The present invention relates generally to use of Toll-like receptor 5 (TLR5) and agonists thereof for immunological based therapies.

BACKGROUND OF THE INVENTION Inhibition of innate immune functions has been long recognized as a therapeutic strategy to inhibit inflammation. As innate immunity is primarily initiated by Toll like receptors (TLRs), inhibition of TLR signaling remains a major focus for several pharmaceutical industries. In contrast, activation of TLRs is generally not desirable due to potential inflammatory responses elicited by TLR ligands. While recent understanding of the diverse nature of TLRs and differential responses to their ligands indicates that activation of specific TLRs can confer beneficial effects, there is an ongoing need for improved compositions and methods for TLR activation in the therapeutic context.

BRIEF DESCRIPTION OF THE DRAWINGS Fig.l provides a visual representation of construction and structure of secreted flagellin derivative. A. Three dimensional structure of flagellin, location of structural (DO, Dl, D2, and D3) and functional (CI and C2) domains, and design of CBLB502 polypeptide, (b) Sequence of CBLB502 polypeptide. The sequence of the linker region is in pink; N- and C- term fragments of flagellin are colored green and blue respectively. Fig. 2 provides a map of single-color lentiviral TR vector. Transcription factor-specific transcriptional response elements (TRE) can be cloned into polylinker upstream of mCMV promoter to provide transcriptional factor-dependent expression of GFP reporter.

Fig.3. provides a summary of data obtained using NFkB-GFP reporter-293 cells, (a) Induction of GFP in Nf-kB reporter clone- 3 by TNF and IL1 Control + CBLB502 +TNF. (b) Lack of GFP induction by CBLB502 in NF-kB reporter clone-3. (c). Selection of TLR5 responding clones: reporter clone- 3 was transduced with lentivirus expressing TLR5 and after 48h of transduction single cell cloning was done by limiting dilution. Several clones were expanded and induced with CBLB502 (5ng/ml). Percentage of GFP positive cells were assessed by flow cytometry. Clone- 14 showed over 50 fold induction of GFP.

Fig. 4 provides a map of pBASP lentiviral vector as one embodiment of the invention.

Fig. 5 illustrates sequences used for optimization of the secretion signal for the TLR5 agonist.

Fig. 6 provides a graphical representation of results obtained from assessment of secretion of CBLB502. HEK-293 NFkB-GFP reporter cells were incubated with indicated dilutions of the conditioned media from cells expressing SEAPCBLB502 or cells expressing empty vector and percentage of GFP expressing cells was assessed by flow cytometry. Fig. 7 shows nucleotide and amino acid sequence for representative flagellin variants that can be used in the invention. The pRSETb leader sequence is shown in Italic (leader includes Met, which is also amino acid 1 of FliC). The N terminal constant domain is underlined. The amino acid linker sequence is in Bold. The C terminal constant domain is underlined. GST, if present, is highlighted. Fig. 8 shows the nucleic acid and amino acid sequence for a human Toll-like receptor 5 protein.

Fig. 9 depicts a recombinant adenovirus encoding a TLR5, but not a TLR5 agonist. This embodiment is provided in the context of the pCD515-CMV -hTLR5 expression vector.

Fig. 10 depicts a recombinant adenovirus encoding a secretable TLR5 agonist, but not a TLR5. This embodiment is provided in the context of the pCD515-CMV -Sseap-502 expression vector.

Fig. 1 1 provides a graphical depiction of the domain structure and approximate boundaries (amino acid coordinates) of selected flagellin derivatives.

Fig. 12 provides a graphical summary of results obtained by measuring the ratio of tumor volume in mice over a number of days in tumor cells (A549) transduced with a control vector (without TLR5) or vector expressing TLR5 wherein the mice are treated three days with either CBLB502 or PBS. DESCRIPTION OF THE INVENTION

The present invention provides methods for use in immunotherapy, and particularly for immunotherapy of cancer. Certain aspects of the invention are based in part on our discovery that, by conferring tumor cells with the capability to express a Toll-like receptor (TLR), the cells become sensitized to certain TLR agonists such that the immunosuppressive quality of the tumor environment is inhibited and/or reversed. Certain aspects of research related to the invention are described in WO/2011/044246, the entire disclosure of which is incorporated herein by reference.

In general, in one aspect, the invention provides compositions comprising polynucleotide sequences encoding a TLR5. In particular embodiments, the polynucleotides are provided in an expression vector that is adapted for use in therapeutic methods. In one embodiment, the expression vector encodes a TLR5, but does not encode a TLR5 agonist. In another embodiment, the invention provides an expression vector which encodes a secretable agonist of TLR5, but does not encode a TLR5. Methods of using each of these types of expression vectors for prophylaxis and/or therapy, particularly for individuals in need of therapy for cancer, are also provided.

In preferred embodiments, the expression vector encoding a TLR used in the invention encodes a human Toll-like receptor 5 (TLR5) or a derivative thereof. A representative nucleic acid and its encoded amino acid sequence for human Toll-like receptor 5 protein is presented in Figure 8. All nucleotide sequences encoding the amino acid sequence presented in Figure 8 are encompassed within the scope of this invention. Further, all modifications to the human TLR5 amino acid sequence which do not materially affect the function of the TLR5 are included within the scope of the invention, as are all nucleotide sequences which encode such modified TLR5 amino acid sequences. Modified TLR5 proteins which can be encoded by expression vectors and used in the method of the invention, and therefore are considered to not have materially altered function, include but are not necessarily limited to TLR5 proteins which recognize bacterial flagellin, or fragments or derivatives thereof. Thus, any TLR5 for which bacterial flagellin can act as an agonist is included within the scope of the present invention. In one embodiment, a TLR5 that is suitable for use in the invention can be identified by determining that binding of a known TLR5 agonist to it mobilizes the nuclear factor NF-κΒ in the cell that expresses the TLR5, and/or stimulates tumor necrosis factor-alpha production by such a cell. In certain embodiments, the TLR5 agonist used in the invention can comprise or consist of the sequences described in Figure 1 or Figure 7.

In various embodiments, the nucleic acids provided by the invention and which encode a TLR5 or a secreatable TLR5 agonist are present in viral vectors which are suitable for insertion into mammalian cells. In one embodiment, the viral vectors are suitable for direct injection into an area of tissue where a localized therapeutic or prophylactic effect is desired. Examples of suitable vectors include but are not limited to vectors derived from adenovirus, adeno-associated virus, retroviruses (e.g, lentiviruses, Rhabdoviruses, murine leukemia virus), herpes virus, and the like. The tropism of the viral vectors can be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses, or by modifying the virus to include any other component that is desirable for targeted expression of the TLR5 or the secreted TLR5 agonist.

Compositions comprising DNA polynucleotides which encode a TLR5 or an agonist of it can be prepared using any acceptable technique, such as by amplification using appropriate primers and inserting the amplification products into expression vectors. Suitable expression vectors for using the DNA polynucleotides which encode the TLR5 or an agonist of it can comprise appropriate eukaryotic and/or vial transcription and translation signals, and may contain additional elements, such as polyadenylation sites, internal ribosome entry sites, etc. In one embodiment, the polynucleotide sequence encoding the TLR5 is not present in a bicistronic region of a polynucleotide. In another embodiment, the polynucleotide sequence encoding the TLR5 agonist is not present in a bicistronic region of a polynucleotide.

In one embodiment, an expression vector encoding a TLR5 or a TLR5 agonist is provided in a recombinant adenovirus context. A particular embodiment of a recombinant adenovirus encoding a TLR5 suitable for use in the method of the invention is depicted in Figure 9, which provides a graphical representation of the pCD515-CMV -hTLR5 expression vector. This recombinant adenovirus construct expresses human TLR5 comprising the sequence presented in Figure 8. The adenovirus construct comprises a strong cytomegalovirus (CMV) promoter cloned upstream of the human TLR5 (hTLR5) cassette and thus is suitable for facilitating constitutively high expression of TLR5 in cells into which this representative construct is introduced. A similar expression vector configuration, but adapted for expressing a secretable form of TLR5 agonist (CBLB502) is presented in Figure 10. This vector is termed pCD515- CMV -Sseap-502. Expression vectors encoding the TLR5 or the TLR5 agonist can be formulated in any pharmaceutically acceptable preparation for administration to individuals in need of prophylaxis or therapy of any disease or disorder, infection, etc., wherein localized expression of TLR5 or a secreted form of TLR5 agonist is desired. Accordingly, the DNA polynucleotides of the present invention can be administered by any means known in the art. For treatment of cancer, a preferred route is intratumoral injection. However, other delivery techniques or any reagent or composition that can assist in targeting delivery of the nucleic acids to a site where expression of TLR5 or a TLR5 agonist is desired can be used. In various embodiments, suitable delivery reagents for administration of the compositions in the invention include but are not limited to the Minis Transit TKO lipophilic reagent; lipofectin; lipofectamine; cellfectin; or polycations (e.g., polylysine), liposomes and microsomes or expression vectors based on retroviruses, lentiviruses, adenoviruses and adenoassociated viruses.

The invention can be used for prophylactic purposes (i.e., vaccination) or therapeutic (i.e., treatment of disease and/or infection or other disorders). In various embodiments, the invention provides methods for enhancing cancer vaccines.

In one embodiment, the invention is used to inhibit or treat ischemia-reperfusion injury.

In one aspect the invention involves introducing a recombinant polynucleotide encoding a TLR5 to one or more tumor cells in an individual in need of cancer prophylaxis and/or therapy and, concurrently or subsequently administering to the individual a TLR5 agonist. In this regard, the design and use of the representative expression vectors disclosed herein demonstrates that recombinant expression of a TLR5 can sensitize cells to a TLR5 agonist. (See, for example, Figure 12). Thus, by performing the method of the invention to selectively engineer tumor cells to express TLR5 (i.e., by intratumoral injection of an adenovirus encoding TLR5), the tumor cells are sensitized to a TLR5 agonist, and this aspect of the invention along with data summarized in Figure 12 support the feasibility of therapeutic approaches that utilize exogenously provided TLR5 agonist polypeptides as an immunotherapeutic modality.

In certain embodiments, the TLR5 agonist is provided as a polypeptide component of a pharmaceutical preparation. The TLR5 agonist can comprise or consist of a flagellin protein or a fragment or derivative thereof. Non-limiting examples of flagellin proteins and derivatives thereof are known in the art. For example, suitable flagellin proteins and derivatives thereof are described in U.S. Patent No. 7,638,485, the entire disclosure of which is incorporated herein by reference. In one embodiment, the flagellin protein derivative that is used in the invention is the Salmonella flagellin derivative known in the art as CBLB502, which is also described in U.S. Patent No. 7,638,485 and is presented here in Figure 1.

As an alternative to providing a TLR5 agonist as a polypeptide component of a pharmaceutical preparation, the invention provides for administering to cancer cells an expression vector which encodes a secretable form of a TLR5 agonist. Thus, in one aspect, the invention provides for conferring the capability of tumor cells, and/or cells proximal to tumor cells, to express a secreted form of a TLR agonist, thereby facilitating activation of TLRs via local TLR-stimulation, which will result in inhibition and/or reversal of the immunosuppressive tumor environment. Figure 10 provides a representation of an illustrative expression vector that is suitable for such use.

With respect to conferring to cells the capability to secrete a TLR5 agonist, it is expected that any peptide signal that facilitates secretion of a protein can be operably linked to the TLR5 agonist to arrive at biologically active secreted (extracellular) peptides (BASP) that would be suitable for use in the invention. Non-limiting examples of such secretion signals include the IL-1 -signal sequence and derivatives thereof, the CD 14 signal sequence and derivatives thereof, and secreted placental alkaline phosphatase (SEAP). In one embodiment, the secretion signal is a truncated form of SEAP, whereby removal of the transmembrane domain of the protein allows it to be secreted from the cells into the surrounding environment.

With respect to the TLR5 agonist CBLB502, it is currently in Phase I human trials and is being developed as radiomitigator and radioprotector for biodefense applications. Without intending to be constrained by theory, our work shows that CBLB502, binds to TLR5 and activates the NF-κΒ pro-survival pathway, which protects from IR damage. Signal transduction by TLR5 involves interaction of its cytoplasmic TIR (Toll IL-1 -receptor homology) domain with the TIR domain of the cytoplasmic adaptor protein MyD88. MyD88 signals through other adaptor molecules, such as IRAK and TRAF6, and causes activation of ΙκΒ kinase (IKK), which in turn leads to NF-kB activation and transcriptional upregulation of pro-survival and inflammatory effector genes. In the classical NF-κΒ activation pathway, the p65 (RelA)/p50 heterodimer is sequestered in the cytoplasm by ΙκΒ. Phosphorylation of ΙκΒ by IKK and the subsequent degradation of ΙκΒ leads to NF-κΒ nuclear translocation and transcriptional activation of NF-κΒ controlled genes. The protective role of NF-κΒ is mediated by transcriptional activation of multiple genes coding for: a) anti-apoptotic proteins that block major apoptotic pathways, b) cytokines and growth factors that induce proliferation and survival of BP and other stem cells, and c) potent ROS-scavenging antioxidant proteins, such as MnSOD (SOD-2).

For administration, compositions of the invention can be combined with standard pharmaceutical carriers. Acceptable pharmaceutical carriers for use with proteins are described in Remington's Pharmaceutical Sciences (18th Edition, A. R. Gennaro et al. Eds., Mack Publishing Co., Easton, Pa., 1990.

The presently provided compositions can be mixed with chemotherapeutic agents, adjuvants, and/or combined with other compositions and used with other techniques, such as surgical or radiological interventions for treatment of a variety of disease, including cancers.

Description of secretable TLR5 agonist. Salmonella flagellin is a strong activator of antiapoptotic NF-κΒ pathway. Based on the analysis of the crystal structure of the F41 proteolytic fragment, Salmonella flagellin is a boomerang-shaped protein with four major domains: DO, Dl, D2 and D3. The DO (random coil) and Dl (a helix) domains located at the N- and C-terminus are mainly responsible for motility and signaling, and are comprised of highly conserved stretches of amino acids (CI and C2 domain in Fig. la). As demonstrated by several research groups the central D2 and D3 domains are highly hypervariable and can be removed without any significant effect on proinflammatory activity induced by flagellin. The D0-D1 domains are conformationally flexible structures that are stabilized by extended interactions between three a-helices in the Dl domain. The TLR5 extracellular domain contains 19-25 copies of a leucine-rich repeat motif (LRR). Based on these structural features of flagellin, we engineered CBLB502 by fusion of the minimal N-terminal fragment comprising of amino acid residues 1-176 and C- terminal fragment comprising of amino acid residues 402-505 using a flexible 15 amino acid linker. The sequence of CBLB502 is depicted in Fig. IB.

Development of Nf-kB transcriptional reporter cell line. In order to develop a readout system for assessment of functional activity of CBLB502 and to facilitate functional screening of new TLR5 agonists, we developed NF-kB transcriptional reporter cell line. First, we have developed a basic, single-color lentiviral transcriptional reporter (TR) vector with destabilized copGFP reporter gene under the control of minimum CMV (mCMV) promoter (Fig.2 ), and utilized this vector for the construction of stable TR cell lines. The lentiviral TR vector is based on third generation, self-inactivated HIV -based vectors, and comprises all necessary functional elements (GAG, RRE, cPPT) necessary for transcription of viral RNA, and packaging of TR constructs in the form of viral genomic RNA into pseudoviral particles. After transduction of these particles into target cells, the viral genomic RNA TR construct is reverse-transcribed into double-stranded DNA, and integrated into host cell genomic DNA, providing long-term expression of the reporter construct in the host cell.

In order to develop the TLR5 -dependent, NF-kB-responsive reporter cell line, we cloned four repeats of the 10 bp NF-kB consensus response element upstream of the mCMV promoter and transduced a packaged pTR-NF-kB-mCMV-dsGFP construct into HEK 293 cells. Then we single cell cloned the transduced cells and selected a clone that exhibited very low basal NFkB activity, but robust activation of NFkB driven GFP induction upon treatment with TNF or IL-1 (Fig.3a). This clone (clone 3) did not respond to CBLB502 (Fig. 3b) due to lack of TLR5 expression in these cells. In order to provide TLR5 -dependent modulation of NF-kB signaling pathway, we cloned the TLR5 cDNA into lentiviral vector and transduced the TLR5 construct into the 293-NF-kB-GFP reporter cells. We next measured the basal and activated levels of GFP reporter expression in HEK293-TLR5-NF-kB-GFP reporter cells after treatment with CBLB502 in several selected clones by flow cytometry. We observed that the HEK293-TLR5NF-kB-GFP TR cell line (clone 14) demonstrates high CBLB502-dependent activation of the GFP reporter gene (Fig 3 c).

Construction of lentiviral vector expressing secreted form for CBLB502 For effective use of CBLB502 as a pharmacological agent for immunotherapy, in one embodiment, CBLB502 can be expressed in the tumor microenvironment such that it is accessible to TLR5 receptors on cell surface. This can be achieved by fusion of a secretion signal to CBLB502. In order to select an optimal signal sequence for peptide secretion, we developed four novel lentiviral secretion vectors containing an IL-1 -signal sequence (SI), an improved mutant form of the IL-1 -signal sequence (S2), a secreted alkaline phosphatase (S3), and a CD 14 signal sequence (S5) in Xbal/BamHI sites of a pR-CMV vector downstream of CMV promoter followed by Kozak sequence and an ATG initiation codon. We then cloned the full-length cDNAs of TNF Da, IL-1 a, and flagellin (CBLB502) in-frame into EcoRI/Sall sites downstream of each of the four lentiviral secretion vectors (Fig. 4, Fig. 5). We transduced HEK293 cells with all 12 packaged constructs, replaced the media after 24h and, after one passage (to ensure that all residual virus particles were removed), the plates were seeded with 293-NFkB-GFP reporter cells. After 24h, NF-kB activation in 293-NFkB-GFP by the control proteins (TNF, IL-1, and CBLB502) secreted by HEK293 cells was analyzed by fluorescence microscopy (GFP induction). The pR- CMV-S3 vector with the secreted alkaline phosphatase signal sequence (SEAP) provided the most efficient secretion of all three proteins. Thus we chose cells expressing CBLB502 fused to

SEAP sequence for further analysis. As shown in Fig. 6, supernatant from cells expressing

SEAP-CBLB502 stimulated NFkB-GFP -reporter cells in a dose dependent manner, while supernatant from cells transduced in parallel with control secretary vector did not induce GFP. In order to establish the efficacy of TLR5 agonists for cancer immunotherapy, we have also constructed adenoviral vectors that express secreted form for CBLB502 for in vivo experiments.

Although the invention has been described in detail for the purposes of illustration, it is understood that such detail is solely for that purpose, and variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention which is defined by the following claims.