IL-21, IL-15, AND IL-12 POLYPEPTIDES, COMPOSITIONS COMPRISING THE SAME, AND METHODS OF USE THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

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

SEQUENCE LISTING

This application contains a Sequence Listing filed electronically as an XML file named

“260034.000/02 ST.26 Sequence Listing XML”, which was created on September 13, 2023 and is 120,860 bytes in size. The Sequence Listing is hereby incorporated by reference in its entirety.

FIELD

Embodiments provided herein relate to polypeptides comprising a IL-21 polypeptide, a IL- 15 polypeptide and a IL- 12 polypeptide, compositions comprising the same and immune checkpoint inhibitors, and methods of using the same.

BACKGROUND

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

Immune checkpoint molecules are accessory molecules that either 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). PD-1 ligands (PD-L ₁ and PD-L ₂ ) expressed on cancer and other cells interact with PD-1 on T-cells to suppress T-cell killing of the 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 therapy has been developed against both PD-1 (Opdivo®) and CTLA-4 (Yervoy®) and have shown promise in pre-clinical and clinical studies for the treatment of tumors. However, the majority of patients do not develop sustained anti-tumor responses using these therapies. Thus, there is a need for improved therapies that take advantage of this critical immune checkpoint signaling nexus.

The present embodiments fulfill these needs as well as others.

SUMMARY

Polypeptides comprising IL-21, IL-12 and IL-15 are provided. In some embodiments, polypeptides are provided comprising the formula of: X ₁ -L ₁ -X ₂ -L ₂ -X ₃ -L ₃ -X ₄ -L ₄ -X ₅ , wherein X ₁ , X ₂ , X ₃ , X ₄ , and X ₅ are each, independently, a IL-21 polypeptide, IL- 15 polypeptide, IL- 12 p40 polypeptide, IL-12 p35 polypeptide, or IL-15Rα polypeptide, provided each of X ₁ , X ₂ , X ₃ , X ₄ , and X ₅ are different; and L ₁ , L ₂ , L ₃ , and L ₄ are each, independently, a polypeptide linker that comprise the same or different polypeptide sequences. In some embodiments, one or more of L ₁ , L ₂ , L ₃ , and L ₄ are each, independently, a cleavable linker or a non-cleavable linker. In some embodiments, X ₁ is a IL-21 polypeptide; X ₂ is a IL-15 polypeptide; X ₃ is a IL-12 p40 polypeptide; X ₄ is a IL- 12 p35 polypeptide; X ₅ is a IL-15Rα polypeptide; L ₂ is a furin cleavable linker; and L ₁ , I.. ₃ , and L. ₄ are non-cleavable linkers.

In some embodiments, polypeptides are provided comprising the formula of: X1-L ₁ -X ₂ - L ₂ -X ₃ -L ₃ -X ₄ -L ₄ -X ₅ -L ₅ -X ₆ , wherein X ₁ , X ₂ , X ₃ , X ₄ , X ₅ , and X ₆ are each, independently, a IL-21 polypeptide, IL-15 polypeptide, IL-12 p40 polypeptide, IL-12 p35 polypeptide, IL-15Rα polypeptide, a first fragment of IL-15Rα polypeptide, or a second fragment of IL-15Rα polypeptide, provided each of X ₁ , X ₂ , X ₃ , X ₄ , X ₅ , and X ₆ are different; and L ₁ , L ₂ , L ₃ , L ₄ , and L ₅ are each, independently, a polypeptide linker that comprise the same or different polypeptide sequences.

Compositions comprising multiple polypeptides are also provided. In some embodiments, compositions are provided comprising: a first polypeptide comprising the formula of: X ₁ - L ₁ -X ₂ - L ₂ -X ₃ -L ₃ -X ₄ -L ₄ -X ₅ wherein X ₁ , X ₂ , X ₃ , X ₄ , and X ₅ are each, independently, IL-21 polypeptide, IL-L ₅ polypeptide, IL-12 p40 polypeptide, IL-12 p35 polypeptide, or IL-15Rα polypeptide, provided each of X ₁ , X ₂ , X ₃ , X ₄ , and X ₅ are different; and L ₁ , L ₂ , L ₃ , and L ₄ are each, independently, a polypeptide linker that comprise the same or different polypeptide sequences; and 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- L ₁ polypeptide, a PD-L ₂ polypeptide, a TIMS polypeptide, a LAGS 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 CD 155 polypeptide, a HHLA2 polypeptide, a BTN3A1 polypeptide, a B7.1 polypeptide, a B7.2 polypeptide, or an active fragment thereof.

In some embodiments, one or more of L ₁ , L ₂ , L ₃ , and L ₄ are each, independently, a cleavable linker or a non-cleavable linker. In some embodiments, X ₁ is a IL-21 polypeptide; X ₂ is a IL-15 polypeptide; X ₃ is a IL-12 p40 polypeptide; X ₄ is a IL-12 p35 polypeptide; X ₅ is a IL- 15Rα polypeptide; L ₂ is a furin cleavable linker; and L ₁ , L ₃ , and L ₁ are non-cleavable linkers.

In some embodiments, compositions are provided comprising: a first polypeptide comprising the formula of: X1-L ₁ -X ₂ -L ₂ -X ₃ -L ₃ -X ₄ -L ₄ -X ₅ -L ₅ -X ₆ , wherein X ₁ , X ₂ , X ₃ , X ₄ , X ₅ , and X ₆ are each, independently, a IL-21 polypeptide, IL-15 polypeptide, IL-12 p40 polypeptide, IL- 12 p35 polypeptide, IL-15Rα polypeptide, a first fragment of IL-15Rα polypeptide, or a second fragment of IL-15Rα polypeptide, provided each of X ₁ , X ₂ , X ₃ , X ₄ , X ₅ , and X ₆ are different; and L ₁ , L ₂ , L ₃ , L ₄ , and L ₅ are each, independently, a polypeptide linker that comprise the same or different polypeptide sequences; and 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- Ll polypeptide, a PD-L 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 CD 155 polypeptide, a HHLA2 polypeptide, a BTN3A1 polypeptide, a B7.1 polypeptide, a B7.2 polypeptide, or an active fragment thereof.

In some embodiments, the second polypeptide comprises, from the N -terminus to the C- terminus, a formula of X _A -L _A -X _B or a formula of 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-L ₁ polypeptide, a PD-L 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 CD 155 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 a Fc domain.

In some embodiments, compositions are provided that further comprise 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).

In some embodiments, nucleic acid molecules are provided that encode for any of the polypeptides provided herein. In some embodiments, compositions are provided comprising a first nucleic acid molecule encoding for a first polypeptide, and a second nucleic acid molecule encoding for a second polypeptide. In some embodiments, the provided compositions further comprise a third nucleic acid molecule.

In some embodiments, vectors, compositions of vectors, plasmids, compositions of plasmids, viruses, liposomes, and pharmaceutical compositions are all provided, each of which independently comprise any of the polypeptides or nucleic acid molecules provided for herein.

In some embodiments, cells and methods of producing cells are provided herein. In some embodiments, a method of producing a cell comprises contacting the cell with any vectors, compositions of vectors, plasmids, compositions of plasmids, viruses, liposomes, and pharmaceutical compositions provided for herein. In some embodiments, the cells are produced in vivo.

In some embodiments, methods of modifying an immune response in a subject are provided, the method comprising administering to the subject a vector, plasmid, virus, liposome, or compositions provided for herein. In some embodiments, methods of treating cancer in a subject are provided, the method comprising administering to the subject a vector, plasmid, virus, liposome, or compositions provided for herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts IL-21 signals of IL-21/IL-15/IL-12 polypeptides with and without a dual checkpoint inhibitor polypeptide.

FIG. 2 depicts IL-15, IL-12, and CTLA-4 signals of IL-21/IL-15/IL-12 polypeptides with and without a dual checkpoint inhibitor polypeptide.

FIG. 3 depicts levels of antigen reactive T cells for IL-21/1L-15/IL-12 polypeptides with and without a dual checkpoint inhibitor polypeptide and/or an antigen. FIG. 4 depicts levels of CD8+ percentage for IL-21/IL-15/IL-12 polypeptides with and without a dual checkpoint inhibitor polypeptide and/or an antigen.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms have the same meaning as is commonly understood by one of ordinary skill in the art to which the embodiments disclosed belongs. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The use of “or” means “and/or” unless stated otherwise. The use of the term “including,” as well as other forms, such as “includes” and “included,” is not limiting.

As used herein, the terms “a” or “an” means that “at least one” or “one or more” unless the context clearly indicates otherwise.

As used herein, the term “about” means that the numerical value is approximate and small variations would not significantly affect the practice of the disclosed embodiments. Where a numerical limitation is used, unless indicated otherwise by the context, “about” means the numerical value can vary by ±10% and remain within the scope of the disclosed embodiments.

As used herein, the term “individual” or “subject,” or “patient” used interchangeably, means any animal, including mammals, such as mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, such as humans.

As used herein, the terms “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”), are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. Any step or composition that uses the transitional phrase of “comprise” or “comprising” can also be said to describe the same with the transitional phase of “consisting of” or “consists.”

As used herein, the term “contacting” means bringing together of two elements in an in vitro system or an in vivo system. For example, “contacting” a vector with a cell or with an individual or patient or cell includes the administration of the vector to an individual or patient, such as a human, as well as, for example, introducing a compound into a sample containing a cellular- or purified preparation containing the cell. A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate. In contrast, a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.

“Effective amount” or “therapeutically effective amount” are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological result or provides a therapeutic or prophylactic benefit. Such results may include, but are not limited to, an amount that when administered to a mammal, causes a detectable level of immune cell activation compared to the immune cell activation detected in the absence of the composition. The immune response can be readily assessed by a plethora of art-recognized methods. The skilled artisan would understand that the amount of the composition administered herein varies and can be readily determined based on a number of 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, and the like.

“Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

“Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., Sendai viruses, lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

As used herein, the term “fused” or “linked” when used in reference to a protein having different domains or heterologous sequences means that the protein domains are part of the same peptide chain that are connected to one another with either peptide bonds or other covalent bonding. The domains or section can be linked or fused directly to one another, or another domain or peptide sequence can be between the two domains or sequences and such sequences would still be considered to be fused or linked to one another. In some embodiments, the various domains or proteins provided for herein are linked or fused directly to one another or a linker sequences, such as a glycine/serine sequence link the two domains together.

“Heterologous” as used herein refers to a non-native nucleic acid or amino acid sequence that is 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.

“Identity” as used herein refers to the subunit sequence identity between two polymeric 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 residues at the same positions, e.g., if a position in each of two polypeptide molecules is occupied by an Arginine, then they are identical at that position. The identity or extent to which two amino acid or two nucleic acid sequences have the same residues at the same positions in an alignment is often 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, e.g., if half of the positions in two sequences are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 of 10), are matched or identical, the two amino acids sequences are 90% identical.

As used herein “located” is meant to give positional clarity in an amino acid or nucleic acid sequence. For example, a sequence X that is said to be located between a first portion A and a second portion B would yield the potential formulas A-X-B or B-X-A.

In a similar manner, the term “upstream” is meant 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 located upstream of a first portion A would indicate that the sequence X is located prior to portion A such that the formula would read 5’-X-A-3'.

By "substantially identical" is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.

Sequence identity can be measured/determined using sequence analysis software (for example, 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 degrees 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 approach to determining the degree of identity, a BLAST program may be used, with a probability score between e3 and el 00 indicating a closely related sequence. In some embodiments, sequence identity is determined by using BLAST with the default settings.

To the extent embodiments provided for herein, includes composition comprising various proteins, these proteins may, in some instances, comprise amino acid sequences that have sequence identity to the amino acid sequences disclosed herein. Therefore, in certain embodiments, depending on the particular sequence, the degree of sequence identity is preferably greater than 50% (e.g., 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%; or more) to the SEQ ID NOs disclosed herein. These proteins may include homologs, orthologues, allelic variants and functional mutants. Typically, 50%- identity or more between two polypeptide sequences is considered to be 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.

These proteins may, compared to the disclosed proteins, include one or more (e.g., 1, 2, 3,4, 5, 6, 7, 8, 9, 10, etc.) conservative amino acid replacements i.e., replacements of one amino acid with another which has a related side chain. Genetically encoded amino acids are generally divided into four families: (1) acidic i.e. aspartate, glutamate; (2) basic i.e. lysine, arginine, histidine; (3) non polar i.e. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar i.e. glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. In general, Substitution of single amino acids within these families does not have a major effect on the biological activity. The proteins 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 sequences. The proteins 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) relative to the disclosed protein sequences.

As used herein, the phrase “in vivo” in reference to a cell being transduced, transfected or transformed in vivo, refers to a cell being transduced, transfected or transformed in the subject without the cells being removed from the subject before such cells are transduced, transfected or transformed.

“Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

By the term “modified” as used herein, is meant a changed state or structure of a molecule or cell as provided herein. Molecules may be modified in many ways, including chemically, structurally, and functionally, such as mutations, substitutions, insertions, or deletions (e.g. internal deletions truncations). Cells may be modified through the introduction of nucleic acids or the expression of heterologous proteins.

By the term “modulating,” as used herein, is meant mediating an increase or decrease in the level of a response in a subject compared with the level of a response in the subject in the absence of a treatment or compound, and/or compared with the level of a response in an otherwise identical but untreated subject. The term encompasses perturbing and/or affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject, such as, a human.

/Xs used herein, the following abbreviations for the commonly occurring nucleic acid bases are used: “A” refers to adenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.

Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some versions contain an intron(s).

The term “oligonucleotide” typically refers to short polynucleotides. It will 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 (i.e., A, U, C, G) in which “U” replaces “T.”

The term “polynucleotide” as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, the terms “nucleic acids” and “polynucleotides” as used herein are interchangeable. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any methods available in the art, including, without limitation, recombinant methods, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using cloning technology and PCR, and the like, and by synthetic means.

As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of a plurality of amino acid residues covalently linked by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

The term “subject” includes living organisms, including those in which an immune response can be elicited (e.g., mammals). As used herein, the term “subject” or “patient” or “individual” may be used interchangeably. A “subject”, as used herein, may be a human or non- human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, non-human primates, feline and murine mammals. In some embodiments, the subject is human.

As used herein, the phrase “in need thereof” means that the subject (animal or mammal) has been identified as having a need for the particular method or treatment. In some embodiments, the identification can be by any means of diagnosis. In any of the methods and treatments described herein, the animal or mammal can be in need thereof. In some embodiments, the animal or mammal is in an environment or will be traveling to an environment in which a particular disease, disorder, or condition is prevalent.

The terms “substituting,” “substituted,” “mutating,” or “mutated” as used herein refer to altering, deleting, or inserting one or more amino acids or nucleotides in a polypeptide or polynucleotide sequence to generate a variant of that sequence.

The term “therapeutic” as used herein means a treatment and/or prophylaxis. A therapeutic effect is obtained by suppression, remission, or eradication of a disease state.

The term “transfected” or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into a cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed, or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny. In some embodiments, the transfection, transformation, or transduction is performed or occurs in vivo.

To “treat” a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

As used herein, the term “variant” when used in conjunction to an amino acid sequence refers to a sequence that is at least, or about, 85%', 90%, 91 %, 92%, 93%, 94%, 95%', 96%, 97%, 98%, or 99% identical to the reference sequence. In some embodiments, the variant comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions. In some embodiments, the substitution is a conservative substitution.

A “vector” is a composition of matter which comprises an isolated nucleic acid encoding a protein or a peptide. Numerous 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 viral vectors, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.

Ranges: throughout this disclosure, various aspects of the embodiments can be presented in a 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 subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 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 breadth of the range. Unless otherwise explicitly stated to the contrary, a range that is disclosed also includes the endpoints of the range.

IL-12, IL-15, and IL-21 Polypeptides

Described herein are polypeptides comprising a IL-21 polypeptide, a IL- 12 polypeptide and a IL-15 polypeptide. In some embodiments, the polypeptide comprises a IL-21 polypeptide, a IL-12 p40 polypeptide, an IL-12 p35 polypeptide, an IL-15 polypeptide, and a IL-15Rα polypeptide. In some embodiments, the polypeptide comprises a IL-21 polypeptide, a IL- 12 p40 polypeptide, an IL-12 p35 polypeptide, an IL-15 polypeptide, a IL- 15Rα polypeptide, and/or a first or second fragment of a IL-15Rα polypeptide. In some embodiments, the IL-12 p40 polypeptide is from a human. In some embodiments, the IL-12 p40 polypeptide is from a mouse. In some embodiments, the IL-12 p35 polypeptide is from a human. In some embodiments, the IL-12 p35 polypeptide is from a mouse. In some embodiments, the IL-21 polypeptide is from a mouse. In some embodiments, the polypeptide can be used to stimulate an immune response. In some embodiments, the polypeptides can be used to activate by NK cells or CD8+ T cells. In some embodiments, the polypeptides can be used to treat cancer, such as those provided for herein, viral infections, bacterial infections, such as, but not limited to tuberculosis, listeriosis, and the like, and fungal infections.

In some embodiments, the 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 the IL- 12 polypeptide or can be linked through the use of a linker to link the p35 and p40 polypeptides together. In some embodiments, the linker is a polypeptide linker, such as those provided herein.

In some embodiments, the IL- 12 p40 polypeptide comprises an amino acid sequence of: MCHQQLV1SWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGI TW1TDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDG1WSTDIL KDQKEPKNKTFLRCE AKN YSGRFTCW WLTTISTDLTFS VKS SRGS S DPQG VTCG AATLS AERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPD PPKNLQLKI’LKNSRQVEVSWEYPDI'WSTPHSYFSLTFCVQVQGKSKREKKDRVFTD KT SATVICRKNASISVRAQDRYYSSSWSEWASVPCS (SEQ ID NO: 1), or an active fragment thereof. The IL- 12 p40 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-12 p40 polypeptide, or active fragment thereof, comprises an amino acid sequence of:

IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGn'WTLDQSSEVLGSGKTLTIQV K EFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGR FTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLS AERVRGDNKEYEYSVECQEDSA CPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDnKPDPPKNLQLKPLKNSRQVEVSWEY PDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSS SWSEWASVPCS (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 be the IL- 12 p40 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 be the IL- 12 p40 polypeptide. In some embodiments, 1, 2, 3, 4, or 5 amino acid residues are deleted from the N-terminus and/or the C-terminus of SEQ ID NO: 2 to be the IL-12 p40 polypeptide. In some embodiments, SEQ ID NOs: 1 and 2 are or are derived from human IL-12 p40.

In some embodiments, the IL- 12 p40 polypeptide comprises an amino acid sequence of: MCPQKLTISWFAIVLLVSPLMAMWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDI TWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILK NFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKV 1TDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPK N LQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLV EKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRS (SEQ ID NO: 3), or an active fragment thereof. The IL- 12 p40 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- 12 p40 polypeptide, or active fragment thereof, comprises an amino acid sequence of: MWELEKDVYVVEVDWTPDAPGETVNLTCDI’PEEDDITWTSDQRHGVIGSGKTLTITV K EFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCS WLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCP TAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDS WSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQD RY YNSSCSKWACVPCRVRS (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 be the IL- 12 p40 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 be the IL-12 p40 polypeptide. In some embodiments, 1, 2, 3, 4, or 5 amino acid residues are deleted from the N-terminus and/or the C-terminus of SEQ ID NO: 4 to be the IL-12 p40 polypeptide. In some embodiments, SEQ ID NOs: 3 and 4 are or are derived from mouse IL- 12 p-40.

In some embodiments, the IL- 12 p35 polypeptide comprises an amino acid sequence of: MCPARSLLLVATLVLLDHLSLARNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQT LEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSF M MALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETV PQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS (SEQ ID NO: 5), or an active fragment thereof. The IL- 12 p35 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- 12 p35 polypeptide, or active fragment thereof, comprises an amino acid sequence of: RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTST VEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSTYEDLKMYQVEFKTM NAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHA FRIRAVTIDRVMSYLNAS (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 be the IL-12 p35 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 be the IL-12 p35 polypeptide. In some embodiments, 1, 2, 3, 4, or 5 amino acid residues are deleted from the N-terminus and/or the C-terminus of SEQ ID NO: 6 to be the IL-12 p35 polypeptide. In some embodiments, SEQ ID NOs: 5 and 6 are or are derived from human IL-12 p35.

In some embodiments, the IL- 12 p35 polypeptide comprises an amino acid sequence of: MCQSRYLLFLATLALLNHLSLARVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYS CTAED1DHED1TRDQTSTLKTCLPLELHKNESCLATRETSSITRGSCLPPQKTSLMMTLC L GS1YEDLKMYQTEFQAINAALQNHNHQQ11LDKGMLVAIDELMQSLNHNGETLRQKPPV GEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSA (SEQ ID NO: 7), or an active fragment thereof. The IL- 12 p35 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- 12 p35 polypeptide, or active fragment thereof, comprises an amino acid sequence of: RVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCL PLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQ NHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTR VVTINRVMGYLSSA (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 be the IL- 12 p35 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 be the IL-12 p35 polypeptide. In some embodiments, 1 , 2, 3, 4, or 5 amino acid residues are deleted from the N-terminus and/or the C-terminus of SEQ ID NO: 8 to be the IL-12 p35 polypeptide. In some embodiments, SEQ ID NOs: 7 and 8 are or are derived from mouse IL- 12 p35.

In some embodiments, the IL-15 polypeptide comprises an amino acid sequence of: MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKI ED LIQSMHID ATL YTESD VHPSCK VT AM KCFLLELQ VISLES GD A SIHDTVENLI1L ANN S LS S NGNVTESGCKECEELEEKN1KEFLQSFVHIVQMFINTS (SEQ ID NO: 9), or an active fragment thereof. 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 active fragment thereof, comprises an amino acid sequence of: GIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMK CFLLELQVISLESGDAS1HDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQ SF VHIVQMFINTS (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 be 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 be the IL-15 polypeptide. In some embodiments, 1, 2, 3, 4, or 5 amino acid residues are deleted from the N-terminus and/or the C-terminus of SEQ ID NO: 6 to be the IL-15 polypeptide.

In some embodiments, the IL-15Rα polypeptide comprises an amino acid sequence of: LQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWT TPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIV P GSQLMPSKSPSTGTTE1SSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAI S TSTVLLCGLSAVSLLACYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL (SEQ ID NO: 11), or an active fragment thereof. In some embodiments, the IL-15Rα polypeptide, or active fragment thereof, comprises an amino acid sequence of:

TTCPPPMS VEHADIW VKS YSLYSRERYICNSGFKRKAGTSSLTEC VLNKATNVAHWTTPS LKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGS Q LMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTS T VLLCGLSAVSLLACYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL (SEQ ID NO: 12). In some embodiments, the first fragment of the IL-15Rα polypeptide comprises an amino acid sequence of:

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWT TPS LKCIRDPALVHQR (SEQ ID NO: 13). In some embodiments, the second fragment of the II..- 15Rα polypeptide comprises an amino acid sequence of:

PAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPST GTTEI SSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLA CYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL (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 be the 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 be the IL-15Rα. In some embodiments, 1 , 2, 3, 4, or 5 amino acid residues are deleted from the N-terminus and/or the C- terminus of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14 to be the IL- 15RA.

There are other isoforms of IL-15Rα that can be modified to produce an active polypeptide. The sequences of such isoforms can be found, for example at GenBank Accession Nos.: NP. 002180.1, AEP26933.1, AAP69528.1, AAH74726.1, NP.001243694.1,

XP 016871684.1, XP.011517767.1, NP„ 001230468.1, and AA107778.1, each of which is hereby incorporated by reference in its entirety. In some embodiments, the lL-15Rα polypeptide or transmembrane fragment thereof can be replaced with or substituted for other transmembrane domains known in the art.

In some embodiments, the IL-21 polypeptide comprises an amino acid sequence of: MERTLVCLVVIFLGTVAHKSSPQGPDRLLIRLRHLID1VEQLKIYENDLDPELLSAPQDV K GHCEHAAFACFQKAKLKPSNPGNNKTFIIDLVAQLRRRLPARRGGKKQKHIAKCPSCDS YEKRTPKEFLERLKWLLQKMIHQHLS (SEQ ID NO: 71), or an active fragment thereof. 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 be the 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 be the IL-21 polypeptide. In some embodiments, 1, 2, 3, 4, or 5 amino acid residues are deleted from the N-terminus and/or the C-terminus of SEQ ID NO: 71 to be the IL-21 polypeptide.

Although the sequences provided herein are human and mouse sequences, other orthologs can also be used because certain orthologs of the same proteins have the same or similar activity in a different species. For example, mouse IL- 12 is active on human cells, and, therefore, the p40 and/or p35 human subunits of IL- 12 can be replaced with the mouse orthologs provided herein. Similarly, IL-15, IL-15Rα and IL-21 polypeptides provided herein can be used in other animals because of the high degree of similarity (identity), regardless if the polypeptide is from a human or a mouse. Likewise, any of the polypeptides provided herein may be replaced with an ortholog from another animal. For example, the IL-21 polypeptides disclosed herein can be replaced with a IL-21 polypeptide from another animal, such as human IL-21.

In some embodiments, polypeptides comprising the formula of: X i-L i -X--1.2-X 3-L ₃ -X ₄ - L ₄ -X ₅ are provided, wherein: X ₁ is a interleukin 21 (IL-21) polypeptide, interleukin 15 (IL- 15) polypeptide, interleukin 12 p40 subunit (IL- 12 p40) polypeptide, interleukin 12 p35 subunit (IL- 12 p35) polypeptide, or interleukin 15 receptor (IL-15Rα) polypeptide; X ₂ is a IL-21 polypeptide, IL-15 polypeptide, IL-12 p40 polypeptide, IL-12 p35 polypeptide, or IL-15Rα polypeptide; X ₃ is a IL-21 polypeptide, IL-15 polypeptide, IL-12 p40 polypeptide, IL-12 p35 polypeptide, or IL-15Rα polypeptide; X ₄ is a IL-21 polypeptide, IL-15 polypeptide, IL-12 p40 polypeptide, IL-12 p35 polypeptide, or IL-15Rα polypeptide; X ₅ is a IL-21 polypeptide, IL-15 polypeptide, IL-12 p40 polypeptide, IL-12 p35 polypeptide, or IL-15Rα polypeptide; L ₁ , L ₂ , L ₃ , and L ₄ are each, independently, a polypeptide linker that comprise the same or different polypeptide sequences, and provided each of X ₁ , X ₂ , X ₃ , X ₄ , and X ₅ are different.

In some embodiments, X ₁ is a interleukin 21 (IL-21) polypeptide. In some embodiments, X ₁ is a IL-21 polypeptide; X ₂ is a IL-15 polypeptide; X ₃ is a IL-12 p40 polypeptide; X ₄ is a IL- 12 p35 polypeptide; and X ₅ is a IL-15Rα polypeptide. In some embodiments, X ₁ is a IL-21 polypeptide; X ₂ is a IL-15 polypeptide; X?, is a IL-12 p35 polypeptide; X ₄ is a IL-12 p40 polypeptide; and X ₅ is a IL-15Rα polypeptide. In some embodiments, X ₁ is a IL-21 polypeptide; X2 is a IL- 12 p40 polypeptide; X ₃ is a IL- 15 polypeptide; X ₄ is a IL- 12 p35 polypeptide; and X ₅ is a IL-15Rα polypeptide. In some embodiments, X ₁ is a IL-21 polypeptide; X ₂ is a IL-12 p40 polypeptide; X ₃ is a IL-12 p35 polypeptide; X ₄ is a IL-15 polypeptide; and X ₅ is a IL-15Rα polypeptide. In some embodiments, X ₁ is a IL-21 polypeptide; X ₂ is a IL-12 p35 polypeptide; X ₃ is a IL-12 p40 polypeptide; X ₄ is a IL-15 polypeptide; and X ₅ is a IL-15Rα polypeptide. In some embodiments, X ₁ is a IL-21 polypeptide; X ₂ . is a IL-12 p35 polypeptide; X ₃ is a IL-15 polypeptide; X ₄ is a IL-12 p40 polypeptide; and X ₅ is a IL-15Rα polypeptide. In some embodiments, X ₁ is a IL-21 polypeptide; X ₂ . is a IL-12 p40 polypeptide; X ₃ is a IL-12 p35 polypeptide; X ₄ is a IL-15 polypeptide; and X ₅ is a IL-15Rα polypeptide. In some embodiments, X ₁ is a IL-21 polypeptide; X ₂ is a IL-12 p40 polypeptide; X ₃ is a IL-15 polypeptide; X ₄ is a IL- 12 p35 polypeptide; and X ₅ is a IL-15Rα polypeptide. In some embodiments, X ₁ is a IL-21 polypeptide; X ₂ is a IL-12 p35 polypeptide; X ₃ is a IL-12 p40 polypeptide; X ₄ is a IL-15 polypeptide; and X ₅ is a IL-15Rα polypeptide. In some embodiments, X ₁ is a IL-21 polypeptide; X ₂ is a IL-12 p35 polypeptide; X ₃ is a IL-15 polypeptide; X ₄ is a IL-12 p40 polypeptide; and X ₅ is a lL-15Rα polypeptide.

In some embodiments, polypeptides comprising the formula of: X1-L ₁ -X ₂ -L ₂ -X ₃ -L ₃ -X ₄ - L ₄ -X ₅ -L ₅ -X6 are provided, wherein: X ₁ is a interleukin 21 (IL-21) polypeptide, interleukin 15 (IL-15) polypeptide, interleukin 12 p40 subunit (IL-12 p40) polypeptide, interleukin 12 p35 subunit (IL-12 p35) polypeptide, interleukin 15 receptor (IL-15Rα) polypeptide, a first fragment of IL-15Rα polypeptide, or a second fragment of IL-15Rα polypeptide; X ₂ is a IL-21 polypeptide, IL-15 polypeptide, IL-12 p40 polypeptide, IL-12 p35 polypeptide, IL-15Rα polypeptide, a first fragment of IL-11Rα polypeptide, or a second fragment of IL-15Rα polypeptide; X ₃ is a IL-21 polypeptide, IL-15 polypeptide, IL-12 p40 polypeptide, IL-12 p35 polypeptide, IL-15Rα polypeptide, a first fragment of IL-15Rα polypeptide, or a second fragment of IL-15Rα polypeptide;

X ₄ is a IL-21 polypeptide, IL- 15 polypeptide, IL- 12 p40 polypeptide, IL- 12 p35 polypeptide, IL-15Rα polypeptide, a first fragment of lL-15Rα polypeptide, or a second fragment of IL-15Rα polypeptide; X ₅ is a IL-21 polypeptide, IL-15 polypeptide, IL-12 p40 polypeptide, IL-12 p35 polypeptide, IL-15Rα polypeptide, a first fragment of IL-15Rα polypeptide, or a second fragment of IL-15Rα polypeptide;

X ₆ is a IL-21 polypeptide, IL-15 polypeptide, IL-12 p40 polypeptide, IL-12 p35 polypeptide, IL-15R(X polypeptide, a first fragment of IL-15Rα polypeptide, or a second fragment of IL-15Rα polypeptide; L ₁ , L ₂ , L ₃ , L ₄ , and L ₅ are each, independently, a polypeptide linker that comprise the same or different polypeptide sequences, provided each of X ₁ , X ₂ , X ₃ , X ₄ , X ₅ , and Xs are different.

In some embodiments, X ₁ is a interleukin 21 (IL-21) polypeptide. In some embodiments, X ₁ is a IL-21 polypeptide; X ₂ is a IL-15 polypeptide; X ₃ is a first fragment of IL-15Rα polypeptide; X ₄ is a IL-12 p40 polypeptide; X ₅ is a IL-12 p35 polypeptide; and Xs is a second fragment of IL-15Rα polypeptide. In some embodiments, X ₁ is a IL-21 polypeptide; X ₂ is a IL- 15 polypeptide; X ₃ is a first fragment of IL- 15Rα polypeptide; X ₄ is a IL- 12 p35 polypeptide; X ₅ is a IL- 12 p40 polypeptide; and Xs is a second fragment of IL-15Rα polypeptide. In some embodiments, X ₁ is a IL-21 polypeptide; X ₂ is a IL-15 polypeptide; X ₃ is a IL-12 p40 polypeptide: X ₄ is a first fragment of IL-15R.0C polypeptide; X ₅ is a IL- 12 p35 polypeptide; and Xs is a second fragment of IL-15Rα polypeptide. In some embodiments, X ₁ is a IL-21 polypeptide; X ₂ is a IL-15 polypeptide; X ₃ is a IL-12 p40 polypeptide; X ₄ is a IL- 12 p35 polypeptide; X ₅ is a first fragment of IL-15Rα polypeptide; and X6 is a second fragment of IL- 15Rα polypeptide. In some embodiments, X ₁ is a IL-21 polypeptide; X ₂ is a IL-15 polypeptide; X ₃ is a IL- 12 p35 polypeptide; X ₄ is a IL-12 p40 polypeptide; X ₅ is a first fragment of IL-15Rα polypeptide; and X ₆ is a second fragment of IL-15Rα polypeptide. In some embodiments, X ₁ is a IL-21 polypeptide; X ₂ is a IL- 15 polypeptide; X ₃ is a IL- 12 p35 polypeptide; X ₄ is a first fragment of IL-15RCX polypeptide; X ₅ is a IL- 12 p40 polypeptide; and Xs is a second fragment of IL- 15Rα polypeptide. In some embodiments, X ₁ is a IL-21 polypeptide; X ₂ is a first fragment of IL-15Rα. polypeptide; X ₃ is a IL- 15 polypeptide; X ₄ is a IL- 12 p40 polypeptide; X ₅ is a IL- 12 p35 polypeptide; and Xs is a second fragment of IL-15Rα polypeptide. In some embodiments, X ₁ is a IL-21 polypeptide; X ₂ is a first fragment of IL-15Rα polypeptide; X ₃ is a IL-15 polypeptide; X ₄ is a IL- 12 p35 polypeptide; X ₅ is a IL-12 p40 polypeptide; and Xs is a second fragment of IL-15Rα polypeptide. In some embodiments, X ₁ is a IL-21 polypeptide; X 2 is a first fragment of IL-15Rα polypeptide; X ₃ is a IL-12 p40 polypeptide; X ₄ is a IL-15 polypeptide; X ₅ is a IL- 12 p35 polypeptide; and Xs is a second fragment of IL-15Rα polypeptide. In some embodiments, X ₁ is a IL-21 polypeptide; X ₂ is a first fragment of IL-15Rα polypeptide; X ₃ is a IL-12 p40 polypeptide; X ₄ is a IL-12 p35 polypeptide; X ₅ is a IL-15; and Xs is a second fragment of IL-15Rα polypeptide. In some embodiments, X ₁ is a IL-21 polypeptide; X ₂ is a first fragment of IL-15Rα polypeptide; X ₃ is a IL-12 p35 polypeptide; X ₄ is a IL- 12 p40 polypeptide; X ₅ is a IL-15; and Xs is a second fragment of IL-15Rα polypeptide. In some embodiments, X] is a IL-21 polypeptide; X ₂ is a first fragment of IL-15Rα polypeptide; X ₃ is a IL- 12 p35 polypeptide; X ₄ is a IL-15; X ₅ is a IL-12 p40 polypeptide; and Xs is a second fragment of IL-15Rα polypeptide. In some embodiments, X] is a IL-21 polypeptide; X ₂ is a IL-12 p40 polypeptide; X ₃ is a IL-15 polypeptide; X ₄ is a first fragment of IL-15Rα polypeptide; X ₅ is a IL-12 p35 polypeptide; and Xs is a second fragment of IL-15Rα polypeptide. In some embodiments, X ₁ is a IL-21 polypeptide; X ₂ is a IL-12 p40 polypeptide; X ₃ is a IL-15 polypeptide; X ₄ is a IL-12 p35 polypeptide; X ₅ is a first fragment of IL-15R(X polypeptide; and Xs is a second fragment of IL- 15Rα polypeptide. In some embodiments, X ₁ is a IL-21 polypeptide; X ₂ is a IL- 12 p40 polypeptide: X ₃ is a first fragment of IL-15R.0C polypeptide; X ₄ is a IL- 15 polypeptide; X ₅ is a IL- 12 p35 polypeptide; and X ₆ is a second fragment of IL-15Rα polypeptide. In some embodiments, X ₁ is a IL-21 polypeptide; X ₂ is a IL-12 p40 polypeptide; X ₃ is a first fragment of IL-15Rα polypeptide; X ₄ is a IL-12 p35 polypeptide; X ₅ is a IL-15; and X& is a second fragment of IL- 15Rα polypeptide. In some embodiments, X ₁ is a IL-21 polypeptide; X ₂ is a IL-12 p40 polypeptide; X ₃ is a IL-12 p35 polypeptide; X ₄ is a first fragment of IL-15Rα polypeptide; X ₅ is a IL-15; and X ₆ is a second fragment of IL-15Rα polypeptide. In some embodiments, X ₁ is a IL- 21 polypeptide; X ₂ is a IL-12 p40 polypeptide; X ₃ is a IL-12 p35 polypeptide; X ₄ is a IL-15; X ₅ is a first fragment of IL-15Rα polypeptide; and X& is a second fragment of IL-15Rα polypeptide. In some embodiments, X ₁ is a IL-21 polypeptide; X ₂ is a IL-12 p35 polypeptide; X ₃ is a IL-15 polypeptide; X ₄ is a first fragment of TL-15R(X polypeptide; X ₅ is a IL-12 p40 polypeptide; and X ₆ is a second fragment of IL-15Rα polypeptide. In some embodiments, X ₁ is a IL-21 polypeptide; X ₂ is a IL-12 p35 polypeptide; X ₃ is a IL-15 polypeptide; X ₄ is a IL-12 p40 polypeptide; X ₅ is a first fragment of IL-15Rα polypeptide; and X ₆ is a second fragment of IL- 15Rα polypeptide. In some embodiments, X-. is a IL-21 polypeptide; X ₂ is a IL-12 p35 polypeptide; X ₃ is a first fragment of IL-15Rα polypeptide; X ₄ is a IL-15 polypeptide; X ₅ is a IL- 12 p40 polypeptide; and X ₆ is a second fragment of IL-15Rα polypeptide. In some embodiments, X ₁ is a IL-21 polypeptide; X ₂ is a IL-12 p35 polypeptide; X ₃ is a first fragment of IL-15Rα polypeptide; X ₄ is a IL- 12 p40 polypeptide; Xs is a IL- 15; and X ₆ is a second fragment of IL- 15Rα polypeptide. In some embodiments, X ₁ is a IL-21 polypeptide; X ₂ is a IL- 12 p35 polypeptide; X ₃ is a IL- 12 p40 polypeptide; X ₄ is a first fragment of IL-15Rα. polypeptide; X ₅ is a IL-15; and X ₆ is a second fragment of IL-15Rα polypeptide. In some embodiments, X-. is a IL- 21 polypeptide; X ₂ is a IL-12 p35 polypeptide; X ₃ is a IL-12 p40 polypeptide; X ₄ is a IL-15; Xs is a first fragment of IL-15Rα polypeptide; and X ₆ is a second fragment of IL-15Rα polypeptide.

In some embodiments, any polypeptide described herein may have one or more elements, polypeptides, or linkers absent. For example, in some embodiments, polypeptides having the formula of X :-l..: - X -l .'-X -.-l .i-X.i. are provided, wherein: X ₁ is a interleukin 21 (IL-21) polypeptide, interleukin 15 (IL- 15) polypeptide, interleukin 12 p40 subunit (IL- 12 p40) polypeptide, interleukin 12 p35 subunit (IL-12 p35) polypeptide, or interleukin 15 receptor (IL-15Rα) polypeptide; X ₂ is a IL-21 polypeptide, IL- 15 polypeptide, IL- 12 p40 polypeptide, IL- 12 p35 polypeptide, or IL-15Rα polypeptide; X ₃ is a IL-21 polypeptide, IL- 15 polypeptide, IL- 12 p40 polypeptide, IL- 12 p35 polypeptide, or IL-15Rα polypeptide; X ₄ is a IL-21 polypeptide, IL- 15 polypeptide, IL- 12 p40 polypeptide, IL- 12 p35 polypeptide, or IL-15Rα polypeptide; L ₁ , L ₂ , and L ₃ , are each, independently, a polypeptide linker that comprise the same or different polypeptide sequences, and provided each of X ₁ , X ₂ , X ₃ , and X ₄ are different.

In some embodiments, polypeptides having the formula of X1-L ₁ -X ₂ -L ₂ -X ₃ , are provided, wherein: X ₁ is a interleukin 21 (IL-21) polypeptide, interleukin 15 (IL- 15) polypeptide, interleukin 12 p40 subunit (IL- 12 p40) polypeptide, interleukin 12 p35 subunit (IL-12 p35) polypeptide, or interleukin 15 receptor (IL-15Rα) polypeptide;

X ₂ is a IL-21 polypeptide, IL-15 polypeptide, IL- 12 p40 polypeptide, IL-12 p35 polypeptide, or IL-15Rα polypeptide; X ₃ is a IL-21 polypeptide, IL- 15 polypeptide, IL- 12 p40 polypeptide, IL-12 p35 polypeptide, or IL-15Rα polypeptide; L ₁ and I..2, are each, independently, a polypeptide linker that comprise the same or different polypeptide sequences, and provided each of X ₁ , X ₂ , and X ₃ are different.

In some embodiments, polypeptides having the formula of X1-L ₁ -X ₂ , are provided, wherein:

X ₁ . is a interleukin 21 (IL-21) polypeptide, interleukin 15 (IL-15) polypeptide, interleukin 12 p40 subunit (IL-12 p40) polypeptide, interleukin 12 p35 subunit (IL-12 p35) polypeptide, or interleukin 15 receptor (IL-15Rα) polypeptide; X ₂ is a IL-21 polypeptide, IL- 15 polypeptide, IL- 12 p40 polypeptide, IL- 12 p35 polypeptide, or IL- 15Rα polypeptide; L ₁ is a polypeptide linker that comprise the same or different polypeptide sequences, and provided each of X ₁ and X ₂ are different.

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

In some embodiments, the IL-12 p35 polypeptide comprises an amino acid having of 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 to a IL-12 p35 polypeptide as provided for herein. In some embodiments, the IL-12 p35 polypeptide comprises an amino acid sequence of SEQ ID NO: 5, 6, 7, or 8, or a IL- 12 p35 polypeptide as provided for herein.

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 to an IL-15 polypeptide as provided for herein. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence of SEQ ID NO: 9 or 10, or an IL-15 polypeptide as provided for herein.

In some embodiments, the IL-15Rα polypeptide comprises an amino acid sequence having of 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 to an IL-15Rα polypeptide as provided for herein. In some embodiments, the IL-15Rα polypeptide comprises an amino acid sequence of SEQ ID NO: 1 1 SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14 or an IL-15Rα polypeptide as provided for herein.

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

The linkers that are referenced as L ₁ , L ₂ , L ₃ , L ₄ , or L ₅ are linkers that can be used to link the different polypeptides provided for herein. Although the linkers of L ₁ , L ₂ , L ₃ , L ₄ , and L ₅ 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, L ₁ , L ₂ , L ₃ , L ₄ , and L ₅ are the same, or each is different, or some are the same and others are different. In some embodiments, L ₁ , Lz, L ₃ , L ₄ , and L ₅ are peptide linkers. In some embodiments, the peptide linker is a cleavable linker. Without being bound by theory, cleavable linkers comprise at least one cleavage site capable of being recognized and cleaved by an enzyme, which can be referred to a protease. For example, in some embodiments, the enzyme furin recognizes the cleavage site with 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 the place of a 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). Nonlimiting examples of cleavable linkers are provided for herein and below.

Accordingly, in some embodiments, one or more of L ₁ ,L ₂ , L ₃ , L ₄ , or L ₅ are each, independently, a cleavable linker. In some embodiments at least one of L ₁ , L ₂ , L ₃ , L ₄ , or L ₅ are a cleavable linker. In some embodiments, L ₁ is a cleavable linker and the remaining linkers are non-cleavable linkers or not known to be cleavable. In some embodiments, L ₂ is a cleavable linker and the remaining linkers are non-cleavable linkers or not known to be cleavable. In some embodiments, L ₃ is a cleavable linker and the remaining linkers are non-cleavable linkers or not known to be cleavable. In some embodiments L ₁ and L ₂ are cleavable linkers, and the remaining linkers are non-cleavable linkers or not known to be cleavable. In some embodiments, none of L ₁ , L ₂ , L ₃ , L ₄ , or L ₅ 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 illustrated in the table below and can be chosen individually from such table. In some embodiments, one or more of L ₁ , L ₄ , L ₃ , L ₄ , and L ₅ comprise 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), (AEEEK)n (SEQ ID NO: 21), wherein each n is, independently, from 1 and 5, or a combination thereof. 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 comprises a sequence of GSGSGGGSGSGGGSGSGG (SEQ ID NO: 22). In some embodiments, each of L ₁ , L ₂ , L ₃ , L ₄ , and L ₅ comprises a sequence of GSGSGGGSGSGGGSGSGG (SEQ ID NO: 22).

Other non-limiting examples of linkers include a glycine/serine linker can be, or comprise, a sequence of GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 23) or comprise a sequence of GGGGSGGGGSGGGGS (SEQ ID NO: 24). This is simply a non-limiting example, and the linker can have varying number of GGGGS (SEQ ID NO: 17) repeats as provided for herein. In some embodiments, the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the GGGGS (SEQ ID NO: 17) repeats. 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 illustrated in the following table:

In some embodiments, the polypeptide can comprise a leader peptide on the N-tenninus of the polypeptide. The leader peptide can be used, without being bound to any particular theory, to facilitate in the expression and trafficking of the polypeptide as it is generated by the cell so that, for example, it can be expressed on the surface of the cell. In some embodiments, the leader peptide comprises a sequence of vesicular stomatitis virus G protein (VSV-G). In some embodiments, the leader peptide comprises, consists, or consists essentially of a sequence of MRISKPHLRSISIQCYLCLLLNSHFLTEA (SEQ ID NO: 15).

In some embodiments, the polypeptide comprises a sequence that is at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 % identical or is identical to a polypeptide comprising the sequence of:

MERTLVCLVVIFLGTVAHKSSPQGPDRLLIRLRHLID1VEQLKIYENDLDPELLSAP QDVK GHCEHAAFACFQKAKLKPSNPGNNKTFIIDLVAQLRRRLPARRGGKKQKH1AKCPSCDS YEKRTPKEFLERLKWLLQKMIHQHLSSGGGPGGGGRAKRGGGGPSGGGSLQGIHVFILG CFSAGLPKTEANWVNV1SDLKKIEDL ₁ QSMHIDATLYTESDVHPSCKVTAMKCFLLELQV ISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQM FI NTSSGGGPGGGGRAKRGGGGPSGGGSLQIWELKKDVYVVELDWYPDAPGEMVVLTCD TPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGI WSTD1LKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTC GAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFI RDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDR VFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGSGSGGGSGSGGGSGSGSR NLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVE ACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNA KLLMDPKRQIFLDQNMLAVTDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFR IRAVTIDRVMSYLNASGSGGSGSGGSGSGGSITCPPPMSVEHADIWVKSYSLYSRERYIC NSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQ PESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQT TA KNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVE MEAMEALPVTWGTSSRDEDLENCSHHL (SEQ ID NO: 72).

In some embodiments, the polypeptide comprises a sequence that is at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 % identical or is identical to a polypeptide comprising the sequence of: MERTLVCL V VIFLGTVAHKSSPQGPDRLLIRLRHLIDIVEQLKIYEN DLDPELLS APQD VK GHCEHAAFACFQKAKLKPSNPGNNKTFIIDLVAQLRRRLPARRGGKKQKHIAKCPSCDS YEKRTPKEFLERLKWLLQKMIHQHLSSGGGPGGGGRAKRGGGGPSGGGSLQGIHVFILG CFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATEYTESDVHPSCKVTAMKCFLLELQV ISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQM FI NTSSGGGPGGGGRAKRGGGGPSGGGSLQMWELEKDVYVVEVDWTPDAPGETVNLTC DTPEEDDITWTSDQRHGVIGSGKTLTITVKEH..DAGQYTCHKGGETLSHSHLLLHKKEN GIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCG MASES AEKVTLDQRDYEKYSVSCQEDVTCPTAEETL.PIEL.AL.EARQQNKYENYSTSFFIR DIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEG CNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRSGSGSGGGSG SGGGSGSGSRVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRD QTSTLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTE FQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKL CILLHAFSTRVVTINRVMGYLSSAGSGGSGSGGSGSGGSITCPPPMSVEHADIWVKSYSL YSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVT TAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESS HG TPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQT PPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL (SEQ ID NO: 73).

In some embodiments, the polypeptide comprises a sequence that is at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 % identical or is identical to a polypeptide comprising the sequence of: MERTLVCLVVIFLGTVAHKSSPQGPDRLLIRLRHLIDIVEQLKIYENDLDPELLSAPQDV K GHCEHAAFACFQKAKLKPSNPGNNKTF11DLVAQLRRRLPARRGGKKQKHIAKCPSCDS YEKRTPKEFLERLKWLLQKMIHQHLSSGGGPGGGGSAGSGGGGPSGGGSLQGIHVFILG CFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQV ISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQM F1 NTSSGGGPGGGGRAKRGGGGPSGGGSLQ1WELKKDVYVVELDWYPDAPGEMVVLTCD TPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGI WSTDILKDQKEPKNKI'FLRCEAKNYSGRFTCWWLITISTDLTFSVKSSRGSSDPQGVTC GAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFI RDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDR VFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGSGSGGGSGSGGGSGSGSR NLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVE ACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNA KLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFR IRAVTIDRVMSYLNASGSGGSGSGGSGSGGSITCPPPMSVEHADIWVKSYSLYSRERYIC NSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQ PESLSPSGKEPAASSPSSNNTA ATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTA KNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVE MEAMEALPVTWGTSSRDEDLENCSHHL (SEQ ID NO: 74).

In some embodiments, the polypeptide comprises a sequence that is at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 % identical or is identical to a polypeptide comprising the sequence of: MERTLVCLVVIFLGTVAHKSSPQGPDRLLIRLRHLIDIVEQLKIYENDLDPELLSAPQDV K GHCEHAAFACFQKAKLKPSNPGNNKTFIIDLVAQLRRRLPARRGGKKQKHIAKCPSCDS YEKRTPKEFLERLKWLLQKMMQHLSSGGGPGGGGSAGSGGGGPSGGGSLQGIHVFILG CFSAGLPKTEANWVNVISDLKKIEDL ₁ QSMHIDATLYTESDVHPSCKVTAMKCFLLELQV ISLESGDAS1HDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVH1VQM FI NTSSGGGPGGGGRAKRGGGGPSGGGSLQMWELEKDVYVVEVDWTPDAPGETVNLTC DTPEEDD1TWTSDQRHGVIGSGKTLTI1VKEFLDAGQYTCHKGGETLSHSHLLLHKKEN GIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFN1KSSSSSPDSRAVTCG MASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIR DIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEG CNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRSGSGSGGGSG SGGGSGSGSRVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRD QTSTLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGS1YEDLKMYQTE FQA1NAALQNHNHQQIILDKGMLVAIDELMQSLNHNGE1TRQKPPVGEADPYRVKMKL CILLHAFSTRVVTINRVMGYLSSAGSGGSGSGGSGSGGSITCPPPMSVEHADIWVKSYSL YSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVT TAGVTPQPESLSPSGKEPAASSPSSNNTAATTAA1VPGSQLMPSKSPSTGTTEISSHESS HG TPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQT PPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL (SEQ ID NO: 75).

In some embodiments, the polypeptide comprises a sequence that is at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 % identical or is identical to a polypeptide comprising the sequence of: MERTLVCLVVIFLGTVAHKSSPQGPDRLLIRLRHLIDIVEQLKIYENDLDPELLSAPQDV K GHCEHAAFACFQKAKLKPSNPGNNKTFIIDLVAQLRRRLPARRGGKKQKHIAKCPSCDS YEKRTPKEFLERLKWLLQKMIHQHLSSGGGPGGGGRAKRGGGGPSGGGSLQGIHVFILG CFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATT.YTESDVHPSCKVTAMKCFLLELQ V ISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQM FI NTSSGGGSGGGGSGGGGSGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNS GFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRGSGGSGSGGSGSGGSGS GGSMWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTI TVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGR FTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQED VTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWE Y PDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQ AQDRYYNSSCSKWACVPCRVRSGSGSGGGSGSGGGSGSGGRVIPVSGPARCLSQSRNLL KTrDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETSST TRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQI1LDKGMLVAID ELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSASGG GGPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTG T TEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSD1TVAISTSTVLLCGLSAVS L LACYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL (SEQ ID NO: 76). In some embodiments, the polypeptide comprises a sequence that is at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 % identical or is identical to a polypeptide comprising the sequence of:

MERTLVCLVVIFLGTVAHKSSPQGPDRLLIRLRHLIDIVEQLKIYENDLDPELLSAP QDVK GHCEHAAFACFQKAKLKPSNPGNNKTF11DLVAQLRRRLPARRGGKKQKHIAKCPSCDS YEKRTPKEFLERLKWLLQKMIHQHLSSGGGPGGGGRAKRGGGGPSGGGSLQGIHVFILG CFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVl'AMKCFLLELQ V ISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQM FI NTSSGGGSGGGGSGGGGSGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNS GFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRGSGGSGSGGSGSGGSGS GGSIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTI QVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKN YSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQ EDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEV S WEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRY YSSSWSEWASVPCSGSGSGGGSGSGGGSGSGGRNLPVATPDPGMFPCLHHSQNLLRAV SNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNG S CI,ASRKTSFMMALCL.SSIYEDLKMYQVEFKTMNAKIJ..MDPKRQIFLDQNMI,AVID EL.M QALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASSGGGGPAP P STVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISS HE SSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLK SRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL (SEQ ID NO: 77).

In some embodiments, the polypeptide comprises a sequence that is at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 % identical or is identical to a polypeptide comprising the sequence of:

MERTLVCLVVIFLGTVAHKSSPQGPDRLLIRLRHLID1VEQLKIYENDLDPELLSAP QDVK GHCEHAAFACFQKAKLKPSNPGNNKTFIIDLVAQLRRRLPARRGGKKQKHIAKCPSCDS YEKRTPKEFLERLKWLLQKMIHQHLSSGGGPGGGGSAGSGGGGPSGGGSLQGIHVF1LG CFSAGLPKTEANWVNVISDLKKIEDL ₁ QSMHIDATLYTESDVHPSCKVTAMKCFLLELQV ISLESGDAS1HDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVH1VQM FI NTSSGGGSGGGGSGGGGSGGGSGGGSLQITCPPPMSVEHADrWVKSYSLYSRERYICNS GFKRKAGTSSLTECVLNKATNVAHWTTPSLKC1RDPALVHQRGSGGSGSGGSGSGGSGS GGSMWELEKDVYVVEVDWTPDAPGEI'VNLTCDTPEEDDITWTSDQRHGVIGSGKTLTl 1VKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGR FTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKV1WQRDYEKYSVSCQED VTCPTAEETLPJELALEARQQNKYENYSTSFFIRDllKPDPPKNLQMKPLKNSQVEVSWE Y PDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQ AQDRYYNSSCSKWACVPCRVRSGSGSGGGSGSGGGSGSGGRVIPVSGPARCLSQSRNLL KTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETSST TRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAID ELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSASGG GGPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTG T TEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVS L LACYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL (SEQ ID NO: 78),

In some embodiments, the polypeptide comprises a sequence that is at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 % identical or is identical to a polypeptide comprising the sequence of: MERTL.VCL,VVIFLGTVAHKSSPQGPDRL,LIRI,RHUDIVEQL,KIYENDL,DPEIJ. ,SAPQDVK GHCEHAAFACFQKAKLKPSNPGNNKTFIIDLVAQLRRRLPARRGGKKQKHIAKCPSCDS YEKRTPKEFLERLKWLLQKMIHQHLSSGGGPGGGGSAGSGGGGPSGGGSLQGIHVFILG CFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQV ISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQM FI NTSSGGGSGGGGSGGGGSGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNS GFKRKAGTSSLTECVLNKATNVAirWTTPSLKCIRDPALVHQRGSGGSGSGGSGSGGSGS GGSIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTI QVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGTWSTDILKDQKEPKNKTFLRCEAKN YSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQ EDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEV S WEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRY YSSSWSEWASVPCSGSGSGGGSGSGGGSGSGGRNLPVATPDPGMFPCLHHSQNLLRAV SNMLQKARQTLEFYPCTSEEIDHEDFrKDKTSTVEACLPLELTKNESCLNSRETSFITNG S CLASRKTSFMMALCLSSIYEDLKMYQVEFXTMNAKLLMDPKRQIFWQNMLAVIDELM QALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASSGGGGPAP P STV1TAGVTPQPESLSPSGKEPAASSPSSNNTAA1TAAIVPGSQLMPSKSPSTG1TEISS HE SSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLK SRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL (SEQ ID NO: 79). 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 illustrated with specific peptide linkers, these linkers can be modified or replaced with other peptide linkers, such as, but not limited to as those provided for herein.

The polypeptides described herein also encompass variants of the peptides provided for herein. In some embodiments, the polypeptides comprise a sequence of amino acids 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 for herein. These variants include those that are described herein with 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 substitution is a conservative substitution. In some embodiments, the conservative substitution is selected based upon the following tables:

The percent identity of two amino acid or two nucleic acid sequences can be determined by visual inspection and mathematical calculation, or for example, the comparison is done by comparing sequence information using a computer program. An exemplary computer program is the Genetics Computer Group (GCG; Madison, Wis.) Wisconsin package version 10.0 program, GAP (Devereux et al. (1984), Nucleic Acids Res. 12: 387-95). The preferred default parameters for the GAP program includes: (1) The GCG implementation of a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted amino acid comparison matrix of Gribskov and Burgess, ((1986) Nucleic Acids Res. 14: 6745) as described in Atlas of Polypeptide Sequence and Structure, Schwartz and Dayhoff, eds., National Biomedical Research Foundation, pp. 353-358 (1979) or other comparable comparison matrices; (2) a penalty of 8 for each gap and an additional penalty of 2 for each symbol in each gap for amino acid sequences, or a penalty of 50 for each gap and an additional penalty of 3 for each symbol in each gap for nucleotide sequences; (3) no penalty for end 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.

In some embodiments, a nucleic acid molecule (e.g., DNA or RNA) encoding a polypeptide provided for herein are provided. In some embodiments, the nucleic acid molecule comprises of a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%' or 100% identity to ATGGAGAGGACACTTGTTTGTCTCGTAGTGATATTCCTGGGGACCGTGGCACACAAGTCC TCTC CACAAGGACCCGACCGCCTTCTCATCAGACTGCGACACCTTATAGATATTGTGGAGCAAC TCAA GATTTATGAGAATGATTTGGACCCCGAACTCTTGTCTGCTCCTCAAGACGTTAAAGGCCA TTGT GAACATGCCGCGTTTGCTTGCTTTCAGAAGGCTAAGCTCAAGCCAAGTAATCCTGGCAAC AACA AGACGTTCATAATTGATCTTGTCGCTCAGCTTAGGCGCCGCCTGCCCGCACGACGGGGGG GAAA AAAGCAGAAGCATATCGCCAAGTGCCCAAGCTGTGATTCTTATGAAAAACGAACTCCTAA AGAA TTTTTGGAACGCCTCAAATGGCTTCTCCAAAAAATGATACATCAACACCTTTCCTCAGGA GGCG GGCCAGGTGGTGGGGGTAGGGCTAAGAGGGGTGGTGGGGGCCCAAGTGGGGGCGGCTCCT TGCA GGGCATACACGTCTTTATTCTTGGATGTTTTTCTGCGGGGTTGCCTAAGACAGAAGCAAA TTGG G T AAAC G T C AT C AG T G AT T T G AAAAAG AT C GAG GAT C T CAT T GAG AG CAT G GAG AT T GAG G C T A CGTTGTATACTGAATCCGACGTTCACCCGTCATGTAAAGTCACTGCCATGAAATGTTTCC TCTT GGAGCTCCAGGTCATCTCCCTGGAAAGCGGCGACGCTAGTATTCATGATACAGTTGAAAA CCTG ATCATTCTTGCAAACAATAGCCTCAGTAGTAACGGCAACGTGACCGAGTCCGGCTGTAAG GAGT G T G AAG AAC T C G AG G AG AAG AAC AT C AAAG AAT T C T T G C AG AG T T T C G T T C AT AT T G T G C AAAT GTTCATCAACACTTCCTCAGGAGGCGGGCCAGGTGGTGGGGGTAGGGCTAAGAGGGGTGG TGGG GGCCCAAGTGGGGGCGGCTCCTTGCAGATGTGGGAATTGGAGAAAGACGTTTATGTTGTG GAGG TGGACTGGACTCCAGACGCGCCCGGTGAGACAGTAAACCTGACTTGCGACACCCCTGAGG AGGA CGACATTACATGGACATCAGACCAGAGGCACGGGGTTATAGGATCCGGTAAGACCCTCAC TATT ACCGTGAAAGAGTTCCTTGATGCAGGTCAGTACACTTGTCATAAGGGAGGGGAAACCCTT TCCC ACAGTCACCTTCTGCTGCACAAGAAAGAGAATGGTATATGGTCCACTGAAATCCTTAAAA ATTT TAAGAATAAAACGTTTCTCAAATGTGAGGCCCCAAACTATAGCGGGAGGTTCACGTGCTC TTGG T T GGT C C AAAGAAAT AT G G AT T T G AAAT T C AAC AT T AAAT CATC TAG T AG C T C AC C AGAC AGT A GGGCTGTGACTTGCGGCATGGCATCACTGTCCGCTGAAAAAGTGACCCTTGATCAACGAG ACTA CGAAAAATATTCTGTCAGCTGCCAGGAGGATGTGACCTGCCCGACTGCTGAAGAGACGCT GCCC ATCGAACTGGCGCTGGAGGCGAGGCAACAGAATAAATATGAAAATTACAGTACTTCATTC TTTA T AC GGG AC AT T AT C AAGC C AGAC C C T C C C AAG AAC C T C C AAAT G AAAC C T C T C AAAAAC AG T C A GGTAGAGGTCTCATGGGAATACCCGGATAGTTGGTCTACACCCCATTCTTATTTCTCCCT TAAA TTTTTTGTTAGGATACAACGGAAGAAGGAGAAGATGAAAGAAACGGAAGAAGGTTGTAAC CAGA AGGGCGCATTTCTCGTAGAAAAAACTTCAACCGAGGTACAATGTAAAGGAGGGAACGTTT GTGT CCAAGCTCAAGATAGGTATTATAACAGCTCATGTAGCAAGTGGGCGTGCGTCCCGTGTAG GGTT CGCAGTGGATCAGGTAGTGGCGGCGGATCCGGGAGTGGCGGGGGCAGCGGGTCTGGTTCC CGGG TCATTCCAGTTTCAGGGCCGGCCAGGTGTCTTTCCCAAAGTCGGAACCTTCTCAAGACAA CGGA TGACATGGTCAAGACTGCGAGGGAAAAGCTTAAACACTACTCTTGTACCGCCGAGGACAT CGAC CATGAAGATATTACGCGGGATCAAACCAGTACGTTGAAAACCTGTCTCCCTCTTGAGCTT CATA AGAATGAGTCTTGTCTGGCAACGAGGGAAACATCATCAACTACACGAGGTAGTTGCTTGC CGCC T GAG AAG AC T T C AC T C AT G AT G AC G C T C T G T C T C G G G T C T AT AT AC G AAG AC T T G AAAAT G T AT C AG AC T G AAT T T C AGG C T AT T AAC G C C GC C C T G C AAAAT CAT AAC C AC C AAC AAAT T AT C C T C G ACAAGGGAATGCTCGTTGCAATCGATGAATTGATGCAAAGTTTGAATCATAACGGGGAGA CACT CCGACAGAAACCACCGGTAGGTGAGGCGGACCCGTACCGAGTGAAAATGAAACTTTGTAT CTTG CTCCACGCGTTCAGCACTCGGGTGGTCACGATAAATCGAGTCATGGGATATTTGTCATCT GCCG GGTCCGGTGGCAGTGGAAGTGGAGGCAGTGGATCCGGTGGAAGTATAACCTGTCCCCCTC CTAT

GTCAGTGGAACACGCGGATATCTGGGTGAAGAGTTACTCACTGTACTCCAGAGAGCG GTATATC TGTAACTCAGGCTTTAAGCGGAAAGCGGGAACCTCAAGCTTGACTGAGTGTGTTCTCAAC AAA.G CAACCAACGTCGCACATTGGACTACTCCGAGTTTGAAGTGTATACGAGATCCGGCATTGG TTCA CCAACGCCCGGCACCACCTAGTACCGTAACCACTGCCGGAGTCACCCCGCAGCCAGAAAG TCTG TCTCCCAGCGGGAAA.GAACCAGCGGCATCATCTCCTTCCTCCAATAATACCGCGGCAAC GACTG CCGCAATCGTGCCTGGATCACAGTTGATGCCCTCTAAGTCCCCATCAACGGGGACAACAG AGAT CAGCTCTCATGAGAGCTCACACGGCACTCCGAGCCAAACTACTGCCAAGAATTGGGAACT TACC GCGTCCGCGAGCCACCAGCCGCCCGGAGTGTACCCTCAAGGCCATTCTGACACCACCGTA GCAA TTAGCACATCAACTGTGTTGCTCTGCGGTTTGTCCGCCGTTTCACTTCTTGCTTGTTACC TTAA GAGTCGCCAGACTCCTCCCCTGGCTTCCGTTGAAATGGAGGCTATGGAGGCCCTTCCCGT TACC TGGGGTACCTCTAGTAGAGATGAGGACCTCGAGAATTGCTCCCATCACCTGTAA (SEQ ID NO: 80).

In some embodiments, a nucleic acid molecule (e.g., DNA or RNA) encoding a polypeptide provided for herein are provided. In some embodiments, the nucleic acid molecule comprises of a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to ATGGAGAGGACACTTGTTTGTCTCGTAGTGATATTCCTGGGGACCGTGGCACACAAGTCC TCTC CACAAGGACCCGACCGCCTTCTCATCAGACTGCGACACCTTATAGATATTGTGGAGCAAC TCAA GATTTATGAGAATGATTTGGACCCCGAACTCTTGTCTGCTCCTCAAGACGTTAAAGGCCA TTGT GAACATGCCGCGTTTGCTTGCTTTCAGAAGGCTAAGCTCAAGCCAAGTAATCCTGGCAAC AACA AGACGTTCATAATTGATCTTGTCGCTCAGCTTAGGCGCCGCCTGCCCGCACGACGGGGGG GAAA AAAGCAGAAGCATATCGCCAAGTGCCCAAGCTGTGATTCTTATGAAAAACGAACTCCTAA AGAA TTTTTGGAACGCCTCAAATGGCTTCTCCAAAAAATGATACATCAACACCTTTCCTCAGGA GGCG GGCCAGGTGGTGGGGGTAGTGCTGGTTCCGGTGGTGGGGGCCCAAGTGGGGGCGGCTCCT TGCA GGGCATACACGTCTTTATTCTTGGATGTTTTTCTGCGGGGTTGCCTAAGACAGAAGCAAA TTGG GTAAACGTCATCAGTGATTTGAAAAAGATCGAGGATCTCATTCAGAGCATGCACATTGAC GCTA CGTTGTATACTGAATCCGACGTTCACCCGTCATGTAAAGTCACTGCCATGAAATGTTTCC TCTT GGAGCTCCAGGTCATCTCCCTGGAAAGCGGCGACGCTAGTATTCATGATACAGTTGAAAA CCTG ATCATTCTTGCAAACAATAGCCTCAGTAGTAACGGCAACGTGACCGAGTCCGGCTGTAAG GAGT GTGAAGAACTCGAGGAGAAGAACATCAAAGAATTCTTGCAGAGTTTCGTTCATATTGTGC AAAT GTTCATCAACACTTCCTCAGGAGGCGGGCCAGGTGGTGGGGGTAGGGCTAAGAGGGGTGG TGGG GGCCCAAGTGGGGGCGGCTCCTTGCAGATGTGGGAATTGGAGAAAGACGTTTATGTTGTG GAGG TGGACTGGACTCCAGACGCGCCCGGTGAGACAGTAAACCTGACTTGCGACACCCCTGAGG AGGA CGACATTACATGGACATCAGACCAGAGGCACGGGGTTATAGGATCCGGTAAGACCCTCAC TATT ACCGTGAAAGAGTTCCTTGATGCAGGTCAGTACACTTGTCATAAGGGAGGGGAAACCCTT TCCC ACAGTCACCTTCTGCTGCACAAGAAAGAGAATGGTATATGGTCCACTGAAATCCTTAAAJ kATTT TAAGAATAAAACGTTTCTCAAATGTGAGGCCCCAAACTATAGCGGGAGGTTCACGTGCTC TTGG TTGGTCCAAAGAAATATGGATTTGAAATTCAACATTAAATCATCTAGTAGCTCACCAGAC AGTA GGGCTGTGACTTGCGGCATGGCATCACTGTCCGCTGAAAAAGTGACCCTTGATCAACGAG ACTA CGAAAAATATTCTGTCAGCTGCCAGGAGGATGTGACCTGCCCGACTGCTGAAGAGACGCT GCCC ATCGAACTGGCGCTGGAGGCGAGGCAACAGAATAAATATGAAAATTACAGTACTTCATTC TTTA T AC G G G AC AT T AT C AAGC C AG AC C C T C C C AAG AAC C T C C AAAT G AAAC C T C T C AAAAAC AG T C A GGTAGAGGTCTCATGGGAATACCCGGATAGTTGGTCTACACCCCATTCTTATTTCTCCCT TAAA TTTTTTGTTAGGATACAACGGAAGAAGGAGAAGATGAAAGAAACGGAAGAAGGTTGTAAC CAGA AGGGCGCATTTCTCGTAGAAAAAACTTCAACCGAGGTACAATGTAAAGGAGGGAACGTTT GTGT CCAAGCTCAAGATAGGTATTATAACAGCTCATGTAGCAAGTGGGCGTGCGTCCCGTGTAG GGTT CGCAGTGGATCAGGTAGTGGCGGCGGATCCGGGAGTGGCGGGGGCAGCGGGTCTGGTTCC CGGG TCATTCCAGTTTCAGGGCCGGCCAGGTGTCTTTCCCAAAGTCGGAACCTTCTCAAGACAA CGGA TGACATGGTCAAGACTGCGAGGGAAAAGCTTAAACACTACTCTTGTACCGCCGAGGACAT CGAC CATGAAGATATTACGCGGGATCAAACCAGTACGTTGAAAACCTGTCTCCCTCTTGAGCTT CATA AGAATGAGTCTTGTCTGGCAACGAGGGAAACATCATCAACTACACGAGGTAGTTGCTTGC CGCC TCAGAAGACTTCACTCATGATGACGCTCTGTCTCGGGTCTATATACGAAGACTTGAAAAT GTAT C AG AC T G AAT T T C AGG C T AT T AAC G C C GC C C T G C AAAAT CAT AAC C AC C AAC AAAT T AT C C T C G ACAAGGGAATGCTCGTTGCAATCGATGAATTGATGCAAAGTTTGAATCATAACGGGGAGA CACT CCGACAGAAACCACCGGTAGGTGAGGCGGACCCGTACCGAGTGAAAATGAAACTTTGTAT CTTG CTCCACGCGTTCAGCACTCGGGTGGTCACGATAAATCGAGTCATGGGATATTTGTCATCT GCCG GGTCCGGTGGCAGTGGAAGTGGAGGCAGTGGATCCGGTGGAAGTATAACCTGTCCCCCTC CTAT GTCAGTGGAACACGCGGATATCTGGGTGAAGAGTTACTCACTGTACTCCAGAGAGCGGTA TATC TGTAACTCAGGCTTTAAGCGGAAAGCGGGAACCTCAAGCTTGACTGAGTGTGTTCTCAAC AAAG CAACCAACGTCGCACATTGGACTACTCCGAGTTTGAAGTGTATACGAGATCCGGCATTGG TTCA CCAACGCCCGGCACCACCTAGTACCGTAACCACTGCCGGAGTCACCCCGCAGCCAGAAAG TCTG TCTCCCAGCGGGAAAGAACCAGCGGCATCATCTCCTTCCTCCAATAATACCGCGGCAACG ACTG CCGCAATCGTGCCTGGATCACAGTTGATGCCCTCTAAGTCCCCATCAACGGGGACAACAG AGAT C AG C T C T C AT G AG AG C T C AC AC G G C AC T C C GAG C C AAAC T AC T G C C AAG AAT T G G G AAC T T AC C GCGTCCGCGAGCCACCAGCCGCCCGGAGTGTACCCTCAAGGCCATTCTGACACCACCGTA GCAA TTAGCACATCAACTGTGTTGCTCTGCGGTTTGTCCGCCGTTTCACTTCTTGCTTGTTACC TTAA GAGTCGCCAGACTCCTCCCCTGGCTTCCGTTGAAATGGAGGCTATGGAGGCCCTTCCCGT TACC TGGGGTACCTCTAGTAGAGATGAGGACCTCGAGAATTGCTCCCATCACCTGTAA (SEQ ID NO: 81).

In some embodiments, a nucleic acid molecule (e.g., DNA or RNA) encoding a polypeptide provided for herein are provided. In some embodiments, the nucleic acid molecule comprises of a nucleic acid sequence having at least 80%, 85%, 90%-, 95%, 96%, 97%, 98%, 99% or 100% identity to ATGGAGAGGACACTTGTTTGTCTCGTAGTGATATTCCTGGGGACCGTGGCACACAAGTCC TCTC CACAAGGACCCGACCGCCTTCTCATCAGACTGCGACACCTTATAGATATTGTGGAGCAAC TCAA GATTTATGAGAA.TGATTTGGACCCCGAACTCTTGTCTGCTCCTCAA.GACGTTAAAGGC CATTGT GAACATGCCGCGTTTGCTTGCTTTCAGAAGGCTAAGCTCAAGCCAAGTAATCCTGGCAAC AACA AGACGTTCATAATTGATCTTGTCGCTCAGCTTAGGCGCCGCCTGCCCGCACGACGGGGGG GAAA AAAGCAGAAGCATATCGCCAAGTGCCCAAGCTGTGATTCTTATGAAAAACGAACTCCTAA AGAA TTTTTGGAACGCCTCAAATGGCTTCTCCAAAAAATGATACATCAACACCTTTCCTCAGGA GGCG GGCCAGGTGGTGGGGGTAGGGCTAAGAGGGGTGGTGGGGGCCCAAGTGGGGGCGGCTCCT TGCA GGGTATCCACGTTTTCATACTCGGGTGCTTCTCCGCTGGGTTGCCCAAAACAGAGGCTAA CTGG GTGAATGTTATCAGCGACCTCAAAAAGATCGAGGACTTGATACAGAGTATGCATATAGAT GCCA CCCTTTATACCGAGAGTGATGTTCATCCGAGTTGTAAGGTAACTGCTATGAAGTGTTTTC TGCT TGAACTTCAGGTAATAAGCCTTGAATCCGGAGACGCAAGCATTCATGATACGGTGGAAAA CCTG ATTATTCTCGCTAACAACTCCCTTTCAAGTAATGGCAACGTAACCGAATCTGGCTGTAAA GAGT GCGAGGAGCTTGAAGAGAAAAACATCAAAGAGTTTTTGCAATCCTTTGTTCATATTGTTC AAAT GTTTATAAACACGTCATCAGGCGGAGGCAGCGGAGGCGGTGGCAGTGGAGGAGGAGGATC TGGA GGCGGATCCGGAGGAGGGAGTCTTCAGATCACGTGCCCACCGCCAATGAGTGTCGAGCAC GCGG ATATCTGGGTTAAATCCTATAGCCTCTACTCCCGGGAGAGGTACATATGCAACAGCGGCT TTAA ACGAAAAGCGGGAACCTCCTCACTCACGGAGTGCGTCTTGAACAAAGCGACTAACGTGGC ACAC TGGACTACTCCTTCTCTCAAATGCATCCGAGACCCTGCGCTGGTACATCAGAGAGGAAGT GGGG GCAGTGGAAGCGGAGGAAGTGGCAGCGGCGGATCTGGCTCTGGTGGGAGTATGTGGGAAT TGGA GAAGGACGTATATGTGGTGGAGGTAGACTGGACCCCAGATGCTCCGGGAGAGACCGTCAA TCTT ACATGCGACACTCCAGAGGAAGATGACATAACTTGGACATCTGACCAACGCCATGGTGTG ATTG G AT C C G G AAAAAC C C T C AC G AT T AC G G T C AAG G AAT TTCTGGATGCAGGCCAGTATACTTGCCA CAAGGGAGGAGAAACGTTGTCACATTCTCATCTTTTGCTTCACAAGAAAGAAAATGGTAT CTGG TCTACAGAvAATCCTCAAGAACTTTAvAGAATAAAACCTTCCTGAAATGCGAAGCTCCCA ATTACA GTGGGCGATTTACATGTTCTTGGCTTGTACAAAGGAACATGGACTTGAAGTTTAATATTA AGAG CAGTAGCTCATCACCGGACTCACGAGCTGTAACATGTGGAATGGCGTCACTGTCCGCAGA GAAG GTAACGCTTGACCAGCGAGATTACGAAAAATACAGCGTTAGTTGTCAAGAAGATGTCACT TGTC CTACGGCGGAGGAGACACTTCCAATTGAGCTTGCATTGGAAGCTCGACAGCAGAACAAAT ATGA AAACTACAGTACGAGTTTCTTTATTAGGGACATAATTAAGCCGGATCCCCCTAAGAATTT GCAG ATGAAACCTCTGAAAAACAGCCAGGTCGAAGTTAGCTGGGAATATCCTGACTCTTGGTCA ACCC C C C AC T C AT AC T T C T C T C T GAAG TTCTTTGTGAGGATTCAGCG G AAG AAAGAAAAAAT G AAG G A AACTGAGGAAGGGTGCAATCAAAAAGGTGCCTTTCTGGTGGAGAAAACGTCTACGGAGGT ACAG TGTAAGGGTGGAAATGTCTGTGTTCAAGCGCAAGATAGATATTACAACTCATCCTGCTCT AAGT GGGCCTGCGTCCCCTGCCGGGTAAGGAGTGGTTCTGGGTCTGGCGGTGGATCTGGGAGTG GCGG CGGCAGTGGCAGTGGAGGGCGGGTAATTCCGGTGAGTGGTCCCGCTCGATGTCTGTCCCA ATCC AGAAACTTGCTCAAGACCACTGACGATATGGTTAAGACGGCCCGGGAGAAACTGAAGCAC TACT C C T GT AC AG C AG AAG AC AT T GAC C AT GAAG AT AT C AC GAG AG AT C AAAC AT C C AC T C T T AAAAC GTGTTTGCCGCTGGAGCTTCATAAAAACGAGTCTTGTTTGGCTACACGCGAGACCTCCAG TACT ACGCGAGGTAGTTGTCTTCCACCACAAAAGACTAGTCTCATGATGACGTTGTGTTTGGGT TCTA TCTACGAAGACCTCAAGATGTATCAAACTGAATTTCAGGCAATTAATGCCGCCCTGCAAA ATCA TAATCATCAACAGATCATTCTTGATAAGGGGATGCTCGTTGCAATTGATGAACTCATGCA ATCA CTTAATCACAACGGTGAGACGTTGCGACAGAAACCACCAGTTGGTGAGGCCGACCCGTAT CGAG TCAAAATGAAACTTTGTATACTCTTGCATGCATTCAGTACGCGAGTAGTGACAATTAATA GGGT CATGGGCTATCTTAGCAGCGCATCCGGCGGAGGCGGACCAGCTCCGCCCAGTACAGTAAC TACG GCCGGGGTTACGCCCCAACCAGAGAGTCTCAGTCCAAGTGGTAAAGAACCTGCGGCGAGT AGCC CGAGCTCCAACAACACCGCCGCGACTACAGCAGCGATTGTTCCGGGGAGCCAGTTGATGC CCAG C AAG T C T C C T AG T AC T G G C AC AAC T G AAAT AT C AAG T C AC G AAAG CTCTCATGGGACTCCTTCC CAGACAACGGCTAAGAACTGGGAGTTGACAGCAAGTGCGTCACATCAACCACCAGGCGTC TACC CACAAGGCCATTCTGACACTACTGTCGCCATTAGTACGTCCACCGTTTTGTTGTGTGGCC TCAG TGCTGTGAGTTTGTTGGCCTGTTACCTGAAAAGTAGACAGACTCCACCATTGGCGTCTGT TGAG ATGGAGGCCATGGAGGCGCTCCCGGTGACCTGGGGTACGTCCAGCCGCGATGAAGACCTT GAGA ATTGTAGTCATCATTTGTAA (SEQ ID NO: 82).

In some embodiments, a nucleic acid molecule (e.g., DNA or RNA) encoding a polypeptide provided for herein are provided. In some embodiments, the nucleic acid molecule comprises of a nucleic acid sequence having at least 80%, 85%, 90%- , 95%, 96%, 97%, 98%, 99% or 100% identity to

ATGGAGAGGACACTTGTTTGTCTCGTAGTGATATTCCTGGGGACCGTGGCACACAAG TCCTCTC

CACAAGGACCCGACCGCCTTCTCATCAGACTGCGACACCTTATAGATATTGTGGAGC AACTCAA

GATTTATGAGAATGATTTGGACCCCGAACTCTTGTCTGCTCCTCAAGACGTTAAAGG CCATTGT

GAACATGCCGCGTTTGCTTGCTTTCAGAAGGCTAAGCTCAAGCCAAGTAATCCTGGC AACAACA

AGACGTTCATAATTGATCTTGTCGCTCAGCTTAGGCGCCGCCTGCCCGCACGACGGG GGGGAAA

AAAGCAGAAGCATATCGCCAAGTGCCCAAGCTGTGATTCTTATGAAAAACGAACTCC TAAAGAA

TTTTTGGAACGCCTCAAATGGCTTCTCCAAAAAATGATACATCAACACCTTTCCTCA GGAGGCG

GGCCAGGTGGTGGGGGTAGTGCTGGTTCCGGTGGTGGGGGCCCAAGTGGGGGCGGCT CCTTGCA

GGGTATCCACGTTTTCATACTCGGGTGCTTCTCCGCTGGGTTGCCCAAAACAGAGGC TAACTGG

GTGAAT GTTATC AGCG AC C T C AAAAAGAT 0 GAG GAC T T G AT AC AGAG T ATGCAT ATAGAT G C C A

CCCTTTATACCGAGAGTGATGTTCATCCGAGTTGTAAGGTAACTGCTATGAAGTGTT TTCTGCT

TGAACTTCAGGTAATAAGCCTTGAATCCGGAGACGCAAGCATTCATGATACGGTGGA AAACCTG ATTATTCTCGCTAACAACTCCCTTTCAAGTAATGGCAACGTAACCGAATCTGGCTGTAAA GAGT GCGAGGAGCTTGAAGAGAAAAACATCAAAGAGTTTTTGCAATCCTTTGTTCATATTGTTC AAAT GTTTATAAACACGTCATCAGGCGGAGGCAGCGGAGGCGGTGGCAGTGGAGGAGGAGGATC TGGA GGCGGATCCGGAGGAGGGAGTCTTCAGATCACGTGCCCACCGCCAATGAGTGTCGAGCAC GCGG ATATCTGGGTTAAATCCTATAGCCTCTACTCCCGGGAGAGGTACATATGCAACAGCGGCT TTAA ACGAAAAGCGGGAACCTCCTCACTCACGGAGTGCGTCTTGAACAAAGCGACTAACGTGGC ACAC TGGACTACTCCTTCTCTCAAATGCATCCGAGACCCTGCGCTGGTACATCAGAGAGGAAGT GGGG GCAGTGGAAGCGGAGGAAGTGGCAGCGGCGGATCTGGCTCTGGTGGGAGTATGTGGGAAT TGGA GAAGGACGTATATGTGGTGGAGGTAGACTGGACCCCAGATGCTCCGGGAGAGACCGTCAA TCTT ACATGCGACACTCCAGAGGAAGATGACATAACTTGGACATCTGACCAACGCCATGGTGTG ATTG GATCCGGAAAAACCCTCACGATTACGGTCAAGGAATTTCTGGATGCAGGCCAGTATACTT GCCA CAAGGGAGGAGAAACGTTGTCACATTCTCATCTTTTGCTTCACAAGAAAGAAAATGGTAT CTGG TCTACAGAAATCCTCAAGAACTTTAAGAATAAAACCTTCCTGAAATGCGAAGCTCCCAAT TACA GTGGGCGATTTACATGTTCTTGGCTTGTACAAAGGAACATGGACTTGAAGTTTAATATTA AGAG CAGTAGCTCATCACCGGACTCACGAGCTGTAACATGTGGAATGGCGTCACTGTCCGCAGA GAAG GTAACGCTTGACCAGCGAGATTACGAAAAATACAGCGTTAGTTGTCAAGAAGATGTCACT TGTC C T AC G G C G G AG G AG AC AC T T C C AAT T G AG C T T G C AT T G G AAG C T C G AC AG C AG AAC AAAT AT G A AAAC TAG AG TAG GAG T T T C T T TAT T AGGG AC AT AAT T AAG C C G GAT C C C C C T AAGAAT T T G C AG ATGAAACCTCTGAAAAACAGCCAGGTCGAAGTTAGCTGGGAATATCCTGACTCTTGGTCA ACCC CCCACTCATACTTCTCTCTGAAGTTCTTTGTGAGGATTCAGCGGAAGAAAGAAAAAATGA AGGA AACTGAGGAAGGGTGCAATCAAAAAGGTGCCTTTCTGGTGGAGAAAACGTCTACGGAGGT ACAG TGTAAGGGTGGAAATGTCTGTGTTCAAGCGCAAGATAGATATTACAACTCATCCTGCTCT AAGT GGGCCTGCGTCCCCTGCCGGGTAAGGAGTGGTTCTGGGTCTGGCGGTGGATCTGGGAGTG GCGG CGGCAGTGGCAGTGGAGGGCGGGTAATTCCGGTGAGTGGTCCCGCTCGATGTCTGTCCCA ATCC AGAAACTTGCTCAAGACCACTGACGATATGGTTAAGACGGCCCGGGAGAAACTGAAGCAC TACT C C T GT AC AGC AG AAGAC AT T GAG CAT GAAG AT AT C AC GAGAG AT C AAAC AT C C AC T C T T AAAAC GTGTTTGCCGCTGGAGCTTCATAAAAACGAGTCTTGTTTGGCTACACGCGAGACCTCCAG TACT ACGCGAGGTAGTTGTCTTCCACCACAAAAGACTAGTCTCATGATGACGTTGTGTTTGGGT TCTA T C TAG G AAGAC C T C AAGAT GT AT C AAAC T G AAT T T C AGG C AAT T AAT G C C GC C C T G CAAAATCA TAATCATCAACAGATCATTCTTGATAAGGGGATGCTCGTTGCAATTGATGAACTCATGCA ATCA CTTAATCACAACGGTGAGACGTTGCGACAGAAACCACCAGTTGGTGAGGCCGACCCGTAT CGAG TCAAAATGAAACTTTGTATACTCTTGCATGCATTCAGTACGCGAGTAGTGACAATTAATA GGGT CATGGGCTATCTTAGCAGCGCATCCGGCGGAGGCGGACCAGCTCCGCCCAGTACAGTAAC TACG GCCGGGGTTACGCCCCAACCAGAGAGTCTCAGTCCAAGTGGTAAAGAACCTGCGGCGAGT AGCC CGAGCTCCAACAACACCGCCGCGACTACAGCAGCGATTGTTCCGGGGAGCCAGTTGATGC CCAG C AAGT C T C C T AG T AC T G G C AC AAC T G AAAT AT C AAG T C AC G AAAG C T C T C AT G G G AC T C C T T C C CAGACAACGGCTAAGAACTGGGAGTTGACAGCAAGTGCGTCACATCAACCACCAGGCGTC TACC CACAAGGCCATTCTGACACTACTGTCGCCATTAGTACGTCCACCGTTTTGTTGTGTGGCC TCAG TGCTGTGAGTTTGTTGGCCTGTTACCTGAAAAGTAGACAGACTCCACCATTGGCGTCTGT TGAG ATGGAGGCCATGGAGGCGCTCCCGGTGACCTGGGGTACGTCCAGCCGCGATGAAGACCTT GAGA ATTGTAGTCATCATTTGTAA (SEQ ID NO: 83).

The sequence of SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, and SEQ ID NO: 83 are merely exemplary sequences that encode for polypeptides described herein. Due to the degenerate nature of 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 an eukaryotic system or cell. In some embodiments, the nucleic acid molecule is a DNA or RNA molecule that encodes a polypeptide as provided for herein. In some embodiments, the RNA molecule is a mRNA molecule.

The nucleic acid molecule can be prepared by synthesis or other traditional techniques known to one of skill in the art once provided a sequence, which can be either the sequence of the polypeptide that is to be encoded by the nucleic acid molecule or the nucleic acid sequence itself.

Another method of constructing a DNA sequence encoding a polypeptide as provided for herein would be chemical synthesis. This for example includes direct synthesis of a peptide by chemical means of the protein sequence encoding for a polypeptide as provided for herein. This method may incorporate both natural and unnatural amino acids at various positions. Alternatively, a nucleic acid molecule which encodes a desired protein may 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 favored in the cell in which the recombinant variant will be produced. It is well recognized that the genetic code is degenerate, i.e., that an amino acid may be coded for by more than one codon. Accordingly, it will be appreciated that for a given DNA sequence encoding a particular polypeptide as provided for herein there will be many DNA degenerate sequences that will code for that polypeptide. Accordingly, in some embodiments, a nucleic acid molecule is provided that encodes the polypeptides provided for herein. The nucleic acid molecule can be DNA or RNA.

In some embodiments, the nucleic acid molecule will encode a signal sequence or leader peptide sequence, such as provided for herein. A signal sequence can be chosen based upon the cell that will be expressed in. In some embodiments, if the host cell is prokaryotic, the nucleic acid molecule does not comprise a signal sequence. In some embodiments, if the host cell is a eukaryotic cell, the signal sequence can be used. In some embodiments, the signal sequence or leader sequence is as provided for herein. The signal or leader sequence of the protein can also be from the immature proteins.

“Recombinant” as it applies to polypeptides or proteins, means that the production of the protein is dependent on at least one step in which nucleic acids, which may or may not encode the protein, are introduced into a cell in which they are not naturally found. The nucleic acid molecule can also be referred to as a heterologous molecule when it is added to the cell or system exogenously. Various host (animals 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 yeasts), plant, insect, mammal, or other appropriate animal cells or cell lines, as well as transgenic animals or plants. In some embodiments, these hosts may include well known eukaryotic and prokaryotic hosts, such as strains of E. coli, Pseudomonas, Bacillus, Streptomyces, fungi, yeast, insect cells such as Spodoptera ffugiperda (Sf9), animal cells such as Chinese hamster ovary (CHO) and mouse cells such as NS/O, African green monkey cells such as COS 1, 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 cultures and particularly the CHO cell line CHO (DHFR-) or the HKB line may be used. The proteins can also be expressed in vivo by delivering a nucleic acid molecule to a cell in vivo and having the cell express the protein, whereby the polypeptide as provided will traffic to the surface of the cell. In some embodiments, the nucleic acid molecule is delivered with a vector, such a viral vector, including but not limited to adenoviral vectors. In some embodiments, the nucleic acid molecule is encapsulated in a nanoparticle, such as a lipid nanoparticle to deliver the nucleic acid molecule to the cell. In some embodiments, the encapsulated nucleic acid molecule is a RNA molecule. In some embodiments, the encapsulated nucleic acid molecule is a DNA molecule.

One of skill in the art may make a selection among various vectors, expression control sequences and hosts without undue experimentation. For example, in selecting a vector, a host can be considered because the vector must be able replicate in it or the polypeptide must be able to be transcribed and/or translated from the vector in the specific host. The vectors 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.

Checkpoint Inhibitor Polypeptides

Without being bound to any particular theory, the embodiments provided for herein have been found to simultaneously inhibit multiple immune checkpoint proteins with the use of a single polypeptide molecule. 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 treatment using these single immune checkpoint targeting protein molecules has demonstrated increased clinical benefit compared to mono-therapy treatments, the risk of severe or dose-limiting adverse or long term systemic off- target immunological events are also increased. By utilizing a single molecule, the adverse and off target events may be controlled. Further, incorporation of binding domains into the polypeptide of embodiments provided for herein will provide targeted delivery of the immune checkpoint inhibitor polypeptide, further increasing efficacy and decreasing undesired events.

Accordingly, described herein are polypeptide molecules comprising one or more immune checkpoint inhibitors. In some embodiments, a polypeptide is provided, the polypeptide comprising from the N-terminus to the C-terminus a formula of X _A -L _A -X _B or a formula of X _B - L _A -X _A , wherein X _A and X _B are each, independently, immune checkpoint inhibiting polypeptides, and L ₁ is a polypeptide linker. In some embodiments, X _A and X _B comprise the same immune checkpoint inhibiting polypeptide. In some embodiments, X _A and X _B comprise different immune checkpoint inhibiting polypeptides. In some embodiments, X _A and X _B can be any polypeptide or fragment thereof that would inhibit 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 are selected from the group including, but not limited to, CTLA-4, PD-1, PD-L ₁ , TIM3, LAG3, VISTA, SIGLEC7, SIGLEC9, TIGIT, CD96, BTLA, B7H3, B7H4, CD155, HHLA2, and BTN3A1.

In some embodiments, the polypeptide comprises from the N-terminus to the C-terminus a formula of X _A -L _A -X _B . In some embodiments, the polypeptide comprises from the N-terminus to the C-terminus a formula of X _B -L _A -X _A - In some embodiments, the polypeptide comprises from the N-terminus to the C-terminus a formula of X _A -L _A -X _A - In some embodiments, the polypeptide comprises from the N-terminus to the C-terminus a formula of X _B -L _A -X _B .

In some embodiments, X _A is a peptide selected from the group comprising a CTLA-4 polypeptide, a PD-1 polypeptide, PD-L ₁ polypeptide, a PD-L ₂ 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 CD 155 polypeptide, a HHLA2 polypeptide, a BTN3A1 polypeptide, or an active fragment thereof. In some embodiments, X _B is a peptide selected from the group comprising a CTLA-4 polypeptide, a PD-1 polypeptide, PD-L ₁ polypeptide, a PD-L ₂ 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, both X _A and X _B are each, independently, peptides selected from the group comprising a PD-L ₁ polypeptide, a PD-L ₂ 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.

In some embodiments, X _A is a PD-L ₁ polypeptide, and X _B is a B7.1 polypeptide. In some embodiments, X _A is a PD-L ₁ polypeptide, and X _B is a B7.2 polypeptide. In some embodiments, X _A is a PD-L ₂ polypeptide, and X _B is a B7.1 polypeptide. In some embodiments, X _A is a PD-L ₂ polypeptide, and X _B is a B7.2 polypeptide.

In some embodiments, the PD-L ₁ polypeptide comprises an amino acid sequence of SEQ ID NO: 101:

MRIFAVFIFMTY^THLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVY WE MEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMI SYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVL SGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPP NE RTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET (SEQ ID NO: 101) or an active fragment thereof. In any of the embodiments herein, the PD-LI polypeptide may comprise an amino acid sequence corresponding to the entire PD-L ₁ protein, or to any fragment thereof. Further, it is to be understood that the PD-LI polypeptide is not limited to a specific isoform, but rather the PD-Li polypeptide as recited herein encompasses all PD-LI isoforms. The PD-LI polypeptide as presented herein can comprise the wild type PD-LI protein, or it can comprise any PD-LI mutant protein known in the art. Further, it is to be understood that the PD-LI polypeptide is not limited to the human PD-LI polypeptide, but rather encompasses PD-LI polypeptide sequences from other relevant species, including but not limited to mouse, rat, non-human primate, bovine, equine, and the like.

In some embodiments, the PD-LI polypeptide comprises a PD-LI extracellular domain. In some embodiments, the PD-LI polypeptide comprises an amino acid sequence substantially similar to SEQ ID NO: 102: FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQ HSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYN KINQR1LVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTS TLRINTTTNEIFYCTFRRLDPEENHTAELV1PELPLAHPPNER (SEQ ID NO: 102). In some embodiments, the PD-L ₁ polypeptide comprises an active fragment of SEQ ID NO: 102. In some embodiments, the PD-L ₁ polypeptide comprises an amino acid sequence having at least 60% identity to SEQ ID NO: 102. In some embodiments, the PD-L 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: 102. In some embodiments, the PD-L ₁ polypeptide comprises an amino acid sequence identical to SEQ ID NO: 102.

In some embodiments, the PD-L ₂ polypeptide comprises an amino acid sequence of SEQ ID NO: 103:

MIFLLLMLSLELQLHQTAALFTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITA SLQK VENDTSPHRERATLLEEQLPLGKASFHIPQVQVRDEGQYQCIIIYGVAWDYKYLTLKVK ASYRKINTHn.KVPETDEVELTCQATGYPLAEVSWPNVSVPANTSHSRTPEGLYQVTSVL RLKPPPGRNFSCVFWNTHVRELTI.ASIDLQSQMEPRTHPTWIXHIFIPFCIIAFIFIAT VIAL RKQLCQKLYSSKDTTKRPVTTTKREVNSAI (SEQ ID NO: 103) or an active fragment thereof. In any of the embodiments herein, the PD-L ₂ polypeptide may comprise an amino acid sequence corresponding to the entire PD-L ₂ protein, or to any fragment thereof. Further, it is to be understood that the PD-L ₂ polypeptide is not limited to a specific isoform, but rather the PD- L ₂ polypeptide as recited herein encompasses all PD-L ₂ isoforms. The PD-L ₂ polypeptide as presented herein can comprise the wild type PD-L ₂ protein, or it can comprise any PD-L ₂ mutant protein known in the art. Further, it is to be understood that the PD-L ₂ polypeptide is not limited to the human PD-L ₁ polypeptide, but rather encompasses PD-L ₂ polypeptide sequences from other relevant species, including but not limited to mouse, rat, non-human primate, bovine, equine, and the like.

In some embodiments, the PD-L ₂ polypeptide comprises a PD-L ₂ extracellular domain. In some embodiments, the PD-L ₂ polypeptide comprises an amino acid sequence substantially similar to SEQ ID NO: 104: LFTVTVTKELYIIEHGSNVTLECNFDTGSHVNLGAn'ASLQKVENDTSPHRERAlTLEEQ L PLGKASFHIPQVQVRDEGQYQCIIIYGVAWDYKYLTLKVKASYRKINTHILKVPETDEVE LTCQATGYPLAEVSWPNVSVPANTSHSRTPEGLYQVTSVLRLKPPPGRNFSCVFWNTHV RELTLASIDLQSQMEPRTHPT (SEQ ID NO: 104). In some embodiments, the PD-L ₂ polypeptide comprises an active fragment of SEQ ID NO: 104. In some embodiments, the PD-L ₂ polypeptide comprises an amino acid sequence having at least 60% identity to SEQ ID NO: 104. In some embodiments, the PD-L 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: 104. In some embodiments, the PD-L ₂ polypeptide comprises an amino acid sequence identical to SEQ ID NO: 104.

In some embodiments, the B7.1 polypeptide comprises an amino acid sequence of SEQ ID NO: 105: MGHT'RRQGTSPSKCPYLNFFQLLVLAGLSHFCSGVIHVTKEVKEVATLSCGHNVSVEEL AQTRIYWQKE.KKMVLTMMSGDMNIWPEYKNRTIFDITNNL,SIVIL.ALRPSDEGTYEC VV LKYEKDAFKREHLAEVTI.SVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLE NGE Fd,NAINTTVSQDPETEEYAVSSKEDFNMTTNHSFMCUKYGHERVNQTFNWNTTKQEHF PDNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV (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 to any fragment thereof. Further, it is to be understood that the B7.1 polypeptide is not limited to a specific isoform, but rather the B7.1 polypeptide as recited herein encompasses all B7.1 isoforms. The B7.1 polypeptide as presented herein can comprise the wild type B7.1 protein, or it can comprise any B7.1 mutant protein known in the art. Further, it is to be understood that the B7.1 polypeptide is not limited to the human B7.1 polypeptide, but rather encompasses B7.1 polypeptide sequences from other relevant species, including but not limited to mouse, rat, nonhuman primate, bovine, equine, and the like.

In some embodiments, the B7.1 polypeptide comprises a B7.1 extracellular domain. In some embodiments, the B7.1 polypeptide comprises an amino acid sequence substantially similar to SEQ ID NO: 106: VIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRT iroiTNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFE IPT SN1RRHCSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFM CLIKYGHLRVNQI'FNWNTTKQEHFPDN (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 ieast 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 an amino acid sequence identical to SEQ ID NO: 106.

In some embodiments, the B7.2 polypeptide comprises an amino acid sequence of SEQ ID NO: 107: MDPQCTMGLSNILFVMAFLLSGAAPLKIQAYFNETADLPCQFANSQNQSLSELVVFWQD QENLVLNEVYLGKEKFDSVHSKYMGRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPT GMIRIHQMNSELSVLANFSQPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNS TI EYDGVMQKSQDNVTELYDVSISLSVSFPDVTSNMTIFCILETDKTRLLSSPFSIELEDPQ PP PDHIPWITAVLPTVIICVMVFCLILWKWKKKKRPRNSYKCGTNTMEREESEQTKKREKIH IPERSDEAQRVFKSSKTSSCDKSDTCF (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 to any fragment thereof. Further, it is to be understood that the B7.2 polypeptide is not limited to a specific isoform, but rather the B7.2 polypeptide as recited herein encompasses all B7.2 isoforms. The B7.2 polypeptide as presented herein can comprise the wild type B7.2 protein, or it can comprise any B7.2 mutant protein known in the art. Further, it is to be understood that the B7.2 polypeptide is not limited to the human B7.2 polypeptide, but rather encompasses B7.2 polypeptide sequences from other relevant species, including but not limited to mouse, rat, non-human primate, bovine, equine, and the like.

In some embodiments, the B7.2 polypeptide comprises a B7.2 extracellular domain. In some embodiments, the B7.2 polypeptide comprises an amino acid sequence substantially similar to SEQ ID NO: 108: APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVHSK YMGRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKI’TGMIRIHQMNSELSVLANFSQ PEI VPISNITENVY1NLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGVMQKSQDNVTELYDVSI S LSVSFPDVTSNMTIFCILETDKTRLLSSPFSIELEDPQPPPDH1P (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 an amino acid sequence identical to SEQ ID NO: 108.

The linker, L ₁ , can comprise any peptide linker. In some embodiments, the peptide linker comprises an amino acid sequence of GSGSGGGSGSGGGSGSGSG (SEQ ID NO: 109). In some embodiments, the peptide linker comprises an amino acid sequence of substantially similar to SEQ ID NO: 109. In some embodiment, the peptide linker comprises a fragment of SEQ ID NO: 109, or comprises n repeats of SEQ ID NO: 109, wherein 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 illustrated in the following table: | | | | | |

In some embodiments, the polypeptide provided 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- L.l . In some embodiments, the leader sequence comprises a polypeptide sequence corresponding to the cell membrane targeting domain of PD-L ₂ . 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 will deliver the provided polypeptide to the cell surface.

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 or is identical to SEQ ID NO: 110: MIFLLLMLSLELQLHQIAA (SEQ ID NO: 110) or is an active fragment thereof.

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 or is identical to SEQ ID NO: 111: MRIFAVFIFMTYWHLLNA (SEQ ID NO: 111) or is an active fragment thereof.

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 or is identical to SEQ ID NO: 112; MGHTRRQGTSPSKCPYLNFFQLLVLAG (SEQ ID NO: 112) or is an active fragment thereof.

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 or is identical to SEQ ID NO: 113: MDPQCTMGLSNILFVMAFLLSGA (SEQ ID NO: 113) or is an active fragment thereof.

In some embodiments, the provided polypeptide further comprises a targeting domain. The targeting domain can comprise any protein that will direct 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, and the like. In some embodiments, the targeting domain comprises a protein from the group including, but not limited to, a cytokine, a cytokine trap, a receptor ligand, a complement protein, a T-cell receptor, an antibody, an Fc domain, a chimeric antigen receptor, an antigen, a tumor antigen, and the like, or any fragment thereof.

In some embodiments, a polypeptide is provided, the polypeptide comprising from the N- terminus to the C-terminus a formula of selected from the group comprising LS-X _A -L _A -X _B -L _B - Xc, LS-X _A -L _A -X _C -L _B -X _B , LS-X _B -L _A -X _A -L _B -X _C , or LS-X _B -L _A -X _C -L _B -X _A , wherein Ls is a leader sequence, X _A and X ₂ are each, independently, immune checkpoint inhibiting polypeptides, Xc is a targeting domain, and L _A and L _B are each, independently, polypeptide linkers. In some embodiments, X _A and X _B comprise the same immune checkpoint inhibiting polypeptide. In some embodiments, X _A and X _B comprise different immune checkpoint inhibiting polypeptides. In some embodiments, X _A and X _B can be any polypeptide or fragment thereof that would inhibit 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 including, but not limited to, CTLA-4, PD-1, PD-L ₁ , 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 as provided for herein. In some embodiments, the targeting domain Xc can comprise any protein that will direct 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, and the like. In some embodiments, the targeting domain comprises a protein from the group including, but not limited to, a cytokine, a cytokine trap, a receptor ligand, a complement protein, a T-cell receptor, an antibody, an Fc domain, a chimeric antigen receptor, an antigen, a tumor antigen, and the like, or any fragment thereof.

In some embodiments, the polypeptide comprises from the N-terminus to the C-terminus a formula of 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 a formula of 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 a formula of L _S -X _B -L _A -X _A - L _B -X _C .

In some embodiments, the polypeptide comprises from the N-terminus to the C-terminus a formula of L _S -X _B -L _A -X _C -L _B -X _A - In some embodiments, L _s is a leader sequence as provided for herein. In some embodiments, X _A comprises a PD-L ₁ polypeptide or a PD-L ₂ polypeptide as provided for herein. In some embodiments, X _B comprises a B7.1 polypeptide or a B7.2 polypeptide as provided for herein. In some embodiments, the targeting domain X _c is a targeting domain as provided for herein. In some embodiments, L _A and L _B are each, independently, absent or a polypeptide linker as provided for herein.

In some embodiments, X _A is a PD-L ₁ polypeptide as provided for herein; X _B is a B7.1 polypeptide as provided for herein; Xc is an antibody Fc domain; L _A is a polypeptide linker as provided for herein; and L _B is a polypeptide linker as provided for herein or absent.

In some embodiments, X _A is a PD-L ₁ polypeptide as provided for herein; X _B is a B7.2 polypeptide as provided for herein; Xc is an antibody Fc domain; L _A is a polypeptide linker as provided for herein; and L _B is a polypeptide linker as provided for herein or absent. In some embodiments, X _A is a PD-L ₂ polypeptide as provided for herein; X _B is a B7.1 polypeptide as provided for herein; Xc is an antibody Fc domain; L _A is a polypeptide linker as provided for herein; and L _B is a polypeptide linker as provided for herein or absent.

In some embodiments, X _A is a PD-L ₂ polypeptide as provided for herein; X _B is a B7.2 polypeptide as provided for herein; Xc is an antibody Fc domain; L _A is a polypeptide linker as provided for herein; and L _B is a polypeptide linker as provided for herein or absent.

In some embodiments, the PD-L ₁ polypeptide comprises a PD-L ₁ extracellular domain as provided for herein. In some embodiments, the PD-L ₁ polypeptide comprises an amino acid sequence substantially similar to SEQ ID NO: 102. In some embodiments, the PD-L ₁ polypeptide comprises an amino acid sequence having at least 60% identity to SEQ ID NO: 102. In some embodiments, the PD-L 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: 102. In some embodiments, the PD-L ₁ polypeptide comprises an amino acid sequence identical to SEQ ID NO: 102.

In some embodiments, the PD-L ₂ polypeptide comprises a PD-L ₂ extracellular domain as provided for herein. In some embodiments, the PD-L ₂ polypeptide comprises an amino acid sequence substantially similar to SEQ ID NO: 104. In some embodiments, the PD-L ₂ polypeptide comprises an amino acid sequence having at least 60% identity to SEQ ID NO: 104. In some embodiments, the PD-L 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: 104. In some embodiments, the PD-L ₂ polypeptide comprises an amino acid sequence identical to SEQ ID NO: 104.

In some embodiments, the B7.1 polypeptide comprises a B7.1 extracellular domain as provided for herein. In some embodiments, the B7.1 polypeptide comprises an amino acid sequence substantially similar to 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 an amino acid sequence identical to SEQ ID NO: 106.

In some embodiments, the B7.2 polypeptide comprises a B7.2 extracellular domain as provided for herein. In some embodiments, the B7.2 polypeptide comprises an amino acid sequence substantially similar to 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 an amino acid sequence identical to SEQ ID NO: 108.

In some embodiments, the homodimerization domain comprises any immunoglobulin fold containing protein. In some embodiments, the immunoglobulin fold containing protein comprises any human Fc domain. In some embodiments, the Fc domain is selected from the group including, but not limited to IgG, IgM, IgA, IgE, or IgD, or any subclass thereof, including but not limited to IgGi, IgG?., IgGs, IgG4, IgAi or IgA?. 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 including, but not limited to FcyR, FcaR, FcgR, FcyRI, FcyRIIA, FcyRIIBl, FcyRIIB2, FcyRIIIA, FcyRIIIB, FcsRI, FcsRII, FcaRI, FcaR/μR, or FcRn. Specific engineering/point mutations of an Fc domain to enhance binding to a specific FcR are known in the art and are within the scope of this application.

In some embodiments, the Fc domain is a human IgGl Fc domain. In some embodiments, the Fc domain comprises an amino acid sequence substantially similar to SEQ ID NO: 114:

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 1 14) 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 an amino acid sequence identical to SEQ ID NO: 114.

In some embodiments, the polypeptide comprising from the N-terminus to the C-terminus a formula selected from the group comprising of X _A -L _A -X _B or X _B -L _A -X _A comprises an amino acid sequence substantially similar to SEQ ID NO: 115: MIFLLLMLSLELQLHQIAALFTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITASLQ K VENDTSPHRERATLLEEQLPLGKASFHIPQVQVRDEGQYQCIIIYGVAWDYKYLTLKVK ASYRKINTHILKVPETDEVELTCQATGYPLAEVSWPNVSVPANTSHSR1TEGLYQVTSVL RLKPPPGRNFSCVFWNTHVRELTLASIDLQSQMEPRTHPTGSGSGGGSGSGGGSGSGSG LSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIW PEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPT P SISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLD FNM TTNHSFMCI JKYGHLRVNQTFNWNTTKQEHFPD (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 an amino acid sequence identical to SEQ ID NO: 115.

In some embodiments, the polypeptide comprising from the N-terminus to the C-terminus a formula selected from the group comprising of X _A -L _A -X _B or X _B -L _A -X _A comprises an amino acid sequence substantially similar to SEQ ID NO: 1 16: MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWE MEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMI SYGGADYKRITVKVN APYNKJNQRILVVDP VTSEHELTCQAEGYPK AEVIWTSSDHQVL SGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTA ELVIPELPLAHPPNE RGSGSGGGSGSGGGSGSGSAPLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENL VLNEVYLGKEKFDSVHSKYMGRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKI’TGM IR IHQMNSELSVLANFSQPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEY DG VMQKSQDNVTELYDVSISLSVSFPDVTSNMTIFCILETDKTRLLSSPFSIELEDPQPPPD HIP (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 an amino acid sequence identical to SEQ ID NO: 116.

In some embodiments, the polypeptide comprising from the N-terminus to the C-terminus a formula selected from the group comprising of LS-X _A -L _A -X _B -L _B -X _C , LS-X _B -L _A -X _A -L _B -X _C , LS- X _A -L _A -X _C -L _B -X _B , or LS-X _B -L _A -X _C -L _B -X _A comprises an amino acid sequence substantially similar to SEQ ID NO: 117: MIFLLLMLSLELQLHQIAALFTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITASLQ K VENDTSPHRERATLLEEQLPLGKASFHIPQ VQ VRDEGQYQCIIIYG VAWD YKYLTLKVK ASYRKINTHILKVPETDEVELTCQATGYPLAEVSWPNVSVPANTSHSRTPEGLYQVTSVL RLKPPPGRNFSCVFWNTHVRELTLASIDLQSQMEPRTHPTGSGSGGGSGSGGGSGSGSG LSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIW PEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPT P SISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLD FNM TTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV SL.TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL.DSDGSFFLYSKLTVDKSRWQQGN V FSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 117) or an active fragment thereof. In some embodiments, the polypeptide comprises an amino acid sequence having at least 60% identity to SEQ ID NO: 117. 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: 117. In some embodiments, the polypeptide comprises an amino acid sequence identical to SEQ ID NO: 1 17.

In some embodiments, the polypeptide comprising from the N-terminus to the C-terminus a formula selected from the group comprising of LS-X _A -L _A -X _B -L _B -X _C , LS-X _B -L _A -X _A -L _B -X _C , LS- X _A -L _A -X _C -L _B -X _B , or LS-X _B -L _A -X _C -L _B -X _A comprises an amino acid sequence substantially similar to SEQ ID NO: 118:

MRIFA VFIFMT YWHLLNAFTVT VPKDL YV VE YGSNMTIECKFPVEKQLDLA ALIV Y WE MEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMI SYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVL SGKTITTNSKREEKLFNVTSTLRINTITNEIFYCTFRRLDPEENHTAELVU’ELPLAH PPNE RGSGSGGGSGSGGGSGSGSAPLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENL VLNEVYLGKEKFDSVHSKYMGRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKl’TGM IR IHQMNSELSVLANFSQPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEY DG VMQKSQDNVTELYDVSISLSVSFPDVTSNMTIFCILETDKTRLLSSPFSIELEDPQPPPD HIP DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 118) or an active fragment thereof. In some embodiments, the polypeptide comprises an amino acid sequence having at least 60% identity to SEQ ID NO: 118. 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: 118. In some embodiments, the polypeptide comprises an amino acid sequence identical to SEQ ID NO: 118.

In some embodiments a nucleic acid molecule is provided. In some embodiments, the nucleic acid molecule encodes for a polypeptide as provided for herein. In some embodiments, the nucleic acid molecule encoding for the polypeptide comprises a nucleic acid sequence substantially similar to SEQ ID NO: 119: ATGATTTTTCTCTTGCTTATGCTTTCTCTTGAGTTGCAACTTCATCAGATTGCCGCCCTT TTTA CCGTCACAGTCCCTAAAGAACTTTACATAATCGAGCACGGTAGCAACGTAACGCTTGAAT GTAA CTTTGACACGGGGAGTCACGTCAATCTTGGAGCTATTACGGCGTCTCTTCAGAAGGTGGA AAAC GATACGAGCCCTCACCGGGAGCGCGCTACCCTGCTTGAAGAACAGTTGCCGCTTGGAAAA GCAT CCTTTCACATACCGCAGGTTCAAGTTCGAGATGAAGGCCAATACCAATGCATAATTATTT ATGG AGTCGCCTGGGACTATAAGTACCTGACATTGAAGGTTAAAGCGAGCTATAGGAAGATCAA TACT CACATACTGAAGGTCCCGGAGACTGATGAAGTGGAATTGACTTGTCAGGCAACAGGGTAT CCTT TGGCGGAGGTTAGTTGGCCTAATGTTTCCGTGCCCGCGAACACATCTCATTCAAGGACAC CAGA GGGCCTGTATCAGGTGACCTCCGTTCTGCGCCTGAAGCCACCGCCAGGTAGGAACTTTAG CTGC G T C T T C T GG AAT AC T CAT GT T C G AGAAC T C AC AC T T GC T T C AAT C GAG C T T GAG T C C GAG AT G G AGCCTAGAACTCATCCTACCGGATCAGGTAGTGGCGGCGGAAGCGGGAGTGGCGGAGGCT CCGG GTCTGGTTCCGGCCTGTCACACTTTTGTTCTGGCGTAATCCACGTCACTAAGGAGGTTAA AGAG GTAGCCACCCTTTCTTGTGGTCATAATGTCTCTGTGGAAGAACTTGCTCAAACAAGAATC TACT GGCAAAAGGAGAAGAAAATGGTCTTGACGATGATGAGCGGTGACATGAACATATGGCCAG AGTA CAAGAATCGCACCATTTTCGACATAACCAACAATCTTTCAATCGTAATACTGGCCCTCAG GCCG TCTGATGAAGGTACGTATGAGTGCGTTGTCTTGAAATACGAAAAAGACGCCTTCAAGCGA GAAC ATCTGGCGGAGGTGACACTTTCTGTTAAAGCCGATTTCCCAACTCCTTCTATTTCAGATT TTGA AATCCCGACCTCTAACATCCGGAGAATTATCTGCAGCACGTCAGGGGGGTTTCCTGAACC ACAT TTGTCTTGGCTTGAGAATGGCGAAGAATTGAACGCCATCAACACCACCGTTAGTCAAGAC CCAG AGACGGAACTGTATGCCGTTTCAAGTAAGCTCGATTTTAATATGACAACTAACCACTCCT TCAT GTGTCTGATAAAATATGGACATCTGCGGGTAAATCAGACATTTAACTGGAACACGACAAA ACAA GAGCATTTCCCAGAT (SEQ ID NO: 119) or an active fragment thereof. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least 60% identity to SEQ ID NO: 119. In some embodiments, the nucleic acid molecule comprises a nucleic 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: 119. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence identical to SEQ ID NO: 119. The sequence of SEQ ID NO: 119 (as well as other nucleotide sequences provided for herein) is an exemplary sequence and is not meant to be limiting in any way. Due to the degenerate nature of 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 that encodes a polypeptide as provided for herein. In some embodiments, the RNA molecule is a mRNA molecule.

In some embodiments a nucleic acid molecule is provided. In some embodiments, the nucleic acid molecule encodes for a polypeptide as provided for herein. In some embodiments, the nucleic acid molecule encoding for the polypeptide comprises a nucleic acid sequence substantially similar to SEQ ID NO: 120: ATGCGCATATTCGCAGTCTTTATATTTATGACTTATTGGCATCTTCTTAATGCATTTACT GTCA CTGTTCCCAAGGATCTGTACGTTGTAGAGTACGGCTCCAACATGACTATAGAGTGCAAAT TTCC AGTCGAGAAACAGCTCGACTTGGCAGCACTCATTGTGTACTGGGAAATGGAAGACAAAAA CATC ATTCAGTTCGTCCACGGGGAGGAGGACTTGAAAGTTCAACACTCATCCTATCGCCAACGA GCCC GCCTGTTGAAGGACCAGCTTTCTCTCGGTAACGCTGCACTTCAGATTACGGATGTTAAGT TGCA GGATGCAGGAGTCTACCGATGTATGATTAGTTACGGCGGTGCCGATTACAAGAGGATCAC TGTT AAAGTCAACGCTCCTTACAATAAAATAAACCAACGGATTCTCGTGGTAGATCCTGTGACT TCCG AGCACGAATTGACGTGCCAAGCAGAAGGATACCCGAAAGCCGAGGTGATCTGGACAAGCT CCGA TCATCAAGTCCTCTCAGGGAAGACGACGACCACGAACTCTAAGAGGGAAGAGAAGCTGTT CAAC GTAACATCAACGCTCCGCATTAACACGACTACTAATGAGATATTCTACTGCACTTTCCGG CGAC TCGATCCGGAGGAAAACCATACAGCGGAGCTTGTGATTCCGGAACTTCCACTCGCGCATC CTCC GAACGAGCGGGGATCAGGTAGTGGCGGCGGATCCGGGAGTGGCGGGGGCAGCGGGTCTGG TTCC GCACCGTTGAAAATACAAGCGTACTTCAACGAAACTGCAGACCTTCCGTGTCAATTCGCG AACT CACAAAATCAATCCCTTAGTGAGCTTGTAGTATTTTGGCAGGACCAGGAAAATCTCGTCC TGAA CGAAGTTTATCTCGGTAAAGAGAAGTTCGACAGCGTACATAGTAAATACATGGGCAGAAC TTCT TTCGATAGTGATAGTTGGACGCTCAGGTTGCATAACCTCCAGATCAAGGATAAAGGCCTG TATC AAT GT AT TAT C GAG C AC AAAAAAC C GAG AG GC AT GAT AC GGAT AC AT C AAAT G AAC T C T GAAC T GTCAGTCTTGGCAAATTTCAGTCAACCTGAGATTGTACCTATCAGCAACATTACCGAGAA TGTC TACATTAATCTCACTTGCTCCAGCATACATGGGTATCCAGAACCTAAGAAAATGTCCGTA CTCC T T AGAAC G AAG AAC T C AAC T AT T G AG T AC G AC G GAG T AAT G C AAAAAT C C C AAG AT AAT GT C AC AGAGC T T TAT GAT GT GAG TAT AT C AC T GAG T G TAT CAT T C C C T GAT GT G AC T TC AAAT AT G AC T ATTTTCTGTATATTGGAGACAGATAAAACAAGGCTGTTGAGTAGTCCTTTCTCAATAGAG TTGG AGGACCCACAACCGCCACCCGACCACATCCCC (SEQ ID NO: 120) or an active fragment thereof. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least 60% identity to SEQ ID NO: 120. In some embodiments, the nucleic acid molecule comprises a nucleic 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: 120. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence identical to SEQ ID NO: 120. The sequence of SEQ ID NO: 120 is an exemplary sequence and is not meant to be limiting in any way. Due to the degenerate nature of 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 that encodes a polypeptide as provided for herein. In some embodiments, the RNA molecule is a mRNA molecule.

In some embodiments a nucleic acid molecule is provided. In some embodiments, the nucleic acid molecule encodes for a polypeptide as provided for herein. In some embodiments, the nucleic acid molecule encoding for the polypeptide comprises a nucleic acid sequence substantially similar to SEQ ID NO: 121:

ATGATTTTTCTCTTGCTTATGCTTTCTCTTGAGTTGCAACTTCATCAGATTGCCGCC CTTTTTA CCGTCACAGTCCCTAAAGAACTTTACATAATCGAGCACGGTAGCAACGTAACGCTTGAAT GTAA CTTTGACACGGGGAGTCACGTCAATCTTGGAGCTATTACGGCGTCTCTTCAGAAGGTGGA AAAC GATACGAGCCCTCACCGGGAGCGCGCTACCCTGCTTGAAGAACAGTTGCCGCTTGGAAAA GCAT CCTTTCACATACCGCAGGTTCAAGTTCGAGATGAAGGCCAATACCAATGCATAATTATTT ATGG AGTCGCCTGGGACTATAAGTACCTGACATTGAAGGTTAAAGCGAGCTATAGGAAGATCAA TACT CACATACTGAAGGTCCCGGAGACTGATGAAGTGGAATTGACTTGTCAGGCAACAGGGTAT CCTT TGGCGGAGGTTAGTTGGCCTAATGTTTCCGTGCCCGCGAACACATCTCATTCAAGGACAC CAGA GGGCCTGTATCAGGTGACCTCCGTTCTGCGCCTGAAGCCACCGCCAGGTAGGAACTTTAG CTGC GTCTTCTGGAATACTCATGTTCGAGAACTCACACTTGCTTCAATCGACCTTCAGTCCCAG ATGG AGCCTAGAACTCATCCTACCGGATCAGGTAGTGGCGGCGGAAGCGGGAGTGGCGGAGGCT CCGG GTCTGGTTCCGGCCTGTCACACTTTTGTTCTGGCGTAATCCACGTCACTAAGGAGGTTAA AGAG GTAGCCACCCTTTCTTGTGGTCATAATGTCTCTGTGGAAGAACTTGCTCAAACAAGAATC TACT GGCAAAAGGAGAAGAAAATGGTCTTGACGATGATGAGCGGTGACATGAACATATGGCCAG AGTA CAAGAAT C G C AC CAT T T T C GAC AT AAC C AAC AAT C T T T C AAT 0 GT AAT AC T GGC C C T C AG G C C G TCTGATGAAGGTACGTATGAGTGCGTTGTCTTGAAATACGAAAAAGACGCCTTCAAGCGA GAAC AT C T G G C G G AG G T GAC ACTTTCTGT T AAAG C C G AT T T C C C AAC T C C T T C T AT T T C AG AT T T T GA AATCCCGACCTCTAACATCCGGAGAATTATCTGCAGCACGTCAGGGGGGTTTCCTGAACC ACAT TTGTCTTGGCTTGAGAATGGCGAAGAATTGAACGCCATCAACACCACCGTTAGTCAAGAC CCAG AG AC G G AAC T GT AT GC C G T T T C AAG T AAGC T C G AT T T TAAT AT GAC AAC T AAC C AC T C C T T CAT GTGTCTGATAAAATATGGACATCTGCGGGTAAATCAGACATTTAACTGGAACACGACAAA ACAA GAGCATTTCCCAGATGATAAAACCCATACCTGTCCTCCTTGCCCTGCCCCCGAACTGCTG GGTG

GGCCTAGCGTGTTCCTTTTCCCGCCCAAGCCGAAGGACACACTCATGATCAGTCGAA CGCCCGA AGTTACGTGCGTGGTCGTAGATGTGTCCCACGAAGATCCAGAGGTGAAGTTTAATTGGTA CGTT GATGGTGTAGAGGTACATAATGCCAAAACAAAGCCACGCGAAGAGCAGTATAATAGCACC TATA GAGTAGTTAGCGTTCTGACAGTTCTCCACCAGGATTGGCTTAACGGCAAAGAGTACAAGT GTAA AGTATCAAACAAAGCACTTCCTGCACCCATTGAAAAAACTATATCTAAGGCCAAAGGCCA ACCT AGGGAACCGCAGGTCTACACGCTGCCCCCGAGCAGAGACGAGCTTACGAAGAACCAAGTC TCAC TGACGTGTCTTGTCAAGGGTTTTTACCCCAGTGACATTGCAGTAGAGTGGGAATCAAATG GGCA AC CAGAAAAC AAC TAT AAAAC TAG AC C AC GAG T C T T GGAC AG T GAT GG AT CAT TCTTCTTG TAT TCAAAACTTACGGTTGACAAGAGCCGCTGGCAACAGGGCAACGTGTTCAGCTGCTCCGTG ATGC ATGAGGCGCTTCACAACC ATTACACCCAAAAGAGTCTTTCACTCTCTCCCGGAAAA (SEQ ID NO: 121) or an active fragment thereof. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least 60% identity to SEQ ID NO: 121. In some embodiments, the nucleic acid molecule comprises a nucleic 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: 121. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence identical to SEQ ID NO: 121. The sequence of SEQ ID NO: 121 is an exemplary sequence and is not meant to be limiting in any way. Due to the degenerate nature of 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 that encodes a polypeptide as provided for herein. In some embodiments, the RNA molecule is a mRNA molecule.

In some embodiments a nucleic acid molecule is provided. In some embodiments, the nucleic acid molecule encodes for a polypeptide as provided for herein. In some embodiments, the nucleic acid molecule encoding for the polypeptide comprises a nucleic acid sequence substantially similar to SEQ ID NO: 122: ATGCGCATATTCGCAGTCTTTATATTTATGACTTATTGGCATCTTCTTAATGCATTTACT GTCA CTGTTCCCAAGGATCTGTACGTTGTAGAGTACGGCTCCAvACATGACTATAGAGTGCAAA TTTCC AGTCGAGAAACAGCTCGACTTGGCAGCACTCATTGTGTACTGGGAAATGGAAGACAAAAA CATC ATTCAGTTCGTCCACGGGGAGGAGGACTTGAAAGTTCAACACTCATCCTATCGCCAACGA GCCC GCCTGTTGAAGGACCAGCTTTCTCTCGGTAACGCTGCACTTCAGATTACGGATGTTAAGT TGCA GGATGCAGGAGTCTACCGATGTATGATTAGTTACGGCGGTGCCGATTACAAGAGGATCAC TGTT AAAGTCAACGCTCCTTACAATAAAATAAACCAACGGATTCTCGTGGTAGATCCTGTGACT TCCG AGCACGAATTGACGTGCCAAGCAGAAGGATACCCGAAAGCCGAGGTGATCTGGACAAGCT CCGA TCATCAAGTCCTCTCAGGGAAGACGACGACCACGAACTCTAAGAGGGAAGAGAAGCTGTT CAAC GTAACATCAACGCTCCGCATTAACACGACTACTAATGAGATATTCTACTGCACTTTCCGG CGAC T C G AT C C G GAG G AAAAC CAT AC AG C G G AG C T T G T G AT T C C G G AAC T T C C AC T C G C G C AT C C T C C GAACGAGCGGGGATCAGGTAGTGGCGGCGGATCCGGGAGTGGCGGGGGCAGCGGGTCTGG TTCC GCACCGTTGAAA.ATACAAGCGTACTTCAA.CGAAACTGCAGACCTTCCGTGTCAATTCG CGAACT CACAAAATCAATCCCTTAGTGAGCTTGTAGTATTTTGGCAGGACCAGGAAAATCTCGTCC TGAA CGAAGTTTATCTCGGTAAAGAGAAGTTCGACAGCGTACATAGTAAATACATGGGCAGAAC TTCT TTCGATAGTGATAGTTGGACGCTCAGGTTGCATAACCTCCAGATCAAGGATAAAGGCCTG TATC AATGTATTATCCACCACAAAAAACCCACAGGCATGATACGGATACATCAAATGAACTCTG AACT GTCAGTCTTGGCAAATTTCAGTCAACCTGAGATTGTACCTATCAGCAACATTACCGAGAA TGTC T AC AT T AAT C T C AC T T GC T C C AG C AT AC AT GG GT AT C C AG AAC C T AAG AAAAT G T C C GT AC T C C TTAGAACGAAGAACTCAACTATTGAGTACGACGGAGTAATGCAAAAATCCCAAGATAATG TCAC AG AGC T T TAT GAT GT GAG TAT AT C AC T GAG T GT AT CAT T C C C T GAT G T GAC T T C AAAT AT G AC T ATTTTCTGTATATTG GAG AC AGAT AAAAC AAG G C T G T T G AG T AG T C C T T T C T C AAT AG AG T T G G AGGACCCACAACCGCCACCCGACCACATCCCCGATAAAACCCATACCTGTCCTCCTTGCC CTGC CCCCGAACTGCTGGGTGGGCCTAGCGTGTTCCTTTTCCCGCCCAAGCCGAAGGACACACT CATG ATCAGTCGAACGCCCGAAGTTACGTGCGTGGTCGTAGATGTGTCCCACGAAGATCCAGAG GTGA AGTTTAATTGGTACGTTGATGGTGTAGAGGTACATAATGCCAAAACAAAGCCACGCGAAG AGCA GTATAATAGCACCTATAGAGTAGTTAGCGTTCTGACAGTTCTCCACCAGGATTGGCTTAA CGGC AAAG AG TAG AAG T G T AAAG T AT C AAAC AAAG C AC T T C C T G C AC C C AT T G AAAAAAC TAT AT C TA AGGCCAAAGGCCAACCTAGGGAACCGCAGGTCTACACGCTGCCCCCGAGCAGAGACGAGC TTAC GAAGAACCAAGTCTCACTGACGTGTCTTGTCAAGGGTTTTTACCCCAGTGACATTGCAGT AGAG TGGGAATCAAATGGGCAACCAGAAAACAACTATAAAACTACACCACCAGTCTTGGACAGT GATG GATCATTCTTCTTGTATTCAAAACTTACGGTTGACAAGAGCCGCTGGCAACAGGGCAACG TGTT CAGCTGCTCCGTGATGCATGAGGCGCTTCACAACCATTACACCCAAAAGAGTCTTTCACT CTCT CCCGGAAAA (SEQ ID NO: 122) or an active fragment thereof. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least 60% identity to SEQ ID NO: 122. In some embodiments, the nucleic acid molecule comprises a nucleic 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: 122. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence identical to SEQ ID NO: 122. The sequence of SEQ ID NO: 122 is an exemplary sequence and is not meant to be limiting in any way. Due to the degenerate nature of 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 that encodes a polypeptide as provided for herein. In some embodiments, the RNA molecule is a mRNA molecule.

In some embodiments, a vector is provided comprising a nucleic acid molecule as provided for herein. In some embodiments, the vector is a plasmid. In some embodiments, the vector is a virus. In some embodiments, a plasmid is provided comprising a nucleic acid molecule as provided for herein. In some embodiments, a composition is provided comprising an encapsulated nucleic acid molecule as provided for herein.

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, or 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 El gene, E3 gene, E4 gene, E4 promoter, hexon gene, penton gene, fiber gene, or combination thereof. In some embodiments, the replicationincompetent recombinant virus comprises a defective or modified El gene.

In some embodiments, the recombinant virus comprises a nucleic acid molecule as provided for 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 for a polypeptide as provided for 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' adenoviral ITR and a 3’ adenoviral ITR.

In some embodiments, a cell is provided. In some embodiments, the cell comprises a polypeptide molecule as provided for 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 for a polypeptide as provided for 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 cell further comprises a chimeric antigen receptor (“CAR”). CARs 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 a polypeptide as provided for herein with the CAR. Thus, in some embodiments, a cell is provided comprising a CAR and a polypeptide as provided for herein.

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, a NK cell, a dendritic cell, a monocyte, a B-cell, a myeloid cell, and the like.

In some embodiments, a host cell is provided. In some embodiments, the host cell contains a competent El gene, E3 gene, E4 gene, E4 promoter, hexon gene, penton gene, fiber gene or a combination thereof to complement any defective or modified gene in the recombinant virus. In some embodiments, the host cell contains a competent El gene to complement the defective or modified El gene in the recombinant virus. In some embodiments, the competent El gene, E3 gene, E4 gene, E4 promoter, hexon gene, penton gene, fiber gene or a combination thereof are provided to the host cell via contacting the host cell transiently. In some embodiments, the host cell contains, within its genome, the competent El gene, E3 gene, E4 gene, E4 promoter, hexon gene, penton gene, fiber gene or a combination thereof.

Combination Compositions

In some embodiments, compositions are provided herein that comprise any of the interleukin polypeptides and any of the immune checkpoint polypeptides provided for herein. In some embodiments, a composition comprises a first polypeptide and a second polypeptide, wherein the first polypeptide is any of the interleukin polypeptides provided for here and wherein the second polypeptide is any of the immune checkpoint polypeptides provided for herein.

In some embodiments, the composition further comprises a third polypeptide, such as an antigen. In some embodiments, the antigen is any antigen provided for herein. 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).

In some embodiments, the compositions comprise nucleic acid molecules that encode for any of the polypeptides provided for herein. In some embodiments, the composition comprises a first nucleic acid molecule encoding for a first polypeptide as provided for herein. In some embodiments, the composition comprises a second nucleic acid molecule encoding for a second polypeptide as provided for herein. In some embodiments, the composition comprises a third nucleic acid molecule encoding for a third polypeptide as provided for herein.

Vectors, Plasmids, Liposomes, Pharmaceutical Compositions, and Host Cells

Accordingly, in some embodiments, vectors encoding the polypeptides described herein are provided, as well as host cells transformed or transduced with such vectors. Any nucleic acids encoding the proteins described herein may be contained in one or more vectors, which can, for example, comprise a selectable marker and an origin of replication, for propagation in a host. In some embodiments, the vectors further include suitable transcriptional or translational regulatory sequences, such as those derived from a mammalian, microbial, viral, or insect genes, operably linked to the nucleic acid molecule encoding the protein. Examples of such regulatory sequences include transcriptional promoters, operators, or enhancers, mRNA ribosomal binding sites, and appropriate sequences that control transcription and translation. Nucleotide sequences are operably linked when the regulatory sequence functionally relates 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.

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

Thus, in some embodiments, a vector comprises a first nucleic acid molecule as provided for herein, a second nucleic acid molecule as provided for herein, and, optionally , a third nucleic acid molecule as provided for herein. In some embodiments, compositions herein comprise a first vector comprising the first nucleic acid molecule, and a second vector comprising the second nucleic acid molecule. In some embodiments, the first vector or the second vector further comprise a third nucleic acid molecule as provided for herein. In some embodiments, the composition comprises a third vector comprises the third nucleic acid molecule. In some embodiments, the first, the second, and/or the third nucleic acid molecules are present in their own independent vectors. For example, in some embodiments, the composition comprises a first vector comprising the first nucleic acid molecule, a second vector comprising the second nucleic acid molecule, and optionally a third vector comprising the third nucleic acid molecule. In some embodiments, each vector is in a separate composition.

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

In some embodiments, a recombinant virus comprises a first nucleic acid molecule as provided for herein, a second nucleic acid molecule as provided for herein, and, optionally, a third nucleic acid molecule as provided for herein. In some embodiments, the recombinant virus may contain any one or more vectors or plasmids described herein. In some embodiments, the recombinant virus is a adenovirus or a lend virus. In some embodiments, the recombinant virus is replication-competent or replication-incompetent. In some embodiments, the recombinant virus is a replication-incompetent adenovirus.

The nucleic acid molecules, vectors, and plasmids described herein can also be encompassed by lipids or liposomes. Thus, in some embodiments, a liposome comprises a first nucleic acid molecule as provided for herein, a second nucleic acid molecule as provided for herein, and, optionally, and optionally, a third nucleic acid molecule as provided for herein. In some embodiments, the liposomes may contain an one or more of the vectors or plasmids described herein, in some embodiments, the lipids and liposomes comprises a cationic lipid, such as, but not limited to, l,2-Dioleoyl-3-Trimethylammonium- Propane (DOTAP), 1,2,-dioleoyl-sn- glycero-3-phosphochiline (DOPC), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), N- [l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 5- carboxyspermylglycinedioctadecylamide (DOGS), 2,3-dioleyloxy-N-[2(spermine- carboxamido)ethyl]-N,N-dimethyl-l-propanamin- ium (DOSPA), 1 ,2-Dioleoyl-3- Dimetliylammomum- Propane (DODAP), l,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1 ,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), 1 ,2-dilinoleyloxy-N,N- dimethyl-3-aminopropane (DLinDMA), heptatriaconta-6,9,28,3 l-tetraen!9-yl 4- (dimethylamino)butanoate (DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]- dioxolane (DLin-KC2-DMA), 1 ,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA), N-dioleyl-N,N-dimethyl ammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N~( 1 ,2-dimyristyioxyprop-3-yi)-N,N -dimethyl -N-hydroxyethyl ammonium bromide (DMRIE), 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-l-(ci s, cis-9,12- octadecadienoxy)propane (CLinDMA), 2-[5’-(cholest-5-en-3-beta-oxy)-3’-oxapentoxy)-3- dimethyl- 1 -(cis,cis-9‘, 1 - -2'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4- dioleyloxybenzylamine (DMOBA), l,2-N,N'-dioleylcarbamyl-3-diniethylaminopropane (DOcarbD AP), 2,3-Dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N’- DilinoleyIcarbamyl-3-dimethylaminopropane (DLincarbDAP), l,2-Dilinoleoylcarbamyl-3- dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4-dimeihylaminoethyl-[l ,3]-dioxolane (DLin-K-XTC2-DMA), or Cl 2-200.

In another aspect, the present embodiments provide compositions, e.g., pharmaceutically acceptable compositions, which include a polypeptide as provided for herein or a nucleic acid molecule encoding the same, which can be, for example, be formulated together 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 glycols, propylene glycol, PEG 400, glycerin, DMA, ethanol, benzyl alcohol, citric acid/sodium citrate (pH3), citric acid/sodium citrate (pH5), tris(hydroxymethyl)amino methane HC1 (pH7.0), 0.9% saline, and 1.2% saline, and any combination thereof.

In some embodiments, a pharmaceutical composition is provided that comprises a cell comprising a vector, polypeptide, or nucleic acid molecule as provided for herein. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In some embodiments, suitable pharmaceutically acceptable carries include, but are not limited to, water, silicone, waxes, petroleum jelly, polyethylene glycol, propylene glycol, liposomes, a lipid such as cholesterol, cationic lipids such as 1,2,- dioleoyl-3-trimethylammonium propane (DOTAP), 1 ,2,-dioleoyl-sn-glycero-3-phosphochiline (DOPC), and l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sugars such as mannitol and lactose, and other materials depending on the specific type of formulation used. In some embodiments, suitable pharmaceutically acceptable carries include, but are not limited to, nanoparticles such as gold or metallic nanoparticles.

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

The compositions may be in a variety of forms. These include, for example, liquid, semisolid 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. Typical compositions are in the form of injectable or infusible solutions. 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 by enteral, sublingual, inhalation, or intranasal. In some embodiments, the composition is administered locally, e.g., by injection, or topical application, to a target site. For example, the pharmaceutical compositions can be lyophilized and reconstituted for use prior to administration to the patient.

The phrases “parenteral administration” and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

Compositions, such as pharmaceutical compositions, typically are 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. Sterile injectable solutions can be prepared by incorporating the active compound (i.e., therapeutic molecule, nucleic acid molecule, cell, polypeptide, vector, etc.) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated 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 that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. In certain embodiments, the active compound may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, poly anhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

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

In some embodiments, administration of the polypeptides, nucleic acids, vectors, plasmids, viruses, liposomes, and compositions provided for herein can be administered in separate doses or formulations. For example, a first nucleic acid, vector, or plasmid can be administered separately from a second or a third nucleic acid, vector, or plasmid as described herein. In some embodiments, administration can be simultaneously, sequentially, or in parallel. In some embodiments, the administration of each component will comprise separate compositions, formulations, dosage regimens, methods of administration, and concentrations, as appropriate, to achieve the desired response. Likewise, in some embodiments, one or more of the polypeptides, nucleic acids, vectors, plasmids, viruses, liposomes, and compositions provided for herein can 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 for herein can be administered in the same dose or formulation, while other polypeptides, nucleic acids, vectors, plasmids, viruses, liposomes, and compositions provided for herein can be administered in separate doses or formulations.

Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

An exemplary, non - limiting range for a therapeutically or prophylactically effective amount of a therapeutic compound is 0.1 -30 mg/kg, more preferably 1-25 mg/kg. Dosages and therapeutic regimens of the therapeutic compound can be determined by a skilled artisan. In certain embodiments, the therapeutic compound is administered by injection (e.g., subcutaneously or intravenously) at a dose of about 1 to 40 mg/kg, e.g., 1 to 30 mg/kg, e.g., about 5 to 25 mg/kg, about 10 to 20 mg/kg, about 1 to 5 mg/kg, 1 to 10 mg/kg, 5 to 15 mg/kg, 10 to 20 mg/kg, 15 to 25 mg/kg, or about 3 mg/kg. The dosing schedule can vary from e.g., once a week to once every 2, 3, or 4 weeks, or, in some embodiments, the dosing schedule can be, once every month, every 2 months, every 3 months, or every 6 months. In one embodiment, the therapeutic compound is administered at a dose from about 10 to 20 mg/kg every other week. The therapeutic compound can be administered by intravenous infusion at a rate of more than 20 mg/min, e.g., 20-40 mg/min, and typically greater than or equal to 40 mg/min to reach a dose of about 35 to 440 mg/m2, typically about 70 to 310 mg/m2, and more typically, about 110 to 130 mg/m2. In embodiments, the infusion rate of about 110 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 of less than 10 mg/min, e.g., less than or equal to 5 mg/min to reach a dose of about 1 to 100 mg/m2, e.g., about 5 to 50 mg/m2, about 7 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 is to be noted that dosage values may vary with the type and severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.

The pharmaceutical compositions may include a “therapeutically effective amount” or a “prophylactic-ally effective amount” of the compositions, vectors, cells, polypeptides, or nucleic acid molecules encoding the same. A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of an active ingredient or molecule may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the therapeutic compound to elicit a desired response in the individual. A “therapeutically effective dosage” can, for example, inhibit a measurable parameter, e.g., tumor growth, by at least about 20%, by at least about 40%, by at least about 60%, and by at least about 80% relative to untreated subjects. 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 a composition can be evaluated by examining the ability of the compound to inhibit, such inhibition in vitro by assays known to the skilled practitioner.

A “prophylactic ally effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophy lactically effective amount can be, but is not necessarily, less than the therapeutically effective amount.

Also provided herein are kits comprising compositions, cells, vectors, nucleic acid molecules, or polypeptides as described herein. The kit can include one or more other elements including: instructions for use: other reagents, e.g., a label, a therapeutic agent, or an agent useful for chelating, or otherwise coupling, a molecule to a label or other therapeutic agent, or a radioprotective composition; devices or other materials for preparing the molecule for administration; pharmaceutically acceptable carriers; and devices or other materials for administration to a subject.

In some embodiments, a cell is provided that comprises a polypeptide as provided for herein. In some embodiments, a cell is provided that comprises a genomic nucleic acid molecule comprising the nucleic acid molecule as provided for herein. A genomic nucleic acid molecule refers to a heterologous nucleic acid molecule that is integrated into the genome of the host cell. In some embodiments, the cell further comprises a chimeric antigen receptor. Chimeric antigen receptors, 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 a polypeptide as provided for herein with the CAR. Thus, in some embodiments, a cell is provided comprising a CAR and a polypeptide as provided for herein.

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, a NK cell, a dendritic cell, and the like.

The cell can be produced according to any known methods. For example, in some embodiments, the method of producing a cell that is provided comprises contacting the cell with a vector, plasmid, virus, liposome, or any composition provided for herein, comprising a nucleic acid molecule encoding for a polypeptide as provided for herein. This can be done, for example, under conditions that are suitable to express the polypeptide in the cell and/or to express on the surface of the cell. In some embodiments, the vector that is used is a plasmid or a virus. In some embodiments, the virus is an adenovirus or lentivirus. In some embodiments, the adenovirus is a repl ication- incompetent or replication-competent adenovirus. In some embodiments, the contacting comprises transducing or transfecting the cell with the vector. In some embodiments, the vector further comprises a nucleic acid molecule encoding for a chimeric antigen receptor or a tumor antigen.

In some embodiments, methods of producing a cell comprising a polypeptide as provided herein in vivo are provided, the methods comprising administering to a subject a vector encoding for the polypeptide to a subject, wherein the vector transduces or transfects a cell in vivo to produce the cell comprising a polypeptide as provided for herein. In some embodiments, the cell is an immune cell. In some embodiments, the immune cell is, but not limited to a T cell, a NK cell, a dendritic cell, and the like. In some embodiments, the vector further comprises a nucleic acid molecule encoding for 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 lentivirus. In some embodiments, the adenovirus is a replication-incompetent or replicalion- competent adenovirus .

Methods

“Treatment” of any disease mentioned herein encompasses an alleviation of at least one symptom of the disease, a reduction in the severity of the disease, or the delay or prevention of disease progression to more serious symptoms that may, in some cases, accompany the disease or to at least one other disease. Treatment need not mean that the disease is totally cured. A useful therapeutic agent needs only to reduce the severity of a disease, reduce the severity of symptom(s) associated with the disease or its treatment, or delay the onset of more serious symptoms or a more serious disease that can occur with some frequency following the treated condition. For example, if the disease is a tumor, the composition may reduce the growth or spread of the tumor, or the tumors effect on the tissue in which it is present. A patient's condition can be assessed by standard techniques. Suitable procedures vary according to the patient's condition and symptoms.

In some embodiments, the compositions provided for herein can be used to modify an immune response in a patient. In some embodiments, the methods comprise administering to the patient a polypeptide or a vector comprising a nucleic acid molecule encoding for a polypeptide as provided for herein. In some embodiments, the immune response is an activated immune response, such as in activating NK and/or CD8+ T cells.

In some embodiments, the compositions provided for herein can be used to treat cancer in a subject (patient). In some embodiments, the methods comprise administering to the patient a vector comprising a nucleic acid molecule encoding for a polypeptide as provided for herein. In some embodiments, the cancer is lymphoma, leukemia, nasopharyngeal, gastric, cervical, hepatocellular, polyoma, anal, head and neck tumor. In some embodiments, the tumor is a lung cancer tumor. In some embodiments, the tumor is benign and metastatic forms of cancer, for example, ovarian cancer (e.g. ovarian carcinoma), reproductive cancers (breast, cervical, testicular', uterine, and placental cancers), lung cancer, gastric cancer, hepatic cancer, pancreatic cancer, bile duct cancer, cancer of the urinary bladder, kidney cancer, colon cancer, small bowel cancer, skin cancer, brain cancer, head and neck cancer, sarcoma, and germ cell tumors, among others.

In some embodiments, diseases that can be treated with the compositions provided for herein also include myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). In some embodiments, the subject has MDS including Fanconi Anemia, refractory anemia, refractory neutropenia, refractory thrombocytopenia, refractory anemia with ringed sideroblasts (RARS), refractory cytopenia with multilineage dysplasia (RCMD), refractory anemia with multilineage dysplasia and ringed sideroblasts (RCMD-RS), refractory anemia with excess blasts I and II (RAEB), myelodysplastic syndrome, unclassified (MDS-IJ), MDS associated with isolated del(5q)-syndrome, chronic myelomonocytic leukemia (CMML), juvenile myelomonocytic leukemia (JMML), refractory cytopenia of childhood, or a combination 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 in 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 in chromosome 3), AML (megakaryoblastic) with a translocation between chromosomes 1 and 22, AML with myelodysplasia-related changes, AML related to previous chemotherapy or radiation (alkylating agent-related ANIL, topoisomerase II inhibitor-related AML), /XML not otherwise categorized (AML minimally differentiated (MO), AML with minimal maturation (Ml), 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 leukemias (also known as mixed phenotype acute leukemias), or a combination thereof. In some embodiments, administration of the compositions provided for herein to a subject decreases the incidence of one or more symptoms associated with MDS or AML or decreases one or more markers of viability of MDS or AML cells. In some embodiments, the one or more symptoms associated with MDS or AML include decreasing marrow failure, immune dysfunction, transformation to overt leukemia, or a combination thereof in the subject, or wherein the marker of viability of MDS or AML cells includes survival over time, proliferation, growth, migration, formation of colonies, chromatic assembly, DNA binding, RNA metabolism, cell migration, cell adhesion, inflammation, or a combination thereof.

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 PD-1 antagonist antibodies. In some embodiments, the tumor is also treated with a PD-L ₁ inhibitor, such as a PD-L ₁ antagonist, such as PD-L ₁ antagonist antibodies. In some embodiments, the tumor is also treated with a CTLA-4 inhibitor, such as a CTLA-4 antagonist, such as CTLA-4 antagonist antibodies.

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

In some embodiments, the methods comprise administering a pharmaceutical composition comprising the polypeptides provided herein or a nucleic acid molecule encoding the same as provided for herein to the subject. In some embodiments, the subject is a subject in need thereof. Any of the above-described can be administered in the form of a compositions (e.g. pharmaceutical compositions) that are described herein.

To treat the disease of interest, the compositions comprising the cells, vectors, nucleic acid molecules, or polypeptides described herein can be administered by any appropriate method including, but not limited to, parenteral, topical, oral, nasal, vaginal, rectal, or pulmonary (by inhalation) administration. If injected, the composition(s) can be administered intra-articularly, intravenously, intraarterially, intramuscularly, intravesicularly, intraperitoneally, or subcutaneously by bolus injection or continuous infusion. Localized administration, that is, at the site of disease, is contemplated, as are transdernial 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 aerosol form, and the like. Administration via a suppository inserted into a body cavity can be accomplished, for example, by inserting a solid form of the composition in a chosen body cavity and allowing it to dissolve. Other alternatives include eyedrops, oral preparations such as pills, lozenges, syrups, and chewing gum, and topical preparations such as lotions, gels, sprays, and ointments.

In the performance of the methods of treatment, the compositions described herein can be administered as described herein and above. For example, the composition can be administered at any dosage, frequency, and duration that can be effective to treat the condition being treated. The dosage depends on the molecular nature of the active ingredient and the nature of the disorder being treated. Treatment may be continued as long as necessary to achieve the desired results. The compositions provided for herein can be administered as a single dosage or as a series of dosages given periodically, including multiple times per day, daily, every other day, twice a week, three times per week, weekly, every other week, and monthly dosages, among other possible dosage regimens. The periodicity of treatment may or may not be constant throughout the duration of the treatment. For example, treatment may initially occur at weekly intervals and later occur every other week. Treatments having durations of days, weeks, months, or years are encompassed by the embodiments provided for herein. Treatment may be discontinued and then restarted. Maintenance doses may or may not be administered after an initial treatment.

Dosage may be measured as milligrams per kilogram of body weight (mg/kg) or as milligrams per square meter of skin surface (mg/m ² ) or as a fixed dose, irrespective of height or weight. All of these are standard dosage units in the art. A person's skin surface area is calculated from her height and weight using a standard formula.

Also provided herein are methods of acti vating CD8+ T cells. In some embodiments, methods provided herein can be used to activate NK cell. In some embodiments, the methods comprise administering to a subject in need thereof a therapeutically effective amount of a polypeptide or a nucleic acid molecule encoding the same, or vector comprising the same or as otherwise described herein or a pharmaceutic ai composition comprising the same.

As used herein, the phrase “in need thereof” means that the subject (animal or mammal) has been identified as having a need for the particular method or treatment. In some embodiments, the identification can be by any means of diagnosis. In any of the methods and treatments described herein, the animal or mammal can be in need thereof. In some embodiments, the animal or mammal is in an environment or will be traveling to an environment in which a particular disease, disorder, or condition is prevalent.

The compositions provided herein can also be administered in conjunction with other agents useful for treating the condition with which the patient is suffering from. Examples of such agents include both proteinaceous and non-proteinaceous drugs. When multiple therapeutics are co-administered, dosages may be adjusted accordingly, as is recognized in the pertinent art. “Co- administration” and combination therapy are not limited to simultaneous administration, but also include treatment regimens in compositions provided for herein are administered at least once during a course of treatment that involves administering at least one other therapeutic agent to the patient, such as an anti-cancer therapeutic, including but not limited to chemotherapy, radiation therapy, targeted therapy, immunotherapy, stem cell or bone marrow transplant therapy, hormone therapy, photodynamic therapy, or any combination thereof.

In some embodiments, the compositions provided herein can be combined, linked, or fused to at least one additional molecule. In some embodiments, the additional molecule is a biologically active molecule, for example, but not limited to, a protein, a polypeptide, a nucleic acid, a lipid, carbohydrate, or any combination thereof. In some embodiments, the additional molecule is a targeting moiety, for example, but not limited to, an antibody, an antigen, a ligand, a ligand trap such as a receptor domain, or any combination thereof. In some embodiments, the additional molecule is a therapeutic molecule, for example, but not limited to, an immunotherapeutic molecule, a checkpoint inhibitor, an immune system activator, an oncological therapeutic, an antibody, or any combination thereof.

In some embodiments, the compositions provided herein can be attached to the at least one additional molecule at the C-terminus of the composition. In some embodiments, the compositions provided herein can be attached to the at least one additional molecule at the N- terminus of the composition. In some embodiments, the compositions provided herein can be attached to the at least one additional molecule at any linker of the composition. In some embodiments, the compositions provided herein can be attached to the at least one additional molecule before any cleavable linkers have been cleaved. In some embodiments, the compositions provided herein can be attached to the at least one additional molecule after the composition has been cleaved at a cleavable linker.

Embodiments

In some embodiments, embodiments provided herein also include, but are not limited to:

1. A polypeptide comprising the formula of: X1-L ₁ -X ₂ -L ₂ -X ₃ -L ₃ -X ₄ -L ₄ -X ₅ , wherein: X ₁ is a interleukin 21 (IL-21) polypeptide, interleukin 15 (IL-15) polypeptide, interleukin 12 p40 subunit (IL- 12 p40) polypeptide, interleukin 12 p35 subunit (IL- 12 p35) polypeptide, or interleukin 15 receptor (IL- 15R a) polypeptide; X ₂ . is a IL-21 polypeptide, IL-15 polypeptide, IL-12 p40 polypeptide, IL-12 p35 polypeptide, or IL-15Rα polypeptide; X ₃ is a IL-21 polypeptide, IL-15 polypeptide, IL-12 p40 polypeptide, IL-12 p35 polypeptide, or IL-15Rα polypeptide; X ₄ is a IL-21 polypeptide, IL-15 polypeptide, IL-12 p40 polypeptide, IL-12 p35 polypeptide, or IL-15Rα polypeptide; X ₅ is a IL-21 polypeptide, IL-15 polypeptide, IL-12 p40 polypeptide, IL-12 p35 polypeptide, or IL-15Rα polypeptide; L ₁ , L ₂ , L'3, and L ₄ tire each, independently, a polypeptide linker that comprise the same or different polypeptide sequences, and provided each of X ₁ , X ₂ , X ₃ , X ₄ , and X ₅ are different.

2. The polypeptide of embodiment 1, wherein X ₁ is a interleukin 21 (IL-21) polypeptide.

3. The polypeptide of embodiments 1 or 2, wherein: X ₁ is a IL-21 polypeptide; X ₂ is a IL- 15 polypeptide; X ₃ is a IL-12 p40 polypeptide; X ₄ is a IL-12 p35 polypeptide; and X ₅ is a IL-15Rα polypeptide. ypeptide of embodiments 1 or embodiment 2, wherein: X ₁ is a IL-21 polypeptide; X ₂ is a IL- 15 polypeptide; X ₃ is a IL-12 p35 polypeptide; X ₁ is a IL- 12 p40 polypeptide; and X ₅ is a IL-15Rα polypeptide. ypeptide of embodiments 1 or 2, wherein: X ₁ is a IL-21 polypeptide; X ₂ is a IL- 12 p40 polypeptide; X ₃ is a IL- 15 polypeptide; X ₄ is a IL-12 p35 polypeptide; and X ₅ is a IL- 15Rα polypeptide. ypeptide of embodiments 1 or 2, wherein:

Xi is a IL-21 polypeptide; X ₂ is a IL-12 p40 polypeptide; X ₃ is a IL-12 p35 polypeptide; X ₄ is a IL- 15 polypeptide; and

Xs is a IL-15Rα polypeptide. ypeptide of embodiments 1 or 2, wherein: X ₁ is a IL-21 polypeptide: X ₂ is a IL-12 p35 polypeptide; X ₃ is a IL- 12 p40 polypeptide; X ₄ is a IL- 15 polypeptide; and X ₅ is a IL- 15Rα polypeptide. ypeptide of embodiments 1 or 2. wherein: X ₁ is a IL-21 polypeptide; X ₂ is a IL-12 p35 polypeptide; X ₃ is a IL- 15 polypeptide; X ₄ is a IL- 12 p40 polypeptide; and X ₅ is a IL-15Rα polypeptide. ypeptide of embodiments 1 or 2, wherein: X ₁ is a IL-21 polypeptide; X ₂ is a IL- 12 p40 polypeptide; X ₃ is a IL-12 p35 polypeptide; X ₄ is a IL- 15 polypeptide; and X ₅ is a IL-15Rα polypeptide. ypeptide of embodiments 1 or 2, wherein: X ₁ is a IL-21 polypeptide;

X ₂ is a IL-12 p40 polypeptide; X ₃ is a IL- 15 polypeptide; X ₄ is a IL-12 p35 polypeptide; and X ₅ is a IL-15Rα polypeptide. ypeptide of embodiments 1 or 2, wherein:

X-. is a IL-21 polypeptide; X ₂ is a IL-12 p35 polypeptide; X ₃ is a IL-12 p40 polypeptide; X ₄ is a IL- 15 polypeptide; and X ₅ is a IL-15Rα polypeptide. ypeptide of embodiments 1 or 2, wherein: X ₁ is a IL-21 polypeptide; X ₂ is a IL-12 p35 polypeptide; X ₃ is a IL- 15 polypeptide; X ₄ is a IL-12 p40 polypeptide; and X ₅ is a IL- 15Rα polypeptide.

13. The polypeptide of any of the preceding 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 for herein.

14. The polypeptide of any of the preceding embodiments, wherein the IL-21 polypeptide comprises an amino acid sequence of SEQ ID NO: 71, or as otherwise provided for herein.

15. The polypeptide of any of the preceding embodiments, wherein the IL- 12 p40 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 for herein.

16. The polypeptide of any of the preceding embodiments, wherein the IL-12 p40 polypeptide comprises an amino acid sequence of SEQ ID NO: 1, 2, 3, or 4, or as otherwise provided for herein.

17. The polypeptide of any of the preceding embodiments, wherein the IL-12 p35 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 for herein.

18. The polypeptide of any of the preceding embodiments, wherein the IL- 12 p35 polypeptide comprises an amino acid sequence of SEQ ID NO: 5, 6, 7, or 8, or as otherwise provided for herein.

19. The polypeptide of any of the preceding 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 for herein.

20. The polypeptide of any of the preceding embodiments, wherein the IL- 15 polypeptide comprises an amino acid sequence of SEQ ID NO: 9 or 10 or as otherwise provided for herein.

21. The polypeptide of any of the preceding 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 for herein.

22. The polypeptide of any of the preceding embodiments, wherein the IL- 15Rα polypeptide comprises an amino acid sequence of SEQ ID NO: 11 or 12 or as otherwise provided for herein.

23. The polypeptide of any of the preceding embodiments, wherein one or more of L ₁ , L ₂ , L<, and L ₄ are each, independently, a cleavable linker.

24. The polypeptide of embodiment 23, wherein L ₁ , L ₃ , and 1,4 are non-cleavable linkers.

The polypeptide of embodiments 23 or 24, wherein the cleavable linker is a furin cleavable linker, a VaLCit linker, a Val-Gly linker, a Gly-Gly linker, an Ala-Ala-Asn linker, or a linker comprising polyethylene glycol (PEG).

26. The polypeptide of any of the preceding embodiments, wherein one of Lj , L ₄ , L ₃ , and L ₄ comprise 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), wherein each n is, independently, 1, 2, 3, 4, or 5, or a combination thereof.

27. The polypeptide of embodiment 26, wherein n is 3.

28. The polypeptide of embodiments 26 or 27, wherein one or more of L ₁ , L ₂ , L ₃ , and L ₄ , comprise a sequence 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, RXXR where X is any amino acid, or a combination thereof.

29. The polypeptide of any one of embodiments 25-28, wherein each of L ₁ , L ₂ , L ₃ , and L<i, comprises a sequence 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, RXXR where X is any amino acid, or a combination thereof.

30. The polypeptide of any preceding embodiment, wherein the polypeptide comprises the sequence of SEQ ID NO: 72, or a variant thereof, or as otherwise provide for herein.

31. The polypeptide of any preceding embodiment, wherein the polypeptide comprises the sequence of SEQ ID NO: 73, or a variant thereof, or as otherwise provide for herein.

32. The polypeptide of any preceding embodiment, wherein the polypeptide comprises the sequence of SEQ ID NO: 74, or a variant thereof, or as otherwise provide for herein.

33. The polypeptide of any preceding embodiment, wherein the polypeptide comprises the sequence of SEQ ID NO: 75, or a variant thereof, or as otherwise provide for herein.

34. The polypeptide of any preceding embodiment, wherein the polypeptide comprises the sequence of SEQ ID NO: 76, or a variant thereof, or as otherwise provide for herein.

35. The polypeptide of any preceding embodiment, wherein the polypeptide comprises the sequence of SEQ ID NO: 77, or a variant thereof, or as otherwise provide for herein. 36. The polypeptide of any preceding embodiment, wherein the polypeptide comprises the sequence of SEQ ID NO: 78, or a variant thereof, or as otherwise provide for herein.

37. The polypeptide of any preceding embodiment, wherein the polypeptide comprises the sequence of SEQ ID NO: 79, or a variant thereof, or as otherwise provide for herein.

38. The polypeptide of any preceding embodiment, wherein the first polypeptide is linked or fused with 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 of embodiment 1, wherein: X ₁ is a IL-21 polypeptide;

X ₂ is a IL-15 polypeptide; X ₃ is a IL-12 p40 polypeptide; X ₄ is a IL-12 p35 polypeptide; X ₅ is a IL-15Rα polypeptide;

1,2 is a furin cleavable linker; and L ₁ , L ₃ , and L ₄ are non-cleavable linkers.

40. The polypeptide embodiment 39, wherein the first polypeptide is linked or fused with 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 for the polypeptide of any one of embodiments 1-40.

42. A vector comprising the nucleic acid of embodiment 41

43. A plasmid comprising the nucleic acid of embodiment 41. 44. A virus, such as a recombinant virus, comprising nucleic acid of embodiment 41.

45. The virus of embodiment 44, wherein the recombinant virus is a adenovirus or lentivirus.

46. The virus of embodiment 44 or 45, where the virus is replication-incompetent adenovirus.

47. A liposome comprising nucleic acid of embodiment 41.

48. The liposome of embodiment 47, wherein the liposome comprises any lipid provided for herein.

49. A pharmaceutical composition comprising nucleic acid of embodiment 41 and a pharmaceutically acceptable carrier.

50. A cell comprising the composition of any one of embodiments 1-40, wherein the cell is an immune cell .

51. A composition (e.g., liposome, virus, encapsulation, and the like) comprising: a first polypeptide comprising the formula of: X1-L ₁ -X ₂ -L ₂ -X ₃ -L ₃ -X ₄ -L ₄ -X ₅ , wherein:

X-. is a interleukin 21 (IL-21) polypeptide, interleukin 15 (IL-15) polypeptide, interleukin 12 p40 subunit (IL-12 p40) polypeptide, interleukin 12 p35 subunit (IL-12 p35) polypeptide, or interleukin 15 receptor (IL-15R&) polypeptide; X ₂ is a IL-21 polypeptide, IL- 15 polypeptide, IL- 12 p40 polypeptide, IL- 12 p35 polypeptide, or IL- 15Rα polypeptide: X ₃ is a IL-21 polypeptide, IL- 15 polypeptide, IL- 12 p40 polypeptide, IL- 12 p35 polypeptide, or IL- 15Rα polypeptide: X ₄ is a IL-21 polypeptide, IL- 15 polypeptide, IL- 12 p40 polypeptide, IL- 12 p35 polypeptide, or IL-15Rα polypeptide: X ₅ is a IL-21 polypeptide, IL- 15 polypeptide, IL- 12 p40 polypeptide, IL- 12 p35 polypeptide, or IL-15Rα polypeptide: L ₁ , L ₂ , L ₃ , and L ₄ are each, independently, a polypeptide linker that comprise the same or different polypeptide sequences, provided each of X ₁ , X ₂ , X ₃ , X ₄ , and X ₅ are different; and/or 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- L ₁ polypeptide, a PD-L ₂ polypeptide, a TIM3 polypeptide, a LAG3 polypeptide, a VISTA polypeptide, a SINGLEC7 polypeptide, a SINGLEC9 polypeptide, a T1GIT polypeptide, a CD96 polypeptide, a BTLA polypeptide, a B7H3 polypeptide, a B7H4 polypeptide, a CD 155 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.

52. The composition of embodiment 51 , wherein X ₁ is a interleukin 21 (IL-21 ) polypeptide.

53. The composition of embodiments 51 or 52, wherein: X ₁ is a IL-21 polypeptide; X ₂ . is a IL- 15 polypeptide; X ₃ is a IL- 12 p40 polypeptide; X ₄ is a IL-12 p35 polypeptide; and X ₅ is a IL-15Rα polypeptide.

54. The composition of embodiments 51 or 52, wherein: X ₁ is a IL-21 polypeptide; X ₂ is a IL- 15 polypeptide; X ₃ is a IL-12 p35 polypeptide; X ₄ is a IL- 12 p40 polypeptide; and X ₅ is a IL-15Rα polypeptide.

55. The composition of embodiments 51 or 52, wherein: X ₁ is a IL-21 polypeptide; X ₂ is a IL- 12 p40 polypeptide; X ₃ is a IL- 15 polypeptide; X ₄ is a IL-12 p35 polypeptide; and X ₅ is a IL-15Rα polypeptide. The composition of embodiments 51 or 52, wherein: X ₁ is a IL-21 polypeptide; X ₂ is a IL- 12 p40 polypeptide; X ₃ is a IL-12 p35 polypeptide;

X?. is a IL- 15 polypeptide; and X ₅ is a IL-15Rα polypeptide. The composition of embodiments 51 or 52, wherein:

X] is a IL-21 polypeptide; X ₂ is a IL- 12 p35 polypeptide; X ₃ is a IL- 12 p40 polypeptide; X ₄ is a IL- 15 polypeptide; and X ₅ is a IL-15Rα polypeptide. The composition of embodiments 51 or 52, wherein: X ₁ is a IL-21 polypeptide; X ₂ . is a IL-12 p35 polypeptide; X ₃ is a IL- 15 polypeptide; X ₄ is a IL-12 p40 polypeptide; and X ₅ is a IL-15Rα polypeptide. The composition of embodiments 51 or 52, wherein: X ₁ is a IL-21 polypeptide; X ₂ is a IL- 12 p40 polypeptide; X ₃ is a IL-12 p35 polypeptide; X ₄ is a IL- 15 polypeptide; and Xs is a IL-15Rα polypeptide.

60. The composition of embodiments 51 or 52, wherein: X ₁ is a IL-21 polypeptide; X ₂ is a IL- 12 p40 polypeptide; X ₃ is a IL- 15 polypeptide; X ₄ is a IL-12 p35 polypeptide; and

Xs is a IL-15Rα polypeptide.

61. The composition of embodiments 51 or 52, wherein: X ₁ is a IL-21 polypeptide;

X ₂ is a IL- 12 p35 polypeptide; X ₃ is a IL- 12 p40 polypeptide; X ₄ is a IL- 15 polypeptide; and X ₅ is a IL- 15Rα polypeptide.

62. The composition of embodiments 51 or 52, wherein:

X] is a IL-21 polypeptide; X ₂ is a IL- 12 p35 polypeptide; X ₃ is a IL- 15 polypeptide; X ₄ is a IL- 12 p40 polypeptide; and X ₅ is a IL-15Rα polypeptide.

63. The composition of any one of embodiments 51-62, 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 for herein.

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

65. The composition of any one of embodiments 51-64, wherein the IL-12 p40 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 for herein.

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

67. The composition of any one of embodiments 51-66, wherein the IL- 12 p35 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 for herein.

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

69. The composition of any one of embodiments 51-68, 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 for herein.

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

71. The composition of any one of embodiments 51-70, 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: 1 1 or 12 or as otherwise provided for herein.

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

73. The composition of any one of embodiments 51-72, wherein one or more of L ₁ , Lz, L ₃ , and L ₄ are each, independently, a cleavable linker. 74. The polypeptide of embodiment 72, wherein L ₁ , Lo, and L ₄ are non-cleavable linkers.

75. The composition of 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).

76. The composition of any one of embodiments 51-75, wherein one of L ₁ , L ₂ , L ₃ , and L ₄ comprise 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), wherein each n is, independently, 1, 2, 3, 4, or 5, or a combination thereof.

77. The composition of embodiment 76, wherein n is 3.

78. The composition of embodiment 76 or 77, wherein one or more of L ₁ , I.,?., I..3, and L ₄ , comprise a sequence 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, RXXR where X is any amino acid, or a combination thereof.

79. The composition of any one of embodiments 76-78, wherein each of L ₁ , L ₂ , L ₃ , and L ₄ , comprises a sequence 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, RXXR where X is any amino acid, or a combination thereof. 80. The composition of any one of embodiments 51-79, wherein the first polypeptide comprises the sequence of SEQ ID NO: 72, or a variant thereof, or as otherwise provide for herein.

81. The composition of any one of embodiments 51-80, wherein the first polypeptide comprises the sequence of SEQ ID NO: 73, or a variant thereof, or as otherwise provide for herein.

82. The composition of any one of embodiments 51-81, wherein the first polypeptide comprises the sequence of SEQ ID NO: 74, or a variant thereof, or as otherwise provide for herein.

83. The composition of any one of embodiments 51-82, wherein the first polypeptide comprises the sequence of SEQ ID NO: 75, or a variant thereof, or as otherwise provide for herein.

84. The composition of any one of embodiments 51-83, wherein the first polypeptide comprises the sequence of SEQ ID NO: 76, or a variant thereof, or as otherwise provide for herein.

85. The composition of any one of embodiments 51 -84, wherein the first polypeptide comprises the sequence of SEQ ID NO: 77, or a variant thereof, or as otherwise provide for herein.

86. The composition of any one of embodiments 51 -85, wherein the first polypeptide comprises the sequence of SEQ ID NO: 78, or a variant thereof, or as otherwise provide for herein.

87. The composition of any one of embodiments 51-86, wherein the first polypeptide comprises the sequence of SEQ ID NO: 79, or a variant thereof, or as otherwise provide for herein. 88. The composition of any one of embodiments 51-87. wherein the first polypeptide is linked or fused with 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.

89. The composition of embodiment 51, wherein: X ₁ is a IL-21 polypeptide; X ₂ is a IL- 15 polypeptide; X ₃ is a IL- 12 p40 polypeptide: X ₄ is a IL-12 p35 polypeptide; X ₅ is a IL-15Rα polypeptide; L ₂ is a furin cleavable linker: and L ₁ , L ₃ , and L ₄ are non-cleavable linkers.

90. The composition of embodiment 89, wherein the first polypeptide is linked or fused with 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.

91. The composition of any preceding embodiment, wherein the second polypeptide comprises, from the N-terminus to the C-terminus, a formula of X _A -L _A -X _B or a formula of 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-L ₁ polypeptide, a PD-L ₂ polypeptide, a T1M3 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. 92. The composition of embodiment 91 , wherein the second polypeptide comprising from the N-terminus to the C-terminus a formula of X _A -L _A -X _B .

93. The composition of embodiment 91, wherein the polypeptide comprising from the N- terminus to the C-terminus a formula of X _B -L _A -X _A .

94. The composition of any one of embodiments 91-93, wherein: X _A is a PD-L ₁ polypeptide; and X _B is a B7.1 polypeptide.

95. The composition of any one of embodiments 91-93, wherein: X _A is a PD-L ₁ polypeptide; and X _B is a B7.2 polypeptide.

96. The composition of any one of embodiments 91-93, wherein: X _A is a PD-L ₂ polypeptide; and X _B is a B7.1 polypeptide.

97. The composition of any one of embodiments 91-93, wherein: X _A is a PD-L ₂ polypeptide; and X _B is a B7.2 polypeptide.

98. The composition of any one of embodiments 91-95, wherein the PD-L ₁ polypeptide comprises a PD-L ₁ extracellular domain.

99. The composition of embodiment 98, wherein the PD-L 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: 102 or comprises the sequence of SEQ ID NO: 102. 100. The composition of any one of embodiments 91-93 and 96-97, wherein the PD-L ₂ polypeptide comprises a PD-L ₂ extracellular domain.

101. The composition of embodiment 100, wherein the PD-L 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: 104 or comprises the sequence of SEQ ID NO: 104.

102. The composition of any one of embodiments 91-94 and 96, wherein the B7.1 polypeptide comprises a B7.1 extracellular domain.

103. The composition of 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.

104. The composition of any one of embodiments 91-93, 95, and 97, wherein the B7.2 polypeptide comprises a B7.2 extracellular domain.

105. The composition of 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.

106. The composition of any one of embodiments 91 -105, wherein the second polypeptide comprises a leader sequence.

107. The composition of embodiment 106, wherein the leader sequence is at the N-terminus of the second polypeptide.

108. The composition of embodiment 106 or 107, wherein the leader sequence is a cell membrane targeting polypeptide.

109. The composition of any one of embodiments 106-108, wherein the leader sequence is a sequence of SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, or combinations thereof.

110. The composition of any one of embodiments 91-109, wherein the second polypeptide further comprises a Fc domain.

111. The composition of embodiment 110, wherein the Fc domain is an Ig Fc domain selected from the group consisting essentially of human IgG, IgGl, IgG2, IgG3, IgG4, IgM, IgA, IgAl, IgA2, IgE, or IgD.

112. The composition of embodiment 110 or 1 1 1, 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 of any one of embodiments 91-112, wherein L _A comprises a sequence of SEQ ID NO: 109, or a combination thereof.

114. The composition of any preceding embodiment, wherein the second polypeptide comprises, from the N-terminus to the C-terminus, a formula of LS-X _A -L _A -X _B -L _B -X _C , LS-X _B -L _A - X _A -L _B -X _C , LS-X _A -L _A -X _C -L _B -X _B , or LS-X _B -L _A -X _C -L _B -X _A wherein:

Ls is a leader sequence; 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-L ₁ polypeptide, a PD-L ₂ polypeptide, a T1M3 polypeptide, a LAG3 polypeptide, a VISTA polypeptide, a SINGLEC7 polypeptide, a SINGLEC9 polypeptide, a TIG1T 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;

Xc is a binder, such as a Fc domain, TCR, cytokine, cytokine trap, receptor ligand, complement protein; L _A is absent or a polypeptide linker; and L _B is absent or a polypeptide linker.

115. The composition of embodiment 114 , wherein the Fc domain is an Ig Fc domain selected from the group consisting essentially of human IgG, IgGl, IgG2, IgG3, IgG4, IgM, IgA, IgAl, Ig A2, IgE, or IgD,

116. The composition of 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.

117. The composition of any one of embodiments 51-116, 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: 1 17.

118. The composition of any one of embodiments 51-117, 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 of any one of embodiments 51 -1 18, wherein the composition further comprises an antigen.

120. The composition of embodiment 119, wherein the antigen is a tumor antigen, a viral antigen, a bacterial antigen, or a microbial antigen.

121. The composition of embodiment 119 or 120, wherein the antigen is ovalbumin (OVA). 122. The composition of any one of claims 51-121, further comprising: a first nucleic acid molecule encoding for the first polypeptide of any one of embodiments 51-90; and/or a second nucleic acid molecule encoding for the second polypeptide of any one of embodiments 51, and 91-121.

123. The composition of embodiment 122, further comprising a third nucleic acid molecule encoding for the antigen of embodiment 119-121.

124. A vector comprising the first nucleic acid molecule and/or the second nucleic acid molecule of embodiment 122.

125. The vector of embodiment 124, further comprising the third nucleic acid molecule of embodiment 123.

126. A composition comprising: a first vector comprising the first nucleic acid molecule of embodiment 122; and a second vector comprising the second nucleic acid molecule of embodiment 122.

127. The composition of embodiment 126, wherein the first vector further comprises the third nucleic acid molecule of embodiment 123.

128. The composition of embodiment 126, wherein the second vector further comprises the third nucleic acid molecule of embodiment 123.

129. The composition of embodiment 126, further comprising a third vector that comprises the third nucleic acid molecule of embodiment 123.

130. A plasmid comprising the first nucleic acid molecule and the second nucleic acid molecule of embodiment 122. 131. The plasmid of embodiment 130, further comprising the third nucleic acid molecule of embodiment 123.

132. A composition comprising: a first plasmid comprising the first nucleic acid molecule of embodiment 122; and a second plasmid comprising the second nucleic acid molecule of embodiment 122.

133. The composition of embodiment 132, wherein the first plasmid further comprises the third nucleic acid molecule of embodiment 123.

134. The composition of embodiment 132, wherein the second plasmid further comprises the third nucleic acid molecule of embodiment 123.

135. The composition of embodiment 132, further comprising a third plasmid that comprises the third nucleic acid molecule of embodiment 123.

136. A recombinant virus comprising the first nucleic acid molecule and the second nucleic acid molecule of embodiment 123.

137. The recombinant virus of embodiment 136, further comprising the third nucleic acid molecule of embodiment 123.

138. The recombinant virus of embodiment 136 or 137, wherein the recombinant virus is a adenovirus or lentivirus.

139. The recombinant virus of embodiment 138, where the recombinant virus is replicationincompetent adenovirus.

140. A liposome comprising the first nucleic acid molecule and the second nucleic acid molecule of embodiment 122. 141. The liposome of embodiment 140, further comprising the third nucleic acid molecule of embodiment 123.

142. The liposome of embodiment 140 or 141, wherein the liposome comprises any lipid provided for herein.

143. A pharmaceutical composition comprising; the first nucleic acid molecule and the second nucleic acid molecule of embodiment 122; and a pharmaceutically acceptable carrier.

144. The pharmaceutical composition of embodiment 143, further comprising the third nucleic acid molecule of embodiment 123.

145. A cell comprising the composition of any one of embodiments 51-123.

146. The cell of embodiment 145, wherein the cell further comprises a chimeric antigen receptor (CAR).

147. The cell of embodiment 145 or 146, wherein the cell is an immune cell.

148. The cell of embodiment 147, wherein the immune cell is a T-cell, a NK cell, or a dendritic cell.

149. A method of producing the cell of any one of embodiments 145-148, the method comprising contacting the cell with: the vector of embodiment 124-125; the composition of any one of embodiments 126-129; the plasmid of embodiment 130-131; the composition of any one of embodiments 132-134; the recombinant virus of any one of embodiments 135-139; the liposome of any one of embodiments 140-142; or pharmaceutical composition of any one of embodiments 143-144.

150. The method of embodiment 149, wherein the contacting comprising transducing or transfecting the cell with the vector, the plasmid, the recombinant virus, the liposome, or one of the compositions.

151. A method of producing the cell of any one of embodiments 145-148 in vivo, the method comprising administering to a subject; the vector of embodiment 124-125; the composition of any one of embodiments 126-129; the plasmid of embodiment 130-131; the composition of any one of embodiments 132-134; the recombinant virus of any one of embodiments 135-139; the liposome of any one of embodiments 140-142; or pharmaceutical composition of any one of embodiments 143-144; wherein the vector, the plasmid, the recombinant virus, the liposome, or the composition transduced or transfects a cell in vivo to produce the cell of any one of embodiments 145-148.

152. A method of modifying an immune response in a subject, the method comprising administering to the subject: the vector of embodiment 124-125; the composition of any one of embodiments 126-129; the plasmid of embodiment 130-131 ; the composition of any one of embodiments 132-134; the recombinant virus of any one of embodiments 135-139; the liposome of any one of embodiments 140-142; or pharmaceutical composition of any one of embodiments 143-144.

153. The method of embodiment 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 of embodiment 124-125: the composition of any one of embodiments 126-129; the plasmid of embodiment 130-131: the composition of any one of embodiments 132-134; the recombinant virus of any one of embodiments 135-139; the liposome of any one of embodiments 140-142: or pharmaceutical composition of any one of embodiments 143-144.

155. The method of embodiment 154, wherein the cancer is a cancer provided for herein.

156. The method of embodiment 154 or 155, wherein the method further comprises the use of at least one additional therapeutic, such as an anti-cancer therapeutic, including but not limited to chemotherapy, radiation therapy, targeted therapy, immunotherapy, stem cell or bone marrow transplant therapy, hormone therapy, photodynamic therapy, or any combination thereof.

157. A method of treating a disease or disorder, such a viral infection, bacterial infection, or fungal infection, such as those provided for herein, in a subject in need thereof, the method comprising administering to the subject: the vector of embodiment 124-125; the composition of any one of embodiments 126-129; the plasmid of embodiment 130-131; the composition of any one of embodiments 132-134; the recombinant virus of any one of embodiments 135-139; the liposome of any one of embodiments 140-142; or pharmaceutical composition of any one of embodiments 143-144.

158. A polypeptide comprising the formula of: X1-L ₁ -X ₂ -L ₂ -X ₃ -L ₃ -X ₄ -L ₄ -X ₅ -L ₅ -X6, wherein: X ₁ is a interleukin 21 (IL-21) polypeptide, interleukin 15 (IL-15) polypeptide, interleukin 12 p40 subunit (IL- 12 p40) polypeptide, interleukin 12 p35 subunit (IL- 12 p35) polypeptide, interleukin 15 receptor (IL-15Rα) polypeptide, a first fragment of IL-15Rα polypeptide, or a second fragment of TL-15R(X polypeptide; X ₂ is a IL-21 polypeptide, IL-15 polypeptide, IL- 12 p40 polypeptide, IL-12 p35 polypeptide, IL-15Rα polypeptide, a first fragment of IL- 15Rα polypeptide, or a second fragment of IL-15Rα polypeptide; X ₃ is a IL-21 polypeptide, IL-15 polypeptide, IL-12 p40 polypeptide, IL-12 p35 polypeptide, IL-15R(X polypeptide, a first fragment of IL-15Rα polypeptide, or a second fragment of IL-15Rα polypeptide; X ₄ is a IL-21 polypeptide, IL-15 polypeptide, IL-12 p40 polypeptide, IL-12 p35 polypeptide, IL-15Rα polypeptide, a first fragment of IL-15Rα polypeptide, or a second fragment of IL-15Rα polypeptide; X ₅ is a IL-21 polypeptide, IL- 15 polypeptide, IL- 12 p40 polypeptide, IL- 12 p35 polypeptide, IL-15Rα polypeptide, a first fragment of IL-15RCX polypeptide, or a second fragment of IL-15Rα polypeptide; X ₆ is a IL-21 polypeptide, IL-15 polypeptide, IL-12 p40 polypeptide, IL-12 p35 polypeptide, IL-15Rα polypeptide, a first fragment of IL-15Rα polypeptide, or a second fragment of IL-15Rα polypeptide; L ₁ , L ₂ , L ₃ , L ₄ , and L ₅ are each, independently, a polypeptide linker that comprise the same or different polypeptide sequences, provided each of X ₁ , X ₂ , X ₃ , X ₄ , X ₅ , and X.6 are different.

159. The polypeptide of embodiment 158, wherein X ₁ is a interleukin 21 (IL-21) polypeptide.

160. The polypeptide of embodiment 158 or 159, wherein: X ₁ is a IL-21 polypeptide; X ₂ . is a IL- 15 polypeptide; X ₃ is a first fragment of IL-15Rα polypeptide; X ₄ is a IL- 12 p40 polypeptide; Xs is a IL-12 p35 polypeptide; and

X ₆ is a second fragment of IL-15Rα polypeptide. The polypeptide of embodiment 158 or 159, wherein: X ₁ is a IL-21 polypeptide; X ₂ is a IL- 15 polypeptide;

Xs is a first fragment of IL-15Rα polypeptide;

X/i is a IL- 12 p35 polypeptide;

Xs is a IL- 12 p40 polypeptide; and Xs is a second fragment of IL-15Rα polypeptide. The polypeptide of embodiment 158 or 159, wherein: X ₁ is a IL-21 polypeptide; X ₂ is a IL- 15 polypeptide; X ₃ is a IL- 12 p40 polypeptide;

X. _: is a first fragment of IL-15Rα polypeptide; X ₅ is a IL-12 p35 polypeptide; and

Xs is a second fragment of IL-15Rα polypeptide. The polypeptide of embodiment 158 or 159, wherein: X ₁ is a IL-21 polypeptide; X ₂ . is a IL- 15 polypeptide; X ₃ is a IL- 12 p40 polypeptide; X ₄ is a IL-12 p35 polypeptide; X ₅ is a first fragment of IL-15Rα polypeptide; and

Xs is a second fragment of IL-15Rα polypeptide. The polypeptide of embodiment 158 or 159, wherein: X ₁ is a IL-21 polypeptide; X ₂ is a IL- 15 polypeptide; X ₃ is a IL- 12 p35 polypeptide; X ₄ is a IL- 12 p40 polypeptide; Xs is a first fragment of lL-15Rα polypeptide; and Xe is a second fragment of 1L-15RCX polypeptide. The polypeptide of embodiment 158 or 159, wherein: X ₁ is a IL-21 polypeptide; X ₂ is a IL- 15 polypeptide; X ₃ is a IL-12 p35 polypeptide; X ₄ is a first fragment of IL-15Rα polypeptide; X ₅ is a IL- 12 p40 polypeptide; and

X ₆ is a second fragment of IL-15Rα polypeptide. The poiypeptide of embodiment 158 or 159, wherein: X ₁ is a IL-21 polypeptide; X ₂ is a first fragment of lL-15Rα polypeptide; X ₃ is a IL- 15 polypeptide; X ₄ is a IL- 12 p40 polypeptide; X ₅ is a IL-12 p35 polypeptide; and X ₆ is a second fragment of IL-15Rα polypeptide. The polypeptide of embodiment 158 or 159, wherein: X ₁ is a IL-21 polypeptide; X ₂ is a first fragment of IL-15Rα polypeptide; X ₃ is a IL- 15 polypeptide; X ₄ is a IL-12 p35 polypeptide; X ₅ is a IL- 12 p40 polypeptide; and X ₆ is a second fragment of IL-15Rα polypeptide. The polypeptide of embodiment 158 or 159, wherein: X ₁ is a IL-21 polypeptide; X ₂ is a first fragment of IL-15Rα polypeptide; X ₃ is a IL- 12 p40 polypeptide; X ₄ is a IL- 15 polypeptide;

Xs is a IL-12 p35 polypeptide; and X ₆ is a second fragment of IL-15Rα polypeptide. The polypeptide of embodiment 158 or 159, wherein: X ₁ is a IL-21 polypeptide; X ₂ . is a first fragment of IL-15Rα polypeptide; X ₃ is a IL- 12 p40 polypeptide; X ₄ is a IL-12 p35 polypeptide; X ₅ is a IL- 15; and

X ₆ is a second fragment of IL-15Rα polypeptide. The polypeptide of embodiment 158 or 159, wherein: X ₁ is a IL-21 polypeptide; X ₂ is a first fragment of IL-15Rα polypeptide; X ₃ is a IL-12 p35 polypeptide; X ₄ is a IL- 12 p40 polypeptide; X ₅ is a IL- 15; and

X ₆ is a second fragment of IL-15Rα polypeptide. The polypeptide of embodiment 158 or 159, wh erein: X ₁ is a IL-21 polypeptide; X ₂ is a first fragment of lL-15Rα polypeptide; X ₃ is a IL-12 p35 polypeptide; X ₄ is a IL- 15; X ₅ is a IL-12 p40 polypeptide; and

X ₆ is a second fragment of IL-15Rα polypeptide. The polypeptide of embodiment 158 or 159, wherein: X ₁ is a IL-21 polypeptide; X ₂ . is a IL- 12 p40 polypeptide; X ₃ is a IL- 15 polypeptide; X ₄ is a first fragment of IL-15Rα polypeptide; X ₅ is a IL- 12 p35 polypeptide; and

X ₆ , is a second fragment of IL-15Rα polypeptide. The polypeptide of embodiment 158 or 159, wherein:

X ₁ is a IL-21 polypeptide; X ₂ is a IL- 12 p40 polypeptide; X ₃ is a IL- 15 polypeptide; X ₄ is a IL- 12 p35 polypeptide; X ₅ is a first fragment of IL-15Rα polypeptide; and

X ₆ is a second fragment of IL-15Rα polypeptide. The polypeptide of embodiment 158 or 159, wherein: X ₁ is a IL-21 polypeptide; X ₂ is a IL- 12 p40 polypeptide; X ₃ is a first fragment of IL-15Rα polypeptide; X ₄ is a IL- 15 polypeptide; X ₅ is a IL-12 p35 polypeptide; and X ₆ is a second fragment of IL-15Rα polypeptide. The polypeptide of embodiment 158 or 159, wherein: X ₁ is a IL-21 polypeptide; X ₂ . is a IL- 12 p40 polypeptide; X ₃ is a first fragment of IL- 15 Rot polypeptide; X ₄ is a IL-12 p35 polypeptide; X ₅ is a IL- 15; and

Xs is a second fragment of IL-15Rα polypeptide. The polypeptide of embodiment 158 or 159, wherein: X ₁ is a IL-21 polypeptide; X ₂ is a IL- 12 p40 polypeptide; X ₃ is a IL-12 p35 polypeptide: X ₄ is a first fragment of lL-15Rα polypeptide; X ₅ is a IL- 15; and X ₆ is a second fragment of IL-15Rα polypeptide. The polypeptide of embodiment 158 or 159, wherein: X ₁ is a IL-21 polypeptide; X ₂ . is a IL- 12 p40 polypeptide; X ₃ is a IL- 12 p35 polypeptide; X ₄ is a IL- 15; X ₅ is a first fragment of IL-15Rα polypeptide; and X ₆ is a second fragment of IL-15Rα polypeptide. The polypeptide of embodiment 158 or 159, wherein: X ₁ is a IL-21 polypeptide; X ₂ is a IL-12 p35 polypeptide: X ₃ is a IL- 15 polypeptide: X ₄ is a first fragment of IL-15Rα polypeptide: X ₅ is a IL- 12 p40 polypeptide; and X ₆ is a second fragment of IL-15Rα polypeptide. The polypeptide of embodiment 158 or 159, wherein:

X] is a IL-21 polypeptide; X ₂ is a IL- 12 p35 polypeptide; X ₃ is a IL- 15 polypeptide;

X ₄ is a IL- 12 p40 polypeptide; X ₅ is a first fragment of IL-15Rα polypeptide; and X ₆ is a second fragment of IL-15Rα polypeptide. The polypeptide of embodiment 158 or 159, wherein: X ₁ is a IL-21 polypeptide; X ₂ is a IL-12 p35 polypeptide; X ₃ is a first fragment of lL-15Rα polypeptide; X ₄ is a IL- 15 polypeptide; X ₅ is a IL- 12 p40 polypeptide; and

X ₆ , is a second fragment of IL-15Rα polypeptide. The polypeptide of embodiment 158 or 159, wherein:

X ₁ is a IL-21 polypeptide; X ₂ is a IL- 12 p35 polypeptide; X ₃ is a first fragment of IL-15Rα polypeptide; X ₄ is a IL- 12 p40 polypeptide; X ₅ is a IL- 15; and

X ₆ is a second fragment of IL-15Rα polypeptide. The polypeptide of embodiment 158 or 159, wherein: X ₁ is a IL-21 polypeptide; X ₂ is a IL-12 p35 polypeptide; X ₃ is a IL- 12 p40 polypeptide; X ₄ is a first fragment of IL-15Rα polypeptide; X ₅ is a IL- 15; and X ₆ is a second fragment of IL-15Rα polypeptide. The polypeptide of embodiment 158 or 159, wherein: X ₁ is a IL-21 polypeptide; X ₂ is a IL-12 p35 polypeptide; X ₃ is a IL- 12 p40 polypeptide;

X ₄ is a IL- 15; X ₅ is a first fragment of IL- 15 Rot polypeptide; and

X ₆ is a second fragment of IL-15Rα polypeptide. 184. The polypeptide of any one of embodiments 158-183, 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 for herein.

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

186. The polypeptide of any one of embodiments 158-185, wherein the IL-12 p40 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 for herein.

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

188. The polypeptide of any one of embodiments 158-187, wherein the IL- 12 p35 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 for herein.

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

190. The polypeptide of any one of embodiments 158-189, 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 for herein.

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

192. The polypeptide of any one of embodiments 158-191, 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 for herein.

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

194. The polypeptide of any one of embodiments 158-193, wherein one or more of L ₁ , L ₂ , L ₃ , L ₄ , and L ₅ are each, independently, a cleavable linker.

195. The polypeptide of embodiment 194, wherein L ₁ , L ₃ , L ₄ , and L ₅ are non-cleavable linkers.

196. The polypeptide of 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).

197. The polypeptide of any one of embodiments 158-196, wherein one of L ₁ , L ₂ , L ₃ , L ₄ , and L ₅ comprise 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), wherein each n is, independently, 1 , 2, 3, 4, or 5, or a combination thereof.

198. The polypeptide of embodiment 197, wherein n is 3.

199. The polypeptide of embodiments 197 or 198, wherein one or more of L-., L ₂ , L ₃ , L ₄ , and L ₅ comprise a sequence 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, RXXR where X is any amino acid, or a combination thereof. 200. The polypeptide of any one of embodiments 197-199, wherein each of L ₁ , L ₂ , L ₃ , L ₄ , and L ₅ comprises a sequence 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, RXXR where X is any amino acid, or a combination thereof.

201. The polypeptide of any one of embodiments 158-200, wherein the first polypeptide comprises the sequence of SEQ ID NO: 72, or a variant thereof, or as otherwise provide for herein.

202. The polypeptide of any one of embodiments 158-201, wherein the first polypeptide comprises the sequence of SEQ ID NO: 73, or a variant thereof, or as otherwise provide for herein.

203. The polypeptide of any one of embodiments 158-202, wherein the first polypeptide comprises the sequence of SEQ ID NO: 74, or a variant thereof, or as otherwise provide for herein.

204. The polypeptide of any one of embodiments 158-203, wherein the first polypeptide comprises the sequence of SEQ ID NO: 75, or a variant thereof, or as otherwise provide for herein.

205. The polypeptide of any one of embodiments 158-204, wherein the first polypeptide comprises the sequence of SEQ ID NO: 76, or a variant thereof, or as otherwise provide for herein.

206. The polypeptide of any one of embodiments 158-205, wherein the first polypeptide comprises the sequence of SEQ ID NO: 77, or a variant thereof, or as otherwise provide for herein.

207. The polypeptide of any one of embodiments 158-206, wherein the first polypeptide comprises the sequence of SEQ ID NO: 78, or a variant thereof, or as otherwise provide for herein.

208. The polypeptide of any one of embodiments 158-207, wherein the first polypeptide comprises the sequence of SEQ ID NO: 79, or a variant thereof, or as otherwise provide for herein.

209. The polypeptide of any one of embodiments 158-208, wherein the first polypeptide is linked or fused with 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. The polypeptide of embodiment 158, wherein: X ₁ is a IL-21 polypeptide; X ₂ . is a IL- 15 polypeptide; X ₃ is a first fragment of IL-15Rα polypeptide; X ₄ is a IL- 12 p40 polypeptide; X ₅ is a IL- 12 p35 polypeptide; and

Xs is a second fragment of IL-15Rα polypeptide; and L ₁ , L ₂ , L ₃ , L ₄ , and L ₅ are non-cleavable linkers.

211. The polypeptide embodiment 210, wherein the first polypeptide is linked or fused with 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 for the polypeptide of any one of embodiments 158-211. 213. A vector comprising the nucleic acid of embodiment 212.

214. A plasmid comprising the nucleic acid of embodiment 212.

215. A recombinant virus comprising nucleic acid of embodiment 212.

216. The recombinant vims of embodiment 215, wherein the recombinant vims is a adenovirus or lentivims.

217. The recombinant virus of embodiment 215 or 216, where the recombinant virus is replication-incompetent ad eno vims .

218. A liposome comprising nucleic acid of embodiment 212.

219. The liposome of embodiment 218, wherein the liposome comprises any lipid provided for herein.

220. A pharmaceutical composition comprising nucleic acid of embodiment 212 and a pharmaceutically acceptable carrier.

221. A cell comprising the composition of any one of embodiments 158-21 1.

222. A compositing comprising: a first polypeptide comprising the formula of: X1-L ₁ -X ₂ -L ₂ -X ₃ -L ₃ -X ₄ -L ₄ -X ₅ are provided, wherein: X ₁ is a interleukin 21 (IL-21) polypeptide, interleukin 15 (IL- 15) polypeptide, interleukin 12 p40 subunit (IL-12 p40) polypeptide, interleukin 12 p35 subunit (IL-12 p35) polypeptide, interleukin 15 receptor (IL-15Rα) polypeptide, a first fragment of IL- 15Rα polypeptide, or a second fragment of IL-15RαC polypeptide: X ₂ is a IL-21 polypeptide, IL- 15 polypeptide, IL- 12 p40 polypeptide, IL- 12 p35 polypeptide, IL-15Rα polypeptide, a first fragment of IL-15Rα polypeptide, or a second fragment of IL-15Rα polypeptide: X ₃ is a IL-21 polypeptide, IL-15 polypeptide, IL-12 p40 polypeptide, IL-12 p35 polypeptide, IL-15Rα polypeptide, a first fragment of IL-15Rα polypeptide, or a second fragment of IL-15Rα polypeptide: X ₄ is a IL-21 polypeptide, IL-15 polypeptide, IL-12 p40 polypeptide, IL-12 p35 polypeptide, IL-15Rα polypeptide, a first fragment of IL-15Rα polypeptide, or a second fragment of IL-15Rα polypeptide; X ₅ is a IL-21 polypeptide, IL-15 polypeptide, IL-12 p40 polypeptide, IL-12 p35 polypeptide, IL-15Rα polypeptide, a first fragment of IL-15Rα polypeptide, or a second fragment of IL-15R(X polypeptide;

X ₆ is a IL-21 polypeptide, IL- 15 polypeptide, IL- 12 p40 polypeptide, IL- 12 p35 polypeptide, IL-15Rα polypeptide, a first fragment of IL-15Rα polypeptide, or a second fragment of IL-15Rα polypeptide; L ₁ , L ₂ , L ₃ , L ₄ , and L ₅ are each, independently, a polypeptide linker that comprise the same or different polypeptide sequences, provided each of X ₁ , X ₂ , X ₃ , X ₄ , X ₅ , and Xr-, are different: and/or 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- L ₁ polypeptide, a PD-L 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 CD 155 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 of embodiment 222, wherein X ₁ is a interleukin 21 (IL-21) polypeptide. The composition of embodiment 222 or 223, wherein: X ₁ is a IL-21 polypeptide; X ₂ is a IL- 15 polypeptide; X ₃ is a first fragment of IL-15Rα polypeptide;

X ₄ is a IL- 12 p40 polypeptide; X ₅ is a IL-12 p35 polypeptide; and X ₆ is a second fragment of IL-15Rα polypeptide. The polypeptide of embodiment 222 or 223, wherein: X ₁ is a IL-21 polypeptide; X ₂ . is a IL- 15 polypeptide; X ₃ is a first fragment of IL-15Rα polypeptide; X ₄ is a IL-12 p35 polypeptide; X ₅ is a IL-12 p40 polypeptide; and X ₆ is a second fragment of IL-15Rα polypeptide. The polypeptide of embodiment 222 or 223, wherein: X ₁ is a IL-21 polypeptide; X ₂ is a IL- 15 polypeptide; X ₃ is a IL- 12 p40 polypeptide:

X ₄ is a first fragment of IL-15Rα polypeptide; X ₅ is a IL-12 p35 polypeptide; and

X ₆ is a second fragment of IL-15Rα polypeptide. The polypeptide of embodiment 222 or 223, wherein: X ₁ is a IL-21 polypeptide; X ₂ . is a IL- 15 polypeptide; X ₃ is a IL- 12 p40 polypeptide; X ₄ is a IL-12 p35 polypeptide; X ₅ is a first fragment of IL- 15 Rot polypeptide; and

X ₆ is a second fragment of IL-15Rot polypeptide. The polypeptide of embodiment 222 or 223, wherein: X ₁ is a IL-21 polypeptide; X ₂ is a IL- 15 polypeptide; X ₃ is a IL- 12 p35 polypeptide; X ₄ is a IL- 12 p40 polypeptide; X ₅ is a first fragment of IL-15Rα polypeptide; and X ₆ is a second fragment of IL-15Rα polypeptide. The polypeptide of embodiment 222 or 223, wherein: X ₁ is a IL-21 polypeptide; X ₂ . is a IL- 15 polypeptide; X ₃ is a IL- 12 p35 polypeptide; X ₄ is a first fragment of IL-15R(X polypeptide; X ₅ is a IL- 12 p40 polypeptide; and X ₆ is a second fragment of IL-15Rα polypeptide. The polypeptide of embodiment 222 or 223, wherein: X ₁ is a IL-21 polypeptide; X ₂ is a first fragment of IL-15Rα polypeptide; X ₃ is a IL- 15 polypeptide; X ₄ is a IL-12 p40 polypeptide; X ₅ is a IL- 12 p35 polypeptide; and X ₆ is a second fragment of IL-15Rα polypeptide. The polypeptide of embodiment 222 or 223, wherein: X ₁ is a IL-21 polypeptide; X ₂ . is a first fragment of IL- 15 Rot polypeptide; X ₃ is a IL- 15 polypeptide;

Xi is a IL- 12 p35 polypeptide; X ₅ is a IL- 12 p40 polypeptide; and X ₆ is a second fragment of IL-15Rα polypeptide. The polypeptide of embodiment 222 or 223, wherein: X ₁ is a IL-21 polypeptide; X ₂ is a first fragment of IL-15Rα polypeptide; X ₃ is a IL- 12 p40 polypeptide; X ₄ is a IL- 15 polypeptide; X ₅ is a IL- 12 p35 polypeptide; and X ₆ is a second fragment of IL-15Rα polypeptide. The polypeptide of embodiment 222 or 223, wherein:

X] is a IL-21 polypeptide; X ₂ . is a first fragment of IL-15Rα polypeptide; X ₃ is a IL- 12 p40 polypeptide; X ₄ is a IL- 12 p35 polypeptide; X ₅ is a IL- 15; and

X ₆ is a second fragment of IL-15Rα polypeptide. The polypeptide of embodiment 222 or 223, wherein: X ₁ is a IL-21 polypeptide; X ₂ is a first fragment of IL-15Rα polypeptide; X ₃ is a IL-12 p35 polypeptide;

X ₄ is a IL- 12 p40 polypeptide; X ₅ is a IL- 15; and

Xs is a second fragment of IL-15Rα polypeptide. The polypeptide of embodiment 222 or 223, wherein: X ₁ is a IL-21 polypeptide; X ₂ is a first fragment of IL-15Rα polypeptide; X ₃ is a IL- 12 p35 polypeptide; X ₄ is a IL- 15; X ₅ is a IL- 12 p40 polypeptide; and X ₆ is a second fragment of IL-15Rα polypeptide. The polypeptide of embodiment 222 or 223, wherein: X ₁ is a IL-21 polypeptide; X ₂ is a IL- 12 p40 polypeptide: X ₃ is a IL- 15 polypeptide; X ₄ is a first fragment of IL-15Rα polypeptide; X ₅ is a IL- 12 p35 polypeptide; and

X ₆ is a second fragment of 1L-15RCX polypeptide. The polypeptide of embodiment 222 or 223, wherein: X ₁ is a IL-21 polypeptide; X ₂ is a IL- 12 p40 polypeptide; X ₃ is a IL- 15 polypeptide; X ₄ is a IL-12 p35 polypeptide; X ₅ is a first fragment of IL-15Rα polypeptide; and

Xs is a second fragment of IL-15Rα polypeptide. The polypeptide of embodiment 222 or 223, wherein: X ₁ is a IL-21 polypeptide; X ₂ is a IL-12 p40 polypeptide; X ₃ is a first fragment of IL-15Rα polypeptide; X ₄ is a IL- 15 polypeptide; X ₅ is a IL-12 p35 polypeptide; and

Xs is a second fragment of IL-15Rα polypeptide. The polypeptide of embodiment 222 or 223, wherein: X ₁ is a IL-21 polypeptide; X ₂ is a IL- 12 p40 polypeptide; X ₃ is a first fragment of IL-15Rα polypeptide; X ₄ is a IL-12 p35 polypeptide; Xs is a IL- 15; and

X ₆ is a second fragment of IL-15Rα polypeptide. The polypeptide of embodiment 222 or 223, wherein: X ₁ is a IL-21 polypeptide; X ₂ is a IL-12 p40 polypeptide; X ₃ is a IL- 12 p35 polypeptide: X ₄ is a first fragment of IL-15Rα polypeptide; X ₅ is a IL- 15; and

Xs is a second fragment of IL-15Rα polypeptide. The polypeptide of embodiment 222 or 223, wherein: X ₁ is a IL-21 polypeptide; X ₂ is a IL- 12 p40 polypeptide: X ₃ is a IL-12 p35 polypeptide; X ₄ is a IL- 15; X ₅ is a first fragment of IL-15Rα polypeptide; and

Xs is a second fragment of IL-15Rα polypeptide. The polypeptide of embodiment 222 or 223, wherein: X ₁ is a IL-21 polypeptide; X ₂ is a IL- 12 p35 polypeptide; X ₃ is a IL- 15 polypeptide; X ₄ is a first fragment of IL-15Rα polypeptide; X ₅ is a IL- 12 p40 polypeptide; and

Xs is a second fragment of IL-15Rα polypeptide. The polypeptide of embodiment 222 or 223, wherein: X ₁ is a IL-21 polypeptide; X ₂ is a IL- 12 p35 polypeptide; X ₃ is a IL- 15 polypeptide; X ₄ is a IL- 12 p40 polypeptide; X ₅ is a first fragment of IL-15Rα polypeptide: and X ₆ is a second fragment of IL-15Rα polypeptide. The polypeptide of embodiment 222 or 223, wherein: X ₁ is a IL-21 polypeptide; X ₂ . is a IL-12 p35 polypeptide; X ₃ is a first fragment of IL-15Rα polypeptide; X ₄ is a IL- 15 polypeptide: X ₅ is a IL- 12 p40 polypeptide; and

X.6 is a second fragment of IL-15Rα polypeptide. The polypeptide of embodiment 222 or 223, wherein: X ₁ is a IL-21 polypeptide: X ₂ is a IL-12 p35 polypeptide: X ₃ is a first fragment of lL-15Rα polypeptide; X ₄ is a IL-12 p40 polypeptide; X ₅ is a IL- 15; and X ₆ is a second fragment of IL-15Rα polypeptide. The polypeptide of embodiment 222 or 223, wherein:

X ₁ is a IL-21 polypeptide; X ₂ is a IL- 12 p35 polypeptide; X ₃ is a IL- 12 p40 polypeptide; X ₄ is a first fragment of IL- 15 Ra polypeptide; X ₅ is a IL- 15; and

X ₆ is a second fragment of IL-15Rα polypeptide. The polypeptide of embodiment 222 or 223, wherein: X ₁ is a IL-21 polypeptide; X ₂ is a IL-12 p35 polypeptide; Xs is a IL- 12 p40 polypeptide;

X ₄ is a IL-15;

X ₅ is a first fragment of lL-15Rα polypeptide; and X ₆ , is a second fragment of IL-15Rα polypeptide.

247. The composition of any one of embodiments 222-246, 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 to SEQ ID NO: 71, or as otherwise provided for herein.

248. The composition of any one of embodiments 222-247, wherein the IL-21 polypeptide comprises an amino acid sequence of SEQ ID NO: 71, or as otherwise provided for herein.

249. The composition of any one of embodiments 222-248, wherein the IL- 12 p40 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 for herein.

250. The composition of any one of embodiments 222-249, wherein the IL- 12 p40 polypeptide comprises an amino acid sequence of SEQ ID NO: 1, 2, 3, or 4, or as otherwise provided for herein.

251. The composition of any one of embodiments 222-250, wherein the IL- 12 p35 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 for herein.

252. The composition of any one of embodiments 222-251, wherein the IL- 12 p35 polypeptide comprises an amino acid sequence of SEQ ID NO: 5, 6, 7, or 8, or as otherwise provided for herein.

253. The composition of any one of embodiments 222-252, 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 for herein. 254. The composition of any one of embodiments 222-253, wherein the IL-15 polypeptide comprises an amino acid sequence of SEQ ID NO: 9 or 10 or as otherwise provided for herein.

255. The composition of any one of embodiments 222-254, 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 for herein.

256. The composition of any one of embodiments 222-255, wherein the IL-15Rα polypeptide comprises an amino acid sequence of SEQ ID NO: 11 or 12 or as otherwise provided for herein.

257. The composition of any one of embodiments 222-256, wherein one or more of L ₁ , La, L ₃ , L ₄ , and L ₅ are each, independently, a cleavable linker.

258. The polypeptide of embodiment 257, wherein L ₁ , La, L ₃ , L ₄ , and L ₅ are non-cleavable linkers.

259. The composition of 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).

260. The composition of any one of embodiments 222-259, wherein one of Lj , L ₄ , L ₃ , L ₄ , and L ₅ comprise 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), wherein each n is, independently, 1, 2, 3, 4, or 5, or a combination thereof.

261. The composition of embodiment 260, wherein n is 3.

262. The composition of embodiment 260 or 261, wherein one or more of L ₁ , L ₂ , L ₃ , L ₄ , and

L ₅ comprise a sequence 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, RXXR where X is any amino acid, or a combination thereof.

263. The composition of any one of embodiments 260-262, wherein each of L ₁ , Li, L ₃ , L ₄ , and L ₅ comprises a sequence 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, RXXR where X is any amino acid, or a combination thereof.

264. The composition of any one of embodiments 222-263, wherein the first polypeptide comprises the sequence of SEQ ID NO: 72, or a variant thereof, or as otherwise provide for herein.

265. The composition of any one of embodiments 222-263, wherein the first polypeptide comprises the sequence of SEQ ID NO: 73, or a variant thereof, or as otherwise provide for herein.

266. The composition of any one of embodiments 222-263, wherein the first polypeptide comprises the sequence of SEQ ID NO: 74, or a variant thereof, or as otherwise provide for herein.

267. The composition of any one of embodiments 222-263, wherein the first polypeptide comprises the sequence of SEQ ID NO: 75, or a variant thereof, or as otherwise provide for herein.

268. The composition of any one of embodiments 222-263, wherein the first polypeptide comprises the sequence of SEQ ID NO: 76, or a variant thereof, or as otherwise provide for herein.

269. The composition of any one of embodiments 222-263, wherein the first polypeptide comprises the sequence of SEQ ID NO: 77, or a variant thereof, or as otherwise provide for herein.

270. The composition of any one of embodiments 222-263, wherein the first polypeptide comprises the sequence of SEQ ID NO: 78, or a variant thereof, or as otherwise provide for herein.

271. The composition of any one of embodiments 222-263, wherein the first polypeptide comprises the sequence of SEQ ID NO: 79, or a variant thereof, or as otherwise provide for herein.

272. The composition of any one of embodiments 222-271, wherein the first polypeptide is linked or fused with 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.

273. The composition of embodiment 222, wherein: X ₁ is a IL-21 polypeptide; X ₂ _ is a IL- 15 polypeptide;

X ₃ is a IL-12 p40 polypeptide; X ₄ is a IL- 12 p35 polypeptide; X ₅ is a IL-15Rα polypeptide; and L ₁ , L ₂ , L ₃ , L ₄ , and L ₅ are non-cleavable linkers.

274. The composition of embodiment 273, wherein the first polypeptide is linked or fused with 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.

275. The composition of any preceding embodiment, wherein the second polypeptide comprises, from the N-terminus to the C-terminus, a formula of X _A -L _A -X _B or a formula of 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-L ₁ polypeptide, a PD-L 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; X _B is a B7.1 polypeptide, or a B7.2 polypeptide; and L _A is a polypeptide linker.

276. The composition of embodiment 275, wherein the second polypeptide comprising from the N-terminus to the C-terminus a formula of X _A -L _A -X _B .

277. The composition of embodiment 275, wherein the polypeptide comprising from the N- terminus to the C-terminus a formula of X _B -L _A -X _A .

278. The composition of any one of embodiments 275-277, wherein: X _A is a PD-L ₁ polypeptide; and X _B is a B7.1 polypeptide.

279. The composition of any one of embodiments 275-277, wherein: X _A is a PD-L ₁ polypeptide; and X _B is a B7.2 polypeptide.

280. The composition of any one of embodiments 275-277, wherein: X _A is a PD-L ₂ polypeptide; and X _B is a B7.1 polypeptide. 281. The composition of any one of embodiments 275-277, wherein: X _A is a PD-L ₂ polypeptide; and X _B is a B7.2 polypeptide.

282. The composition of any one of embodiments 275-279, wherein the PD-L ₁ polypeptide comprises a PD-L ₁ extracellular- domain.

283. The composition of embodiment 282, wherein the PD-L 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: 102 or comprises the sequence of SEQ ID NO: 102.

284. The composition of any one of embodiments 275-277 and 280-281, wherein the PD-L ₂ polypeptide comprises a PD-L.2 extracellular domain.

285. The composition of embodiment 284, wherein the PD-L 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: 104 or comprises the sequence of SEQ ID NO: 104.

286. The composition of any one of embodiments 275-278 and 280, wherein the B7.1 polypeptide comprises a B7.1 extracellular domain.

287. The composition of 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.

288. The composition of any one of embodiments 275-277, 279, and 281, wherein the B7.2 polypeptide comprises a B7.2 extracellular domain. 289. The composition of embodiment 288, wherein the B7.2 polypeptide comprises an amino acid sequence having at ieast 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.

290. The composition of any one of embodiments 275-289, wherein the second polypeptide comprises a leader sequence.

291. The composition of embodiment 290, wherein the leader sequence is at the N-terminus of the second polypeptide.

292. The composition of embodiment 290 or 291, wherein the leader sequence is a cell membrane targeting polypeptide.

293. The composition of any one of embodiments 290-292, wherein the leader sequence is a sequence of SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, or combinations thereof.

294. The composition of any one of embodiments 275-293, wherein the second polypeptide further comprises a Fc domain.

295. The composition of embodiment 294, wherein the Fc domain is an Ig Fc domain selected from the group consisting essentially of human IgG, IgGl, IgG2, IgG3, IgG4, IgM, IgA, IgAl, IgA2, IgE, or IgD.

294. The composition of embodiment 294-295, wherein the Fc domain comprises an amino acid sequence having at ieast 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. 295. The composition of any one of embodiments 275-294, wherein L _A comprises a sequence of SEQ ID NO: 109, or a combination thereof.

296. The composition of any preceding embodiment, wherein the second polypeptide comprises, from the N-terminus to the C-terminus, a formula of LS-X _A -L _A -X _B -L _B -X _C , LS-X _B -L _A - X _A -L _B -X _C , LS-X _A -L _A -X _C -L _B -X _B , or LS-X _B -L _A -X _C -L _B ~X _A wherein:

Ls is a leader sequence; 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-L ₁ polypeptide, a PD-L 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; X _B is a B7.1 polypeptide or a B7.2 polypeptide;

Xc is a binder, such as a Fc domain, TCR, cytokine, cytokine trap, receptor ligand, complement protein; L _A is absent or a polypeptide linker; and L _B is absent or a polypeptide linker.

297. The composition of embodiment 296, wherein the Fc domain is an Ig Fc domain selected from the group consisting essentially of human IgG, IgGl , IgG2, IgG3, IgG4, IgM, IgA, IgAl, IgA2, IgE, or IgD.

298. The composition of 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.

299. The composition of any one of embodiments 222-298, 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. 300. The composition of any one of embodiments 222-299, 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.

301. The composition of any one of embodiments 222-300, wherein the composition further comprises an antigen.

302. The composition of embodiment 301, wherein the antigen is a tumor antigen, a viral antigen, a bacterial antigen, or a microbial antigen.

303. The composition of embodiment 301-302, wherein the antigen is ovalbumin (OVA).

304. A composition of any one of claims 222-303 further comprising: a first nucleic add molecule encoding for the first polypeptide of any one of embodiments 275-274; and/or a second nucleic acid molecule encoding for the second polypeptide of any one of embodiments 275, and 274-300.

305. The composition of embodiment 304, further comprising a third nucleic acid molecule encoding for the antigen of embodiment 301 -303.

306. A vector comprising the first nucleic acid molecule and the second nucleic acid molecule of embodiment 304.

307. The vector of embodiment 306, further comprising the third nucleic acid molecule of embodiment 305.

308. A composition comprising: a first vector comprising the first nucleic acid molecule of embodiment 304; and a second vector comprising the second nucleic acid molecule of embodiment 304.

309. The composition of embodiment 308, wherein the first vector further comprises the third nucleic acid molecule of embodiment 305.

310. The composition of embodiment 308, wherein the second vector further comprises the third nucleic acid molecule of embodiment 305.

311. The composition of embodiment 308, further comprising a third vector that comprises the third nucleic acid molecule of embodiment 305.

312. A plasmid comprising the first nucleic acid molecule and the second nucleic acid molecule of embodiment 304.

313. The plasmid of embodiment 312, further comprising the third nucleic acid molecule of embodiment 305.

314. A composition comprising: a first plasmid comprising the first nucleic acid molecule of embodiment 304; and a second plasmid comprising the second nucleic acid molecule of embodiment 304.

315. The composition of embodiment 314, wherein the first plasmid further comprises the third nucleic acid molecule of embodiment 305.

316. The composition of embodiment 314, wherein the second plasmid further comprises the third nucleic acid molecule of embodiment 305.

317. The composition of embodiment 314, further comprising a third plasmid that comprises the third nucleic acid molecule of embodiment 305.

318. A recombinant virus comprising the first nucleic acid molecule and the second nucleic acid molecule of embodiment 304.

319. The recombinant vims of embodiment 318, further comprising the third nucleic acid molecule of embodiment 305.

320. The recombinant virus of embodiment 318 or 319, wherein the recombinant virus is a adenovirus or lentivirus.

321. The recombinant virus of embodiment 320, where the recombinant virus is replication- incompetent adenovirus.

322. A liposome comprising the first nucleic acid molecule and the second nucleic acid molecule of embodiment 304.

323. The liposome of embodiment 322, further comprising the third nucleic acid molecule of embodiment 305.

324. The liposome of embodiment 322 or 323, wherein the liposome comprises any lipid provided for herein.

325. A pharmaceutical composition comprising: the first nucleic acid molecule and the second nucleic acid molecule of embodiment 304; and a pharmaceutically acceptable carrier.

326. The pharmaceutical composition of embodiment 325, further comprising the third nucleic acid molecule of embodiment 305.

327. A cell comprising the composition of any one of embodiments 275-303.

328. The cell of embodiment 327, wherein the cell further comprises a chimeric antigen receptor (CAR).

329. The cell of embodiment 327 or 328, wherein the cell is an immune cell.

330. The cell of embodiment 329, wherein the immune cell is a T-cell, a NK cell, or a dendritic cell.

331. A method of producing the cell of any one of embodiments 327-330, the method comprising contacting the cell with: the vector of embodiment 306-307 ; the composition of any one of embodiments 308-311; the plasmid of embodiment 312-313; the composition of any one of embodiments 314-317; the recombinant virus of any one of embodiments 318-321; the liposome of any one of embodiments 322-324; or pharmaceutical composition of any one of embodiments 325-326.

332. The method of embodiment 331, wherein the contacting comprising transducing or transfecting the cell with the vector, the plasmid, the recombinant virus, the liposome, or one of the compositions.

333. A method of producing the cell of any one of embodiments 327-330 in vivo, the method comprising administering to a subject: the vector of embodiment 306-307; the composition of any one of embodiments 308-311; the pl asmid of embodiment 312-313; the composition of any one of embodiments 314-317; the recombinant virus of any one of embodiments 318-321; the liposome of any one of embodiments 322-324; or pharmaceutical composition of any one of embodiments 325-326; wherein the vector, the plasmid, the recombinant virus, the liposome, or the composition transduced or transfects a cell in vivo to produce the cell of any one of embodiments 327-330.

334. A method of modifying an immune response in a subject, the method comprising administering to the subject: the vector of embodiment 306-307 ; the composition of any one of embodiments 308-311; the plasmid of embodiment 312-313; the composition of any one of embodiments 314-317; the recombinant virus of any one of embodiments 318-321; the liposome of any one of embodiments 322-324; or pharmaceutical composition of any one of embodiments 325-326.

335. The method of embodiment 334, wherein the immune response is activated.

336. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject: the vector of embodiment 306-307; the composition of any one of embodiments 308-311; the plasmid of embodiment 312-313; the composition of any one of embodiments 314-317; the recombinant virus of any one of embodiments 318-321; the liposome of any one of embodiments 322-324; or pharmaceutical composition of any one of embodiments 325-326.

337. The method of embodiment 336, wherein the cancer is a cancer provided for herein.

338. The method of embodiment 336 or 337, wherein the method further comprises the use of at least one additional therapeutic, such as an anti-cancer therapeutic, including but not limited to chemotherapy, radiation therapy, targeted therapy, immunotherapy, stem cell or bone marrow transplant therapy, hormone therapy, photodynamic therapy, or any combination thereof. 339. A method of treating a disease or disorder, such a viral infection, bacterial infection, or fungal infection, such as those provided for herein, in a subject in need thereof, the method comprising administering to the subject: the vector of embodiment 306-307 ; the composition of any one of embodiments 308-311; the plasmid of embodiment 312-313; the composition of any one of embodiments 314-317; the recombinant virus of any one of embodiments 318-321; the liposome of any one of embodiments 322-324; or pharmaceutical composition of any one of embodiments 325-326.

EXAMPLES

The following examples are illustrative, but not limiting, of the compounds, compositions and methods described herein. Other suitable modifications and adaptations known to those skilled in the art are within the scope of the following embodiments.

Example I: measurement of active IL-21, IL-12, and IL-15 using polypeptide constructs

The following polypeptide constructs listed in Table 1 were expressed in HEK cells to determine the relative activity of IL-21, IL-12 and IL-15. The effect of adding a dual checkpoint inhibitor with the IL-12/IL-15/IL-21 polypeptides was also measured. Unless otherwise noted, any L listed in the sequence map in Table 1 can be any linker described herein.

Table 1: IL-12/IL-15/IL-21 and dual checkpoint expression constructs

The activity of IL-21, IL- 15, and IL- 12 was determined by cytokine-responsive reporter cellular assays designed specifically to demonstrate IL-21, IL-15, and IL-12 activities. FIG 1 depicts IL-21 signals of the m21-01 and m21-03 polypeptides with and without the SynTauR dual checkpoint polypeptide. FIG. 2 depicts IL-15, IL-12, and CTLA-4 signals of the m21-01 and m21-03 polypeptides with and without the SynTauR dual checkpoint polypeptide. In FIG. 2, 01 and 03 controls are identical to the m21-01 and m21-03 polypeptides, but with the IL-21 sequence and first linker removed. The results show that both the m21-01 and m21-02 polypeptides are capable of activating the IL-21, IL- 15, and IL- 12 pathways.

Example 2: IL-12/IL-157IL-21 and dual checkpoint polypeptides show synergy in vivo

To determine T-cell responses in vivo, C57BL/6 mice were administered compositions comprising nucleic acids that encoded for m21-Ol, m21-O3, and/or SynTauR dual checkpoint polypeptides described above. The engineered genes encoding fusion proteins were subcloned into a vector bearing CMV promoter/enhancer elements and poly A termination signals along with a cassette for expressing the model antigen ovalbumin (OVA) where noted. The animals were acclimated a minimum of 3 days prior to the start of the study, and were housed in microisolators in a 12: 12 light/dark cycle. Animals were maintained with a standard rodent chow diet with water and food provided ad libitum.

Results shows that the various immunostimulant elements synergize to maximize splenic antigen reactive T cell responses. In FIG. 3, animals were given intermuscular injections of vehicle control, antigen (OVA) only control, 01 (IL-15 and IL-12 only) + antigen, SynTaur dual checkpoint inhibitor + antigen. 01 + SynTaur + antigen, and finally m21-01 + SynTaur + antigen. Initial injections occurred on day 0, with a intermuscular injection boost on day 21, before spleens were harvested on day 28. Injections were 50 uL per mouse, with n ~ 2. In FIG. 4, animals were given intravenous injections of vehicle control, antigen (OVA) only control, 01 + antigen, SynTaur dual checkpoint inhibitor + antigen, 01 + SynTaur + antigen, and finally m21- 01 + SynTaur + antigen, along with a Ad5 viral control for comparison. Initial injections again occurred on day 0, with an intravenous injection boost on day 21 and spleen harvesting on day 28. The increasing levels of antigen reactive T cells (FIG. 3) and CD8+ % (FIG. 4) indicate that the presence of IL-21, IL-15, IL-12, OVA, and the immune checkpoint inhibitor surprisingly work much stronger all together than their individual effects would suggest.

Immunology Assay methods

Isolation of single cell suspensions: Organs of interest were harvest from immunized mice on Day 29. Single cell suspensions were isolated from spleen and liver by mechanical disruption and differential centrifugation.

Flow cytometry assays: Immune cell populations were identified using flow' cytometry and antibodies directed against CD3, CD4, CD8, and NK1.1. Memory and effector T cell populations were identified with antibodies directed against CD44 and CD62L.

ELISpot: Functional responses from antigen-specific T cells were evaluated by ELISpot. In short, single cell suspensions were co-cultured with ovalbumin peptide overnight. Cytokine (IFN-y and IL-4) secreting cells were enumerated using cytokine-specific antibodies.

Example 3: Treatment of cancer using vectors encoding polypeptides.

A therapeutic composition comprising a vector comprising a nucleic acid molecule encoding a polypeptide as provided for herein is administered to a patient with cancer. The subject’s immune system is activated and the cancer is treated. The vector can also comprise a nucleic acid molecule encoding for a tumor antigen. The vector can also comprise a nucleic acid molecule encoding for a CAR.

Example 4: Treatment of cancer using vectors encoding polypeptides and a liposome A therapeutic composition comprising a vector comprising a nucleic acid molecule encoding a polypeptide as provided for herein is administered as a formulation with a DOTAP/Cholesterol admixture to a patient with cancer. Idle subject’s immune system is activated and the cancer is treated. The vector can also comprise a nucleic acid molecule encoding for a tumor antigen. The admixture can also comprise the pharmaceutical formulations provided for herein.

Example 5: Treatment of cancer using adoptive cell therapy such as modified leukocytes,

A T-cell comprising a polypeptide as provided for herein and a chimeric antigen receptor is administered to a subject with a cancer, such as leukemia, and the cancer is treated.

Example 6: Treatment of cancer using biologic fusions.

A therapeutic composition comprising a polypeptide as provided for herein administered to a patient with cancer. In some embodiments, the polypeptide is linked or fused to a biologically active partner, such as a protein, polypeptide, nucleic acid, lipid, carbohydrate, or any combination thereof. In some embodiments, a polypeptide is linked or fused to a targeting partner such as an antibody, an antigen, a ligand, or a ligand trap, such as a receptor domain. In some embodiments, the poly is linked or fused to an immunotherapy such as a checkpoint inhibitor. The subject’s immune system is activated and the cancer is treated.

In summary, the embodiments and examples provided herein demonstrate that the polypeptides provided for herein can be used to enhance an immune response to treat a tumor or infection as provided for herein.

This specification contains numerous citations to patents, patent applications, accession numbers, and/or publications. Each is hereby incorporated by reference for all purposes.