WO2022094482A1 - Two-domain multimeric fusion proteins - Google Patents

Abstract

Provided are conjugates of TRAIL and another Tumor Necrosis Factor (TNF) superfamily ligand, and related compositions and methods of use thereof. Also provided are conjugates of two trimeric polypeptides linked through a genetically encoded linker or chemical linkers and related compositions and methods of use thereof.

Description

TWO-DOMAIN MULTIMERIC FUSION PROTEINS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This Application claims priority to, and the benefit of, U.S. Provisional Patent Application Serial No. 63/108,789 filed November 2, 2020, entitled “TWO AND THREE DOMAIN FUSION PROTEINS”, and claims priority to, and the benefit of, U.S. Provisional Patent Application Serial No. 63/246,247 filed September 20, 2021 entitled “TWO-DOMAIN MULTIMERIC FUSION PROTEINS”, and claims priority to, and the benefit of, U.S. Provisional Patent Application Serial No. 63/253,501 filed October 7, 2021, entitled “TWO- DOMAIN MULTIMERIC FUSION PROTEINS”, the entire contents of each application are incorporated herein by reference in their entirety.

FIELD

[0002] The present disclosure relates to conjugates of a Tumor Necrosis Factor TNF) related apoptosis-inducing ligand (TRAIL) and CD137L or OX40L, where the fusion protein self- assembles into trimeric proteins, or its oligomer (e.g., hexamer) and related compositions and methods of use thereof. The present disclosure also relates to conjugates of two trimeric polypeptides, or nucleic acids encoding these fusion polypeptides and related compositions and methods of use thereof.

BACKGROUND

[0003] Activation of cell surface death receptors of the tumor necrosis factor (TNF) receptor superfamily by the appropriate ligands represents an attractive therapeutic strategy to induce cell death by apoptosis in cancer cells (see, for e.g.: Palacios etal., Curr Pharm Des. 2014;20(17):2819- 33). As an example, TRAIL possesses the ability to induce apoptosis selectively in cancer cells and has demonstrated robust anticancer activity in a number of preclinical studies. TRAIL, is a trimeric protein, expressed as a type-II transmembrane protein and plays a physiological role in anti-tumor immune surveillance (see, for e.g:. LeBlanc HN and Ashkenazi A. Cell Death Differ 10:66-75 (2003)). TRAIL induces apoptotic cell death upon binding Death Receptor-4 (DR4 or TRAIL-R1) or Death Receptor-5 (DR5 or TRAIL-R2) receptors (see, for e.g:. Pan et al, Science 277:815-8 (1997)). Biologically active soluble TRAIL can be generated after cleavage at the stalk domain. TRAIL receptor agonists, soluble recombinant TRAIL and antibodies against DR4 and DR5 receptors, have been pursued as a promising anti-cancer strategy and showed favorable activity in pre-clinical studies (see, for e.g:. Trivedi R and Mishra DP., Front Oncol. 5:69 (2015)). In early clinical trials these agents showed good safety profile but had limited efficacy (see, for e.g. : Lemke J. et al., Cell Death Differ. 21: 1350-1364 (2014)). [0004] One of the challenges hindering development of recombinant human TRAIL (rhTRAIL) is its very short half-life (3-5 minutes in mice and 30-60 minutes in primates (Kelley SK et al., J Pharmacol Exp Ther. 299:31-38 (2001)). Several groups addressed this issue through protein engineering (e.g. PEGylation, single chain Fc fusion, etc.) with the resulting molecules having improved PK with differential effect on activity ranging from less potent to more potent than the unmodified rhTRAIL trimer (see, for e.g. : Pan LQ et al., Biomaterials. 34:9115-23 (2013)).

[0005] Many cancer cells are intrinsically resistant to TRAIL-induced apoptosis. Resistance can arise due to surface levels of TRAIL receptors (low levels of functional receptors and/or high levels of decoy receptors), modulation of pro- and anti-apoptotic signaling molecules such as cellular FLICE inhibitory protein (c-FLIP), inhibitors of apoptosis proteins (IAPS) and caspase 8 (see, for e.g. : LeBlanc HN, Ashkenazi A. Cell Death Differ. 10:66-75 (2003)).

[0006] CD137 (4-1-BB; TNFRSF9) is a member of tumor necrosis factor receptor superfamily, found primarily on activated T cells, B cells and natural killer (NK) cells, and is a potent co-stimulator of antitumor immune responses. CD 137 has one known endogenous ligand, CD137 ligand (CD137L; 4-1BBL; TNFSF9). CD137L plays an important role during hematopoiesis and in immune regulation. CD137L is expressed by antigen presenting cells (APC), such as dendritic cells, monocytes/macrophages, and B cells, and its expression is upregulated during activation of these cells and CD137L reverse signaling into APC enhances their activity. CD137-CD137L interactions drive type 1, cell-mediated immune responses, CD137 agonists enhance antitumor immune responses. CD137L provides co-stimulatory signaling to CD4+ and CD8+ T cells, induces expansion and survival of T cells, protects them from activation induced cell death and leads to establishment of long-term memory ((Croft, Nat Rev Immunol.9: 271-285 (2009); Shao Z, Schwarz H. J Leukoc Biol. 89:21-29 (2011); Dharmadhikari et al., Oncoimmunology. (2016)). In addition, CD137L signaling can inhibit transformation of naive CD4+ T cells into regulatory T cells (Treg). The co-stimulation of CD 137 can reduce of T cell exhaustion (Long et al. Nat Med. 21:581-590 ( 2015)).

[0007] CD 137 agonistic antibody, urelumab, showed promising anticancer activity in preclinical studies, but failed in clinic due to hepatotoxicity as efficacious doses were above maximum tolerated dose (Dubrot etal. Cancer Immunol. Immunother. 59, 1223-1233 (2010)). Several groups are working on developing CD 137 agonists with potent costimulatory properties and favorable toxicity profiles.

[0008] 0X40 (CD 134; TNFRSF4) is a member of tumor necrosis factor receptor superfamily found primarily on activated CD4+ and CD8+ T-cells, Treg and NK cells (Croft et al., Immunol Rev. 229: 173-91(2009)). 0X40 has one known endogenous ligand, 0X40 ligand (OX40L; CD152; TNFSF4), that exists in a trimeric form and can cluster 0X40 resulting in potent cell signaling events within T cells (Croft et al., Immunol Rev. 229: 173-91 (2009)). Signaling through 0X40 on activated CD4+ and CD8+ T cells leads to enhanced cytokine production, granzyme and perforin release and expansion of effector and memory T cells (Jensen et al., Semin Oncol. 37:524-32 (2010)). In addition, 0X40 signaling on Treg cells inhibits expansion of Tregs, shuts down the induction of Tregs and blocks Treg-suppressive function (see, for e.g. : Voo et al., J Immunol. 191:3641-50 (2013)).

[0009] 0X40 is expressed on T cells infdtrating a broad range of human cancers (see, for e.g. :

Baruah et al., Immunobiology 217:668-675 (2011)). 0X40 expression on tumor-infiltrating lymphocytes correlates with longer survival in several human cancers, suggesting that 0X40 signals may play a critical role in establishing an antitumor immune response (see, for e.g. : Ladanyi et al., Clin Cancer Res. 10:521-30 (2004)).

[0010] In a variety of nonclinical mouse tumor models, agonists of 0X40, including antibodies and 0X40 ligand fusion proteins, have been used successfully with promising results (see, for e.g. : Kjaergaard e/a/., Cancer Res. 60:5514-21 (2000)). Co-stimulating T cells through 0X40 promoted anti-tumor activity that in some cases was durable, providing long-lasting protection against subsequent tumor challenge (Weinberg et al., J Immunol. 164:2160-9 (2000)). Treg cell inhibition and co-stimulation of effector T cells were shown to be necessary for tumor growth inhibition of 0X40 agonists (Piconese et al., J Exp Med. 205:825-39 (2008)). Many strategies and technologies have been explored to enhance the anti-tumor effect of 0X40 agonist therapy through combinations with vaccines, chemotherapy, radiotherapy, and immunotherapy (see, for e.g. : Jensen et al., Semin Oncol. 37:524-32 (2010)).

SUMMARY

[0011] In an aspect, a conjugate is disclosed comprising: TRAIL that is covalently linked to CD137L through either C- or N-terminus. In another aspect, a conjugate is disclosed comprising: TRAIL that is covalently linked to OX40L through either C- or N-terminus. In another aspect, a conjugate is disclosed comprising: TRAIL that is covalently linked to another TNF superfamily ligand through either the C- or N-terminus.

[0012] In embodiments, the TNF superfamily ligand comprises, consists, or consists essentially of an amino acid sequence that is at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from Table T1. In embodiments, the TNF superfamily ligand is a trimeric or homotrimeric polypeptide. In embodiments, TRAIL and CD137L are separated by a linker, optionally a physiologically-stable linker. In embodiments, TRAIL and OX40L are separated by a linker, optionally a physiologically-stable linker. In embodiments, the linker is a peptide linker, optionally a flexible peptide linker or a rigid peptide linker. In embodiments, the peptide linker is about 1-100 amino acids, about 1-90 amino acids, about 1-80 amino acids, about 1-70 amino acids, about 1-80 amino acids, about 1-50 amino acids, about 1-40 amino acids, about 1-30 amino acids, about 1-20 amino acids, about 1-10 amino acids, or about 1-5 amino acids in length, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 ,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, or 100 amino acids in length. In embodiments, the peptide linker is selected from Table L1. In embodiments, the conjugate is a fusion polypeptide. In embodiments, TRAIL is fused to the N-terminus or C-terminus of CD137L, optionally separated by a linker. In embodiments, TRAIL is fused to the N-terminus or C-terminus of OX40L, optionally separated by a linker. In embodiments, the linker is a non-peptide linker. In embodiments, the conjugate has improved pharmacokinetic, physical, and/or biological properties relative to the TRAIL alone and/or CD137L alone and/or OX40L alone, optionally selected from one or more of increased stability, increased serum half-life, increased bioavailability, increased biological activity, increased exposure, and decreased clearance. In embodiments, the conjugate has increased stability and/or serum half-life relative to the TNF superfamily ligand alone, optionally wherein the stability and/or serum half-life relative of the conjugate is increased by about or at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000% or more relative to the TNF superfamily ligand alone. In embodiments, the conjugate has increased biological activity relative to TRAIL alone and/or OX40L alone and/or CD137L alone, optionally wherein the biological activity of the conjugate is increased by about or at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000% or more relative to the TRAIL alone and/or OX40L alone and/or CD137L alone, or optionally wherein the biological activity is increased synergistically relative to the TRAIL alone and/or CD137L alone and/or OX40L alone. In embodiments, the biological activity is induction of cell death or apoptosis in cancer cells, which is optionally increased relative to TRAIL alone. In embodiments, the cancer cells are optionally selected from one or more of breast cancer cells, colon cancer cells, ovarian cancer cells, multiple myeloma and NSCLC cells.

[0013] In another aspect, a conjugate is disclosed comprising: two functional components, a trimeric polypeptide that is covalently linked to another trimeric polypeptide. Each polypeptide can be at either N- or C- terminus. In embodiments, the trimeric polypeptide is a homotrimeric polypeptide. In embodiments, the trimeric polypeptide is selected from a TNF superfamily ligand. [0014] In another aspect, an isolate polynucleotide is disclosed wherein the polynucleotide comprises a conjugate as disclosed herein. [0015] In another aspect, a therapeutic composition is disclosed wherein the composition comprises a conjugate as disclosed herein, and a pharmaceutically acceptable carrier or antioxidant. In another aspect, a therapeutic composition is disclosed wherein the composition comprises one or more pharmaceutically acceptable polysaccharides.

[0016] In another aspect, a therapeutic composition is disclosed wherein the composition comprises a conjugate a disclosed herein, and a pharmaceutically acceptable carrier or excipient. In embodiments, the conjugate is at least about 95% pure and less than about 5% aggregated. In another aspect, the excipients include one or more pharmaceutically acceptable preservatives and/or buffering agents.

[0017] In another aspect, a method of treating, ameliorating the symptoms of, or reducing the progression of a cancer in a subject in need thereof, comprising administering to the subject a therapeutic composition as detailed herein. In embodiments, the cancer is selected from one or more of breast cancer (including triple negative), ovarian cancer, colorectal cancer, non-small cell lung cancer (NSCLC), kidney cancer, hepatocellular carcinoma (HCC), multiple myeloma, melanoma, metastatic melanoma, pancreatic cancer, prostate cancer, small cell lung cancer, mesothelioma, leukemia (including lymphocytic leukemia, chronic myelogenous leukemia, lymphoma, acute myeloid leukemia, and relapsed acute myeloid leukemia), hepatoma, sarcoma, B-cell malignancy, gastric cancer, glioma (e.g., astrocytoma, oligodendroglioma, ependymoma, or a choroid plexus papilloma), glioblastoma multiforme (e.g., giant cell gliobastoma or a gliosarcoma), meningioma, pituitary adenoma, vestibular schwannoma, primary CNS lymphoma, primitive neuroectodermal tumor (medulloblastoma), bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancers, cervical cancer, testicular cancer, and stomach cancer. [0018] In an aspect, a pharmaceutical composition is provided comprising a therapeutically effective amount of any of the conjugates described herein.

[0019] In an aspect, any of the conjugates described herein can be used as a method of treating cancer.

[0020] In an aspect, any of the conjugates described herein can be used as a method of treating cancers, inflammatory diseases and conditions, immunodeficiency diseases, autoimmune disease, microbial infection, protozoan infection, viral infection, graft versus host disease, a condition associated with HIV infection, or pre-cancerous cells.

[0021] In an aspect, a method of making a conjugate comprising TNF superfamily ligands including TRAIL that is covalently linked to CD137L or OX40L through either C- or N-terminus is provided comprising transforming into a host cell exogenous nucleic acids that encode one or more of TRAIL or a portion thereof, CD137L or a portion thereof, and OX40L or a portion thereof; and culturing the host cell under conditions that allow the conjugate to be expressed. In embodiments, the CD137L or a portion thereof comprises the receptor binding domain of CD137L. In embodiments, the OX40L or portion thereof comprises the receptor binding domain of OX40L. In embodiments, the method further comprises purifying the conjugate in a host cell.

[0022] In an aspect, a method is provided in which a cell-free system is used to synthesize a conjugate comprising TNF superfamily ligands including TRAIL that is covalently linked to CD137L or OX40L through either C- or N-terminus. In embodiments, the method comprises culturing cells of interest; lysing the cells; preparing cell extracts from the lysed cells that includes the transcription and translation machinery of the cells; and adding to the cell extracts one or more nucleic acids that encodes TRAIL or a portion thereof, CD137L or a portion thereof, and OX40L or a portion thereof. In embodiments, the cells are lysed using homogenization, sonication, or through use of a freeze/thaw cycles. In embodiments, the cells are lysed using any technique known in the art that is capable of lysing cells. In embodiments, the cell extracts are prepared through centrifugation or sonification techniques that removes cellular membranes and cell debris from the cells. In embodiments, the cell extracts are prepared through using any technique known in the art capable of preparing cell extracts. In embodiments, the cell-free expression system utilizes bacterial cells. In embodiments, the cell-free expression system utilizes mammalian cells.

[0023] In an aspect, a method of stimulating a T cell response is provided comprising administering to a cell a fusion protein comprising TRAIL and at least one of a CD 137 agonist and an 0X40 agonist. In embodiments, the CD 137 agonist comprises a CD137L or a portion thereof. In embodiments, the 0X40 agonist comprises an OX40L or a portion thereof. In embodiments, the CD137L or a portion thereof comprises a receptor binding domain of CD137L. In embodiments, the OX40L or a portion thereof comprises a receptor binding domain of OX40L. In embodiments, TRAIL comprises the DR4 receptor binding domain of TRAIL, the DR5 receptor binding domain of TRAIL, or both the DR4 receptor binding domain of TRAIL and the DR5 receptor binding domain of TRAIL. In embodiments, the fusion protein comprises a receptor binding domain of TRAIL and a receptor binding domain of CD 137L. In embodiments, the fusion protein comprises a receptor binding domain of TRAIL and a receptor binding domain of OX40L. [0024] In an aspect, an article of manufacture is provided comprising a pharmaceutical composition of any of the conjugates described herein, a container, and a package insert or label indicating that the pharmaceutical composition can be used to treat cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] FIG. 1 depicts a schematic representation of a trimeric fusion protein TRAIL-CD137L or TRAIL-OX40L. [0026] FIG. 2 depicts a schematic representation of Cancer-T cell immune synapse mediated by TRAIL-CD137L or TRAIL-OX40L fusion protein.

[0027] FIG. 3 depicts the effects of exemplary AB001 (TRAIL-OX40L) versus AB002 (OX40L-TRAIL) fusion polypeptides versus rhTRAIL on caspase 3/7 induction (FIGs. 3A-3B) and relative cell viability (FIGs. 3C-3D) in HCT116 (FIGs. 3A and 3C) and Colo 205 cell lines (FIGs. 3B and 3D).

[0028] FIG. 4 depicts the effects of exemplary AB001 (TRAIL-OX40L) versus AB002 (OX40L -TRAIL) fusion polypeptides versus rhOX40L on induction of NF-kB luciferase reporter in 0X40 expressing HEK293 reporter cell line.

[0029] FIG. 5 depicts induction of cell death in RPML8226 cells by exemplary fusion polypeptides TRAIL-OX40L (AB001), OX40L-TRAIL (AB002), TRAIL-CD137L polypeptides (AB007-L1, AB007-L2, AB007-L3,) and CD137L-TRAIL (AB008-L3) as well as rhCD137L and rhOX40L, each with and without equimolar rhTRAIL.

[0030] FIGs. 6A-6H depict the effects of exemplary TRAIL-CD137L fusion polypeptides AB007-L1, AB007-L2, AB007-L3 and CD137-TRAIL fusion polypeptide AB008-L3 versus rhTRAIL on caspase 3/7 induction (FIGs. 6A, 6C, 6E, and 6G) and relative cell viability (FIGs. 6B, 6D, 6F, and 6H) in RPMI-8226 (FIGs. 6A and 6B), Colo205 (FIGs. 6C and 6D), HCT116 (FIGs. 6E and 6F), and Caov-3 (FIGs. 6G and GH) cell lines.

[0031] FIG. 7 depicts the effects of exemplary TRAIL-CD137L fusion polypeptides AB007- Ll, AB007-L2, AB007-L3 and CD137L-TRAIL fusion polypeptide AB008-L3 versus rhCD137L on induction of NF-kB luciferase reporter in CD 137 expressing HEK293 reporter cell line.

[0032] FIG. 8 depicts effect of exemplary fusion polypeptides TRAIL-OX40L (AB001), OX40L-TRAIL (AB002), TRAIL-CD137L polypeptides (AB007-L1, AB007-L2, AB007-L3), and CD137L-TRAIL (AB008-L3) as well as rhCD137L and rhOX40L by themselves or in combination with equimolar amounts of rhTRAIL on T cell proliferation (FIG. 8A) and TNFa secretion (FIG.8B) in a co-culture of pre-activated T cells and RPMI-8226 cancer cells.

DETAILED DESCRIPTION

Definitions

[0033] In respect of the present disclosure, and unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the disclosure belongs. Although any methods, materials, compositions, reagents, cells, similar or equivalent similar or equivalent to those described herein can be used in the practice or testing of the subject matter of the present disclosure, preferred methods and materials are described. All publications and references, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference in their entirety as if each individual publication or reference were specifically and individually indicated to be incorporated by reference herein as being fully set forth. Any patent application to which this application claims priority is also incorporated by reference herein in its entirety in the manner described above for publications and references.

[0034] The practice of the present disclosure will employ, unless indicated specifically to the contrary, conventional methods of virology, immunology, microbiology, molecular biology and recombinant DNA techniques within the skill of the art, many of which are described below for the purpose of illustration. Such techniques are explained fully in the literature. See, e.g., Current Protocols in Protein Science, Current Protocols in Molecular Biology or Current Protocols in Immunology, John Wiley & Sons, New York, N.Y. (2009); Ausubel et al., Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995; Sambrook and Russell, Molecular Cloning: A Laboratory Manual (3rd Edition, 2001); Maniatis et al. Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985); Transcription and Translation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984) and other like references.

[0035] Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. These and related techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. Unless specific definitions are provided, the nomenclature utilized in connection with, and the laboratory procedures and techniques of, molecular biology, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques may be used for recombinant technology, molecular biological, microbiological, chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0036] For the purposes of the present disclosure, the following terms are defined below. [0037] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element or “polypeptide subunit” is understood to represent one or more polypeptide subunits. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.

[0038] By “about” is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0039] An “agonist” refers to biological structure or chemical agent that increases or enhances the physiological action of another agent or molecule. In some instances, the agonist specifically binds to the other agent or molecule. Included are full and partial agonists.

[0040] As used herein, the term “amino acid” is intended to mean both naturally occurring and non-naturally occurring amino acids as well as amino acid analogs and mimetics. Naturally occurring amino acids include the 20 (L)-amino acids utilized during protein biosynthesis as well as others such as 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine, homocysteine, citrulline and ornithine, for example. Non-naturally occurring amino acids include, for example, (D)-amino acids, norleucine, norvaline, p-fluorophenylalanine, ethionine and the like, which are known to a person skilled in the art. Amino acid analogs include modified forms of naturally and non-naturally occurring amino acids. Such modifications can include, for example, substitution or replacement of chemical groups and moieties on the amino acid or by derivatization of the amino acid. Amino acid mimetics include, for example, organic structures which exhibit functionally similar properties such as charge and charge spacing characteristic of the reference amino acid. For example, an organic structure which mimics Arginine (Arg or R) would have a positive charge moiety located in similar molecular space and having the same degree of mobility as the e-amino group of the side chain of the naturally occurring Arg amino acid. Mimetics also include constrained structures so as to maintain optimal spacing and charge interactions of the amino acid or of the amino acid functional groups. Those skilled in the art know or can determine what structures constitute functionally equivalent amino acid analogs and amino acid mimetics.

[0041] “Biocompatible” refers to materials or compounds which are generally not injurious to biological functions and which will not result in any degree of unacceptable toxicity, including allergenic and disease states.

[0042] By “coding sequence” is meant any nucleic acid sequence that contributes to the code for the polypeptide product of a gene. By contrast, the term “non-coding sequence” refers to any nucleic acid sequence that does not directly contribute to the code for the polypeptide product of a gene.

[0043] Throughout this disclosure, unless the context requires otherwise, the words “comprise,” “comprises,” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

[0044] By “consisting of’ is meant including, and limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of’ is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of’ indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they materially affect the activity or action of the listed elements.

[0045] The term “conjugate” refers to an entity formed as a result of covalent or non-covalent attachment or linkage of at least two separate polypeptides (for example, a first polypeptide and a second polypeptide), as described herein. One example of a conjugate polypeptide is a “fusion protein” or “fusion polypeptide,” that is, a polypeptide that is created through the joining of two or more coding sequences, which originally coded for separate polypeptides; translation of the joined coding sequences results in a single, fusion polypeptide, typically with functional properties derived from each of the separate polypeptides.

[0046] The “half-life” of a conjugate or polypeptide can refer to the time it takes for the conjugate or polypeptide to lose half of its pharmacologic, physiologic, or other activity, relative to such activity at the time of administration into the serum or tissue of an organism, or relative to any other defined time-point. “Half-life” can also refer to the time it takes for the amount or concentration of a conjugate or polypeptide to be reduced by half of a starting amount administered into the serum or tissue of an organism, relative to such amount or concentration at the time of administration into the serum or tissue of an organism, or relative to any other defined time-point. The half-life can be measured in serum and/or any one or more selected tissues.

[0047] The terms “modulating” and “altering” include “increasing,” “enhancing” or “stimulating,” as well as “decreasing” or “reducing,” typically in a statistically significant or a physiologically significant amount or degree relative to a control. An “increased,” “stimulated” or “enhanced” amount is typically a “statistically significant” amount, and may include an increase that is 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and ranges in between e.g., 1.5, 1.6, 1.7. 1.8, etc.) the amount produced by no composition (e.g., the absence of agent) or a control composition. A “decreased” or “reduced” amount is typically a “statistically significant” amount, and may include a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18% , 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% decrease (including all integers and ranges in between) in the amount produced by no composition (e.g., the absence of an agent) or a control composition. Examples of comparisons and “statistically significant” amounts are described herein.

[0048] The terms “polypeptide,” “protein” and “peptide” are used interchangeably and mean a polymer of amino acids not limited to any length. The terms include modifications such as myristoylation, sulfation, glycosylation, phosphorylation and addition or deletion of signal sequences. The terms “polypeptide” or “protein” means one or more chains of amino acids, wherein each chain comprises amino acids covalently linked by peptide bonds, and wherein said polypeptide or protein can comprise a plurality of chains non-covalently and/or covalently linked together by peptide bonds, having the sequence of native proteins, that is, proteins produced by naturally-occurring and specifically non-recombinant cells, or genetically-engineered or recombinant cells, and comprise molecules having the amino acid sequence of the native protein, or molecules having deletions from, additions to, and/or substitutions of one or more amino acids of the native sequence. In certain embodiments, the polypeptide is a “recombinant” polypeptide, produced by recombinant cell that comprises one or more recombinant DNA molecules, which are typically made of heterologous polynucleotide sequences or combinations of polynucleotide sequences that would not otherwise be found in the cell.

[0049] As used herein, the term “polypeptide” is intended to encompass a singular “polypeptide” as well as plural “polypeptides,” and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term “polypeptide” refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, “protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids are included within the definition of “polypeptide,” and the term “polypeptide” can be used instead of, or interchangeably with any of these terms. The term “polypeptide” is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-standard amino acids. A polypeptide can be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It can be generated in any manner, including by chemical synthesis. [0050] A “protein” as used herein can refer to a single polypeptide, i.e., a single amino acid chain as defined above, but can also refer to two or more polypeptides that are associated, e.g, by disulfide bonds, hydrogen bonds, or hydrophobic interactions, to produce a multimeric protein. As used herein, the term “polypeptide subunit” refers to a polypeptide chain of amino acids which can interact with other polypeptide subunits, either identical or different, to form a multimeric protein, e.g., a trimeric protein as described herein.

[0051] A polypeptide as disclosed herein can be of a size of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids. Polypeptides can have a defined three-dimensional structure, although they do not necessarily have such structure. Polypeptides with a defined three- dimensional structure are referred to as folded, and polypeptides that do not possess a defined three- dimensional structure, but rather can adopt a large number of different conformations, and are referred to as unfolded.

[0052] By an “isolated” polypeptide or a fragment, variant, or derivative thereof is intended a polypeptide that is not in its natural milieu. No particular level of purification is required. For example, an isolated polypeptide can be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated as disclosed herein, as are native or recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technique.

[0053] Other polypeptides disclosed herein are fragments, derivatives, analogs, or variants of the foregoing polypeptides, and any combination thereof. The terms “fragment,” “variant,” “derivative” and “analog” when referring to polypeptide subunit or multimeric protein as disclosed herein can include any polypeptide or protein that retain at least some of the activities of the complete polypeptide or protein, but which is structurally different. Fragments of polypeptides include, for example, proteolytic fragments, as well as deletion fragments. Variants include fragments as described above, and also polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions. Variants can occur spontaneously or be intentionally constructed. Intentionally constructed variants can be produced using art-known mutagenesis techniques. Variant polypeptides can comprise conservative or non-conservative amino acid substitutions, deletions or additions. Derivatives are polypeptides that have been altered so as to exhibit additional features not found on the native polypeptide. Examples include fusion proteins. Variant polypeptides can also be referred to herein as “polypeptide analogs.” As used herein a “derivative” refers to a subject polypeptide having one or more amino acids chemically derivatized by reaction of a functional side group. Also included as “derivatives” are those peptides that contain one or more standard or synthetic amino acid derivatives of the twenty standard amino acids. For example, 4-hydroxyproline can be substituted for proline; 5 -hydroxy lysine can be substituted for lysine; 3-methylhistidine can be substituted for histidine; homoserine can be substituted for serine; and ornithine can be substituted for lysine.

[0054] The term “isolated” polypeptide or protein referred to herein means that a subject protein (1) is free of at least some other proteins with which it would typically be found in nature, (2) is essentially free of other proteins from the same source, e.g., from the same species, (3) is expressed by a cell from a different species, (4) has been separated from at least about 50 percent of polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature, (5) is not associated (by covalent or non-covalent interaction) with portions of a protein with which the “isolated protein” is associated in nature, (6) is operably associated (by covalent or non-covalent interaction) with a polypeptide with which it is not associated in nature, or (7) does not occur in nature. Such an isolated protein can be encoded by genomic DNA, cDNA, mRNA or other RNA, or may be of synthetic origin, or any combination thereof. In certain embodiments, the isolated protein is substantially free from proteins or polypeptides or other contaminants that are found in its natural environment that would interfere with its use (therapeutic, diagnostic, prophylactic, research or otherwise).

[0055] In certain embodiments, the “purity” of any given agent (for example, a conjugate) in a composition may be specifically defined. For instance, certain compositions may comprise a conjugate that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% pure, including all decimals and ranges in between, as measured, for example and by no means limiting, by high performance liquid chromatography (HPLC), a well-known form of column chromatography used frequently in biochemistry and analytical chemistry to separate, identify, and quantify compounds. In some instances, the purity of a composition is characterized by the degree of aggregation. For instance, the degree of aggregation of a conjugate (for example, fusion protein) can be determined by Size-exclusion chromatography (SEC), which separates particles based on size. It is a generally accepted method for determining the tertiary structure and quaternary structure of purified proteins. SEC is used primarily for the analysis of large molecules such as proteins or polymers. SEC works by trapping these smaller molecules in the pores of a particle. The larger molecules simply pass by the pores as they are too large to enter the pores. Larger molecules therefore flow through the column quicker than smaller molecules, that is, the smaller the molecule, the longer the retention time. Certain compositions are also substantially free of aggregates (greater than about 95% appearing as a single peak by SEC HPLC). Certain embodiments are free of aggregates with greater than about 96%, about 97%, about 98%, or about 99%, appearing as a single peak by SEC HPLC. [0056] The term “reference sequence” refers generally to a nucleic acid coding sequence, or amino acid sequence, to which another sequence is being compared. All polypeptide and polynucleotide sequences described herein are included as references sequences, including those described by name and those described in the Tables and the informal or formal Sequence Listing. [0057] The terms “sequence identity” or, for example, comprising a “sequence 50% identical to,” as used herein, refer to the extent that sequences are identical on a nucleotide-by -nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a “percentage of sequence identity” may be calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g, A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Vai, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (z. e. , the window size), and multiplying the result by 100 to yield the percentage of sequence identity. Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, Wis., USA) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al., Nucl. Acids Res. 25:3389 (1997). [0058] A “conservative amino acid substitution” is one in which one amino acid is replaced with another amino acid having a similar side chain. Families of amino acids having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g, threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, substitution of a phenylalanine for a tyrosine is a conservative substitution. Methods of identifying nucleotide and amino acid conservative substitutions which do not eliminate protein activity are well-known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1 187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA 94: 412-417 (1997)).

[0059] As used herein, a “coding region” is a portion of nucleic acid comprising codons translated into amino acids. Although a “stop codon” (TAG, TGA, or TAA) is not translated into an amino acid, it can be considered to be part of a coding region, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, and the like, are not part of a coding region. Two or more coding regions can be present in a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g, on separate (different) vectors. Furthermore, any vector can contain a single coding region, or can comprise two or more coding regions, e.g., a single vector can separately encode an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region. In addition, a vector, polynucleotide, or nucleic acid can encode heterologous coding regions, either fused or unfused to a nucleic acid encoding a polypeptide subunit or fusion protein as provided herein. Heterologous coding regions include without limitation specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain.

[0060] In certain embodiments, the polynucleotide or nucleic acid is DNA. In the case of DNA, a polynucleotide comprising a nucleic acid that encodes a polypeptide normally can include a promoter and/or other transcription or translation control elements operably associated with one or more coding regions. An operable association or linkage is when a coding region for a gene product, e.g., a polypeptide, is associated with one or more regulatory sequences in such a way as to place expression of the gene product under the influence or control of the regulatory sequence(s). Two DNA fragments (such as a polypeptide coding region and a promoter associated therewith) are “operably associated” or “operably linked” if induction of promoter function results in the transcription of mRNA encoding the desired gene product and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed. Thus, a promoter region would be operably associated with a nucleic acid encoding a polypeptide if the promoter was capable of effecting transcription of that nucleic acid. The promoter can be a cell-specific promoter that directs substantial transcription of the DNA only in predetermined cells. Other transcription control elements, besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can be operably associated with the polynucleotide to direct cell-specific transcription. Suitable promoters and other transcription control regions are disclosed herein.

[0061] A variety of transcription control regions are known to those skilled in the art. These include, without limitation, transcription control regions that function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (the immediate early promoter, in conjunction with intron-A), simian virus 40 (the early promoter), and retroviruses (such as Rous sarcoma virus). Other transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone and rabbit [3-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers as well as lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins). [0062] Similarly, a variety of translation control elements are known to those of ordinary skill in the art. These include, but are not limited to ribosome binding sites, translation initiation and termination codons, and elements derived from picornaviruses (particularly an internal ribosome entry site, or IRES, also referred to as a CITE sequence).

[0063] In other embodiments, a polynucleotide can be RNA, for example, in the form of messenger RNA (mRNA).

[0064] Polynucleotide and nucleic acid coding regions can be associated with additional coding regions that encode secretory or signal peptides, which direct the secretion of a polypeptide encoded by a polynucleotide as disclosed herein, e.g, a polynucleotide encoding a polypeptide subunit provided herein. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence that is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Those of ordinary skill in the art are aware that polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the complete or “full length” polypeptide to produce a secreted or “mature” form of the polypeptide. In certain embodiments, the native signal peptide, e.g., an immunoglobulin heavy chain or light chain signal peptide is used, or a functional derivative of that sequence that retains the ability to direct the secretion of the polypeptide that is operably associated with it. Alternatively, a heterologous mammalian signal peptide, or a functional derivative thereof, can be used. For example, the wild-type leader sequence can be substituted with the leader sequence of human tissue plasminogen activator (TPA) or mouse [3-glucuronidase.

[0065] A “vector” is nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell. A vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector can also include one or more selectable marker gene and other genetic elements known in the art.

[0066] A “transformed” cell, or a “host” cell, is a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. As used herein, the term transformation encompasses all techniques by which a nucleic acid molecule can be introduced into such a cell, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration. A transformed cell or a host cell can be a bacterial cell or a eukaryotic cell.

[0067] By “specifically binds,” it is generally meant that a molecule, e.g., a CD137L, an OX40L, TRAIL or receptor-binding fragment thereof, binds to another molecule, e.g., CD137, 0X40 or DR4, DR5, via its receptor-binding domain, and that the binding entails some complementarity between the antigen binding domain and the epitope. According to this definition, a ligand is said to “specifically bind” to a receptor when it binds to that receptor, via its receptor- binding domain more readily than it would bind to a random, unrelated molecule. The term “specificity” is used herein to qualify the relative affinity by which a certain ligand binds to a certain receptor. For example, ligand “A” can be deemed to have a higher specificity for a given receptor than ligand “B,” or ligand “A” can be said to bind to receptor “C” with a higher specificity than it has for related receptor “D.”

[0068] By “a receptor-binding domain,” it is intended a binding domain comprised in a ligand, e.g, a CD137L, or an OX40L or TRAIL as disclosed herein.

[0069] The term “solubility” refers to the property of an agent (for example, a conjugate) described herein to dissolve in a liquid solvent and form a homogeneous solution. Solubility is typically expressed as a concentration, either by mass of solute per unit volume of solvent (g of solute per kg of solvent, g per dL (100 mL), mg/mL, etc.), molarity, molality, mole fraction or other similar descriptions of concentration. The maximum equilibrium amount of solute that can dissolve per amount of solvent is the solubility of that solute in that solvent under the specified conditions, including temperature, pressure, pH, and the nature of the solvent. In certain embodiments, solubility is measured at physiological pH, or other pH, for example, at pH 5.0, pH 6.0, pH 7.0, pH 7.4, pH 7.6, pH 7.8, or pH 8.0 (e.g., about pH 5-8). In certain embodiments, solubility is measured in water or a physiological buffer such as PBS or NaCl (with or without NaP). In specific embodiments, solubility is measured at relatively lower pH (e.g., pH 6.0) and relatively higher salt (e.g., 500mM NaCl and lOmM NaP). In certain embodiments, solubility is measured in a biological fluid (solvent) such as blood or serum. In certain embodiments, the temperature can be about room temperature (e.g., about 20, 21, 22, 23, 24, 25°C) or about body temperature (37°C). In certain embodiments, an agent has a solubility of at least about 0. 1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90 or 100 mg/mL at room temperature or at 37°C.

[0070] A “subject” or a “subject in need thereof’ or a “patient” or a “patient in need thereof’ includes a mammalian subject such as a human subject.

[0071] “Substantially” or “essentially” means nearly totally or completely, for instance, 95%, 96%, 97%, 98%, 99% or greater of some given quantify.

[0072] By “statistically significant,” it is meant that the result was unlikely to have occurred by chance. Statistical significance can be determined by any method known in the art. Commonly used measures of significance include the p-value, which is the frequency or probability with which the observed event would occur, if the null hypothesis were true. If the obtained p-value is smaller than the significance level, then the null hypothesis is rejected. In simple cases, the significance level is defined at a p-value of 0.05 or less. [0073] “Therapeutic response” refers to improvement of symptoms (whether or not sustained) based on administration of one or more therapeutic agents, for example, conjugates.

[0074] As used herein, “treatment” of a subject (e.g., a mammal, such as a human) or a cell is any type of intervention used in an attempt to alter the natural course of the individual or cell. Treatment includes, but is not limited to, administration of a pharmaceutical composition, and may be performed either prophylactically or after the initiation of a pathologic event or contact with an etiologic agent. Also included are “prophylactic” treatments, which can be directed to reducing the rate of progression of the disease or condition being treated, delaying the onset of that disease or condition, or reducing the severity of its onset. “Treatment” or “prophylaxis” does not necessarily indicate complete eradication, cure, or prevention of the disease or condition, or associated symptoms thereof.

[0075] The term “wild-type” refers to a gene or gene product (e.g., a polypeptide) that is most frequently observed in a population and is thus arbitrarily designed the “normal” or “wild-type” form of the gene. Each embodiment in this specification is to be applied mutatis mutandis to every other embodiment unless expressly stated otherwise.

Aspects and Embodiments of the Present Disclosure

[0076] Aspects of the present disclosure include conjugates comprising of any one or more of the TNF superfamily of ligands. In embodiments, the conjugates comprise any one or more of the TNF superfamily of ligands known in the art. In embodiments, the conjugates comprise any one more the ligands disclosed in Table T1. In embodiments, the conjugates comprise at least one (1), at least two (2), at least three (3), at least four (4), at least five (5), at least 6 (six), at least 7 (seven), at least eight (8), at least nine (9), or at least ten (10) of the TNF superfamily of ligands. In embodiments, the conjugates comprise more than ten (10) of the TNF superfamily of ligands.

[0077] In embodiments, the conjugates comprise TRAIL that is covalently linked to either CD137L or OX40L. In some embodiments the conjugate is comprised of covalently linked TRAIL and CD137L. In some embodiments the conjugate is comprised of covalently linked TRAIL and OX40L.

[0078] Disclosed herein are polypeptide subunits, each including, as a fusion polypeptide, DR4 or DR5 receptor binding domain of TRAIL, a linker, the receptor-binding domain of CD 137 Ligand (CD137L) or 0X40 Ligand (OX40L), which are capable of forming a stable trimer or trimer multimers, e.g., hexameric proteins.

[0079] In embodiments, the compositions and methods disclosed herein are useful in treating diseases. In embodiments, the compositions and methods disclosed herein are useful in treating cancer (immuno)therapy. [0080] In an aspect, the disclosure provides a single-chain polypeptide subunit that includes: TRAIL DR4 or DR5 binding domain, a receptor binding domain of CD137L or OX40L. In certain aspects, a polypeptide subunit as provided can self-assemble into a trimeric or a hexameric protein. In some embodiments, TRAIL comprises, consists, or consists essentially of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence 1 or 2 from Table T2. In embodiments, TRAIL comprises, consists, or consists essentially of an amino acid sequence that is less than 80% identical to a sequence 1 or 2 from Table T2. In some embodiments, OX40L comprises, consists, or consists essentially of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence 5 from Table T2. In embodiments, OX40L comprises, consists, or consists essentially of an amino acid sequence that is less than 80% identical to a sequence 5 from Table T2. In some embodiments, CD137L comprises, consists, or consists essentially of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence 7 or 8 from Table T2. In embodiments, CD137L comprises, consists, or consists essentially of an amino acid sequence that is less than 80% identical to a sequence 7 or 8 from Table T2.

[0081] In some embodiments, the TNF superfamily ligand is a trimeric or homotrimeric polypeptide. In some embodiments CD137L is a trimeric polypeptide and comprises, consists, or consists essentially of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7.

[0082] In some embodiments, polypeptides of the fusion protein, TRAIL, CD137L or OX40L are separated by a linker, optionally a physiologically-stable linker. In some embodiments, the linker is a peptide linker, optionally a flexible peptide linker or a rigid peptide linker. In some embodiments, the peptide linker is about 1-100 amino acids, about 1-90 amino acids, about 1-80 amino acids, about 1-70 amino acids, about 1-80 amino acids, about 1-50 amino acids, about 1-40 amino acids, about 1-30 amino acids, about 1-20 amino acids, about 1-10 amino acids, or about 1- 5 amino acids in length, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 ,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, or 100 amino acids in length. In some embodiments, the peptide linker is greater than 100 amino acids. In some embodiments, the peptide linker is selected from Table L1.

[0083] In some embodiments, the linker is 5 amino acids in length. In some embodiments, the linker is 12 amino acids in length. In some embodiments, the linker is 15 amino acids in length. In some embodiments, the linker is 25 amino acids in length.

[0084] In some embodiments, fusion proteins described herein retain their ability to reduce cell viability in cancer cells regardless of the length of a linker contained in the fusion proteins. [0085] In some embodiments, the conjugate is a fusion polypeptide. In some embodiments, TRAIL is fused to the N-terminus of CD137L, optionally separated by a linker. In some embodiments, the TRAIL is fused to the C-terminus of CD137L, optionally separated by a linker. In some embodiments, TRAIL is fused to the N-terminus of OX40L, optionally separated by a linker. In some embodiments, the TRAIL is fused to the C-terminus of OX40L, optionally separated by a linker.

[0086] In some embodiments, the conjugate has improved pharmacokinetic, physical, and/or biological properties relative to either trimeric polypeptide alone (TRAIL and/or OX40L and/or CD137L), optionally selected from one or more of increased stability, increased serum half-life, increased bioavailability, increased biological activity, increased exposure, and decreased clearance.

[0087] In some embodiments, conjugation of the two or three trimeric and/or hexameric polypeptides increases the stability and/or serum half-life of either or each trimeric polypeptide, optionally by about or at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000% or more relative to either trimeric and/or hexameric polypeptide alone. In embodiments, the two or three trimeric and/or hexameric polypeptides increases the stability and/or serum half-life of either or each trimeric polypeptide, optionally by about or at least less than 10% or more than 1000%.

[0088] In some embodiments, the conjugate has increased biological activity relative to TRAIL alone and/or CD137L or OX40L alone, optionally wherein the biological activity of the conjugate is increased by about or at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000% or more relative to the TRAIL alone and/or CD137L or OX40L alone, or optionally wherein the biological activity is increased synergistically relative to TRAIL alone and/or CD137L or OX40L alone. In some embodiments, the biological activity is an induction of cell death or apoptosis in cancer cells, which is optionally increased synergistically relative to TRAIL alone. In some embodiments, the conjugate is attached to cancer cells through its TRAIL moiety thereby localizing CD137L or OX40L moiety to the tumor site. In some embodiments, the biological activity is activation of immune cells, such as T cells which is optionally increased synergistically relative to CD137L or OX40L alone. In some embodiments, the biological activity is redirection by the fusion protein to bring T cells and cancer cells together indicative of immune synapse formation between immune cells, such as T cells, and cancer cells, culminating in T cell activation and effector function (T cell mediated killing of cancer cells). Synapse formation also leads to CD 137 or 0X40 clustering on T cells and TRAIL receptors (DR4 and/or DR5) clustering on cancer cells; receptor clustering results in signal amplification with each moiety having a greater activity than in the absence of receptor clustering.

[0089] In some embodiments, the cancer cells are DR4 or DR5 expressing cells, which are optionally selected from one or more of leukemic cells, breast, colon, lung, ovarian, multiple myeloma, pancreatic, prostate and renal cancer cells.

[0090] In an aspect conjugates are provided. In embodiments, the conjugates comprise a trimeric polypeptide that is covalently linked to the same or different trimeric polypeptide and/or a hexameric polypeptide.

[0091] In some embodiments, the trimeric polypeptide is a homotrimeric polypeptide or a homohexameric polypeptide that can specifically bind to human TRAIL receptors DR4 (TRAIL- Rl) and DR5 (TRAIL-R2) and/or human CD 137 or 0X40. In some embodiments, the trimeric polypeptide and/or the second trimeric polypeptide is selected from a TNF superfamily ligand, optionally as described herein. In some embodiments, the first trimeric polypeptide is covalently linked to the N-terminus of the second trimeric polypeptide.

[0092] In some embodiments, the conjugate is a fusion polypeptide.

[0093] In additional aspects of the polypeptide subunit provided by the disclosure, the DR4 and DR5 receptor binding domain can include amino acids 114 to 281 of human TRAIL (SEQ ID NO: 2). In some embodiments, the DR4 and DR5 receptor binding domain can include amino acids 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119 to 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, or 285 of human TRAIL. In some embodiments, the OX40L receptor binding domain can include amino acids 51 to 183 of human OX40L (SEQ ID NO: 5). In some embodiments, the OX40L receptor binding domain can include amino acids 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, or 56 to 178, 179, 180, 181, 182, 183, 184, 185, 186, or 187 of OX40L. In some embodiments, CD137L can include amino acids 71 to 254 of human CD137L (SEQ ID NO: 7). In some embodiments, CD137L can include amino acids 50, 52, 55, 57, 60, 62, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, or 76 to 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, or 259 of human CD137L (SEQ ID NO: 8).

[0094] Certain embodiments relate to isolated polynucleotides which encode a conjugate described herein, wherein the conjugate is a fusion protein. Also included are expression vectors that comprises the isolated polynucleotide, and host cells that comprise the isolated polynucleotide or the expression vector.

[0095] In an aspect, therapeutic compositions are provided. In embodiments, the therapeutic compositions comprise any conjugate described herein and a pharmaceutically acceptable carrier or excipient. In particular embodiments, the conjugate forms a trimeric complex of TRAIL-OX40L or OX40L-TRAIL, or TRAIL-CD137L, and/or CD137L-TRAIL, conjugates, optionally as fusion proteins (see, for example, FIG. 1). In some embodiments, the conjugate or composition is at least about 95% pure. In some embodiments, the conjugate or composition is at least 85%, at least 86%, at least 87%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, or at least 94% pure. In some embodiments methods are provided of treating, ameliorating the symptoms of, or reducing the progression of a cancer in a subject in need thereof, comprising administering to the subject a conjugate or therapeutic composition as described herein either by itself or in combination with another therapeutic.

[0096] In some embodiments, the cancer is selected from one or more of colorectal cancer, breast cancer (including triple negative breast cancer), pancreatic cancer, prostate cancer, lymphocytic leukemia, chronic myelogenous leukemia, lymphoma, sarcoma, leukemia, acute myeloid leukemia, relapsed acute myeloid leukemia, B-cell malignancy, multiple myeloma, ovarian cancer, gastric cancer, lung cancer, kidney cancer, bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancers, cervical cancer, testicular cancer, and stomach cancer. In some embodiments, the cancer is any cancer known in the art.

[0097] In certain aspects, a trimeric protein as provided. In some embodiments, the trimeric protein can specifically bind receptors of any TNF superfamily member. In some embodiments, the trimeric protein can specifically bind receptors of any TNF superfamily member listed in Table T1. In some embodiments, the trimeric protein can specifically bind to TRAIL receptors DR4 and/or DR5. In some embodiments, binding of the trimeric protein to TRAIL receptors DR4 and/or DR5 can induce dose-dependent apoptotic cell death.

[0098] In certain aspects, a trimeric protein as provided by the disclosure can specifically bind to any member of the tumor necrosis factor (TNF) receptor family. In some embodiments, the TNF receptor is CD 137. In some embodiments, CD 137 is expressed on activated T cells. In some embodiments, the activated T cells derive from human, cynomolgus monkey, and/or rhesus monkey. For example, the fusion protein can specifically bind to CD 137 as expressed on primary activated T cells from human, cynomolgus monkey, and/or rhesus monkey.

[0099] In certain aspects, a trimeric protein as provided by the disclosure can specifically bind to 0X40 as expressed on activated T cells from human, cynomolgus monkey, and/or rhesus monkey. For example, the fusion protein can specifically bind to 0X40 as expressed on primary activated T cells from human, cynomolgus monkey, and/or rhesus monkey.

[0100] In certain aspects a trimeric protein as provided by the disclosure can induce dose- dependent proliferation of activated T cells and dose-dependent cytokine release from activated T cells.

[0101] In certain aspects a trimeric protein as provided by the disclosure can activate the NFKB pathway in CD137 expressing T cells or a reporter cell line. For example, the CD137-expressing cells can be CD137-expressing HEK293 NFKB-luciferase reporter cells that produce luciferase in response to stimulation of the NFKB signaling pathway. This fusion protein mediated activation can be further amplified in the presence of DR4 or DR5 expressing cells. This phenomenon is specific to the fusion protein such as TRAIL-CD137L, and not to CD137L alone.

[0102] In certain aspects a trimeric protein as provided by the disclosure can activate the NFKB pathway in 0X40 expressing T cells or a reporter cell line. For example, the OX40-expressing reporter cells can be OX40-expressing HEK293 NFKB-luciferase reporter cells that produce luciferase in response to stimulation of the NFKB signaling pathway. This fusion protein, mediated activation can be further amplified in the presence of DR4 or DR5 expressing cells. This phenomenon is specific to the fusion protein such as TRAIL-OX40L, and not to OX40L alone.

[0103] In certain aspects a trimeric protein as provided by the disclosure, when administered as an effective dose to a subject in need of cancer treatment, can inhibit tumor growth in the subject. In certain aspects, tumor growth can be inhibited by at least 10%, at least 20%, at least 30%, at least 40%, and least 50%, at least 60%, or at least 70% compared to administration of a vehicle control.

[0104] The disclosure further provides a vector that incorporates the polynucleotide as provided and a host cell that incorporates the polynucleotide or the vector as provided. In a related aspect, the disclosure provides a method of producing a polypeptide subunit of as provided herein or a trimeric protein as provided herein, where the method includes culturing the provided host cell under conditions in which the polypeptide subunit or trimeric protein encoded by the polynucleotide is expressed, and recovering the polypeptide subunit or trimeric protein.

[0105] In certain aspects, the disclosure provides a method to redirect activated T cells to DR4 and/or DR5 expressing cancer cells by the trimeric fusion protein through interaction of TRAIL receptor binding domain with a cancer cell expressing DR4 and/or DR5 receptors and contacting activated T cells through CD 137 or 0X40 binding domain. The disclosure further provides a method to promote survival or proliferation of activated T cells, of inducing cytokine release from activated T cells. In certain aspects, the cytokine can be IFNy, TNFa, IL-2, IL-5, IL- 10, IL-4, IL- 13, IL-8, IL- 12 p70, and/or IL- 1(3. In certain aspects the activated T cells are activated CD4+ T cells and/or activated CD8+ T cells.

[0106] In further aspects, the disclosure provides a method of stimulating a T cell response. In embodiments, the method comprises administering to a cell a fusion protein comprising TRAIL, and at least one of a CD 137 agonist and an 0X40 agonist. In embodiments, the CD 137 agonist comprises a CD 137L or a portion thereof. In embodiments, the 0X40 agonist comprises an OX40L or a portion thereof. In embodiments, the CD137L or a portion thereof comprises the receptor binding domain of CD137L. In embodiments, the OX40L or a portion thereof comprises the receptor binding domain of OX40L.

[0107] In embodiments, the method of stimulating a T cell response comprises administering a fusion polypeptide comprising CD137L or portion thereof and TRAIL or a portion thereof. In embodiments, the fusion polypeptide comprises at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% of any one or more of SEQ ID NOs: 9-12.

[0108] In embodiments, the method of stimulating a T cell response comprises administering a fusion polypeptide comprising OX40L or a portion thereof and TRAIL or a portion thereof. In embodiments, the fusion polypeptide comprises at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% of any one or more of SEQ ID NOs: 13 and 14.

[0109] In embodiments, the method of stimulating the T cell response comprises administering a fusion polypeptide comprising the receptor binding domain of CD137L and TRAIL, or a fusion polypeptide comprising the receptor binding domain of OX40L and TRAIL.

[0110] In further aspects, the disclosure provides a method to reduce regulatory T cell (Treg)- mediated suppression of activated T cell proliferation. In embodiments, the method includes contacting a mixture of activated T cells and Treg cells with the trimeric protein as disclosed herein. In embodiments, the trimeric protein can specifically bind to CD 137 or 0X40 on the surface of the T cells.

[0111] In certain aspects, the methods provided above include administering an effective amount of the trimeric protein or composition as provided by the disclosure to a subject in need of treatment. In a related aspect, the disclosure provides a method of treating cancer in a subject, where the method includes administering to a subject in need of treatment an effective amount of a trimeric fusion protein as provided herein. In certain aspects, the cancer is a solid tumor. In certain aspects, administration of the trimeric or hexameric protein or composition can inhibit tumor growth, can promote tumor reduction, or both. In certain aspects tumor growth inhibition is achieved in the presence of T-cells. In embodiments, provides a method of treating any cancer described herein.

[0112] In yet another aspect, the disclosure provides a method of enhancing an immune response in a subject, where the method includes administering to a subject in need thereof a therapeutically effective amount of a trimeric fusion protein as provided by the disclosure, or a composition as provided by the disclosure. For any of the treatment methods provided in this disclosure, the subject can be a human subject. For any of the treatment methods provided in this disclosure, the subject can be an animal subject.

[0113] In an aspect, methods of promoting lymphocyte activation are provided. In embodiments, the methods promote T cell activation. In embodiments, the method comprises contacting T cells with a trimeric or hexameric protein as provided herein. In embodiments, the protein is a TRAIL-CD137L or a TRAIL-OX40L fusion protein. In embodiments, the trimeric protein can specifically bind to CD 137 or 0X40 on the surface of the T cells. In certain aspects the contacting occurs in the presence of antigen, e.g., a tumor antigen. In certain aspects the method further comprising redirecting T cells to cancer cells by the trimeric protein through binding of CD 137 or 0X40 on activated T cell by the CD137L or OX40L moiety of the fusion protein and interaction of TRAIL component of the fusion protein with a cancer cell expressing DR4 and/or DR5 receptor (see, for e.g. : Figure 2). In certain aspects, the T cell activation can be measured through stimulation of the NFKB signal transduction pathway. In embodiments, T cell activation is measured through the production of cytokines. In embodiments, the cytokines include any one or more of IL-2, IFN-y, TNFa, IL-4, IL- 10, and IL- 13. In embodiments, the cytokines include any cytokine produced by T cells. In certain aspects the contacting is in vitro. In certain aspects the contacting is in vivo, e.g., via administration of an effective dose of the fusion protein to a subject in need of treatment.

Polynucleotides Encoding TRAIL-CD137L and CD137L-TRAIL- Fusion Proteins

[0114] In an aspect, the disclosure further provides a polynucleotide comprising a nucleic acid that encodes TRAIL-CD137L or CD137L-TRAIL fusion polypeptide subunit, or a trimeric protein as provided herein. The disclosure also provides a trimeric protein as provided herein, e.g., TRAIL- OX40L or OX40L-TRAIL fusion protein. In certain aspects, nucleic acid sequences encoding TRAIL, and CD137L are joined in a 5' to 3' orientation, e.g., contiguously linked in a 5' to 3' orientation. In certain aspects, nucleic acid sequences encoding TRAIL, and OX40L are joined in a 5' to 3' orientation, e.g., contiguously linked in a 5' to 3' orientation.

Polypeptide Sequences of Extracellular Domains of Human TRAIL and CD137L Fusion Proteins [0115] SEQ ID NO: 9 TRAIL-/zwter7-CD137L

MVRERGPQRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHSFLSNLHL RNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSA RNSCWSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLIDMDHEASFFGAFLVGGG GGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSY KEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVD LPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPE

IPAGLPSPRSEHHHHHH

[0116] SEQ ID NO: 10 TRAIL-/zzzter2-CD137L

MVRERGPQRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHSFLSNLHL

RNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSA

RNSCWSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLIDMDHEASFFGAFLVGGG

GGSGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLA GVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAA GAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGA TVLGLFRVTPEIPAGLPSPRSEHHHHHH

[0117] SEQ ID NO: 11 TRAIL-/zzzter3-CD137L

MVRERGPQRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHSFLSNLHL

RNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSA

RNSCWSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLIDMDHEASFFGAFLVGGG

GGSGGGGSGGGGSGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGP

LSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLA

LHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARAR HAWOLTOGATVLGLFRVTPEIPAGLPSPRSEHHHHHH

[0118] SEQ ID NO: 12 CD137L-/zzzter3-TRAIL

MHHHHHHREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLT

GGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAAL

ALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGL

FRVTPEIPAGLPSPRSEGGGGSGGGGSGGGGSGGGGSGGGGSVRERGPQRVAAHITGTR

GRSNTLSSPNSKNEKALGRKINSWESSRSGHSFLSNLHLRNGELVIHEKGFYYIYSQT YFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQ GGIFELKENDRIFVSVTNEHLIDMDHEASFFGAFLVG

[0119] An exemplary polypeptide sequences of TRAIL-OX40L and OX40L-TRAIL fusion proteins are represented by SEQ ID NOs: 13 and 14.

[0120] SEQ ID NO: 13: TRAIL -/z«ter-OX40L

VRERGPQRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHSFLSNLHLR

NGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSAR NSCWSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLIDMDHEASFFGAFLVGGGG SGGGSGGGSQVSHRYPRIQSIKVQFTEYKKEKGFILTSQK EDEIMKVQNNSVIINCDGFYL ISLKGYFSQEVNISLHYQKDEEPLFQLKK VRSVNSLMVASLTYKDKVYLNVTTDNTSLD

DFHVNGGELILIHQNPGEFCVL [0121] SEQ ID NO: 14: OX40L-/zwter-TRAIL

QVSHRYPRIQSIKVQFTEYKKEKGFILTSQK EDEIMKVQNNSVIINCDGFYLISLKGYFSQ EVNISLHYQKDEEPLFQLKK VRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGE LILIHQNPGEFCVLGGGSGGGSGGGSVRERGPQRVAAHITGTRGRSNTLSSPNSKNEKA LGRKINSWESSRSGHSFLSNLHLRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKND KQMVQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGGIFELKENDRIFVSV TNEHLIDMDHEASFFGAFLVG

[0122] In other aspects, the provided polynucleotide can further comprise a signal sequence encoding, e.g., a secretory signal peptide or membrane localization sequence. An example of secretory signal peptide is artificial secrecon MWWRLWWLLLLLLLLWPMVWA or MEWSWVFLFFLSVTTGVHS. In embodiments, any secretory signal peptides can be used.

[0123] In an aspect, polynucleotides encoding a TRAIL-CD137L or TRAIL-OX40L fusion polypeptide subunit include deoxyribonucleotides (DNA, cDNA) or ribodeoxynucleotides (RNA) sequences, or modified forms of either nucleotide, which encode the fusion polypeptides are provided. The term includes single and double stranded forms of DNA and/or RNA.

[0124] Also provided are polynucleotides comprising nucleic acid sequences comprising one or a small number of deletions, additions and/or substitutions. Such changes can be contiguous or can be distributed at different positions in the nucleic acid. A substantially identical nucleic acid sequence can, for example, have 1, or 2, or 3, or 4, or even more nucleotide deletions, additions and/or substitutions. In certain aspects, the one or more deletions, additions and/or substitutions do not alter the reading frame encoded by the polynucleotide sequence, such that a modified (“mutant”) but substantially identical polypeptide is produced upon expression of the nucleic acid. [0125] The similarity between amino acid (and/or nucleic acid) sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity); the higher the percentage, the more similar are the primary activated structures of the two sequences. “Percent (%) identity” is defined herein as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in a selected sequence, after aligning the sequences and introducing gaps in the candidate and/or selected sequence, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative amino acid substitutions as part of the sequence identity.

[0126] In embodiments, a polynucleotide comprising a nucleic acid that encodes a TRAIL- CDI37L, CD137L-TRAIL, TRAIL-OX40L or OX40L-TRAIL, fusion polypeptide subunit, can be at least about 80%, or 90%, or about 95%, or at least 96%, frequently at least 97%, 98%, or 99% identical to SEQ ID NOs: 9-14 or to at least one subsequence thereto. Alignment for purposes of determining percent homology (i.e., sequence similarity) or percent identity can be achieved in various ways that are within the skill in the art, for instance, using publicly or proprietary algorithms. For instance, sequence similarity can be determined using pairwise alignment methods, e.g, BLAST, BLAST-2, ALIGN, or ALIGN-2 or multiple sequence alignment methods such as Megalign (DNASTAR), ClustalW or T-Coffee software. Those skilled in the art can determine appropriate scoring functions, e.g., gap penalties or scoring matrices for measuring alignment, including any algorithms needed to achieve optimal alignment quality over the full-length of the sequences being compared. In addition, sequence alignment can be achieved using structural alignment methods (e.g., methods using secondary or tertiary structure information to align two or more sequences), or hybrid methods combining sequence, structural, and phylogenetic information to identify and optimally align candidate protein sequences.

[0127] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403 (1990)) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, Md.) and on the internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. A description of how to determine sequence identity using this program is available on the NCBI website on the internet.

[0128] Thus, a polypeptide sequence or a nucleic acid that encodes this sequence that is substantially identical, or substantially similar to SEQ ID NOs: 9 -14 is encompassed within the present disclosure. A sequence is substantially identical to SEQ ID NOs: 9-14 if the sequence is identical with at least a subsequence of the reference sequence (e.g., SEQ ID NO: 2, 5 and/or 7). Such polypeptides can include, e.g., insertions, deletions, and substitutions relative to SEQ ID NOs: 9-14. For example, nucleic acids encoding these polypeptides can be at least about 70%, 80%, 90%, 95%, 96%, 97%, 98% or even 99% identical to a reference nucleic acid, or encode a polypeptide at least about 80%, 90%, 95%, 96%, 97%, 98% or even 99% identical to the reference polypeptide sequence, e.g., SEQ ID NOs: 9-14.

[0129] Additionally, a polynucleotide comprising a nucleic acid encoding a TRAIL and either one or both a CD137L or an OX40L fusion polypeptide subunits can also include polynucleotide sequences, such as expression regulatory sequences and/or vector sequences that facilitate the expression or replication of the nucleic acids. Similarly, a polynucleotide comprising a nucleic acid encoding a TRAIL and either one or both CD137L and OX40L fusion polypeptide subunits, can include additional coding sequences that confer functional attributes on the encoded polypeptide. Such sequences include secretory signal sequences and membrane localization signals.

[0130] A polynucleotide comprising a nucleic acid encoding a TRAIL and either one or both a CD137L and/or an OX40L fusion polypeptide subunit, can be introduced into a vector by conventional techniques. In embodiments, the vector is a eukaryotic expression vector. Accordingly, the disclosure provides a vector comprising a polynucleotide as provided herein. An expression vector is designed to permit the transcription of the polynucleotide sequence encoding a TRAIL and either one or both a CD137L or an OX40L fusion polypeptide subunit, in cells by providing regulatory sequences that initiate and enhance the transcription of the cDNA and ensure its proper splicing and polyadenylation. Numerous expression vectors are known to those of skill in the art, and are available commercially, or can be assembled from individual components according to conventional molecular biology procedures.

[0131] The choice of expression control sequence and expression vector will depend upon the choice of host cell. A wide variety of expression host/vector combinations can be employed. Useful expression vectors for eukaryotic hosts, include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from E. coli, including pCR 1, pBR322, pMB9 and their derivatives, wider host range plasmids, such as M13 and fdamentous single-stranded DNA phages.

[0132] Suitable host cells for expression of a TRAIL and either one or both a CD137L or an OX40L fusion polypeptide subunit, or a trimeric protein as provided herein, are higher eukaryotic cells under the control of appropriate promoters. Higher eukaryotic cells include established cell lines of mammalian origin as described below. Cell-free translation systems could also be employed.

[0133] Also provided is a host cell comprising a polynucleotide or vector as provided herein. Various mammalian or insect cell culture systems can be advantageously employed to express polypeptide subunits, trimeric proteins provided herein. Expression of recombinant proteins in mammalian cells can be performed because such proteins are generally correctly folded, appropriately modified and completely functional. Examples of suitable mammalian host cell lines include HEK-293 and HEK-293T, the COS-7 lines of monkey kidney cells, described by Gluzman (Cell 23: 175, 1981), and other cell lines including, for example, L cells, C127, 3T3, Chinese hamster ovary (CHO), HeLa and BHK cell lines. Mammalian expression vectors can comprise nontranscribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5' or 3' flanking nontranscribed sequences, and 5' or 3' nontranslated sequences, such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and transcriptional termination sequences. Baculovirus systems for production of heterologous proteins in insect cells are reviewed by Luckow and Summers, BioTechnology 6:47 (1988).

[0134] The expression and purification of proteins, such as TRAIL-CD137L or TRAIL- OX40L fusion polypeptide subunit can be performed using standard laboratory techniques. Examples of such methods are discussed or referenced herein. After expression, purified proteins have many uses, including for instance functional analyses and diagnostics, as well as the prophylactic and therapeutic uses described below.

[0135] A TRAIL-CD137L or TRAIL-OX40L fusion polypeptide produced by a transformed host, can be purified according to any suitable method. Such standard methods include chromatography (e.g., ion exchange, affinity and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for protein purification. Affinity tags such as hexahistidine, maltose binding domain, influenza coat sequence and glutathione-S-transferase can be attached to the protein to allow easy purification by passage over an appropriate affinity column. Isolated proteins can also be physically characterized using such techniques as proteolysis, nuclear magnetic resonance and x-ray crystallography.

[0136] For example, supernatants from systems that secrete recombinant protein into culture media can be first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. Following the concentration step, the concentrate can be applied to a suitable purification matrix. Alternatively, an anion exchange resin can be employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) groups. The matrices can be acrylamide, agarose, dextran, cellulose or other types commonly employed in protein purification. Alternatively, a cation exchange step can be employed. Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups. Finally, one or more reversed-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify an influenza B/Yamagata virus-binding molecule. Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a homogeneous recombinant protein.

[0137] In an aspect, conjugation (for example, fusion) of ligands described herein improve the pharmacokinetics and/or biological activity of the conjugate relative to either or each of the components alone, and in many instances does so synergistically. In embodiments, conjugation (for example, fusion) of Tumor Necrosis Factor (TNF) superfamily ligand (TNFSL), for example, TRAIL, to another TNFSL, for example, CD137L or OX40L, or both improves the pharmacokinetics and/or biological activity of the conjugate relative to either or each of the components alone, and in many instances does so synergistically. In embodiments, conjugation of a trimeric or homotrimeric polypeptide to another trimeric or homotrimeric, or hexameric or homohexameric polypeptide improves the pharmacokinetics and/or biological activity of the conjugate relative to either of the components alone. In embodiments, conjugation of more than one polypeptide improves the pharmacokinetics and/or biological activity of the conjugate relative to one or more of the components alone. In embodiments, conjugation of a first trimeric polypeptide to a trimeric or hexameric polypeptide (which differs from the first) improves the pharmacokinetics and/or biological activity of the conjugate relative to one or both of the components alone. In some instances, each component of the conjugate improves the pharmacokinetics and/or biological activity of the other component.

[0138] Certain embodiments relate to conjugates that include components that derive from the TNF superfamily of ligands. In embodiment, the conjugates comprise TRAIL that is covalently linked to CD137L and/or OX40L, each of which is described in greater detail herein. In some embodiments, the TRAIL is conjugated to the N-terminus of CD137L. In some embodiments, the TRAIL is conjugated to the C-terminus of CD137L. In some embodiments, the TRAIL is conjugated to the N-terminus of OX40L. In some embodiments, the TRAIL is conjugated to the C- terminus of OX40L.

[0139] Also included are conjugates, comprising a first trimeric polypeptide that is covalently linked to a second trimeric polypeptide which differs from the first trimeric polypeptide. In some instances, TRAIL and OX40L are separated by (fused through) another trimeric or hexameric polypeptide. Examples of the hexameric or homohexameric polypeptide include, for example adiponectin or collagen-like domain thereof, which are described in greater detail herein. Adiponectin is a 244-amino acid protein composed of an amino-terminal signal peptide, a collagen- like domain at the N-terminus, and a globular domain at the C-terminus. Adiponectin self- associates into larger structures, for example, adiponectin molecules bind together via the collagen- like domain to form homotrimers, and in some instances the trimers continue to self-associate and form hexamers.

[0140] In some instances, the trimeric polypeptide is a homotrimeric polypeptide. Examples of the first trimeric or homotrimeric polypeptide include adiponectin or a collagen-like domain thereof, T4 fibritin or a trimerization domain thereof (foldon), C-propeptide of procollagen, surfactant protein A (SP-A), and mannose-binding protein A (MBP-A). As noted above, in some instances adiponectin or the collagen-liked domain thereof self-associates into trimers. Bacteriophage T4 fibritin is a triple-stranded, parallel, segmented alpha-helical coiled-coil protein. The C-terminal globular domain (foldon) of T4 fibritin is essential for correct trimerization and folding of the protein, however foldon is capable of trimerization in the absence of the coiled-coil part of fibritin (see Letarov et al., Biochemistry (Mose). 64(7):817-23, 1999). The C-propeptides of fibrillar procollagens play crucial roles in tissue growth and repair by controlling both the intracellular assembly of procollagen molecules and the extracellular assembly of collagen fibrils, and are responsible for the selective formation of homotrimers and certain heterotrimers between various procollagens (see, e.g., Bourhis et al., Nat Struct Mol Biol. 19(10): 1031-1036 (2012)). Surfactant protein A (SP-A), one of four proteins associated with pulmonary surfactant, binds with high affinity to alveolar phospholipid membranes, positioning the protein at the first line of defense against inhaled pathogens. SP-A exhibits both calcium-dependent carbohydrate binding, a characteristic of the collectin family, and specific interactions with lipid membrane components. The carbohydrate recognition domain (CRD) of SP-A forms trimeric structure with the neck domain (see, e.g., J. Biol Chem. 278(44):43254-60 (2003)). Mannose-binding proteins (MBPs) are C-type (Ca(2+)-dependent) animal lectins found in serum. They recognize cell-surface oligosaccharide structures characteristic of pathogenic bacteria and fungi, and trigger the neutralization of these organisms. The carbohydrate-recognition domain (CRD) of MBP and the neck domain that links the carboxy -terminal CRD to the collagen-like portion of the intact molecule form trimeric structures (see, e.g., Weis and Drickamer, Structure. 2(12): 1227-40 (1994)). Thus, any of the foregoing trimeric or hexametric polypeptides or fragments/domains thereof can be employed as a trimeric or hexametric polypeptide connecting TRAIL and CD137L polypeptides, or TRAIL and OX40L polypeptides.

[0141] In some embodiments, the trimeric or homotrimeric polypeptides are TNF superfamily ligands, which are described in greater detail herein.

[0142] In some embodiments, the first trimeric polypeptide is covalently linked to the N- terminus of the second trimeric polypeptide. In some embodiments, the first trimeric polypeptide is covalently linked to the C-terminus of the second trimeric polypeptide. In some embodiments, the first and second trimeric polypeptide are covalently linked to each other and a hexameric polypeptide. In some embodiments, the first and second trimeric polypeptide are each covalently linked to a hexameric polypeptide, one through N-terminus and the other through C-terminus of a hexameric polypeptide.

[0143] In some instances, the conjugate is a fusion protein, for example, where the covalent linkage between the two components of the conjugate is composed entirely of peptide bonds. In some instances, the conjugate is a non-fusion protein, for example, where the covalent linkage between the components of the conjugate comprises at least one non-peptide bond, or where the covalent linkage is chemically -reacted after each polypeptide of the conjugate has been separately produced (e.g., recombinantly produced) and optionally purified.

[0144] In some embodiments, the conjugate comprises a linker between each component of the conjugate, for example, a physiologically-stable linker. General examples of linkers include peptide linkers (for example, flexible and rigid peptide linkers) and non-peptide linkers. Exemplary linkers are described in greater detail herein. [0145] In some instances, as noted above, at least one component of the conjugate improves one or more properties of the other component of the conjugate, and in some instances, the conjugate does so synergistically. In some instances, each component improves one or more properties of the other component of the conjugate. In some instances, the conjugate has one or more improved properties relative to one, two or all three of the components alone. Exemplary properties include physical and/or pharmacokinetic properties such as protein stability, solubility, serum half-life, bioavailability, exposure, and clearance. Also included are biological properties or activities.

[0146] In specific embodiments, the conjugate comprises a TNF superfamily member ligand (for example, TRAIL, TNF-a, FasL) that is covalently linked to another TNF superfamily ligand such as OX40L or CD 137L (4- 1BBL), or CD40L and the conjugate has improved pharmacokinetic, physical, and/or biological properties relative to each component alone. As noted above, exemplary pharmacokinetic and physical properties include increased stability, increased serum half-life, increased bioavailability, increased exposure, and decreased clearance. In some instances, the conjugate has increased stability and/or serum half-life relative to TRAIL alone, CD137L and/or OX40L alone. In particular instances, the stability and/or serum half-life of the conjugate is increased by about or at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000% or more relative to TRAIL alone and/or OX40L alone and/or CD137L alone.

[0147] In some instances, the conjugate has increased biological activity relative to TRAIL alone, CD137L alone and/or OX40L alone. In particular instances, the biological activity of the conjugate is increased by about or at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000% or more relative to TRAIL alone, CD137L alone and/or OX40L alone. In some instances, the increase in biological activity is a synergistic increase relative to TRAIL alone and/or CD137L or OX40L alone. In some instances, the increase in biological activity is an additive increase relative to TRAIL alone and/or CD137L or OX40L alone. In particular embodiments, the biological activity is induction of cell death or apoptosis in cancer cells and/or activation of immune cells such as T cells.

[0148] In specific instances, the conjugate has increased (for example, by about at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000% or more) or synergistically-increased tumor cell -killing activity relative to TRAIL alone and/or CD137L or OX40L alone. In particular instances, the cancer cells are selected from one or more of breast cancer cells, colon cancer cells, non-small lung cell cancer (NSCLC) cells, multiple myeloma, ovarian cancer cells, pancreatic cancer cells, renal cancer cells, Burkitt's Lymphoma cells, glioblastoma cancer cells, leukemic cells, melanoma cancer cells, prostate cancer cells, and hepatocellular carcinoma cells.

[0149] In some embodiments, the conjugate comprises a trimeric (for example, homotrimeric) polypeptide that is covalently linked to another trimeric (for example, homotrimeric) polypeptide, and in some instances two different trimeric polypeptides are linked through another trimeric or a hexameric polypeptide. In some instances, the conjugate has increased physical, pharmacokinetic, and/or biological properties relative to one or each of the components alone. In specific instances, the conjugate has increased stability and/or serum half-life relative to one or both the components alone, for example, where the stability and/or serum half-life of the conjugate is increased by about at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000% or more relative to one or both the components alone. In some instances, the conjugate has increased biological activity relative to one or both the components alone, for example, where the biological activity of the conjugate is increased by about at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000% or more relative to one or both the components alone.

[0150] The individual components of the conjugates are described in greater detail below.

TNF Superfamily Ligands

[0151] Certain conjugates comprise one or more TNF superfamily ligands, also referred to as TNF superfamily ligand polypeptides. The Tumor Necrosis Factor receptor superfamily (TNFRSF) is a protein superfamily of cytokine receptors characterized by the ability to bind tumor necrosis factors (TNFs) via an extracellular cysteine-rich domain. With the exception of nerve growth factor (NGF), all TNFs are homologous to the archetypal TNF-a. TNF receptors are primarily involved in apoptosis and inflammation, but also regulate other signal transduction pathways, such as cell proliferation, survival, and differentiation. The term death receptor refers to those members of the TNF receptor superfamily that contain a death domain, examples of which include TNFR1, the Fas receptor, Death Receptor 4 (DR4), and Death Receptor 5 (DR5).

[0152] An illustrative list of TNF superfamily receptors and their corresponding ligands is provided in Table T1 below.

[0153] Thus, in certain embodiments, one, two or three components of the fusion polypeptides is the TNF superfamily ligand selected from a ligand polypeptide in Table T1. In certain embodiments, the TNF superfamily ligand is a human polypeptide ligand selected from Table T1. [0154] In some embodiments, the TNF superfamily ligand is a trimeric or homotrimeric polypeptide. As noted above, according to one non-limiting theory the trimeric or homotrimeric TNF superfamily ligand component of the conjugate can stabilize the other trimeric or homotrimeric component of the conjugate. In certain embodiments, the TNF superfamily ligand is a trimeric or homotrimeric polypeptide ligand selected from Table T1. [0155] In some embodiments, the TNF superfamily ligand induces apoptosis in cancer cells, for example, by binding to a death domain or death receptor of a TNF superfamily receptor. Thus, in some embodiments, TNF superfamily ligand (e.g., trimeric or homotrimeric ligand) binds to at least one TNF death receptor, or a TNF superfamily receptor that contains at least one death domain. Examples of TNF superfamily death receptors include TNFR1, Fas receptor, DR4, and DR5. Particular examples of death receptor ligands include TRAIL, TNF-a, and FasL. Thus, in certain embodiments, one TNF superfamily ligand component of the conjugate is selected from one or more of TRAIL, TNF-a, and FasL, optionally a human TRAIL, human TNF-a, or human FasL.

[0156] The amino acid sequences of human TRAIL, human TNF-a, human FasL, OX40L, CD40L and CD137L(4-1BBL) are provided in Table T2 below.

[0157] In embodiments, in the CD40L full length sequence, the start amino acid can be amino acid 51, amino acid 113, or amino acid 116. In embodiments, in the CD137L sequence, the start amino acid can be within its extracellular domain, e.g., amino acid 52, amino acid 71, amino acid 80, or amino acid 85. In embodiments, the start amino acid can be at other amino acids in the extracellular domain.

[0158] In some embodiments, the TNF superfamily ligand component of the conjugate comprises, consists, or consists essentially of an amino acid sequence selected from Table T2, or an active variant or fragment thereof. Particular examples of variants and fragments comprise, consist, or consist essentially of an amino acid sequence that is at least 80%, 95%, 90%, 95%, 96%,

97%, 98%, or 99% identical to a sequence selected from Table T2. Additional examples of active polypeptide “variants” and “fragments” are described elsewhere herein.

[0159] In specific embodiments, one TNF superfamily ligand component of the conjugate is a human TNF -related apoptosis-inducing ligand (TRAIL) polypeptide, or a variant or fragment thereof. TRAIL is a cytokine that is produced and secreted by most normal tissue cells. It causes apoptosis in tumor cells, for example, by binding to certain death receptors. The predicted 281 amino acid TRAIL protein has the characteristic structure of a type II membrane protein, with a single internal hydrophobic domain and no signal sequence. The extracellular C-terminal domain of TRAIL shares 22 to 28% identity with the C-terminal domains of other TNF family members. Formation of a complex between TRAIL and its signaling receptors, DR4 and DR5, triggers apoptosis by inducing the oligomerization of intracellular death domains.

[0160] In certain embodiments, the TRAIL component of the conjugate comprises, consists, or consists essentially of a TRAIL sequence from Table T2, or a variant or fragment thereof. Specific examples of TRAIL variants include those having any one or more of the following substitutions: S96C, S 101C, S 111C, R170C, and K179C. In some embodiments, the TRAIL variant has a set of amino acid substitutions at the residue position selected from one or more of Y189Q, R191K, Q193R; H264R, I266L, D267Q; Y189Q, R191K, Q193R; and Y189Q, R191K, Q193R, I266L (see U.S. Application Nos. 2013/0165383; and 2012/0165267, incorporated by reference). Particular examples of TRAIL fragments include residues 114-281 (extracellular domain, SEQ ID NO: 2), residues 95-281, residues 92-281, residues 91-281, residues 41-281, residues 39-281, residues 15-281, residues 119-281, and residues 1-281 of the full-length sequence (SEQ ID NO: 1). Additional examples of polypeptide “variants” and “fragments” are described elsewhere herein. [0161] TRAIL can be combined with any one or more of the TNF superfamily ligands described herein, to form a conjugate, for example, a fusion protein.

[0162] In specific embodiments, one component of the fusion protein, e.g., TRAIL, induces cancer cell apoptotic death, while another component, e.g., CD137L or OX40L activates and redirects immune response towards cancer cells that can bind the first component (e.g., TRAIL). In some embodiments, one component binds a cancer cells and another component binds an immune cell, e.g., an activated T lymphocyte, which leads to receptor clustering on one or both cell types (a cancer cell and/or an immune cell).

Linkers

[0163] In an aspect, certain conjugates comprise one or more linker groups. The term “linkage,” “linker,” “linker moiety,” or “L” is used herein to refer to a linker. In embodiments, the linker can be used to separate one polypeptide component of a conjugate from another polypeptide component. In embodiments, the linker separates components of the ligands that make up the conjugates. In embodiments, the linker separates components of conjugates that are made up of ligands that derive from the TNF superfamily of ligands. In embodiments, the linker separates TRAIL from CD137L and/or OX40L. The linker may be physiologically stable or may include a releasable linker such as a labile linker or an enzymatically degradable linker (e.g., proteolytically cleavable linkers). In certain aspects, the linker is a peptide linker. In some aspects, the linker is a non-peptide linker or non-proteinaceous linker.

[0164] Certain embodiments comprise one or more peptide linkers, for example, two (2) linkers, three (3) linkers, four (4) linkers, five (5) linkers, or more. Such a peptide linker sequence can be incorporated into a conjugate, for example, a fusion polypeptide, using standard techniques in the art.

[0165] Certain peptide linker sequences may be chosen based on the following exemplary factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; (3) their physiological stability; and (4) the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes, or other features. See, e.g., George and Heringa, J Protein Eng. 15:871-879 (2002). In some embodiments, the peptide linker is a rigid linker. In some embodiments, the peptide linker is a flexible linker. In particular embodiments, flexible linkers can be rationally designed using a computer program capable of modeling the peptides themselves (see, for e.g. : Desjarlais & Berg, PNAS. 90:2256-2260 (1993); and PNAS 91: 11099-11103 (1994)) or by phage display methods.

[0166] In some embodiments, the peptide linker sequence is from 1 to about 200 amino acids in length. In some embodiments, the linker is greater than 200 amino acids in length, for example 210 amino acids, 220 amino acids, 230 amino acids, 240 amino acids 250 amino acids, 260 amino acids, 270 amino acids, 280 amino acids, 290 amino acids, 300 amino acids, or longer. Exemplary linkers can have an overall amino acid length of about 1-200 amino acids, 1-150 amino acids, 1- 100 amino acids, 1-90 amino acids, 1-80 amino acids, 1-70 amino acids, 1-60 amino acids, 1-50 amino acids, 1-40 amino acids, 1-30 amino acids, 1-20 amino acids, 1-10 amino acids, 1-5 amino acids, 1-4 amino acids, 1-3 amino acids, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 ,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 or more amino acids.

[0167] A peptide linker may employ any one or more naturally-occurring amino acids, non- naturally occurring amino acid(s), amino acid analogs, and/or amino acid mimetics as described elsewhere herein and known in the art. Certain amino acid sequences which may be usefully employed as linkers include those disclosed in Maratea et al., Gene 40:39-46 (1985); Murphy et al., PNAS USA. 83:8258-8262 (1986); U.S. Pat. No. 4,935,233 and U.S. Pat. No. 4,751,180. Particular peptide linker sequences contain Gly, Ser, and/or Asn residues. Other near neutral amino acids, such as Thr and Ala may also be employed in the peptide linker sequence, if desired. [0168] Certain exemplary peptide linkers are provided in Table L1 below.

Table L1. Linker Sequences

[0169] Thus, in certain embodiments, a conjugate, for example, a fusion polypeptide, comprises one or more peptide linkers selected from Table L1.

[0170] In some embodiments, for example, in non-fusion or chemically-linked conjugates, the linker is a non-peptide linker. For example, in some embodiments the linker is an organic moiety constructed to contain an alkyl, or aryl backbone, and contains an amide, ether, ester, hydrazone, disulfide linkage or any combination thereof. Linkages containing amino acid, ether and amide bound components are stable under conditions of physiological pH, normally 7.4 in serum. Also included are linkages that contain esters or hydrazones and are stable at serum pH.

[0171] In some instances, a linker includes a spacer that increases the distance between the two joined atoms. A spacer may further add flexibility and/or length to the linker. Spacers may include, but are not be limited to, alkyl groups, alkenyl groups, alkynyl groups, aryl groups, aralkyl groups, aralkenyl groups, aralkynyl groups; each of which can contain one or more heteroatoms, heterocycles, amino acids, nucleotides, and saccharides.

[0172] In some embodiments, the linker is about 1 to about 30 atoms in length, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 0, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 atoms in length, including all ranges in between. In certain embodiments, the linker is about 1 to 30 atoms in length with carbon chain atoms which may be substituted by heteroatoms independently selected from the group consisting of O, N. or S. In some embodiments, from 1- 4 or from 5 to 15 of the C atoms are substituted with a heteroatom independently selected from O, N, S.

[0173] In certain embodiments, the linker comprises or consists of a structure selected from the following: —O— , — NH— , — S— , — C(O)— , C(O)— NH, NH— C(O)— NH, O— C(O)— NH, — C(S)— , — CH₂— , — CH₂— CH₂— , — CH₂— CH₂— CH₂— , — CH₂— CH₂— CH₂— CH₂— , — O— CH₂— , — CH₂ —O— , —O— CH₂— CH₂— , — CH₂—O— CH₂— , — CH₂— CH₂—O— , —O— CH₂— CH₂— CH₂— , — CH₂—O— CH₂— CH₂— , — CH₂— CH₂—O— CH₂— , — CH₂— CH₂— CH₂—O— , —O— CH₂— CH₂— CH₂— CH₂— , — CH₂—O— CH₂— CH₂— CH₂— , —CH₂— CH₂ —O— CH₂— CH₂— , — CH₂— CH₂— CH₂—O— CH₂— , —CH₂—CH₂—CH₂—CH₂—O— — C(O)— NH— CH₂— , — C(O)— NH— CH₂— CH₂— , — CH₂— C(O)— NH— CH₂— , — CH₂— CH₂— C(O)— NH— , — C(O)— NH— CH₂— CH₂— CH₂— , — CH₂— C(O)— NH— CH₂— CH₂— , — CH₂— CH₂— C(O)— NH— CH₂— , — CH₂— CH₂— CH₂— C(O)— NH— , —C(O)—NH— CH₂— CH₂— CH₂— CH₂— , — CH₂— C(O)— NH— CH₂— CH₂— CH₂— , — CH₂— CH₂— C(O)— NH— CH₂— CH₂— , — CH₂— CH₂— CH₂— C(O)— NH— CH₂— , — CH₂— CH₂— CH₂— C(O)— NH— CH₂— CH₂— , — CH₂— CH₂— CH₂— CH₂— C(O)— NH — , — NH— C(O)— CH₂— , — CH₂— NH— C(O)— CH₂— , — CH₂— CH₂— NH— C(O)— CH₂— , — NH— C(O)— CH₂— CH₂— , — CH₂— NH— C(O)— CH₂— CH₂, — CH₂— CH₂— NH— C(O)— CH₂— CH₂, — C(O)— NH— CH₂— , — C(O)— NH— CH₂— CH₂— , —O— C(O)— NH— CH₂— , —O— C(O)— NH— CH₂— CH₂— , — NH— CH₂— , — NH— CH₂— CH₂— , — CH₂— NH— CH₂— , — CH₂— CH₂— NH— CH₂— , — C(O)— CH₂— , — C(O)— CH₂— CH₂— , — CH₂— C(O)— CH₂— , — CH₂— CH₂— C(O)— CH₂— , — CH₂— CH₂— C(O)— CH₂— CH₂— , — CH₂— CH₂— C(O)— , —CH₂—CH₂— CH₂— C(O)— NH— CH₂— CH₂— NH— , — CH₂— CH₂— CH₂— C(O)— NH— CH₂— CH₂— NH— C(O)— — CH₂— CH₂— CH₂— C(O)— NH— CH₂— CH₂— NH— C(O)— CH₂— , bivalent cycloalkyl group, — N(R⁶) — , R⁶ is H or an organic radical selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl and substituted aryl.

[0174] In some embodiments, the linker is a stable linker. In some embodiments, the stable linker is selected from the group consisting of: succinimide, propionic acid, carboxymethylate linkages, ethers, carbamates, amides, amines, carbamides, imides, aliphatic C-C bonds, and thio ethers. In some embodiments, the linker group is hydrophilic, for instance, to enhance the solubility of the conjugate in body fluids. In certain embodiments, the foregoing linkers are optional.

Polypeptide Variants

[0175] Certain embodiments include “variants” and “fragments” of the reference sequences described herein, whether described by name or by reference to a Table or sequence identifier. Examples include any of the TNF superfamily ligand polypeptides, and fusion polypeptides described herein. A “variant” sequence refers to a polypeptide or polynucleotide sequence that differs from a reference sequence by one or more substitutions, deletions (e.g., truncations), additions, and/or insertions. Variant polypeptides are biologically active, that is, they continue to possess the enzymatic or binding activity of a reference polypeptide. Such variants may result from, for example, genetic polymorphism and/or from human manipulation.

[0176] In many instances, a biologically active variant will contain one or more conservative substitutions. A “conservative substitution” is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. As described above, modifications may be made in the structure of the polynucleotides and polypeptides of the present disclosure and still obtain a functional molecule that encodes a variant or derivative polypeptide with desirable characteristics. When it is desired to alter the amino acid sequence of a polypeptide to create an equivalent, or even an improved, variant or portion of a polypeptide described herein, one skilled in the art will typically change one or more of the codons of the encoding DNA sequence.

[0177] For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity. Since it is the interactive capacity and nature of a protein that defines that protein’s biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated that various changes may be made in the peptide sequences of the disclosed compositions, or corresponding DNA sequences which encode said peptides without appreciable loss of their utility. [0178] In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte & Doolittle (1982), incorporated herein by reference). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte & Doolittle (1982)). These values are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (- 0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (- 3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those within ± 1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0179] It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Patent 4,554,101 (specifically incorporated herein by reference in its entirety), states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. As detailed in U. S. Patent 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0180] As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

[0181] Amino acid substitutions may further be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine. Other groups of amino acids that may represent conservative changes include: (1) ala, pro, gly, glu, asp, gin, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. [0182] A variant may also, or alternatively, contain non-conservative changes. In a preferred embodiment, variant polypeptides differ from a native or reference sequence by substitution, deletion or addition of fewer than about 10, 9, 8, 7, 6, 5, 4, 3, 2 amino acids, or even 1 amino acid. Variants may also (or alternatively) be modified by, for example, the deletion or addition of amino acids that have minimal influence on the immunogenicity, secondary structure, enzymatic activity, and/or hydropathic nature of the polypeptide.

[0183] In certain embodiments, a polypeptide sequence is about, at least about, or up to about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280,

290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480,

490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680,

690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 800, 810, 820, 830, 840, 850, 860, 870,

880, 890, 900, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 or more contiguous amino acids in length, including all integers in between, and which may comprise all or a portion of a reference sequence (see, e.g., Tables or an informal or formal Sequence Listing).

[0184] In some embodiments, a polypeptide sequence consists of about or no more than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280,

290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480,

490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680,

690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800. 800, 810, 820, 830, 840, 850, 860, 870,

880, 890, 900, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 or more contiguous amino acids, including all integers in between, and which may comprise all or a portion of a reference sequence (see, e.g., Tables or an informal or formal Sequence Listing).

[0185] In certain embodiments, a polypeptide sequence is about 10-1000, 10-900, 10-800, 10- 700, 10-600, 10-500, 10-400, 10-300, 10-200, 10-100, 10-50, 10-40, 10-30, 10-20, 20-1000, 20- 900, 20-800, 20-700, 20-600, 20-500, 20-400, 20-300, 20-200, 20-100, 20-50, 20-40, 20-30, 50- 1000, 50-900, 50-800, 50-700, 50-600, 50-500, 50-400, 50-300, 50-200, 50-100, 100-1000, 100- 900, 100-800, 100-700, 100-600, 100-500, 100-400, 100-300, 100-200, 200-1000, 200-900, 200- 800, 200-700, 200-600, 200-500, 200-400, or 200-300 contiguous amino acids, including all ranges in between, and comprises all or a portion of a reference sequence. In certain embodiments, the C- terminal or N-terminal region of any reference polypeptide may be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or 800 or more amino acids, or by about 10-50, 20-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400- 450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, 750-800 or more amino acids, including all integers and ranges in between (e.g., 101, 102, 103, 104, 105), so long as the truncated polypeptide retains the binding properties and/or activity of the reference polypeptide. Typically, the biologically-active fragment has no less than about 1%, about 5%, about 10%, about 25%, or about 50% of an activity of the biologically-active reference polypeptide from which it is derived. [0186] In certain instances, variants will display at least about 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% similarity or sequence identity or sequence homology to a reference polypeptide sequence. Moreover, sequences differing from the native or parent sequences by the addition (e.g., C-terminal addition, N-terminal addition, both), deletion, truncation, insertion, or substitution (e.g., conservative substitution) of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids (including all integers and ranges in between) but which retain the properties or activities of a parent or reference polypeptide sequence are contemplated.

[0187] In some embodiments, variant polypeptides differ from reference sequence by at least one but by less than 50, 40, 30, 20, 15, 10, 8, 6, 5, 4, 3 or 2 amino acid residue(s). In certain embodiments, variant polypeptides differ from a reference sequence by at least 1% but less than 20%, 15%, 10% or 5% of the residues. (If this comparison requires alignment, the sequences should be aligned for maximum similarity. “Looped” out sequences from deletions or insertions, or mismatches, are considered differences.) [0188] Calculations of sequence similarity or sequence identity between sequences (the terms are used interchangeably herein) are performed as follows. To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g. , gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In certain embodiments, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.

[0189] The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0190] The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch, (J. Mol. Biol. 48: 444-453, 1970) algorithm which has been incorporated into the GAP program in the GCG software package, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package, using aNWSgapdn CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used unless otherwise specified) are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

[0191] The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller (Cabios. 4: 11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

[0192] The nucleic acid and protein sequences described herein can be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al., (J. Mol. Biol, 215: 403-10 (1990)). BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to nucleic acid molecules described herein. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (Nucleic Acids Res. 25: 3389-3402 (1997)). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0193] In some embodiments, as noted above, polynucleotides and/or polypeptides can be evaluated using a BLAST alignment tool. A local alignment consists simply of a pair of sequence segments, one from each of the sequences being compared. A modification of Smith- Waterman or Sellers algorithms will find all segment pairs whose scores cannot be improved by extension or trimming, called high-scoring segment pairs (HSPs). The results of the BLAST alignments include statistical measures to indicate the likelihood that the BLAST score can be expected from chance alone.

[0194] The raw score, S, is calculated from the number of gaps and substitutions associated with each aligned sequence wherein higher similarity scores indicate a more significant alignment. Substitution scores are given by a look-up table (see PAM, BLOSUM).

[0195] Gap scores are typically calculated as the sum of G, the gap opening penalty and L, the gap extension penalty. For a gap of length n, the gap cost would be G+Ln. The choice of gap costs, G and L is empirical, but it is customary to choose a high value for G (10-15), e.g, 11, and a low value for L (1-2) e.g., 1.

[0196] The bit score, S’, is derived from the raw alignment score S in which the statistical properties of the scoring system used have been taken into account. Bit scores are normalized with respect to the scoring system, therefore they can be used to compare alignment scores from different searches. The terms “bit score” and “similarity score” are used interchangeably. The bit score gives an indication of how good the alignment is; the higher the score, the better the alignment.

[0197] The E-Value, or expected value, describes the likelihood that a sequence with a similar score will occur in the database by chance. It is a prediction of the number of different alignments with scores equivalent to or better than S that are expected to occur in a database search by chance. The smaller the E-Value, the more significant the alignment. For example, an alignment having an E value of e ¹¹⁷ means that a sequence with a similar score is very unlikely to occur simply by chance. Additionally, the expected score for aligning a random pair of amino acids is required to be negative, otherwise long alignments would tend to have high score independently of whether the segments aligned were related. Additionally, the BLAST algorithm uses an appropriate substitution matrix, nucleotide or amino acid and for gapped alignments uses gap creation and extension penalties. For example, BLAST alignment and comparison of polypeptide sequences are typically done using the BLOSUM62 matrix, a gap existence penalty of 11 and a gap extension penalty of 1.

[0198] In some embodiments, sequence similarity scores are reported from BLAST analyses done using the BLOSUM62 matrix, a gap existence penalty of 11 and a gap extension penalty of 1.

[0199] In a particular embodiment, sequence identity/similarity scores provided herein refer to the value obtained using GAP Version 10 (GCG, Accelrys, San Diego, Calif.) using the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino acid sequence using GAP Weight of 8 and Length Weight of 2, and the BLOSUM62 scoring matrix (Henikoff and Henikoff, PNAS USA. 89: 10915-10919 (1992)). GAP uses the algorithm of Needleman and Wunsch (J Mol Biol. 48:443-453 (1970)) to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps.

[0200] In particular embodiments, the variant polypeptide comprises an amino acid sequence that can be optimally aligned with a reference polypeptide sequence (see, e.g., Sequence Listing) to generate a BLAST bit scores or sequence similarity scores of at least about 50, 60, 70, 80, 90, 100, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280,

290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480,

490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680,

690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880,

890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, or more, including all integers and ranges in between, wherein the BLAST alignment used the BLOSUM62 matrix, a gap existence penalty of 11, and a gap extension penalty of 1.

[0201] As noted above, a reference polypeptide may be altered in various ways including amino acid substitutions, deletions, truncations, additions, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of a reference polypeptide can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel (PNAS USA. 82: 488-492 (1985)); Kunkel et al., (Methods in Enzymol. 154: 367-382 (1987)), U.S. Pat. No. 4,873,192, Watson, J. D. et al., (“Molecular Biology of the Gene,” Fourth Edition, Benjamin/Cummings, Menlo Park, Calif.(1987)) and the references cited therein. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.). [0202] Methods for screening gene products of combinatorial libraries made by such modifications, and for screening cDNA libraries for gene products having a selected property are known in the art. Such methods are adaptable for rapid screening of the gene libraries generated by combinatorial mutagenesis of reference polypeptides. As one example, recursive ensemble mutagenesis (REM), a technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify polypeptide variants (Arkin and Yourvan, PNAS USA 89: 7811-7815 (1992); Delgrave et al., Protein Engineering. 6: 327-331 (1993)).

Polypeptide Modifications

[0203] Certain embodiments include conjugates that comprise at least one “modifying agent,” examples of which included but are not limited to macromolecule polymers, proteins, peptides, polysaccharides, and other compounds. In embodiments, the modifying agent is attached to a component of any of the conjugates described herein. In some instances, the modifying agent is attached to the TRAIL component of a conjugate, or CD137L component of a conjugate, or OX40L component of a conjugate, or all components simultaneously. The conjugate and the modifying agent may be linked by either covalent bonds or non-covalent interaction to form a stable conjugate or a stable composition to achieve a desired effect. In certain embodiments, the modified conjugate retains the biological activity of a corresponding unmodified conjugate (e.g., of the same or similar sequence) and has a longer half-life in vivo, and lower antigenicity than the corresponding unmodified conjugate. In certain embodiments, the modified conjugate retains at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the biological activity of the corresponding unmodified conjugate. In embodiments, the modified conjugate retains biological activity sufficient for therapeutic use.

[0204] In some embodiments, the modifying agent is a polymer or a protein or a fragment thereof that is biocompatible and increases the half-life of the conjugate in blood. The modifying agent can be either chemically coupled to the conjugate or a component thereof or where applicable, linked to the conjugate or a component thereof via fusion protein expression.

[0205] Macromolecule polymers may include a non-peptide macromolecule polymer, which in certain embodiments, may have its own bioactivity. Suitable polymers include, but are not limited to, polyenol compounds, polyether compounds, polyvinylpyrrolidone, poly amino acids, copolymer of divinyl ether and maleic anhydride, N-(2-hydroxypropyl)-methacrylamide, polysaccharide, polyoxyethylated polyol, heparin or its fragment, poly-alkyl-ethylene glycol and its derivatives, copolymers of poly-alkyl-ethylene glycol and its derivatives, poly(vinyl ethyl ether), a,P-Poly[(2-hydroxyethyl)-DL-aspartamide], polycarboxylates, poly oxyethylene- oxymethylenes, polyacryloyl morpholines, copolymer of amino compounds and oxyolefin, poly hyaluronic acid, polyoxiranes, copolymer of ethanedioic acid and malonic acid, poly (1,3- dioxolane), ethylene and maleic hydrazide copolymer, poly sialic acid, cyclodextrin, etc. In certain embodiments, the polymer is polyethylene glycol.

[0206] The polyenol compounds as used herein include, but are not limited to, polyethylene glycol (including monomethoxy polyethylene glycol, monohydroxyl polyethylene glycol), polyvinyl alcohol, polyallyl alcohol, polybutenol and the like, and their derivatives, such as lipids. [0207] The polyether compounds include, but are not limited to poly alkylene glycol (HO((CH₂)xO)nH), polypropylene glycol, polyoxyrehylene (HO((CH₂)2O)nH), polyvinyl alcohol ((CH₂CHOH)n).

[0208] Poly amino acids include, but are not limited to, polymers of one type of amino acid or copolymers of two or more types of amino acids, for example, polyalanine or polylysine, or block co-polymers thereof.

[0209] Polysaccharides include but are not limited to, glucosan and its derivatives, for example dextran sulfate, cellulose and its derivatives (including methyl cellulose and carboxymethyl cellulose), starch and its derivatives, polysucrose, etc.

[0210] Polynucleotides. Expression Vectors, and Host Cells

[0211] Certain embodiments relate to polynucleotides that encode a conjugate, for example, a fusion polypeptide, as described herein. Also included are polynucleotides that encode any one or more of the individual TRAIL polypeptide described herein, alone or in combination with polynucleotide that encode any one or more of the individual CD137L, alone or in combination with polynucleotide that encode any one or more of the individual OX40L or trimeric polypeptides described herein. Thus, certain embodiments include a polynucleotide that encodes any one or more of the individual TRAIL polypeptides, any one or more of the individual CD 137L, any one or more of the individual OX40L, or a fusion polypeptide described herein, for example, a fusion polypeptide that comprise TRAIL and CD137L or OX40L polypeptides.

[0212] Among other uses, these and related embodiments may be utilized to recombinantly produce a fusion polypeptide or an individual component thereof (TRAIL, OX40L, CD137L, trimeric polypeptide) in a host cell. It will be appreciated by those of ordinary skill in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode a polypeptide described herein. Some of these polynucleotides may bear minimal homology to the nucleotide sequence of any native gene. Nonetheless, polynucleotides that vary due to differences in codon usage are specifically contemplated, for example, polynucleotides that are optimized for human, yeast or bacterial codon selection. [0213] As will be recognized by the skilled artisan, polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA or synthetic) or RNA molecules. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide of the present disclosure, and a polynucleotide may, but need not, be linked to other molecules and/or support materials.

[0214] Polynucleotides may comprise a native sequence (i.e., an endogenous sequence that encodes a fusion polypeptide or a component thereof) or may comprise a variant, or a biological functional equivalent of such a sequence. Polynucleotide variants may contain one or more substitutions, additions, deletions and/or insertions, as described herein, preferably such that the activity of the variant polypeptide is not substantially diminished relative to the unmodified polypeptide.

[0215] Additional coding or non-coding sequences may, but need not, be present within a polynucleotide, and a polynucleotide may, but need not, be linked to other molecules and/or support materials. Hence, the polynucleotides, regardless of the length of the coding sequence itself, may be combined with other DNA or RNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably.

[0216] The polynucleotide sequences may also be of mixed genomic, cDNA, RNA, and that of synthetic origin. For example, a genomic or cDNA sequence encoding a leader peptide may be joined to a genomic or cDNA sequence encoding the polypeptide, after which the DNA or RNA sequence may be modified at a site by inserting synthetic oligonucleotides encoding the desired amino acid sequence for homologous recombination in accordance with well-known procedures or preferably generating the desired sequence by PCR using suitable oligonucleotides. In some embodiments a signal sequence can be included before the coding sequence. This sequence encodes a signal peptide N-terminal to the coding sequence which communicates to the host cell to direct the polypeptide to the cell surface or secrete the polypeptide into the media. Typically, the signal peptide is clipped off by the host cell before the protein leaves the cell. Signal peptides can be found in variety of proteins in prokaryotes and eukaryotes.

[0217] One or multiple polynucleotides can encode the TRAIL, CD137L, OX40L or another TNF superfamily ligand, trimeric, and/or fusion polypeptides described herein. Moreover, the polynucleotide sequence can be manipulated for various reasons. Examples include but are not limited to the incorporation of preferred codons to enhance the expression of the polynucleotide in various organisms (see generally Nakamura et al., Nuc. Acid. Res. 28:292 (2000)). In addition, silent mutations can be incorporated in order to introduce, or eliminate restriction sites, decrease the density of CpG dinucleotide motifs (see for example, Kameda et al., Biochem. Biophys. Res. Commun. 349: 1269-1277 (2006)) or reduce the ability of single stranded sequences to form stem- loop structures (see, e.g., Zuker M., Nucl. Acid Res. 31:3406-3415 (2003)). In addition, mammalian expression can be further optimized by including a Kozak consensus sequence (i.e., (a/g)cc(a/g)ccATGg) at the start codon. Kozak consensus sequences useful for this purpose are known in the art (Mantyh et al., PNAS 92: 2662-2666 (1995); Mantyh et al., Prot. Exp. & Purif. 6: 124 (1995)).

[0218] Also included are expression vectors that comprise the polynucleotides, and host cells that comprise the polynucleotides and/or expression vectors. Polypeptides and conjugates, for example, fusion polypeptides, can be produced by expressing a DNA or RNA sequence encoding the polypeptide in a suitable host cell by well-known techniques. The term “host cell” is used to refer to a cell into which has been introduced, or which is capable of having introduced into it, a nucleic acid sequence encoding one or more of the polypeptides described herein, and which further expresses or is capable of expressing a polypeptide of interest, such as a polynucleotide encoding any herein described polypeptide. The term includes the progeny of the parent cell, whether or not the progeny are identical in morphology or in genetic make-up to the original parent, so long as the selected gene is present. Host cells may be chosen for certain characteristics, for instance, the expression of a formylglycine generating enzyme (FGE) to convert a cysteine or serine residue within a sulfatase motif into a formylglycine (FGly) residue, or the expression of aminoacyl tRNA synthetase(s) that can incorporate unnatural amino acids into the polypeptide, including unnatural amino acids with an azide side-chain, alkyne side-chain, or other desired side-chain, to facilitate chemical conjugation or modification.

[0219] In some instances, a polynucleotide or expression vector comprises additional non- coding sequences. For example, the “control elements” or “regulatory sequences” present in an expression vector are non-translated regions of the vector, including enhancers, promoters, 5' and 3' untranslated regions, which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the PBLUESCRIPT phagemid (Stratagene (Millipore/Sigma), La Jolla, Calif.) or PSPORT1 plasmid (Gibco BRL (Thermo Fisher Scientific), Gaithersburg, Md.) and the like may be used. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are generally preferred. If it is necessary to generate a cell line that contains multiple copies of the sequence encoding a polypeptide, vectors based on SV40 or EBV may be advantageously used with an appropriate selectable marker. [0220] In mammalian host cells, a number of expression systems are well known in the art and commercially available. Exemplary mammalian vector systems include for example, pCEP4, pREP4, and pREP7 from Invitrogen (Thermo Fisher Scientific), the PerC6 system from Crucell, and Lentiviral based systems such as pLPl from Invitrogen, and others. For example, in cases where an adenovirus is used as an expression vector, sequences encoding a polypeptide of interest may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome may be used to obtain a viable virus which is capable of expressing the polypeptide in infected host cells (Logan & Shenk, PNAS USA. 81:3655-3659 (1984)). In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.

[0221] Examples of useful mammalian host cell lines include monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells sub-cloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44- 68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2). Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., PNAS USA. 77:4216 (1980)); and myeloma cell lines such as NSO and Sp2/0. For a review of certain mammalian host cell lines suitable for protein production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B. K.C Lo, ed., Humana Press, Totowa, N.J. (2003)), pp.255-268. Certain preferred mammalian cell expression systems include CHO and HEK293-cell based expression systems. Mammalian expression systems can utilize attached cell lines, for example, in T-flasks, roller bottles, or cell factories, or suspension cultures, for example, in IL and 5L spinners, 5L, 14L, 40L, 100L and 200L stir tank bioreactors, or 20/50L and 100/200L WAVE bioreactors, among others known in the art.

[0222] In addition, a host cell strain may be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion. Such modifications of the polypeptide include, but are not limited to, post-translational modifications such as acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation, or the insertion of non-naturally occurring amino acids (see generally US Patent Nos. 7,939,496; 7,816,320; 7,947,473; 7,883,866; 7,838,265; 7,829,310; 7,820,766; 7,820,766; 7,7737,226, 7,736,872; 7,638,299; 7,632,924; and 7,230,068). Post-translational processing which cleaves a “prepro” form of the protein may also be used to facilitate correct insertion, folding and/or function. Different host cells such as yeast, CHO, HeLa, MDCK, HEK293, and W138, in addition to bacterial cells, which have or even lack specific cellular machinery and characteristic mechanisms for such post-translational activities, may be chosen to ensure the correct modification and processing of the foreign protein.

Exemplary Methods for Conjugation

[0223] Conjugation or coupling of a first polypeptide (e.g., TRAIL, trimeric polypeptide) to a second polypeptide (e.g, CD137L or OX40L, trimeric polypeptide) or more can be carried out using standard chemical, biochemical, and/or molecular techniques. It will be apparent how to make a conjugate in light of the present disclosure using available art-recognized methodologies. In some instances, it will generally be preferred when coupling the primary components of a conjugate that the techniques employed and the resulting linking chemistries do not substantially disturb the desired functionality or activity of the individual components of the conjugate.

[0224] In certain embodiments, the conjugate is a fusion polypeptide or fusion protein. In some instances, a fusion polypeptide is expressed as a recombinant polypeptide in an expression system, as described herein and known in the art. Fusion polypeptides can contain one or multiple copies of a polypeptide sequence and may contain one or multiple copies of a polypeptide-based agent of interest, present in any desired arrangement.

[0225] For fusion proteins, DNA sequences encoding the fusion polypeptide components and optionally the peptide linker components may be assembled separately, and then ligated into an appropriate expression vector. The 3’ end of the DNA sequence encoding one polypeptide component is ligated, with or without a peptide linker, to the 5 ’ end of a DNA sequence encoding the other polypeptide component s) so that the reading frames of the sequences are in phase. The ligated DNA sequences are operably linked to suitable transcriptional or translational regulatory elements. The regulatory elements responsible for expression of DNA are located only 5’ to the DNA sequence encoding the first polypeptides. Similarly, stop codons required to end translation and transcription termination signals are only present 3’ to the DNA sequence encoding the most C -terminal polypeptide. This permits translation into a single fusion polypeptide that retains the biological activity of both component polypeptides.

[0226] Similar techniques, mainly the arrangement of regulatory elements such as promoters, stop codons, and transcription termination signals, can be applied to the recombinant production of non-fusion polypeptides, for instance, polypeptides for the production of non-fusion conjugates (e.g., chemically-coupled conjugates).

[0227] Polynucleotides and fusion polynucleotides of the disclosure can contain one or multiple copies of a nucleic acid encoding a polypeptide sequence, and/or may contain one or multiple copies of a nucleic acid encoding a polypeptide agent.

[0228] In some embodiments, a polynucleotide encoding a polypeptide and/or fusion polypeptide are introduced directly into a host cell, and the cell incubated under conditions sufficient to induce expression of the encoded polypeptide(s). The polypeptide sequences of this disclosure may be prepared using standard techniques well known to those of skill in the art in combination with the polypeptide and nucleic acid sequences provided herein.

[0229] Therefore, according to certain embodiments, there is provided a recombinant host cell that comprises a polynucleotide or a fusion polynucleotide which encodes a polypeptide or fusion polypeptide described herein. Expression of a polypeptide or a fusion polypeptide in the host cell may be achieved by culturing under appropriate conditions recombinant host cells containing the polynucleotide. Following production by expression, the polypeptide(s) may be isolated and/or purified using any suitable technique, and then used as desired. Exemplary polynucleotides, expression vectors, and host cells are described elsewhere herein.

[0230] The polypeptides, for example, fusion polypeptides, produced by a recombinant cell can be purified and characterized according to a variety of techniques known in the art. Exemplary systems for performing protein purification and analyzing protein purity include fast protein liquid chromatography (FPLC) (e.g., AKTA and Bio-Rad FPLC systems), high-performance liquid chromatography (HPLC) (e.g., Beckman and Waters HPLC). Exemplary chemistries for purification include ion exchange chromatography (e.g., Q, S), size exclusion chromatography, salt gradients, affinity purification (e.g., Ni, Co, FLAG, maltose, glutathione, protein A/G), gel filtration, reverse-phase, ceramic HYPERD® ion exchange chromatography, and hydrophobic interaction columns (HIC), among others known in the art.

[0231] In some embodiments, the conjugate is a non-fusion polypeptide, for example, a conjugate produced by chemically-linking or coupling a first polypeptide (e.g., TRAIL, trimeric polypeptide) to a second polypeptide (e.g., CD137L or OX40L, trimeric polypeptide) or more. The particular coupling chemistry employed will depend upon the structure of the polypeptides, the potential presence of multiple functional groups within the biologically active agent, the need for protection/deprotection steps, chemical stability of the agent, and the like, and will be readily determined by one skilled in the art. Illustrative coupling chemistry useful for preparing the conjugates of the disclosure can be found, for example, in Wong (1991), “Chemistry of Protein Conjugation and Crosslinking”, CRC Press, Boca Raton, Fla.; and Brinkley “A Brief Survey of Methods for Preparing Protein Conjugates with Dyes, Haptens, and Crosslinking Reagents,” in Bioconjug. Chem., 3:2013 (1992). Preferably, the binding ability and/or activity of the conjugate is not substantially reduced as a result of the conjugation technique employed, for example, relative to the unconjugated polypeptides.

[0232] In certain embodiments, a first polypeptide (e.g., TRAIL, trimeric polypeptide) is coupled to a second polypeptide ((e.g., CD137L or OX40L, trimeric polypeptide) either directly or indirectly. A direct reaction between two polypeptides of interest is possible when each possesses a substituent capable of reacting with the other. For example, a nucleophilic group, such as an amino or sulfhydryl group, on one may be capable of reacting with a carbonyl-containing group, such as an anhydride or an acid halide, or with an alkyl group containing a good leaving group (e.g, a halide) on the other.

[0233] Alternatively, it may be desirable to indirectly couple a first polypeptide (e.g, TRAIL, trimeric polypeptide) and a second polypeptide (e.g., CD137L or OX40L, trimeric polypeptide) of interest via a linker group, as described herein, including non-peptide linkers and peptide linkers, as described herein. A linker group can also function as a spacer to distance a first and second polypeptide in order to avoid interference with binding capabilities, targeting capabilities or other functionalities. A linker group can also serve to increase the chemical reactivity of a substituent on a polypeptide, and thus increase the coupling efficiency. An increase in chemical reactivity may also facilitate the use of agents, or functional groups on agents, which otherwise would not be possible. Examples of linking groups include, for example, disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups and esterase labile groups. In other illustrative embodiments, the conjugates include linking groups such as those disclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 Bl, and Chari et al., Cancer Research. 52: 127-131, 1992. Additional exemplary linkers are described herein.

[0234] In certain exemplary embodiments, a reaction between a polypeptide comprising a succinimidyl ester functional group and a polypeptide comprising an amino group forms an amide linkage; a reaction between a polypeptide comprising a oxycarbonylimidizaole functional group and a polypeptide comprising an amino group forms an carbamate linkage; a reaction between a polypeptide comprising a p-nitrophenyl carbonate functional group and a polypeptide comprising an amino group forms an carbamate linkage; a reaction between a polypeptide comprising a trichlorophenyl carbonate functional group and a polypeptide comprising an amino group forms an carbamate linkage; a reaction between a polypeptide comprising a thioester functional group and a polypeptide comprising an n-terminal amino group forms an amide linkage; a reaction between a polypeptide comprising a proprionaldehyde functional group and a polypeptide comprising an amino group forms a secondary amine linkage. [0235] In some exemplary embodiments, a reaction between a polypeptide comprising a butyraldehyde functional group and a polypeptide comprising an amino group forms a secondary amine linkage; a reaction between a polypeptide comprising an acetal functional group and a polypeptide comprising an amino group forms a secondary amine linkage; a reaction between a polypeptide comprising a piperidone functional group and a polypeptide comprising an amino group forms a secondary amine linkage; a reaction between a polypeptide comprising a methylketone functional group and a polypeptide comprising an amino group forms a secondary amine linkage; a reaction between a polypeptide comprising a tresylate functional group and a polypeptide comprising an amino group forms a secondary amine linkage; a reaction between a polypeptide comprising a maleimide functional group and a polypeptide comprising an amino group forms a secondary amine linkage; a reaction between a polypeptide comprising a aldehyde functional group and a polypeptide comprising an amino group forms a secondary amine linkage; and a reaction between a polypeptide comprising a hydrazine functional group and a polypeptide comprising an carboxylic acid group forms a secondary amine linkage.

[0236] In particular exemplary embodiments, a reaction between a polypeptide comprising a maleimide functional group and a polypeptide comprising a thiol group forms a thio ether linkage; a reaction between a polypeptide comprising a vinyl sulfone functional group and a polypeptide comprising a thiol group forms a thio ether linkage; a reaction between a polypeptide comprising a thiol functional group and a polypeptide comprising a thiol group forms a di-sulfide linkage; a reaction between a polypeptide comprising a orthopyridyl disulfide functional group and a polypeptide comprising a thiol group forms a di-sulfide linkage; and a reaction between a polypeptide comprising an iodoacetamide functional group and a polypeptide comprising a thiol group forms a thio ether linkage.

[0237] In a specific embodiment, an amine-to-sulfhydryl crosslinker is used for preparing a conjugate. In one preferred embodiment, for example, the crosslinker is succinimidyl-4-(N- maleimidomethyljcyclohexane- 1 -carboxylate (SMCC) (Thermo Scientific), which is a sulfhydryl crosslinker containing NHS-ester and maleimide reactive groups at opposite ends of a medium- length cyclohexane-stabilized spacer arm (8.3 angstroms). SMCC is a non-cleavable and membrane permeable crosslinker that can be used to create sulfhydryl-reactive, maleimide- activated agents (e.g., polypeptides) for subsequent reaction with the components of the conjugate. NHS esters react with primary amines at pH 7-9 to form stable amide bonds. Maleimides react with sulfhydryl groups at pH 6.5-7.5 to form stable thioether bonds. Thus, the amine reactive NHS ester of SMCC crosslinks rapidly with primary amines of a polypeptide and the resulting sulfhydryl- reactive maleimide group is then available to react with cysteine residues of the other polypeptide to yield specific conjugates of interest. [0238] In certain specific embodiments, a polypeptide is modified to contain exposed sulfhydryl groups to facilitate crosslinking, e.g., to facilitate crosslinking to a maleimide-activated polypeptide. In some specific embodiments, a polypeptide is modified with a reagent which modifies primary amines to add protected thiol sulfhydryl groups. In some embodiments, the reagent N-succinimidyl-S-acetylthioacetate (SATA) (Thermo Scientific) is used to produce thiolated polypeptides.

[0239] In certain embodiments, a maleimide-activated polypeptide is reacted under suitable conditions with a thiolated polypeptides to produce a conjugate. It will be understood that by manipulating the ratios of SMCC, SATA, agent, and polypeptides in these reactions it is possible to produce conjugates having differing stoichiometries, molecular weights and properties.

[0240] In some illustrative embodiments, conjugates are made using bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4- (N-maleimidomethyl)cyclohexane-l -carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p- azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)- ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4-dinitrobenzene). Particular coupling agents include N- succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (Carlsson et al., Biochem. J. 173:723-737 (1978)) and N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for a disulfide linkage.

[0241] The specific crosslinking strategies discussed herein are but a few of many examples of suitable conjugation strategies that may be employed in producing the conjugates described herein. It will be evident to those skilled in the art that a variety of other bifunctional or polyfunctional reagents, both homo- and hetero-functional (such as those described in the catalog of the Pierce Chemical Co., Rockford, IL), may be employed as the linker group. Coupling may be affected, for example, through amino groups, carboxyl groups, sulfhydryl groups or oxidized carbohydrate residues. There are numerous references describing such methodology, e.g., U.S. Patent No. 4,671,958, to Rodwell et al.

[0242] Conjugates can also be prepared by a various “click chemistry” techniques, including reactions that are modular, wide in scope, give very high yields, generate mainly inoffensive byproducts that can be removed by non-chromatographic methods, and can be stereospecific but not necessarily enantioselective (see Kolb et al., Angew Chem Int Ed Engl. 40:2004-2021, 2001). Particular examples include conjugation techniques that employ the Huisgen 1,3-dipolar cycloaddition of azides and alkynes, also referred to as “azide-alkyne cycloaddition” reactions (see Hein etal., Pharm Res. 25:2216-2230, 2008). Non-limiting examples of azide-alkyne cycloaddition reactions include copper-catalyzed azide-alkyne cycloaddition (CuAAC) reactions and ruthenium- catalyzed azide-alkyne cycloaddition (RuAAC) reactions.

[0243] CuAAC works over a broad temperature range, is insensitive to aqueous conditions and a pH range over 4 to 12, and tolerates a broad range of functional groups (see Himo et al, J Am Chem Soc. 127:210-216 (2005)). The active Cu(I) catalyst can be generated, for example, from Cu(I) salts or Cu(II) salts using sodium ascorbate as the reducing agent. This reaction forms 1,4- substituted products, making it region-specific (see Hein et al., supra).

[0244] RuAAC utilizes pentamethylcyclopentadienyl ruthenium chloride [Cp*RuCl] complexes that are able to catalyze the cycloaddition of azides to terminal alkynes, regioselectively leading to 1,5-disubstituted 1,2, 3 -triazoles (see Rasmussen et al., Org. Lett. 9:5337-5339 (2007)). Further, and in contrast to CuAAC, RuAAC can also be used with internal alkynes to provide fully substituted 1,2,3-triazoles.

[0245] Any one or more of the fusion or non-fusion techniques can be employed in the preparation of a conjugate, as described herein.

Pharmaceutical

and Administration Methods

[0246] In an aspect, methods of preparing and administering a trimeric fusion protein as provided herein, e.g., TRAIL-CD137L or TRAIL-OX40L as provided herein, to a subject in need thereof, e.g., to enhance an immune response in a cancer patient, e.g., to inhibit or reduce tumor growth, or induce a tumor shrinkage are well known to or can be readily determined by those skilled in the art. The route of administration of a TRAIL-CD137L or TRAIL-OX40L fusion protein can be, for example, oral, parenteral, by inhalation, rectally, vaginally or topical. The term parenteral as used herein includes, e.g., intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration. While all these forms of administration are clearly contemplated as suitable forms, another example of a form for administration would be a solution for injection, in particular for intravenous or intraarterial injection or drip or subcutaneous administration. Usually, a suitable pharmaceutical composition can comprise, without limitation, a buffer (e.g., acetate, phosphate or citrate buffer), a surfactant (e.g., polysorbate), a stabilizer agent (e.g., human albumin), etc. In other methods compatible with the teachings herein, a trimeric protein as provided herein, e.g., TRAIL-CD137L or TRAIL-OX40L fusion protein as provided herein can be delivered directly to the site of the adverse cellular population thereby increasing the exposure of the diseased tissue to the therapeutic agent.

[0247] Certain pharmaceutical compositions provided herein can be orally administered in an acceptable dosage form including, e.g., capsules, tablets, aqueous suspensions or solutions. Certain pharmaceutical compositions also can be administered by nasal aerosol or inhalation. Such compositions can be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, and/or other conventional solubilizing or dispersing agents.

[0248] The amount of a trimeric fusion protein TRAIL-CD137L or TRAIL-OX40L as provided herein can be combined with carrier materials to produce a single dosage form will vary depending upon the subject treated and the particular mode of administration. The composition can be administered as a single dose, multiple doses or over an established period of time in an infusion. Dosage regimens also can be adjusted to provide the optimum desired response (e.g, a therapeutic or prophylactic response).

[0249] By “therapeutically effective dose or amount” or “effective amount” is intended an amount of a TRAIL-CD137L or TRAIL-OX40L fusion protein, that when administered by itself or in combination with another drug brings about a positive therapeutic response with respect to treatment of a patient with a disease or condition to be treated.

[0250] Effective doses of compositions for treatment of cancer vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Usually, the patient is a human but non-human mammals including transgenic mammals can also be treated. Treatment dosages can be titrated using routine methods known to those skilled in the art to optimize safety and efficacy.

[0251] The compositions of the disclosure can be administered by any suitable method, e.g, parenterally, intraventricularly, orally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term “parenteral” as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrastemal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.

[0252] The disclosure further provides a method of enhancing an immune response in a subject comprising administering to a subject in need thereof a therapeutically effective amount of a trimeric protein as provided herein, e.g., TRAIL-CD137L or TRAIL-OX40L Fusion Protein, or a composition or formulation comprising the trimeric protein.

[0253] The subject to be treated can be any animal, e.g., mammal, in need of treatment, in certain aspects, subject is a human subject.

[0254] In its simplest form, a preparation to be administered to a subject is a trimeric protein as provided herein, e.g., TRAIL-CD137L or TRAIL-OX40L Fusion Protein, administered in conventional dosage form, and in some aspects, combined with a pharmaceutical excipient, carrier or diluent as described elsewhere herein. [0255] TRAIL-CD137L or TRAIL-OX40L fusion protein as provided herein, can be administered by any suitable method as described elsewhere herein, e.g, via IV infusion or subcutaneous administration. In certain aspects, a trimeric protein as provided herein, e.g., TRAIL- CD137L or TRAIL-OX40L Fusion Protein, can be introduced into a tumor, or in its vicinity.

[0256] All types of tumors are potentially amenable to treatment by this approach including, without limitation, carcinoma of the breast, colon, lung, pancreas, ovary, kidney, and bladder, as well as melanomas, sarcomas and lymphomas.

[0257] This disclosure employs, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Sambrook et al., ed. (1989) Molecular Cloning A Laboratory Manual (2nd ed.; Cold Spring Harbor Laboratory Press); Sambrook et al., ed. (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); D. N. Glover ed., (1985) DNA Cloning, Volumes I and II; Gait, ed. (1984) Oligonucleotide Synthesis; Mullis et al. U.S. Pat. No. 4,683,195; Hames and Higgins, eds. (1984) Nucleic Acid Hybridization; Hames and Higgins, eds. (1984) Transcription And Translation; Freshney (1987) Culture Of Animal Cells (Alan R. Liss, Inc.); Immobilized Cells And Enzymes (IRL Press) (1986); Perbal (1984) A Practical Guide To Molecular Cloning; the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Miller and Calos eds. (1987) Gene Transfer Vectors For Mammalian Cells, (Cold Spring Harbor Laboratory); Wu et al., eds., Methods In Enzymology, Vols. 154 and 155; Mayer and Walker, eds. (1987) Immunochemical Methods In Cell And Molecular Biology (Academic Press, London); Weir and Blackwell, eds., (1986) Handbook Of Experimental Immunology, Volumes I-IV; Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1986); and in Ausubel et al. (1989) Current Protocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.).

[0258] General principles of protein engineering are set forth in Rickwood et al., eds. (1995) Protein Engineering, A Practical Approach (IRL Press at Oxford Univ. Press, Oxford, Eng.). Additionally, standard methods in immunology known in the art and not specifically described are generally followed as in Current Protocols in Immunology, John Wiley & Sons, New York; Stites et al., eds. (1994) Basic and Clinical Immunology (8th ed; Appleton & Lange, Norwalk, Conn.) and Mishell and Shiigi (eds) (1980) Selected Methods in Cellular Immunology (W.H. Freeman and Co., NY).

[0259] Standard reference works setting forth general principles of immunology include Current Protocols in Immunology, John Wiley & Sons, New York; Klein (1982) J., Immunology: The Science of Self-Nonself Discrimination (John Wiley & Sons, NY); Kennett et al., eds. (1980) Monoclonal Antibodies, Hybridoma: A New Dimension in Biological Analyses (Plenum Press, NY); Campbell (1984) “Monoclonal Antibody Technology” in Laboratory Techniques in Biochemistry and Molecular Biology, ed. Burden et al., (Elsevier, Amsterdam); Goldsby etal., eds. (2000) Kuby Immunology (4th ed.; H. Freemand & Co.); Roitt et al. (2001) Immunology (6th ed.; London: Mosby); Abbas et al. (2005) Cellular and Molecular Immunology (5th ed.; Elsevier Health Sciences Division); Kontermann and Dubel (2001) Antibody Engineering (Springer Verlag); Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press); Lewin (2003) Genes VIII (Prentice Hall 2003); Harlow and Lane (1988) Antibodies: A Laboratory Manual (Cold Spring Harbor Press); Dieffenbach and Dveksler (2003) PCR Primer (Cold Spring Harbor Press).

[0260] All of the references cited above, as well as all references cited herein, are incorporated herein by reference in their entireties.

[0261] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei- Show, 2nd ed. (2002), CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed. (1999), Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised (2000), Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.

[0262] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various aspects or aspects of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0263] The terms “inhibit,” “block,” and “suppress” are used interchangeably herein and refer to any statistically significant decrease in biological activity, including full blocking of the activity. For example, “inhibition” can refer to a decrease of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% in biological activity.

[0264] As used herein, the term “affinity” refers to a measure of the strength of the binding of a ligand to its cognate receptor. As used herein, the term “avidity” refers to the overall stability of the complex between a population of ligands and receptors, that is, the functional combining strength of a combination of ligands and receptors, e.g., interaction of a trimeric TRAIL-CD137L or a trimeric TRAIL-OX40L Fusion Protein with cell surface CD 137 or 0X40 and TRAIL receptors DR4 and DR5. Avidity is related to both the affinity of individual receptor binding domains in the population with specific receptors, and also the valences of the ligands and the receptors.

[0265] As used herein, the term “CD137L” is a transmembrane glycoprotein that is encoded by the TNFSF9 gene. Also included within the definition of CD137L are CD137 ligand variants which vary in amino acid sequence from naturally occurring CD 137 ligand (SEQ ID NO: 8), but which retain the ability to specifically bind to CD137.

[0266] CD137, or “CD137 receptor” is a protein (also variously termed 4-1BB, tumor necrosis factor receptor superfamily member 9) expressed on the surface of activated T cells, B cells and NK cells; naive T cells do not express CD137 (Pollok, K.E et al. J. Immunol. 150, 771-781(1993)).

[0267] 0X40, or “0X40 receptor” is a protein (also variously termed CD 134, tumor necrosis factor receptor superfamily member 4, and ACT-35) expressed on the surface of activated T cells, e.g., CD4⁺ and CD8⁺ T-cells, as well as on Foxp3⁺ CD4⁺ regulatory T cells (Tregs) and NK cells. Naive CD4⁺ and CD8⁺ T cells do not express 0X40 (Croft, M., Ann Rev Immunol 28:57-78 (2010)).

[0268] “0X40 ligand” (“OX40L”) (also variously termed tumor necrosis factor ligand superfamily member 4, gp34, TAX transcriptionally-activated glycoprotein- 1, and CD252) is found largely on antigen presenting cells (APCs), and can be induced on activated B cells, dendritic cells (DCs), Langerhans cells, plamacytoid DCs, and macrophages (Croft, M., Ann Rev Immunol 28:57-78 (2010) ). Other cells, including activated T cells, NK cells, mast cells, endothelial cells, and smooth muscle cells can express OX40L in response to inflammatory cytokines. OX40L specifically binds to the 0X40 receptor. A functionally active soluble form of OX40L can be produced by deleting the intracellular and transmembrane domains. A functionally active form of OX40L is a form that retains the capacity to bind specifically to 0X40, that is, that possesses an 0X40 “receptor binding domain.” An example is amino acids 51 to 183 of SEQ ID NO: 5, human OX40L.

[0269] As used herein, the term “OX40L” includes the entire 0X40 ligand, soluble 0X40 ligand, and functionally active portions of the 0X40 ligand. Also included within the definition of OX40L are 0X40 ligand variants which vary in amino acid sequence from naturally occurring 0X40 ligand molecules, but which retain the ability to specifically bind to an 0X40 receptor.

[0270] A “trimerization domain” is an amino acid sequence within a polypeptide that promotes assembly of the polypeptide into trimers. For example, a trimerization can promote assembly into trimers via associations with other trimerization domains (of additional polypeptides with the same or a different amino acid sequence). The term is also used to refer to a polynucleotide that encodes such a peptide or polypeptide. [0271] As used herein, the terms “linked,” “fused” or “fusion” can be used interchangeably. These terms refer to the joining together of two more elements or components, by whatever means including chemical conjugation or recombinant means. An “in-frame fusion” refers to the joining of two or more polynucleotide open reading frames (ORFs) to form a continuous longer ORF, in a manner that maintains the correct translational reading frame of the original ORFs. Thus, a recombinant fusion protein is a single protein containing two or more segments that correspond to polypeptides encoded by the original ORFs (which segments are not normally so joined in nature.), e.g., TRAIL-CD137L or TRAIL-OX40L fusion protein as provided herein. Although the reading frame is thus made continuous throughout the fused segments, the segments can be physically or spatially separated by, for example, in-frame linker sequence.

[0272] In the context of polypeptides, a “linear sequence” or a “sequence” is an order of amino acids in a polypeptide in an amino to carboxyl terminal direction in which amino acids that neighbor each other in the sequence are contiguous in the primary activated structure of the polypeptide.

[0273] The term “expression” as used herein refers to a process by which a gene produces a biochemical, for example, a polypeptide. The process includes any manifestation of the functional presence of the gene within the cell including, without limitation, gene knockdown as well as both transient expression and stable expression. It includes without limitation transcription of the gene into messenger RNA (mRNA), and the translation of such mRNA into polypeptide(s). If the final desired product is a biochemical, expression includes the creation of that biochemical and any precursors. Expression of a gene produces a “gene product.” As used herein, a gene product can be either a nucleic acid, e.g., a messenger RNA produced by transcription of a gene, or a polypeptide which is translated from a transcript. Gene products described herein further include nucleic acids with post transcriptional modifications, e.g., polyadenylation, or polypeptides with post translational modifications, e.g., methylation, glycosylation, the addition of lipids, association with other protein subunits, proteolytic cleavage, and the like.

[0274] As used herein the terms “treat,” “treatment,” or “treatment of’ (e.g., in the phrase “treating a cancer patient”) refers to reducing the potential for disease pathology, reducing the occurrence of disease symptoms, e.g., to an extent that the subject has a longer survival rate or reduced discomfort. For example, treating can refer to the ability of a therapy when administered to a subject, to reduce disease symptoms, signs, or causes. Treating also refers to mitigating or decreasing at least one clinical symptom and/or inhibition or delay in the progression of the condition and/or prevention or delay of the onset of a disease or illness.

[0275] By “subject” or “individual” or “animal” or “patient” or “mammal,” is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans, domestic animals, farm animals, sports animals, and zoo animals, including, e.g, humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, bears, and so on.

[0276] The term “pharmaceutical composition” refers to a preparation that is in such form as to permit the biological activity of the active ingredient to be effective, and that contains no additional components that are unacceptably toxic to a subject to which the composition would be administered. Such composition can be sterile.

[0277] Engagement of the CD 137 or 0X40 receptor on T cells, during, or shortly after, priming by an antigen results in an increased response of the T cells to the antigen. In the context of the present disclosure, the term “engagement” refers to binding to and stimulation of at least one activity mediated by the CD137 or the 0X40 receptor. For example, engagement of the CD137 or the 0X40 receptor on antigen specific T cells results in increased T cell proliferation as compared to the response to antigen alone, and increased cytokine production. The elevated response to the antigen can be maintained for a period of time substantially longer than in the absence of CD 137 or 0X40 receptor engagement. Thus, stimulation via the 0X40 receptor enhances the antigen specific immune response by boosting T-cell recognition of antigens, e.g., tumor antigens.

[0278] CD137 and 0X40 agonists can enhance antigen specific immune responses in a subject, such as a human subject, when administered to the subject during or shortly after priming of T- cells by an antigen. CD137 agonists include CD137 ligand (“CD137L”), such as soluble CD137L fusion proteins and anti-CD137 antibodies or fragments thereof. 0X40 agonists include 0X40 ligand (“OX40L”), such as soluble OX40L fusion proteins and anti-OX40 antibodies or fragments thereof. A specific example is a fusion polypeptide subunit comprising the receptor binding domain of CD137L and TRAIL or OX40L and TRAIL, where the polypeptide subunit self-assembles into a multimeric (e.g., trimeric or hexameric) fusion protein. Also described are nucleic acids including polynucleotide sequences that encode such fusion polypeptides. This disclosure also provides methods for enhancing an antigen specific immune response as well as antigen non-specific immune response in a subject using the multimeric TRAIL-CD137L or TRAIL-OX40L fusion polypeptides. The antigen specific immune response is achieved through stimulation of CD 137 or 0X40 receptor on activated T cells. Antigen non-specific activity can be achieved after TRAIL- CD137L or TRAIL-OX40L mediated formation of immune synapse following simultaneous binding of cancer cells through TRAIL domain of the fusion protein and T cell via CD137L or OX40L domain of the fusion protein. The term ‘redirects’ refers to ability of the fusion protein to bind T cells with one domain (e.g. CD137L or OX40L) and cancer cells with another (e.g., TRAIL) as shown in example depicted in FIG. 2. The compositions and methods disclosed herein with respect to TRAIL-CD 137L or TRAIL-OX40L fusion proteins can be more generally applied to the production and use of multimeric (e.g., trimeric) receptor-binding fusion proteins. [0279] The terms “cancer”, “tumor”, “cancerous”, and “malignant” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancers include but are not limited to, carcinoma including adenocarcinomas, lymphomas, blastomas, melanomas, sarcomas, and leukemias. More particular examples of such cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, Hodgkin's and non-Hodgkin's lymphoma, pancreatic cancer, glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer such as hepatic carcinoma and hepatoma, bladder cancer, breast cancer (including triple negative breast cancer), colon cancer, endometrial carcinoma, myeloma (multiple myeloma), salivary gland carcinoma, kidney cancer such as renal cell carcinoma and Wilms' tumors, basal cell carcinoma, melanoma, prostate cancer, vulval cancer, thyroid cancer, testicular cancer, esophageal cancer, various types of head and neck cancer including, but not limited to, squamous cell cancers, and cancers of mucinous origins, such as, mucinous ovarian cancer, cholangiocarcinoma (liver) and renal papillary carcinoma.

[0280] Some embodiments are directed to a method of preventing or treating a cancer in a subject in need thereof, comprising administering to the subject an effective amount of TRAIL- CD137L or TRAIL-OX40L fusion protein as provided herein, a composition or formulation comprising the trimeric protein, or a polynucleotide, a vector, or a host cell as described herein.

[0281] Some embodiments are directed to a method of preventing or treating a cancer in a subject in need thereof, comprising administering to the subject an effective amount of TRAIL- ED 137L and TRAIL-OX40L fusion protein as provided herein, a composition or formulation comprising the trimeric protein, or a polynucleotide, a vector, or a host cell as described herein.

[0282] Some embodiments are directed to a method of preventing or treating a cancer in a subject in need thereof, comprising first administering to the subject an effective amount of TRAIL- ED 137L fusion protein as provided herein, a composition or formulation comprising the trimeric protein, or a polynucleotide, a vector, or a host cell as described herein and then administering to the subject an effective amount of and TRAIL-OX40L fusion protein as provided herein, a composition or formulation comprising the trimeric protein, or a polynucleotide, a vector, or a host cell as described herein.

[0283] Some embodiments are directed to a method of preventing or treating a cancer in a subject in need thereof, comprising first administering to the subject an effective amount of TRAIL- OX40L fusion protein as provided herein, a composition or formulation comprising the trimeric protein, or a polynucleotide, a vector, or a host cell as described herein and then administering to the subject an effective amount of and TRAIL-ED137L fusion protein as provided herein, a composition or formulation comprising the trimeric protein, or a polynucleotide, a vector, or a host cell as described herein. [0284] A trimerization domain promotes self-assembly into a trimeric fusion protein. In one embodiment, the trimerization domain is a leucine zipper domain. An exemplary leucine zipper domain is the engineered yeast GCN4 leucine variant described by Harbury et al. (1993) Science 262: 1401-1407, the disclosure of which is incorporated herein for all purposes. Exemplary trimerization domains include: TNF receptor-associated factor-2 (TRAF2) (GENBANK® Accession No. Q12933 [gi:23503103]; amino acids 310-349); Thrombospondin 1 (Accession No. P07996 [gi: 135717]; amino acids 291-314); Matrilin-4 (Accession No. 095460 [gi: 14548117]; amino acids 594-618; cartilage matrix protein (matrilin-1) (Accession No. NP002370 [gi:4505111]; amino acids 463-496; Heat shock transcription factor (HSF) (Accession No. AAX42211 [gi:61362386]; amino acids 165-191; and Cubilin (Accession No. NP001072 [gi:4557503]; amino acids 104-138. In certain aspects, the trimerization domain comprises amino acids 310 to 349 of human TRAF2.

[0285] TRAIL and/or CD137L and/or OX40L subunit polypeptides as provided herein can contain one or more conservative amino acid changes, e.g., up to ten conservative changes (e.g., two substituted amino acids, three substituted amino acids, four substituted amino acids, or five substituted amino acids, etc.), provided that the changes can be made in the polypeptide without changing a biochemical function of the TRAIL and/or CD137L and/or OX40L fusion polypeptide subunit or trimeric protein.

[0286] For example, one or more conservative changes can be made in a TRAIL and a CD 137L or an OX40L receptor binding domain without changing its ability to bind to DR4/DR5 receptors and CD 137 or 0X40 correspondingly. Similarly, one or more conservative changes can be made in trimerization domain without altering its ability to trimerize.

[0287] Additionally, part of a polypeptide domain can be deleted without impairing or eliminating all of its functions. Similarly, insertions or additions can be made in the polypeptide chain, for example, adding epitope tags, without impairing or eliminating its functions, as described below. Other modifications that can be made without materially impairing one or more functions of a polypeptide include, for example, in vivo or in vitro chemical and biochemical modifications that incorporate unusual amino acids. Such modifications include, for example, acetylation, carboxylation, phosphorylation, glycosylation, labeling, e.g., with radionuclides, and various enzymatic modifications, as will be readily appreciated by those of ordinary skill in the art. A variety of methods for labeling polypeptides, and labels useful for such purposes, are well known in the art, and include radioactive isotopes such as ³²P, fluorophores, chemiluminescent agents, enzymes, and anti-ligands.

[0288] In certain aspects, the heterologous agent can be chemically conjugated to the polypeptide subunit. Exemplary heterologous agents that can be chemically conjugated to the polypeptide subunit include, without limitation, linkers, drugs, toxins, imaging agents, radioactive compounds, organic and inorganic polymers, and any other compositions which might provide a desired activity that is not provided by the polypeptide subunit itself. Specific agents include, without limitation, polyethylene glycol (PEG), a cytotoxic agent, a radionuclide, an imaging agent, biotin.

Methods of Use and Compositions

[0289] In an aspect, disclosed herein are methods of using the conjugates described herein for treating a subject in need thereof. In an aspect, disclosed herein compositions comprising the conjugates. For example, certain embodiments include methods of treating, ameliorating the symptoms of, or inhibiting the progression of, a cancer in a subject in need thereof, comprising administering to the subject a conjugate described herein, or a composition comprising the conjugate.

[0290] In embodiments, the methods and compositions described herein can be used in the treatment of any variety of cancers. In embodiments, the methods and compositions described herein can be used to treat any cancer known in the art.

[0291] In embodiments, the trimeric fusion protein or composition can inhibit tumor growth, can promote tumor reduction, or both. In certain aspects, the tumor growth inhibition is achieved in the presence of T-cells and/or another drug administered to a patient. In certain aspects, the cancer is a solid tumor. In some embodiments, the cancer is selected from one or more of pancreatic cancer, colorectal cancer, breast cancer (including triple negative breast cancer), prostate cancer, small cell lung cancer, mesothelioma, lymphocytic leukemia, chronic myelogenous leukemia, lymphoma, hepatoma, sarcoma, leukemia, acute myeloid leukemia, relapsed acute myeloid leukemia, B-cell malignancy, myeloma (multiple myeloma), ovarian cancer, gastric cancer, non- small cell lung cancer (NSCLC), kidney cancer, bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancers, cervical cancer, testicular cancer, and stomach cancer. In embodiments, the cancer is any cancer known in the art.

[0292] In some embodiments, the methods or compositions described herein increase median survival time of a patient by 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 15 weeks, 20 weeks, 25 weeks, 30 weeks, 40 weeks, or longer. In certain embodiments, the methods or compositions described herein increase median survival time of a patient by 1 year, 2 years, 3 years, or longer. In some embodiments, the methods or compositions described herein increase progression-free survival by 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks or longer. In certain embodiments, the methods or compositions described herein increase progression-free survival by 1 year, 2 years, 3 years, or longer. [0293] In certain embodiments, the composition administered is sufficient to result in tumor regression, as indicated by a statistically significant decrease in the amount of viable tumor, for example, at least a 10%, 20%, 30%, 40%, 50% or greater decrease in tumor mass, or by altered (e.g., decreased with statistical significance) scan dimensions. In certain embodiments, the composition administered is sufficient to result in stable disease. In certain embodiments, the composition administered is sufficient to result in stabilization or clinically relevant reduction in symptoms of a particular disease indication known to the skilled clinician.

[0294] The methods or compositions for treating cancers can be combined with other therapeutic modalities. For example, a composition described herein can be administered to a subject before, during, or after other therapeutic interventions, including symptomatic care, chemotherapy, radiotherapy, surgery, transplantation, hormone therapy, photodynamic therapy, antibiotic therapy, or any combination thereof. Symptomatic care includes administration of corticosteroids, to reduce cerebral edema, headaches, cognitive dysfunction, and emesis, and administration of anti-convulsants, to reduce seizures. Radiotherapy includes whole-brain irradiation, fractionated radiotherapy, and radiosurgery, such as stereotactic radiosurgery, which can be further combined with traditional surgery.

[0295] Methods for identifying subjects with one or more of the diseases or conditions described herein are known in the art.

[0296] For in vivo use, for instance, for the treatment of human disease or testing, the conjugates described herein are generally incorporated into one or more pharmaceutical or therapeutic compositions prior to administration. In some instances, a pharmaceutical or therapeutic composition comprises one or more of the conjugates described herein in combination with a physiologically acceptable carrier or excipient. In some instances, a pharmaceutical or therapeutic composition comprises one or more additional pharmaceutical agents.

[0297] To prepare a pharmaceutical or therapeutic composition, an effective or desired amount of one or more conjugates is mixed with any pharmaceutical carrier(s) or excipient known to those skilled in the art to be suitable for the particular conjugate and/or mode of administration. A pharmaceutical carrier may be liquid, semi-liquid or solid. Solutions or suspensions used for parenteral, intradermal, subcutaneous or topical application may include, for example, a sterile diluent (such as water), saline solution (e.g., phosphate buffered saline; PBS), fixed oil, polyethylene glycol, glycerin, propylene glycol or other synthetic solvent; antimicrobial agents (such as benzyl alcohol and methyl parabens); antioxidants (such as ascorbic acid and sodium bisulfite) and chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); buffers (such as acetates, citrates and phosphates). If administered intravenously (e.g., by IV infusion), suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, polypropylene glycol and mixtures thereof.

[0298] In certain aspects, the pH of the composition is near physiological pH or about pH 7.4, including about pH 6.5, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.5, or any range thereof. In specific embodiments, the composition has one or more of the following determinations of purity: less than about 1 EU endotoxin/mg protein, less that about 100 ng host cell protein/mg protein, less than about 10 pg host cell DNA/mg protein, and/or greater than about 95% single peak purity by SEC HPLC.

[0299] Administration may be achieved by a variety of different routes, including oral, parenteral, intranasal, intravenous, intradermal, intramuscular, intrathecal, subcutaneous, sublingual, buccal, rectal, vaginal, and topical. Preferred modes of administration depend upon the nature of the condition to be treated or prevented. Particular embodiments include administration by IV infusion.

[0300] Carriers can include, for example, pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as polysorbate 20 (TWEEN™) polyethylene glycol (PEG), and poloxamers (PLURONICS™), and the like.

[0301] In some embodiments, one or more conjugates can be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methyhnethacylate)microcapsules, respectively), in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington’s Pharmaceutical Sciences, 16th edition, Oslo, A., Ed., (1980). The particle(s) or liposomes may further comprise other therapeutic or diagnostic agents.

[0302] Typical routes of administering these and related pharmaceutical compositions thus include, without limitation, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrastemal injection or infusion techniques. Certain pharmaceutical or therapeutic compositions are formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a patient. Compositions that will be administered to a subject or patient may take the form of one or more dosage units, where for example, a tablet may be a single dosage unit, and a container of a herein described conjugate in aerosol form may hold a plurality of dosage units. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000). The composition to be administered will typically contain a therapeutically effective amount of a conjugate described herein, for treatment of a disease or condition of interest.

[0303] A pharmaceutical or therapeutic composition may be in the form of a solid or liquid. In one embodiment, the carrier(s) are particulate, so that the compositions are, for example, in tablet or powder form. The carrier(s) may be liquid, with the compositions being, for example, an oral oil, injectable liquid or an aerosol, which is useful in, for example, inhalatory administration. When intended for oral administration, the pharmaceutical composition is preferably in either solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid.

[0304] As a solid composition for oral administration, the pharmaceutical composition may be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like. Such a solid composition will typically contain one or more inert diluents or edible carriers. In addition, one or more of the following may be present: binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, Primogel, com starch and the like; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; a flavoring agent such as peppermint, methyl salicylate or orange flavoring; and a coloring agent. When the pharmaceutical composition is in the form of a capsule, for example, a gelatin capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or oil.

[0305] The pharmaceutical or therapeutic composition may be in the form of a liquid, for example, an elixir, syrup, solution, emulsion or suspension. The liquid may be for oral administration or for delivery by injection, as two examples. When intended for oral administration, preferred composition contain, in addition to the present compounds, one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer. In a composition intended to be administered by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent may be included. [0306] The liquid pharmaceutical or therapeutic compositions, whether they be solutions, suspensions or other like form, may include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer’s solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Physiological saline is a preferred adjuvant. An injectable pharmaceutical composition is preferably sterile.

[0307] A liquid pharmaceutical or therapeutic composition intended for either parenteral or oral administration should contain an amount of a conjugate such that a suitable dosage will be obtained. Typically, this amount is at least 0.01% of the conjugate of interest in the composition. When intended for oral administration, this amount may be varied to be between 0. 1 and about 70% of the weight of the composition. Certain oral pharmaceutical compositions contain between about 4% and about 75% of the conjugate of interest. In certain embodiments, pharmaceutical compositions and preparations are prepared so that a parenteral dosage unit contains between 0.01 to 10% by weight of the conjugate of interest prior to dilution.

[0308] The pharmaceutical composition may be intended for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment or gel base. The base, for example, may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, bee wax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. Thickening agents may be present in a pharmaceutical composition for topical administration. If intended for transdermal administration, the composition may include a transdermal patch or iontophoresis device.

[0309] The pharmaceutical composition may be intended for rectal administration, in the form, for example, of a suppository, which will melt in the rectum and release the drug. The composition for rectal administration may contain an oleaginous base as a suitable nonirritating excipient. Such bases include, without limitation, lanolin, cocoa butter, and polyethylene glycol.

[0310] The pharmaceutical composition may include various materials, which modify the physical form of a solid or liquid dosage unit. For example, the composition may include materials that form a coating shell around the active ingredients. The materials that form the coating shell are typically inert, and may be selected from, for example, sugar, shellac, and other enteric coating agents. Alternatively, the active ingredients may be encased in a gelatin capsule. The pharmaceutical composition in solid or liquid form may include a component that binds to the conjugate and thereby assists in the delivery of the conjugate. Suitable components that may act in this capacity include monoclonal or polyclonal antibodies, one or more proteins or a liposome.

[0311] The pharmaceutical composition may consist essentially of dosage units that can be administered as an aerosol. The term aerosol is used to denote a variety of systems ranging from those of colloidal nature to systems consisting of pressurized packages. Delivery may be by a liquefied or compressed gas or by a suitable pump system that dispenses the active ingredients. Aerosols may be delivered in single phase, bi-phasic, or tri-phasic systems in order to deliver the active ingredient(s). Delivery of the aerosol includes the necessary container, activators, valves, subcontainers, and the like, which together may form a kit. One of ordinary skill in the art, without undue experimentation may determine preferred aerosols.

[0312] The compositions described herein may be prepared with carriers that protect the conjugates against rapid elimination from the body, such as time release formulations or coatings. Such carriers include controlled release formulations, such as, but not limited to, implants and microencapsulated delivery systems, and biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid and others known to those of ordinary skill in the art.

[0313] The pharmaceutical compositions may be prepared by methodology well known in the pharmaceutical art. For example, a pharmaceutical composition intended to be administered by injection may comprise one or more of salts, buffers and/or stabilizers, with sterile, distilled water so as to form a solution. A surfactant may be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that non-covalently interact with the conjugate so as to facilitate dissolution or homogeneous suspension of the conjugate in the aqueous delivery system.

[0314] The compositions may be administered in a therapeutically effective amount, which will vary depending upon a variety of factors including the activity of the specific compound employed; the metabolic stability and length of action of the compound; the age, body weight, general health, sex, and diet of the patient; the mode and time of administration; the rate of excretion; the drug combination; the severity of the particular disorder or condition; and the subject undergoing therapy.

[0315] The precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by testing the compositions in model systems known in the art and extrapolating therefrom. Controlled clinical trials may also be performed. Dosages may also vary with the severity of the condition to be alleviated. A pharmaceutical composition is generally formulated and administered to exert a therapeutically useful effect while minimizing undesirable side effects. The composition may be administered one time, or may be divided into a number of smaller doses to be administered at intervals of time. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need.

[0316] In some embodiments, a therapeutically effective amount or therapeutic dosage of a composition described herein is an amount that is effective to reduce or stabilize tumor growth. In certain instances, treatment is initiated with small dosages which can be increased by small increments until the optimum effect under the circumstances is achieved. In some instances, a therapeutically effective daily dose is (for a 70 kg mammal) from about 0.001 mg/kg (i.e., ~ 0.07 mg) to about 100 mg/kg (i.e., ~ 7.0 g); preferably a therapeutically effective dose is (for a 70 kg mammal) from about 0.01 mg/kg (i.e., ~ 0.7 mg) to about 50 mg/kg (i.e., ~ 3.5 g); more preferably a therapeutically effective dose is (for a 70 kg mammal) from about 1 mg/kg (i.e., ~ 70 mg) to about 25 mg/kg (i.e., ~ 1.75 g).

[0317] In some embodiments, a dosage is administered from about once a day to about once every two or three weeks. For example, in certain embodiments, a dosage is administered about once every 1, 2, 3, 4, 5, 6, or 7 days, or about once a week, or about twice a week, or about three times a week, or about once every two or three weeks.

[0318] In some embodiments, the dosage is from about 0.1 mg/kg to about 20 mg/kg, or to about 10 mg/kg, or to about 5 mg/kg, or to about 3 mg/kg. In some embodiments, the dosage is about 0. 10 mg/kg, 0.15 mg/kg, 0.20 mg/kg, 0.25 mg/kg, 0.30 mg/kg, 0.35 mg/kg, 0.40 mg/kg, 0.45 mg/kg, 0.50 mg/kg, 0.55 mg/kg, 0.60 mg/kg, 0.65 mg/kg, 0.70 mg/kg, 0.75 mg/kg, 0.80 mg/kg, 0.85 mg/kg, 0.90 mg/kg, 0.95 mg/kg, 1.0 mg/kg, 1.5 mg/kg, 2.0 mg/kg, 2.5 mg/kg, 3.0 mg/kg, 3.5 mg/kg, 4.0 mg/kg, 4.5 mg/kg, 5.0 mg/kg, 5.5 mg/kg, 6.0 mg/kg, 6.5 mg/kg. 7.0 mg/kg, 7.5 mg/kg, 8.0 mg/kg, 8.5 mg/kg, 9.0 mg/kg, 9.5 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg, 19 mg/kg, or 20 mg/kg, including all integers and ranges in between. In specific embodiments, the dosage is about 1 mg/kg once a week as a 2 ml intravenous injection to about 20 mg/kg once every 3 days.

[0319] Also included are patient care kits, comprising one or more conjugates or compositions described herein. Certain kits also comprise one or more pharmaceutically acceptable diluents or solvents, such as water (e.g., sterile water). In some embodiments, the conjugates are stored in vials, cartridges, dual chamber syringes, and/or pre-filled mixing systems.

[0320] The kits herein may also include a one or more additional therapeutic agents (e.g, conjugates) or other components suitable or desired for the indication being treated, or for the desired diagnostic application. The kits herein can also include one or more syringes or other components necessary or desired to facilitate an intended mode of delivery (e.g, stents, implantable depots, etc.).

[0321] All publications, patent applications, and issued patents cited in this specification are herein incorporated by reference as if each individual publication, patent application, or issued patent were specifically and individually indicated to be incorporated by reference.

[0322] Although the foregoing has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this disclosure that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. The following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of noncritical parameters that could be changed or modified to yield essentially similar results.

[0323] A TRAIL-CD137L or TRAIL-OX40L fusion polypeptide produced by a transformed host, can be purified according to any suitable method. Such standard methods include chromatography (e.g., ion exchange, affinity and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for protein purification. Affinity tags such as hexahistidine, maltose binding domain, influenza coat sequence and glutathione-S-transferase can be attached to the protein to allow easy purification by passage over an appropriate affinity column. Isolated proteins can also be physically characterized using such techniques as proteolysis, nuclear magnetic resonance and x-ray crystallography.

[0324] For example, supernatants from systems that secrete recombinant protein into culture media can be first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. Following the concentration step, the concentrate can be applied to a suitable purification matrix. Alternatively, an anion exchange resin can be employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) groups. The matrices can be acrylamide, agarose, dextran, cellulose or other types commonly employed in protein purification. Alternatively, a cation exchange step can be employed. Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups. Finally, one or more reversed-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify an influenza B/Yamagata virus-binding molecule. Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a homogeneous recombinant protein. EXAMPLES

[0325] The expression and purification of proteins, such as TRAIL-CD137L or TRAIL- OX40L (See, e.g., FIG. 1) fusion polypeptide subunit are performed using standard laboratory techniques.

[0326] A variety of expression vector/host systems are known and may be utilized to contain and express polynucleotide sequences. These include, but are not limited to, microorganisms such as bacteria transformed with an expression vector, for example, a recombinant bacteriophage, plasmid, or cosmid DNA expression vector; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus); plant cell systems transformed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems, including mammalian cell and more specifically human cell systems transformed with viral, plasmid, episomal, integrating, or other expression vectors. Certain embodiments therefore include an expression vector, comprising a polynucleotide sequence that encodes a polypeptide described herein, for example, a fusion polypeptide. Also included are host cells that comprise the polynucleotides and/or expression vectors.

[0327] Certain embodiments may employ E. coli-based expression systems (see, e.g., Structural Genomics Consortium et al., Nature Methods. 5: 135-146, 2008). These and related embodiments may rely partially or totally on ligation-independent cloning (LIC) to produce a suitable expression vector. In specific embodiments, protein expression may be controlled by a T7 RNA polymerase (e.g., pET vector series), or modified pET vectors with alternate promoters, including for example the TAC promoter. These and related embodiments may utilize the expression host strain BL21(DE3), a 1DE3 lysogen of BL21 that supports T7-mediated expression and is deficient in Ion and ompT proteases for improved target protein stability. Also included are expression host strains carrying plasmids encoding tRNAs rarely used in E. coli, such as ROSETTA™ (DE3) and Rosetta 2 (DE3) strains. In some embodiments other E. coli strains may be utilized, including other E. coli K-12 strains such as W3110 (F- lambda- IN(rmD-rmE)l rph- 1), and UT5600 (F, araC14, leuB6(Am), secA206(aziR), lacYl, proC14, tsx67, A(ompTfepC)266, entA403, glnX44(AS), X-, trpE38, rfbCl, rpsL109(strR), xylA5, mtl-1, thiEl), which can result in reduced levels of post-translational modifications during fermentation. Cell lysis and sample handling may also be improved using reagents sold under the trademarks BENZONASE® nuclease and BUGBUSTER® Protein Extraction Reagent. For cell culture, auto-inducing media can improve the efficiency of many expression systems, including high-throughput expression systems. Media of this type (e.g., OVERNIGHT EXPRESS™ Autoinduction System) gradually elicit protein expression through metabolic shift without the addition of artificial inducing agents such as IPTG.

[0328] Particular embodiments employ hexahistidine tags (such as those sold under the trademark HIS*TAG® fusions), followed by immobilized metal affinity chromatography (IMAC) purification, or related techniques. In certain aspects, however, clinical grade proteins can be isolated from E. coli inclusion bodies, without or without the use of affinity tags (see, e.g., Shimp et al., Protein Expr Purif. 50:58-67, 2006). As a further example, certain embodiments may employ a cold-shock induced E. coli high-yield production system, because over-expression of proteins in Escherichia coli at low temperature improves their solubility and stability (see, e.g., Qing et al., Nature Biotechnology. 22:877-882, 2004).

[0329] Also included are high-density bacterial fermentation systems. For example, high cell density cultivation of Ralstonia eutropha allows protein production at cell densities of over 150 g/L, and the expression of recombinant proteins at titers exceeding 10 g/L. In the yeast Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH may be used. For reviews, see Ausubel et al. (supra) and Grant et al., Methods Enzymol. 153:516-544, 1987. Also included are Pichia pandoris expression systems (see, e.g., Li et al., Nature Biotechnology. 24, 210-215, 2006; and Hamilton et al., Science, 301: 1244, 2003). Certain embodiments include yeast systems that are engineered to selectively glycosylate proteins, including yeast that have humanized N-glycosylation pathways, among others (see, e.g., Hamilton et al., Science. 313: 1441-1443, 2006; Wildt et al., Nature Reviews Microbiol. 3: 119-28, 2005; and Gemgross et al., Nature-Biotechnology. 22: 1409 -1414, 2004; U.S. Patent Nos. 7,629,163; 7,326,681; and 7,029,872). Merely by way of example, recombinant yeast cultures can be grown in Fembach Flasks or 15L, 50L, 100L, and 200L fermentors, among others.

[0330] In cases where plant expression vectors are used, the expression of sequences encoding polypeptides may be driven by any of a number of promoters. For example, viral promoters such as the 35 S and 19S promoters of CaMV may be used alone or in combination with the omega leader sequence from TMV (Takamatsu, EMBO J. 6:307-311,1987). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used (Coruzzi et al., EMBO J. 3: 1671-1680, 1984; Broglie et al., Science. 224:838-843, 1984; and Winter et al., Results Probl. Cell Differ. 17:85-105, 1991). These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. Such techniques are described in a number of generally available reviews (see, e.g., Hobbs in McGraw Hill, Yearbook of Science and Technology, pp. 191-196, 1992).

[0331] Mammalian cell culture systems are advantageously employed to express trimeric or hexametric proteins provided and detailed herein. Expression of recombinant proteins in mammalian cells are performed because such proteins are generally correctly folded, appropriately modified and completely functional. Examples of preferred mammalian host cell lines include human embryonic kidney line (HEK-293 and HEK-293T) and Chinese hamster ovary (CHO) cell lines. Mammalian expression systems utilize attached cell lines, for example, in T-flasks, roller bottles, or cell factories, or suspension cultures, for example, in IL and 5L spinners, 5L, 14L, 40L, 100L and 200L stir tank bioreactors, or 20/50L and 100/200L WAVE bioreactors, among others known in the art.

[0332] Mammalian expression vectors comprise non-transcribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5' or 3' flanking non-transcribed sequences, and 5' or 3' non-translated sequences, such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and transcriptional termination sequences.

[0333] Expression vectors are transfected into the above-mentioned mammalian host cell lines, e.g. HEK293 or CHO cells. Fusion proteins are expressed in the host cell lines, collected and purified.

[0334] The fusion proteins are expressed with and without tags. Properly folded TRAIL moiety allows purification of the fusion proteins on Ni-NTA column. This a unique property of TRAIL (which extends to its fusion proteins); Ni-NTA column is usually used to purify his-tagged proteins.

[0335] Fusion proteins between the extracellular domains of human TRAIL (residues 114-281) and OX40L (residues 51-183) (SEQ ID NOs: 13 and 14) were cloned with a signal peptide (for extracellular secretion), expressed in CHO cells, and purified using IMAC (properly folded rhTRAIL gets retained on Ni-NTA column) followed by SEC polishing. Fusion proteins consisting of the extracellular domains of human TRAIL (residues 114-281) and CD137L (residues 51-183) (SEQ ID NOs: 9-12) were cloned with His-tag, expressed in E.coli, and purified according to routine techniques (IMAC followed by SEC).

Example 2. Methods to evaluate functionality of fusion proteins

[0336] The fusion proteins were tested for direct anti-cancer activity that should arise from their TRAIL moiety. To assess effect of the fusion proteins on cancer cell growth, viability, and apoptosis induction, various cancer cell lines were plated in 96-well plates and treated with the fusion proteins. The suspension cell lines were treated right after plating, while the adherent cells were allowed to attach overnight prior to addition of an investigational protein therapeutic or a control treatment to the cultures. [0337] Relative cell viability was calculated by dividing the cell viability signal from a test sample by that of a non-treated control. Cell viability was determined with CellTiter-Glo 2.0 (Promega) and luminescence measured using a plate reader. For evaluation of apoptosis induction, caspase 3/7 activation was assessed using Promega’s caspase 3/7 Gio reagent and luminescence readout by a plate reader. See FIGs. 3 and 6.

[0338] Alternatively, viable and dead cells were analyzed after treatment using flow cytometry after staining cells with SYTOX™ AADvanced™ (ThermoFisher Scientific) according to manufacturer’s instructions. Data reporting may include dead cells or live cells as a percentage of total (all cells, dead and live). See FIG. 5.

[0339] Activity of OX40L and CD137L moieties were determined in HEK293 NF-KB luciferase reporter cell lines expressing 0X40 and CD137 respectively. Cells were seeded into white clear-bottom 96-well microplates and allowed to attach overnight. The next day test protein or reference control were added to the cells and the cells were incubated at 37°C and 5% CO2 for 5-6 hours. After treatment incubation, cells were lysed, and the luciferase assay was performed using ONE-Step luciferase assay system. Luminescence was measured using a luminometer. The fold induction in the presence of each treatment agent (e.g., fusion protein) was calculated according to the following equation: Fold induction = (L-Lb)/(Lt-Lb), where L= the luminescence intensity in the presence of treatment, Lb= the luminescence intensity in the absence of cells, and Lt = the luminescence intensity in the absence of treatment. See FIGs. 4 and 7.

[0340] Fusion proteins containing both TRAIL and either CD137L or OX40L moieties can be tested in a co-culture assay of primary T cells and cancer cells an example of which is described below.

[0341] Co-culture assay: Cancer cells expressing TRAIL receptors DR4 and/or DR5 (e.g. RPMI-8226, Colo205, HCT116, Jurkat) are cultured with purified T cells or PBMCs at various ratios of cancer (target) cells and effector cells (primary immune cells such as T cells or PBMCs) in a multi-well plate. Prior to addition to the co-culture T cells (purified or within PBMCs) are pre- activated with anti-CD3 and anti-CD28 antibodies or phytohemagglutinin or another activating agent for two days. Serially diluted Fusion Protein or control proteins are added to the co-culture system for 24-72 hours. Readouts may include measurements of cancer cell viability, cytokines secreted into the medium and T cell proliferation after 3 day incubation.

[0342] Results shown in FIGs. 3A-3D depict the effects of the exemplary TRAIL-OX40L (AB001) and OX40L-TRAIL (AB002) fusion polypeptides (SEQ ID NOs: 13 and 14) on caspase 3/7 induction (Fig. 3A-3B) and relative cell viability (Figs. 3C-3D) in HCT116 (Figs. 3A and 3C) and Colo 205 (Figs. 3B and 3D) colon cancer cell lines, relative to rhTRAIL. This data demonstrate that TRAIL activity is improved in TRAIL-OX40L fusion protein versus OX40L-TRAIL; both were less potent than control rhTRAIL.

[0343] Data in FIG. 4 illustrate OX40L-related activity in the exemplary TRAIL-OX40L (AB001) and OX40L-TRAIL (AB002) fusion polypeptides (SEQ ID NO: 13 and 14) versus rhOX40L. Fusion proteins were superior (both potency and efficacy) in activating 0X40 receptor compared to the control rhOX40L. TRAIL-OX40L (AB001; SEQ ID NO: 13) induced greater maximum activation of 0X40 receptor than OX40L-TRAIL (AB002; SEQ ID NO: 14).

[0344] FIG. 5 illustrates ability of the exemplary fusion proteins versus control proteins to induce cell death in RPMI-8226 cell line. The exemplary fusion proteins tested were TRAIL-OX40L (AB001; SEQ ID NO: 13), OX40L-TRAIL (AB002; SEQ ID NO: 14), TRAIL-CD137L (AB007-L1, AB007-L2, AB007-L3; SEQ ID NOs:9-ll) and CD137L-TRAIL (AB008-L3; SEQ ID NO: 12). Control proteins rhOX40L and rhCD137L had no effect on cell viability (data points overlap with one another). Addition of rhTRAIL in equimolar amounts to either OX40L or CD137L (two concentrations tested) resulted in induction of cell death consistent with the activity of TRAIL-OX40L (AB001) fusion protein. Dose response curves of TRAIL-CD137L (AB007-L1, AB007-L2, AB007-L3) and CD137L-TRAIL (AB008-L3) fusion proteins overlap with one another indicating that linker length or placement of TRAIL moiety at N- or C- terminus did not affect TRAIL-associated activity of the TRAIL-CD137L fusion protein. TRAIL-CD137L fusion proteins were more potent than TRAIL-OX40L fusion proteins in RPMI-8226 cell killing.

[0345] FIGs. 6A-6H illustrate the effects of the exemplary TRAIL-CD137L with varied linker length (AB007-L1-L3; SEQ ID NOs:9-l 1) and CD137L-TRAIL fusion polypeptides (AB008-L3; SEQ ID NO: 12) on caspase 3/7 induction (FIGs. 6A, 6C, 6E and 6G) and relative cell viability (FIGs. 6B, 6D, 6F, and 6H) relative to rhTRAIL in RPMI-8226 (FIGs. 6A and 6B), Colo205 (FIGs. 6C and 6D), HCT116 (FIGs. 6E and 6F), Caov-3 (FIG. 6G and 6H) cell lines. This data demonstrate that placement of TRAIL moiety at N- or C- terminus or flexible linker length within TRAIL-CD137 fusion polypeptide (AB007) did not noticeably affect TRAIL-associated activity. AB007 proteins with varied linker lengths had similar potencies; the linker lengths examined were 5 amino acids (AB007-L1), 15 amino acids (AB007-L2) and 25 amino acids (AB007-L3). TRAIL-CD137 and CD137-TRAIL fusion proteins reduced relative cell viability with potency and efficacy similar to one another and to rhTRAIL in the tested cancer cell lines.

[0346] Data in FIG. 7 illustrate CD137L-related activity in the exemplary TRAIL-CD137L and CD137L-TRAIL fusion polypeptides (SEQ ID NO: 9 -12) versus rhCD137L. Activity of the fusion proteins were similar to one another, and a lot more potent (10-15 fold) than control rhCD137L in activating CD137 receptor. This data demonstrate that the linker length and position of each moiety (N- versus C- terminus) within the fusion polypeptide did not largely affect activity of CD137L moiety.

[0347] FIG. 8 illustrates effect of exemplary fusion polypeptides TRAIL-QX40L (AB001), QX40L-TRAIL (AB002), TRAIL-CD137L polypeptides (AB007-L1, AB007-L2, AB007-L3), and CD137L-TRAIL (AB008-L3) as well as rhCD137L andrhQX40L by themselves or in combination with equimolar amounts of rhTRAIL on pre-activated T cells when co-cultured together with RPMI-8226 cancer cells. T cells from a healthy donor were pre-activated with anti-CD3 and anti- CD28 antibody agonists for 2 days. Pre-activated T cells were stained with CellTrace Violet (Thermo Fisher Scientific) according to manufacturer’s instructions and combined with RPMI 8226 cancer cells at 10: 1 ratio (T:cancer cells). The co-cultures were treated with serially diluted fusion proteins or control ligands (rhCD137L or rhOX40L with and without equimolar amounts of rhTRAIL). TNFa secretion into the media was measured after 24h incubation (FIG.8B) with an ELISA kit from BioLegend according to manufacturer’s instructions. T cell proliferation was evaluated after 72h treatment by flow cytometry (FIG. 8A). Pre-activation of T cells of a healthy donor resulted in their robust proliferation which was slightly increased by the fusion proteins in a concentration dependent manner, this increase was somewhat more pronounced than that induced by the control ligands. There was also a small increase in TNFa secretion after 24h treatment with the highest tested concentrations of the fusion proteins.

Claims

WHAT IS CLAIMED IS:

1. A conjugate comprising: TNF superfamily ligands comprising TRAIL that is covalently linked to CD137L through either C- or N-terminus.

2. A conjugate comprising: TNF superfamily ligands comprising TRAIL that is covalently linked to OX40L through either C- or N-terminus.

3. The conjugates of any one of claim 1-2, wherein the TNF superfamily ligand comprises, consists, or consists essentially of an amino acid sequence that is at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from Table T2.

4. The conjugate of any one of claims 1-3, wherein the TNF superfamily ligand is a trimeric or homotrimeric polypeptide.

5. The conjugate of any one of claims 1-4, wherein TRAIL and CD137L are separated by a linker, optionally a physiologically-stable linker.

6. The conjugate of any one of claims 1-4, wherein TRAIL and OX40L are separated by a linker, optionally a physiologically-stable linker.

7. The conjugate of any one of claims 5-6, wherein the linker is a peptide linker, optionally a flexible peptide linker or a rigid peptide linker.

8. The conjugate of claim 7, wherein the peptide linker is about 1-100 amino acids, about 1- 90 amino acids, about 1-80 amino acids, about 1-70 amino acids, about 1-80 amino acids, about 1-50 amino acids, about 1-40 amino acids, about 1-30 amino acids, about 1-20 amino acids, about 1-10 amino acids, or about 1-5 amino acids in length, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 ,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, or 100 amino acids in length.

9. The conjugate of any one of claim 5-8, wherein the peptide linker is selected from Table L1.

10. The conjugate of any one of claims 1-9, wherein the conjugate is a fusion polypeptide.

11. The conjugate of claim 10, wherein TRAIL is fused to the N-terminus or C-terminus of CD137L, optionally separated by a linker.

12. The conjugate of claim 10, wherein TRAIL is fused to the N-terminus or C-terminus of OX40L, optionally separated by a linker.

13. The conjugates of claim 5-6, wherein the linker is a non-peptide linker.

14. The conjugate of any one of claims 1-13, wherein the conjugate has improved pharmacokinetic, physical, and/or biological properties relative to the TRAIL alone and/or OX40L alone and/or CD137L alone, optionally selected from one or more of increased stability, increased serum half-life, increased bioavailability, increased biological activity, increased exposure, and decreased clearance. The conjugate of claim 14, wherein the conjugate has increased stability and/or serum half- life relative to the TNF superfamily ligand alone, optionally wherein the stability and/or serum half-life relative of the conjugate is increased by about or at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000% or more relative to the TNF superfamily ligand alone. The conjugate of claim 14, wherein the conjugate has increased biological activity relative to TRAIL alone and/or OX40L alone and/or CD137L alone, optionally wherein the biological activity of the conjugate is increased by about or at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000% or more relative to the TRAIL alone and/or OX40L alone and/or CD137L alone, or optionally wherein the biological activity is increased synergistically relative to the TRAIL alone and/or CD137L alone and/or OX40L alone. The conjugate of claim 16, wherein the biological activity is induction of cell death or apoptosis in cancer cells, which is optionally increased relative to TRAIL alone. The conjugate of claim 17, wherein the cancer cells are optionally selected from one or more of breast cancer cells, colon cancer cells, multiple myeloma cells, pancreatic cancer cells, ovarian cancer cells and NSCLC cells. A conjugate comprising of two functional components, a trimeric polypeptide that is covalently linked to another trimeric polypeptide. Each polypeptide can be at either N- , C- terminus. The conjugate of the claims 19, wherein the trimeric polypeptide is a homotrimeric polypeptide. The conjugate the claim 19 or 20, wherein the trimeric polypeptide is selected from a Tumor Necrosis Factor (TNF) superfamily ligand. An isolated polynucleotide which encodes a conjugate of any one of claims 1-21. A therapeutic composition, comprising a conjugate of any one of claims 1-21, and a pharmaceutically acceptable carrier or excipient. The therapeutic composition of claim 23, where the conjugate as is at least about 95% pure and less than about 5% aggregated. A method of treating, ameliorating the symptoms of, or reducing the progression of a cancer in a subject in need thereof, comprising administering to the subject the therapeutic composition of claim 23 or 24.

26. The method of claim 25, wherein the cancer is selected from one or more of breast cancer (including triple negative), ovarian cancer, colorectal cancer, non-small cell lung cancer (NSCLC), kidney cancer, hepatocellular carcinoma (HCC), melanoma, metastatic melanoma, multiple myeloma, pancreatic cancer, prostate cancer, small cell lung cancer, mesothelioma, leukemia (including lymphocytic leukemia, chronic myelogenous leukemia, acute myeloid leukemia, relapsed acute myeloid leukemia), lymphoma, hepatoma, sarcoma, B-cell malignancy, gastric cancer, glioma (e.g., astrocytoma, oligodendroglioma, ependymoma, or a choroid plexus papilloma), glioblastoma multiforme (e.g., giant cell gliobastoma or a gliosarcoma), meningioma, pituitary adenoma, vestibular schwannoma, primary CNS lymphoma, primitive neuroectodermal tumor (medulloblastoma), bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancers, cervical cancer, testicular cancer, and stomach cancer.

T1. A pharmaceutical composition comprising a therapeutically effective amount of a conjugate of any one of claims 1-21.

28. A conjugate according to any one of claims 1-21, for use in a method for treating cancer.

29. A method of making a conjugate comprising TNF superfamily ligands comprising TRAIL that is covalently linked to CD 137L or OX40L through either C- or N-terminus, the method comprising:

(i) transforming into a host cell exogenous nucleic acids that encode one or more of CD137L or a portion thereof, OX40L or a portion thereof, and TRAIL or a portion thereof; and

(ii) culturing the host cell under conditions that allow the conjugate to be expressed.

30. The method of claim 29, wherein the CD137L or a portion thereof comprises the receptor binding domain of CD137L, and wherein the OX40L or portion thereof comprises the receptor binding domain of OX40L.

31. The method of claim 29, further comprising purifying the conjugate in the host cell.

32. The method of claim 29, further comprising purifying the conjugate in a cell-free system.

33. A method of stimulating a T cell response comprising: administering to a cell a fusion protein comprising TRAIL and at least one of (i) a CD 137 agonist and (ii) an 0X40 agonist.

34. The method of claim 33, wherein the CD137 agonist comprises a CD137L or a portion thereof, and wherein the 0X40 agonist comprises an OX40L or a portion thereof.

35. The method of claim 34, wherein the CD137L or a portion thereof comprises a receptor binding domain of CD137L, and wherein the OX40L or a portion thereof comprises a receptor binding domain of OX40L.

36. The method of claim 33, wherein the fusion protein comprises a receptor binding domain of TRAIL and a receptor binding domain of CD137L.

37. The method of claim 33, wherein the fusion protein comprises a receptor binding domain of TRAIL and a receptor binding domain of OX40L. 38. An article of manufacture comprising a pharmaceutical composition of claim 27, a container, and a package insert or label indicating that the pharmaceutical composition can be used to treat cancer.