PD-L1 polypeptide comprises an active fragment of SEQ ID NO: 102. In some embodiments, the PD-L1 polypeptide comprises an amino acid sequence having at least 60% identity to SEQ ID NO: 102. In some embodiments, the PD-L1 polypeptide comprises an amino acid sequence having at least 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 102. In some embodiments, the PD-L1 polypeptide comprises the same amino acid sequence as SEQ ID NO: 102.

[0159] In some embodiments, the PD-L2 polypeptide comprises the amino acid sequence of SEQ ID NO: 103: MIFLLLMLSLELQLHQIAALFTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITASLQKVENDTSPHRERATLLEEQLPLGKASFHIPQVQVRDEGQYQCIIIYGVAWDYKYLTLKVKASYRKINTHILKVPETDEVELTCQATGYPLAEVSWPNVSVPANTSHSRTPEGLYQVTSVLRLKPPPGRNFSCVFWNTHVRELTLASIDLQSQMEPRTHPTWLLHIFIPFCIIAFIFIATVIALRKQLCQKLYSSKDTTKRPVTTTKREVNSAI (SEQ ID NO: 103) or an active fragment thereof. In any of the embodiments herein, the PD-L2 polypeptide may comprise the amino acid sequence corresponding to the entire PD-L2 protein or any fragment thereof. Additionally, it should be understood that the PD-L2 polypeptide is not limited to a particular isotype, but rather the PD-L2 polypeptides as described herein encompass all PD-L2 isotypes. The PD-L2 polypeptides presented herein may comprise the wild-type PD-L2 protein, or it may comprise any PD-L2 mutant protein known in the art. Additionally, it should be understood that the PD-L2 polypeptide is not limited to the human PD-L1 polypeptide, but rather encompasses PD-L2 polypeptide sequences from other related species, including but not limited to mice, rats, non-human primates, cattle, horses, etc.

[0160] In some embodiments, the PD-L2 polypeptide comprises the extracellular domain of PD-L2. In some embodiments, the PD-L2 polypeptide comprises an amino acid sequence substantially similar to SEQ ID NO: 104: LFTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITASLQKVENDTSPHRERATLLEEQLPLGKASFHIPQVQVRDEGQYQCIIIYGVAWDYKYLTLKVKASYRKINTHILKVPETDEVELTCQATGYPLAEVSWPNVSVPANTSHSRTPEGLYQVTSVLRLKPPPGRNFSCVFWNTHVRELTLASIDLQSQMEPRTHPT (SEQ ID NO: 104). In some embodiments, the PD-L2 polypeptide comprises an active fragment of SEQ ID NO: 104. In some embodiments, the PD-L2 polypeptide comprises an amino acid sequence having at least 60% identity to SEQ ID NO: 104. In some embodiments, the PD-L2 polypeptide comprises an amino acid sequence having at least 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 104. In some embodiments, the PD-L2 polypeptide comprises the same amino acid sequence as SEQ ID NO: 104.

[0161] In some embodiments, the B7.1 polypeptide comprises the amino acid sequence of SEQ ID NO:105: MGHTRRQGTSPSKCPYLNFFQLLVLAGLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV (SEQ ID NO:105) or an active fragment thereof. In any of the embodiments herein, the B7.1 polypeptide may comprise an amino acid sequence corresponding to the entire B7.1 protein or any fragment thereof. Further, it should be understood that the B7.1 polypeptide is not limited to a particular isotype, but rather the B7.1 polypeptides as described herein encompass all B7.1 isotypes. The B7.1 polypeptides presented herein may comprise the wild-type B7.1 protein, or it may comprise any B7.1 mutant protein known in the art. Additionally, it should be understood that the B7.1 polypeptide is not limited to human B7.1 polypeptides, but rather encompasses B7.1 polypeptide sequences from other related species, including but not limited to mice, rats, non-human primates, cattle, horses, etc.

[0162] In some embodiments, the B7.1 polypeptide comprises the B7.1 extracellular domain. In some embodiments, the B7.1 polypeptide comprises an amino acid sequence substantially similar to SEQ ID NO:106: VIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDN (SEQ ID NO:106). In some embodiments, the B7.1 polypeptide comprises an active fragment of SEQ ID NO:106. In some embodiments, the B7.1 polypeptide comprises an amino acid sequence having at least 60% identity to SEQ ID NO:106. In some embodiments, the B7.1 polypeptide comprises an amino acid sequence having at least 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:106. In some embodiments, the B7.1 polypeptide comprises the same amino acid sequence as SEQ ID NO:106.

[0163] In some embodiments, the B7.2 polypeptide comprises the amino acid sequence of SEQ ID NO: 107: MDPQCTMGLSNILFVMAFLLSGAAPLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVHSKYMGRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVLANFSQPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGVMQKSQDNVTELYDVSISLSVSFPDVTSNMTIFCILETDKTRLLSSPFSIELEDPQPPPDHIPWITAVLPTVIICVMVFCLILWKWKKKKRPRNSYKCGTNTMEREESEQTKKREKIHIPERSDEAQRVFKSSKTSSCDKSDTCF (SEQ ID NO: 107) or an active fragment thereof. In any of the embodiments herein, the B7.2 polypeptide may comprise an amino acid sequence corresponding to the entire B7.2 protein or any fragment thereof. Additionally, it should be understood that the B7.2 polypeptide is not limited to a particular isotype, but rather the B7.2 polypeptides as described herein encompass all B7.2 isotypes. The B7.2 polypeptides presented herein may comprise the wild-type B7.2 protein, or it may comprise any B7.2 mutant protein known in the art. Additionally, it should be understood that the B7.2 polypeptide is not limited to human B7.2 polypeptides, but rather encompasses B7.2 polypeptide sequences from other related species, including but not limited to mice, rats, non-human primates, cattle, horses, etc.

[0164] In some embodiments, the B7.2 polypeptide comprises the B7.2 extracellular domain. In some embodiments, the B7.2 polypeptide comprises an amino acid sequence substantially similar to SEQ ID NO: 108: APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVHSKYMGRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVLANFSQPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGVMQKSQDNVTELYDVSISLSVSFPDVTSNMTIFCILETDKTRLLSSPFSIELEDPQPPPDHIP (SEQ ID NO: 108). In some embodiments, the B7.2 polypeptide comprises an amino acid sequence having at least 60% identity to SEQ ID NO: 108. In some embodiments, the B7.2 polypeptide comprises an amino acid sequence having at least 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 108. In some embodiments, the B7.2 polypeptide comprises the same amino acid sequence as SEQ ID NO: 108.

[0165] Linker L1 can comprise any peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of GSGSGGGSGSGGGSGSGSG (SEQ ID NO: 109). In some embodiments, the peptide linker comprises an amino acid sequence substantially similar to SEQ ID NO: 109. In some embodiments, the peptide linker comprises a fragment of SEQ ID NO: 109, or comprises n repeats of SEQ ID NO: 109, where n is an integer from 1-5. In some embodiments, the linker is a flexible linker. In some embodiments, the linker is a rigid linker. In some embodiments, the linker can be as described herein or as shown in the table below:

[0166] Type Sequence Flexible GGGGS Flexible <![CDATA[(GGGGS)3]]> Flexible <![CDATA[(GGGGS) n (n = 1, 2, 3, 4)]]> Flexible <![CDATA[(Gly)8]]> Flexible <![CDATA[(Gly)6]]> Rigid <![CDATA[(EAAAK)3]]> Rigid <![CDATA[(EAAK) n (n = 1 - 3)]]> Rigid <![CDATA[A(EAAAK)4ALEA(EAAAK)4A]]> Rigid AEAAAKEAAAKA Rigid PAPAP Rigid <![CDATA[(Ala-Pro) n (10-34aa)]]> Cleavable Disulfide Cleavable VSQTSKLTRAETVFPDV Cleavable PLGLWA Cleavable RVLAEA Cleavable EDVVCCSMSY Cleavable GGIEGRGS Cleavable TRHRQPRGWE Cleavable AGNRVRRSVG Cleavable RRRRRRRRR Cleavable GFLG Dipeptide LE

[0167] In some embodiments, the provided polypeptide further comprises a leader sequence. In some embodiments, the leader sequence is located at the N-terminus of the polypeptide. In some embodiments, the leader sequence is a targeting polypeptide sequence. In some embodiments, the targeting sequence is used to direct the polypeptide to a specific subcellular compartment or organelle. In some embodiments, the targeting sequence is used to direct the polypeptide to the nucleus. In some embodiments, the leader sequence is a cell membrane targeting sequence. In some embodiments, the leader sequence is native to the polypeptide comprising the N-terminus of the polypeptide. In some embodiments, the leader sequence is not native to the polypeptide comprising the N-terminus of the polypeptide. In some embodiments, the leader sequence comprises a polypeptide sequence corresponding to the cell membrane targeting domain of PD-L1. In some embodiments, the leader sequence comprises a polypeptide sequence corresponding to the cell membrane targeting domain of PD-L2. In some embodiments, the leader sequence comprises a polypeptide sequence corresponding to the cell membrane targeting domain of B7.1. In some embodiments, the leader sequence comprises a polypeptide sequence corresponding to the cell membrane targeting domain of B7.2. In some embodiments, the leader sequence comprises a polypeptide sequence corresponding to any cell membrane targeting domain that delivers the provided polypeptide to the cell surface.

[0168] In some embodiments, the leader sequence comprises an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 110: MIFLLLMLSLELQLHQIAA (SEQ ID NO: 110), or is the same as SEQ ID NO: 110, or is an active fragment thereof.

[0169] In some embodiments, the leader sequence comprises an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 111: MRIFAVFIFMTYWHLLNA (SEQ ID NO: 111), or is the same as SEQ ID NO: 111, or is an active fragment thereof.

[0170] In some embodiments, the leader sequence comprises an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:112: MGHTRRQGTSPSKCPYLNFFQLLVLAG (SEQ ID NO:112), or is the same as SEQ ID NO:112, or is an active fragment thereof.

[0171] In some embodiments, the leader sequence comprises an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:113: MDPQCTMGLSNILFVMAFLLSGA (SEQ ID NO:113), or is the same as SEQ ID NO:113, or is an active fragment thereof.

[0172] In some embodiments, the provided polypeptide further comprises a targeting domain. The targeting domain can comprise any protein that directs the provided polypeptide to its intended cellular target. In some embodiments, the targeting domain directs the provided polypeptide to an antigen-presenting cell, an immune cell, a cancer cell, an epithelial cell, a mesenchymal cell, a neuron, etc. In some embodiments, the targeting domain comprises a protein from the group including but not limited to: cytokines, cytokine traps, receptor ligands, complement proteins, T cell receptors, antibodies, Fc domains, chimeric antigen receptors, antigens, tumor antigens, etc., or any fragment thereof.

[0173] In some embodiments, a polypeptide is provided that comprises, from the N-terminus to the C-terminus, a formula selected from the group consisting of: L S -X A -L A -X B -L B -X C 、L S -X A -L A -X C -L B -X B 、L S -X B -L A -X A -L B -XC or L S -X B -L A -X C -L B -X A , wherein L S is a leader sequence, X A and X2 are each independently an immune checkpoint inhibitory polypeptide, X C is a targeting domain, and A and L B are each independently a polypeptide linker. In some embodiments, X A and X B comprise the same immune checkpoint inhibitory polypeptide. In some embodiments, X A and X B comprise different immune checkpoint inhibitory polypeptides. In some embodiments, X A and X B can be any polypeptide or fragment thereof that inhibits an immune checkpoint protein. In some embodiments, X A and X B target the same immune checkpoint protein. In some embodiments, X A and X B target different immune checkpoint proteins. In some embodiments, X A and X B target immune checkpoint proteins selected from the group consisting of, but not limited to, the following: CTLA-4, PD-1, PD-L1, TIM3, LAG3, VISTA, SIGLEC7, SIGLEC9, TIGIT, CD96, BTLA, B7H3, B7H4, CD155, HHLA2, and BTN3A1. In some embodiments, L A and L B comprise the same polypeptide linker. In some embodiments, L A and L B comprise different polypeptide linkers. In some embodiments, L A and L B are each independently a polypeptide linker provided herein. In some embodiments, the targeting domain X CAny protein that can direct the provided polypeptide to its intended cellular target may be included. In some embodiments, the targeting domain directs the provided polypeptide to antigen-presenting cells, immune cells, cancer cells, epithelial cells, mesenchymal cells, neurons, etc. In some embodiments, the targeting domain comprises a protein from the group including but not limited to: cytokines, cytokine traps, receptor ligands, complement proteins, T cell receptors, antibodies, Fc domains, chimeric antigen receptors, antigens, tumor antigens, etc. or any fragment thereof.

[0174] In some embodiments, the polypeptide comprises, from the N-terminus to the C-terminus, formula L S -X A -L A -X B -L B -X C 。In some embodiments, the polypeptide comprises, from the N-terminus to the C-terminus, formula L S -X A -L A -X C -L B -X B 。In some embodiments, the polypeptide comprises, from the N-terminus to the C-terminus, formula L S -X B -L A -X A -L B -X C 。

[0175] In some embodiments, the polypeptide comprises, from the N-terminus to the C-terminus, formula L S -X B -L A -X C -L B -X A 。In some embodiments, L S is the leader sequence provided herein. In some embodiments, X A comprises the PD-L1 polypeptide or PD-L2 polypeptide provided herein. In some embodiments, X B comprises the B7.1 polypeptide or B7.2 polypeptide provided herein. In some embodiments, the targeting domain X C is the targeting domain provided herein. In some embodiments, L A and L B each independently is absent or is the polypeptide linker provided herein.

[0176] In some embodiments, X A is the PD-L1 polypeptide provided herein; X B is the B7.1 polypeptide provided herein; X Cis the antibody Fc domain; L A is the polypeptide linker provided herein; and L B is the polypeptide linker provided herein or is absent.

[0177] In some embodiments, X A is the PD-L1 polypeptide provided herein; X B is the B7.2 polypeptide provided herein; X C is the antibody Fc domain; L A is the polypeptide linker provided herein; and L B is the polypeptide linker provided herein or is absent.

[0178] In some embodiments, X A is the PD-L2 polypeptide provided herein; X B is the B7.1 polypeptide provided herein; X C is the antibody Fc domain; L A is the polypeptide linker provided herein; and L B is the polypeptide linker provided herein or is absent.

[0179] In some embodiments, X A is the PD-L2 polypeptide provided herein; X B is the B7.2 polypeptide provided herein; X C is the antibody Fc domain; L A is the polypeptide linker provided herein; and L B is the polypeptide linker provided herein or is absent.

[0180] In some embodiments, the PD-L1 polypeptide comprises the PD-L1 extracellular domain provided herein. In some embodiments, the PD-L1 polypeptide comprises an amino acid sequence that is substantially similar to SEQ ID NO:102. In some embodiments, the PD-L1 polypeptide comprises an amino acid sequence that has at least 60% identity to SEQ ID NO:102. In some embodiments, the PD-L1 polypeptide comprises an amino acid sequence that has at least 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:102. In some embodiments, the PD-L1 polypeptide comprises the same amino acid sequence as SEQ ID NO:102.

[0181] In some embodiments, the PD-L2 polypeptide comprises the PD-L2 extracellular domain provided herein. In some embodiments, the PD-L2 polypeptide comprises an amino acid sequence that is substantially similar to SEQ ID NO:104. In some embodiments, the PD-L2 polypeptide comprises an amino acid sequence that has at least 60% identity to SEQ ID NO:104. In some embodiments, the PD-L2 polypeptide comprises an amino acid sequence that has at least 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:104. In some embodiments, the PD-L2 polypeptide comprises the same amino acid sequence as SEQ ID NO:104.

[0182] In some embodiments, the B7.1 polypeptide comprises the B7.1 extracellular domain provided herein. In some embodiments, the B7.1 polypeptide comprises an amino acid sequence that is substantially similar to SEQ ID NO:106. In some embodiments, the B7.1 polypeptide comprises an amino acid sequence that has at least 60% identity to SEQ ID NO:106. In some embodiments, the B7.1 polypeptide comprises an amino acid sequence that has at least 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:106. In some embodiments, the B7.1 polypeptide comprises the same amino acid sequence as SEQ ID NO:106.

[0183] In some embodiments, the B7.2 polypeptide comprises the B7.2 extracellular domain provided herein. In some embodiments, the B7.2 polypeptide comprises an amino acid sequence that is substantially similar to SEQ ID NO:108. In some embodiments, the B7.2 polypeptide comprises an amino acid sequence that has at least 60% identity to SEQ ID NO:108. In some embodiments, the B7.2 polypeptide comprises an amino acid sequence that has at least 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:108. In some embodiments, the B7.2 polypeptide comprises the same amino acid sequence as SEQ ID NO:108.

[0184] In some embodiments, the homodimerization domain comprises any protein containing an immunoglobulin fold. In some embodiments, the protein containing an immunoglobulin fold comprises any human Fc domain. In some embodiments, the Fc domain is selected from the group consisting of, but not limited to, IgG, IgM, IgA, IgE, or IgD, or any subclass thereof, including but not limited to IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2. In some embodiments, the Fc domain can be engineered to enhance binding to a specific target protein. For example, in some embodiments, the Fc domain is bispecific. In some embodiments, the Fc domain is engineered to enhance binding to a specific Fc receptor (FcR). In some embodiments, the FcR is selected from the group consisting of, but not limited to, FcγR, FcαR, FcεR, FcγRI, FcγRIIA, FcγRIIB1, FcγRIIB2, FcγRIIIA, FcγRIIIB, FcεRI, FcεRII, FcαRI, FcαR / μR, or FcRn. Specific engineering / point mutations of the Fc domain that enhance binding to a specific FcR are known in the art and are within the scope of the present application.

[0185] In some embodiments, the Fc domain is a human IgG1 Fc domain. In some embodiments, the Fc domain comprises an amino acid sequence substantially similar to SEQ ID NO: 114: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 114) or an active fragment thereof. In some embodiments, the Fc domain comprises an amino acid sequence having at least 60% identity to SEQ ID NO: 114. In some embodiments, the Fc domain comprises an amino acid sequence having at least 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 114. In some embodiments, the Fc domain comprises the same amino acid sequence as SEQ ID NO: 114.

[0186] In some embodiments, a polypeptide of the formula comprising, from the N-terminus to the C-terminus, a group selected from the group consisting of X A -L A -X B or X B -L A -X A comprises an amino acid sequence that is substantially similar to SEQ ID NO: 115: MIFLLLMLSLELQLHQIAALFTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITASLQKVENDTSPHRERATLLEEQLPLGKASFHIPQVQVRDEGQYQCIIIYGVAWDYKYLTLKVKASYRKINTHILKVPETDEVELTCQATGYPLAEVSWPNVSVPANTSHSRTPEGLYQVTSVLRLKPPPGRNFSCVFWNTHVRELTLASIDLQSQMEPRTHPTGSGSGGGSGSGGGSGSGSGLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPD (SEQ ID NO: 115) or an active fragment thereof. In some embodiments, the polypeptide comprises an amino acid sequence having at least 60% identity to SEQ ID NO: 115. In some embodiments, the polypeptide comprises an amino acid sequence having at least 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 115. In some embodiments, the polypeptide comprises the same amino acid sequence as SEQ ID NO: 115.

[0187] In some embodiments, a polypeptide of the formula comprising, from the N-terminus to the C-terminus, a group selected from the group consisting of X A -L A -X B or X B -L A -X AThe polypeptides of the group of formulae comprise an amino acid sequence substantially similar to SEQ ID NO:116: MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERGSGSGGGSGSGGGSGSGSAPLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVHSKYMGRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVLANFSQPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGVMQKSQDNVTELYDVSISLSVSFPDVTSNMTIFCILETDKTRLLSSPFSIELEDPQPPPDHIP(SEQ ID NO:116) or an active fragment thereof. In some embodiments, the polypeptide comprises an amino acid sequence having at least 60% identity to SEQ ID NO:116. In some embodiments, the polypeptide comprises an amino acid sequence having at least 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:116. In some embodiments, the polypeptide comprises the same amino acid sequence as SEQ ID NO:116.

[0188] In some embodiments, from the N-terminus to the C-terminus, it comprises a selection from the group consisting of L S -X A -L A -X B -L B -X C L S -X B -L A -X A -L B -X C L S -X A -L A-X C -L B -X B or L S -X B -L A -X C -L B -X AA polypeptide of the formula of the group contains an amino acid sequence substantially similar to SEQ ID NO: 117: MIFLLLMLSLELQLHQIAALFTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITASLQKVENDTSPHRERATLLEEQLPLGKASFHIPQVQVRDEGQYQCIIIYGVAWDYKYLTLKVKASYRKINTHILKVPETDEVELTCQATGYPLAEVSWPNVSVPANTSHSRTPEGLYQVTSVLRLKPPPGRNFSCVFWNTHVRELTLASIDLQSQMEPRTHPTGSGSGGGSGSGGGSGSGSGLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 117) or an active fragment thereof. In some embodiments, the polypeptide contains an amino acid sequence having at least 60% identity to SEQ ID NO: 117. In some embodiments, the polypeptide contains an amino acid sequence having at least 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 117. In some embodiments, the polypeptide contains the same amino acid sequence as SEQ ID NO: 117.

[0189] In some embodiments, from the N-terminus to the C-terminus, it contains an L selected from the group consisting of S -XA -L A -X B -L B -X C L S -X B -L A -X A -L B -X C L S -X A -L A -X C -L B -X B or L S -X B -L A -X C -L B -X APolypeptides of the formula of the group contain an amino acid sequence substantially similar to SEQ ID NO:118: MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERGSGSGGGSGSGGGSGSGSAPLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVHSKYMGRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVLANFSQPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGVMQKSQDNVTELYDVSISLSVSFPDVTSNMTIFCILETDKTRLLSSPFSIELEDPQPPPDHIPDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:118) or an active fragment thereof. In some embodiments, the polypeptide contains an amino acid sequence having at least 60% identity to SEQ ID NO:118. In some embodiments, the polypeptide contains an amino acid sequence having at least 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:118. In some embodiments, the polypeptide contains the same amino acid sequence as SEQ ID NO:118.

[0190]

[0191]

[0192]

[0193]

[0194] In some embodiments, a vector is provided that comprises a nucleic acid molecule provided herein. In some embodiments, the vector is a plasmid. In some embodiments, the vector is a virus. In some embodiments, a plasmid is provided that comprises a nucleic acid molecule provided herein. In some embodiments, a composition is provided that comprises an encapsulated nucleic acid molecule provided herein.

[0195] In some embodiments, a virus is provided. In some embodiments, the virus is a recombinant virus. In some embodiments, the recombinant virus is selected from the group consisting of lentivirus, adenovirus, adeno-associated virus, and the like. In some embodiments, the recombinant virus is a recombinant adenovirus. In some embodiments, the recombinant adenovirus is replication-competent. In some embodiments, the recombinant adenovirus is replication-incompetent. In some embodiments, the replication-incompetent recombinant virus further comprises a defective or modified E1 gene, E3 gene, E4 gene, E4 promoter, hexon gene, penton gene, fiber gene, or a combination thereof. In some embodiments, the replication-incompetent recombinant virus comprises a defective or modified E1 gene.

[0196] In some embodiments, the recombinant virus comprises a nucleic acid molecule provided herein. In some embodiments, the nucleic acid molecule is selected from the group consisting of SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, and SEQ ID NO:122. In some embodiments, the recombinant virus comprises a nucleic acid molecule encoding a polypeptide provided herein. In some embodiments, the polypeptide is selected from the group consisting of SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, and SEQ ID NO:118. In some embodiments, the nucleic acid molecule is flanked by a 5' adenovirus ITR and a 3' adenovirus ITR.

[0197] In some embodiments, a cell is provided. In some embodiments, the cell comprises a polypeptide molecule provided herein. In some embodiments, the polypeptide molecule is selected from the group consisting of SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, and SEQ ID NO:118. In some embodiments, the cell comprises a nucleic acid molecule encoding a polypeptide provided herein. In some embodiments, the nucleic acid molecule is selected from the group consisting of SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, and SEQ ID NO:122.

[0198] In some embodiments, the cell further comprises a chimeric antigen receptor (“CAR”). The CAR can be used to treat cancer or tumors in a subject. In some embodiments, the activity of the CAR can be enhanced by co-expressing the polypeptide provided herein with the CAR. Thus, in some embodiments, a cell comprising the CAR and the polypeptide provided herein is provided.

[0199] The cell can be any type of suitable cell. In some embodiments, the cell is an immune cell, such as but not limited to a T cell, NK cell, dendritic cell, monocyte, B cell, myeloid cell, etc.

[0200] In some embodiments, a host cell is provided. In some embodiments, the host cell contains a competent E1 gene, E3 gene, E4 gene, E4 promoter, hexon gene, penton gene, fiber gene, or a combination thereof to complement any defective or modified genes in the recombinant virus. In some embodiments, the host cell contains a competent E1 gene to complement a defective or modified E1 gene in the recombinant virus. In some embodiments, the competent E1 gene, E3 gene, E4 gene, E4 promoter, hexon gene, penton gene, fiber gene, or a combination thereof is provided to the host cell by transient contact with the host cell. In some embodiments, the host cell contains the competent E1 gene, E3 gene, E4 gene, E4 promoter, hexon gene, penton gene, fiber gene, or a combination thereof within its genome.

[0201] Combination composition

[0202] In some embodiments, a composition comprising any interleukin polypeptide provided herein and any immune checkpoint polypeptide provided herein is provided. In some embodiments, the composition comprises a first polypeptide and a second polypeptide, wherein the first polypeptide is any interleukin polypeptide provided herein, and wherein the second polypeptide is any immune checkpoint polypeptide provided herein.

[0203] In some embodiments, the composition further comprises a third polypeptide, such as an antigen. In some embodiments, the antigen is any antigen provided herein. In some embodiments, the antigen is a tumor antigen, viral antigen, bacterial antigen, or microbial antigen. In some embodiments, the antigen is ovalbumin (OVA).

[0204] In some embodiments, the composition comprises a nucleic acid molecule encoding any polypeptide provided herein. In some embodiments, the composition comprises a first nucleic acid molecule encoding the first polypeptide provided herein. In some embodiments, the composition comprises a second nucleic acid molecule encoding the second polypeptide provided herein. In some embodiments, the composition comprises a third nucleic acid molecule encoding the third polypeptide provided herein.

[0205] Vectors, plasmids, liposomes, pharmaceutical compositions, and host cells

[0206] Thus, in some embodiments, vectors encoding the polypeptides described herein, and host cells transformed or transduced with such vectors are provided. Any nucleic acid encoding the proteins described herein can be included in one or more vectors, which can, for example, contain selectable markers and origins of replication for propagation in a host. In some embodiments, the vector also includes suitable transcriptional or translational regulatory sequences operably linked to the nucleic acid molecule encoding the protein, such as those derived from mammalian, microbial, viral, or insect genes. Examples of such regulatory sequences include transcriptional promoters, operators or enhancers, mRNA ribosome binding sites, and appropriate sequences that control transcription and translation. A nucleotide sequence is operably linked when the regulatory sequence is functionally related to the DNA encoding the target protein. Thus, a promoter nucleotide sequence is operably linked to a nucleic acid molecule if the promoter nucleotide sequence directs the transcription of the nucleic acid molecule.

[0207] In some embodiments, a vector is provided that contains the nucleic acid molecule provided herein. In some embodiments, the vector is a plasmid. In some embodiments, the vector is an encapsulated vector, such as provided herein. In some embodiments, the vector is a viral vector, such as, but not limited to, an adenovirus, an adeno-associated virus, or a lentivirus. In some embodiments, the adenovirus is replication-incompetent. In some embodiments, the adenovirus is replication-competent.

[0208] Thus, in some embodiments, the vector contains the first nucleic acid molecule provided herein, the second nucleic acid molecule provided herein, and optionally the third nucleic acid molecule provided herein. In some embodiments, the compositions herein contain a first vector containing the first nucleic acid molecule and a second vector containing the second nucleic acid molecule. In some embodiments, the first vector or the second vector further contains the third nucleic acid molecule provided herein. In some embodiments, the composition contains a third vector that contains the third nucleic acid molecule. In some embodiments, the first, second, and / or third nucleic acid molecules are present in their own separate vectors. For example, in some embodiments, the composition contains a first vector containing the first nucleic acid molecule, a second vector containing the second nucleic acid molecule, and optionally a third vector containing the third nucleic acid molecule. In some embodiments, each vector is in a separate composition.

[0209] In some embodiments, the plasmid comprises the first nucleic acid molecule provided herein, the second nucleic acid molecule provided herein, and optionally the third nucleic acid molecule provided herein. In some embodiments, the compositions herein comprise a first plasmid containing the first nucleic acid molecule and a second plasmid containing the second nucleic acid molecule. In some embodiments, the first plasmid or the second plasmid further comprises the third nucleic acid molecule provided herein. In some embodiments, the composition comprises a third plasmid, which third plasmid comprises the third nucleic acid molecule. In some embodiments, the first, second, and / or third nucleic acid molecules are present in their own separate plasmids. For example, in some embodiments, the composition comprises a first plasmid containing the first nucleic acid molecule, a second plasmid containing the second nucleic acid molecule, and optionally a third plasmid containing the third nucleic acid molecule. In some embodiments, each plasmid is in a separate composition.

[0210] In some embodiments, the recombinant virus comprises the first nucleic acid molecule provided herein, the second nucleic acid molecule provided herein, and optionally the third nucleic acid molecule provided herein. In some embodiments, the recombinant virus can contain any one or more of the vectors or plasmids described herein. In some embodiments, the recombinant virus is an adenovirus or a lentivirus. In some embodiments, the recombinant virus is replication-competent or replication-incompetent. In some embodiments, the recombinant virus is a replication-incompetent adenovirus.

[0211] The nucleic acid molecules, vectors, and plasmids described herein may also be encapsulated by lipids or liposomes. Thus, in some embodiments, a liposome comprises a first nucleic acid molecule provided herein, a second nucleic acid molecule provided herein, and optionally a third nucleic acid molecule provided herein. In some embodiments, a liposome may contain one or more of the vectors or plasmids described herein.In some embodiments, the lipids and liposomes comprise cationic lipids such as, but not limited to, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 5-carboxyspermine-glycine bis(octadecylamide) (DOGS), 2,3-dioleoyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propylamine (DOSPA), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), 1,2-distearoyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dioleoyloxy-N,N-dimethyl-3-aminopropane (DODMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLenDMA), N-dioleoyl-N,N-dimethylammonium chloride (DODAC), N,N-distearoyl-N,N-dimethylammonium bromide (DDAB), N-(1,2-dimyristoyloxypropyl-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), 3-dimethylamino-2-(cholest-5-en-3-β-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadienyloxy)propane (CLinDMA), 2-[5'-(cholest-5-en-3-β-oxy)-3'-oxapentyloxy)-3-dimethyl-1-(cis,cis-9',12'-octadecadienyloxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleoyloxybenzylamine (DMOBA), 1,2-N,N'-dioleoylaminocarbonyl-3-dimethylaminopropane (DOcarbDAP), 2,3-dilinoleyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N'-dilinoeylaminocarbonyl-3-dimethylaminopropane (DLincarbDAP), 1,2-dilinoeylaminocarbonyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-XTC2-DMA) or C12-200.

[0212] In another aspect, embodiments of the present invention provide compositions, such as pharmaceutically acceptable compositions, that comprise the polypeptides provided herein or nucleic acid molecules encoding them, which can be formulated, for example, with one or more excipients. In some embodiments, suitable excipients include, but are not limited to, purified water, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, polymers such as polyethylene glycol, propylene glycol, PEG 400, glycerol, DMA, ethanol, benzyl alcohol, citric acid / sodium citrate (pH 3), citric acid / sodium citrate (pH 5), tris(hydroxymethyl)aminomethane HCl (pH 7.0), 0.9% saline, and 1.2% saline, and any combination thereof.

[0213] In some embodiments, there are provided pharmaceutical compositions comprising cells that comprise the vectors, polypeptides, or nucleic acid molecules provided herein.

[0214] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In some embodiments, suitable pharmaceutically acceptable carriers include, but are not limited to, water, silicone, wax, petrolatum, polyethylene glycol, propylene glycol, liposomes, lipids such as cholesterol, cationic lipids such as 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)), sugars (such as mannitol and lactose), and other materials depending on the particular type of formulation used. In some embodiments, suitable pharmaceutically acceptable carriers include, but are not limited to, nanoparticles, such as gold or metal nanoparticles.

[0215] In some embodiments, the pharmaceutically acceptable carrier may be suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, topical, transdermal, spinal, or epidermal administration (e.g., by injection or infusion). In some embodiments, the pharmaceutical composition comprises a carrier that comprises a nucleic acid molecule encoding the polypeptide provided herein. In some embodiments, the nucleic acid molecule is a DNA molecule or an RNA molecule. In some embodiments, the carrier is a virus, such as those provided herein.

[0216] The composition can be in various forms. These dosage forms include, for example, liquid, semi-solid, and solid dosage forms such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes, and suppositories. The preferred form depends on the intended mode of administration and therapeutic application. A typical composition is in the form of an injectable or infusible solution. In some embodiments, the mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intradermal, intramuscular, intravesicular). In some embodiments, the composition is administered by intravenous infusion or injection. In some embodiments, the composition is administered by intramuscular or subcutaneous injection. In some embodiments, the composition is administered enterally, sublingually, by inhalation, or intranasally. In some embodiments, the composition is administered locally, e.g., by injection or topical application to the target site. For example, the pharmaceutical composition can be lyophilized and reconstituted for use prior to administration to a patient.

[0217] As used herein, the phrases "parenteral administration" and "parenterally administered" mean a mode of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

[0218] Compositions, such as pharmaceutical compositions, are generally sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for a high concentration of the active ingredient. A sterile injectable solution can be prepared by incorporating the required amount of the active compound (i.e., therapeutic molecule, nucleic acid molecule, cell, polypeptide, vector, etc.) into a suitable solvent, with one or a combination of the ingredients listed above as needed, followed by filtration sterilization. Generally, a dispersion is prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze drying, which yield a powder of the active ingredient plus any desired additional ingredients from its previously sterile filtered solution. The proper fluidity of the solution can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of a dispersion, and by the use of surfactants. Prolonged absorption of the injectable composition can be achieved by including agents that delay absorption (e.g., monostearate and gelatin) in the composition.

[0219] As will be appreciated by those skilled in the art, the route and / or mode of administration will vary depending on the desired results. In certain embodiments, the active compound may be prepared with a carrier that protects the compound from rapid release, such as a controlled-release formulation, including implants, transdermal patches, and microencapsulation delivery systems. Biodegradable, biocompatible polymers such as ethylene-vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Many methods of preparing such formulations are patented or are well known to those skilled in the art. See, for example, Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson et al., Marcel Dekker, Inc., New York, 1978.

[0220] In certain embodiments, the pharmaceutical composition may be administered orally, for example, with an inert diluent or an assimilable edible carrier. The compounds (and other ingredients, if desired) may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or directly incorporated into the diet of the subject. For oral therapeutic administration, the compounds may be combined with excipients and used in ingestible tablets, buccal tablets, lozenges, capsules, elixirs, suspensions, syrups, cachets, etc. To administer the compositions provided herein by parenteral administration, it may be necessary to coat the compositions with, or co-administer the compositions with, a material to prevent their inactivation. The compositions may also be administered using medical devices known in the art.

[0221] In some embodiments, the administration of the polypeptides, nucleic acids, vectors, plasmids, viruses, liposomes, and compositions provided herein may be administered in separate doses or formulations. For example, a first nucleic acid, vector, or plasmid may be administered separately from a second or third nucleic acid, vector, or plasmid as described herein. In some embodiments, the administration may be simultaneous, sequential, or parallel. In some embodiments, the administration of each component will include separate compositions, formulations, dosage regimens, methods of administration, and concentrations to appropriately achieve the desired response. Similarly, in some embodiments, one or more of the polypeptides, nucleic acids, vectors, plasmids, viruses, liposomes, and compositions provided herein may be administered in the same dose or formulation. In some embodiments, two or more of the polypeptides, nucleic acids, vectors, plasmids, viruses, liposomes, and compositions provided herein may be administered in the same dose or formulation, while other polypeptides, nucleic acids, vectors, plasmids, viruses, liposomes, and compositions provided herein may be administered in separate doses or formulations.

[0222] Adjust the dosage regimen to provide the optimal desired response (e.g., therapeutic response). For example, a single bolus may be administered, several separate doses may be administered over time, or the dose may be proportionally decreased or increased depending on the urgency of the treatment situation. It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suitable as a single dose for a subject to be treated; each unit contains a predetermined quantity of the active compound which is calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0223] Exemplary non-limiting ranges for a therapeutically or prophylactically effective amount of the therapeutic compound are from 0.1 mg / kg to 30 mg / kg, more preferably from 1 mg / kg to 25 mg / kg. The dosage and treatment regimen of the therapeutic compound can be determined by a person skilled in the art. In certain embodiments, the therapeutic compound is administered by injection (e.g., subcutaneously or intravenously) at a dose of about 1 mg / kg to 40 mg / kg, such as 1 mg / kg to 30 mg / kg, such as about 5 mg / kg to 25 mg / kg, about 10 mg / kg to 20 mg / kg, about 1 mg / kg to 5 mg / kg, 1 mg / kg to 10 mg / kg, 5 mg / kg to 15 mg / kg, 10 mg / kg to 20 mg / kg, 15 mg / kg to 25 mg / kg, or about 3 mg / kg. The dosing schedule can vary from, for example, once a week to once every 2, 3, or 4 weeks, or in some embodiments, the dosing schedule can be once a month, once every 2 months, once every 3 months, or once every 6 months. In one embodiment, the therapeutic compound is administered every other week at a dose of about 10 mg / kg to 20 mg / kg. The therapeutic compound can be administered by intravenous infusion at a rate greater than 20 mg / min, such as 20 mg / min - 40 mg / min, and generally greater than or equal to 40 mg / min, to achieve a dose of about 35 mg / m2 to 440 mg / m2, generally about 70 mg / m2 to 310 mg / m2, and more generally about 110 mg / m2 to 130 mg / m2. In an embodiment, an infusion rate of about 110 mg / m2 to 130 mg / m2 achieves a level of about 3 mg / kg. In other embodiments, the therapeutic compound can be administered by intravenous infusion at a rate less than 10 mg / min, such as less than or equal to 5 mg / min, to achieve a dose of about 1 mg / m2 to 100 mg / m2, such as about 5 mg / m2 to 50 mg / m2, about 7 mg / m2 to 25 mg / m2, or about 10 mg / m2. In some embodiments, the therapeutic compound is infused over a period of about 30 min. It should be noted that the dose values can vary with the type and severity of the condition to be alleviated. It should also be understood that for any particular subject, the specific dosage regimen should be adjusted over time according to individual needs and the professional judgment of the person administering or supervising the administration of the composition, and the dosage ranges listed herein are only exemplary and are not intended to limit the scope or practice of the claimed composition.

[0224] A pharmaceutical composition may comprise a "therapeutically effective amount" or a "prophylactically effective amount" of a composition, a vector, a cell, a polypeptide, or a nucleic acid molecule encoding them. A "therapeutically effective amount" refers to an amount that is effective to achieve the desired therapeutic outcome at the required dosage and for the required period of time. The therapeutically effective amount of the active ingredient or molecule may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the therapeutic compound to elicit the desired response in the individual. A "therapeutically effective dose" may, for example, inhibit a measurable parameter (e.g., tumor growth) by at least about 20%, at least about 40%, at least about 60%, and at least about 80% relative to an untreated subject. The ability of a compound to inhibit a measurable parameter (e.g., tumor growth) can be evaluated in an animal model system predictive of efficacy in tumor growth. Alternatively, this property of the composition can be evaluated by examining the inhibitory ability of the compound, such inhibition being performed in vitro by assays known to a skilled practitioner.

[0225] A "prophylactically effective amount" refers to an amount that is effective to achieve the desired prophylactic outcome at the required dosage and for the required period of time. Generally, since prophylactic doses are used in subjects before or at an early stage of a disease, a prophylactically effective amount can be, but is not necessarily, less than a therapeutically effective amount.

[0226] Also provided herein are kits comprising a composition, a cell, a vector, a nucleic acid molecule, or a polypeptide as described herein. The kit may include one or more other elements, including: instructions for use; other reagents, such as labels, therapeutic agents, or agents that can be used to chelate or otherwise conjugate a molecule to a label or other therapeutic agent, or radioprotective compositions; devices or other materials for preparing the molecule for administration; a pharmaceutically acceptable carrier; and devices or other materials for administration to a subject.

[0227] In some embodiments, cells comprising the polypeptides provided herein are provided. In some embodiments, cells comprising a genomic nucleic acid molecule comprising the nucleic acid molecule provided herein are provided. A genomic nucleic acid molecule refers to a heterologous nucleic acid molecule integrated into the genome of a host cell. In some embodiments, the cell further comprises a chimeric antigen receptor. A chimeric antigen receptor or "CAR" can be used to treat cancer or tumors in a subject. In some embodiments, the activity of the CAR can be enhanced by co-expressing the polypeptides provided herein with the CAR. Thus, in some embodiments, cells comprising a CAR and the polypeptides provided herein are provided.

[0228] The cell can be any type of suitable cell. In some embodiments, the cell is an immune cell, such as, but not limited to, a T cell, an NK cell, a dendritic cell, etc.

[0229] Cells can be produced according to any known method. For example, in some embodiments, the provided methods for producing cells include contacting the cells with a vector, plasmid, virus, liposome, or any composition provided herein that contains a nucleic acid molecule encoding a polypeptide provided herein. This can be done, for example, under conditions suitable for expressing the polypeptide in the cell and / or expressing it on the cell surface. In some embodiments, the vector used is a plasmid or a virus. In some embodiments, the virus is an adenovirus or a lentivirus. In some embodiments, the adenovirus is a replication - incompetent or replication - competent adenovirus. In some embodiments, the contacting includes transducing or transfecting the cells with the vector. In some embodiments, the vector further contains a nucleic acid molecule encoding a chimeric antigen receptor or a tumor antigen.

[0230] In some embodiments, methods for producing cells that contain a polypeptide provided herein in vivo are provided, and these methods include administering to a subject a vector encoding the polypeptide, wherein the vector transduces or transfects cells in vivo to produce cells that contain a polypeptide provided herein. In some embodiments, the cells are immune cells. In some embodiments, the immune cells are, but are not limited to, T cells, NK cells, dendritic cells, etc. In some embodiments, the vector further contains a nucleic acid molecule encoding a chimeric antigen receptor or a tumor antigen. In some embodiments, the vector is a plasmid or a virus. In some embodiments, the virus is an adenovirus or a lentivirus. In some embodiments, the adenovirus is a replication - incompetent or replication - competent adenovirus.

[0231] Method

[0232] "Treatment" of any disease mentioned herein encompasses the alleviation of at least one symptom of the disease, a reduction in the severity of the disease, or a delay or prevention of the progression of the disease to more severe symptoms, which more severe symptoms may, in some cases, accompany the disease or at least one other disease. Treatment does not necessarily mean that the disease is completely cured. The available therapeutic agents only need to reduce the severity of the disease, reduce the severity of the symptoms associated with the disease or its treatment, or delay the onset of more severe symptoms or a more severe disease, which more severe symptoms or more severe disease may occur at a certain frequency after treating the condition. For example, if the disease is a tumor, the composition can reduce the growth or spread of the tumor, or the impact of the tumor on the tissue in which it is present. The condition of the patient can be estimated by standard techniques. Suitable methods vary according to the condition and symptoms of the patient.

[0233] In some embodiments, the compositions provided herein can be used to alter the immune response of a patient. In some embodiments, these methods include administering to the patient a polypeptide or a vector that contains a nucleic acid molecule encoding a polypeptide provided herein. In some embodiments, the immune response is an activated immune response, such as in activating NK and / or CD8 + T cells.

[0234] In some embodiments, the compositions provided herein can be used to treat cancer in a subject (patient). In some embodiments, these methods include administering to the patient a vector comprising a nucleic acid molecule encoding a polypeptide provided herein. In some embodiments, the cancer is lymphoma, leukemia, nasopharyngeal carcinoma, gastric cancer, cervical cancer, hepatocellular carcinoma, polyomavirus, anal cancer, head and neck cancer. In some embodiments, the tumor is a lung cancer tumor. In some embodiments, the tumors are in the benign and metastatic forms of cancer, such as, ovarian cancer (e.g., ovarian carcinoma), germ cell cancer (breast cancer, cervical cancer, testicular cancer, uterine cancer, and placental cancer), lung cancer, gastric cancer, liver cancer, pancreatic cancer, bile duct cancer, bladder cancer, kidney cancer, colon cancer, small intestine cancer, skin cancer, brain cancer, head and neck cancer, sarcoma, and germ cell tumor, etc.

[0235] In some embodiments, diseases treatable with the compositions provided herein also include myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML). In some embodiments, the subject has MDS, including Fanconi anemia, refractory anemia, refractory neutropenia, refractory thrombocytopenia, primary sideroblastic anemia (RARS), refractory cytopenia with multilineage dysplasia (RCMD), refractory anemia with multilineage dysplasia and ring sideroblasts (RCMD-RS), refractory anemia with excess blasts I and II (RAEB), myelodysplastic syndrome, unclassified (MDS-U), MDS with isolated del(5q)-syndrome, chronic myelomonocytic leukemia (CMML), juvenile myelomonocytic leukemia (JMML), refractory cytopenia of childhood, or combinations thereof. In some embodiments, the subject has AML, including AML with recurrent genetic abnormalities (AML with translocation between chromosomes 8 and 21, AML with translocation or inversion of chromosome 16, AML with translocation between chromosomes 9 and 11, APL (M3) with translocation between chromosomes 15 and 17, AML with translocation between chromosomes 6 and 9, AML with translocation or inversion of chromosome 3), AML with translocation between chromosomes 1 and 22 (megakaryoblastic), AML with changes related to myelodysplasia, AML related to prior chemotherapy or radiation (alkylator-related AML, topoisomerase II inhibitor-related AML), AML not otherwise classified (AML minimally differentiated (M0), AML with minimal maturation (M1), AML with maturation (M2), acute myelomonocytic leukemia (M4), acute monocytic leukemia (M5), acute erythroid leukemia (M6), acute megakaryoblastic leukemia (M7), acute basophilic leukemia, acute panmyelosis with fibrosis), myeloid sarcoma (also known as granulocytic sarcoma, chloroma, or extramedullary myeloblastoma), undifferentiated and biphenotypic acute leukemia (also known as mixed phenotype acute leukemia), or combinations thereof. In some embodiments, administering the compositions provided herein to the subject reduces the incidence of one or more symptoms associated with MDS or AML, or reduces one or more markers of MDS or AML cell viability. In some embodiments, one or more symptoms associated with MDS or AML include reduced bone marrow failure, immunodysfunction, transformation to overt leukemia, or combinations thereof in the subject, or wherein markers of the viability of MDS or AML cells include survival, proliferation, growth, migration, colony formation, chromatin assembly, DNA binding, RNA metabolism, cell migration, cell adhesion, inflammation, or combinations thereof over time.

[0236] In some embodiments, the tumor is also treated with a checkpoint inhibitor. In some embodiments, the tumor is also treated with a PD-1 inhibitor, such as a PD-1 antagonist, such as a PD-1 antagonist antibody. In some embodiments, the tumor is also treated with a PD-L1 inhibitor, such as a PD-L1 antagonist, such as a PD-L1 antagonist antibody. In some embodiments, the tumor is also treated with a CTLA-4 inhibitor, such as a CTLA-4 antagonist, such as a CTLA-4 antagonist antibody.

[0237] In some embodiments, the compositions provided herein can be used to treat viral, bacterial, or fungal infections in a subject (patient).

[0238] In some embodiments, the methods include administering to the subject a pharmaceutical composition comprising a polypeptide provided herein or a nucleic acid molecule encoding a polypeptide provided herein. In some embodiments, the subject is a subject in need thereof. Any of the foregoing can be administered in the form of a composition (e.g., a pharmaceutical composition) described herein.

[0239] To treat a disease of interest, a composition comprising a cell, vector, nucleic acid molecule, or polypeptide described herein can be administered by any suitable method, including but not limited to parenteral, topical, oral, nasal, vaginal, rectal, or pulmonary (by inhalation) administration. If by injection, the composition can be administered by intra-articular, intravenous, intra-arterial, intramuscular, intracapsular, intraperitoneal, or bolus injection or continuous infusion. Local administration is contemplated, i.e., at the site of the disease, such as transdermal delivery and sustained release from implants, skin patches, or suppositories. Delivery by inhalation includes, for example, nasal or oral inhalation, use of a nebulizer, inhalation in the form of an aerosol, etc. Administration by suppository inserted into a body cavity can be achieved, for example, by inserting a solid form of the composition into the selected body cavity and allowing it to dissolve. Other alternatives include eye drops, oral formulations (such as pills, lozenges, syrups, and chewing gums), and topical formulations (such as lotions, gels, sprays, and ointments).

[0240] In the practice of the methods of treatment, the compositions described herein may be administered as described herein and above. For example, the compositions may be administered in any dose, frequency, and duration effective to treat the condition being treated. The dose depends on the molecular nature of the active ingredient and the nature of the disorder being treated. Treatment may continue as long as the desired result is to be achieved. The compositions provided herein may be administered as a single dose or as a series of doses administered periodically, including multiple times per day, daily, every other day, twice a week, three times a week, once a week, once every other week, and once a month doses, as well as other possible dosing regimens. The periodicity of treatment may or may not be constant throughout the duration of treatment. For example, treatment may initially occur at weekly intervals and subsequently every other week. The embodiments provided herein encompass treatments lasting for days, weeks, months, or years. Treatment may be interrupted and then restarted. A maintenance dose may or may not be administered after the initial treatment.

[0241] Doses may be measured in milligrams per kilogram body weight (mg / kg) or milligrams per square meter of skin surface (mg / m 2 ) or as a fixed dose, regardless of height or weight. All of these are standard dosing units in the art. The skin surface area of a human is calculated from their height and weight using standard formulas.

[0242] Also provided herein are methods of activating CD8+ T cells. In some embodiments, the methods provided herein can be used to activate NK cells. In some embodiments, the methods include administering to a subject in need thereof a therapeutically effective amount of a polypeptide or a nucleic acid molecule encoding the polypeptide, or a vector comprising the nucleic acid molecule or as otherwise described herein, or a pharmaceutical composition comprising the vector.

[0243] As used herein, the phrase "in need thereof" means that a subject (animal or mammal) has been identified as in need of a particular method or treatment. In some embodiments, the identification can be by any diagnostic means. In any of the methods and treatments described herein, an animal or mammal may be in need thereof. In some embodiments, the animal or mammal is in an environment where a particular disease, disorder, or condition is endemic or will travel to an environment where a particular disease, disorder, or condition is endemic.

[0244] The compositions provided herein may also be administered in combination with other agents for treating the disorder(s) afflicting the patient. Examples of such agents include protein and non-protein drugs. When multiple therapeutic agents are co-administered, the dosages may be adjusted accordingly, as recognized in the relevant art. "Co-administered" and combination therapy are not limited to simultaneous administration, but also include administering at least one course of treatment from the compositions provided herein during the course of treatment, which involves administering to the patient at least one other therapeutic agent, such as an anti-cancer therapeutic agent, including but not limited to chemotherapy, radiotherapy, targeted therapy, immunotherapy, stem cell or bone marrow transplantation therapy, hormone therapy, photodynamic therapy, or any combination thereof.

[0245] In some embodiments, the compositions provided herein may be combined, linked, or fused with at least one additional molecule. In some embodiments, the additional molecule is a bioactive molecule, such as but not limited to a protein, polypeptide, nucleic acid, lipid, carbohydrate, or any combination thereof. In some embodiments, the additional molecule is a targeting moiety, such as but not limited to an antibody, antigen, ligand, ligand trap such as a receptor domain, or any combination thereof. In some embodiments, the additional molecule is a therapeutic molecule, such as but not limited to an immunotherapeutic molecule, checkpoint inhibitor, immune system activator, tumor therapeutic agent, antibody, or any combination thereof.

[0246] In some embodiments, the compositions provided herein may be linked to at least one additional molecule at the C-terminus of the composition. In some embodiments, the compositions provided herein may be linked to at least one additional molecule at the N-terminus of the composition. In some embodiments, the compositions provided herein may be linked to at least one additional molecule at any linker of the composition. In some embodiments, the compositions provided herein may be linked to at least one additional molecule before any cleavable linker is cleaved. In some embodiments, the compositions provided herein may be linked to at least one additional molecule after the composition has been cleaved at the cleavable linker.

[0247] Embodiment

[0248] In some embodiments, the embodiments provided herein further include but are not limited to:

[0249] 1. A polypeptide comprising the formula: X1-L1-X2-L2-X3-L3-X4-L4-X5, wherein:

[0250] X1 is an interleukin-21 (IL-21) polypeptide, an interleukin-15 (IL-15) polypeptide, an interleukin-12p40 subunit (IL-12p40) polypeptide, an interleukin-12p35 subunit (IL-12p35) polypeptide, or an interleukin-15 receptor (IL-15Rα) polypeptide;

[0251] X2 is an IL-21 polypeptide, an IL-15 polypeptide, an IL-12p40 polypeptide, an IL-12p35 polypeptide, or an IL-15Rα polypeptide;

[0252] X3 is an IL-21 polypeptide, an IL-15 polypeptide, an IL-12p40 polypeptide, an IL-12p35 polypeptide, or an IL-15Rα polypeptide;

[0253] X4 is an IL-21 polypeptide, an IL-15 polypeptide, an IL-12p40 polypeptide, an IL-12p35 polypeptide, or an IL-15Rα polypeptide;

[0254] X5 is an IL-21 polypeptide, an IL-15 polypeptide, an IL-12p40 polypeptide, an IL-12p35 polypeptide, or an IL-15Rα polypeptide;

[0255] L1, L2, L3, and L4 are each independently a polypeptide linker comprising the same or different polypeptide sequences, and

[0256] provided that each of X1, X2, X3, X4, and X5 is different.

[0257] 2. The polypeptide according to embodiment 1, wherein X1 is an interleukin 21 (IL-21) polypeptide.

[0258] 3. The polypeptide according to embodiment 1 or 2, wherein:

[0259] X1 is an IL-21 polypeptide;

[0260] X2 is an IL-15 polypeptide;

[0261] X3 is an IL-12p40 polypeptide;

[0262] X4 is an IL-12p35 polypeptide; and

[0263] X5 is an IL-15Rα polypeptide.

[0264] 4. The polypeptide according to embodiment 1 or embodiment 2, wherein:

[0265] X1 is an IL-21 polypeptide;

[0266] X2 is an IL-15 polypeptide;

[0267] X3 is an IL-12p35 polypeptide;

[0268] X4 is an IL-12p40 polypeptide; and

[0269] X5 is an IL-15Rα polypeptide.

[0270] 5. The polypeptide according to embodiment 1 or 2, wherein:

[0271] X1 is an IL-21 polypeptide;

[0272] X2 is an IL-12p40 polypeptide;

[0273] X3 is an IL-15 polypeptide;

[0274] X4 is an IL-12p35 polypeptide; and

[0275] X5 is an IL-15Rα polypeptide.

[0276] 6. The polypeptide according to embodiment 1 or 2, wherein:

[0277] X1 is an IL-21 polypeptide;

[0278] X2 is an IL-12p40 polypeptide;

[0279] X3 is an IL-12p35 polypeptide;

[0280] X4 is an IL-15 polypeptide; and

[0281] X5 is an IL-15Rα polypeptide.

[0282] 7. The polypeptide according to embodiment 1 or 2, wherein:

[0283] X1 is an IL-21 polypeptide;

[0284] X2 is an IL-12p35 polypeptide;

[0285] X3 is an IL-12p40 polypeptide;

[0286] X4 is an IL-15 polypeptide; and

[0287] X5 is an IL-15Rα polypeptide.

[0288] 8. The polypeptide according to embodiment 1 or 2, wherein:

[0289] X1 is an IL-21 polypeptide;

[0290] X2 is an IL-12p35 polypeptide;

[0291] X3 is an IL-15 polypeptide;

[0292] X4 is an IL-12p40 polypeptide; and

[0293] X5 is an IL-15Rα polypeptide.

[0294] 9. The polypeptide according to embodiment 1 or 2, wherein:

[0295] X1 is an IL-21 polypeptide;

[0296] X2 is an IL-12p40 polypeptide;

[0297] X3 is an IL-12p35 polypeptide;

[0298] X4 is an IL-15 polypeptide; and

[0299] X5 is an IL-15Rα polypeptide.

[0300] 10. The polypeptide according to embodiment 1 or 2, wherein:

[0301] X1 is an IL-21 polypeptide;

[0302] X2 is an IL-12p40 polypeptide;

[0303] X3 is an IL-15 polypeptide;

[0304] X4 is an IL-12p35 polypeptide; and

[0305] X5 is an IL-15Rα polypeptide.

[0306] 11. The polypeptide according to embodiment 1 or 2, wherein:

[0307] X1 is an IL-21 polypeptide;

[0308] X2 is an IL-12p35 polypeptide;

[0309] X3 is an IL-12p40 polypeptide;

[0310] X4 is an IL-15 polypeptide; and

[0311] X5 is an IL-15Rα polypeptide.

[0312] 12. The polypeptide according to embodiment 1 or 2, wherein:

[0313] X1 is an IL-21 polypeptide;

[0314] X2 is an IL-12p35 polypeptide;

[0315] X3 is an IL-15 polypeptide;

[0316] X4 is an IL-12p40 polypeptide; and

[0317] X5 is an IL-15Rα polypeptide.

[0318] 13. The polypeptide according to any one of the foregoing embodiments, wherein the IL-21 polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:71, or as otherwise provided herein.

[0319] 14. The polypeptide according to any one of the foregoing embodiments, wherein the IL-21 polypeptide comprises the amino acid sequence of SEQ ID NO:71, or as otherwise provided herein.

[0320] 15. The polypeptide according to any one of the foregoing embodiments, wherein the IL-12p40 polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:1, 2, 3 or 4, or as otherwise provided herein.

[0321] 16. The polypeptide according to any one of the foregoing embodiments, wherein the IL-12p40 polypeptide comprises the amino acid sequence of SEQ ID NO:1, 2, 3 or 4, or as otherwise provided herein.

[0322] 17. The polypeptide according to any one of the foregoing embodiments, wherein the IL-12p35 polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:5, 6, 7 or 8, or as otherwise provided herein.

[0323] 18. The polypeptide according to any one of the foregoing embodiments, wherein the IL-12p35 polypeptide comprises the amino acid sequence of SEQ ID NO:5, 6, 7 or 8, or as otherwise provided herein.

[0324] 19. The polypeptide according to any one of the foregoing embodiments, wherein the IL-15 polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:9 or 10, or as otherwise provided herein.

[0325] 20. The polypeptide according to any one of the foregoing embodiments, wherein the IL-15 polypeptide comprises the amino acid sequence of SEQ ID NO:9 or 10, or as otherwise provided herein.

[0326] 21. The polypeptide according to any one of the foregoing embodiments, wherein the IL-15Rα polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 11 or 12, or as otherwise provided herein.

[0327] 22. The polypeptide according to any one of the foregoing embodiments, wherein the IL-15Rα polypeptide comprises the amino acid sequence of SEQ ID NO: 11 or 12, or as otherwise provided herein.

[0328] 23. The polypeptide according to any one of the foregoing embodiments, wherein one or more of L1, L2, L3 and L4 are each independently a cleavable linker.

[0329] 24. The polypeptide according to embodiment 23, wherein L1, L3 and L4 are non-cleavable linkers.

[0330] 25. The polypeptide according to embodiment 23 or 24, wherein the cleavable linker is a furin-cleavable linker, a Val-Cit linker, a Val-Gly linker, a Gly-Gly linker, an Ala-Ala-Asn linker or a linker comprising polyethylene glycol (PEG).

[0331] 26. The polypeptide according to any one of the foregoing embodiments, wherein one of L1, L2, L3 and L4 comprises the sequence of (GSGSGG)n (SEQ ID NO: 16), (GGGGS)n (SEQ ID NO: 17), (GGGGA)n (SEQ ID NO: 18), (GGGSE)n (SEQ ID NO: 19), (GGGSK)n (SEQ ID NO: 20) or (AEEEK)n (SEQ ID NO: 21) or a combination thereof, wherein each n is independently 1, 2, 3, 4 or 5.

[0332] 27. The polypeptide according to embodiment 26, wherein n is 3.

[0333] 28. The polypeptide according to embodiment 26 or 27, wherein one or more of L1, L2, L3 and L4 comprises the sequence RXXR of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:70 or a combination thereof, wherein X is any amino acid.

[0334] 29. The polypeptide according to any one of embodiments 25 to 28, wherein each of L1, L2, L3 and L4 comprises the sequence RXXR of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:70 or a combination thereof, wherein X is any amino acid.

[0335] 30. The polypeptide according to any of the foregoing embodiments, wherein the polypeptide comprises the sequence of SEQ ID NO:72 or a variant thereof, or as otherwise provided herein.

[0336] 31. The polypeptide according to any of the preceding embodiments, wherein the polypeptide comprises the sequence of SEQ ID NO:73 or a variant thereof, or as otherwise provided herein.

[0337] 32. The polypeptide according to any of the preceding embodiments, wherein the polypeptide comprises the sequence of SEQ ID NO:74 or a variant thereof, or as otherwise provided herein.

[0338] 33. The polypeptide according to any of the preceding embodiments, wherein the polypeptide comprises the sequence of SEQ ID NO:75 or a variant thereof, or as otherwise provided herein.

[0339] 34. The polypeptide according to any of the preceding embodiments, wherein the polypeptide comprises the sequence of SEQ ID NO:76 or a variant thereof, or as otherwise provided herein.

[0340] 35. The polypeptide according to any of the preceding embodiments, wherein the polypeptide comprises the sequence of SEQ ID NO:77 or a variant thereof, or as otherwise provided herein.

[0341] 36. The polypeptide according to any of the preceding embodiments, wherein the polypeptide comprises the sequence of SEQ ID NO:78 or a variant thereof, or as otherwise provided herein.

[0342] 37. The polypeptide according to any of the preceding embodiments, wherein the polypeptide comprises the sequence of SEQ ID NO:79 or a variant thereof, or as otherwise provided herein.

[0343] 38. The polypeptide according to any of the preceding embodiments, wherein the first polypeptide is linked or fused to at least one additional molecule selected from the group consisting of: a polypeptide, an antigen, a ligand, an antibody, a checkpoint inhibitor, a lipid, a carbohydrate, a nucleic acid, or any combination thereof.

[0344] 39. The polypeptide according to embodiment 1, wherein:

[0345] X1 is an IL-21 polypeptide;

[0346] X2 is an IL-15 polypeptide;

[0347] X3 is an IL-12p40 polypeptide;

[0348] X4 is an IL-12p35 polypeptide;

[0349] X5 is an IL-15Rα polypeptide;

[0350] L2 is a furin-cleavable linker; and

[0351] L1, L3, and L4 are non-cleavable linkers.

[0352] 40. The polypeptide according to embodiment 39, wherein the first polypeptide is linked or fused to at least one additional molecule selected from the group consisting of: a polypeptide, an antigen, a ligand, an antibody, a checkpoint inhibitor, a lipid, a carbohydrate, a nucleic acid, or any combination thereof.

[0353] 41. A nucleic acid encoding the polypeptide according to any one of embodiments 1 to 40.

[0354] 42. A vector comprising the nucleic acid according to embodiment 41.

[0355] 43. A plasmid comprising the nucleic acid according to embodiment 41.

[0356] 44. A virus, such as a recombinant virus, comprising the nucleic acid according to embodiment 41.

[0357] 45. The virus according to embodiment 44, wherein the recombinant virus is an adenovirus or a lentivirus.

[0358] 46. The virus according to embodiment 44 or 45, wherein the virus is a replication-incompetent adenovirus.

[0359] 47. A liposome comprising the nucleic acid according to embodiment 41.

[0360] 48. The liposome according to embodiment 47, wherein the liposome comprises any lipid provided herein.

[0361] 49. A pharmaceutical composition comprising the nucleic acid according to embodiment 41 and a pharmaceutically acceptable carrier.

[0362] 50. A cell comprising the composition according to any one of embodiments 1 to 40, wherein the cell is an immune cell.

[0363] 51. A composition (e.g., liposome, virus, encapsulation, etc.) comprising:

[0364] A first polypeptide having the formula: X1-L1-X2-L2-X3-L3-X4-L4-X5, wherein:

[0365] X1 is an interleukin 21 (IL-21) polypeptide, an interleukin 15 (IL-15) polypeptide, an interleukin 12p40 subunit (IL-12p40) polypeptide, an interleukin 12p35 subunit (IL-12p35) polypeptide, or an interleukin 15 receptor (IL-15Rα) polypeptide;

[0366] X2 is an IL-21 polypeptide, an IL-15 polypeptide, an IL-12p40 polypeptide, an IL-12p35 polypeptide, or an IL-15Rα polypeptide;

[0367] X3 is an IL-21 polypeptide, an IL-15 polypeptide, an IL-12p40 polypeptide, an IL-12p35 polypeptide, or an IL-15Rα polypeptide;

[0368] X4 is an IL-21 polypeptide, an IL-15 polypeptide, an IL-12p40 polypeptide, an IL-12p35 polypeptide, or an IL-15Rα polypeptide;

[0369] X5 is an IL-21 polypeptide, an IL-15 polypeptide, an IL-12p40 polypeptide, an IL-12p35 polypeptide, or an IL-15Rα polypeptide;

[0370] L1, L2, L3, and L4 are each independently a polypeptide linker comprising the same or different polypeptide sequences,

[0371] provided that each of X1, X2, X3, X4, and X5 is different; and / or

[0372] a second polypeptide comprising at least one immune checkpoint polypeptide or an active fragment thereof, such as but not limited to a CTLA-4 polypeptide, a PD-1 polypeptide, a PD-L1 polypeptide, a PD-L2 polypeptide, a TIM3 polypeptide, a LAG3 polypeptide, a VISTA polypeptide, a SINGLEC7 polypeptide, a SINGLEC9 polypeptide, a TIGIT polypeptide, a CD96 polypeptide, a BTLA polypeptide, a B7H3 polypeptide, a B7H4 polypeptide, a CD155 polypeptide, an HHLA2 polypeptide, a BTN3A1 polypeptide, a B7.1 polypeptide, a B7.2 polypeptide, or an active fragment thereof;

[0373] or one or more nucleic acid molecules encoding the first polypeptide and the second polypeptide.

[0374] 52. The composition according to embodiment 51, wherein X1 is an interleukin 21 (IL-21) polypeptide.

[0375] 53. The composition according to embodiment 51 or 52, wherein:

[0376] X1 is an IL-21 polypeptide;

[0377] X2 is an IL-15 polypeptide;

[0378] X3 is an IL-12p40 polypeptide;

[0379] X4 is an IL-12p35 polypeptide; and

[0380] X5 is an IL-15Rα polypeptide.

[0381] 54. The composition according to embodiment 51 or 52, wherein:

[0382] X1 is an IL-21 polypeptide;

[0383] X2 is an IL-15 polypeptide;

[0384] X3 is an IL-12p35 polypeptide;

[0385] X4 is an IL-12p40 polypeptide; and

[0386] X5 is an IL-15Rα polypeptide.

[0387] 55. The composition according to embodiment 51 or 52, wherein:

[0388] X1 is an IL-21 polypeptide;

[0389] X2 is an IL-12p40 polypeptide;

[0390] X3 is an IL-15 polypeptide;

[0391] X4 is an IL-12p35 polypeptide; and

[0392] X5 is an IL-15Rα polypeptide.

[0393] 56. The composition according to embodiment 51 or 52, wherein:

[0394] X1 is an IL-21 polypeptide;

[0395] X2 is an IL-12p40 polypeptide;

[0396] X3 is an IL-12p35 polypeptide;

[0397] X4 is an IL-15 polypeptide; and

[0398] X5 is an IL-15Rα polypeptide.

[0399] 57. The composition according to embodiment 51 or 52, wherein:

[0400] X1 is an IL-21 polypeptide;

[0401] X2 is an IL-12p35 polypeptide;

[0402] X3 is an IL-12p40 polypeptide;

[0403] X4 is an IL-15 polypeptide; and

[0404] X5 is an IL-15Rα polypeptide.

[0405] 58. The composition according to embodiment 51 or 52, wherein:

[0406] X1 is an IL-21 polypeptide;

[0407] X2 is an IL-12p35 polypeptide;

[0408] X3 is an IL-15 polypeptide;

[0409] X4 is an IL-12p40 polypeptide; and

[0410] X5 is an IL-15Rα polypeptide.

[0411] 59. The composition according to embodiment 51 or 52, wherein:

[0412] X1 is an IL-21 polypeptide;

[0413] X2 is an IL-12p40 polypeptide;

[0414] X3 is an IL-12p35 polypeptide;

[0415] X4 is an IL-15 polypeptide; and

[0416] X5 is an IL-15Rα polypeptide.

[0417] 60. The composition according to embodiment 51 or 52, wherein:

[0418] X1 is an IL-21 polypeptide;

[0419] X2 is an IL-12p40 polypeptide;

[0420] X3 is an IL-15 polypeptide;

[0421] X4 is an IL-12p35 polypeptide; and

[0422] X5 is an IL-15Rα polypeptide.

[0423] 61. The composition according to embodiment 51 or 52, wherein:

[0424] X1 is an IL-21 polypeptide;

[0425] X2 is an IL-12p35 polypeptide;

[0426] X3 is an IL-12p40 polypeptide;

[0427] X4 is an IL-15 polypeptide; and

[0428] X5 is an IL-15Rα polypeptide.

[0429] 62. The composition according to embodiment 51 or 52, wherein:

[0430] X1 is an IL-21 polypeptide;

[0431] X2 is an IL-12p35 polypeptide;

[0432] X3 is an IL-15 polypeptide;

[0433] X4 is an IL-12p40 polypeptide; and

[0434] X5 is an IL-15Rα polypeptide.

[0435] 63. The composition according to any one of embodiments 51 to 62, wherein the IL-21 polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 71, or as otherwise provided herein.

[0436] 64. The composition according to any one of embodiments 51 to 63, wherein the IL-21 polypeptide comprises the amino acid sequence of SEQ ID NO: 71, or as otherwise provided herein.

[0437] 65. The composition according to any one of embodiments 51 to 64, wherein the IL-12p40 polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1, 2, 3 or 4, or as otherwise provided herein.

[0438] 66. The composition according to any one of embodiments 51 to 65, wherein the IL-12p40 polypeptide comprises the amino acid sequence of SEQ ID NO: 1, 2, 3 or 4, or as otherwise provided herein.

[0439] 67. The composition according to any one of embodiments 51 to 66, wherein the IL-12p35 polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 5, 6, 7 or 8, or as otherwise provided herein.

[0440] 68. The composition according to any one of embodiments 51 to 67, wherein the IL-12p35 polypeptide comprises the amino acid sequence of SEQ ID NO: 5, 6, 7 or 8, or as otherwise provided herein.

[0441] 69. The composition according to any one of embodiments 51 to 68, wherein the IL-15 polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 9 or 10, or as otherwise provided herein.

[0442] 70. The composition according to any one of embodiments 51 to 69, wherein the IL-15 polypeptide comprises the amino acid sequence of SEQ ID NO: 9 or 10, or as otherwise provided herein.

[0443] 71. The composition according to any one of embodiments 51 to 70, wherein the IL-15Rα polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 11 or 12, or as otherwise provided herein.

[0444] 72. The composition according to any one of embodiments 51 to 71, wherein the IL-15Rα polypeptide comprises the amino acid sequence of SEQ ID NO: 11 or 12, or as otherwise provided herein.

[0445] 73. The composition according to any one of embodiments 51 to 72, wherein one or more of L1, L2, L3 and L4 are each independently a cleavable linker.

[0446] 74. The polypeptide according to embodiment 72, wherein L1, L3 and L4 are non-cleavable linkers.

[0447] 75. The composition according to embodiment 73 or 74, wherein the cleavable linker is a furin-cleavable linker, a Val-Cit linker, a Val-Gly linker, a Gly-Gly linker, an Ala-Ala-Asn linker or a linker comprising polyethylene glycol (PEG).

[0448] 76. The composition according to any one of embodiments 51 to 75, wherein one of L1, L2, L3 and L4 comprises the sequence of (GSGSGG)n (SEQ ID NO: 16), (GGGGS)n (SEQ ID NO: 17), (GGGGA)n (SEQ ID NO: 18), (GGGSE)n (SEQ ID NO: 19), (GGGSK)n (SEQ ID NO: 20) or (AEEEK)n (SEQ ID NO: 21) or a combination thereof, wherein each n is independently 1, 2, 3, 4 or 5.

[0449] 77. The composition according to embodiment 76, wherein n is 3.

[0450] 78. The composition according to embodiment 76 or 77, wherein one or more of L1, L2, L3, and L4 comprise the sequence RXXR of SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 70, or a combination thereof, wherein X is any amino acid.

[0451] 79. The composition according to any one of embodiments 76 to 78, wherein each of L1, L2, L3, and L4 comprises the sequence RXXR of SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 70, or a combination thereof, wherein X is any amino acid.

[0452] 80. The composition according to any one of embodiments 51 to 79, wherein the first polypeptide comprises the sequence of SEQ ID NO: 72 or a variant thereof, or as otherwise provided herein.

[0453] 81. The composition according to any one of embodiments 51 to 80, wherein the first polypeptide comprises the sequence of SEQ ID NO: 73 or a variant thereof, or as otherwise provided herein.

[0454] 82. The composition according to any one of embodiments 51 to 81, wherein the first polypeptide comprises the sequence of SEQ ID NO: 74 or a variant thereof, or as otherwise provided herein.

[0455] 83. The composition according to any one of embodiments 51 to 82, wherein the first polypeptide comprises the sequence of SEQ ID NO: 75 or a variant thereof, or as otherwise provided herein.

[0456] 84. The composition according to any one of embodiments 51 to 83, wherein the first polypeptide comprises the sequence of SEQ ID NO: 76 or a variant thereof, or as otherwise provided herein.

[0457] 85. The composition according to any one of embodiments 51 to 84, wherein the first polypeptide comprises the sequence of SEQ ID NO: 77 or a variant thereof, or as otherwise provided herein.

[0458] 86. The composition according to any one of embodiments 51 to 85, wherein the first polypeptide comprises the sequence of SEQ ID NO: 78 or a variant thereof, or as otherwise provided herein.

[0459] 87. The composition according to any one of embodiments 51 to 86, wherein the first polypeptide comprises the sequence of SEQ ID NO: 79 or a variant thereof, or as otherwise provided herein.

[0460] 88. The composition according to any one of embodiments 51 to 87, wherein the first polypeptide is linked or fused to at least one additional molecule selected from the group consisting of: a polypeptide, an antigen, a ligand, an antibody, a checkpoint inhibitor, a lipid, a carbohydrate, a nucleic acid, or any combination thereof.

[0461] 89. The composition according to embodiment 51, wherein:

[0462] X1 is an IL-21 polypeptide;

[0463] X2 is an IL-15 polypeptide;

[0464] X3 is an IL-12p40 polypeptide;

[0465] X4 is an IL-12p35 polypeptide;

[0466] X5 is an IL-15Rα polypeptide;

[0467] L2 is a furin-cleavable linker; and

[0468] L1, L3, and L4 are non-cleavable linkers.

[0469] 90. The composition according to embodiment 89, wherein the first polypeptide is linked or fused to at least one additional molecule selected from the group consisting of: a polypeptide, an antigen, a ligand, an antibody, a checkpoint inhibitor, a lipid, a carbohydrate, a nucleic acid, or any combination thereof.

[0470] 91. The composition according to any one of the preceding embodiments, wherein the second polypeptide comprises, from the N-terminus to the C-terminus, the formula X A -L A -X B or the formula X B -L A -X A , wherein:

[0471] X A is an immune checkpoint polypeptide or an active fragment thereof, such as, but not limited to, a CTLA-4 polypeptide, a PD-1 polypeptide, a PD-L1 polypeptide, a PD-L2 polypeptide, a TIM3 polypeptide, a LAG3 polypeptide, a VISTA polypeptide, a SINGLEC7 polypeptide, a SINGLEC9 polypeptide, a TIGIT polypeptide, a CD96 polypeptide, a BTLA polypeptide, a B7H3 polypeptide, a B7H4 polypeptide, a CD155 polypeptide, a HHLA2 polypeptide, a BTN3A1 polypeptide, or an active fragment thereof;

[0472] X B is a B7.1 polypeptide or a B7.2 polypeptide; and

[0473] L A is a polypeptide linker.

[0474] 92. The composition according to embodiment 91, wherein the second polypeptide comprises, from the N-terminus to the C-terminus, the formula X A -L A -X B .

[0475] 93. The composition according to embodiment 91, wherein the polypeptide comprises, from the N-terminus to the C-terminus, the formula X B -L A -X A .

[0476] 94. The composition according to any one of embodiments 91 to 93, wherein:

[0477] X A is a PD-L1 polypeptide; and

[0478] X B is a B7.1 polypeptide.

[0479] 95. The composition according to any one of embodiments 91 to 93, wherein:

[0480] X A is a PD-L1 polypeptide; and

[0481] X B is a B7.2 polypeptide.

[0482] 96. The composition according to any one of embodiments 91 to 93, wherein:

[0483] X A is a PD-L2 polypeptide; and

[0484] X B is a B7.1 polypeptide.

[0485] 97. The composition according to any one of embodiments 91 to 93, wherein:

[0486] X A is a PD-L2 polypeptide; and

[0487] X B is a B7.2 polypeptide.

[0488] 98. The composition according to any one of embodiments 91 to 95, wherein the PD-L1 polypeptide comprises a PD-L1 extracellular domain.

[0489] 99. The composition according to embodiment 98, wherein the PD-L1 polypeptide comprises an amino acid sequence having at least 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 102 or comprises the sequence of SEQ ID NO: 102.

[0490] 100. The composition according to any one of embodiments 91 to 93 and 96 to 97, wherein the PD-L2 polypeptide comprises a PD-L2 extracellular domain.

[0491] 101. The composition according to embodiment 100, wherein the PD-L2 polypeptide comprises an amino acid sequence having at least 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 104 or comprises the sequence of SEQ ID NO: 104.

[0492] 102. The composition according to any one of embodiments 91 to 94 and 96, wherein the B7.1 polypeptide comprises a B7.1 extracellular domain.

[0493] 103. The composition according to embodiment 102, wherein the B7.1 polypeptide comprises an amino acid sequence having at least 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 106 or comprises the sequence of SEQ ID NO: 106.

[0494] 104. The composition according to any one of embodiments 91 to 93, 95 and 97, wherein the B7.2 polypeptide comprises a B7.2 extracellular domain.

[0495] 105. The composition according to embodiment 104, wherein the B7.2 polypeptide comprises an amino acid sequence having at least 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 108 or comprises the sequence of SEQ ID NO: 108.

[0496] 106. The composition according to any one of embodiments 91 to 105, wherein the second polypeptide comprises a leader sequence.

[0497] 107. The composition according to embodiment 106, wherein the leader sequence is located at the N-terminus of the second polypeptide.

[0498] 108. The composition according to embodiment 106 or 107, wherein the leader sequence is a cell membrane targeting polypeptide.

[0499] 109. The composition according to any one of embodiments 106 to 108, wherein the leader sequence is the sequence of SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113 or a combination thereof.

[0500] 110. The composition according to any one of embodiments 91 to 109, wherein the second polypeptide further comprises an Fc domain.

[0501] 111. The composition according to embodiment 110, wherein the Fc domain is an Ig Fc domain selected from the group consisting of substantially human IgG, IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgA1, IgA2, IgE or IgD.

[0502] 112. A composition according to embodiment 110 or 111, wherein the Fc domain comprises an amino acid sequence that is at least 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:114 or a sequence comprising SEQ ID NO:114.

[0503] 113. A composition according to any one of embodiments 91 to 112, wherein L A A sequence comprising SEQ ID NO: 109 or a combination thereof.

[0504] 114. A composition according to any of the preceding embodiments, wherein the second polypeptide comprises from N-terminus to C-terminus the formula L S -X A -L A -X B -L B -X C , L S -X B -L A -X A -L B -X C , L S -X A -L A -X C -L B -X B or L S -X B -L A -X C -L B -X A ,in:

[0505] L S is the leading sequence;

[0506] X A It is an immune checkpoint polypeptide or an active fragment thereof, such as but not limited to a CTLA-4 polypeptide, a PD-1 polypeptide, a PD-L1 polypeptide, a PD-L2 polypeptide, a TIM3 polypeptide, a LAG3 polypeptide, a VISTA polypeptide, a SINGLEC7 polypeptide, a SINGLEC9 polypeptide, a TIGIT polypeptide, a CD96 polypeptide, a BTLA polypeptide, a B7H3 polypeptide, a B7H4 polypeptide, a CD155 polypeptide, a HHLA2 polypeptide, a BTN3A1 polypeptide, or an active fragment thereof;

[0507] X B is a B7.1 polypeptide or a B7.2 polypeptide;

[0508] XC is a binder such as an Fc domain, TCR, cytokine, cytokine trap, receptor ligand, complement protein;

[0509] L A is absent or is a polypeptide linker; and

[0510] L B is absent or is a polypeptide linker.

[0511] 115. The composition according to embodiment 114, wherein the Fc domain is an Ig Fc domain selected from the group consisting essentially of human IgG, IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgA1, IgA2, IgE or IgD.

[0512] 116. The composition according to embodiment 115, wherein the Fc domain comprises an amino acid sequence having at least 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 114 or comprises the sequence of SEQ ID NO: 114.

[0513] 117. The composition according to any one of embodiments 51 to 116, wherein the second polypeptide comprises an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 117 or comprises the sequence of SEQ ID NO: 117.

[0514] 118. The composition according to any one of embodiments 51 to 117, wherein the second polypeptide comprises an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 118 or comprises the sequence of SEQ ID NO: 118.

[0515] 119. The composition according to any one of embodiments 51 to 118, wherein the composition further comprises an antigen.

[0516] 120. The composition according to embodiment 119, wherein the antigen is a tumor antigen, viral antigen, bacterial antigen or microbial antigen.

[0517] 121. The composition according to embodiment 119 or 120, wherein the antigen is ovalbumin (OVA).

[0518] 122. The composition according to any one of claims 51 to 121, the composition further comprising:

[0519] A first nucleic acid molecule encoding a first polypeptide according to any one of embodiments 51 to 90; and / or

[0520] A second nucleic acid molecule encoding a second polypeptide according to any one of embodiments 51 and 91 to 121.

[0521] 123. The composition according to embodiment 122, the composition further comprising a third nucleic acid molecule encoding an antigen according to any one of embodiments 119 to 121.

[0522] 124. A vector comprising the first nucleic acid molecule and / or the second nucleic acid molecule according to embodiment 122.

[0523] 125. The vector according to embodiment 124, the vector further comprising the third nucleic acid molecule according to embodiment 123.

[0524] 126. A composition comprising:

[0525] A first vector comprising the first nucleic acid molecule according to embodiment 122; and

[0526] A second vector comprising the second nucleic acid molecule according to embodiment 122.

[0527] 127. The composition according to embodiment 126, wherein the first vector further comprises the third nucleic acid molecule according to embodiment 123.

[0528] 128. The composition according to embodiment 126, wherein the second vector further comprises the third nucleic acid molecule according to embodiment 123.

[0529] 129. The composition according to embodiment 126, the composition further comprising a third vector comprising the third nucleic acid molecule according to embodiment 123.

[0530] 130. A plasmid comprising the first nucleic acid molecule and the second nucleic acid molecule according to embodiment 122.

[0531] 131. The plasmid according to embodiment 130, wherein the plasmid further comprises a third nucleic acid molecule according to embodiment 123.

[0532] 132. A composition comprising:

[0533] a first plasmid comprising a first nucleic acid molecule according to embodiment 122; and

[0534] a second plasmid comprising a second nucleic acid molecule according to embodiment 122.

[0535] 133. The composition according to embodiment 132, wherein the first plasmid further comprises a third nucleic acid molecule according to embodiment 123.

[0536] 134. The composition according to embodiment 132, wherein the second plasmid further comprises a third nucleic acid molecule according to embodiment 123.

[0537] 135. The composition according to embodiment 132, further comprising a third plasmid comprising a third nucleic acid molecule according to embodiment 123.

[0538] 136. A recombinant virus comprising a first nucleic acid molecule and a second nucleic acid molecule according to embodiment 123.

[0539] 137. The recombinant virus according to embodiment 136, wherein the recombinant virus further comprises a third nucleic acid molecule according to embodiment 123.

[0540] 138. The recombinant virus according to embodiment 136 or 137, wherein the recombinant virus is an adenovirus or a lentivirus.

[0541] 139. The recombinant virus according to embodiment 138, wherein the recombinant virus is a replication - incompetent adenovirus.

[0542] 140. A liposome comprising a first nucleic acid molecule and a second nucleic acid molecule according to embodiment 122.

[0543] 141. The liposome according to embodiment 140, wherein the liposome further comprises a third nucleic acid molecule according to embodiment 123.

[0544] 142. The liposome according to embodiment 140 or 141, wherein the liposome comprises any lipid provided herein.

[0545] 143. A pharmaceutical composition comprising:

[0546] The first nucleic acid molecule and the second nucleic acid molecule according to embodiment 122; and

[0547] A pharmaceutically acceptable carrier.

[0548] 144. The pharmaceutical composition according to embodiment 143, further comprising a third nucleic acid molecule according to embodiment 123.

[0549] 145. A cell comprising the composition according to any one of embodiments 51 to 123.

[0550] 146. The cell according to embodiment 145, wherein the cell further comprises a chimeric antigen receptor (CAR).

[0551] 147. The cell according to embodiment 145 or 146, wherein the cell is an immune cell.

[0552] 148. The cell according to embodiment 147, wherein the immune cell is a T cell, an NK cell or a dendritic cell.

[0553] 149. A method for generating a cell according to any one of embodiments 145 to 148, the method comprising contacting the cell with:

[0554] The carrier according to embodiments 124 to 125;

[0555] The composition according to any one of embodiments 126 to 129;

[0556] The plasmid according to embodiments 130 to 131;

[0557] The composition according to any one of embodiments 132 to 134;

[0558] The recombinant virus according to any one of embodiments 135 to 139;

[0559] The liposome according to any one of embodiments 140 to 142; or

[0560] The pharmaceutical composition according to any one of embodiments 143 to 144.

[0561] 150. The method according to embodiment 149, wherein the contacting comprises transducing or transfecting the cell with the carrier, the plasmid, the recombinant virus, the liposome or one of these compositions.

[0562] 151. A method for generating a cell according to any one of embodiments 145 to 148 in vivo, the method comprising administering to a subject:

[0563] A vector according to embodiments 124 to 125;

[0564] A composition according to any one of embodiments 126 to 129;

[0565] A plasmid according to embodiments 130 to 131;

[0566] A composition according to any one of embodiments 132 to 134;

[0567] A recombinant virus according to any one of embodiments 135 to 139;

[0568] A liposome according to any one of embodiments 140 to 142; or

[0569] A pharmaceutical composition according to any one of embodiments 143 to 144;

[0570] wherein the vector, plasmid, recombinant virus, liposome or composition transduces or transfects cells in vivo to produce a cell according to any one of embodiments 145 to 148.

[0571] 152. A method of altering an immune response in a subject, the method comprising administering to the subject:

[0572] A vector according to embodiments 124 to 125;

[0573] A composition according to any one of embodiments 126 to 129;

[0574] A plasmid according to embodiments 130 to 131;

[0575] A composition according to any one of embodiments 132 to 134;

[0576] A recombinant virus according to any one of embodiments 135 to 139;

[0577] A liposome according to any one of embodiments 140 to 142; or

[0578] A pharmaceutical composition according to any one of embodiments 143 to 144.

[0579] 153. The method according to embodiment 152, wherein the immune response is activated.

[0580] 154. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject:

[0581] A vector according to embodiments 124 to 125;

[0582] A composition according to any one of embodiments 126 to 129;

[0583] A plasmid according to embodiments 130 to 131;

[0584] A composition according to any one of embodiments 132 to 134;

[0585] A recombinant virus according to any one of embodiments 135 to 139;

[0586] A liposome according to any one of embodiments 140 to 142; or

[0587] A pharmaceutical composition according to any one of embodiments 143 to 144.

[0588] 155. The method according to embodiment 154, wherein the cancer is a cancer provided herein.

[0589] 156. The method according to embodiment 154 or 155, wherein the method further comprises using at least one additional therapeutic agent, such as an anti-cancer therapeutic agent, including but not limited to chemotherapy, radiotherapy, targeted therapy, immunotherapy, stem cell or bone marrow transplantation therapy, hormone therapy, photodynamic therapy, or any combination thereof.

[0590] 157. A method of treating a disease or disorder in a subject in need thereof, the disease or disorder such as a viral infection, a bacterial infection, or a fungal infection, such as those provided herein, the method comprising administering to the subject:

[0591] A vector according to embodiments 124 to 125;

[0592] A composition according to any one of embodiments 126 to 129;

[0593] A plasmid according to embodiments 130 to 131;

[0594] A composition according to any one of embodiments 132 to 134;

[0595] A recombinant virus according to any one of embodiments 135 to 139;

[0596] A liposome according to any one of embodiments 140 to 142; or

[0597] A pharmaceutical composition according to any one of embodiments 143 to 144.

[0598] 158. A polypeptide comprising the formula: X1-L1-X2-L2-X3-L3-X4-L4-X5-L5-X6, wherein:

[0599] X1 is interleukin-21 (IL-21) polypeptide, interleukin-15 (IL-15) polypeptide, interleukin-12p40 subunit (IL-12p40) polypeptide, interleukin-12p35 subunit (IL-12p35) polypeptide, interleukin-15 receptor (IL-15Rα) polypeptide, the first fragment of IL-15Rα polypeptide or the second fragment of IL-15Rα polypeptide;

[0600] X2 is IL-21 polypeptide, IL-15 polypeptide, IL-12p40 polypeptide, IL-12p35 polypeptide, IL-15Rα polypeptide, the first fragment of IL-15Rα polypeptide or the second fragment of IL-15Rα polypeptide;

[0601] X3 is IL-21 polypeptide, IL-15 polypeptide, IL-12p40 polypeptide, IL-12p35 polypeptide, IL-15Rα polypeptide, the first fragment of IL-15Rα polypeptide or the second fragment of IL-15Rα polypeptide;

[0602] X4 is IL-21 polypeptide, IL-15 polypeptide, IL-12p40 polypeptide, IL-12p35 polypeptide, IL-15Rα polypeptide, the first fragment of IL-15Rα polypeptide or the second fragment of IL-15Rα polypeptide;

[0603] X5 is IL-21 polypeptide, IL-15 polypeptide, IL-12p40 polypeptide, IL-12p35 polypeptide, IL-15Rα polypeptide, the first fragment of IL-15Rα polypeptide or the second fragment of IL-15Rα polypeptide;

[0604] X6 is IL-21 polypeptide, IL-15 polypeptide, IL-12p40 polypeptide, IL-12p35 polypeptide, IL-15Rα polypeptide, the first fragment of IL-15Rα polypeptide or the second fragment of IL-15Rα polypeptide;

[0605] L1, L2, L3, L4 and L5 are each independently a polypeptide linker containing the same or different polypeptide sequences,

[0606] provided that each of X1, X2, X3, X4, X5 and X6 is different.

[0607] 159. The polypeptide according to embodiment 158, wherein X1 is interleukin-21 (IL-21) polypeptide.

[0608] 160. The polypeptide according to embodiment 158 or 159, wherein:

[0609] X1 is IL-21 polypeptide;

[0610] X2 is IL-15 polypeptide;

[0611] X3 is the first fragment of the IL-15Rα polypeptide;

[0612] X4 is the IL-12p40 polypeptide;

[0613] X5 is the IL-12p35 polypeptide; and

[0614] X6 is the second fragment of the IL-15Rα polypeptide.

[0615] 161. The polypeptide according to embodiment 158 or 159, wherein:

[0616] X1 is the IL-21 polypeptide;

[0617] X2 is the IL-15 polypeptide;

[0618] X3 is the first fragment of the IL-15Rα polypeptide;

[0619] X4 is the IL-12p35 polypeptide;

[0620] X5 is the IL-12p40 polypeptide; and X6 is the second fragment of the IL-15Rα polypeptide.

[0621] 162. The polypeptide according to embodiment 158 or 159, wherein:

[0622] X1 is the IL-21 polypeptide;

[0623] X2 is the IL-15 polypeptide;

[0624] X3 is the IL-12p40 polypeptide;

[0625] X4 is the first fragment of the IL-15Rα polypeptide;

[0626] X5 is the IL-12p35 polypeptide; and

[0627] X6 is the second fragment of the IL-15Rα polypeptide.

[0628] 163. The polypeptide according to embodiment 158 or 159, wherein:

[0629] X1 is the IL-21 polypeptide;

[0630] X2 is the IL-15 polypeptide;

[0631] X3 is the IL-12p40 polypeptide;

[0632] X4 is the IL-12p35 polypeptide;

[0633] X5 is the first fragment of the IL-15Rα polypeptide; and

[0634] X6 is the second fragment of the IL-15Rα polypeptide.

[0635] 164. The polypeptide according to embodiment 158 or 159, wherein:

[0636] X1 is an IL-21 polypeptide;

[0637] X2 is an IL-15 polypeptide;

[0638] X3 is an IL-12p35 polypeptide;

[0639] X4 is an IL-12p40 polypeptide;

[0640] X5 is the first fragment of the IL-15Rα polypeptide; and

[0641] X6 is the second fragment of the IL-15Rα polypeptide.

[0642] 165. The polypeptide according to embodiment 158 or 159, wherein:

[0643] X1 is an IL-21 polypeptide;

[0644] X2 is an IL-15 polypeptide;

[0645] X3 is an IL-12p35 polypeptide;

[0646] X4 is the first fragment of the IL-15Rα polypeptide;

[0647] X5 is an IL-12p40 polypeptide; and

[0648] X6 is the second fragment of the IL-15Rα polypeptide.

[0649] 166. The polypeptide according to embodiment 158 or 159, wherein:

[0650] X1 is an IL-21 polypeptide;

[0651] X2 is the first fragment of the IL-15Rα polypeptide;

[0652] X3 is an IL-15 polypeptide;

[0653] X4 is an IL-12p40 polypeptide;

[0654] X5 is an IL-12p35 polypeptide; and

[0655] X6 is the second fragment of the IL-15Rα polypeptide.

[0656] 167. The polypeptide according to embodiment 158 or 159, wherein:

[0657] X1 is an IL-21 polypeptide;

[0658] X2 is a first fragment of an IL-15Rα polypeptide;

[0659] X3 is an IL-15 polypeptide;

[0660] X4 is an IL-12p35 polypeptide;

[0661] X5 is an IL-12p40 polypeptide; and

[0662] X6 is a second fragment of an IL-15Rα polypeptide.

[0663] 168. The polypeptide according to embodiment 158 or 159, wherein:

[0664] X1 is an IL-21 polypeptide;

[0665] X2 is a first fragment of an IL-15Rα polypeptide;

[0666] X3 is an IL-12p40 polypeptide;

[0667] X4 is an IL-15 polypeptide;

[0668] X5 is an IL-12p35 polypeptide; and

[0669] X6 is a second fragment of an IL-15Rα polypeptide.

[0670] 169. The polypeptide according to embodiment 158 or 159, wherein:

[0671] X1 is an IL-21 polypeptide;

[0672] X2 is a first fragment of an IL-15Rα polypeptide;

[0673] X3 is an IL-12p40 polypeptide;

[0674] X4 is an IL-12p35 polypeptide;

[0675] X5 is IL-15; and

[0676] X6 is a second fragment of an IL-15Rα polypeptide.

[0677] 170. The polypeptide according to embodiment 158 or 159, wherein:

[0678] X1 is an IL-21 polypeptide;

[0679] X2 is a first fragment of an IL-15Rα polypeptide;

[0680] X3 is an IL-12p35 polypeptide;

[0681] X4 is an IL-12p40 polypeptide;

[0682] X5 is IL-15; and

[0683] X6 is a second fragment of an IL-15Rα polypeptide.

[0684] 171. The polypeptide according to embodiment 158 or 159, wherein:

[0685] X1 is an IL-21 polypeptide;

[0686] X2 is a first fragment of an IL-15Rα polypeptide;

[0687] X3 is an IL-12p35 polypeptide;

[0688] X4 is IL-15;

[0689] X5 is an IL-12p40 polypeptide; and

[0690] X6 is a second fragment of an IL-15Rα polypeptide.

[0691] 172. The polypeptide according to embodiment 158 or 159, wherein:

[0692] X1 is an IL-21 polypeptide;

[0693] X2 is an IL-12p40 polypeptide;

[0694] X3 is an IL-15 polypeptide;

[0695] X4 is a first fragment of an IL-15Rα polypeptide;

[0696] X5 is an IL-12p35 polypeptide; and

[0697] X6 is a second fragment of an IL-15Rα polypeptide.

[0698] 173. The polypeptide according to embodiment 158 or 159, wherein:

[0699] X1 is an IL-21 polypeptide;

[0700] X2 is an IL-12p40 polypeptide;

[0701] X3 is an IL-15 polypeptide;

[0702] X4 is an IL-12p35 polypeptide;

[0703] X5 is a first fragment of an IL-15Rα polypeptide; and

[0704] X6 is a second fragment of an IL-15Rα polypeptide.

[0705] The polypeptide according to embodiment 158 or 159, wherein:

[0706] X1 is an IL-21 polypeptide;

[0707] X2 is an IL-12p40 polypeptide;

[0708] X3 is a first fragment of an IL-15Rα polypeptide;

[0709] X4 is an IL-15 polypeptide;

[0710] X5 is an IL-12p35 polypeptide; and

[0711] X6 is a second fragment of an IL-15Rα polypeptide.

[0712] The polypeptide according to embodiment 158 or 159, wherein:

[0713] X1 is an IL-21 polypeptide;

[0714] X2 is an IL-12p40 polypeptide;

[0715] X3 is a first fragment of an IL-15Rα polypeptide;

[0716] X4 is an IL-12p35 polypeptide;

[0717] X5 is IL-15; and

[0718] X6 is a second fragment of an IL-15Rα polypeptide.

[0719] The polypeptide according to embodiment 158 or 159, wherein:

[0720] X1 is an IL-21 polypeptide;

[0721] X2 is an IL-12p40 polypeptide;

[0722] X3 is an IL-12p35 polypeptide;

[0723] X4 is a first fragment of an IL-15Rα polypeptide;

[0724] X5 is IL-15; and

[0725] X6 is a second fragment of an IL-15Rα polypeptide.

[0726] The polypeptide according to embodiment 158 or 159, wherein:

[0727] X1 is an IL-21 polypeptide;

[0728] X2 is an IL-12p40 polypeptide;

[0729] X3 is an IL-12p35 polypeptide;

[0730] X4 is IL-15;

[0731] X5 is a first fragment of an IL-15Rα polypeptide; and

[0732] X6 is a second fragment of an IL-15Rα polypeptide.

[0733] 178. The polypeptide according to embodiment 158 or 159, wherein:

[0734] X1 is an IL-21 polypeptide;

[0735] X2 is an IL-12p35 polypeptide;

[0736] X3 is an IL-15 polypeptide;

[0737] X4 is a first fragment of an IL-15Rα polypeptide;

[0738] X5 is an IL-12p40 polypeptide; and

[0739] X6 is a second fragment of an IL-15Rα polypeptide.

[0740] 179. The polypeptide according to embodiment 158 or 159, wherein:

[0741] X1 is an IL-21 polypeptide;

[0742] X2 is an IL-12p35 polypeptide;

[0743] X3 is an IL-15 polypeptide;

[0744] X4 is an IL-12p40 polypeptide;

[0745] X5 is a first fragment of an IL-15Rα polypeptide; and

[0746] X6 is a second fragment of an IL-15Rα polypeptide.

[0747] 180. The polypeptide according to embodiment 158 or 159, wherein:

[0748] X1 is an IL-21 polypeptide;

[0749] X2 is an IL-12p35 polypeptide;

[0750] X3 is a first fragment of an IL-15Rα polypeptide;

[0751] X4 is an IL-15 polypeptide;

[0752] X5 is an IL-12p40 polypeptide; and

[0753] X6 is a second fragment of an IL-15Rα polypeptide.

[0754] 181. The polypeptide according to embodiment 158 or 159, wherein:

[0755] X1 is an IL-21 polypeptide;

[0756] X2 is an IL-12p35 polypeptide;

[0757] X3 is a first fragment of an IL-15Rα polypeptide;

[0758] X4 is an IL-12p40 polypeptide;

[0759] X5 is IL-15; and

[0760] X6 is a second fragment of an IL-15Rα polypeptide.

[0761] 182. The polypeptide according to embodiment 158 or 159, wherein:

[0762] X1 is an IL-21 polypeptide;

[0763] X2 is an IL-12p35 polypeptide;

[0764] X3 is an IL-12p40 polypeptide;

[0765] X4 is a first fragment of an IL-15Rα polypeptide;

[0766] X5 is IL-15; and

[0767] X6 is a second fragment of an IL-15Rα polypeptide.

[0768] 183. The polypeptide according to embodiment 158 or 159, wherein:

[0769] X1 is an IL-21 polypeptide;

[0770] X2 is an IL-12p35 polypeptide;

[0771] X3 is an IL-12p40 polypeptide;

[0772] X4 is IL-15;

[0773] X5 is a first fragment of an IL-15Rα polypeptide; and

[0774] X6 is a second fragment of an IL-15Rα polypeptide.

[0775] 184. The polypeptide according to any one of embodiments 158 to 183, wherein the IL-21 polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 71, or as otherwise provided herein.

[0776] 185. The polypeptide according to any one of embodiments 158 to 184, wherein the IL-21 polypeptide comprises the amino acid sequence of SEQ ID NO: 71, or as otherwise provided herein.

[0777] 186. The polypeptide according to any one of embodiments 158 to 185, wherein the IL-12p40 polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1, 2, 3 or 4, or as otherwise provided herein.

[0778] 187. The polypeptide according to any one of embodiments 158 to 186, wherein the IL-12p40 polypeptide comprises the amino acid sequence of SEQ ID NO: 1, 2, 3 or 4, or as otherwise provided herein.

[0779] 188. The polypeptide according to any one of embodiments 158 to 187, wherein the IL-12p35 polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 5, 6, 7 or 8, or as otherwise provided herein.

[0780] 189. The polypeptide according to any one of embodiments 158 to 188, wherein the IL-12p35 polypeptide comprises the amino acid sequence of SEQ ID NO: 5, 6, 7 or 8, or as otherwise provided herein.

[0781] 190. The polypeptide according to any one of embodiments 158 to 189, wherein the IL-15 polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 9 or 10, or as otherwise provided herein.

[0782] 191. The polypeptide according to any one of embodiments 158 to 190, wherein the IL-15 polypeptide comprises the amino acid sequence of SEQ ID NO: 9 or 10, or as otherwise provided herein.

[0783] 192. The polypeptide according to any one of embodiments 158 to 191, wherein the IL-15Rα polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 11 or 12, or as otherwise provided herein.

[0784] 193. The polypeptide according to any one of embodiments 158 to 192, wherein the IL-15Rα polypeptide comprises the amino acid sequence of SEQ ID NO: 11 or 12, or as otherwise provided herein.

[0785] 194. The polypeptide according to any one of embodiments 158 to 193, wherein one or more of L1, L2, L3, L4 and L5 are each independently a cleavable linker.

[0786] 195. The polypeptide according to embodiment 194, wherein L1, L3, L4 and L5 are non-cleavable linkers.

[0787] 196. The polypeptide according to embodiment 194 or 195, wherein the cleavable linker is a furin-cleavable linker, a Val-Cit linker, a Val-Gly linker, a Gly-Gly linker, an Ala-Ala-Asn linker or a linker comprising polyethylene glycol (PEG).

[0788] 197. The polypeptide according to any one of embodiments 158 to 196, wherein one of L1, L2, L3, L4 and L5 comprises a sequence of (GSGSGG)n (SEQ ID NO: 16), (GGGGS)n (SEQ ID NO: 17), (GGGGA)n (SEQ ID NO: 18), (GGGSE)n (SEQ ID NO: 19), (GGGSK)n (SEQ ID NO: 20) or (AEEEK)n (SEQ ID NO: 21) or a combination thereof, wherein each n is independently 1, 2, 3, 4 or 5.

[0789] 198. The polypeptide according to embodiment 197, wherein n is 3.

[0790] The polypeptide according to embodiment 197 or 198, wherein one or more of L1, L2, L3, L4 and L5 comprises the sequence RXXR of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:70 or a combination thereof, wherein X is any amino acid.

[0791] The polypeptide according to any one of embodiments 197 to 199, wherein each of L1, L2, L3, L4 and L5 comprises the sequence RXXR of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:70 or a combination thereof, wherein X is any amino acid.

[0792] The polypeptide according to any one of embodiments 158 to 200, wherein the first polypeptide comprises the sequence of SEQ ID NO:72 or a variant thereof, or as otherwise provided herein.

[0793] 202. The polypeptide according to any one of embodiments 158 to 201, wherein the first polypeptide comprises the sequence of SEQ ID NO: 73 or a variant thereof, or as otherwise provided herein.

[0794] 203. The polypeptide according to any one of embodiments 158 to 202, wherein the first polypeptide comprises the sequence of SEQ ID NO: 74 or a variant thereof, or as otherwise provided herein.

[0795] 204. The polypeptide according to any one of embodiments 158 to 203, wherein the first polypeptide comprises the sequence of SEQ ID NO: 75 or a variant thereof, or as otherwise provided herein.

[0796] 205. The polypeptide according to any one of embodiments 158 to 204, wherein the first polypeptide comprises the sequence of SEQ ID NO: 76 or a variant thereof, or as otherwise provided herein.

[0797] 206. The polypeptide according to any one of embodiments 158 to 205, wherein the first polypeptide comprises the sequence of SEQ ID NO: 77 or a variant thereof, or as otherwise provided herein.

[0798] 207. The polypeptide according to any one of embodiments 158 to 206, wherein the first polypeptide comprises the sequence of SEQ ID NO: 78 or a variant thereof, or as otherwise provided herein.

[0799] 208. The polypeptide according to any one of embodiments 158 to 207, wherein the first polypeptide comprises the sequence of SEQ ID NO: 79 or a variant thereof, or as otherwise provided herein.

[0800] 209. The polypeptide according to any one of embodiments 158 to 208, wherein the first polypeptide is linked or fused to at least one additional molecule selected from the group consisting of: a polypeptide, an antigen, a ligand, an antibody, a checkpoint inhibitor, a lipid, a carbohydrate, a nucleic acid, or any combination thereof.

[0801] 210. The polypeptide according to embodiment 158, wherein:

[0802] X1 is an IL-21 polypeptide;

[0803] X2 is an IL-15 polypeptide;

[0804] X3 is a first fragment of an IL-15Rα polypeptide;

[0805] X4 is an IL-12p40 polypeptide;

[0806] X5 is an IL-12p35 polypeptide; and

[0807] X6 is the second fragment of the IL-15Rα polypeptide; and

[0808] L1, L2, L3, L4, and L5 are non-cleavable linkers.

[0809] 211. The polypeptide according to embodiment 210, wherein the first polypeptide is linked or fused to at least one additional molecule selected from the group consisting of: a polypeptide, an antigen, a ligand, an antibody, a checkpoint inhibitor, a lipid, a carbohydrate, a nucleic acid, or any combination thereof.

[0810] 212. A nucleic acid encoding the polypeptide according to any one of embodiments 158 to 211.

[0811] 213. A vector comprising the nucleic acid according to embodiment 212.

[0812] 214. A plasmid comprising the nucleic acid according to embodiment 212.

[0813] 215. A recombinant virus comprising the nucleic acid according to embodiment 212.

[0814] 216. The recombinant virus according to embodiment 215, wherein the recombinant virus is an adenovirus or a lentivirus.

[0815] 217. The recombinant virus according to embodiment 215 or 216, wherein the recombinant virus is a replication-incompetent adenovirus.

[0816] 218. A liposome comprising the nucleic acid according to embodiment 212.

[0817] 219. The liposome according to embodiment 218, wherein the liposome comprises any lipid provided herein.

[0818] 220. A pharmaceutical composition comprising the nucleic acid according to embodiment 212 and a pharmaceutically acceptable carrier.

[0819] 221. A cell comprising the composition according to any one of embodiments 158 to 211.

[0820] 222. A composition comprising:

[0821] There is provided a first polypeptide having the formula: X1-L1-X2-L2-X3-L3-X4-L4-X5, wherein:

[0822] X1 is interleukin-21 (IL-21) polypeptide, interleukin-15 (IL-15) polypeptide, interleukin-12 p40 subunit (IL-12p40) polypeptide, interleukin-12 p35 subunit (IL-12p35) polypeptide, interleukin-15 receptor (IL-15Rα) polypeptide, the first fragment of IL-15Rα polypeptide or the second fragment of IL-15Rα polypeptide;

[0823] X2 is IL-21 polypeptide, IL-15 polypeptide, IL-12p40 polypeptide, IL-12p35 polypeptide, IL-15Rα polypeptide, the first fragment of IL-15Rα polypeptide or the second fragment of IL-15Rα polypeptide;

[0824] X3 is IL-21 polypeptide, IL-15 polypeptide, IL-12p40 polypeptide, IL-12p35 polypeptide, IL-15Rα polypeptide, the first fragment of IL-15Rα polypeptide or the second fragment of IL-15Rα polypeptide;

[0825] X4 is IL-21 polypeptide, IL-15 polypeptide, IL-12p40 polypeptide, IL-12p35 polypeptide, IL-15Rα polypeptide, the first fragment of IL-15Rα polypeptide or the second fragment of IL-15Rα polypeptide;

[0826] X5 is IL-21 polypeptide, IL-15 polypeptide, IL-12p40 polypeptide, IL-12p35 polypeptide, IL-15Rα polypeptide, the first fragment of IL-15Rα polypeptide or the second fragment of IL-15Rα polypeptide;

[0827] X6 is IL-21 polypeptide, IL-15 polypeptide, IL-12p40 polypeptide, IL-12p35 polypeptide, IL-15Rα polypeptide, the first fragment of IL-15Rα polypeptide or the second fragment of IL-15Rα polypeptide;

[0828] L1, L2, L3, L4 and L5 are each independently a polypeptide linker containing the same or different polypeptide sequences,

[0829] provided that each of X1, X2, X3, X4, X5 and X6 is different; and / or

[0830] A second polypeptide, the second polypeptide comprising at least one immune checkpoint polypeptide or an active fragment thereof, such as but not limited to a CTLA-4 polypeptide, a PD-1 polypeptide, a PD-L1 polypeptide, a PD-L2 polypeptide, a TIM3 polypeptide, a LAG3 polypeptide, a VISTA polypeptide, a SINGLEC7 polypeptide, a SINGLEC9 polypeptide, a TIGIT polypeptide, a CD96 polypeptide, a BTLA polypeptide, a B7H3 polypeptide, a B7H4 polypeptide, a CD155 polypeptide, a HHLA2 polypeptide, a BTN3A1 polypeptide, a B7.1 polypeptide, a B7.2 polypeptide or an active fragment thereof;

[0831] or one or more nucleic acid molecules encoding the first polypeptide and the second polypeptide.

[0832] 223. The composition according to embodiment 222, wherein X1 is an interleukin 21 (IL-21) polypeptide.

[0833] 224. The composition according to embodiment 222 or 223, wherein:

[0834] X1 is an IL-21 polypeptide;

[0835] X2 is an IL-15 polypeptide;

[0836] X3 is a first fragment of an IL-15Rα polypeptide;

[0837] X4 is an IL-12p40 polypeptide;

[0838] X5 is an IL-12p35 polypeptide; and

[0839] X6 is a second fragment of an IL-15Rα polypeptide.

[0840] 225. The polypeptide according to embodiment 222 or 223, wherein:

[0841] X1 is an IL-21 polypeptide;

[0842] X2 is an IL-15 polypeptide;

[0843] X3 is a first fragment of an IL-15Rα polypeptide;

[0844] X4 is an IL-12p35 polypeptide;

[0845] X5 is an IL-12p40 polypeptide; and X6 is a second fragment of an IL-15Rα polypeptide.

[0846] 226. The polypeptide according to embodiment 222 or 223, wherein:

[0847] X1 is an IL-21 polypeptide;

[0848] X2 is an IL-15 polypeptide;

[0849] X3 is an IL-12p40 polypeptide;

[0850] X4 is a first fragment of an IL-15Rα polypeptide;

[0851] X5 is an IL-12p35 polypeptide; and

[0852] X6 is a second fragment of an IL-15Rα polypeptide.

[0853] 227. The polypeptide according to embodiment 222 or 223, wherein:

[0854] X1 is an IL-21 polypeptide;

[0855] X2 is an IL-15 polypeptide;

[0856] X3 is an IL-12p40 polypeptide;

[0857] X4 is an IL-12p35 polypeptide;

[0858] X5 is a first fragment of an IL-15Rα polypeptide; and

[0859] X6 is a second fragment of an IL-15Rα polypeptide.

[0860] 228. The polypeptide according to embodiment 222 or 223, wherein:

[0861] X1 is an IL-21 polypeptide;

[0862] X2 is an IL-15 polypeptide;

[0863] X3 is an IL-12p35 polypeptide;

[0864] X4 is an IL-12p40 polypeptide;

[0865] X5 is a first fragment of an IL-15Rα polypeptide; and

[0866] X6 is a second fragment of an IL-15Rα polypeptide.

[0867] 229. The polypeptide according to embodiment 222 or 223, wherein:

[0868] X1 is an IL-21 polypeptide;

[0869] X2 is an IL-15 polypeptide;

[0870] X3 is an IL-12p35 polypeptide;

[0871] X4 is a first fragment of an IL-15Rα polypeptide;

[0872] X5 is an IL-12p40 polypeptide; and

[0873] X6 is a second fragment of an IL-15Rα polypeptide.

[0874] 230. The polypeptide according to embodiment 222 or 223, wherein:

[0875] X1 is an IL-21 polypeptide;

[0876] X2 is a first fragment of an IL-15Rα polypeptide;

[0877] X3 is an IL-15 polypeptide;

[0878] X4 is an IL-12p40 polypeptide;

[0879] X5 is an IL-12p35 polypeptide; and

[0880] X6 is a second fragment of an IL-15Rα polypeptide.

[0881] 231. The polypeptide according to embodiment 222 or 223, wherein:

[0882] X1 is an IL-21 polypeptide;

[0883] X2 is a first fragment of an IL-15Rα polypeptide;

[0884] X3 is an IL-15 polypeptide;

[0885] X4 is an IL-12p35 polypeptide;

[0886] X5 is an IL-12p40 polypeptide; and

[0887] X6 is a second fragment of an IL-15Rα polypeptide.

[0888] 232. The polypeptide according to embodiment 222 or 223, wherein:

[0889] X1 is an IL-21 polypeptide;

[0890] X2 is a first fragment of an IL-15Rα polypeptide;

[0891] X3 is an IL-12p40 polypeptide;

[0892] X4 is an IL-15 polypeptide;

[0893] X5 is an IL-12p35 polypeptide; and

[0894] X6 is a second fragment of an IL-15Rα polypeptide.

[0895] The polypeptide according to embodiment 222 or 223, wherein:

[0896] X1 is an IL-21 polypeptide;

[0897] X2 is a first fragment of an IL-15Rα polypeptide;

[0898] X3 is an IL-12p40 polypeptide;

[0899] X4 is an IL-12p35 polypeptide;

[0900] X5 is IL-15; and

[0901] X6 is a second fragment of an IL-15Rα polypeptide.

[0902] The polypeptide according to embodiment 222 or 223, wherein:

[0903] X1 is an IL-21 polypeptide;

[0904] X2 is a first fragment of an IL-15Rα polypeptide;

[0905] X3 is an IL-12p35 polypeptide;

[0906] X4 is an IL-12p40 polypeptide;

[0907] X5 is IL-15; and

[0908] X6 is a second fragment of an IL-15Rα polypeptide.

[0909] The polypeptide according to embodiment 222 or 223, wherein:

[0910] X1 is an IL-21 polypeptide;

[0911] X2 is a first fragment of an IL-15Rα polypeptide;

[0912] X3 is an IL-12p35 polypeptide;

[0913] X4 is IL-15;

[0914] X5 is an IL-12p40 polypeptide; and

[0915] X6 is a second fragment of an IL-15Rα polypeptide.

[0916] The polypeptide according to embodiment 222 or 223, wherein:

[0917] X1 is an IL-21 polypeptide;

[0918] X2 is an IL-12p40 polypeptide;

[0919] X3 is an IL-15 polypeptide;

[0920] X4 is a first fragment of an IL-15Rα polypeptide;

[0921] X5 is an IL-12p35 polypeptide; and

[0922] X6 is a second fragment of an IL-15Rα polypeptide.

[0923] 236. The polypeptide according to embodiment 222 or 223, wherein:

[0924] X1 is an IL-21 polypeptide;

[0925] X2 is an IL-12p40 polypeptide;

[0926] X3 is an IL-15 polypeptide;

[0927] X4 is an IL-12p35 polypeptide;

[0928] X5 is a first fragment of an IL-15Rα polypeptide; and

[0929] X6 is a second fragment of an IL-15Rα polypeptide.

[0930] 237. The polypeptide according to embodiment 222 or 223, wherein:

[0931] X1 is an IL-21 polypeptide;

[0932] X2 is an IL-12p40 polypeptide;

[0933] X3 is a first fragment of an IL-15Rα polypeptide;

[0934] X4 is an IL-15 polypeptide;

[0935] X5 is an IL-12p35 polypeptide; and

[0936] X6 is a second fragment of an IL-15Rα polypeptide.

[0937] 238. The polypeptide according to embodiment 222 or 223, wherein:

[0938] X1 is an IL-21 polypeptide;

[0939] X2 is an IL-12p40 polypeptide;

[0940] X3 is a first fragment of an IL-15Rα polypeptide;

[0941] X4 is an IL-12p35 polypeptide;

[0942] X5 is IL-15; and

[0943] X6 is the second fragment of the IL-15Rα polypeptide.

[0944] 239. The polypeptide according to embodiment 222 or 223, wherein:

[0945] X1 is an IL-21 polypeptide;

[0946] X2 is an IL-12p40 polypeptide;

[0947] X3 is an IL-12p35 polypeptide;

[0948] X4 is the first fragment of the IL-15Rα polypeptide;

[0949] X5 is IL-15; and

[0950] X6 is the second fragment of the IL-15Rα polypeptide.

[0951] 240. The polypeptide according to embodiment 222 or 223, wherein:

[0952] X1 is an IL-21 polypeptide;

[0953] X2 is an IL-12p40 polypeptide;

[0954] X3 is an IL-12p35 polypeptide;

[0955] X4 is IL-15;

[0956] X5 is the first fragment of the IL-15Rα polypeptide; and

[0957] X6 is the second fragment of the IL-15Rα polypeptide.

[0958] 241. The polypeptide according to embodiment 222 or 223, wherein:

[0959] X1 is an IL-21 polypeptide;

[0960] X2 is an IL-12p35 polypeptide;

[0961] X3 is an IL-15 polypeptide;

[0962] X4 is the first fragment of the IL-15Rα polypeptide;

[0963] X5 is an IL-12p40 polypeptide; and

[0964] X6 is the second fragment of the IL-15Rα polypeptide.

[0965] 242. The polypeptide according to embodiment 222 or 223, wherein:

[0966] X1 is an IL-21 polypeptide;

[0967] X2 is an IL-12p35 polypeptide;

[0968] X3 is an IL-15 polypeptide;

[0969] X4 is an IL-12p40 polypeptide;

[0970] X5 is a first fragment of an IL-15Rα polypeptide; and

[0971] X6 is a second fragment of an IL-15Rα polypeptide.

[0972] 243. The polypeptide according to embodiment 222 or 223, wherein:

[0973] X1 is an IL-21 polypeptide;

[0974] X2 is an IL-12p35 polypeptide;

[0975] X3 is a first fragment of an IL-15Rα polypeptide;

[0976] X4 is an IL-15 polypeptide;

[0977] X5 is an IL-12p40 polypeptide; and

[0978] X6 is a second fragment of an IL-15Rα polypeptide.

[0979] 244. The polypeptide according to embodiment 222 or 223, wherein:

[0980] X1 is an IL-21 polypeptide;

[0981] X2 is an IL-12p35 polypeptide;

[0982] X3 is a first fragment of an IL-15Rα polypeptide;

[0983] X4 is an IL-12p40 polypeptide;

[0984] X5 is an IL-15; and

[0985] X6 is a second fragment of an IL-15Rα polypeptide.

[0986] 245. The polypeptide according to embodiment 222 or 223, wherein:

[0987] X1 is an IL-21 polypeptide;

[0988] X2 is an IL-12p35 polypeptide;

[0989] X3 is an IL-12p40 polypeptide;

[0990] X4 is the first fragment of the IL-15Rα polypeptide;

[0991] X5 is IL-15; and

[0992] X6 is the second fragment of the IL-15Rα polypeptide.

[0993] 246. The polypeptide according to embodiment 222 or 223, wherein:

[0994] X1 is an IL-21 polypeptide;

[0995] X2 is an IL-12p35 polypeptide;

[0996] X3 is an IL-12p40 polypeptide;

[0997] X4 is IL-15;

[0998] X5 is the first fragment of the IL-15Rα polypeptide; and

[0999] X6 is the second fragment of the IL-15Rα polypeptide.

[1000] 247. The composition according to any one of embodiments 222 to 246, wherein the IL-21 polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 71, or as otherwise provided herein.

[1001] 248. The composition according to any one of embodiments 222 to 247, wherein the IL-21 polypeptide comprises the amino acid sequence of SEQ ID NO: 71, or as otherwise provided herein.

[1002] 249. The composition according to any one of embodiments 222 to 248, wherein the IL-12p40 polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 1, 2, 3 or 4, or as otherwise provided herein.

[1003] 250. The composition according to any one of embodiments 222 to 249, wherein the IL-12p40 polypeptide comprises the amino acid sequence of SEQ ID NO: 1, 2, 3 or 4, or as otherwise provided herein.

[1004] 251. A composition according to any one of embodiments 222 to 250, wherein the IL-12p35 polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:5, 6, 7 or 8, or as otherwise provided herein.

[1005] 252. A composition according to any one of embodiments 222 to 251, wherein the IL-12p35 polypeptide comprises the amino acid sequence of SEQ ID NO:5, 6, 7 or 8, or as otherwise provided herein.

[1006] 253. A composition according to any one of embodiments 222 to 252, wherein the IL-15 polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:9 or 10, or as otherwise provided herein.

[1007] 254. A composition according to any one of embodiments 222 to 253, wherein the IL-15 polypeptide comprises the amino acid sequence of SEQ ID NO:9 or 10, or as otherwise provided herein.

[1008] 255. A composition according to any one of embodiments 222 to 254, wherein the IL-15Rα polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:11 or 12, or as otherwise provided herein.

[1009] 256. A composition according to any one of embodiments 222 to 255, wherein the IL-15Rα polypeptide comprises the amino acid sequence of SEQ ID NO:11 or 12, or as otherwise provided herein.

[1010] 257. A composition according to any one of embodiments 222 to 256, wherein one or more of L1, L2, L3, L4 and L5 are each independently a cleavable linker.

[1011] 258. The polypeptide according to embodiment 257, wherein L1, L2, L3, L4 and L5 are non-cleavable linkers.

[1012] 259. The composition according to embodiment 257 or 258, wherein the cleavable linker is a furin-cleavable linker, a Val-Cit linker, a Val-Gly linker, a Gly-Gly linker, an Ala-Ala-Asn linker or a linker comprising polyethylene glycol (PEG).

[1013] 260. A composition according to any one of embodiments 222 to 259, wherein one of L1, L2, L3, L4, and L5 comprises the sequence of (GSGSGG)n (SEQ ID NO:16), (GGGGS)n (SEQ ID NO:17), (GGGGA)n (SEQ ID NO:18), (GGGSE)n (SEQ ID NO:19), (GGGSK)n (SEQ ID NO:20), or (AEEEK)n (SEQ ID NO:21), or a combination thereof, wherein each n is independently 1, 2, 3, 4, or 5.

[1014] 261. The composition according to embodiment 260, wherein n is 3.

[1015] 262. The composition according to embodiment 260 or 261, wherein one or more of L1, L2, L3, L4, and L5 comprises the sequence RXXR of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:70, or a combination thereof, wherein X is any amino acid.

[1016] 263. The composition according to any one of embodiments 260 to 262, wherein each of L1, L2, L3, L4, and L5 comprises the sequence RXXR of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:70 or a combination thereof, wherein X is any amino acid.

[1017] 264. The composition according to any one of embodiments 222 to 263, wherein the first polypeptide comprises the sequence of SEQ ID NO:72 or a variant thereof, or as otherwise provided herein.

[1018] 265. The composition according to any one of embodiments 222 to 263, wherein the first polypeptide comprises the sequence of SEQ ID NO:73 or a variant thereof, or as otherwise provided herein.

[1019] 266. The composition according to any one of embodiments 222 to 263, wherein the first polypeptide comprises the sequence of SEQ ID NO:74 or a variant thereof, or as otherwise provided herein.

[1020] 267. The composition according to any one of embodiments 222 to 263, wherein the first polypeptide comprises the sequence of SEQ ID NO:75 or a variant thereof, or as otherwise provided herein.

[1021] 268. The composition according to any one of embodiments 222 to 263, wherein the first polypeptide comprises the sequence of SEQ ID NO:76 or a variant thereof, or as otherwise provided herein.

[1022] 269. The composition according to any one of embodiments 222 to 263, wherein the first polypeptide comprises the sequence of SEQ ID NO:77 or a variant thereof, or as otherwise provided herein.

[1023] 270. A composition according to any one of embodiments 222 to 263, wherein the first polypeptide comprises the sequence of SEQ ID NO: 78 or a variant thereof, or as otherwise provided herein.

[1024] 271. A composition according to any one of embodiments 222 to 263, wherein the first polypeptide comprises the sequence of SEQ ID NO: 79 or a variant thereof, or as otherwise provided herein.

[1025] 272. A composition according to any one of embodiments 222 to 271, wherein the first polypeptide is linked or fused to at least one additional molecule selected from the group consisting of: a polypeptide, an antigen, a ligand, an antibody, a checkpoint inhibitor, a lipid, a carbohydrate, a nucleic acid, or any combination thereof.

[1026] 273. A composition according to embodiment 222, wherein:

[1027] X1 is an IL-21 polypeptide;

[1028] X2 is an IL-15 polypeptide;

[1029] X3 is an IL-12p40 polypeptide;

[1030] X4 is an IL-12p35 polypeptide;

[1031] X5 is an IL-15Rα polypeptide; and

[1032] L1, L2, L3, L4, and L5 are non-cleavable linkers.

[1033] 274. A composition according to embodiment 273, wherein the first polypeptide is linked or fused to at least one additional molecule selected from the group consisting of: a polypeptide, an antigen, a ligand, an antibody, a checkpoint inhibitor, a lipid, a carbohydrate, a nucleic acid, or any combination thereof.

[1034] 275. A composition according to any of the preceding embodiments, wherein the second polypeptide comprises, from the N-terminus to the C-terminus, the formula X A -L A -X B or the formula X B -L A -X A , wherein:

[1035] X Ais an immune checkpoint polypeptide or an active fragment thereof, such as but not limited to CTLA-4 polypeptide, PD-1 polypeptide, PD-L1 polypeptide, PD-L2 polypeptide, TIM3 polypeptide, LAG3 polypeptide, VISTA polypeptide, SINGLEC7 polypeptide, SINGLEC9 polypeptide, TIGIT polypeptide, CD96 polypeptide, BTLA polypeptide, B7H3 polypeptide, B7H4 polypeptide, CD155 polypeptide, HHLA2 polypeptide, BTN3A1 polypeptide or an active fragment thereof;

[1036] X B is a B7.1 polypeptide or a B7.2 polypeptide; and

[1037] L A is a polypeptide linker.

[1038] 276. The composition according to embodiment 275, wherein the second polypeptide comprises the formula X from the N-terminus to the C-terminus A -L A -X B 。

[1039] 277. The composition according to embodiment 275, wherein the polypeptide comprises the formula X from the N-terminus to the C-terminus B -L A -X A 。

[1040] 278. The composition according to any one of embodiments 275 to 277, wherein:

[1041] X A is a PD-L1 polypeptide; and

[1042] X B is a B7.1 polypeptide.

[1043] 279. The composition according to any one of embodiments 275 to 277, wherein:

[1044] X A is a PD-L1 polypeptide; and

[1045] X B is a B7.2 polypeptide.

[1046] 280. The composition according to any one of embodiments 275 to 277, wherein:

[1047] X A is a PD-L2 polypeptide; and

[1048] X B is a B7.1 polypeptide.

[1049] 281. The composition according to any one of embodiments 275 to 277, wherein:

[1050] X A is a PD-L2 polypeptide; and

[1051] X B is a B7.2 polypeptide.

[1052] 282. The composition according to any one of embodiments 275 to 279, wherein the PD-L1 polypeptide comprises a PD-L1 extracellular domain.

[1053] 283. The composition according to embodiment 282, wherein the PD-L1 polypeptide comprises an amino acid sequence having at least 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 102 or comprises the sequence of SEQ ID NO: 102.

[1054] 284. The composition according to any one of embodiments 275 to 277 and 280 to 281, wherein the PD-L2 polypeptide comprises a PD-L2 extracellular domain.

[1055] 285. The composition according to embodiment 284, wherein the PD-L2 polypeptide comprises an amino acid sequence having at least 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 104 or comprises the sequence of SEQ ID NO: 104.

[1056] 286. The composition according to any one of embodiments 275 to 278 and 280, wherein the B7.1 polypeptide comprises a B7.1 extracellular domain.

[1057] 287. The composition according to embodiment 286, wherein the B7.1 polypeptide comprises an amino acid sequence having at least 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 106 or comprises the sequence of SEQ ID NO: 106.

[1058] 288. The composition according to any one of embodiments 275 to 277, 279 and 281, wherein the B7.2 polypeptide comprises a B7.2 extracellular domain.

[1059] 289. The composition according to embodiment 288, wherein the B7.2 polypeptide comprises an amino acid sequence having at least 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 108 or comprises the sequence of SEQ ID NO: 108.

[1060] 290. The composition according to any one of embodiments 275 to 289, wherein the second polypeptide comprises a leader sequence.

[1061] 291. The composition according to embodiment 290, wherein the leader sequence is located at the N-terminus of the second polypeptide.

[1062] 292. The composition according to embodiment 290 or 291, wherein the leader sequence is a cell membrane targeting polypeptide.

[1063] 293. The composition according to any one of embodiments 290 to 292, wherein the leader sequence is the sequence of SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113 or a combination thereof.

[1064] 294. The composition according to any one of embodiments 275 to 293, wherein the second polypeptide further comprises an Fc domain.

[1065] 295. The composition according to embodiment 294, wherein the Fc domain is an Ig Fc domain selected from the group consisting essentially of human IgG, IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgA1, IgA2, IgE or IgD.

[1066] 294. The composition according to embodiments 294 to 295, wherein the Fc domain comprises an amino acid sequence having at least 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 114 or comprises the sequence of SEQ ID NO: 114.

[1067] 295. The composition according to any one of embodiments 275 to 294, wherein L A comprises the sequence of SEQ ID NO: 109 or a combination thereof.

[1068] 296. The composition according to any of the foregoing embodiments, wherein the second polypeptide comprises, from the N-terminus to the C-terminus, the formula L S -XA -L A -X B -L B -X C 、L S -X B -L A -X A -L B -X C 、L S -X A -L A -X C -L B -X B or L S -X B -L A -X C -L B -X A , wherein:

[1069] L S is a leading sequence;

[1070] X A is an immune checkpoint polypeptide or an active fragment thereof, such as but not limited to CTLA-4 polypeptide, PD-1 polypeptide, PD-L1 polypeptide, PD-L2 polypeptide, TIM3 polypeptide, LAG3 polypeptide, VISTA polypeptide, SINGLEC7 polypeptide, SINGLEC9 polypeptide, TIGIT polypeptide, CD96 polypeptide, BTLA polypeptide, B7H3 polypeptide, B7H4 polypeptide, CD155 polypeptide, HHLA2 polypeptide, BTN3A1 polypeptide or an active fragment thereof;

[1071] X B is a B7.1 polypeptide or a B7.2 polypeptide;

[1072] X C is a binder, such as an Fc domain, TCR, cytokine, cytokine trap, receptor ligand, complement protein;

[1073] L A is absent or is a polypeptide linker; and

[1074] L B is absent or is a polypeptide linker.

[1075] 297. The composition according to embodiment 296, wherein the Fc domain is an Ig Fc domain selected from the group consisting essentially of human IgG, IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgA1, IgA2, IgE or IgD.

[1076] 298. The composition according to embodiment 297, wherein the Fc domain comprises an amino acid sequence having at least 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 114 or comprises the sequence of SEQ ID NO: 114.

[1077] 299. The composition according to any one of embodiments 222 to 298, wherein the second polypeptide comprises an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 117 or comprises the sequence of SEQ ID NO: 117.

[1078] 300. The composition according to any one of embodiments 222 to 299, wherein the second polypeptide comprises an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 118 or comprises the sequence of SEQ ID NO: 118.

[1079] 301. The composition according to any one of embodiments 222 to 300, wherein the composition further comprises an antigen.

[1080] 302. The composition according to embodiment 301, wherein the antigen is a tumor antigen, a viral antigen, a bacterial antigen or a microbial antigen.

[1081] 303. The composition according to embodiments 301 to 302, wherein the antigen is ovalbumin (OVA).

[1082] 304. The composition according to any one of claims 222 to 303, the composition further comprises:

[1083] A first nucleic acid molecule encoding a first polypeptide according to any one of embodiments 275 to 274; and / or

[1084] A second nucleic acid molecule encoding a second polypeptide according to any one of embodiments 275 and 274 to 300.

[1085] 305. The composition according to embodiment 304, wherein the composition further comprises a third nucleic acid molecule encoding an antigen according to any one of embodiments 301 to 303.

[1086] 306. A vector, comprising the first nucleic acid molecule and the second nucleic acid molecule according to embodiment 304.

[1087] 307. The vector according to embodiment 306, wherein the vector further comprises a third nucleic acid molecule according to embodiment 305.

[1088] 308. A composition, comprising:

[1089] a first vector, comprising the first nucleic acid molecule according to embodiment 304; and

[1090] a second vector, comprising the second nucleic acid molecule according to embodiment 304.

[1091] 309. The composition according to embodiment 308, wherein the first vector further comprises a third nucleic acid molecule according to embodiment 305.

[1092] 310. The composition according to embodiment 308, wherein the second vector further comprises a third nucleic acid molecule according to embodiment 305.

[1093] 311. The composition according to embodiment 308, wherein the composition further comprises a third vector, comprising the third nucleic acid molecule according to embodiment 305.

[1094] 312. A plasmid, comprising the first nucleic acid molecule and the second nucleic acid molecule according to embodiment 304.

[1095] 313. The plasmid according to embodiment 312, wherein the plasmid further comprises a third nucleic acid molecule according to embodiment 305.

[1096] 314. A composition, comprising:

[1097] a first plasmid, comprising the first nucleic acid molecule according to embodiment 304; and

[1098] a second plasmid, comprising the second nucleic acid molecule according to embodiment 304.

[1099] 315. The composition according to embodiment 314, wherein the first plasmid further comprises a third nucleic acid molecule according to embodiment 305.

[1100] 316. The composition according to embodiment 314, wherein the second plasmid further comprises a third nucleic acid molecule according to embodiment 305.

[1101] 317. The composition according to embodiment 314, the composition further comprising a third plasmid, the third plasmid comprising a third nucleic acid molecule according to embodiment 305.

[1102] 318. A recombinant virus, the recombinant virus comprising a first nucleic acid molecule and a second nucleic acid molecule according to embodiment 304.

[1103] 319. The recombinant virus according to embodiment 318, the recombinant virus further comprising a third nucleic acid molecule according to embodiment 305.

[1104] 320. The recombinant virus according to embodiment 318 or 319, wherein the recombinant virus is an adenovirus or a lentivirus.

[1105] 321. The recombinant virus according to embodiment 320, wherein the recombinant virus is a replication - incompetent adenovirus.

[1106] 322. A liposome, the liposome comprising a first nucleic acid molecule and a second nucleic acid molecule according to embodiment 304.

[1107] 323. The liposome according to embodiment 322, the liposome further comprising a third nucleic acid molecule according to embodiment 305.

[1108] 324. The liposome according to embodiment 322 or 323, wherein the liposome comprises any lipid provided herein.

[1109] 325. A pharmaceutical composition, the pharmaceutical composition comprising:

[1110] A first nucleic acid molecule and a second nucleic acid molecule according to embodiment 304; and

[1111] A pharmaceutically acceptable carrier.

[1112] 326. The pharmaceutical composition according to embodiment 325, the pharmaceutical composition further comprising a third nucleic acid molecule according to embodiment 305.

[1113] 327. A cell, the cell comprising the composition according to any one of embodiments 275 to 303.

[1114] 328. The cell according to embodiment 327, wherein the cell further comprises a chimeric antigen receptor (CAR).

[1115] 329. A cell according to embodiment 327 or 328, wherein the cell is an immune cell.

[1116] 330. A cell according to embodiment 329, wherein the immune cell is a T cell, an NK cell or a dendritic cell.

[1117] 331. A method for producing a cell according to any one of embodiments 327 to 330, the method comprising contacting the cell with:

[1118] A vector according to embodiments 306 to 307;

[1119] A composition according to any one of embodiments 308 to 311;

[1120] A plasmid according to embodiments 312 to 313;

[1121] A composition according to any one of embodiments 314 to 317;

[1122] A recombinant virus according to any one of embodiments 318 to 321;

[1123] A liposome according to any one of embodiments 322 to 324; or

[1124] A pharmaceutical composition according to any one of embodiments 325 to 326.

[1125] 332. A method according to embodiment 331, wherein the contacting comprises transducing or transfecting the cell with the vector, the plasmid, the recombinant virus, the liposome or one of these compositions.

[1126] 333. A method for producing in vivo a cell according to any one of embodiments 327 to 330, the method comprising administering to a subject:

[1127] A vector according to embodiments 306 to 307;

[1128] A composition according to any one of embodiments 308 to 311;

[1129] A plasmid according to embodiments 312 to 313;

[1130] A composition according to any one of embodiments 314 to 317;

[1131] A recombinant virus according to any one of embodiments 318 to 321;

[1132] A liposome according to any one of embodiments 322 to 324; or

[1133] A pharmaceutical composition according to any one of embodiments 325 to 326;

[1134] wherein the vector, plasmid, recombinant virus, liposome or composition transduces or transfects cells in vivo to produce a cell according to any one of embodiments 327 to 330.

[1135] 334. A method of altering an immune response in a subject, the method comprising administering to the subject:

[1136] A vector according to embodiments 306 to 307;

[1137] A composition according to any one of embodiments 308 to 311;

[1138] A plasmid according to embodiments 312 to 313;

[1139] A composition according to any one of embodiments 314 to 317;

[1140] A recombinant virus according to any one of embodiments 318 to 321;

[1141] A liposome according to any one of embodiments 322 to 324; or

[1142] A pharmaceutical composition according to any one of embodiments 325 to 326.

[1143] 335. The method according to embodiment 334, wherein the immune response is activated.

[1144] 336. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject:

[1145] A vector according to embodiments 306 to 307;

[1146] A composition according to any one of embodiments 308 to 311;

[1147] A plasmid according to embodiments 312 to 313;

[1148] A composition according to any one of embodiments 314 to 317;

[1149] A recombinant virus according to any one of embodiments 318 to 321;

[1150] A liposome according to any one of embodiments 322 to 324; or

[1151] A pharmaceutical composition according to any one of embodiments 325 to 326.

[1152] 337. The method according to embodiment 336, wherein the cancer is a cancer provided herein.

[1153] 338. The method according to embodiment 336 or 337, wherein the method further comprises using at least one additional therapeutic agent, such as an anti-cancer therapeutic agent, including but not limited to chemotherapy, radiotherapy, targeted therapy, immunotherapy, stem cell or bone marrow transplantation therapy, hormone therapy, photodynamic therapy, or any combination thereof.

[1154] 339. A method of treating a disease or disorder in a subject in need thereof, the disease or disorder such as a viral infection, a bacterial infection, or a fungal infection, such as those provided herein, the method comprising administering to the subject:

[1155] The vector according to embodiments 306 to 307;

[1156] The composition according to any one of embodiments 308 to 311;

[1157] The plasmid according to embodiments 312 to 313;

[1158] The composition according to any one of embodiments 314 to 317;

[1159] The recombinant virus according to any one of embodiments 318 to 321;

[1160] The liposome according to any one of embodiments 322 to 324; or the pharmaceutical composition according to any one of embodiments 325 to 326.

[1161] Example

[1162] The following examples are illustrative and not limiting of the compounds, compositions, and methods described herein. Other suitable modifications and variations known to those skilled in the art are within the scope of the following embodiments.

[1163] Example 1: Measuring active IL-21, IL-12, and IL-15 using polypeptide constructs

[1164] The following polypeptide constructs listed in Table 1 were expressed in HEK cells to determine the relative activities of IL-21, IL-12, and IL-15. The effect of adding a dual checkpoint inhibitor with the IL-12 / IL-15 / IL-21 polypeptide was also measured. Any L listed in the sequence diagrams in Table 1 can be any linker described herein, unless otherwise specified.

[1165] Table 1: IL-12 / IL-15 / IL-21 and dual checkpoint expression constructs

[1166]

[1167] The activities of IL-21, IL-15 and IL-12 were determined by cytokine response reporter cell assays specifically designed to demonstrate the activities of IL-21, IL-15 and IL-12. Figure 1 IL-21 signals of m21-O1 and m21-O3 polypeptides in the presence and absence of the SynTauR dual checkpoint polypeptide are depicted. Figure 2 IL-15, IL-12 and CTLA-4 signals of m21-O1 and m21-O3 polypeptides in the presence and absence of the SynTauR dual checkpoint polypeptide are depicted. In Figure 2 the O1 and O3 controls are identical to the m21-O1 and m21-O3 polypeptides, but the IL-21 sequence and the first linker are removed. The results show that both the m21-O1 and m21-O2 polypeptides are capable of activating the IL-21, IL-15 and IL-12 pathways.

[1168] Example 2: IL-12 / IL-15 / IL-21 and dual checkpoint polypeptides show in vivo synergy

[1169] To determine the in vivo T cell response, a composition comprising a nucleic acid encoding the m21-O1, m21-O3 and / or SynTauR dual checkpoint polypeptide described above was administered to C57BL / 6 mice. The engineered gene encoding the fusion protein was subcloned into a vector carrying a CMV promoter / enhancer element and a polyA termination signal, and a cassette for expressing the model antigen ovalbumin (OVA) (as indicated). Animals were acclimated for at least 3 days before the start of the study and housed in microisolators on a 12:12 light / dark cycle. Animals were fed a standard rodent chow diet and provided with unlimited water and food.

[1170] The results show that various immune stimulatory elements act synergistically to maximize the splenic antigen-reactive T cell response. In Figure 3 the animals were given an intramuscular injection of a vector control, an antigen-only (OVA) control, O1 (IL-15 and IL-12 only)+antigen, a SynTaur dual checkpoint inhibitor+antigen, O1+SynTaur+antigen and finally m21-O1+SynTaur+antigen. The initial injection was performed on day 0, the intramuscular injection boost was performed on day 21, and then the spleens were harvested on day 28. The injection was 50 uL per mouse, n = 2. In Figure 4Among them, animals were given an intravenous injection of a vector control, an antigen-only (OVA) control, O1 + antigen, a SynTaur dual checkpoint inhibitor + antigen, O1 + SynTaur + antigen, and finally m21 - O1 + SynTaur + antigen, as well as an Ad5 virus control for comparison. The initial injection was repeated on day 0, an intravenous boost injection was given on day 21, and spleens were harvested on day 28. The antigen-reactive T cells ( Figure 3 ) and the increased level of CD8+% ( Figure 4 ) indicate that the presence of IL-21, IL-15, IL-12, OVA, and the immune checkpoint inhibitor surprisingly act together much more strongly than what their individual actions would suggest.

[1171] Immunological assay method

[1172] Isolation of single-cell suspensions: On day 29, the organs of interest were harvested from immunized mice. Single-cell suspensions were isolated from the spleen and liver by mechanical disruption and differential centrifugation.

[1173] Flow cytometry assay: Immunocyte populations were identified using flow cytometry and antibodies against CD3, CD4, CD8, and NK1.1. Memory and effector T cell populations were identified using antibodies against CD44 and CD62L.

[1174] ELISpot: Functional responses from antigen-specific T cells were evaluated by ELISpot. Briefly, single-cell suspensions were co-cultured with ovalbumin peptides overnight. Cytokine (IFN-γ and IL-4) secreting cells were counted using cytokine-specific antibodies.

[1175] Example 3: Treatment of cancer using a vector encoding a polypeptide .

[1176] A therapeutic composition containing a vector is administered to a patient with cancer, the vector containing a nucleic acid molecule encoding the polypeptide provided herein. The immune system of the subject is activated, and the cancer is treated. The vector may also contain a nucleic acid molecule encoding a tumor antigen. The vector may also contain a nucleic acid molecule encoding a CAR.

[1177] Example 4: Treatment of cancer using a vector encoding a polypeptide and liposomes

[1178] A therapeutic composition containing a vector is administered to a patient with cancer as a formulation with a DOTAP / cholesterol admixture, the vector containing a nucleic acid molecule encoding the polypeptide provided herein. The immune system of the subject is activated, and the cancer is treated. The vector may also contain a nucleic acid molecule encoding a tumor antigen. The admixture may also contain the pharmaceutical formulation provided herein.

[1179] Example 5: Treatment of cancer using adoptive cell therapy such as modified white blood cells .

[1180] T cells comprising the polypeptides and chimeric antigen receptors provided herein are administered to a subject having a cancer such as leukemia, and the cancer is treated.

[1181] Example 6: Treatment of cancer using a biofusion 。

[1182] A therapeutic composition comprising the polypeptides provided herein is administered to a patient having a cancer. In some embodiments, the polypeptide is linked or fused to a bioactive moiety such as a protein, polypeptide, nucleic acid, lipid, carbohydrate, or any combination thereof. In some embodiments, the polypeptide is linked or fused to a targeting moiety such as an antibody, antigen, ligand, or ligand trap such as a receptor domain. In some embodiments, the polymer is linked or fused to an immunotherapy such as a checkpoint inhibitor. The immune system of the subject is activated and the cancer is treated.

[1183] Generally speaking, the embodiments and examples provided herein demonstrate that the polypeptides provided herein can be used to enhance the immune response to treat the tumors or infections provided herein.

[1184] This specification contains numerous references to patents, patent applications, accession numbers, and / or publications. Each is hereby incorporated by reference for all purposes.

Claims

1. A polypeptide, the polypeptide comprising the following formula: X1-L1-X2-L2-X3-L3-X4-L4-X5, wherein: X1 is an interleukin-21 (IL-21) polypeptide, an interleukin-15 (IL-15) polypeptide, an interleukin-12p40 subunit (IL-12p40) polypeptide, an interleukin-12p35 subunit (IL-12p35) polypeptide, or an interleukin-15 receptor (IL-15Rα) polypeptide; X2 is an IL-21 polypeptide, an IL-15 polypeptide, an IL-12p40 polypeptide, an IL-12p35 polypeptide, or an IL-15Rα polypeptide; X3 is an IL-21 polypeptide, an IL-15 polypeptide, an IL-12p40 polypeptide, an IL-12p35 polypeptide, or an IL-15Rα polypeptide; X4 is an IL-21 polypeptide, an IL-15 polypeptide, an IL-12p40 polypeptide, an IL-12p35 polypeptide, or an IL-15Rα polypeptide; X5 is an IL-21 polypeptide, an IL-15 polypeptide, an IL-12p40 polypeptide, an IL-12p35 polypeptide, or an IL-15Rα polypeptide; L1, L2, L3, and L4 are each independently a polypeptide linker comprising the same or different polypeptide sequences, and provided that each of X1, X2, X3, X4, and X5 is different.

2. The polypeptide according to claim 1, wherein X1 is an interleukin-21 (IL-21) polypeptide.

3. The polypeptide according to claim 1 or 2, wherein: X1 is an IL-21 polypeptide; X2 is an IL-15 polypeptide; X3 is an IL-12p40 polypeptide; X4 is an IL-12p35 polypeptide; and X5 is an IL-15Rα polypeptide.

4. The polypeptide according to claim 1 or claim 2, wherein: X1 is an IL-21 polypeptide; X2 is an IL-15 polypeptide; X3 is an IL-12p35 polypeptide; X4 is an IL-12p40 polypeptide; and X5 is an IL-15Rα polypeptide.

5. The polypeptide according to claim 1 or 2, wherein: X1 is an IL-21 polypeptide; X2 is an IL-12p40 polypeptide; X3 is an IL-15 polypeptide; X4 is an IL-12p35 polypeptide; and X5 is an IL-15Rα polypeptide.

6. The polypeptide according to claim 1 or 2, wherein: X1 is an IL-21 polypeptide; X2 is an IL-12p40 polypeptide; X3 is an IL-12p35 polypeptide; X4 is an IL-15 polypeptide; and X5 is an IL-15Rα polypeptide.

7. The polypeptide according to claim 1 or 2, wherein: X1 is an IL-21 polypeptide; X2 is an IL-12p35 polypeptide; X3 is an IL-12p40 polypeptide; X4 is an IL-15 polypeptide; and X5 is an IL-15Rα polypeptide.

8. The polypeptide according to claim 1 or 2, wherein: X1 is an IL-21 polypeptide; X2 is an IL-12p35 polypeptide; X3 is an IL-15 polypeptide; X4 is an IL-12p40 polypeptide; and X5 is an IL-15Rα polypeptide.

9. The polypeptide according to claim 1 or 2, wherein: X1 is an IL-21 polypeptide; X2 is an IL-12p40 polypeptide; X3 is an IL-12p35 polypeptide; X4 is an IL-15 polypeptide; and X5 is an IL-15Rα polypeptide.

10. The polypeptide according to claim 1 or 2, wherein: X1 is an IL-21 polypeptide; X2 is an IL-12p40 polypeptide; X3 is an IL-15 polypeptide; X4 is an IL-12p35 polypeptide; and X5 is an IL-15Rα polypeptide.

11. The polypeptide according to claim 1 or 2, wherein: X1 is an IL-21 polypeptide; X2 is an IL-12p35 polypeptide; X3 is an IL-12p40 polypeptide; X4 is an IL-15 polypeptide; and X5 is an IL-15Rα polypeptide.

12. The polypeptide according to claim 1 or 2, wherein: X1 is an IL-21 polypeptide; X2 is an IL-12p35 polypeptide; X3 is an IL-15 polypeptide; X4 is an IL-12p40 polypeptide; and X5 is an IL-15Rα polypeptide.

13. The polypeptide according to any one of the preceding claims, wherein the IL-21 polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:71, or as otherwise provided herein.

14. The polypeptide according to any one of the preceding claims, wherein the IL-21 polypeptide comprises the amino acid sequence of SEQ ID NO:71, or as otherwise provided herein.

15. The polypeptide according to any one of the preceding claims, wherein the IL-12p40 polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:1, 2, 3 or 4, or as otherwise provided herein.

16. The polypeptide according to any one of the preceding claims, wherein the IL-12p40 polypeptide comprises the amino acid sequence of SEQ ID NO:1, 2, 3 or 4, or as otherwise provided herein.

17. The polypeptide according to any one of the preceding claims, wherein the IL-12p35 polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:5, 6, 7 or 8, or as otherwise provided herein.

18. The polypeptide according to any one of the preceding claims, wherein the IL-12p35 polypeptide comprises the amino acid sequence of SEQ ID NO:5, 6, 7 or 8, or as otherwise provided herein.

19. The polypeptide according to any one of the preceding claims, wherein the IL-15 polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:9 or 10, or as otherwise provided herein.

20. The polypeptide according to any one of the preceding claims, wherein the IL-15 polypeptide comprises the amino acid sequence of SEQ ID NO: 9 or 10, or as otherwise provided herein.

21. The polypeptide according to any one of the preceding claims, wherein the IL-15Rα polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 11 or 12, or as otherwise provided herein.

22. The polypeptide according to any one of the preceding claims, wherein the IL-15Rα polypeptide comprises the amino acid sequence of SEQ ID NO: 11 or 12, or as otherwise provided herein.

23. The polypeptide according to any one of the preceding claims, wherein one or more of L1, L2, L3 and L4 are each independently a cleavable linker.

24. The polypeptide according to claim 23, wherein L1, L3 and L4 are non-cleavable linkers.

25. The polypeptide according to claim 23 or 24, wherein the cleavable linker is a furin-cleavable linker, a Val-Cit linker, a Val-Gly linker, a Gly-Gly linker, an Ala-Ala-Asn linker or a linker comprising polyethylene glycol (PEG).

26. The polypeptide according to any one of the preceding claims, wherein one of L1, L2, L3 and L4 comprises the sequence of (GSGSGG)n (SEQ ID NO: 16), (GGGGS)n (SEQ ID NO: 17), (GGGGA)n (SEQ ID NO: 18), (GGGSE)n (SEQ ID NO: 19), (GGGSK)n (SEQ ID NO: 20) or (AEEEK)n (SEQ ID NO: 21) or a combination thereof, wherein each n is independently 1, 2, 3, 4 or 5.

27. The polypeptide according to claim 26, wherein n is 3.

28. The polypeptide according to claim 26 or 27, wherein one or more of L1, L2, L3 and L4 comprise SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:70 of the sequence RXXR or combinations thereof, where X is any amino acid.

29. The polypeptide according to any one of claims 25 to 28, wherein each of L1, L2, L3 and L4 comprises the sequence RXXR of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:70 or combinations thereof, where X is any amino acid.

30. The polypeptide according to any one of the preceding claims, wherein the polypeptide comprises the sequence of SEQ ID NO:72 or a variant thereof, or as otherwise provided herein.

31. The polypeptide according to any one of the preceding claims, wherein the polypeptide comprises the sequence of SEQ ID NO:73 or a variant thereof, or as otherwise provided herein.

32. The polypeptide according to any one of the preceding claims, wherein the polypeptide comprises the sequence of SEQ ID NO:74 or a variant thereof, or as otherwise provided herein.

33. The polypeptide according to any one of the preceding claims, wherein the polypeptide comprises the sequence of SEQ ID NO:75 or a variant thereof, or as otherwise provided herein.

34. The polypeptide according to any one of the preceding claims, wherein the polypeptide comprises the sequence of SEQ ID NO:76 or a variant thereof, or as otherwise provided herein.

35. The polypeptide according to any one of the preceding claims, wherein the polypeptide comprises the sequence of SEQ ID NO:77 or a variant thereof, or as otherwise provided herein.

36. The polypeptide according to any one of the preceding claims, wherein the polypeptide comprises the sequence of SEQ ID NO:78 or a variant thereof, or as otherwise provided herein.

37. The polypeptide according to any one of the preceding claims, wherein the polypeptide comprises the sequence of SEQ ID NO:79 or a variant thereof, or as otherwise provided herein.

38. The polypeptide according to any one of the preceding claims, wherein the first polypeptide is linked or fused to at least one additional molecule selected from the group consisting of: a polypeptide, an antigen, a ligand, an antibody, a checkpoint inhibitor, a lipid, a carbohydrate, a nucleic acid, or any combination thereof.

39. The polypeptide according to claim 1, wherein: X1 is an IL-21 polypeptide; X2 is an IL-15 polypeptide; X3 is an IL-12p40 polypeptide; X4 is an IL-12p35 polypeptide; X5 is an IL-15Rα polypeptide; and L2 is a furin-cleavable linker; and L1, L3, and L4 are non-cleavable linkers.

40. The polypeptide according to claim 39, wherein the first polypeptide is linked or fused to at least one additional molecule selected from the group consisting of: a polypeptide, an antigen, a ligand, an antibody, a checkpoint inhibitor, a lipid, a carbohydrate, a nucleic acid, or any combination thereof.

41. A nucleic acid encoding the polypeptide according to any one of claims 1 to 40.

42. A vector comprising the nucleic acid according to claim 41.

43. A plasmid comprising the nucleic acid according to claim 41.

44. A virus, such as a recombinant virus, comprising the nucleic acid according to claim 41.

45. The virus according to claim 44, wherein the recombinant virus is an adenovirus or a lentivirus.

46. The virus according to claim 44 or 45, wherein the virus is a replication-incompetent adenovirus.

47. A liposome comprising the nucleic acid according to claim 41.

48. The liposome according to claim 47, wherein the liposome comprises any lipid provided herein.

49. A pharmaceutical composition comprising the nucleic acid according to claim 41 and a pharmaceutically acceptable carrier.

50. A cell comprising the composition according to any one of claims 1 to 40, wherein the cell is an immune cell.

51. A composition (e.g., liposome, virus, encapsulation, etc.) comprising: A first polypeptide having the formula: X1-L1-X2-L2-X3-L3-X4-L4-X5, wherein: X1 is an interleukin 21 (IL-21) polypeptide, an interleukin 15 (IL-15) polypeptide, an interleukin 12p40 subunit (IL-12p40) polypeptide, an interleukin 12p35 subunit (IL-12p35) polypeptide, or an interleukin 15 receptor (IL-15Rα) polypeptide; X2 is an IL-21 polypeptide, an IL-15 polypeptide, an IL-12p40 polypeptide, an IL-12p35 polypeptide, or an IL-15Rα polypeptide; X3 is an IL-21 polypeptide, an IL-15 polypeptide, an IL-12p40 polypeptide, an IL-12p35 polypeptide, or an IL-15Rα polypeptide; X4 is an IL-21 polypeptide, an IL-15 polypeptide, an IL-12p40 polypeptide, an IL-12p35 polypeptide, or an IL-15Rα polypeptide; X5 is an IL-21 polypeptide, an IL-15 polypeptide, an IL-12p40 polypeptide, an IL-12p35 polypeptide, or an IL-15Rα polypeptide; L1, L2, L3, and L4 are each independently a polypeptide linker comprising the same or different polypeptide sequences, provided that each of X1, X2, X3, X4, and X5 is different; and / or a second polypeptide, the second polypeptide comprising at least one immune checkpoint polypeptide or an active fragment thereof, such as but not limited to a CTLA-4 polypeptide, a PD-1 polypeptide, a PD-L1 polypeptide, a PD-L2 polypeptide, a TIM3 polypeptide, a LAG3 polypeptide, a VISTA polypeptide, a SINGLEC7 polypeptide, a SINGLEC9 polypeptide, a TIGIT polypeptide, a CD96 polypeptide, a BTLA polypeptide, a B7H3 polypeptide, a B7H4 polypeptide, a CD155 polypeptide, an HHLA2 polypeptide, a BTN3A1 polypeptide, a B7.1 polypeptide, a B7.2 polypeptide, or an active fragment thereof; or one or more nucleic acid molecules encoding the first polypeptide and the second polypeptide.

52. The composition according to claim 51, wherein X1 is an interleukin 21 (IL-21) polypeptide.

53. The composition according to claim 51 or 52, wherein: X1 is an IL-21 polypeptide; X2 is an IL-15 polypeptide; X3 is an IL-12p40 polypeptide; X4 is an IL-12p35 polypeptide; and X5 is an IL-15Rα polypeptide.

54. The composition according to claim 51 or 52, wherein: X1 is an IL-21 polypeptide; X2 is an IL-15 polypeptide; X3 is an IL-12p35 polypeptide; X4 is an IL-12p40 polypeptide; and X5 is an IL-15Rα polypeptide.

55. The composition according to claim 51 or 52, wherein: X1 is an IL-21 polypeptide; X2 is an IL-12p40 polypeptide; X3 is an IL-15 polypeptide; X4 is an IL-12p35 polypeptide; and X5 is an IL-15Rα polypeptide.

56. The composition according to claim 51 or 52, wherein: X1 is an IL-21 polypeptide; X2 is an IL-12p40 polypeptide; X3 is an IL-12p35 polypeptide; X4 is an IL-15 polypeptide; and X5 is an IL-15Rα polypeptide.

57. The composition according to claim 51 or 52, wherein: X1 is an IL-21 polypeptide; X2 is an IL-12p35 polypeptide; X3 is an IL-12p40 polypeptide; X4 is an IL-15 polypeptide; and X5 is an IL-15Rα polypeptide.

58. The composition according to claim 51 or 52, wherein: X1 is an IL-21 polypeptide; X2 is an IL-12p35 polypeptide; X3 is an IL-15 polypeptide; X4 is an IL-12p40 polypeptide; and X5 is an IL-15Rα polypeptide.

59. The composition according to claim 51 or 52, wherein: X1 is an IL-21 polypeptide; X2 is an IL-12p40 polypeptide; X3 is an IL-12p35 polypeptide; X4 is an IL-15 polypeptide; and X5 is an IL-15Rα polypeptide.

60. The composition according to claim 51 or 52, wherein: X1 is an IL-21 polypeptide; X2 is an IL-12p40 polypeptide; X3 is an IL-15 polypeptide; X4 is an IL-12p35 polypeptide; and X5 is an IL-15Rα polypeptide.

61. The composition according to claim 51 or 52, wherein: X1 is an IL-21 polypeptide; X2 is an IL-12p35 polypeptide; X3 is an IL-12p40 polypeptide; X4 is an IL-15 polypeptide; and X5 is an IL-15Rα polypeptide.

62. The composition according to claim 51 or 52, wherein: X1 is an IL-21 polypeptide; X2 is an IL-12p35 polypeptide; X3 is an IL-15 polypeptide; X4 is an IL-12p40 polypeptide; and X5 is an IL-15Rα polypeptide.

63. The composition according to any one of claims 51 to 62, wherein the IL-21 polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 71, or as otherwise provided herein.

64. The composition according to any one of claims 51 to 63, wherein the IL-21 polypeptide comprises the amino acid sequence of SEQ ID NO: 71, or as otherwise provided herein.

65. The composition according to any one of claims 51 to 64, wherein the IL-12p40 polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1, 2, 3 or 4, or as otherwise provided herein.

66. The composition according to any one of claims 51 to 65, wherein the IL-12p40 polypeptide comprises the amino acid sequence of SEQ ID NO: 1, 2, 3 or 4, or as otherwise provided herein.

67. The composition according to any one of claims 51 to 66, wherein the IL-12p35 polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 5, 6, 7 or 8, or as otherwise provided herein.

68. The composition according to any one of claims 51 to 67, wherein the IL-12p35 polypeptide comprises the amino acid sequence of SEQ ID NO: 5, 6, 7 or 8, or as otherwise provided herein.

69. The composition according to any one of claims 51 to 68, wherein the IL-15 polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 9 or 10, or as otherwise provided herein.

70. The composition according to any one of claims 51 to 69, wherein the IL-15 polypeptide comprises the amino acid sequence of SEQ ID NO: 9 or 10, or as otherwise provided herein.

71. The composition according to any one of claims 51 to 70, wherein the IL-15Rα polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 11 or 12, or as otherwise provided herein.

72. The composition according to any one of claims 51 to 71, wherein the IL-15Rα polypeptide comprises the amino acid sequence of SEQ ID NO: 11 or 12, or as otherwise provided herein.

73. The composition according to any one of claims 51 to 72, wherein one or more of L1, L2, L3 and L4 are each independently a cleavable linker.

74. The polypeptide according to claim 72, wherein L1, L3 and L4 are non-cleavable linkers.

75. The composition according to claim 73 or 74, wherein the cleavable linker is a furin-cleavable linker, a Val-Cit linker, a Val-Gly linker, a Gly-Gly linker, an Ala-Ala-Asn linker or a linker comprising polyethylene glycol (PEG).

76. The composition according to any one of claims 51 to 75, wherein one of L1, L2, L3 and L4 comprises the sequence of (GSGSGG)n (SEQ ID NO: 16), (GGGGS)n (SEQ ID NO: 17), (GGGGA)n (SEQ ID NO: 18), (GGGSE)n (SEQ ID NO: 19), (GGGSK)n (SEQ ID NO: 20) or (AEEEK)n (SEQ ID NO: 21) or a combination thereof, wherein each n is independently 1, 2, 3, 4 or 5.

77. The composition according to claim 76, wherein n is 3.

78. The composition according to claim 76 or 77, wherein one or more of L1, L2, L3, and L4 comprise the sequence RXXR of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:70, or a combination thereof, wherein X is any amino acid.

79. The composition according to any one of claims 76 to 78, wherein each of L1, L2, L3, and L4 comprises the sequence RXXR of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:70, or a combination thereof, wherein X is any amino acid.

80. The composition according to any one of claims 51 to 79, wherein the first polypeptide comprises the sequence of SEQ ID NO:72 or a variant thereof, or as otherwise provided herein.

81. The composition according to any one of claims 51 to 80, wherein the first polypeptide comprises the sequence of SEQ ID NO:73 or a variant thereof, or as otherwise provided herein.

82. The composition according to any one of claims 51 to 81, wherein the first polypeptide comprises the sequence of SEQ ID NO: 74 or a variant thereof, or as otherwise provided herein.

83. The composition according to any one of claims 51 to 82, wherein the first polypeptide comprises the sequence of SEQ ID NO: 75 or a variant thereof, or as otherwise provided herein.

84. The composition according to any one of claims 51 to 83, wherein the first polypeptide comprises the sequence of SEQ ID NO: 76 or a variant thereof, or as otherwise provided herein.

85. The composition according to any one of claims 51 to 84, wherein the first polypeptide comprises the sequence of SEQ ID NO: 77 or a variant thereof, or as otherwise provided herein.

86. The composition according to any one of claims 51 to 85, wherein the first polypeptide comprises the sequence of SEQ ID NO: 78 or a variant thereof, or as otherwise provided herein.

87. The composition according to any one of claims 51 to 86, wherein the first polypeptide comprises the sequence of SEQ ID NO: 79 or a variant thereof, or as otherwise provided herein.

88. The composition according to any one of claims 51 to 87, wherein the first polypeptide is linked or fused to at least one additional molecule selected from the group consisting of: polypeptide, antigen, ligand, antibody, checkpoint inhibitor, lipid, carbohydrate, nucleic acid, or any combination thereof.

89. The composition according to claim 51, wherein: X1 is an IL-21 polypeptide; X2 is an IL-15 polypeptide; X3 is an IL-12p40 polypeptide; X4 is an IL-12p35 polypeptide; X5 is an IL-15Rα polypeptide; L2 is a furin-cleavable linker; and L1, L3, and L4 are non-cleavable linkers.

90. The composition according to claim 89, wherein the first polypeptide is linked or fused to at least one additional molecule selected from the group consisting of: polypeptide, antigen, ligand, antibody, checkpoint inhibitor, lipid, carbohydrate, nucleic acid, or any combination thereof.

91. The composition according to any one of the preceding claims, wherein the second polypeptide comprises, from the N-terminus to the C-terminus, the formula X A -L A -X B or the formula X B -L A -X A , wherein: X A is an immune checkpoint polypeptide or an active fragment thereof, such as but not limited to CTLA-4 polypeptide, PD-1 polypeptide, PD-L1 polypeptide, PD-L2 polypeptide, TIM3 polypeptide, LAG3 polypeptide, VISTA polypeptide, SINGLEC7 polypeptide, SINGLEC9 polypeptide, TIGIT polypeptide, CD96 polypeptide, BTLA polypeptide, B7H3 polypeptide, B7H4 polypeptide, CD155 polypeptide, HHLA2 polypeptide, BTN3A1 polypeptide or an active fragment thereof; X B is a B7.1 polypeptide or a B7.2 polypeptide; and L A is a polypeptide linker.

92. The composition according to claim 91, wherein the second polypeptide comprises, from the N-terminus to the C-terminus, the formula X A -L A -X B .

93. The composition according to claim 91, wherein the polypeptide comprises, from the N-terminus to the C-terminus, the formula X B -L A -X A 。 94. The composition according to any one of claims 91 to 93, wherein: X A is a PD-L1 polypeptide; and X B is a B7.1 polypeptide.

95. The composition according to any one of claims 91 to 93, wherein: X A is a PD-L1 polypeptide; and X B is a B7.2 polypeptide.

96. The composition according to any one of claims 91 to 93, wherein: X A is a PD-L2 polypeptide; and X B is a B7.1 polypeptide.

97. The composition according to any one of claims 91 to 93, wherein: X A is a PD-L2 polypeptide; and X B is a B7.2 polypeptide.

98. The composition according to any one of claims 91 to 95, wherein the PD-L1 polypeptide comprises the PD-L1 extracellular domain.

99. The composition according to claim 98, wherein the PD-L1 polypeptide comprises an amino acid sequence having at least 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 102 or comprises the sequence of SEQ ID NO:

102.

100. The composition according to any one of claims 91 to 93 and 96 to 97, wherein the PD-L2 polypeptide comprises the PD-L2 extracellular domain.

101. The composition according to claim 100, wherein the PD-L2 polypeptide comprises an amino acid sequence having at least 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 104 or comprises the sequence of SEQ ID NO:

104.

102. The composition according to any one of claims 91 to 94 and 96, wherein the B7.1 polypeptide comprises the B7.1 extracellular domain.

103. The composition according to claim 102, wherein the B7.1 polypeptide comprises an amino acid sequence having at least 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 106 or comprises the sequence of SEQ ID NO:

106.

104. The composition according to any one of claims 91 to 93, 95 and 97, wherein the B7.2 polypeptide comprises the B7.2 extracellular domain.

105. The composition according to claim 104, wherein the B7.2 polypeptide comprises an amino acid sequence having at least 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 108 or comprises the sequence of SEQ ID NO:

108.

106. The composition according to any one of claims 91 to 105, wherein the second polypeptide comprises a leader sequence.

107. The composition according to claim 106, wherein the leader sequence is located at the N-terminus of the second polypeptide.

108. The composition according to claim 106 or 107, wherein the leader sequence is a cell membrane targeting polypeptide.

109. The composition according to any one of claims 106 to 108, wherein the leader sequence is the sequence of SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113 or a combination thereof.

110. The composition according to any one of claims 91 to 109, wherein the second polypeptide further comprises an Fc domain.

111. The composition according to claim 110, wherein the Fc domain is an Ig Fc domain selected from the group consisting essentially of human IgG, IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgA1, IgA2, IgE or IgD.

112. The composition according to claim 110 or 111, wherein the Fc domain comprises an amino acid sequence having at least 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 114 or comprises the sequence of SEQ ID NO:

114.

113. The composition according to any one of claims 91 to 112, wherein L A comprises the sequence of SEQ ID NO: 109 or a combination thereof.

114. The composition according to any one of the preceding claims, wherein the second polypeptide comprises, from the N-terminus to the C-terminus, the formula L S -X A -L A -X B -L B -X C 、L S -X B -L A -X A -L B -X C 、L S -X A -L A -X C - L B -X B or L S -X B -L A -X C -L B -X A , where: L S is a leading sequence; X A is an immune checkpoint polypeptide or an active fragment thereof, such as but not limited to CTLA-4 polypeptide, PD-1 polypeptide, PD-L1 polypeptide, PD-L2 polypeptide, TIM3 polypeptide, LAG3 polypeptide, VISTA polypeptide, SINGLEC7 polypeptide, SINGLEC9 polypeptide, TIGIT polypeptide, CD96 polypeptide, BTLA polypeptide, B7H3 polypeptide, B7H4 polypeptide, CD155 polypeptide, HHLA2 polypeptide, BTN3A1 polypeptide or an active fragment thereof; X B is a B7.1 polypeptide or a B7.2 polypeptide; X C is a binder, such as an Fc domain, TCR, cytokine, cytokine trap, receptor ligand, complement protein; L A is absent or is a polypeptide linker; and L B Absent or polypeptide linker.

115. The composition according to claim 114, wherein the Fc domain is an Ig Fc domain selected from the group consisting essentially of human IgG, IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgA1, IgA2, IgE or IgD.

116. The composition according to claim 115, wherein the Fc domain comprises an amino acid sequence having at least 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 114 or comprises the sequence of SEQ ID NO:

114.

117. The composition according to any one of claims 51 to 116, wherein the second polypeptide comprises an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 117 or comprises the sequence of SEQ ID NO:

117.

118. The composition according to any one of claims 51 to 117, wherein the second polypeptide comprises an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 118 or comprises the sequence of SEQ ID NO:

118.

119. The composition according to any one of claims 51 to 118, wherein the composition further comprises an antigen.

120. The composition according to claim 119, wherein the antigen is a tumor antigen, a viral antigen, a bacterial antigen or a microbial antigen.

121. The composition according to claim 119 or 120, wherein the antigen is ovalbumin (OVA).

122. A composition comprising: a first nucleic acid molecule, the first nucleic acid molecule encoding a first polypeptide according to any one of claims 51 to 90; and / or a second nucleic acid molecule, the second nucleic acid molecule encoding a second polypeptide according to any one of claims 51 and 91 to 121.

123. The composition according to claim 122, further comprising a third nucleic acid molecule encoding an antigen according to any one of claims 119 to 121.

124. A vector comprising the first nucleic acid molecule and / or the second nucleic acid molecule according to claim 122.

125. The vector according to claim 124, further comprising the third nucleic acid molecule according to claim 123.

126. A composition comprising: a first vector comprising the first nucleic acid molecule according to claim 122; and a second vector comprising the second nucleic acid molecule according to claim 122.

127. The composition according to claim 126, wherein the first vector further comprises the third nucleic acid molecule according to claim 123.

128. The composition according to claim 126, wherein the second vector further comprises the third nucleic acid molecule according to claim 123.

129. The composition according to claim 126, further comprising a third vector comprising the third nucleic acid molecule according to claim 123.

130. A plasmid comprising the first nucleic acid molecule and the second nucleic acid molecule according to claim 122.

131. The plasmid according to claim 130, further comprising the third nucleic acid molecule according to claim 123.

132. A composition comprising: a first plasmid comprising the first nucleic acid molecule according to claim 122; and a second plasmid comprising the second nucleic acid molecule according to claim 122.

133. The composition according to claim 132, wherein the first plasmid further comprises the third nucleic acid molecule according to claim 123.

134. The composition according to claim 132, wherein the second plasmid further comprises the third nucleic acid molecule according to claim 123.

135. The composition according to claim 132, further comprising a third plasmid comprising the third nucleic acid molecule according to claim 123.

136. A recombinant virus comprising the first nucleic acid molecule and the second nucleic acid molecule according to claim 123.

137. The recombinant virus according to claim 136, further comprising the third nucleic acid molecule according to claim 123.

138. The recombinant virus according to claim 136 or 137, wherein the recombinant virus is an adenovirus or a lentivirus.

139. The recombinant virus according to claim 138, wherein the recombinant virus is a replication-incompetent adenovirus.

140. A liposome, the liposome comprising a first nucleic acid molecule and a second nucleic acid molecule according to claim 122.

141. The liposome according to claim 140, the liposome further comprising a third nucleic acid molecule according to claim 123.

142. The liposome according to claim 140 or 141, wherein the liposome comprises any lipid provided herein.

143. A pharmaceutical composition, the pharmaceutical composition comprising: a first nucleic acid molecule and a second nucleic acid molecule according to claim 122; and a pharmaceutically acceptable carrier.

144. The pharmaceutical composition according to claim 143, the pharmaceutical composition further comprising a third nucleic acid molecule according to claim 123.

145. A cell, the cell comprising a composition according to any one of claims 51 to 123.

146. The cell according to claim 145, wherein the cell further comprises a chimeric antigen receptor (CAR).

147. The cell according to claim 145 or 146, wherein the cell is an immune cell.

148. The cell according to claim 147, wherein the immune cell is a T cell, an NK cell or a dendritic cell.

149. A method of producing a cell according to any one of claims 145 to 148, the method comprising contacting the cell with: a carrier according to claims 124 to 125; a composition according to any one of claims 126 to 129; a plasmid according to claims 130 to 131; a composition according to any one of claims 132 to 134; a recombinant virus according to any one of claims 135 to 139; a liposome according to any one of claims 140 to 142; or a pharmaceutical composition according to any one of claims 143 to 144.

150. The method according to claim 149, wherein the contacting comprises transducing or transfecting the cell with one of the carrier, the plasmid, the recombinant virus, the liposome or the composition.

151. A method of producing a cell according to any one of claims 145 to 148 in vivo, the method comprising administering to a subject: a carrier according to claims 124 to 125; a composition according to any one of claims 126 to 129; a plasmid according to claims 130 to 131; a composition according to any one of claims 132 to 134; a recombinant virus according to any one of claims 135 to 139; a liposome according to any one of claims 140 to 142; or a pharmaceutical composition according to any one of claims 143 to 144; wherein the carrier, the plasmid, the recombinant virus, the liposome or the composition transduces or transfects cells in vivo to produce a cell according to any one of claims 145 to 148.

152. A method of altering an immune response in a subject, the method comprising administering to the subject: the vector according to claims 124 to 125; the composition according to any one of claims 126 to 129; the plasmid according to claims 130 to 131; the composition according to any one of claims 132 to 134; the recombinant virus according to any one of claims 135 to 139; the liposome according to any one of claims 140 to 142; or the pharmaceutical composition according to any one of claims 143 to 144.

153. The method according to claim 152, wherein the immune response is activated.

154. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject: the vector according to claims 124 to 125; the composition according to any one of claims 126 to 129; the plasmid according to claims 130 to 131; the composition according to any one of claims 132 to 134; the recombinant virus according to any one of claims 135 to 139; the liposome according to any one of claims 140 to 142; or the pharmaceutical composition according to any one of claims 143 to 144.

155. The method according to claim 154, wherein the cancer is a cancer provided herein.

156. The method according to claim 154 or 155, wherein the method further comprises using at least one additional therapeutic agent, such as an anti-cancer therapeutic agent, including but not limited to chemotherapy, radiotherapy, targeted therapy, immunotherapy, stem cell or bone marrow transplantation therapy, hormone therapy, photodynamic therapy, or any combination thereof.

157. A method of treating a disease or disorder in a subject in need thereof, the disease or disorder such as a viral infection, a bacterial infection, or a fungal infection, such as those provided herein, the method comprising administering to the subject: the vector according to claims 124 to 125; the composition according to any one of claims 126 to 129; the plasmid according to claims 130 to 131; the composition according to any one of claims 132 to 134; the recombinant virus according to any one of claims 135 to 139; the liposome according to any one of claims 140 to 142; or the pharmaceutical composition according to any one of claims 143 to 144.

158. A polypeptide comprising the formula: X1-L1-X2-L2-X3-L3-X4-L4-X5-L5-X6, wherein: X1 is an interleukin 21 (IL-21) polypeptide, an interleukin 15 (IL-15) polypeptide, an interleukin 12p40 subunit (IL-12p40) polypeptide, an interleukin 12p35 subunit (IL-12p35) polypeptide, an interleukin 15 receptor (IL-15Rα) polypeptide, a first fragment of the IL-15Rα polypeptide, or a second fragment of the IL-15Rα polypeptide; X2 is an IL-21 polypeptide, an IL-15 polypeptide, an IL-12p40 polypeptide, an IL-12p35 polypeptide, an IL-15Rα polypeptide, a first fragment of an IL-15Rα polypeptide, or a second fragment of an IL-15Rα polypeptide; X3 is an IL-21 polypeptide, an IL-15 polypeptide, an IL-12p40 polypeptide, an IL-12p35 polypeptide, an IL-15Rα polypeptide, a first fragment of an IL-15Rα polypeptide, or a second fragment of an IL-15Rα polypeptide; X4 is an IL-21 polypeptide, an IL-15 polypeptide, an IL-12p40 polypeptide, an IL-12p35 polypeptide, an IL-15Rα polypeptide, a first fragment of an IL-15Rα polypeptide, or a second fragment of an IL-15Rα polypeptide; X5 is an IL-21 polypeptide, an IL-15 polypeptide, an IL-12p40 polypeptide, an IL-12p35 polypeptide, an IL-15Rα polypeptide, a first fragment of an IL-15Rα polypeptide, or a second fragment of an IL-15Rα polypeptide; X6 is an IL-21 polypeptide, an IL-15 polypeptide, an IL-12p40 polypeptide, an IL-12p35 polypeptide, an IL-15Rα polypeptide, a first fragment of an IL-15Rα polypeptide, or a second fragment of an IL-15Rα polypeptide; L1, L2, L3, L4, and L5 are each independently a polypeptide linker comprising the same or different polypeptide sequences, provided that each of X1, X2, X3, X4, X5, and X6 is different.

159. The polypeptide according to claim 158, wherein X1 is an interleukin 21 (IL-21) polypeptide.

160. The polypeptide according to claim 158 or 159, wherein: X1 is an IL-21 polypeptide; X2 is an IL-15 polypeptide; X3 is a first fragment of an IL-15Rα polypeptide; X4 is an IL-12p40 polypeptide; X5 is an IL-12p35 polypeptide; and X6 is a second fragment of an IL-15Rα polypeptide.

161. The polypeptide according to claim 158 or 159, wherein: X1 is an IL-21 polypeptide; X2 is an IL-15 polypeptide; X3 is a first fragment of an IL-15Rα polypeptide; X4 is an IL-12p35 polypeptide; X5 is an IL-12p40 polypeptide; and X6 is a second fragment of an IL-15Rα polypeptide.

162. The polypeptide according to claim 158 or 159, wherein: X1 is an IL-21 polypeptide; X2 is an IL-15 polypeptide; X3 is an IL-12p40 polypeptide; X4 is a first fragment of an IL-15Rα polypeptide; X5 is an IL-12p35 polypeptide; and X6 is a second fragment of an IL-15Rα polypeptide.

163. The polypeptide according to claim 158 or 159, wherein: X1 is an IL-21 polypeptide; X2 is an IL-15 polypeptide; X3 is an IL-12p40 polypeptide; X4 is an IL-12p35 polypeptide; X5 is a first fragment of an IL-15Rα polypeptide; and X6 is a second fragment of an IL-15Rα polypeptide.

164. The polypeptide according to claim 158 or 159, wherein: X1 is an IL-21 polypeptide; X2 is an IL-15 polypeptide; X3 is an IL-12p35 polypeptide; X4 is an IL-12p40 polypeptide; X5 is a first fragment of an IL-15Rα polypeptide; and X6 is a second fragment of an IL-15Rα polypeptide.

165. The polypeptide according to claim 158 or 159, wherein: X1 is an IL-21 polypeptide; X2 is an IL-15 polypeptide; X3 is an IL-12p35 polypeptide; X4 is a first fragment of an IL-15Rα polypeptide; X5 is an IL-12p40 polypeptide; and X6 is a second fragment of an IL-15Rα polypeptide.

166. The polypeptide according to claim 158 or 159, wherein: X1 is an IL-21 polypeptide; X2 is a first fragment of an IL-15Rα polypeptide; X3 is an IL-15 polypeptide; X4 is an IL-12p40 polypeptide; X5 is an IL-12p35 polypeptide; and X6 is a second fragment of an IL-15Rα polypeptide.

167. The polypeptide according to claim 158 or 159, wherein: X1 is an IL-21 polypeptide; X2 is a first fragment of an IL-15Rα polypeptide; X3 is an IL-15 polypeptide; X4 is an IL-12p35 polypeptide; X5 is an IL-12p40 polypeptide; and X6 is a second fragment of an IL-15Rα polypeptide.

168. The polypeptide according to claim 158 or 159, wherein: X1 is an IL-21 polypeptide; X2 is a first fragment of an IL-15Rα polypeptide; X3 is an IL-12p40 polypeptide; X4 is an IL-15 polypeptide; X5 is an IL-12p35 polypeptide; and X6 is a second fragment of an IL-15Rα polypeptide.

169. The polypeptide according to claim 158 or 159, wherein: X1 is an IL-21 polypeptide; X2 is a first fragment of an IL-15Rα polypeptide; X3 is an IL-12p40 polypeptide; X4 is an IL-12p35 polypeptide; X5 is IL-15; and X6 is a second fragment of an IL-15Rα polypeptide.

170. The polypeptide according to claim 158 or 159, wherein: X1 is an IL-21 polypeptide; X2 is a first fragment of an IL-15Rα polypeptide; X3 is an IL-12p35 polypeptide; X4 is an IL-12p40 polypeptide; X5 is IL-15; and X6 is a second fragment of an IL-15Rα polypeptide.

171. The polypeptide according to claim 158 or 159, wherein: X1 is an IL-21 polypeptide; X2 is a first fragment of an IL-15Rα polypeptide; X3 is an IL-12p35 polypeptide; X4 is IL-15; X5 is an IL-12p40 polypeptide; and X6 is a second fragment of an IL-15Rα polypeptide.

172. The polypeptide according to claim 158 or 159, wherein: X1 is an IL-21 polypeptide; X2 is an IL-12p40 polypeptide; X3 is an IL-15 polypeptide; X4 is a first fragment of an IL-15Rα polypeptide; X5 is an IL-12p35 polypeptide; and X6 is a second fragment of an IL-15Rα polypeptide. The polypeptide according to claim 158 or 159, wherein: X1 is an IL-21 polypeptide; X2 is an IL-12p40 polypeptide; X3 is an IL-15 polypeptide; X4 is an IL-12p35 polypeptide; X5 is a first fragment of an IL-15Rα polypeptide; and X6 is a second fragment of an IL-15Rα polypeptide.

174. The polypeptide according to claim 158 or 159, wherein: X1 is an IL-21 polypeptide; X2 is an IL-12p40 polypeptide; X3 is a first fragment of an IL-15Rα polypeptide; X4 is an IL-15 polypeptide; X5 is an IL-12p35 polypeptide; and X6 is a second fragment of an IL-15Rα polypeptide.

175. The polypeptide according to claim 158 or 159, wherein: X1 is an IL-21 polypeptide; X2 is an IL-12p40 polypeptide; X3 is a first fragment of an IL-15Rα polypeptide; X4 is an IL-12p35 polypeptide; X5 is IL-15; and X6 is a second fragment of an IL-15Rα polypeptide.

176. The polypeptide according to claim 158 or 159, wherein: X1 is an IL-21 polypeptide; X2 is an IL-12p40 polypeptide; X3 is an IL-12p35 polypeptide; X4 is a first fragment of an IL-15Rα polypeptide; X5 is IL-15; and X6 is a second fragment of an IL-15Rα polypeptide.

177. The polypeptide according to claim 158 or 159, wherein: X1 is an IL-21 polypeptide; X2 is an IL-12p40 polypeptide; X3 is an IL-12p35 polypeptide; X4 is IL-15; X5 is a first fragment of an IL-15Rα polypeptide; and X6 is a second fragment of an IL-15Rα polypeptide.

178. The polypeptide according to claim 158 or 159, wherein: X1 is an IL-21 polypeptide; X2 is an IL-12p35 polypeptide; X3 is an IL-15 polypeptide; X4 is a first fragment of an IL-15Rα polypeptide; X5 is an IL-12p40 polypeptide; and X6 is a second fragment of an IL-15Rα polypeptide.

179. The polypeptide according to claim 158 or 159, wherein: X1 is an IL-21 polypeptide; X2 is an IL-12p35 polypeptide; X3 is an IL-15 polypeptide; X4 is an IL-12p40 polypeptide; X5 is a first fragment of an IL-15Rα polypeptide; and X6 is a second fragment of an IL-15Rα polypeptide.

180. The polypeptide according to claim 158 or 159, wherein: X1 is an IL-21 polypeptide; X2 is an IL-12p35 polypeptide; X3 is a first fragment of an IL-15Rα polypeptide; X4 is an IL-15 polypeptide; X5 is an IL-12p40 polypeptide; and X6 is a second fragment of an IL-15Rα polypeptide.

181. The polypeptide according to claim 158 or 159, wherein: X1 is an IL-21 polypeptide; X2 is an IL-12p35 polypeptide; X3 is a first fragment of an IL-15Rα polypeptide; X4 is an IL-12p40 polypeptide; X5 is IL-15; and X6 is the second fragment of the IL-15Rα polypeptide.

182. The polypeptide according to claim 158 or 159, wherein: X1 is an IL-21 polypeptide; X2 is an IL-12p35 polypeptide; X3 is an IL-12p40 polypeptide; X4 is the first fragment of the IL-15Rα polypeptide; X5 is IL-15; and X6 is the second fragment of the IL-15Rα polypeptide.

183. The polypeptide according to claim 158 or 159, wherein: X1 is an IL-21 polypeptide; X2 is an IL-12p35 polypeptide; X3 is an IL-12p40 polypeptide; X4 is IL-15; X5 is the first fragment of the IL-15Rα polypeptide; and X6 is the second fragment of the IL-15Rα polypeptide.

184. The polypeptide according to any one of claims 158 to 183, wherein the IL-21 polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 71, or as otherwise provided herein.

185. The polypeptide according to any one of claims 158 to 184, wherein the IL-21 polypeptide comprises the amino acid sequence of SEQ ID NO: 71, or as otherwise provided herein.

186. The polypeptide according to any one of claims 158 to 185, wherein the IL-12p40 polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1, 2, 3 or 4, or as otherwise provided herein.

187. The polypeptide according to any one of claims 158 to 186, wherein the IL-12p40 polypeptide comprises the amino acid sequence of SEQ ID NO: 1, 2, 3 or 4, or as otherwise provided herein.

188. The polypeptide according to any one of claims 158 to 187, wherein the IL-12p35 polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 5, 6, 7 or 8, or as otherwise provided herein.

189. The polypeptide according to any one of claims 158 to 188, wherein the IL-12p35 polypeptide comprises the amino acid sequence of SEQ ID NO: 5, 6, 7 or 8, or as otherwise provided herein.

190. The polypeptide according to any one of claims 158 to 189, wherein the IL-15 polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 9 or 10, or as otherwise provided herein.

191. A polypeptide according to any one of claims 158 to 190, wherein the IL-15 polypeptide comprises the amino acid sequence of SEQ ID NO: 9 or 10, or as otherwise provided herein.

192. A polypeptide according to any one of claims 158 to 191, wherein the IL-15Rα polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 11 or 12, or as otherwise provided herein.

193. A polypeptide according to any one of claims 158 to 192, wherein the IL-15Rα polypeptide comprises the amino acid sequence of SEQ ID NO: 11 or 12, or as otherwise provided herein.

194. A polypeptide according to any one of claims 158 to 193, wherein one or more of L1, L2, L3, L4 and L5 are each independently a cleavable linker.

195. The polypeptide according to claim 194, wherein L1, L3, L4 and L5 are non-cleavable linkers.

196. The polypeptide according to claim 194 or 195, wherein the cleavable linker is a furin-cleavable linker, a Val-Cit linker, a Val-Gly linker, a Gly-Gly linker, an Ala-Ala-Asn linker or a linker comprising polyethylene glycol (PEG).

197. A polypeptide according to any one of claims 158 to 196, wherein one of L1, L2, L3, L4 and L5 comprises the sequence of (GSGSGG)n (SEQ ID NO: 16), (GGGGS)n (SEQ ID NO: 17), (GGGGA)n (SEQ ID NO: 18), (GGGSE)n (SEQ ID NO: 19), (GGGSK)n (SEQ ID NO: 20) or (AEEEK)n (SEQ ID NO: 21) or a combination thereof, wherein each n is independently 1, 2, 3, 4 or 5.

198. The polypeptide according to claim 197, wherein n is 3.

199. The polypeptide according to claim 197 or 198, wherein one or more of L1, L2, L3, L4, and L5 comprise the sequence RXXR of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:70 or combinations thereof, wherein X is any amino acid.

200. The polypeptide according to any one of claims 197 to 199, wherein each of L1, L2, L3, L4, and L5 comprises the sequence RXXR of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:70 or combinations thereof, wherein X is any amino acid.

201. The polypeptide according to any one of claims 158 to 200, wherein the first polypeptide comprises the sequence of SEQ ID NO:72 or a variant thereof, or as otherwise provided herein.

202. The polypeptide according to any one of claims 158 to 201, wherein the first polypeptide comprises the sequence of SEQ ID NO:73 or a variant thereof, or as otherwise provided herein.

203. A polypeptide according to any one of claims 158 to 202, wherein the first polypeptide comprises the sequence of SEQ ID NO: 74 or a variant thereof, or as otherwise provided herein.

204. A polypeptide according to any one of claims 158 to 203, wherein the first polypeptide comprises the sequence of SEQ ID NO: 75 or a variant thereof, or as otherwise provided herein.

205. A polypeptide according to any one of claims 158 to 204, wherein the first polypeptide comprises the sequence of SEQ ID NO: 76 or a variant thereof, or as otherwise provided herein.

206. A polypeptide according to any one of claims 158 to 205, wherein the first polypeptide comprises the sequence of SEQ ID NO: 77 or a variant thereof, or as otherwise provided herein.

207. A polypeptide according to any one of claims 158 to 206, wherein the first polypeptide comprises the sequence of SEQ ID NO: 78 or a variant thereof, or as otherwise provided herein.

208. A polypeptide according to any one of claims 158 to 207, wherein the first polypeptide comprises the sequence of SEQ ID NO: 79 or a variant thereof, or as otherwise provided herein.

209. A polypeptide according to any one of claims 158 to 208, wherein the first polypeptide is linked or fused to at least one additional molecule selected from the group consisting of: a polypeptide, an antigen, a ligand, an antibody, a checkpoint inhibitor, a lipid, a carbohydrate, a nucleic acid, or any combination thereof.

210. A polypeptide according to claim 158, wherein: X1 is an IL-21 polypeptide; X2 is an IL-15 polypeptide; X3 is a first fragment of an IL-15Rα polypeptide; X4 is an IL-12p40 polypeptide; X5 is an IL-12p35 polypeptide; and X6 is a second fragment of an IL-15Rα polypeptide; and L1, L2, L3, L4, and L5 are non-cleavable linkers.

211. A polypeptide according to claim 210, wherein the first polypeptide is linked or fused to at least one additional molecule selected from the group consisting of: a polypeptide, an antigen, a ligand, an antibody, a checkpoint inhibitor, a lipid, a carbohydrate, a nucleic acid, or any combination thereof.

212. A nucleic acid encoding a polypeptide according to any one of claims 158 to 211.

213. A vector comprising the nucleic acid according to claim 212.

214. A plasmid comprising the nucleic acid according to claim 212.

215. A recombinant virus comprising the nucleic acid according to claim 212.

216. The recombinant virus according to claim 215, wherein the recombinant virus is an adenovirus or a lentivirus.

217. The recombinant virus according to claim 215 or 216, wherein the recombinant virus is a replication-incompetent adenovirus.

218. A liposome comprising the nucleic acid according to claim 212.

219. The liposome according to claim 218, wherein the liposome comprises any lipid provided herein.

220. A pharmaceutical composition comprising the nucleic acid according to claim 212 and a pharmaceutically acceptable carrier.

221. A cell comprising the composition according to any one of claims 158 to 211.

222. A composition comprising: A first polypeptide is provided, the first polypeptide comprising the following formula: X1-L1-X2-L2-X3-L3-X4-L4-X5, wherein: X1 is an interleukin-21 (IL-21) polypeptide, an interleukin-15 (IL-15) polypeptide, an interleukin-12p40 subunit (IL-12p40) polypeptide, an interleukin-12p35 subunit (IL-12p35) polypeptide, an interleukin-15 receptor (IL-15Rα) polypeptide, a first fragment of the IL-15Rα polypeptide or a second fragment of the IL-15Rα polypeptide; X2 is an IL-21 polypeptide, an IL-15 polypeptide, an IL-12p40 polypeptide, an IL-12p35 polypeptide, an IL-15Rα polypeptide, a first fragment of the IL-15Rα polypeptide or a second fragment of the IL-15Rα polypeptide; X3 is an IL-21 polypeptide, an IL-15 polypeptide, an IL-12p40 polypeptide, an IL-12p35 polypeptide, an IL-15Rα polypeptide, a first fragment of the IL-15Rα polypeptide or a second fragment of the IL-15Rα polypeptide; X4 is an IL-21 polypeptide, an IL-15 polypeptide, an IL-12p40 polypeptide, an IL-12p35 polypeptide, an IL-15Rα polypeptide, a first fragment of the IL-15Rα polypeptide or a second fragment of the IL-15Rα polypeptide; X5 is an IL-21 polypeptide, an IL-15 polypeptide, an IL-12p40 polypeptide, an IL-12p35 polypeptide, an IL-15Rα polypeptide, a first fragment of the IL-15Rα polypeptide or a second fragment of the IL-15Rα polypeptide; X6 is an IL-21 polypeptide, an IL-15 polypeptide, an IL-12p40 polypeptide, an IL-12p35 polypeptide, an IL-15Rα polypeptide, a first fragment of the IL-15Rα polypeptide or a second fragment of the IL-15Rα polypeptide; L1, L2, L3, L4 and L5 are each independently a polypeptide linker comprising the same or different polypeptide sequences, provided that each of X1, X2, X3, X4, X5 and X6 is different; and / or A second polypeptide, the second polypeptide comprising at least one immune checkpoint polypeptide or an active fragment thereof, such as but not limited to a CTLA-4 polypeptide, a PD-1 polypeptide, a PD-L1 polypeptide, a PD-L2 polypeptide, a TIM3 polypeptide, a LAG3 polypeptide, a VISTA polypeptide, a SINGLEC7 polypeptide, a SINGLEC9 polypeptide, a TIGIT polypeptide, a CD96 polypeptide, a BTLA polypeptide, a B7H3 polypeptide, a B7H4 polypeptide, a CD155 polypeptide, a HHLA2 polypeptide, a BTN3A1 polypeptide, a B7.1 polypeptide, a B7.2 polypeptide or an active fragment thereof; or one or more nucleic acid molecules encoding the first polypeptide and the second polypeptide.

223. The composition according to claim 222, wherein X1 is an interleukin-21 (IL-21) polypeptide.

224. The composition according to claim 222 or 223, wherein: X1 is an IL-21 polypeptide; X2 is an IL-15 polypeptide; X3 is a first fragment of an IL-15Rα polypeptide; X4 is an IL-12p40 polypeptide; X5 is an IL-12p35 polypeptide; and X6 is a second fragment of an IL-15Rα polypeptide.

225. The polypeptide according to claim 222 or 223, wherein: X1 is an IL-21 polypeptide; X2 is an IL-15 polypeptide; X3 is a first fragment of an IL-15Rα polypeptide; X4 is an IL-12p35 polypeptide; X5 is an IL-12p40 polypeptide; and X6 is a second fragment of an IL-15Rα polypeptide.

226. The polypeptide according to claim 222 or 223, wherein: X1 is an IL-21 polypeptide; X2 is an IL-15 polypeptide; X3 is an IL-12p40 polypeptide; X4 is a first fragment of an IL-15Rα polypeptide; X5 is an IL-12p35 polypeptide; and X6 is a second fragment of an IL-15Rα polypeptide.

227. The polypeptide according to claim 222 or 223, wherein: X1 is an IL-21 polypeptide; X2 is an IL-15 polypeptide; X3 is an IL-12p40 polypeptide; X4 is an IL-12p35 polypeptide; X5 is a first fragment of an IL-15Rα polypeptide; and X6 is a second fragment of an IL-15Rα polypeptide.

228. The polypeptide according to claim 222 or 223, wherein: X1 is an IL-21 polypeptide; X2 is an IL-15 polypeptide; X3 is an IL-12p35 polypeptide; X4 is an IL-12p40 polypeptide; X5 is a first fragment of an IL-15Rα polypeptide; and X6 is a second fragment of an IL-15Rα polypeptide.

229. The polypeptide according to claim 222 or 223, wherein: X1 is an IL-21 polypeptide; X2 is an IL-15 polypeptide; X3 is an IL-12p35 polypeptide; X4 is a first fragment of an IL-15Rα polypeptide; X5 is an IL-12p40 polypeptide; and X6 is a second fragment of an IL-15Rα polypeptide.

230. The polypeptide according to claim 222 or 223, wherein: X1 is an IL-21 polypeptide; X2 is a first fragment of an IL-15Rα polypeptide; X3 is an IL-15 polypeptide; X4 is an IL-12p40 polypeptide; X5 is an IL-12p35 polypeptide; and X6 is a second fragment of an IL-15Rα polypeptide.

231. The polypeptide according to claim 222 or 223, wherein: X1 is an IL-21 polypeptide; X2 is a first fragment of an IL-15Rα polypeptide; X3 is an IL-15 polypeptide; X4 is an IL-12p35 polypeptide; X5 is an IL-12p40 polypeptide; and X6 is a second fragment of an IL-15Rα polypeptide. The polypeptide according to claim 222 or 223, wherein: X1 is an IL-21 polypeptide; X2 is a first fragment of an IL-15Rα polypeptide; X3 is an IL-12p40 polypeptide; X4 is an IL-15 polypeptide; X5 is an IL-12p35 polypeptide; and X6 is a second fragment of an IL-15Rα polypeptide.

233. The polypeptide according to claim 222 or 223, wherein: X1 is an IL-21 polypeptide; X2 is a first fragment of an IL-15Rα polypeptide; X3 is an IL-12p40 polypeptide; X4 is an IL-12p35 polypeptide; X5 is IL-15; and X6 is a second fragment of an IL-15Rα polypeptide.

234. The polypeptide according to claim 222 or 223, wherein: X1 is an IL-21 polypeptide; X2 is a first fragment of an IL-15Rα polypeptide; X3 is an IL-12p35 polypeptide; X4 is an IL-12p40 polypeptide; X5 is IL-15; and X6 is a second fragment of an IL-15Rα polypeptide.

235. The polypeptide according to claim 222 or 223, wherein: X1 is an IL-21 polypeptide; X2 is a first fragment of an IL-15Rα polypeptide; X3 is an IL-12p35 polypeptide; X4 is IL-15; X5 is an IL-12p40 polypeptide; and X6 is a second fragment of an IL-15Rα polypeptide.

236. The polypeptide according to claim 222 or 223, wherein: X1 is an IL-21 polypeptide; X2 is an IL-12p40 polypeptide; X3 is an IL-15 polypeptide; X4 is a first fragment of an IL-15Rα polypeptide; X5 is an IL-12p35 polypeptide; and X6 is a second fragment of an IL-15Rα polypeptide.

237. The polypeptide according to claim 222 or 223, wherein: X1 is an IL-21 polypeptide; X2 is an IL-12p40 polypeptide; X3 is an IL-15 polypeptide; X4 is an IL-12p35 polypeptide; X5 is a first fragment of an IL-15Rα polypeptide; and X6 is a second fragment of an IL-15Rα polypeptide.

238. The polypeptide according to claim 222 or 223, wherein: X1 is an IL-21 polypeptide; X2 is an IL-12p40 polypeptide; X3 is a first fragment of an IL-15Rα polypeptide; X4 is an IL-15 polypeptide; X5 is an IL-12p35 polypeptide; and X6 is a second fragment of an IL-15Rα polypeptide.

239. The polypeptide according to claim 222 or 223, wherein: X1 is an IL-21 polypeptide; X2 is an IL-12p40 polypeptide; X3 is a first fragment of an IL-15Rα polypeptide; X4 is an IL-12p35 polypeptide; X5 is IL-15; and X6 is a second fragment of an IL-15Rα polypeptide.

240. The polypeptide according to claim 222 or 223, wherein: X1 is an IL-21 polypeptide; X2 is an IL-12p40 polypeptide; X3 is an IL-12p35 polypeptide; X4 is a first fragment of an IL-15Rα polypeptide; X5 is IL-15; and X6 is the second fragment of the IL-15Rα polypeptide.

241. The polypeptide according to claim 222 or 223, wherein: X1 is an IL-21 polypeptide; X2 is an IL-12p40 polypeptide; X3 is an IL-12p35 polypeptide; X4 is IL-15; X5 is the first fragment of the IL-15Rα polypeptide; and X6 is the second fragment of the IL-15Rα polypeptide.

242. The polypeptide according to claim 222 or 223, wherein: X1 is an IL-21 polypeptide; X2 is an IL-12p35 polypeptide; X3 is an IL-15 polypeptide; X4 is the first fragment of the IL-15Rα polypeptide; X5 is an IL-12p40 polypeptide; and X6 is the second fragment of the IL-15Rα polypeptide.

243. The polypeptide according to claim 222 or 223, wherein: X1 is an IL-21 polypeptide; X2 is an IL-12p35 polypeptide; X3 is an IL-15 polypeptide; X4 is an IL-12p40 polypeptide; X5 is the first fragment of the IL-15Rα polypeptide; and X6 is the second fragment of the IL-15Rα polypeptide.

244. The polypeptide according to claim 222 or 223, wherein: X1 is an IL-21 polypeptide; X2 is an IL-12p35 polypeptide; X3 is the first fragment of the IL-15Rα polypeptide; X4 is an IL-15 polypeptide; X5 is an IL-12p40 polypeptide; and X6 is the second fragment of the IL-15Rα polypeptide.

245. The polypeptide according to claim 222 or 223, wherein: X1 is an IL-21 polypeptide; X2 is an IL-12p35 polypeptide; X3 is the first fragment of the IL-15Rα polypeptide; X4 is an IL-12p40 polypeptide; X5 is IL-15; and X6 is the second fragment of the IL-15Rα polypeptide.

246. The polypeptide according to claim 222 or 223, wherein: X1 is an IL-21 polypeptide; X2 is an IL-12p35 polypeptide; X3 is an IL-12p40 polypeptide; X4 is the first fragment of the IL-15Rα polypeptide; X5 is IL-15; and X6 is the second fragment of the IL-15Rα polypeptide.

247. The polypeptide according to claim 222 or 223, wherein: X1 is an IL-21 polypeptide; X2 is an IL-12p35 polypeptide; X3 is an IL-12p40 polypeptide; X4 is IL-15; X5 is the first fragment of the IL-15Rα polypeptide; and X6 is the second fragment of the IL-15Rα polypeptide.

248. The composition according to any one of claims 222 to 246, wherein the IL-21 polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO:71, or as otherwise provided herein.

249. The composition according to any one of claims 222 to 247, wherein the IL-21 polypeptide comprises the amino acid sequence of SEQ ID NO:71, or as otherwise provided herein.

250. The composition according to any one of claims 222 to 248, wherein the IL-12p40 polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1, 2, 3 or 4, or as otherwise provided herein.

251. The composition according to any one of claims 222 to 249, wherein the IL-12p40 polypeptide comprises the amino acid sequence of SEQ ID NO: 1, 2, 3 or 4, or as otherwise provided herein.

252. The composition according to any one of claims 222 to 250, wherein the IL-12p35 polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 5, 6, 7 or 8, or as otherwise provided herein.

253. The composition according to any one of claims 222 to 251, wherein the IL-12p35 polypeptide comprises the amino acid sequence of SEQ ID NO: 5, 6, 7 or 8, or as otherwise provided herein.

254. The composition according to any one of claims 222 to 252, wherein the IL-15 polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 9 or 10, or as otherwise provided herein.

255. The composition according to any one of claims 222 to 253, wherein the IL-15 polypeptide comprises the amino acid sequence of SEQ ID NO: 9 or 10, or as otherwise provided herein.

256. The composition according to any one of claims 222 to 254, wherein the IL-15Rα polypeptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 11 or 12, or as otherwise provided herein.

257. The composition according to any one of claims 222 to 255, wherein the IL-15Rα polypeptide comprises the amino acid sequence of SEQ ID NO: 11 or 12, or as otherwise provided herein.

258. The composition according to any one of claims 222 to 256, wherein one or more of L1, L2, L3, L4 and L5 are each independently a cleavable linker.

259. The polypeptide according to claim 257, wherein L1, L2, L3, L4 and L5 are non-cleavable linkers.

260. The composition according to claim 257 or 258, wherein the cleavable linker is a furin-cleavable linker, a Val-Cit linker, a Val-Gly linker, a Gly-Gly linker, an Ala-Ala-Asn linker or a linker comprising polyethylene glycol (PEG).

261. A composition according to any one of claims 222 to 259, wherein one of L1, L2, L3, L4 and L5 comprises a sequence of (GSGSGG)n (SEQ ID NO:16), (GGGGS)n (SEQ ID NO:17), (GGGGA)n (SEQ ID NO:18), (GGGSE)n (SEQ ID NO:19), (GGGSK)n (SEQ ID NO:20) or (AEEEK)n (SEQ ID NO:21) or a combination thereof, wherein each n is independently 1, 2, 3, 4 or 5.

262. A composition according to claim 260, wherein n is 3.

263. A composition according to claim 260 or 261, wherein one or more of L1, L2, L3, L4 and L5 comprises a sequence RXXR of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:70 or a combination thereof, wherein X is any amino acid.

264. The composition according to any one of claims 260 to 262, wherein each of L1, L2, L3, L4 and L5 comprises the sequence RXXR of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:70 or a combination thereof, wherein X is any amino acid.

265. The composition according to any one of claims 222 to 263, wherein the first polypeptide comprises the sequence of SEQ ID NO:72 or a variant thereof, or as otherwise provided herein.

266. The composition according to any one of claims 222 to 263, wherein the first polypeptide comprises the sequence of SEQ ID NO:73 or a variant thereof, or as otherwise provided herein.

267. The composition according to any one of claims 222 to 263, wherein the first polypeptide comprises the sequence of SEQ ID NO:74 or a variant thereof, or as otherwise provided herein.

268. The composition according to any one of claims 222 to 263, wherein the first polypeptide comprises the sequence of SEQ ID NO:75 or a variant thereof, or as otherwise provided herein.

269. The composition according to any one of claims 222 to 263, wherein the first polypeptide comprises the sequence of SEQ ID NO:76 or a variant thereof, or as otherwise provided herein.

270. The composition according to any one of claims 222 to 263, wherein the first polypeptide comprises the sequence of SEQ ID NO:77 or a variant thereof, or as otherwise provided herein.

271. The composition according to any one of claims 222 to 263, wherein the first polypeptide comprises the sequence of SEQ ID NO:78 or a variant thereof, or as otherwise provided herein.

272. The composition according to any one of claims 222 to 263, wherein the first polypeptide comprises the sequence of SEQ ID NO:79 or a variant thereof, or as otherwise provided herein.

273. The composition according to any one of claims 222 to 271, wherein the first polypeptide is linked or fused to at least one additional molecule selected from the group consisting of: a polypeptide, an antigen, a ligand, an antibody, a checkpoint inhibitor, a lipid, a carbohydrate, a nucleic acid, or any combination thereof.

274. The composition according to claim 222, wherein: X1 is an IL-21 polypeptide; X2 is an IL-15 polypeptide; X3 is an IL-12p40 polypeptide; X4 is an IL-12p35 polypeptide; X5 is an IL-15Rα polypeptide; and L1, L2, L3, L4, and L5 are non-cleavable linkers.

275. The composition according to claim 273, wherein the first polypeptide is linked or fused to at least one additional molecule selected from the group consisting of: a polypeptide, an antigen, a ligand, an antibody, a checkpoint inhibitor, a lipid, a carbohydrate, a nucleic acid, or any combination thereof.

276. The composition according to any one of the preceding claims, wherein the second polypeptide comprises, from the N-terminus to the C-terminus, the formula X A -L A -X B or the formula X B -L A -X A , wherein: X A is an immune checkpoint polypeptide or an active fragment thereof, such as but not limited to CTLA-4 polypeptide, PD-1 polypeptide, PD-L1 polypeptide, PD-L2 polypeptide, TIM3 polypeptide, LAG3 polypeptide, VISTA polypeptide, SINGLEC7 polypeptide, SINGLEC9 polypeptide, TIGIT polypeptide, CD96 polypeptide, BTLA polypeptide, B7H3 polypeptide, B7H4 polypeptide, CD155 polypeptide, HHLA2 polypeptide, BTN3A1 polypeptide or an active fragment thereof; X B is a B7.1 polypeptide or a B7.2 polypeptide; and L A is a polypeptide linker.

277. The composition according to claim 275, wherein the second polypeptide comprises, from the N-terminus to the C-terminus, the formula X A -L A -X B .

278. The composition according to claim 275, wherein the polypeptide comprises, from the N-terminus to the C-terminus, the formula X B -L A -X A .

279. The composition according to any one of claims 275 to 277, wherein: X A is a PD-L1 polypeptide; and X B is a B7.1 polypeptide.

280. The composition according to any one of claims 275 to 277, wherein: X A is a PD-L1 polypeptide; and X B is a B7.2 polypeptide.

281. The composition according to any one of claims 275 to 277, wherein: X A is a PD-L2 polypeptide; and X B is a B7.1 polypeptide.

282. The composition according to any one of claims 275 to 277, wherein: X A is a PD-L2 polypeptide; and X B is a B7.2 polypeptide.

283. The composition according to any one of claims 275 to 279, wherein the PD-L1 polypeptide comprises the PD-L1 extracellular domain.

284. The composition according to claim 282, wherein the PD-L1 polypeptide comprises an amino acid sequence having at least 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 102 or comprises the sequence of SEQ ID NO:

102.

285. The composition according to any one of claims 275 to 277 and 280 to 281, wherein the PD-L2 polypeptide comprises the PD-L2 extracellular domain.

286. The composition according to claim 284, wherein the PD-L2 polypeptide comprises an amino acid sequence having at least 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 104 or comprises the sequence of SEQ ID NO:

104.

287. The composition according to any one of claims 275 to 278 and 280, wherein the B7.1 polypeptide comprises the B7.1 extracellular domain.

288. The composition according to claim 286, wherein the B7.1 polypeptide comprises an amino acid sequence having at least 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 106 or comprises the sequence of SEQ ID NO:

106.

289. The composition according to any one of claims 275 to 277, 279 and 281, wherein the B7.2 polypeptide comprises the B7.2 extracellular domain.

290. The composition according to claim 288, wherein the B7.2 polypeptide comprises an amino acid sequence having at least 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 108 or comprises the sequence of SEQ ID NO:

108.

291. The composition according to any one of claims 275 to 289, wherein the second polypeptide comprises a leader sequence.

292. The composition according to claim 290, wherein the leader sequence is located at the N-terminus of the second polypeptide.

293. The composition according to claim 290 or 291, wherein the leader sequence is a cell membrane targeting polypeptide.

294. The composition according to any one of claims 290 to 292, wherein the leader sequence is the sequence of SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113 or a combination thereof.

295. The composition according to any one of claims 275 to 293, wherein the second polypeptide further comprises an Fc domain.

296. The composition according to claim 294, wherein the Fc domain is an Ig Fc domain selected from the group consisting essentially of human IgG, IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgA1, IgA2, IgE or IgD.

297. The composition according to claims 294 to 295, wherein the Fc domain comprises an amino acid sequence having at least 85%, 86%, 97%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 114 or comprises the sequence of SEQ ID NO:

114.

298. The composition according to any one of claims 275 to 294, wherein L A comprises the sequence of SEQ ID NO: 109 or combinations thereof.

299. A composition according to any preceding claim, wherein the second polypeptide comprises, from N-terminus to C-terminus, the formula L S -X A -L A -X B -L B -X C , L S -X B -L A -X A -L B -X C , L S -X A -L A -X C -L B -X B or L S -X B -L A -X C -L B -X A ,in: L S is a leading sequence; X A is an immune checkpoint polypeptide or an active fragment thereof, such as but not limited to CTLA-4 polypeptide, PD-1 polypeptide, PD-L1 polypeptide, PD-L2 polypeptide, TIM3 polypeptide, LAG3 polypeptide, VISTA polypeptide, SINGLEC7 polypeptide, SINGLEC9 polypeptide, TIGIT polypeptide, CD96 polypeptide, BTLA polypeptide, B7H3 polypeptide, B7H4 polypeptide, CD155 polypeptide, HHLA2 polypeptide, BTN3A1 polypeptide or an active fragment thereof; X B is a B7.1 polypeptide or a B7.2 polypeptide; X C is a binder, such as an Fc domain, a TCR, a cytokine, a cytokine trap, a receptor ligand, a complement protein; L A is absent or is a polypeptide linker; and L B Absent or a polypeptide linker.

300. The composition according to claim 296, wherein the Fc domain is an Ig Fc domain selected from the group consisting essentially of human IgG, IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgA1, IgA2, IgE or IgD.

301. The composition according to claim 297, wherein the Fc domain comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 114 or comprises the sequence of SEQ ID NO:

114.

302. The composition according to any one of claims 222 to 298, wherein the second polypeptide comprises an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 117 or comprises the sequence of SEQ ID NO:

117.

303. The composition according to any one of claims 222 to 299, wherein the second polypeptide comprises an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 118 or comprises the sequence of SEQ ID NO:

118.

304. The composition according to any one of claims 222 to 300, wherein the composition further comprises an antigen.

305. The composition according to claim 301, wherein the antigen is a tumor antigen, a viral antigen, a bacterial antigen or a microbial antigen.

306. The composition according to claims 301 to 302, wherein the antigen is ovalbumin (OVA).

307. A composition, the composition comprising: a first nucleic acid molecule encoding a first polypeptide according to any one of claims 275 to 274; and a second nucleic acid molecule encoding a second polypeptide according to any one of claims 275 and 274 to 300.

308. The composition according to claim 304, the composition further comprising a third nucleic acid molecule encoding an antigen according to any one of claims 301 to 303.

309. A vector, the vector comprising the first nucleic acid molecule and the second nucleic acid molecule according to claim 304.

310. The vector according to claim 306, the vector further comprising the third nucleic acid molecule according to claim 305.

311. A composition, the composition comprising: a first vector, the first vector comprising the first nucleic acid molecule according to claim 304; and a second vector, the second vector comprising the second nucleic acid molecule according to claim 304.

312. The composition according to claim 308, wherein the first vector further comprises a third nucleic acid molecule according to claim 305.

313. The composition according to claim 308, wherein the second vector further comprises a third nucleic acid molecule according to claim 305.

314. The composition according to claim 308, the composition further comprising a third vector, the third vector comprising a third nucleic acid molecule according to claim 305.

315. A plasmid, the plasmid comprising a first nucleic acid molecule and a second nucleic acid molecule according to claim 304.

316. The plasmid according to claim 312, the plasmid further comprising a third nucleic acid molecule according to claim 305.

317. A composition, the composition comprising: A first plasmid, the first plasmid comprising a first nucleic acid molecule according to claim 304; and A second plasmid, the second plasmid comprising a second nucleic acid molecule according to claim 304.

318. The composition according to claim 314, wherein the first plasmid further comprises a third nucleic acid molecule according to claim 305.

319. The composition according to claim 314, wherein the second plasmid further comprises a third nucleic acid molecule according to claim 305.

320. The composition according to claim 314, the composition further comprising a third plasmid, the third plasmid comprising a third nucleic acid molecule according to claim 305.

321. A recombinant virus, the recombinant virus comprising a first nucleic acid molecule and a second nucleic acid molecule according to claim 304.

322. The recombinant virus according to claim 318, the recombinant virus further comprising a third nucleic acid molecule according to claim 305.

323. The recombinant virus according to claim 318 or 319, wherein the recombinant virus is an adenovirus or a lentivirus.

324. The recombinant virus according to claim 320, wherein the recombinant virus is a replication - incompetent adenovirus.

325. A liposome, the liposome comprising a first nucleic acid molecule and a second nucleic acid molecule according to claim 304.

326. The liposome according to claim 322, the liposome further comprising a third nucleic acid molecule according to claim 305.

327. The liposome according to claim 322 or 323, wherein the liposome comprises any lipid provided herein.

328. A pharmaceutical composition, the pharmaceutical composition comprising: A first nucleic acid molecule and a second nucleic acid molecule according to claim 304; and A pharmaceutically acceptable carrier.

329. The pharmaceutical composition according to claim 325, the pharmaceutical composition further comprising a third nucleic acid molecule according to claim 305.

330. A cell, the cell comprising the composition according to any one of claims 275 to 303.

331. The cell according to claim 327, wherein the cell further comprises a chimeric antigen receptor (CAR).

332. The cell according to claim 327 or 328, wherein the cell is an immune cell.

333. The cell according to claim 329, wherein the immune cell is a T cell, an NK cell or a dendritic cell.

334. A method for producing a cell according to any one of claims 327 to 330, the method comprising contacting the cell with: The vector according to claims 306 to 307; The composition according to any one of claims 308 to 311; The plasmid according to claims 312 to 313; The composition according to any one of claims 314 to 317; The recombinant virus according to any one of claims 318 to 321; The liposome according to any one of claims 322 to 324; or The pharmaceutical composition according to any one of claims 325 to 326.

335. The method according to claim 331, wherein the contacting comprises transducing or transfecting the cell with one of the composition of the vector, the plasmid, the recombinant virus, the liposome or the composition.

336. A method for producing in vivo a cell according to any one of claims 327 to 330, the method comprising administering to a subject: The vector according to claims 306 to 307; The composition according to any one of claims 308 to 311; The plasmid according to claims 312 to 313; The composition according to any one of claims 314 to 317; The recombinant virus according to any one of claims 318 to 321; The liposome according to any one of claims 322 to 324; or The pharmaceutical composition according to any one of claims 325 to 326; wherein the vector, the plasmid, the recombinant virus, the liposome or the composition transduces or transfects cells in vivo to produce a cell according to any one of claims 327 to 330.

337. A method for altering an immune response in a subject, the method comprising administering to the subject: The vector according to claims 306 to 307; The composition according to any one of claims 308 to 311; The plasmid according to claims 312 to 313; The composition according to any one of claims 314 to 317; The recombinant virus according to any one of claims 318 to 321; The liposome according to any one of claims 322 to 324; or The pharmaceutical composition according to any one of claims 325 to 326.

338. The method according to claim 334, wherein the immune response is activated.

339. A method for treating cancer in a subject in need thereof, the method comprising administering to the subject: The vector according to claims 306 to 307; The composition according to any one of claims 308 to 311; The plasmid according to claims 312 to 313; The composition according to any one of claims 314 to 317; The recombinant virus according to any one of claims 318 to 321; The liposome according to any one of claims 322 to 324; or The pharmaceutical composition according to any one of claims 325 to 326.

340. The method according to claim 336, wherein the cancer is a cancer provided herein.

341. The method according to claim 336 or 337, wherein the method further comprises using at least one additional therapeutic agent, such as an anti-cancer therapeutic agent, including but not limited to chemotherapy, radiotherapy, targeted therapy, immunotherapy, stem cell or bone marrow transplantation therapy, hormone therapy, photodynamic therapy, or any combination thereof.

342. A method of treating a disease or disorder in a subject in need thereof, the disease or disorder such as a viral infection, a bacterial infection, or a fungal infection, such as those provided herein, the method comprising administering to the subject: The vector according to claims 306 to 307; The composition according to any one of claims 308 to 311; The plasmid according to claims 312 to 313; The composition according to any one of claims 314 to 317; The recombinant virus according to any one of claims 318 to 321; The liposome according to any one of claims 322 to 324; or The pharmaceutical composition according to any one of claims 325 to 326.

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