COMPOSITIONS AND METHODS FOR IDENTIFYING AND TREATING CACHEXIA OR PRE-CACHEXIA

Abstract

The invention provides therapeutic, engineered protein/peptide compositions comprising e.g., RAGE antibodies, T-cell receptors to target a RAGE receptor (including soluble forms thereof) directly and/or via differential competition with one or more pre-cachexia and/or cachexia-associated RAGE ligands or markers.

Claims

1. An antibody specific for a RAGE receptor, which antibody competes for binding to the RAGE receptor with a RAGE ligand selected from S100A7, S100A8, S100A9 and/or Calprotectin.

2. Antibody according to claim 1, wherein the antibody is selected from an IgG, IgA, or an antigen binding antibody fragment selected from an antibody single variable domain polypeptide, dAb, FAb, F(ab′)2, an scFv, an Fv, or a disulfide-bonded Fv.

3. Antibody according to claim 1, which binds to a RAGE receptor polypeptide with a dissociation coefficient of 10 m or less.

4. Antibody according to claim 3, which binds to a RAGE receptor polypeptide with a dissociation coefficient of lnm or less.

5. An antibody according to claim 1 which binds to an epitope on the RAGE receptor selected from an epitope which is bound by S100A7, S100A8, S100A9 or Calprotectin.

6. An antibody according to claim 5, which binds to a RAGE epitope bound by S100A7, S100A8, S100A9 and/or Calprotectin by mimicking the linear epitope defined by any one of residues 41-93 in SEQ ID NO: 1 (S100A7); residues 36-89 in SEQ ID NO: 2 (S100A8); or residues 45-98 in SEQ ID NO: 3 (S100A9); or binds to a RAGE epitope bound by S100A7, S100A8, S100A9 and/or Calprotectin by mimicking the conformational epitope represented by helix 2A formed by residues 41-53 in S100A7, residues 43-49 in S100A8 or residues 47-55 in S100A9; does not mimic the epitope defined by residues 43-78 in SEQ ID NO: 9 (S100B).

7. The antibody of claim 1, which binds to a linear epitope which is selected from the linear epitope defined by residues 41-93 in SEQ ID NO: 1 (S100A7), residues 36-89 in SEQ ID NO: 2 (S100A8), residues 45-98 in SEQ ID NO: 3 (S100A9) or residues 43-78 in SEQ ID NO: 9 (S100B) , and does not bind to the S100B epitope defined by residues 43-78 in SEQ ID NO: 9 (S100B), or which binds to the conformational epitope formed by residues 41-53 on S100A7, residues 43-49 on S100A8, residues 47-55 on S100A9 or residues 40-53 on S10A15 and does not bind to the conformational epitope formed by residues 43-78 on S100B.

8. A method of treating pre-cachexia, the method comprising administering to the subject an effective amount of an antibody according to claim 1.

9. A method of inhibiting the progression of pre-cachexia to cachexia in a subject, the method comprising administering to the subject an effective amount of an antibody according to claim 1.

10. A method of treating or preventing undesirable muscle or fat loss in a cancer patient, the method comprising administering to the subject an effective amount of an antibody according to claim 1.

11. A method according to claim 8, wherein the antibody binds to a RAGE epitope bound by S100A7, S100A8, S100A9 and/or Calprotectin by mimicking the linear epitope defined by any one of residues 41-93 in SEQ ID NO: 1 (S100A7); residues 36-89 in SEQ ID NO: 2 (S100A8); or residues 45-98 in SEQ ID NO: 3 (S100A9); or binds to a RAGE epitope bound by S100A7, S100A8, S100A9 and/or Calprotectin by mimicking the conformational epitope represented by helix 2A formed by residues 41-53 in S100A7, residues 43-49 in S100A8 or residues 47-55 in S100A9; does not mimic the epitope defined by residues 43-78 in SEQ ID NO: 9 (S100B); or binds to a linear epitope which is selected from the linear epitope defined by residues 41-93 in SEQ ID NO: 1 (S100A7), residues 36-89 in SEQ ID NO: 2 (S100A8), residues 45-98 in SEQ ID NO: 3 (S100A9) o residues 43-78 in SEQ ID NO: 9 (S100B), and does not bind to the S 100B epitope defined by residues 43-78 in SEQ ID NO: 9 (S100B), or which binds to the conformational epitope formed by residues 41-53 on S100A7, residues 43-49 on S100A8, residues 47-55 on S100A9 or residues 40-53 on S10A15 and does not bind to the conformational epitope formed by residues 43-78 on S100B.

12. The method of claim 8, wherein the subject is pre-selected as having a molecular signature indicative of pre-cachexia or cachexia by detecting an alteration in at least three markers selected from the group consisting of S100A2 or S100A4; S100A8 or S100A9; and S100A7; detecting an alteration in at least four markers selected from the group consisting of: S100A2 or S100A4; S100A8 or S100A9; S100A7; and S100A14; or measuring the level of at least five markers selected from the group consisting of: S100A2 or S100A4; S100A8 or S100A9; S100A7; S100A14; and S100P, thereby pre-selecting the patient as having a molecular signature indicative of pre-cachexia or cachexia.

13. A method of inhibiting the loss of myosin heavy chain in a myocyte, the method comprising contacting the myocyte with an effective amount of an antibody according to claim 1.

14. A method of inhibiting lipolysis in an adipocyte, the method comprising contacting the adipocyte with an effective amount an antibody according to claim 1.

15. A method of inhibiting atrophy in a cell, the method comprising contacting the adipocyte or myocyte with an effective amount ofan antibody according to claim 1.

16. The method of claim 13, wherein the myocyte, adipocyte, or cell is in vitro or in vivo.

17. The method of claim 13, wherein the myocyte, adipocyte, or cell is present in a subject identified as having at least one cancer.

18. The method of claim 17, wherein the cancer includes but is not limited to one or more carcinomas and sarcomas and cancers of the skin, pancreas, stomach, colon, thorax, liver, gallbladder, musculoskeletal system, breast, lung, ovary, uterus, endometrium, prostrate, colon, skin, mouth, salivary, esophagus, head and neck, plus other tumors of the gastrointestinal tract.

19. A method according to claim 13, wherein the antibody binds to a RAGE epitope bound by S100A7, S100A8, S100A9 and/or Calprotectin by mimicking the linear epitope defined by any one of residues 41-93 in SEQ ID NO: 1 (S100A7); residues 36-89 in SEQ ID NO: 2 (S100A8); or residues 45-98 in SEQ ID NO: 3 (S100A9); or binds to a RAGE epitope bound by S100A7 , S100A8, S100A9 and/or Calprotectin by mimicking the conformational epitope represented by helix 2A formed by residues 41-53 in S100A7, residues 43-49 in S100A8 or residues 47-55 in S100A9; does not mimic the epitope defined by residues 43-78 in SEQ ID NO: 9 (S100B); or binds to a linear epitope which is selected from the linear epitope defined by residues 41-93 in SEQ ID NO: 1 (S100A7), residues 36-89 in SEQ ID NO: 2 (S100A8), residues 45-98 in SEQ ID NO: 3 (S100A9) or residues 43-78 in SEQ ID NO: 9 (S100B), and does not bind to the S100B epitope defined by residues 43-78 in SEQ ID NO: 9 (S100B), or which binds to the conformational epitope formed by residues 41-53 on S100A7, residues 43-49 on S100A8, residues 47-55 on S100A9 or residues 40-53 on S100A15 and does not bind to the conformational epitope formed by residues 43-78 on S100B.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0098] FIG. 1 is a graph showing the differential cachexia-inducing activity of r-S100 faimily proteins. All rpooteins are tested in HBSS buffer. Myosin HC is measured in an in-cell Western assay.

[0099] FIG. 2 is a graph showing that S100B has little or no activity in inducing cachexia in the assay used in FIG. 1.

[0100] FIG. 3 is a graph conmparing S100A7, S100A8/9 and HMGB1 in a cachexia assay as in FIG. 1.

[0101] FIG. 4 is a graph comparing S100A12, S100A13, S100A15, S100A5 and S00P in a cachexia assay.

[0102] FIG. 5 is a figure indicating Helix 2A, a unique helix fouind in cachexia-inducing S100 ligands.

[0103] FIG. 6 is a comparison of sequences of S100 family proteins (SEQ ID NOS 1-11, respectively, in order of appearance).

[0104] FIG. 7 is a graph and diagram indicating the effect of anti-RAGE antibodies on cachexia induction by cachexia-inducing media.

[0105] FIG. 8 is a graph showing a comparison of the molecule.

[0106] FIG. 9 illustrates human RAGE antibody which has cachexia-inhibiting activity in mouse skeletal muscle.

[0107] FIG. 10 shows that antibody inhibition of cachexia-inducing S100 proteins has a comparable efficiency in blocking cachexia.

[0108] FIG. 11 is a schematic that illustrates cancer cachexia signaling vs. targeted and/or collateral effects on muscle, fat, and liver.

[0109] FIG. 12 is a schematic of a clinical sample derived phenotypic screening platform for cancer cachexia mediator discovery (Reverse Translational Medicine).

[0110] FIG. 13 is a graph showing the mean tumor-free weight change in cohorts of xenograft mice (n=5 mice per cohort). Human Cancer Cell Lines are classified for Cachexia-inducing Activity in vivo and the most cachexia-inducing cell lines over the 8-week period are shown in red.

[0111] FIG. 14 shows that the in vitro system recapitulates cachexia phenotype in mouse and human muscle. Patient Plasma in vitro muscle signature vs. cachectic patient muscle biopsy signature was compared and the specificity for clinical cachexia is greater than the 99.9% ile. The analysis was performed across 4,815 genetic and small molecule perturbations in Connectivity Map.

[0112] FIG. 15 shows a schematic of Metabolomic Profiling with global high-resolution readout. Metabolomic Profiling Platform analyzes >320 lipid, cationic, and anionic metabolites/sample.

[0113] FIG. 16 shows the Metabolite profiling of mouse plasma shows loss of branched chain AA (an occurrence in late stage cachexia), increased DAGs/TAGs seen clinically. The profiling was conducted via Mass Spectrometry of >320 metabolites.

[0114] FIG. 17 shows that the Metabolite profiling of in vitro models recapitulates many of the features of the in vivo response.

[0115] FIG. 18 shows that a Drug Screen using Cachexia Gene Expression Signature identified a signaling pathway critical to the cachexia phenotype.

[0116] FIG. 19 shows that MEK and ERK inhibitors block cachexia-inducing activity of conditioned media.

[0117] FIG. 20 shows that RAGE ligands are more abundant in cachexia-inducing conditioned media.

[0118] FIG. 21 illustrates that in vitro adipocytes generate relevant lipid metabolites in response to cachexia-inducing conditioned media. This indicates that the induction of cachexia may be targeted to providing certain tumors with the necessary metabolites to sustain growth independent of certain aspects of the cellular machinery establishing a feebback or sustenance cycle.

[0119] FIG. 22 illustrates that soluble RAGE receptor depletes conditioned media of cachexia activity and the eluate reconstitutes cachexia activity further establishing a role for RAGE ligands in pre-cachexia and cachexia.

[0120] FIG. 23 shows that inhibition of RAGE signaling inhibits lipid loss in adipocytes. Inhibition of RAGE signaling is carried out using and anti-RAGE mAb as well as Trametinib (a MEK inhibitor drug).

DETAILED DESCRIPTION OF THE INVENTION

[0121] Disclosed herein are Applicants' novel findings as to the role of particular RAGE ligands in pre-cachexia and/or cachexia. Applicants have demonstrated for the first time the key involvement of specific RAGE ligands of the S100 family in inducing precachexia and/or cachexia in the context of several pathologies, and in particular cancer. Specifically, Applicants have shown that the RAGE ligands S100A7, S100A8, S100A9, and homodimers, heterodimers and heterotetramers of S100A8 and S100A9 (e.g., Calprotectin) are key effector molecules in inducing precachexia and/or cachexia. Importantly, Applicants have discovered that high mobility group box 1 (HMGB1) and S100B do not induce precachexia and/or cachexia. Applicants' novel findings open the door for effectively targeting RAGE in the context of precachexia and cachexia. Applicants disclose herein, inter alia, targeted and efficacious therapeutic engineered biologic compositions for specifically targeting RAGE in a subject at risk of developing pre-cachexia and/or cachexia or a subject suffering from pre-cachexia and/or cachexia. More specifically, the invention disclosed herein provides therapeutic, engineered protein/peptide compositions comprising e.g., anti-RAGE antibodies to target a RAGE receptor (including soluble forms thereof) directly and/or via differential competition with one or more pre-cachexia and/or cachexia-associated RAGE ligands or markers (e.g., a RAGE ligand of the S100 family).

[0122] Notably, patents and published patent publications disclosing anti-RAGE antibodies, some of which are in clinical stage trials, appear to predominantly target HMGB1. Interestingly, a recent publication showed that HMGB1-rage signaling is actually important in maintaining muscle homeostasis. (Riuzzi et al. HMGB1-RAGE regulates muscle satellite cell homeostasis through p38-MAPK- and myogenin-dependent repression of Pax7 transcription. J Cell Sci 2012;125 1440-1454). As noted above, Applicants' functional data shows that HMGB1 is not cachexogenic in muscle. This demonstration by Applicants underscores the key need in the art for and the critical importance for the therapeutic biologic compositions (e.g., anti-RAGE antibodies, anti-RAGE T-cell receptors) disclosed herein, which inhibit target a RAGE receptor directly and/or via differential competition with one or more pre-cachexia and/or cachexia-associated RAGE ligands or markers RAGE ligands (e.g., S100A7, S100A8, S100A9, including homodimers, heterodimers and heterotetramers of S100A8 and S100A9).

[0123] S100 proteins are small (9-13 kDa) acidic regulatory metal-binding proteins and belong to a protein family consisting of at least 24 members functionally distributed into three main subgroups. (see e.g., Donato R. et al. Curr. Mol. Med. 2013 January; 13(1):24-57. Functions of S100 proteins; Marenholz I. et al. Biochem. Biophys. Res. Comm. 2004 Oct. 1; 322(4):1111-22. S100 proteins in mouse and man: from evolution to function and pathology (including an update of the nomenclature)). The functional diversity of S100 proteins is extremely broad, partly due to their ability to adapt to various targets. (see e.g., Permyakov S. E. et al., Mol. BioSyst. 2011 July; 7(7):2164-80. Intrinsic disorder in S100 proteins). The crystal structures of several S100 proteins are known. (see e.g., Brodersen D. E. et al. Biochemistry 1999 Feb. 9; 38(6):1695-704. Zinc-binding site of an S100 protein revealed. Two crystal structures of Ca2+-bound human psoriasin (S100A7) in the Zn2+-loaded and Zn2+-free states; Itou H. et al. J. Mol. Biol. 2002 Feb. 15; 316(2):265-76. The crystal structure of human MRP14 (S100A9), a Ca(2+)-dependent regulator protein in inflammatory process; Ishikawa K. et al., J. Acta Crystallographica Section D Biological Crystallography 2000 May; 56(Pt 5):559-66. The structure of human MRP8, a member of the S100 calcium-binding protein family, by MAD phasing at 1.9 A resolution; Korndorfer I. P. et al. J. Mol. Biol. 2007 Jul. 27; 370(5):887-98. Epub 2007 May 3. The crystal structure of the human (S100A8/S100A9) heterotetramer, calprotectin, illustrates how conformational changes of interacting alpha-helices can determine specific association of two EF-hand proteins; Zhang H. et al. Mol. Biol. 2003 Jan. 24; 325(4):785-94. The crystal structure at 2A resolution of the Ca2+-binding protein S100P) Several S100 proteins bind and activate RAGE. (see e.g., Leclerc E. et al. Biochimica et Biophysica Acta 2009 June; 1793(6):993-1007. Binding of S100 proteins to RAGE: an update; Koch M. et al. Structure 2010 Oct. 13; 18(10):1342-52. Structural basis for ligand recognition and activation of RAGE.).

[0124] Anti-RAGE antibodies are known; see, e.g., WO2008137552, WO2007109747, WO2010019656, WO2009136382, WO 2011053707, WO2011042548, U.S. Pat. No. 8,420,083. Anti-RAGE monoclonal antibodies are known; see, e.g., U.S. Pat. No. 8,420,083, WO2011042548 and WO2008137552. However, these antibodies and monoclonal antibodies compete for binding with a range of RAGE ligands including HMGB1—contrary to and teaching away from, the instant invention. For instance, WO2008137552 specifically involves antibodies that inhibit binding between RAGE and HMGB1. As noted above, Applicant's functional data shows that HMGB1 in not cachexogenic in muscle and therefore HMGB1 is NOT a relevant target RAGE ligand for treating, preventing, alleviating or addressing pre-cachexia and/or cachexia. Accordingly, the anti-RAGE antibodies and monoclonal antibodies of the prior art: are not useful in the practice of the instant invention; fail to teach or suggest the present invention; and moreover, expressly teach away from the instant invention. Again, in embodiments, the present invention provides antibody(ies) specific for a RAGE receptor, which antibody(ies) competes for binding to the RAGE receptor with RAGE ligand(s) S100A7, S100A8, S100A9 and/or Calprotectin, but preferably may not compete for binding to the RAGE receptor with RAGE ligand HMGB1; for example, antibody(ies) that binds to epitope(s) on the RAGE receptor selected from epitope(s) which is/are bound by S100A7, S100A8, S100A9 or Calprotectin, but preferably may not bind to an epitope which is bound by the RAGE ligand HMGB1. The prior art teaches and directs towards antibodies that compete for binding to the RAGE receptor with RAGE ligand HMBG1, and hence, teach away from the instant invention; and the prior art does not teach or suggest antibody(ies) competes for binding to the RAGE receptor with RAGE ligand(s) S100A7, S100A8, S100A9 and/or Calprotectin, but preferably may not compete for binding to the RAGE receptor with RAGE ligand HMGB1, and hence does not teach or suggest the instant invention.

[0125] Thus, as herein discussed, the invention involves an antibody specific for a RAGE receptor, which antibody competes for binding to the RAGE receptor with a RAGE ligand selected from S100A7, S100A8, S100A9 and/or Calprotectin, but preferably may not compete for binding to the RAGE receptor with RAGE ligand HMGB1. The antibody can be is selected from an IgG, IgA, or an antigen binding antibody fragment selected from an antibody single variable domain polypeptide, dAb, FAb, F(ab′).sub.2, an scFv, an Fv, or a disulfide-bonded Fv. The antibody can binds to a RAGE receptor polypeptide with a dissociation coefficient of 1μm or less. The antibody can bind to a RAGE receptor polypeptide with a dissociation coefficient of lnm or less. The antibody can bind to an epitope on the RAGE receptor selected from an epitope, which is bound by S100A7, S100A8, S100A9 or Calprotectin, but preferably may not bind to an epitope which is bound by the RAGE ligand HMGB1. The antibody can bind to a RAGE epitope bound by S100A7, S100A8 or S100A9 and/or calprotectin by mimicking the linear epitope defined by defined by any one of residues 41-93 in SEQ ID NO: 1 (S100A7), residues 36-89 in SEQ ID NO: 2 (S100A8) or residues 45-98 in SEQ ID NO: 3 (S100A9); or by mimicking the conformational epitope represented by helix A2 formed by residues 41-53 in S100A7, residues 43-49 in S100 A8 or residues 47-55 in S100A9; or which does not bind to the epitope defined by residues 43-78 in SEQ ID NO: 9 (S100B). The antibody can bind to residues 41-93 in SEQ ID NO: 1 (S100A7), residues 36-89 in SEQ ID NO: 2 (S100A8) or residues 45-98 in SEQ ID NO: 3 (S100A9), S100A15 or Calprotectin. The antibody binds to a linear epitope which is selected from the linear epitope defined by residues 41-93 in SEQ ID NO: 1 (S100A7), residues 36-89 in SEQ ID NO: 2 (S100A8), residues 45-98 in SEQ ID NO: 3 (S100A9) or residues 43-78 in SEQ ID NO: 9 (S100B)|.sub.[TR1], and does not bind to the S100B epitope defined by residues 43-78 in SEQ ID NO: 9 (S100B), or which binds to the conformational epitope formed by residues 41-53 on S100A7, residues 43-49 on S100A8, residues 47-55 on S100A9 or residues 40-53 on S100A15 and does not bind to the conformational epitope formed by residues 43-78 on S100B.

[0126] As referred to herein, an “epitope” can be a linear epitope, comprising a sequence of contiguous amino acids, or conformational, formed by the three-dimensional juxtaposition of amino acids and/or glycosyl groups in the tertiary structure of a protein. An epitope can comprise or consist of as few as at least 6 amino acids that are unique to a polypeptide or protein sequence. An epitope can be longer; for example, an epitope can comprise, consist essentially of or consist of at least 6 or at least 8, or at least 10, or at least 12, or at least 15 or at least 20 amino acids.

[0127] S100, including S100A7, S100A8, S100A9 and S100B, is, in embodiments of the invention, human S100. Likewise, HMBG1 is, in embodiments of the invention, human HMGB1. Sequences of human S100 proteins, as well as HMBG1, are set forth in FIG. 6 and can be located in the UniProt database under accession numbers P31151, P05109, P06702, P33763, P06703, P29034, P25815, P23297, P04271, Q99584 and P80511.

[0128] The invention also encompasses sequences which are homologous the database sequences recited above. In one embodiment, the sequences according to the invention are at least 95% homologous the sequences referred to above. In other embodiments, they are at least 96%, 97%, 98%, 99% or 100% homologous. Homology can be assessed using any suitable sequence alignment technique. Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Tunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g. the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAST, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).

[0129] The invention comprises a method of treating pre-cachexia and providing treatment to patients in need thereof, the method comprising administering to the subject an effective amount of one or more agents that is an anti-RAGE antibody, a RAGE blocking peptide or small molecule that inhibits binding of RAGE to ligands.

[0130] The invention comprehends a method of inhibiting the progression of pre-cachexia to cachexia in a subject, the method comprising administering to the subject an effective amount of one or more agents that is an anti-RAGE antibody, a RAGE blocking peptide or small molecule that inhibits binding of RAGE to ligands.

[0131] The invention comprehends a method of treating or preventing undesirable muscle or fat loss in a cancer patient in need of treatment thereof, the method comprising administering to the subject an effective amount of one or more agents that is an anti-RAGE antibody, a RAGE blocking peptide or small molecule that inhibits binding of RAGE to ligands.

[0132] The method can involve wherein: the subject is pre-selected as having a molecular signature indicative of pre-cachexia or cachexia by detecting an alteration in at least three markers selected from the group consisting of S100A2 or S100A4; S100A8 or S100A9; and S100A7; detecting an alteration in at least four markers selected from the group consisting of: S100A2 or S100A4; S100A8 or S100A9; S100A7; and S100A14; and/or measuring the level of at least five markers selected from the group consisting of : S100A2 or S100A4; S100A8 or S100A9; S100A7; S100A14; and S100P, thereby pre-selecting the patient as having a molecular signature indicative of pre-cachexia or cachexia. The anti-RAGE antibody can be an antibody which competes for binding to the RAGE receptor with a RAGE ligand selected from S100A7, S100A8, S100A9 and Calprotectin, but preferably may not compete for binding to the RAGE receptor with RAGE ligand HMGB1. The anti-RAGE antibody is an antibody as herein disclosed.

[0133] The invention also comprehends a method of inhibiting the loss of myosin heavy chain in a myocyte, the method comprising contacting the myocyte with an effective amount of one or more agents that is an anti-RAGE antibody, a RAGE blocking peptide or small molecule that inhibits binding of RAGE to ligands.

[0134] The invention also comprehends a method of inhibiting lipolysis in an adipocyte, the method comprising contacting the adipocyte with an effective amount of one or more agents that is an anti-RAGE antibody, a RAGE blocking peptide or small molecule that inhibits binding of RAGE to ligands.

[0135] The invention also involves a method of inhibiting atrophy in a cell, the method comprising contacting the adipocyte, myocyte, or hepatocyte with an effective amount of one or more agents that is an anti-RAGE antibody, a RAGE blocking peptide or small molecule that inhibits binding of RAGE to ligands.

[0136] In inventive methods, the myocyte, adipocyte, or cell is in vitro or in vivo. The myocyte, adipocyte, or cell can be present in a subject identified as having at least one cancer. The cancer includes but is not limited to one or more carcinomas and sarcomas and cancers of the skin, pancreas, stomach, colon, thorax, liver, gallbladder, musculoskeletal system, breast, lung, ovary, uterus, endometrium, prostrate, colon, skin, mouth, salivary, esophagus, head and neck, plus other tumors of the gastrointestinal tract.

[0137] The anti-RAGE antibody can be an antibody, which competes for binding to the RAGE receptor with a RAGE ligand selected from S100A7, S100A8, S100A9 and Calprotectin, but preferably may not compete for binding to the RAGE receptor with RAGE ligand HMGB1 or A100B. The anti-RAGE antibody is an antibody as herein disclosed.

[0138] The invention additionally involves a method of enhancing cancer sensitivity to chemotherapy, the method comprising administering gemcitabine in combination with an anti-RAGE therapy selected from the group consisting of an anti-RAGE antibody, a RAGE blocking peptide, or a small molecule that inhibits binding of RAGE to ligands, thereby enhancing cancer sensitivity to chemotherapy. The cancer can be selected from the group consisting of one or more carcinoma or sarcoma, including but not limited to cancers of the skin, pancreas, stomach, colon, thorax, liver, gallbladder, musculoskeletal system, breast, lung, ovary, uterus, endometrium, prostrate, colon, skin, mouth, salivary, esophagus, head and neck, plus other tumors of the gastrointestinal tract.

[0139] The invention also comprehends a non-naturally-occurring or engineered B-cell that expresses an antibody of the invention.

[0140] The invention also comprehends a vector expressing an antibody or binding fragment thereof of the invention.

[0141] In inventive methods the anti-RAGE antibody can be administered by administering a vector that contains nucleic acid molecule(s) encoding and expresses the antibody or a binding fragment thereof.

Anti-RAGE Antibodies

[0142] The present invention provides antibodies that bind specifically to RAGE, including soluble RAGE and endogenous secretory RAGE, and competitively inhibit binding of RAGE to one or more specific RAGE ligands, in particular RAGE ligands of the S100 family. Antibodies can be expressed in various ways, as herein discussed. Phage display and yeast systems, as well as other vector systems, e.g., vectors for in vivo expression, are amongst the ways to produce antibodies. See generally “Therapeutic Monoclonal Antibodies: From Bench to Clinic,” edited by Zhiqiang An (Wiley 2009) (available from Google Books), e.g., chapters 8, 9, 10, 11, 12 concerning phage, yeast, ribosomal, bacterial and mammalian antibody display systems (for instance sections 9.2, 9.4 9.6, 9.6, 9.6.1.1, 9.6.1.2 concerning yeast display, tumor applications, and intracellular applications), and the documents cited therein.

[0143] The invention includes anti-RAGE antibodies that bind specifically to RAGE-expressing cells in vitro and in vivo, and antibodies that bind to human RAGE with a dissociation constant (Kd) in the range of from at least about 1×10.sup.−7 M to about 1×10.sup.−10 M. Also Included are anti-RAGE antibodies of the invention that bind specifically to the V domain of human RAGE, and anti-RAGE antibodies that block the binding of RAGE to an S100 RAGE ligand.

[0144] Also included in the invention is an antibody that binds specifically to RAGE and blocks the binding of RAGE to RAGE ligands such as S100A7, S100A8, S100A9 and/or Calprotectin.

[0145] The anti-RAGE antibodies of the invention include an anti-RAGE antibody as described above, which binds to RAGE or a RAGE ligand and has the features set forth in the Summary section above or otherwise in this description, or a RAGE-binding fragment which is selected from the group consisting of a chimeric antibody, a humanized antibody, a single chain antibody, a tetrameric antibody, a tetravalent antibody, a multispecific antibody, a domain-specific antibody, a domain-deleted antibody, a fusion protein, an Fab fragment, an Fab′ fragment, an F(ab′)2 fragment, an Fv fragment, an ScFv fragment, an Fd fragment, a single domain antibody, a dAb fragment, a V.sub.HH domain such as a Nanobody™ (see Mulydermans et al., Annual Review of Biochemistry (2013) Vol. 82: 775-797; and an Fc fusion protein (i.e., an antigen binding domain fused to an immunoglobulin constant region). These antibodies can be coupled with a cytotoxic agent, a radiotherapeutic agent, or a detectable label.

[0146] Antibodies of the invention that specifically bind to RAGE also include variants of any of the antibodies described herein, which may be readily prepared using known molecular biology and cloning techniques.

[0147] Antibodies of the invention may also comprise a label attached thereto and able to be detected, (e.g. the label can be a radioisotope, fluorescent compound, enzyme or enzyme co-factor).

[0148] Proteins or polypeptides referred to herein as “recombinant” are proteins or polypeptides produced by the expression of recombinant nucleic acids.

[0149] The term “antibody” is used interchangeably with the term “immunoglobulin” herein, and includes intact antibodies, fragments of antibodies, e.g., Fab, F(ab′)2 fragments, and intact antibodies and fragments that have been mutated either in their constant and/or variable region (e.g., mutations to produce chimeric, partially humanized, or fully humanized antibodies, as well as to produce antibodies with a desired trait, e.g., enhanced IL 13 binding and/or reduced FcR binding). The term “fragment” refers to a part or portion of an antibody or antibody chain comprising fewer amino acid residues than an intact or complete antibody or antibody chain. Fragments can be obtained via chemical or enzymatic treatment of an intact or complete antibody or antibody chain. Fragments can also be obtained by recombinant means. Exemplary fragments include Fab, Fab′, F(ab′)2, Fabc, Fd, dAb, V.sub.HH and scFv and/or Fv fragments.

[0150] The antibody specifically binding to RAGE, or the antigen binding fragments thereof, may include variants of amino acid sequences disclosed herein within a range retaining the ability to specifically recognize RAGE. For example, to enhance the binding affinity and/or other biological properties of the antibody, the amino acid sequences of the antibody may be mutated. For example, such mutations include deletion, insertion, and/or substitution of amino acid sequence residues of the antibody. An amino acid mutation is made based on the relative similarity of the amino acid side chain substituents, for example, with respect to hydrophobic properties, hydrophilic properties, charges, or sizes. For example, arginine, lysine, and histidine are each a positively charged residue; alanine, glycine, and serine have a similar size; and phenylalanine, tryptophan, and tyrosine have a similar shape. Therefore, based on the considerations described above, arginine, lysine, and histidine may be biological functional equivalents; alanine, glycine, and serine may be biological functional equivalents; and phenylalanine, tryptophan, and tyrosine may be biological functional equivalents.

[0151] Amino acid substitution in a protein in which the activity of the molecule is not completely changed is well known in the art. Typical substitutions include Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly substitutions. Considering mutations with biologically equivalent activity, an antibody specifically binding to RAGE or the antigen-binding fragments thereof may also include sequences substantially identical to sequences disclosed herein. In this regard, a substantially identical amino acid sequence may be a sequence with at least 60% homology, at least 70% homology, at least 80% homology, at least 90%, at least 95% homology or 100% homology to a sequence disclosed herein, when the amino acid sequences are aligned to correspond to each other as much as possible. The aligned amino acid sequences are analyzed using an algorithm known in the art. Alignment methods for sequence comparison are well known to one of ordinary skill in the art. For example, a sequence analysis program available on the Internet at the NCBI Basic Local Alignment Search Tool (BLAST) home page, such as blastp, blastx, tblastn, or tblastx, may be used.

[0152] As used herein, a preparation of antibody protein having less than about 50% of non-antibody protein (also referred to herein as a “contaminating protein”), or of chemical precursors, is considered to be “substantially free.” 40%, 30%, 20%, 10% and more preferably 5% (by dry weight), of non-antibody protein, or of chemical precursors is considered to be substantially free. When the antibody protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 30%, preferably less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume or mass of the protein preparation.

[0153] The term “antigen-binding fragment” refers to a polypeptide fragment of an immunoglobulin or antibody that binds antigen or competes with intact antibody (i.e., with the intact antibody from which they were derived) for antigen binding (i.e., specific binding). As such these antibodies or fragments thereof are included in the scope of the invention, provided that the antibody or fragment binds specifically to RAGE, and neutralizes or inhibits one or more RAGE-associated activities (e.g., inhibits binding of RAGE binding ligands to RAGE).

[0154] It is intended that the term “antibody” encompass any Ig class or any Ig subclass (e.g. the IgG1, IgG2, IgG3, and IgG4 subclasses of IgG) obtained from any source (e.g., humans and non-human primates, and in rodents, lagomorphs, caprines, bovines, equines, ovines, etc.).

[0155] The term “Ig class” or “immunoglobulin class”, as used herein, refers to the five classes of immunoglobulin that have been identified in humans and higher mammals, IgG, IgM, IgA, IgD, and IgE. The term “Ig subclass” refers to the two subclasses of IgM (H and L), three subclasses of IgA (IgA1, IgA2, and secretory IgA), and four subclasses of IgG (IgG1, IgG2, IgG3, and IgG4) that have been identified in humans and higher mammals. The antibodies can exist in monomeric or polymeric form; for example, 1 gM antibodies exist in pentameric form, and IgA antibodies exist in monomeric, dimeric or multimeric form.

[0156] The term “IgG subclass” refers to the four subclasses of immunoglobulin class IgG-IgG1, IgG2, IgG3, and IgG4 that have been identified in humans and higher mammals by the heavy chains of the immunoglobulins, V1-γ4, respectively. The term “single-chain immunoglobulin” or “single-chain antibody” (used interchangeably herein) refers to a protein having a two-polypeptide chain structure consisting of a heavy and a light chain, said chains being stabilized, for example, by interchain peptide linkers, which has the ability to specifically bind antigen. The term “domain” refers to a globular region of a heavy or light chain polypeptide comprising peptide loops (e.g., comprising 3 to 4 peptide loops) stabilized, for example, by β pleated sheet and/or intrachain disulfide bond. Domains are further referred to herein as “constant” or “variable”, based on the relative lack of sequence variation within the domains of various class members in the case of a “constant” domain, or the significant variation within the domains of various class members in the case of a “variable” domain. Antibody or polypeptide “domains” are often referred to interchangeably in the art as antibody or polypeptide “regions”. The “constant” domains of an antibody light chain are referred to interchangeably as “light chain constant regions”, “light chain constant domains”, “CL” regions or “CL” domains. The “constant” domains of an antibody heavy chain are referred to interchangeably as “heavy chain constant regions”, “heavy chain constant domains”, “CH” regions or “CH” domains). The “variable” domains of an antibody light chain are referred to interchangeably as “light chain variable regions”, “light chain variable domains”, “VL” regions or “VL” domains). The “variable” domains of an antibody heavy chain are referred to interchangeably as “heavy chain constant regions”, “heavy chain constant domains”, “VH” regions or “VH” domains).

[0157] The term “region” can also refer to a part or portion of an antibody chain or antibody chain domain (e.g., a part or portion of a heavy or light chain or a part or portion of a constant or variable domain, as defined herein), as well as more discrete parts or portions of said chains or domains. For example, light and heavy chains or light and heavy chain variable domains include “complementarity determining regions” or “CDRs” interspersed among “framework regions” or “FRs”, as defined herein.

[0158] The term “conformation” refers to the tertiary structure of a protein or polypeptide (e.g., an antibody, antibody chain, domain or region thereof). For example, the phrase “light (or heavy) chain conformation” refers to the tertiary structure of a light (or heavy) chain variable region, and the phrase “antibody conformation” or “antibody fragment conformation” refers to the tertiary structure of an antibody or fragment thereof.

[0159] “Specific binding” of an antibody means that the antibody exhibits appreciable affinity for a particular antigen or epitope and, generally, does not exhibit significant crossreactivity. The term “anti-RAGE antibody” as used herein refers to an antibody that binds specifically to a RAGE. “Appreciable” binding includes binding with an affinity of at least 25 μM. Antibodies with affinities greater than 1'10.sup.7 M.sup.−1 (or a dissociation coefficient of 1 μm or less or a dissociation coefficient of lnm or less) typically bind with correspondingly greater specificity. Values intermediate of those set forth herein are also intended to be within the scope of the present invention and antibodies of the invention bind to RAGE with a range of affinities, for example, 100 nM or less, 75 nM or less, 50 nM or less, 25 nM or less, for example 10 nM or less, 5 nM or less, 1 nM or less, or in embodiments 500 pM or less, 100 pM or less, 50 pM or less or 25 pM or less. An antibody that “does not exhibit significant crossreactivity” is one that will not appreciably bind to an entity other than its target (e.g., a different epitope or a different molecule). For example, an antibody that specifically binds to RAGE will appreciably bind RAGE but will not significantly react with non-RAGE proteins or peptides. An antibody specific for a particular epitope will, for example, not significantly cross-react with remote epitopes on the same protein or peptide. Specific binding can be determined according to any art-recognized means for determining such binding. Preferably, specific binding is determined according to Scatchard analysis and/or competitive binding assays.

[0160] As used herein, the term “affinity” refers to the strength of the binding of a single antigen-combining site with an antigenic determinant. Affinity depends on the closeness of stereochemical fit between antibody combining sites and antigen determinants, on the size of the area of contact between them, on the distribution of charged and hydrophobic groups, etc. Antibody affinity can be measured by equilibrium dialysis or by the kinetic BIACORE™ method. The dissociation constant, Kd, and the association constant, Ka, are quantitative measures of affinity.

[0161] As used herein, the term “monoclonal antibody” refers to an antibody derived from a clonal population of antibody-producing cells (e.g., B lymphocytes or B cells) which is homogeneous in structure and antigen specificity. The term “polyclonal antibody” refers to a plurality of antibodies originating from different clonal populations of antibody-producing cells which are heterogeneous in their structure and epitope specificity but which recognize a common antigen. Monoclonal and polyclonal antibodies may exist within bodily fluids, as crude preparations, or may be purified, as described herein.

[0162] The term “binding portion” of an antibody (or “antibody portion”) includes one or more complete domains, e.g., a pair of complete domains, as well as fragments of an antibody that retain the ability to specifically bind to RAGE. It has been shown that the binding function of an antibody can be performed by fragments of a full-length antibody. Binding fragments are produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins. Binding fragments include Fab, Fab′, F(ab′)2, Fabc, Fd, dAb, Fv, single chains, single-chain antibodies, e.g., scFv, and single domain antibodies.

[0163] “Humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, FR residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0164] Dose or dosage levels for antibodies in suitable and/or preferred pharmaceutical formulations can be determined in view of the present disclosure and general knowledge of formulation technology, depending upon the intended route of administration, delivery format, and desired dosage. Typically, a physician will determine the actual dosage which will be most suitable for an individual subject.

[0165] The specific dose level and frequency of dosage for any particular patient or subject in need of treatment thereof may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing therapy. For example, the antibody compound or composition may be administered at a dose of between 2 mg/kg and 0.1 mg/kg, depending on its activity.

[0166] The anti-RAGE antibody or fragment thereof may be administered as a fixed dose, independent of a dose per subject weight ratio, or at an appropriate dose in mg/kg body weight with an approximate maximum of 200 mg/kg. The antibody or fragment thereof may be administered to a 70 kg individual in one or more separate, simultaneous or sequential doses of 14,000 mg or less of antibody or fragment thereof, 13,000 mg or less of antibody or fragment thereof, 12,000 mg or less of antibody or fragment thereof, 11,000 mg or less of antibody or fragment thereof, 10,000 mg or less of antibody or fragment thereof, 9000 mg or less of antibody or fragment thereof, 8000 mg or less of antibody or fragment thereof, 7000 mg or less of antibody or fragment thereof, 6000 mg or less of antibody or fragment thereof, 5000 mg or less of antibody or fragment thereof, 4500 mg or less of antibody or fragment thereof, 4000 mg or less of antibody or fragment thereof, 3700 mg or less of antibody or fragment thereof, 3500 mg or less of antibody or fragment thereof, mg or less of antibody or fragment thereof, 3200 mg or less of antibody or fragment thereof, 3000 mg or less of antibody or fragment thereof, 2700 mg or less of antibody or fragment thereof, 2500 mg or less of antibody or fragment thereof, 2200 mg or less of antibody or fragment thereof, or 2100 mg or less of antibody or fragment thereof, 1700 mg or less of antibody or fragment thereof, 1500 mg or less of antibody or fragment thereof, 1200 mg or less of antibody or fragment thereof, or 1100 mg or less of antibody or fragment thereof, 1000 mg or less of antibody or fragment thereof, 700 mg or less of antibody or fragment thereof, 500 mg or less of antibody or fragment thereof, 200 mg or less of antibody or fragment thereof, or 100 mg or less of antibody or fragment thereof. In another embodiment, the antibody or fragment is administered in one or more doses of at least 20 mg of antibody or fragment thereof. Since the total protein concentration in human plasma is 70,000 mg/l, and standard blood transfusion or plasma or albumin infusions routinely deliver tens or even hundreds of grams of protein intravenously, administration of the maximal doses of anti-RAGE antibody are safe and acceptable.

[0167] The anti-RAGE antibody compound or composition may be administered as a fixed dose, independent of a dose per subject weight ratio. The anti-RAGE antibody compound or composition may be administered in one or more separate, simultaneous or sequential parenteral doses of 100 mg or less, of 50 mg or less, 25 mg or less, or 10 mg or less. Alternatively, anti-RAGE antibody compound or composition may be administered in a dose per subject weight ratio as easily determined by one of skill in the art. The component(s) may be formulated into a pharmaceutical composition, such as by mixing with one or more of a suitable carrier, diluent or excipient, by using techniques that are known in the art. Vector delivery systems

[0168] One type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, wherein virally-derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g. retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses). Viral vectors also include polynucleotides carried by a virus for transfection into a host cell. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g. bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as “expression vectors.” Common expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.

[0169] Recombinant expression vectors can comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory elements, which may be selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allows for expression of the nucleotide sequence (e.g. in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).

[0170] Preferably, delivery is in the form of a vector which may be a viral vector, such as a lenti- or baculo- or preferably adeno-viral/adeno-associated viral vectors, but other means of delivery are known (such as yeast systems, microvesicles, gene guns/means of attaching vectors to gold nanoparticles) and are provided. A vector may mean not only a viral or yeast system (for instance, where the nucleic acids of interest may be operably linked to and under the control of (in terms of expression, such as to ultimately provide a processed RNA) a promoter), but also direct delivery of nucleic acids into a host cell. While in herein methods the vector may be a viral vector and this is advantageously an AAV, other viral vectors as herein discussed can be employed, such as lentivirus. For example, baculoviruses may be used for expression in insect cells. These insect cells may, in turn be useful for producing large quantities of further vectors, such as AAV or lentivirus vectors adapted for delivery of the present invention. Also envisaged is a method of delivering the present compositions comprising nucleic acids encoding anti-RAGE antibodies of the invention comprising delivering to a cell mRNA encoding said anti-RAGE antibodies. AAV and lentiviral vectors are preferred. delivering a non-naturally occurring or engineered composition comprising an AAV or lentivirus vector system comprising one or more AAV or lentivirus vectors operably encoding a composition for expression thereof.

[0171] Use of viral vectors for antibody-based protection (commonly referred to as “vectored immunprophylaxis”) is known in the art. For example, Balzas et al. discuss antibody-based protection against HIV infection by vectored immunoprophylaxis (see e.g., Balazs A. B. et al. Nature 2011 Nov. 30; 481(7379):81-4. Antibody-based protection against HIV infection by vectored immunoprophylaxis; Balazs A. B. et al. Nat. Med. 2014 March; 20(3):296-300. Vectored immunoprophylaxis protects humanized mice from mucosal HIV transmission.).

[0172] The invention in some embodiments comprehends a method of preparing the AAV of the invention comprising transfecting plasmid(s) containing or consisting essentially of nucleic acid molecule(s) coding for the AAV into AAV-infected cells, and supplying AAV rep and/or cap obligatory for replication and packaging of the AAV. In some embodiments the AAV rep and/or cap obligatory for replication and packaging of the AAV are supplied by transfecting the cells with helper plasmid(s) or helper virus(es). In some embodiments the helper virus is a poxvirus, adenovirus, herpesvirus or baculovirus. In some embodiments the poxvirus is a vaccinia virus. In some embodiments the cells are mammalian cells. And in some embodiments the cells are insect cells and the helper virus is baculovirus. In other embodiments, the virus is a lentivirus.

[0173] As to AAV, the AAV can be AAV1, AAV2, AAV5 or any combination thereof. One can select the AAV of the AAV with regard to the cells to be targeted; e.g., one can select AAV serotypes 1, 2, 5 or a hybrid or capsid AAV1, AAV2, AAV5 or any combination thereof for targeting brain or neuronal cells; and one can select AAV4 for targeting cardiac tissue. AAV8 is useful for delivery to the liver. The above promoters and vectors are preferred individually. For example, for AAV, the route of administration, formulation and dose can be as in U.S. Pat. No. 8,454,972 and as in clinical trials involving AAV. For Adenovirus, the route of administration, formulation and dose can be as in U.S. Pat. No. 8,404,658 and as in clinical trials involving adenovirus. For plasmid delivery, the route of administration, formulation and dose can be as in U.S. Pat. No. 5,846,946 and as in clinical studies involving plasmids. Doses may be based on or extrapolated to an average 70 kg individual, and can be adjusted for patients, subjects, mammals of different weight and species. Frequency of administration is within the ambit of the medical or veterinary practitioner (e.g., physician, veterinarian), depending on usual factors including the age, sex, general health, other conditions of the patient or subject and the particular condition or symptoms being addressed.

[0174] The viral vectors can be injected into the tissue of interest. The expression of anti-RAGE antibodies can be driven by a cell-type specific promoter. For example, liver-specific expression might use the Albumin promoter and neuron-specific expression might use the Synapsin I promoter.

[0175] In some embodiments, the viral vector is delivered to the tissue of interest by, for example, an intramuscular injection, while other times the viral delivery is via intravenous, transdermal, intranasal, oral, mucosal, or other delivery methods. Such delivery may be either via a single dose, or multiple doses. One skilled in the art understands that the actual dosage to be delivered herein may vary greatly depending upon a variety of factors, such as the vector chose, the target cell, organism, or tissue, the general condition of the subject to be treated, the degree of transformation/modification sought, the administration route, the administration mode, the type of transformation/modification sought, etc.

[0176] Such a dosage may further contain, for example, a carrier (water, saline, ethanol, glycerol, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, etc.), a diluent, a pharmaceutically-acceptable carrier (e.g., phosphate-buffered saline), a pharmaceutically-acceptable excipient, an adjuvant to enhance antigenicity, an immunostimulatory compound or molecule, and/or other compounds known in the art. The adjuvant herein may contain a suspension of minerals (alum, aluminum hydroxide, aluminum phosphate) on which antigen is adsorbed; or water-in-oil emulsion in which antigen solution is emulsified in oil (MF-59, Freund's incomplete adjuvant), sometimes with the inclusion of killed mycobacteria (Freund's complete adjuvant) to further enhance antigenicity (inhibits degradation of antigen and/or causes influx of macrophages). Adjuvants also include immunostimulatory molecules, such as cytokines, costimulatory molecules, and for example, immunostimulatory DNA or RNA molecules, such as CpG oligonucleotides. Such a dosage formulation is readily ascertainable by one skilled in the art. The dosage may further contain one or more pharmaceutically acceptable salts such as, for example, a mineral acid salt such as a hydrochloride, a hydrobromide, a phosphate, a sulfate, etc.; and the salts of organic acids such as acetates, propionates, malonates, benzoates, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, gels or gelling materials, flavorings, colorants, microspheres, polymers, suspension agents, etc. may also be present herein. In addition, one or more other conventional pharmaceutical ingredients, such as preservatives, humectants, suspending agents, surfactants, antioxidants, anticaking agents, fillers, chelating agents, coating agents, chemical stabilizers, etc. may also be present, especially if the dosage form is a reconstitutable form. Suitable exemplary ingredients include microcrystalline cellulose, carboxymethylcellulose sodium, polysorbate 80, phenylethyl alcohol, chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, parachlorophenol, gelatin, albumin and a combination thereof. A thorough discussion of pharmaceutically acceptable excipients is available in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991) which is incorporated by reference herein. In an embodiment herein the delivery is via an adenovirus, which may be at a single booster dose containing at least 1×10.sup.5 particles (also referred to as particle units, pu) of adenoviral vector. In an embodiment herein, the dose preferably is at least about 1×10.sup.6 particles (for example, about 1×10.sup.6-1×10.sup.12 particles), more preferably at least about 1×10.sup.7 particles, more preferably at least about 1×10.sup.8 particles (e.g., about 1×10.sup.8-1×10.sup.11 particles or about 1×10.sup.8-1×10.sup.12 particles), and most preferably at least about 1×10.sup.9 particles (e.g., about 1*×10.sup.9-1×10.sup.10 particles or about 1×10.sup.9-1×10.sup.12 particles), or even at least about 1×10.sup.10 particles (e.g., about 1×10.sup.10-1×10.sup.12 particles) of the adenoviral vector. Alternatively, the dose comprises no more than about 1×10.sup.14 particles, preferably no more than about 1×10.sup.13 particles, even more preferably no more than about 1×10.sup.12 particles, even more preferably no more than about 1×10.sup.11 particles, and most preferably no more than about 1×10.sup.10 particles (e.g., no more than about 1×10.sup.9 articles). Thus, the dose may contain a single dose of adenoviral vector with, for example, about 1×10.sup.6 particle units (pu), about 2×10.sup.6 pu, about 4×10.sup.6 pu, about 1×10.sup.7 pu, about 2×10.sup.7 pu, about 4×10.sup.7 pu, about 1×10.sup.8 pu, about 2×10.sup.8 pu, about 4×10.sup.8 pu, about 1×10.sup.9 pu, about 2×10.sup.9 pu, about 4×10.sup.9 pu, about 1×10.sup.10 pu, about 2×10.sup.10 pu, about 4×10.sup.10 pu, about 1×10.sup.11 pu, about 2×10.sup.11 pu, about 4×10.sup.11 pu, about 1×10.sup.12 pu, about 2×10.sup.12 pu, or about 4×10.sup.12 pu of adenoviral vector. See, for example, the adenoviral vectors in U.S. Pat. No. 8,454,972 B2 to Nabel, et. al., granted on Jun. 4, 2013; incorporated by reference herein, and the dosages at col 29, lines 36-58 thereof. In an embodiment herein, the adenovirus is delivered via multiple doses.

[0177] In an embodiment herein, the delivery is via an AAV. A therapeutically effective dosage for in vivo delivery of the AAV to a human is believed to be in the range of from about 20 to about 50 ml of saline solution containing from about 1×10.sup.10 to about 1×10.sup.10 functional AAV/ml solution. The dosage may be adjusted to balance the therapeutic benefit against any side effects. In an embodiment herein, the AAV dose is generally in the range of concentrations of from about 1×10.sup.5 to 1×10.sup.50 genomes AAV, from about 1×10.sup.8 to 1×10.sup.20 genomes AAV, from about 1×10.sup.10 to about 1×10.sup.16 genomes, or about 1×10.sup.11 to about 1×10.sup.16 genomes AAV. A human dosage may be about 1×10.sup.13 genomes AAV. Such concentrations may be delivered in from about 0.001 ml to about 100 ml, about 0.05 to about 50 ml, or about 10 to about 25 ml of a carrier solution. Other effective dosages can be readily established by one of ordinary skill in the art through routine trials establishing dose response curves. See, for example, U.S. Pat. No. 8,404,658 B2 to Hajjar, et al., granted on Mar. 26, 2013, at col. 27, lines 45-60.

[0178] In an embodiment herein the delivery is via a plasmid. In such plasmid compositions, the dosage should be a sufficient amount of plasmid to elicit a response. For instance, suitable quantities of plasmid DNA in plasmid compositions can be from about 0.1 to about 2 mg, or from about 1 μg to about 10 μg.

[0179] The doses herein are based on an average 70 kg individual. The frequency of administration is within the ambit of the medical or veterinary practitioner (e.g., physician, veterinarian), or scientist skilled in the art.

Lentivirus

[0180] Lentiviruses are complex retroviruses that have the ability to infect and express their genes in both mitotic and post-mitotic cells. The most commonly known lentivirus is the human immunodeficiency virus (HIV), which uses the envelope glycoproteins of other viruses to target a broad range of cell types.

[0181] Lentiviruses may be prepared as follows. After cloning pCasES10 (which contains a lentiviral transfer plasmid backbone), HEK293FT at low passage (p=5) were seeded in a T-75 flask to 50% confluence the day before transfection in DMEM with 10% fetal bovine serum and without antibiotics. After 20 hours, media was changed to OptiMEM (serum-free) media and transfection was done 4 hours later. Cells were transfected with 10 μg of lentiviral transfer plasmid (pCasES10) and the following packaging plasmids: 5 μg of pMD2.G (VSV-g pseudotype), and 7.5 ug of psPAX2 (gag/pol/rev/tat). Transfection was done in 4 mL OptiMEM with a cationic lipid delivery agent (50 μL Lipofectamine 2000 and 100 μl Plus reagent). After 6 hours, the media was changed to antibiotic-free DMEM with 10% fetal bovine serum. Lentivirus may be purified as follows. Viral supernatants were harvested after 48 hours. Supernatants were first cleared of debris and filtered through a 0.45 um low protein binding (PVDF) filter. They were then spun in a ultracentrifuge for 2 hours at 24,000 rpm. Viral pellets were resuspended in 50 μl of DMEM overnight at 4 C. They were then aliquotted and immediately frozen at −80 C.

[0182] In another embodiment, minimal non-primate lentiviral vectors based on the equine infectious anemia virus (EIAV) are also contemplated, especially for ocular gene therapy (see, e.g., Balagaan, J Gene Med 2006; 8: 275-285, Published online 21 Nov. 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jgm.845). In another embodiment, RetinoStat®, an equine infectious anemia virus-based lentiviral gene therapy vector that expresses angiostatic proteins endostain and angiostatin that is delivered via a subretinal injection for the treatment of the web form of age-related macular degeneration is also contemplated (see, e.g., Binley et al., HUMAN GENE THERAPY 23:980-991 (September 2012)) may be modified for expressing antibody(ies) of the present invention.

[0183] In another embodiment, self-inactivating lentiviral vectors with an siRNA targeting a common exon shared by HIV tat/rev, a nucleolar-localizing TAR decoy, and an anti-CCR5-specific hammerhead ribozyme (see, e.g., DiGiusto et al. (2010) Sci Transl Med 2:36ra43) may be used/and or adapted for expressing antibody(ies) of the present invention. A minimum of 2.5×10.sup.6 CD34+ cells per kilogram patient weight may be collected and prestimulated for 16 to 20 hours in X-VIVO 15 medium (Lonza) containing 2 mML-glutamine, stem cell factor (100 ng/ml), Flt-3 ligand (Flt-3L) (100 ng/ml), and thrombopoietin (10 ng/ml) (CellGenix) at a density of 2×106 cells/ml. Prestimulated cells may be transduced with lentiviral at a multiplicity of infection of 5 for 16 to 24 hours in 75-cm.sup.2 tissue culture flasks coated with fibronectin (25 mg/cm2) (RetroNectin, Takara Bio Inc.).

[0184] Lentiviral vectors have been disclosed as in the treatment for Parkinson's Disease, see, e.g., US Patent Publication No. 20120295960 and U.S. Pat. Nos. 7,303,910 and 7,351,585, and for delivery of anti-αβ antibodies for the treatment of Alzheimers disease (Fukuchi et al., Neurobiol Dis. 2006 September; 23(3): 502-511). Lentiviral vectors have also been disclosed for the treatment of ocular diseases, see e.g., US Patent Publication Nos. 20060281180, 20090007284, US20110117189; US20090017543; US20070054961, US20100317109. Lentiviral vectors have also been disclosed for delivery to the brain, see, e.g., US Patent Publication Nos. US20110293571; US20110293571, US20040013648, US20070025970, US20090111106 and U.S. Pat. No. 7,259,015. These vectors can be adapted to express the antibody(ies) of the invention, and can be administered in analogous amounts, albeit advantageously iv or im or to the tumor mass rather than to the brain or eye (unless, of course, the tumor is in the brain or eye).

Pre-Cachexia and Cachexia

[0185] The invention provides compositions and methods for identifying patients with pre-cachexia and/or cachexia; a culture system that reproduces the cachectic process in cells in vitro; and methods of using this system to identify markers associated with pre-cachexia and/or cachexia, as well as for the discovery of therapeutic agents useful for disrupting (slowing, reducing, reversing, or preventing) the progression of pre-cachexia to cachexia.

[0186] The invention is based, at least in part, on the discovery of a molecular signature associated with pre-cachexia and/or cachexia. Accordingly, the invention provides a panel of markers useful for identifying patients that are pre-cachectic. This provides for the identification and treatment of subjects before they develop the progressive weight loss and muscle atrophy that define cachexia, and which has proven refractory to all attempted therapeutic interventions. The invention further provides markers useful for characterizing cachexia.

Cachexia's Effect on Patient Prognosis

[0187] The development of new highly effective oncologic therapies has transformed many cancers into chronically managed diseases. The efficacy of these new treatment regimens does not guarantee an increase in survival. While surgery, radiation or chemotherapy may successfully reduce tumor size, this reduction does not always correlate with an increase in survival. In fact, for many patients, degree of tumor burden does not correlate with prognosis. As shown herein, the difference in survival time for patients with only modest tumor burden is only about eight months more than the survival time of patients with extensive tumor burden. This counter-intuitive result may be attributed, at least in part, to cachexia. The clinical manifestations of cachexia are complex: muscle and fat wasting, multi organ dysfunction (e.g., cardiac, pulmonary, gastrointestinal), and profound metabolic derangement.

[0188] More than twenty-five percent of cancer deaths are caused—not by cancer—but by cachexia. Cachexia associated deaths include death by respiratory failure, cardiac failure, and metabolic derangement. The devastating effects of cachexia are poignantly illustrated in a case study of a sixty-one year old patient with pancreatic cancer. The patient presented with worsening abdominal pain. Upon imaging, a small (3×3×2 cm) tumor was observed in the head of the pancreas. The patient was diagnosed with stage III non-resectable pancreatic cancer. She also had cachexia, having lost 35 lbs (17%) of her body weight. The patient was treated with 5′-FU/Leucovorin/Irinotecan/Oxaliplatin. Treatment successfully reduced the size of the patient's tumor, as well as levels of CA-19.9 a serum marker associated with pancreatic cancer. Despite this positive response, the patient continued to lose weight, ultimately becoming so frail that anti-cancer therapy had to be discontinued.

[0189] While cachexia shares certain phenotypic similarities with food deprivation, in fact cachexia is distinct from starvation. Even where patients with cachexia are provided with total parenteral nutrition, weight loss, including loss of lean body mass, continues. These losses have proven refractory to all therapeutic interventions, except for a complete removal of the cancer, which remains elusive for the vast majority of cancer patients.

In Vitro Cachexia/Pre-Cachexia Model System

[0190] To date, progress in understanding cachexia has been hampered by the lack of an in vitro system that could reproduce the hallmarks of cachexia. The identification of such a system is critical to identifying factors that mediate the tumor-host interaction. In the simplest view of tumor-host interactions, the tumor produces a factor that mediates wasting/dysfunction of target tissues. While conventional wisdom has held that mice “do not get cancer cachexia”. To the contrary, results provided herein demonstrate that human cancer cell lines induce a cachectic state in murine xenograft models. Conventional wisdom also holds to the notion that immune system is required to generate inflammatory cytokines that have been thought to drive cachexia. We have demonstrated that the tumor cells from multiple tumor types grown in isolation produce the mediator(s) that cause target cells (muscle, fat, and liver) to phenocopy clinical cachexia with tremendous specificity, thus the immune is not required. These observations support the premise that tumor cells secrete one or more factors that drive cachexia. The present invention provides an in vitro system that models pre-cachexia and/or cachexia and facilitates the identification of agents that drive cachexia vs. factors that merely contribute to the disease process.

[0191] The present invention provides a culture system, comprising a human target cell and a cachexia-inducing factor. In certain embodiments, the target cell is selected from a myocyte, an adipocyte, and a hepatocyte. In one embodiment, the target cell is a myocyte.

[0192] In certain embodiments, the cachexia-inducing factor is provided in (i) human plasma from a cachexia patient or (ii) cachexia-inducing conditioned media, such as human cancer cell conditioned media. In certain embodiments, the media is conditioned by a human cancer cell line selected from one or more of the following: AsPC-1, A375, CAPAN-1, CAPAN-2, C32, G361, HCT-15, HPAF-II, JHU012, JHU022, LS180, LX1, MKN1, PC9, HCC827 and PANC-1. In particular embodiments, a cachexia-inducing cell line is the human melanoma cell line A375 (ATCC® CRL1619™); the intestinal human colon adenocarcinoma cell line LS180 (ATCC® CL-187™); the squamous cel lung cancer cell line LX1 (Cell vol. 29, 161-169 (1982); the malignant melanoma human cell line G361 (ATCC® CRL-1424™), or the human malignant melanoma cell line C32 (ATCC® CRL-1585™). In certain embodiments, the media is conditioned by a human cancer cell isolated from a patient.

[0193] In certain embodiments, the culture is in a single or a multi-well plate format. In certain embodiments, the invention provides a multi-well plate suitable for use in a high-throughput screening system, the plate having a plurality of wells comprising the culture system described therein. The surface of the multi-well plate may be the surface of a culture well or glass slide, or any other suitable surface. In certain embodiments the multi-well plate contains 384 wells. Cultures can be contained in a multi-well plate having a 96-, 384-, 1536- or more than 1536-well format.

[0194] In certain embodiments, the invention further provides a method for producing the model of the present invention, in a single or multi-well plate format.

Uses of the Cachexia/Pre-cachexia Model System

[0195] The present invention provides methods for characterizing an agent for the ability to induce pre-cachexia or cachexia in a target cell. The method generally involves exposing a target cell to a test agent, and characterizing the effect of the agent on the target cell relative to: (i) a control target cell not exposed to the test agent; or (ii) a transcriptional profile of a non-cachectic cell and/or a transcriptional profile of a cachectic cell, wherein the cell type of the target cell and the cachectic and/or non-cachectic cell is the same.

[0196] The present invention provides a method for predicting the effect of a test agent on a target cell of a patient in vivo, comprising culturing a target cell obtained from a patient in the system of the invention, exposing it to the test agent, and assaying for a pharmacological effect of the test agent on the target cell relative to: (i) a control target cell not treated with the test agent; or (ii) a transcriptional profile of a non-cachectic cell and/or a transcriptional profile of a cachectic cell, wherein the cell type of the target cell and the cachectic and/or non-cachectic cell is the same.

[0197] In certain embodiments, the target cell is isolated from a patient with cancer. “Cancer” is understood in the context it is used in the literature, e.g., Martin, L. et al., J Clin Oncol 31(12): 1539-1547 (2013).

[0198] In certain embodiments, the effect is selected from proliferation, viability, and differentiation, or combinations thereof.

[0199] In certain embodiments, the effect is detected by assessing a change in gene expression profile between the target cell and the control target cell of step (i); or the cachectic or non-cachectic cell of step (ii).

[0200] In certain embodiments, the target cell is selected from a myocyte, an adipocyte, and a hepatocyte. In a preferred embodiment, the target cell is a myocyte.

[0201] The cachectic culture system can be used to screen for test agents (such as solvents, small molecule drugs, peptides, and polynucleotides) or environmental conditions (such as culture conditions or manipulation) that affect the characteristics of cells. Two or more agents can be tested in combination (by exposing to the cells either simultaneously or sequentially), to detect possible drug-drug interactions and/or rescue effects (e.g., by testing a toxin and a potential anti-toxin). Agent(s) and environmental condition(s) can be tested in combination (by treating the cells with a drug either simultaneously or sequentially relative to an environmental condition), to detect possible agent-environment interaction effects.

[0202] In certain embodiments, the assay to determine the characteristics of cells is selected in a manner appropriate to the cell type and agent and/or environmental factor being studied as disclosed in WO 2002/04113, which is hereby incorporated by reference in its entirely. For example, changes in cell morphology may be assayed by standard light, or electron microscopy. Alternatively, the effects of treatments or compounds potentially affecting the expression of cell surface proteins may be assayed by exposing the cells to either fluorescently labeled ligands of the proteins or antibodies to the proteins and then measuring the fluorescent emissions associated with each cell on the plate. As another example, the effects of treatments or compounds which potentially alter the pH or levels of various ions within cells may be assayed using various dyes which change in color at determined pH values or in the presence of particular ions. The use of such dyes is well known in the art. For cells, which have been transformed or transfected with a genetic marker, such as the β-galactosidase, alkaline phosphatase, or luciferase genes, the effects of treatments or compounds may be assessed by assays for expression of that marker. In particular, the marker may be chosen so as to cause spectrophotometrically assayable changes associated with its expression.

[0203] Particular screening applications of this invention relate to the testing of pharmaceutical compounds in drug research. The reader is referred generally to the standard textbook In vitro Methods in Pharmaceutical Research, Academic Press, 1997, and U.S. Pat. No. 5,030,015. In certain aspects of this invention, the culture of the invention is used to grow and differentiate a cachectic target cell to play the role of test cells for standard drug screening and toxicity assays. Assessment of the activity of candidate pharmaceutical compounds generally involves combining the target cell (e.g., a myocyte, an adipocyte or a hepatocyte) with the candidate compound, determining any change in the morphology, marker phenotype, or metabolic activity of the cells that is attributable to the candidate compound (compared with untreated cells or cells treated with an inert compound, such as vehicle), and then correlating the effect of the candidate compound with the observed change. The screening may be done because the candidate compound is designed to have a pharmacological effect on the target cell, or because a candidate compound may have unintended side effects on the target cell. Alternatively, libraries can be screened without any predetermined expectations in hopes of identifying compounds with desired effects.

[0204] Cytotoxicity can be determined in the first instance by the effect on cell viability and morphology. In certain embodiments, toxicity may be assessed by observation of vital staining techniques, ELISA assays, immunohistochemistry, and the like or by analyzing the cellular content of the culture, e.g., by total cell counts, and differential cell counts or by metabolic markers such as MTT and XTT.

[0205] Additional further uses of the culture of the invention include, but are not limited to, its use in research e.g., to elucidate cachectic mechanisms leading to the identification of novel targets for cachectic therapies, and to generate genotype-specific cells for disease modeling, including the generation of new therapies customized to different genotypes. Such customization can reduce adverse drug effects and help identify therapies appropriate to the patient's genotype.

Methods of Identifying a Patient as Having Pre-Cachexia or Cachexia

[0206] As reported herein, within days of contacting target cells with cachexia-inducing media changes in metabolism, metabolite profiles, the differential expression of markers, and changes in cell morphology are observed. Such changes closely phenocopy overt clinical cachexia as induced by cachectic patient plasma and seen in patient biopsy samples, and likely reproduce the alterations present in pre-cachectic humans and mice. Significantly, many of the changes observed in cells in vitro in response to cachexia inducing factors were reversible (e.g., the cachectic gene expression signature, loss of myosin heavy chain, loss of lipid content, and cell atrophy) with novel treatments identified with this discovery platform.

[0207] Accordingly, the invention provides methods for identifying subjects as pre-cachectic. Such methods are particularly advantageous given that treatment of pre-cachexia could slow or even reverse the progression of pre-cachexia to cachexia. Moreover, early identification of pre-cachectic patients could ensure that subjects are sufficiently strong to benefit from therapies delineated herein.

[0208] The invention provides for a transcriptional biomarker profile of a cachectic phenotype (e.g., cachexia, pre-cachexia) suitable for use in diagnosing and/or monitoring a patient with cachexia or pre-cachexia. Accordingly, the present invention provides a method for diagnosing a patient with cachexia or pre-cachexia, comprising (i) obtaining a target cell from the patient; (ii) determining the gene expression profile of that cell; and (iii) comparing the transcriptional profile of that target cell and the transcriptional profile of a cachectic cell, wherein the cell type of the target cell and the cachectic cell is the same.

[0209] The invention provides for a transcriptional biomarker profile of a cachectic phenotype suitable for use in diagnosing and/or monitoring a patient with cachexia or pre-cachexia. Accordingly, the present invention provides a method for diagnosing a patient with cachexia or pre-cachexia, comprising (i) obtaining a target cell from the patient; (ii) determining the gene expression profile of that cell; and (iii) comparing the transcriptional profile of that target cell and the transcriptional profile of a cachectic cell, wherein the cell type of the target cell and the cachectic cell is the same.

[0210] The present invention further provides a method for monitoring a patient with cachexia or pre-cachexia, comprising (i) obtaining a target cell from the patient; (ii) determining the gene expression profile of that cell; and (iii) comparing the transcriptional profile of that target cell and the transcriptional profile of a cachectic cell, wherein the cell type of the target cell and the cachectic cell is the same. Monitoring the cachectic transcriptional profile of a patient is useful, for example, to determine the patient's pharmacological response to a drug or disease progression.

[0211] The invention further provides protein-mediated cachexia-inducing factors, present in media conditioned by certain human cancer cell lines, that induce cellular consequences recapitulated by cachectic-patient-derived plasma samples. For instance, RAGE-based biomarkers were identified herein as associated with the cachexia phenotype. Soluble RAGE (a negative regulator of RAGE signaling that acts as a sink for RAGE ligands) was found to be elevated in non-cachexia-inducing conditioned media in comparison to cachexia-inducing conditioned media. In contrast, levels of certain RAGE ligands, such as S100 proteins, were found to be elevated in cachexia-inducing conditioned media in comparison to non-cachexia-inducing conditioned media.

[0212] The presence and/or level of the cachexia-inducing proteins, e.g. the factors listed in the following Table, in patient plasma may serve as cachectic biomarker(s) useful for diagnosing and/or monitoring a patient with cachexia. Accordingly, the present invention also provides a method for diagnosing a patient with cachexia, comprising (i) obtaining plasma from the patient; and (ii) detecting the presence or level of a cachexia-inducing factor(s) selected from the following Table. The present invention also provides a method for monitoring a patient with cachexia, comprising (i) obtaining plasma from the patient; and (ii) detecting the presence or level of a cachexia-inducing factor(s) selected from the following Table.

TABLE-US-00001 Cachexia-Inducing Factors BCAM NGF BTC PDGFA CCL5 PDGFB CCL27 S100A7 DKK3 S100A8 EGFR S100A9 Follistatin-related peptide 1 sRAGE (soluble RAGE)* IGFBP-1 TNFRSF10C IL6 TNFSF18 Lipolysis-stimulated lipoprotein receptor TYRO3 BCAM (*anti-cachectogenic, and as such, a potential therapeutic)

[0213] In particular embodiments, at least one or more of IGFBP-1, CCL27, AXL, CSF1, ICAM2, PIGF, TMP2, FGF4, KDR and CSF3 are increased in cachexia.

[0214] In other embodiments, a marker of cachexia or pre-cachexia is any one or more of three markers selected from S100A2 or S100A4, S100A8 or S100A9, and S100A7; four markers selected from S100A2 or S100A4, S100A8 or S100A9, S100A7 and S100A14; five markers selected from S100A2 or S100A4, S100A8 or S100A9, S100A7, S100A14, and S100P, which are increased in pre-cachexia or cachexia.

[0215] In other embodiments, a marker of cachexia or pre-cachexia is Basal cell adhesion molecule (BCAM), Buchang-tang (BCT), Chemokine ligand (CCL)5, CCL28, Dickkopf-related protein 3 (DKK3), Epidermal Growth Factor Receptor (EGFR), FASLG (Fas ligand), Fibroblast growth factor 4 (FGF4), Follistatin-related peptide 1, intercellular adhesion molecule (ICAM2), High Mobility Group (HMG1), Insulin Growth Factor-2 (IGF-2), Insulin Growth Factor Binding Protein-2 (IGFBP-2), IGFBP-6, interleukin-6 (IL6, Kinase insert domain receptor (KDR), lipolysis-stimulated receptor (LSR), NM (NME/NM23 Nucleoside Diphosphate Kinase 1), Nerve Growth Factor (NGF), Platelet Derived Growth Factor-A (PDGFA), PDGFB, P1GF (placenta growth factor), tyrosine-protein kinase receptor (TYR03), Plasminogen activator inhibitor 1, tissue inhibitor of metalloproteinases (TIMP2), soluble Receptor for Advanced Glycation Endproducts (sRAGE)*, Tumor necrosis factor receptor superfamily member 10C (TNFRSF10C), and tumor necrosis factor superfamily member 18 (TNFSF18). All of the aforementioned markers are increased, with the exception of sRAGE, which is decreased. In other embodiments, the method further involves measuring an increase in an S100 family member selected from HMGB1, S100P, S100A2, S100A3, S100A4, S100A5, S100A7, S100A7A, S100A8, S100A9, S100A11, S100Al2, S100A13, S100A14, and S100A15.

[0216] In certain embodiments, the patient has cachexia associated with a cancer. In a preferred embodiment, the patient has cachexia associated with a cancer, e.g., an epithelial-derived cancer or a mesenchemal-derived cancer, such as carcinomas and sarcomas and cancers of the skin, pancreas, stomach, colon, thorax, liver, gallbladder, musculoskeletal system, breast, lung, ovary, uterus, endometrium, prostrate, colon, skin, mouth, salivary, esophagus, head and neck, plus other tumors of the gastrointestinal tract.

[0217] In other embodiments, the patient has age-related weight loss or has age-related sarcopenia. In the case of age-related sarcopenia, a patient may present with muscle wasting without evidence of unintended weight loss. As used herein, at least a subset of patients with age-related weight loss or has age-related sarcopenia may be characterized by an increase in one or more S100 RAGE ligands and/or a decrease in soluble RAGE relative to a reference level.

[0218] In other embodiments, the patient has a disease-related cachexia that is not associated with cancer, but that like cancer-induced cachexia fails to respond to treatment with nutritional support and anti-inflammatory therapy. Such disease-related cachexia may, for example, be associated with AIDS, chronic obstructive lung disease, or congestive heart failure and, like cancer-induced cachexia are characterized by an increase in one or more S100 RAGE ligands and/or a decrease in soluble RAGE relative to a reference level.

[0219] In certain embodiments, the target cell is selected from a myocyte, an adipocyte, and a hepatocyte. In a preferred embodiment, the target cell is a myocyte.

[0220] The presence or absence of the herein disclosed marker(s) is measured in a tissue (e.g., biopsy) or bodily fluid from a pre-cachectic or cachectic subject. Bodily fluids used to evaluate the presence or absence of the herein disclosed markers include without limitation blood, serum, plasma, urine, and or saliva. For example, levels of biomarker are measured in the blood or biopsy before and after treatment in a subject.

[0221] Biopsy refers to the removal of a sample of tissue for purposes of diagnosis. For example, a biopsy is from a muscle, fat, a cancer or tumor, including a sample of tissue from an abnormal area or an entire tumor.

[0222] The disclosed methods involve comparing the presence or levels of the disclosed markers in a sample from a subject identified as having cancer or a pre-cancerous condition to the levels of the same markers in a reference (e.g., levels present in a corresponding sample from a healthy control). It is understood a reference includes a concurrently run control, or a standard created by assaying one or more non-cancer cells and collecting the marker data. Thus, the control sample is optionally a standard that is created and used continuously. The standard includes, for example, the average level of a biomarker in a sample from a non-cancer control group.

[0223] Also provided is a method of predicting or monitoring the efficacy of an anti-cachectic agent in a subject. The method comprises acquiring a biological sample, such as tissue or bodily fluid, from the subject after administering the agent to the subject. F or example, the tissue or bodily fluid is collected from the subject 1 to 60 minutes, hours, days, or weeks after administering the agent to the subject. The method further comprises detecting levels of one or more biomarkers delineated herein (e.g., S100 family proteins). A decrease in level(s) of one or more biomarkers is evidence of treatment efficacy. Thus, a decline in said increase or time is evidence of decreasing efficacy. Thus, it is preferred that biological samples be systematically acquired over time to monitor changes in marker levels.

Panels and Arrays for Characterizing Pre-cachexia or Cachexia

[0224] The invention provides panels for characterizing cachexia comprising the aforementioned markers. In one embodiment, the panel is present on a protein array. Protein arrays are solid-phase ligand binding assay systems using immobilized proteins on surfaces which include glass, membranes, microtiter wells, mass spectrometer plates, and beads or other particles. The assays are highly parallel (multiplexed) and often miniaturized (microarrays, protein chips). Their advantages include being rapid and automatable, capable of high sensitivity, economical on reagents, and giving an abundance of data for a single experiment. Bioinformatics support is important; the data handling demands sophisticated software and data comparison analysis. However, the software is adapted from that used for DNA arrays, as can much of the hardware and detection systems.

[0225] One of the chief formats for characterizing cachexia is the capture array, in which ligand-binding reagents, which are usually antibodies, but can also be alternative protein scaffolds, peptides or nucleic acid aptamers, are used to detect target molecules in mixtures such as plasma or tissue extracts. In diagnostics, capture arrays are used to carry out multiple immunoassays in parallel, both testing for several analytes in individual sera for example and testing many serum samples simultaneously. In proteomics, capture arrays are used to quantitate and compare the levels of proteins in different samples in health and disease, i.e. protein expression profiling. Proteins other than specific ligand binders are used in the array format for in vitro functional interaction screens such as protein-protein, protein-DNA, protein-drug, receptor-ligand, enzyme-substrate, etc. The capture reagents themselves are selected and screened against many proteins, optionally in a multiplex array format against multiple protein targets.

[0226] For construction of arrays, sources of proteins include cell-based expression systems for recombinant proteins, purification from natural sources, production in vitro by cell-free translation systems, and synthetic methods for peptides. Many of these methods are automated for high throughput production. For capture arrays and protein function analysis, it is important that proteins be correctly folded and functional; this is not always the case, e.g., where recombinant proteins are extracted from bacteria under denaturing conditions. Nevertheless, arrays of denatured proteins are useful in screening antibodies for cross-reactivity, and selecting ligand binding proteins.

[0227] Protein arrays have been designed as a miniaturization of familiar immunoassay methods such as ELISA and dot blotting, often utilizing fluorescent readout, and facilitated by robotics and high throughput detection systems to enable multiple assays to be carried out in parallel. Physical supports include glass slides, silicon, microwells, nitrocellulose or PVDF membranes, and magnetic and other microbeads. While microdrops of protein delivered onto planar surfaces are the most familiar format, alternative architectures include CD centrifugation devices based on developments in microfluidics (Gyros, Monmouth Junction, N.J.) and specialized chip designs, such as engineered microchannels in a plate (e.g., The Living Chip™, Biotrove, Woburn, Mass.) and tiny 3D posts on a silicon surface (Zyomyx, Hayward Calif.). Particles in suspension are also used as the basis of arrays, providing they are coded for identification; systems include color coding for microbeads (Luminex, Austin, Tex.; Bio-Rad Laboratories), semiconductor nanocrystals (e.g., QDOTS™, Quantum Dot, Hayward, Calif.), barcoding for beads (ULTRAPLEX™ beads, SmartBead Technologies Ltd, Babraham, Cambridge, UK) and multimetal microrods (e.g., NANOBARCODES™ particles, Nanoplex Technologies, Mountain View, Calif.). Beads are optionally assembled into planar arrays on semiconductor chips (LEAPS™ technology, BioArray Solutions, Warren, N.J.).

[0228] Immobilization of proteins involves both the coupling reagent and the nature of the surface being coupled to. A good protein array support surface is chemically stable before and after the coupling procedures, allows good spot morphology, displays minimal nonspecific binding, does not contribute a background in detection systems, and is compatible with different detection systems. The immobilization method used are reproducible, applicable to proteins of different properties (size, hydrophilic, hydrophobic), amenable to high throughput and automation, and compatible with retention of fully functional protein activity. Orientation of the surface-bound protein is recognized as an important factor in presenting it to ligand or substrate in an active state; for capture arrays the most efficient binding results are obtained with orientated capture reagents, which generally require site-specific labeling of the protein.

[0229] Both covalent and noncovalent methods of protein immobilization are used and have various pros and cons. Passive adsorption to surfaces is methodologically simple, but allows little quantitative or orientational control. It may or may not alter the functional properties of the protein, and reproducibility and efficiency are variable. Covalent coupling methods provide a stable linkage, are applied to a range of proteins and have good reproducibility. However, orientation is variable. Furthermore, chemical derivatization may alter the function of the protein and requires a stable interactive surface. Biological capture methods utilizing a tag on the protein provide a stable linkage and bind the protein specifically and in reproducible orientation, but the biological reagent must first be immobilized adequately, and the array may require special handling and have variable stability.

[0230] Several immobilization chemistries and tags have been described for fabrication of protein arrays. Substrates for covalent attachment include glass slides coated with amino- or aldehyde-containing silane reagents. In the VERSALINX™ system (Prolinx, Bothell, Wash.) reversible covalent coupling is achieved by interaction between the protein derivatised with phenyldiboronic acid, and salicylhydroxamic acid immobilized on the support surface. This also has low background binding and low intrinsic fluorescence and allows the immobilized proteins to retain function. Noncovalent binding of unmodified protein occurs within porous structures such as HYDROGEL™ (PerkinElmer, Wellesley, Mass.), based on a 3-dimensional polyacrylamide gel; this substrate is reported to give a particularly low background on glass microarrays, with a high capacity and retention of protein function. Widely used biological coupling methods are through biotin/streptavidin or hexahistidine (SEQ ID NO: 12)/Ni interactions, having modified the protein appropriately. Biotin may be conjugated to a poly-lysine backbone immobilized on a surface such as titanium dioxide (Zyomyx, Inc., Hayward, Calif.) or tantalum pentoxide (Zeptosens, Witterswil, Switzerland).

[0231] Array fabrication methods include robotic contact printing, ink-jetting, piezoelectric spotting and photolithography. A number of commercial arrayers are available [e.g. Packard Biosciences, Affymetrix Inc. and Genetix] as well as manual equipment [e.g., V & P Scientific]. Bacterial colonies are optionally robotically gridded onto PVDF membranes for induction of protein expression in situ.

[0232] At the limit of spot size and density are nanoarrays, with spots on the nanometer spatial scale, enabling thousands of reactions to be performed on a single chip less than 1 mm square. BioForce Nanosciences Inc. and Nanolink Inc., for example, have developed commercially available nanoarrays.

[0233] Fluorescence labeling and detection methods are widely used. The same instrumentation as used for reading DNA microarrays is applicable to protein arrays. For differential display, capture (e.g., antibody) arrays are probed with fluorescently labeled proteins from two different cell states, in which cell lysates are directly conjugated with different fluorophores (e.g. Cy-3, Cy-5) and mixed, such that the color acts as a readout for changes in target abundance. Fluorescent readout sensitivity is amplified 10-100 fold by tyramide signal amplification (TSA) (PerkinElmer Lifesciences). Planar waveguide technology (Zeptosens) enables ultrasensitive fluorescence detection, with the additional advantage of no intervening washing procedures. High sensitivity is achieved with suspension beads and particles, using phycoerythrin as label (Luminex) or the properties of semiconductor nanocrystals (Quantum Dot). A number of novel alternative readouts have been developed, especially in the commercial biotech arena. These include adaptations of surface plasmon resonance (HTS Biosystems, Intrinsic Bioprobes, Tempe, Ariz.), rolling circle DNA amplification (Molecular Staging, New Haven, Conn.), mass spectrometry (Intrinsic Bioprobes; Ciphergen, Fremont, Calif.), resonance light scattering (Genicon Sciences, San Diego, Calif.) and atomic force microscopy [BioForce Laboratories].

[0234] Capture arrays form the basis of diagnostic chips and arrays for expression profiling. They employ high affinity capture reagents, such as conventional antibodies, single domains, engineered scaffolds, peptides or nucleic acid aptamers, to bind and detect specific target ligands in high throughput manner.

[0235] Antibody arrays have the required properties of specificity and acceptable background, and some are available commercially (BD Biosciences, San Jose, Calif.; Clontech, Mountain View, Calif.; BioRad; Sigma, St. Louis, Mo.). Antibodies for capture arrays are made either by conventional immunization (polyclonal sera and hybridomas), or as recombinant fragments, usually expressed in E. coli, after selection from phage or ribosome display libraries (Cambridge Antibody Technology, Cambridge, UK; Biolnvent, Lund, Sweden; Affitech, Walnut Creek, Calif.; Biosite, San Diego, Calif.). In addition to the conventional antibodies, Fab and scFv fragments, single V-domains from camelids or engineered human equivalents (Domantis, Waltham, Mass.) are optionally useful in arrays.

[0236] The term scaffold refers to ligand-binding domains of proteins, which are engineered into multiple variants capable of binding diverse target molecules with antibody-like properties of specificity and affinity. The variants are produced in a genetic library format and selected against individual targets by phage, bacterial or ribosome display. Such ligand-binding scaffolds or frameworks include Affibodies based on S. aureus protein A (Affibody, Bromma, Sweden), Trinectins based on fibronectins (Phylos, Lexington, Mass.) and Anticalins based on the lipocalin structure (Pieris Proteolab, Freising-Weihenstephan, Germany). These are used on capture arrays in a similar fashion to antibodies and have advantages of robustness and ease of production.

[0237] Nonprotein capture molecules, notably the single-stranded nucleic acid aptamers which bind protein ligands with high specificity and affinity, are also used in arrays (SomaLogic, Boulder, Colo.). Aptamers are selected from libraries of oligonucleotides by the Selex™ procedure (SomaLogic, Boulder, Colo.) and their interaction with protein is enhanced by covalent attachment, through incorporation of brominated deoxyuridine and UV-activated crosslinking (photoaptamers). Photocrosslinking to ligand reduces the crossreactivity of aptamers due to the specific steric requirements. Aptamers have the advantages of ease of production by automated oligonucleotide synthesis and the stability and robustness of DNA; on photoaptamer arrays, universal fluorescent protein stains are used to detect binding.

[0238] Protein analytes binding to antibody arrays are detected directly or indirectly, for example, via a secondary antibody. Direct labeling is used for comparison of different samples with different colors. Where pairs of antibodies directed at the same protein ligand are available, sandwich immunoassays provide high specificity and sensitivity and are therefore the method of choice for low abundance proteins such as cytokines; they also give the possibility of detection of protein modifications. Label-free detection methods, including mass spectrometry, surface plasmon resonance and atomic force microscopy, avoid alteration of ligand. What is required from any method is optimal sensitivity and specificity, with low background to give high signal to noise. Since analyte concentrations cover a wide range, sensitivity has to be tailored appropriately. Serial dilution of the sample or use of antibodies of different affinities are solutions to this problem. Proteins of interest are frequently those in low concentration in body fluids and extracts, requiring detection in the pictogram (pg) range or lower, such as cytokines or the low expression products in cells.

[0239] An alternative to an array of capture molecules is one made through molecular imprinting technology, in which peptides (e.g., from the C-terminal regions of proteins) are used as templates to generate structurally complementary, sequence-specific cavities in a polymerizable matrix; the cavities can then specifically capture (denatured) proteins that have the appropriate primary amino acid sequence (ProteinPrint™, Aspira Biosystems, Burlingame, Calif.).

[0240] Another methodology which is useful diagnostically and in expression profiling is the ProteinChipφ array (Ciphergen, Fremont, Calif.), in which solid phase chromatographic surfaces bind proteins with similar characteristics of charge or hydrophobicity from mixtures such as plasma or tumor extracts, and SELDI-TOF mass spectrometry is used to detection the retained proteins.

[0241] Large-scale functional chips have been constructed by immobilizing large numbers of purified proteins and are used to assay a wide range of biochemical functions, such as protein interactions with other proteins, drug-target interactions, enzyme-substrates, etc. Generally they require an expression library, cloned into E. coli, yeast or similar from which the expressed proteins are then purified, e.g., via a His tag and immobilized. Cell free protein transcription/translation is a viable alternative for synthesis of proteins which do not express well in bacterial or other in vivo systems.

[0242] For detecting protein-protein interactions, protein arrays are in vitro alternatives to the cell-based yeast two-hybrid system and are useful where the latter is deficient, such as interactions involving secreted proteins or proteins with disulphide bridges. High-throughput analysis of biochemical activities on arrays has been described for yeast protein kinases and for various functions (protein-protein and protein-lipid interactions) of the yeast proteome, where a large proportion of all yeast open-reading frames was expressed and immobilized on a microarray. Large-scale proteome chips are also useful in identification of functional interactions, drug screening, etc. (Proteometrix, Branford, Conn.).

[0243] As a two-dimensional display of individual elements, a protein array is used to screen phage or ribosome display libraries, in order to select specific binding partners, including antibodies, synthetic scaffolds, peptides and aptamers. In this way, library against library screening is carried out. Screening of drug candidates in combinatorial chemical libraries against an array of protein targets identified from genome projects is another application of the approach.

[0244] Multiplexed bead assays use a series of spectrally discrete particles that are used to capture and quantitate soluble analytes. The analyte is then measured by detection of a fluorescence-based emission and flow cytometric analysis. Multiplexed bead assays generate data that is comparable to ELISA based assays, but in a multiplexed or simultaneous fashion. Concentration of unknowns is calculated for the cytometric bead array as with any sandwich format assay, i.e., through the use of known standards and by plotting unknowns against a standard curve. Further, multiplexed bead assays allow quantification of soluble analytes in samples never previously considered due to sample volume limitations. In addition to the quantitative data, powerful visual images are generated revealing unique profiles or signatures that provide the user with additional information at a glance.

[0245] In some examples of the disclosed methods, when the level of expression of a biomarker(s) is assessed, the level is compared with the level of expression of the biomarker(s) in a reference standard. By reference standard is meant the level of expression of a particular biomarker(s) from a sample or subject lacking a cancer, at a selected stage of cancer, or in the absence of a particular variable such as a therapeutic agent. Alternatively, the reference standard comprises a known amount of biomarker. Such a known amount correlates with an average level of subjects lacking a cancer, at a selected stage of cancer, or in the absence of a particular variable such as a therapeutic agent. A reference standard also includes the expression level of one or more biomarkers from one or more selected samples or subjects as described herein. For example, a reference standard includes an assessment of the expression level of one or more biomarkers in a sample from a subject that does not have a cancer, is at a selected stage of progression of a cancer, or has not received treatment for a cancer. Another exemplary reference standard includes an assessment of the expression level of one or more biomarkers in samples taken from multiple subjects that do not have a cancer, are at a selected stage of progression of a cancer, or have not received treatment for a cancer.

[0246] When the reference standard includes the level of expression of one or more biomarkers in a sample or subject in the absence of a therapeutic agent, the control sample or subject is optionally the same sample or subject to be tested before or after treatment with a therapeutic agent or is a selected sample or subject in the absence of the therapeutic agent. Alternatively, a reference standard is an average expression level calculated from a number of subjects without a particular cancer. A reference standard also includes a known control level or value known in the art. In one aspect of the methods disclosed herein, it is desirable to age-match a reference standard with the subject diagnosed with a cancer.

[0247] In one technique to compare protein levels of expression from two different samples (e.g., a sample from a subject diagnosed with a cancer and a reference standard), each sample is separately subjected to 2D gel electrophoresis. Alternatively, each sample is differently labeled and both samples are loaded onto the same 2D gel. See, e.g., Unlu et al. Electrophoresis, 1997; 18:2071-2077, which is incorporated by reference herein for at least its teachings of methods to assess and compare levels of protein expression. The same protein or group of proteins in each sample is identified by the relative position within the pattern of proteins resolved by 2D electrophoresis. The expression levels of one or more proteins in a first sample is then compared to the expression level of the same protein(s) in the second sample, thereby allowing the identification of a protein or group of proteins that is expressed differently between the two samples (e.g., a biomarker). This comparison is made for subjects before and after they are suspected of having a cancer, before and after they begin a therapeutic regimen, and over the course of that regimen.

[0248] In another technique, the expression level of one or more proteins is in a single sample as a percentage of total expressed proteins. This assessed level of expression is compared to a preexisting reference standard, thereby allowing for the identification of proteins that are differentially expressed in the sample relative to the reference standard.

[0249] There are a variety of sequences related to biomarkers as well as any other protein disclosed herein that are disclosed on GenBank, and these sequences and others are herein incorporated by reference in their entireties as well as for individual subsequences contained therein. Thus, a variety of sequences are provided herein and these and others are found in GenBank at http://www.ncbi.nih.gov/entrez/query.fcgi. Those of skill in the art understand how to resolve sequence discrepancies and differences and to adjust the compositions and methods relating to a particular sequence to other related sequences. Primers and/or probes are designed for any sequence given the information disclosed herein and known in the art.

[0250] Screens for in vivo validation of the RAGE pathway in cachexia, eg. cancer cachexia may be conducted by either targeting RAGE receptors or RAGE ligands, as illustrated in FIGS. 41 and 42. In particular the aspects of the CRISPR system may be used to knock out the RAGE receptor using AAV-Cas9 system (e.g. AAV-SaCas9) or knock out a RAGE ligand by expressing a CRISPR-Cas system with a guide RNA targeting a genomic locus comprising the coding sequence of a particular RAGE ligand. With respect to general information on CRISPR-Cas Systems, components thereof, and delivery of such components, including methods, materials, delivery vehicles, vectors, particles, AAV, and making and using thereof, including as to amounts and formulations, all useful in the practice of the instant invention, reference is made to general information on CRISPR-Cas Systems, mention is made of the following (also hereby incorporated herein by reference): [0251] Multiplex genome engineering using CRISPR/Cas systems. Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D., Wu, X., Jiang, W., Marraffini, L. A., & Zhang, F. Science February 15; 339(6121):819-23 (2013); [0252] RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Jiang W., Bikard D., Cox D., Zhang F, Marraffini L A. Nat Biotechnol March; 31(3):233-9 (2013); [0253] One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering. Wang H., Yang H., Shivalila C S., Dawlaty M M., Cheng A W., Zhang F., Jaenisch R. Cell May 9; 153(4):910-8 (2013); [0254] Optical control of mammalian endogenous transcription and epigenetic states. Konermann S, Brigham M D, Trevino A E, Hsu P D, Heidenreich M, Cong L, Platt R J, Scott D A, Church G M, Zhang F. Nature. 2013 Aug. 22; 500(7463):472-6. doi: 10.1038/Nature12466. Epub 2013 Aug. 23; [0255] Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity. Ran, F A., Hsu, P D., Lin, C Y., Gootenberg, J S., Konermann, S., Trevino, A E., Scott, D A., Inoue, A., Matoba, S., Zhang, Y., & Zhang, F. Cell August 28. pii: S0092-8674(13)01015-5. (2013); [0256] DNA targeting specificity of RNA-guided Cas9 nucleases. Hsu, P., Scott, D., Weinstein, J., Ran, F A., Konermann, S., Agarwala, V., Li, Y., Fine, E., Wu, X., Shalem, O., Cradick, T J., Marraffini, LA., Bao, G., & Zhang, F. Nat Biotechnol doi:10.1038/nbt.2647 (2013); [0257] Genome engineering using the CRISPR-Cas9 system. Ran, F A., Hsu, P D., Wright, J., Agarwala, V., Scott, D A., Zhang, F. Nature Protocols November; 8(11):2281-308. (2013); [0258] Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells. Shalem, O., Sanjana, N E., Hartenian, E., Shi, X., Scott, D A., Mikkelson, T., Heckl, D., Ebert, B L., Root, D E., Doench, J G., Zhang, F. Science December 12. (2013). [Epub ahead of print]; [0259] Crystal structure of cas9 in complex with guide RNA and target DNA. Nishimasu, H., Ran, F A., Hsu, P D., Konermann, S., Shehata, S I., Dohmae, N., Ishitani, R., Zhang, F., Nureki, O. Cell February 27. (2014). 156(5):935-49; [0260] Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells. Wu X., Scott D A., Kriz A J., Chiu A C., Hsu P D., Dadon D B., Cheng A W., Trevino A E., Konermann S., Chen S., Jaenisch R., Zhang F., Sharp P A. Nat Biotechnol. (2014) April 20. doi: 10.1038/nbt.2889, [0261] CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling, Platt et al., Cell 159(2): 440-455 (2014) DOI: 10.1016/j.cell.2014.09.014, [0262] Development and Applications of CRISPR-Cas9 for Genome Engineering, Hsu et al, Cell 157, 1262-1278 (Jun. 5, 2014) (Hsu 2014), [0263] Genetic screens in human cells using the CRISPR/Cas9 system, Wang et al., Science. 2014 Jan. 3; 343(6166): 80-84. doi:10.1126/science.1246981, [0264] Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation, Doench et al., Nature Biotechnology published online 3 Sep. 2014; doi:10.1038/nbt.3026, and [0265] In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9, Swiech et al, Nature Biotechnology published online 19 Oct. 2014; doi:10.1038/nbt.3055, [0266] Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Konermann, et al, Nature published online 10 Dec. 2014, doi: 10.1038/nature14136, [0267] Genome-wide CRISPR Screen in a Mouse Model of tumor Growth and Metastasis. Chen, et al, Cell published 12 Mar. 2015, doi: 10.1016/j.cell.2015.02.038, and [0268] In vivo genome editing using Staphyloccocus aureus Cas9. Ran et al, Nature published online 9 Apr. 2015, doi: 10.1038/nature14299
each of which is incorporated herein by reference.

[0269] In aspects of the invention, screening may be via High-throughput means or in vivo animal model systems in which expression, epigentic state and loss of function metrics are utilized. In a preferred embodiment, a GeCKO library may be utilized in the screen.

Methods of Treating and/or Preventing Pre-cachexia and/or Cachexia

[0270] In one aspect, the present invention provides a method of treating and/or preventing cachexia or pre-cachexia in a patient, comprising administering to a patient in need thereof an effective dose of a compound that inhibits the RAGE signaling pathway, or a pharmaceutically acceptable salt or prodrug thereof. As described herein, activation of the Receptor for Advanced Glycation End-products was associated with the cachectic phenotype induced by multiple cancer types.

[0271] In certain embodiments, the compound inhibits the Receptor for Advanced Glycation Endproducts (RAGE). RAGE is a member of the immunoglobulin supergene family of molecules and was identified herein to be transcriptionally upregulated in muscle cells exposed to a cachexia-inducing factor(s). The extracellular (N-terminal) domain of RAGE includes three immunoglobulin-type regions: one V (variable) type domain followed by two C-type (constant) domains (Neeper et al., J Biol Chem 267:14998-15004 (1992); and Schmidt et al., Circ (Suppl) 96#194 (1997)). A single transmembrane-spanning domain and a short, highly charged cytosolic tail follow the extracellular domain. The N-terminal, extracellular domain can be isolated by proteolysis of RAGE or by molecular biological approaches to generate soluble RAGE (sRAGE) comprising the V and C domains. Soluble RAGE (a negative regulator of RAGE signaling that acts as a sink for RAGE ligands) was also identified herein to be elevated in non-cachexia-inducing conditioned media in comparison to cachexia-inducing conditioned media. Additionally, treatment with a blocking peptide or antibody to RAGE has been shown to inhibit and reverse the cachectic induced loss of myosin heavy chain protein in human muscle cells in vitro. RAGE blocking peptides are known in the art (see, for example, Arumugam et al., Clin Cancer Res. 2012; 18(16): 10.1158/1078-0432.CCR-12-0221), and are commercially available (e.g., RAP; a 10 amino acid sequence from S100P (R&D Systems)). Importantly, in contrast to the prior art, in the present invention antibody(ies) or blocking peptide(s) specific for RAGE compete(s) for binding to RAGE with RAGE ligand(s) S100A7, S100A8, S100A9 and/or Calprotectin, and preferably may not compete for binding to RAGE with RAGE ligand HMGB1.

[0272] RAGE binds to multiple functionally and structurally diverse ligands, such as proteins having β-sheet fibrils characteristic of amyloid deposits and pro-inflammatory mediators, and includes amyloid beta (Aβ), serum amyloid A (SAA), Advanced Glycation End products (AGEs), S100 (a proinflammatory member of the Calgranulin 65 family), carboxymethyl lysine (CML), Mac-1, β2-integrin, CD11b/CD18 and high-mobility group protein 1 (HMG1), which is also known as high-mobility group protein box-1 (HMGB1) or amphoterin (Bucciarelli et al., Cell Mol Life Sci 59: 1117-1128 (2002); Chavakis et al., Microbes Infect 6:1219-1225 (2004); Kokkola et al., Scand J Immunol 61:1-9 (2005); Schmidt et al., J Clin Invest 108:949-955 (2001); Rocken et al., Am J Pathol 162:1213-1220 (2003); Donato et al., Curr Mol Med 13(1): 24-57 (2013)).

[0273] The invention also provides methods for inhibiting RAGE by alternative means, such as with the use of small organic molecules, soluble receptor fragments, fusion proteins, antibodies or peptides. Mention is made of WO2011/042548, WO2007/109747, WO2009/136382, WO2011053707, WO2007/109749, WO2008/137552, WO2004/016229, US2010/0226915A1 and U.S. Pat. Nos. 7,981,424, 7,485,697 and 8,420,083, which are hereby incorporated by reference for the compounds and methods for inhibiting RAGE disclosed therein. Importantly, in contrast to the prior art, in the present invention antibody(ies) small organic molecule(s), soluble receptor fragment(s), fusion protein(s), or peptide(s) specific for RAGE compete(s) for binding to RAGE with RAGE ligand(s) S100A7, S100A8, S100A9 and/or Calprotectin, and preferably may not compete for binding to RAGE with RAGE ligand HMGB1.

[0274] The invention provides therapeutic, engineered protein/peptide compositions comprising e.g., anti-RAGE antibodies, to target a RAGE receptor (including soluble forms thereof and endogenous secretory RAGE), directly and/or via differential competition with one or more pre-cachexia and/or cachexia-associated RAGE ligands or markers. In one aspect, the invention provides an anti-RAGE antibody or antibodies specific for a RAGE receptor, which antibody competes for binding to the RAGE receptor with a RAGE ligand of the S100 family, e.g., S100A7, S100A8, S100A9 and/or Calprotectin, and preferably may not compete for binding to the RAGE receptor with the RAGE ligand HMGB1. In another aspect, the invention provides an anti-RAGE antibody mixture/cocktail. comprising one or more antibodies which compete for ligand binding to the RAGE receptor, wherein each antibody in said mixture/cocktail competes for binding of one or more specific S100 family member/ligand (e.g., S100A7, S100A8, S100A9 and/or Calprotectin) to the RAGE receptor, and preferably may not compete for binding to the RAGE receptor with the RAGE ligand HMGB1. In another aspect, the invention provides an antibody specific for a RAGE receptor, which antibody competes for binding to the RAGE receptor with a RAGE ligand of the S100 family, wherein said competition is via a moiety/epitope (linear and/or conformational) which is bound by one or more specific S100 family members/ligands that induce pre-cachexia and/or cachexia. In one embodiment, the antibody binds to an epitope on the RAGE receptor selected from an epitope, which is bound by S100A7, S100A8, S100A9 and/or Calprotectin, and preferably may not bind to an epitope which is bound by the RAGE ligand HMGB1.

[0275] Suitable antibodies (or small organic molecules, soluble receptor fragments, fusion proteins) may also comprise a label attached thereto, such as a detectable label (e.g., a radioisotope, fluorescent compound, enzyme or enzyme co-factor). Suitable antibodies include whole antibodies and fragments thereof including chimeric antibodies, humanized antibodies, single chain antibodies, tetrameric antibodies, tetravalent antibodies, heteroconjugate antibodies, bispecific antibodies, multispecific antibodies, domain-specific antibodies, domain-deleted antibodies, diabodies, antibody conjugates (e.g., with an Fc domain (e.g., an antigen binding domain fused to an immunoglobulin constant region), PEG, an immunoglobulin domain, etc.), Fab fragments, Fab′ fragments, F(ab′).sub.2 fragments, Fv fragments, ScFv fragments, Fd fragments, single domain antibodies, and dAb fragments, and Fc fusion protein.

[0276] In certain embodiments, the compound inhibits MEK/ERK signaling either directly or downstream of MEK/ERK.

[0277] In certain embodiments, the invention provides methods for the treatment and/or prevention of cachexia or pre-cachexia by administering a low dose(s) of MEK/ERK inhibitors, e.g., as compared to an effective dose of that inhibitor for the treatment of cancer. In certain embodiments, an effective dose of the compound for treating or preventing cachexia or pre-cachexia is at most half the effective therapeutic dose of that compound for the treatment of cancer. In certain embodiments, an effective dose of the compound is half the effective therapeutic dose for the treatment of cancer. In certain embodiments, an effective dose of the compound is less than half the effective therapeutic dose for the treatment of cancer. Accordingly, a dose of a MEK/ERK inhibitor that effectively treats and/or prevents cachexia or pre-cachexia may not be an effective dose for treating cancer. Additionally, because complete inactivation of MEK/ERK in muscle cells can be deleterious, in some embodiments, an effective dose of MEK/ERK inhibitors to treat and/or prevent cachexia results in MEK/ERK pathway activity that is at least 50%, 40%, 30% or 20% of healthy baseline. For example, an effective amount of a MEK/ERK inhibitor to treat and/or prevent cachexia results in MEK/ERK pathway activity that is at least 50%, 40%, 30% or 20% of healthy baseline.

[0278] In certain embodiments, MEK/ERK and/or RAGE signaling is inhibited in a myocyte or an adipocyte. In certain embodiments, the myocyte is a mature or immature myocyte. In certain embodiments, the myocyte is a skeletal myocyte or a cardiac myocyte. In a preferred embodiment, the myocyte is a skeletal myocyte or a cardiac myocyte.

[0279] In certain embodiments, an effective dose of the compound increases myofibrillar protein content and does not inhibit cellular proliferation of the myocyte. In certain embodiments, the myofibrillar protein is selected from myosin, actin, tropomyosin, myosin heavy chain, myosin light chain, troponin, titin, and nebulin. In a preferred embodiment, the myofibrillar protein is a myosin heavy chain protein.

[0280] In certain embodiments, the patient has cachexia or pre-cachexia associated with a cancer. In a preferred embodiment, the patient has cachexia or pre-cachexia associated with a cancer. In one embodiment, the cancer is an epithelial-derived cancer or a mesenchemal-derived cancer. In one embodiment, the cancer is selected from one or more of a carcinoma, a sarcoma, and cancers of the skin, pancreas, stomach, colon, thorax, liver, gallbladder, musculoskeletal system, breast, lung, ovary, uterus, endometrium, prostrate, colon, skin, mouth, salivary, esophagus, head and neck, plus other tumors of the gastrointestinal tract.

[0281] Significantly, no treatment for cancer-induced cachexia was previously available.

[0282] In other embodiments, the patient has age-related weight loss or has age-related sarcopenia. In the case of age-related sarcopenia, unintended weight loss is not necessarily present. In at least a subset of this population, such conditions are characterized by an increase in one or more S100 RAGE ligands and/or a decrease in soluble RAGE relative to a reference level.

[0283] In other embodiments, the patient has disease-associated cachexia that is not associated with cancer, but that like cancer-induced cachexia fails to respond to treatment with nutritional support and anti-inflammatory therapy. Such disease-associated cachexia may, for example, be associated with AIDS, chronic obstructive lung disease, or congestive heart failure and, like cancer-induced cachexia are characterized by an increase in one or more S100 RAGE ligands and/or a decrease in soluble RAGE relative to a reference level.

[0284] The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture” (Freshney, 1987); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Current Protocols in Molecular Biology” (Ausubel, 1987); “PCR: The Polymerase Chain Reaction” (Mullis, 1994); “Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

[0285] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.

EXAMPLES

Example 1

Differential Cachexia induction by S100 and HMGB1 Marker Proteins

[0286] FIGS. 1-4 illustrate the effect of various proteins of the S100 family, as well as HMGB1, as cachexia inducers when conmpared with cachexia-inducing media. Primary human myoblast were plated and differentiated into terminal myotubes as previously described. Recombinant ligands were prepared at the same dose curve in Hanks Balance Salt Solution (buffer) and applied 1:2 v:v to fresh maintenance media each day for 3 days. Cells were assessed for myosin HC content by in-cell Western staining as previously described. S100 A7, A8 and A9 are shown to be inducers of the cachectic phenotype, whilst S100B, HMGB1 and other S100 family members are not.

Example 2

Mapping of Helix 2A in S100

[0287] Crystallographic structures for each S100 protein involved in the induction of cachexia, as well as S100B, were obtained from public databases and the overlaid using standard software, which revealed the uniqure structural motifs shared among S100A7, A8 and A9. We hypothesize that these uniquely shared structural motifs provide unique binding sites to RAGE and are functionally important in RAGE cachexia signaling. Alignment of the protein sequences indicates that S100 A7, A8 and A9 possess residues forming helix A2, which is not present in S100B and other non-cachectic factors.

Example 3

Inhibitory Antibodies Block Cachexia Induction by Conditioned Media

[0288] 7 anti-RAGE Ab targeting various domains of RAGE were analyzed by MyosinHC in-cell Western analysis in Primary human myotubes. All antibodies were dosed at the same dose curve (1, 5, 10 ug/ml) along with conditioned media from either cachexia-inducing melanoma or gastric cancer cell lines. For comparision, 2 other recently reported anti-cachexia antibodies were tested against the conditioned media as well; both were negative for anti-cachexia activity.

[0289] Antibodes tested were as follows:

[0290] 1. IgG-like C2-type 1, Abbiotec 251890; raised against a KLH-conjugated synthetic peptide encompassing a sequence within the center region of human RAGE.

[0291] 2. IgG-like C2-typel, Aviva OAAB04025; specific for the central regain of RAGE.

[0292] 3. V Domain 1, Milipore AB9714; a portion of amino acids 1-60 of human RAGE was used as the immunogen.

[0293] 4. V-Domain 2, Novus NBP2-03950; raised against a peptide representing amino acids 23-54 of RAGE of bovine origin.

[0294] 5. V-Domain 4, Santa Cruz SC-3362; raised against a peptide representing amino acids 23-54 of RAGE of bovine origin.

[0295] 6. V-Domain 5, Santa Cuz SC-365154; RAGE (A-9) is a mouse monoclonal antibody specific for an epitope mapping between amino acids 23-43 at the N-terminus of RAGE of human origin.

[0296] 7. ECD, R&D Systems MAB-1141; raised against rh-RAGE (aa 24-344).

[0297] Antibodies 5 and 7 are effective in inhibiting cachexia.

Other Embodiments

[0298] The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0299] Having thus described in detail preferred embodiments of the present invention, it is to be understood that the invention defined by the above paragraphs is not to be limited to particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope of the present invention.