WO2017139405A1 - Method of reducing tolerization of t cells to tumor antigens - Google Patents

Abstract

Disclosed are methods for reducing T cell tolerization to tumor antigens in a subject in need, the methods comprising administering to the subject a therapeutically effective amount of an enhancer of RGMb interaction with PD-L2. The methods are useful for treating cancer.

Description

METHOD OF REDUCING TOLERIZATION OF T CELLS TO TUMOR ANTIGENS

FIELD

[0001] This invention relates to methods of reducing tolerization of T cells to tumor antigens using enhancers of repulsive guidance molecule family member b (RGMb) interaction with tumor antigen PD-1 ligand 2 (PD-L2). This invention also relates to methods useful for treating cancers.

BACKGROUND

[0002] Cancer therapies are limited by the development of immune tolerance, a state of unresponsiveness of the immune system to tumor cells. This is strongly influenced by T cell tolerization. When T cells become tolerized to tumor antigens, induction of the immune response is inhibited. There is a need in the art for compositions and methods for reducing T cell tolerization in the treatment of cancer.

SUMMARY

[0003] The present disclosure provides a method of reducing tolerization of T cells to one or more tumor antigens in a subject in need, the method comprising administering to the subject a therapeutically effective amount of an enhancer of repulsive guidance molecule family member b (RGMb) interaction with PD-L2.

[0004] In a preferred embodiment, the present disclosure provides a method of using a soluble, chimeric, fusion or pegylated RGMb protein to reduce tolerization of T cells to one or more tumor antigens in a subject in need. [0005] In an aspect, the present disclosure provides a method of treating a cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an enhancer of RGMb interaction with PD-L2.

[0006] In one embodiment, the enhancer increases RGMb in the subj ect for binding with PD- L2. In one embodiment, the enhancer is targeted to one or more RGMb molecules in the subject. In another embodiment, the enhancer is targeted to a cell or tissue type in a subject that is enriched in RGMb in the subj ect. In an embodiment of each of the foregoing aspects, the enhancer of RGMb interaction with PD-L2 is targeted to a cancer cell expressing the one or more tumor antigens.

[0007] In one embodiment, the enhancer of RGMb interaction with PD-L2 is a soluble, chimeric, fusion or pegylated form of RGMb, which binds with tumor antigen PD-1 ligand 2 (PD-L2), preventing PD-L2 interactions with Programmed Cell Death Protein 1 (PD-1). In one aspect of this embodiment the soluble RGMb protein is RGMb.Fc. In an embodiment the Fc is human Fc. The soluble, chimeric, fusion or pegylated forms of RGMb may be targeted to a cancer cell expressing the one or more tumor antigens. In certain embodiments, the soluble RGMb protein is targeted to a cancer cell expressing the one or more tumor antigens.

[0008] In another embodiment, the enhancer of RGMb interaction with PD-L2 is a PD-L2- specific antibody or a fragment thereof. In an embodiment, the antibody or fragment thereof mimics the interaction of RGMb with PD-L2. In an embodiment, the antibody is targeted to a cancer cell expressing the one or more tumor antigens. [0009] In an embodiment, the PD-L2-specific antibody is a bispecific antibody that simultaneously binds PD-L2 and one of the one or more tumor antigens.

[0010] In another embodiment, the enhancer of RGMb interaction with PD-L2 is a nucleic acid encoding RGMb. In an embodiment, the enhancer of RGMb interaction with PD-L2 is a nucleic acid encoding RGMb.Fc. In an embodiment, the nucleic acid encoding RGMb or RGMb.Fc is targeted to a cancer cell expressing the one or more tumor antigens. In an embodiment, the nucleic acid encoding RGMb or RGMb.Fc is conjugated to an antibody that binds one of the one or more tumor antigens. In an embodiment, the nucleic acid encoding RGMb or RGMb.Fc is conjugated to a tumor cell-specific ligand.

[0011] In another embodiment, the enhancer is a soluble, chimeric, fusion, pegylated RGMb protein comprising a mutation in a PD-L2 binding domain. In an embodiment, the mutation increases RGMb binding to PD-L2. In an embodiment, the mutated RGMb protein is targeted to a cancer cell expressing one of the one or more tumor antigens.

[0012] In certain embodiments, the enhancer of RGMb interaction with PD-L2 is administered directly to a tumor. In an embodiment, the enhancer is injected directly into a tumor. [0013] In an embodiment, the enhancer of RGMb interaction with PD-L2 is administered with one or more cancer therapies. In an embodiment, the cancer therapy is an

immunotherapy, a hormone therapy, a signal transduction inhibitor, a gene expression modulator, an apoptosis inducer, an angiogenesis inhibitor, an antibody-drug conjugate, a cancer vaccine, or a gene therapy.

[0014] In an embodiment the immunotherapy is a T cell receptor (TCR) therapy targeting one of the one or more tumor antigens. In an embodiment, the TCR therapy is an isolated recombinant TCR, wherein the recombinant TCR specifically binds to a major

histocompatibility complex (MHC) molecule complexed with one of the one or more tumor antigens. In an embodiment the TCR therapy is a cell expressing a recombinant TCR on the surface, wherein the TCR specifically binds to an MHC molecule complexed with one of the one or more tumor antigens.

[0015] In an embodiment, the immunotherapy is a chimeric antigen receptor (CAR) therapy targeting one of the one or more tumor antigens. In an embodiment, the CAR therapy is a cell expressing a CAR on the surface, wherein the CAR specifically binds to one of the one or more tumor antigens.

[0016] In an embodiment, the immunotherapy is a monoclonal antibody targeting a tumor antigen. In an embodiment, the monoclonal antibody is conjugated to a chemotherapy drug. In an embodiment, the monoclonal antibody is conjugated to a radioactive particle. [0017] In an embodiment, the immunotherapy is an inhibitor of an immune checkpoint protein. In an embodiment, the immune checkpoint protein is PD-1, PD-L1, PD-L2, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), T cell immunoglobulin and mucin protein 3 (TIM-3), lymphocyte-activation protein 3 (LAG-3), carcinoembryonic antigen- related cell adhesion molecule 1 (CEACAMl), glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR), or tumor necrosis factor receptor superfamily, member 4 (TNFRSF4). In an embodiment, the immune checkpoint protein is a member of the indoleamine-pyrrole 2,3-dioxygenase (IDO) pathway. In an embodiment, the inhibitor of the immune checkpoint protein is norharmane, rosmarinic acid, a cyclooxygenase-2 (COX-2) inhibitor, or alpha-methyl tryptophan. In an embodiment, the immune checkpoint inhibitor is a monoclonal antibody. [0018] In an embodiment, the immunotherapy is a bispecific antibody targeting a tumor antigen and an immune-cell specific protein.

[0019] In an embodiment, the cancer therapy is administered with an adjuvant. In an embodiment, the adjuvant is Quillaj a saponin 21 (QS21), Quillaja saponin 7 (QS7), monophosphoryl lipid A, aluminum hydroxide, an oil-in-water emulsion, bryostatin-1 , a tolllike receptor (TLR) agonist, or CpG oligodeoxynucleotides.

[0020] In an embodiment, the cancer is breast cancer, colon cancer, non-small cell lung carcinoma, testicular cancer, ovarian cancer, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, cervical cancer, endometrial cancer, esophageal cancer, gallbladder cancer, kidney cancer, laryngeal cancer, hypopharyngeal cancer, gastrointestinal cancer, liver cancer, lung cancer, oropharyngeal cancer, pancreatic cancer, penile cancer, prostate cancer, stomach cancer, thymus cancer, thyroid cancer, vaginal cancer, vulvar cancer, skin cancer, bone cancer, leukemia, lymphoma, neuronal cancer of the nervous system, or non-neuronal cancer of the nervous system. [0021] In an embodiment, the subject is a human.

[0022] According to any of the embodiments, herein, the enhancer of RGMb interaction with PD-L2 may be administered systemically. According to certain embodiments, the systemic administration is a route of administration selected from the group consisting of intravenous, intramuscular, intraventricular, intrathecal, oral, topical, subcutaneous, subconjunctival, intranasal, intradermal, sublingual, vaginal, rectal and epidural. In some embodiments, administration is intravenous or subcutaneous.

DETAILED DESCRIPTION

[0023] The methods of use disclosed herein will now be described more fully. The methods described should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of this work to those skilled in the art.

[0024] The disclosure provides methods of reducing T cell tolerization to tumor antigens in a subject in need thereof. The methods include administering to the subject a therapeutically effective amount of an enhancer of repulsive guidance molecule family member b (RGMb) protein interaction with PD-L2. The disclosure also provides methods for treating a cancer in a subject in need thereof. According to some embodiments, the cancer is treated with a therapeutically effective amount of an enhancer of RGMb interaction with PD-L2, and may be targeted to a cancer cell. [0025] "RGMb" refers to RGMb and any naturally occurring RGMb homolog and biologically active fragments thereof. RGMb, also known as DRG11 -Responsive Axonal Guidance and Outgrowth of Neurite (DRAGON), is a glycosylphosphatidylinositol (GPI)- anchored member of the repulsive guidance molecule family. RGMb homologs have been identified in human, mouse, and zebrafish. See WO2003/089608, incorporated by reference herein in its entirety. RGMs have been shown to regulate breast and prostate cancer through bone morphogenetic protein (BMP) signaling. See Samad et al. J. Biol. Chem. 2005 280: 14122-14129; Li et al. J. Cell. Biochem. 2012 113:2523-2531; Li et al. Int. J. Oncology 2012 40: 544-550, each of which is incorporated by reference herein in its entirety. RGMb has also been shown to interact with immune checkpoint protein programmed cell death protein ligand 2 (PD-L2). Xiao et al. J. Experimental Med. 2014 211 :943-959.

[0026] The enhancer of RGMb interaction with PD-L2 may be a soluble, chimeric, fusion or pegylated RGMb protein , a PD-L2-specific antibody or fragment thereof, or a nucleic acid molecule encoding RGMb or RGMb.Fc. The soluble RGMb protein may be RGMb.Fc. An enhancer may be an RGMb protein comprising a mutation in a PD-L2 binding domain. In certain embodiments, the mutation in the PD-L2 binding domain would result in higher affinity of the mutant RGMb for PD-L2. In certain embodiments, this higher affinity is at least 50%, 100%, 200%, 250%, 300%, 400%, 500%, or even 1000% higher than wild-type RGMb. In some cases, the PD-L2-specific antibody or fragment thereof mimics the interaction of RGMb with PD-L2. The PD-L2-specific antibody or fragment thereof may be a bispecific antibody that binds PD-L2and a tumor antigen. The enhancer of RGMb interaction with PD-L2 may be targeted to a cancer cell.

[0027] The enhancer of RGMb interaction with PD-L2 may be administered with one or more cancer therapies, including an immunotherapy, a hormone therapy, a signal transduction inhibitor, a gene expression modulator, an apoptosis inducer, an angiogenesis inhibitor, and an antibody-drug conjugate, a cancer vaccine, and a gene therapy. According to some embodiments, the immunotherapy is a T cell receptor (TCR) therapy targeting a tumor antigen associated with the cancer, such as an isolated recombinant TCR. In some cases, the immunotherapy is a chimeric antigen receptor (CAR) therapy targeting the tumor antigen. The immunotherapy may be a monoclonal antibody targeting the tumor antigen, and may be conjugated to a chemotherapy drug or a radioactive particle. According to some embodiments, the immunotherapy is an inhibitor of a checkpoint protein. [0028] "PD-L2" refers to PD-L2 and any naturally occurring PD-L2 homolog and biologically active fragments thereof. Also known as B7-DC and cluster of differentiation 273 (CD273), PD-L2 is a type I transmembrane glycoprotein that binds to programmed cell death protein-1 (PD-1), an immunoglobulin superfamily member that is expressed on T cells and pro-B cells. The interaction of PD-L2 and PD-1 inhibits T cell receptor (TCR)-mediated proliferation and cytokine production, thereby promoting immune tolerance. Latchman et al. Nat. Immun. 2001 2:261-268; Dai et al. Cell. Immun. 2014 290:72-79.

[0029] As used herein, the term "tolerization" refers to a state at which a T cell becomes less responsive or unresponsive to tumor antigens.

[0030] The term "cancer", as used herein, refers to a disease in which abnormal cells divide uncontrollably and destroy body tissue. Cancers include breast cancer, colon cancer, non- small cell lung carcinoma, testicular cancer, ovarian cancer, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, cervical cancer, endometrial cancer, esophageal cancer, gallbladder cancer, kidney cancer, laryngeal cancer, hypopharyngeal cancer, gastrointestinal cancer, liver cancer, lung cancer, oropharyngeal cancer, pancreatic cancer, penile cancer, prostate cancer, stomach cancer, thymus cancer, thyroid cancer, vaginal cancer, vulvar cancer, skin cancer, bone cancer, leukemia, lymphoma, neuronal cancer of the nervous system, and non-neuronal cancer of the nervous system.

[0031] The term "tumor antigen", as used herein, refers to a protein that is expressed on a tumor cell surface, and is recognized by T cells during immune response. A tumor antigen may be expressed only on tumor cells, and not on other cells. Alternatively, a tumor antigen may be expressed at an abnormally high level in tumor cells compared to non-tumor cells.

[0032] The term "targeted to a cancer cell" refers to preferential expression of enhancers in cancer cells, and minimal expression of enhancers in normal, non-cancer cells.

[0033] The term "therapeutically effective amount" refers to an amount of a compound or composition effective to "treat" a disease or disorder in a subject or mammal. [0034] "Treating" or "treatment" or "alleviation" refers to both therapeutic treatment and prophylactic or preventative measures; wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented. A subject or mammal is successfully "treated" for a disease if, after receiving a therapeutic amount of an enhancer of RGMb interaction with PD-L2 according to the methods of the present disclosure, the subject shows observable and/or measurable reduction in or absence of one or more of the following: reduction in tumor size, reduction in metastasis, and/or relief to some extent, of one or more of the symptoms associated with the disease; reduced morbidity and mortality, and improvement in quality of life issues. The above parameters for assessing successful treatment and improvement in the disease are readily measurable by routine procedures familiar to a physician.

[0035] The term "immunotherapy" refers to therapeutic agents that induce the immune system to target cancer cells. As used herein, immunotherapies include monoclonal antibodies, immune checkpoint inhibitors, T cell receptor therapy, and chimeric antigen receptor therapy.

[0036] "Hormone therapy" refers to therapeutic agents, surgeries, and procedures that reduce the levels of hormones that stimulate cancer cell proliferation.

[0037] "Signal transduction inhibitor" refers to therapeutic agents that inhibit proteins that stimulate cancer cell proliferation. Examples include, but are not limited to, epidermal growth factor receptor (EGFR) inhibitors, human epidermal growth factor receptor 2 (HER2) inhibitors, Bcr-Abl tyrosine-kinase inhibitors (TKI), anaplastic lymphoma kinase (ALK) inhibitors, and B-raf inhibitors.

[0038] "Gene expression modulator" refers to a therapeutic agent that reduces the expression of a gene that increases cancer cell proliferation. The gene expression modulator may modulate gene expression at the level of transcription by DNA-binding agents, small molecules, or synthetic oligonucleotides. Alternatively, the gene expression modulator may modulate gene expression post-transcriptionally via RNA interference. [0039] "Apoptosis inducer" refers to therapeutic drugs that induce apoptosis in cancer cells. For example, proteasome inhibitors prevent degradation of proteins that would otherwise induce apoptosis.

[0040] "Angiogenesis inhibitor" refers to therapeutic agents that prevent formation of new blood vessels in tumors. For example, angiogenesis inhibitors may target vascular endothelial growth factor (VEGF).

[0041] The term "antibody" means any immunoglobulin (Ig) molecule comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivative thereof, which retains the essential epitope binding features of an Ig molecule. Such mutant, variant, or derivative antibody formats are known in the art, non-limiting embodiments of which are discussed below.

[0042] The term "human antibody" includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term "human antibody" does not include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. [0043] The term "recombinant human antibody" means human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial human antibody library, antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes, or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

[0044] In certain embodiments, antibodies may be polyclonal or monoclonal; xenogeneic, allogeneic, or syngeneic; or modified forms thereof (e.g., humanized, chimeric, etc.).

"Monoclonal antibody" refers to a population of antibody polypeptides that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of an antigen, whereas "polyclonal antibody" refer to a population of antibody polypeptides that contain multiple species of antigen binding sites capable of interacting with a particular antigen. In addition, antibodies can be "humanized," which includes antibodies made by a non-human cell having variable and constant regions which have been altered to more closely resemble antibodies that would be made by a human cell. For example, by altering the non- human antibody amino acid sequence to incorporate amino acids found in human germline immunoglobulin sequences. The humanized antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs. The term "humanized antibody," as used herein, also includes antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0045] "Antibody-drug conjugate" refers to an antibody or antibody fragment that is chemically and stably linked to a biological active cytotoxic payload or drug.

[0046] The term "cancer vaccines" is used herein to refer to therapeutic agents that target viruses that cause cancers.

[0047] The term "gene therapy," as used herein, refers to administration of oncolytic agents, such as viruses that are genetically engineered to target and destroy cancer cells. [0048] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein to refer to single- or double-stranded RNA, DNA, PNA, or mixed polymers. Polynucleotides may include genomic sequences, extra-genomic and plasmid sequences, and smaller engineered gene segments that express, or may be adapted to express, polypeptides.

[0049] An "isolated nucleic acid" is a nucleic acid that is substantially separated from other genome DNA sequences as well as proteins or complexes such as ribosomes and polymerases, which naturally accompany a native sequence. The term embraces a nucleic acid sequence that has been removed from its naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogues or analogues biologically synthesized by heterologous systems. A substantially pure nucleic acid includes isolated forms of the nucleic acid. Of course, this refers to the nucleic acid as originally isolated and does not exclude genes or sequences later added to the isolated nucleic acid by the hand of man.

[0050] The term "polynucleotide" is used in its conventional meaning, i.e., as a sequence of amino acids. The polypeptides are not limited to a specific length of the product. Peptides, oligopeptides, and proteins are included within the definition of polypeptide, and such terms may be used interchangeably herein unless specifically indicated otherwise. This term also does not refer to or exclude post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like, as well as other modifications known in the art, both naturally occurring and nonnaturally occurring. A polypeptide may be an entire protein, or a subsequence thereof.

[0051] An "isolated peptide" is one that has been identified and separated and/or recovered from a component of its natural environment. In preferred embodiments, the isolated polypeptide will be purified (1) to greater than 95% by weight of polypeptide as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or non- reducing conditions using Coomassie blue or, preferably, silver stain. Isolated polypeptide includes the polypeptide in situ within recombinant cells since at least one component of the polypeptide's natural environment will not be present. Ordinarily, however, isolated polypeptide will be prepared by at least one purification step.

[0052] A "small molecule" is a term of the art and includes molecules that are less than about 1000 molecular weight or less than about 500 molecular weight. In one embodiment, small molecules do not exclusively comprise peptide bonds. In another embodiment, small molecules are not oligomeric. [0053] A polynucleotide "variant," as the term is used herein, is a polynucleotide that typically differs from a polynucleotide specifically disclosed herein in one or more substitutions, deletions, additions and/or insertions. Such variants may be naturally occurring or may be synthetically generated, for example, by modifying one or more of the

polynucleotide sequences of the present disclosure and evaluating one or more biological activities of the encoded polypeptide as described herein and/or using any of a number of techniques well known in the art.

[0054] A polypeptide "variant," as the term is used herein, is a polypeptide that typically differs from a polypeptide specifically disclosed herein in one or more substitutions, deletions, additions and/or insertions. Such variants may be naturally occurring or may be synthetically generated, for example, by modifying one or more of the above polypeptide sequences of the present disclosure and evaluating one or more biological activities of the polypeptide as described herein and/or using any of a number of techniques well known in the art.

[0055] Modifications may be made in the structure of the polynucleotides and polypeptides of the present disclosure and still obtain a functional molecule that encodes a variant or derivative polypeptide with desirable characteristics. When it is desired to alter the amino acid sequence of a polypeptide to create an equivalent, or even an improved, valiant or portion of a polypeptide of the present disclosure, one skilled in the art will typically change one or more of the codons of the encoding DNA sequence.

[0056] For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of its ability to bind other polypeptides (e.g., antigens) or cells. Since it is the binding capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated that various changes may be made in the peptide sequences of the disclosed compositions, or corresponding DNA sequences that encode said peptides without appreciable loss of their biological utility or activity.

[0057] In many instances, a polypeptide variant will contain one or more conservative substitutions. A "conservative substitution" is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged.

[0058] It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e. still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Patent No. 4,554,101 states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein.

[0059] As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their

hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take one or more of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

[0060] Amino acid substitutions may further be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine,

phenylalanine and tyrosine. Other groups of amino acids that may represent conservative changes include: (1) ala, pro, gly, glu, asp, gin, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; ( 4) lys, arg, his; and (5) phe, tyr, trp, his. A variant may also, or alternatively, contain nonconservative changes. In a preferred embodiment, variant polypeptides differ from a native sequence by substitution, deletion or addition of five amino acids or fewer. Variants may also (or alternatively) be modified by, for example, the deletion or addition of amino acids that have minimal influence on the immunogenicity, secondary structure and hydropathic nature of the polypeptide. [0061] Polypeptides may comprise a signal (or leader) sequence at the N-terminal end of the protein, which co-translationally or post-translationally directs transfer of the protein. The polypeptide may also be conjugated to a linker or other sequence for ease of synthesis, purification or identification of the polypeptide (e.g., poly-His), or to enhance binding of the polypeptide to a solid support. For example, a polypeptide may be conjugated to an immunoglobulin Fc region.

[0062] In certain embodiments, polypeptide and polynucleotide variants as described herein are polypeptide or polynucleotide sequences at least 70% identical in to the polypeptide or polynucleotide sequence they vary from. In other embodiments, polypeptide and polynucleotide variants as described herein are polypeptide or polynucleotide sequences that are at least 75, 80, 85, 90, 95, 96, 97, 98 or 99% identical to the polypeptide or

polynucleotide sequence they vary from.

[0063] The phrase "pharmaceutically acceptable" or "therapeutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and preferably do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human. Preferably, as used herein, the term

"pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a State government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia (e.g., Remington's Pharmaceutical Sciences) for use in animals, and more particularly in humans.

[0064] The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one.

Production of Enhancers of RGMb Interaction with PD-L2 Using Recombinant DNA Technology

[0065] In certain embodiments, enhancers of RGMb interaction with PD-L2 include mutated RGMb proteins, soluble RGMb proteins, and nucleic acids that increase RGMb expression. Generation and expression of these RGMb variants and RGMb encoding nucleic acids may be achieved by the use of recombinant technology. A "vector" refers to a nucleic acid molecule, preferably self-replicating, which transfers an inserted nucleic acid molecule into and/or between host cells. Typically, the vector will include an origin of replication (e.g., the ColEl origin of replication) and a selectable marker (e.g., ampicillin or tetracycline resistance), and the necessary control sequences or regulatory elements for expression of the inserted nucleic acid molecule.

[0066] One type of vector is a "plasmid", which refers to a linear or circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. 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. 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". In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present disclosure, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, the present disclosure is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno associated viruses), which serve equivalent functions.

[0067] Recombinant expression vectors comprise a nucleic acid of the present disclosure 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 sequences, 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 sequence(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). The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the present disclosure can be introduced into host cells to thereby produce fusion proteins and variants, derivatives and peptide mimetics derived therefrom of the present disclosure, encoded by nucleic acids as described herein.

[0068] The recombinant expression vectors of the present disclosure can be designed for expression of fusion proteins and variants, derivatives and peptide mimetics derived therefrom of the present disclosure in prokaryotic or eukaryotic cells. For example, fusion proteins and variants, derivatives and peptide mimetics derived therefrom of the present disclosure can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vectors can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

[0069] Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: (1) to increase expression of recombinant protein; (2) to increase the solubility of the recombinant protein; and (3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathione S transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. [0070] Examples of suitable inducible non fusion E. coli expression vectors include pTrc (Amrann et al, (1988) Gene 69:301 315) and pET lid (Studier et al, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89). [0071] One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein. See, Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN

ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 119-128. Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et a/., (1992) Nucleic Acids Res. 20:211 : 1-7, 10-13, 19-34, 45-53, 58-85, 111- 113, 120, 130, 132-134 and 13518). Such alteration of nucleic acid sequences of the present disclosure can be carried out by standard DNA synthesis techniques.

[0072] The expression vector may be a yeast expression vector. Examples of vectors for expression in yeast S. cerevisiae include p Yep Sec 1 (Baldari, et al, (1987) EMBO J 6:229 234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933 943), pJRY88 (Schultz et al, (1987) Gene 54: 113 123), pYES2 (Invitrogen Corporation, San Diego, Calif), and picZ (InVitrogen Corp, San Diego, Calif).

[0073] A nucleic acid of the present disclosure can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith et al. (1983) Mol Cell Biol 3:2156 2165) and the pVL series (Luck:low and Sunmers (1989) Virology 170:31 39).

[0074] Alternatively, a nucleic acid of the present disclosure can be expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed (1987) Nature 329: 840) and pMT2PC (Kaufman et al. (1987) EMBO J 6: 187 195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells. See, e.g., Chapters 16 and 17 of Sambrook et al, MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

[0075] The recombinant mammalian expression vector may be capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue specific regulatory elements are used to express the nucleic acid). Tissue specific regulatory elements are known in the art. Non limiting examples of suitable tissue specific promoters include the albumin promoter (liver specific; Pinkert et al. (1987) Genes Dev 1 :268 277), lymphoid specific promoters (Calame and Eaton (1988) Adv Immunol 43:235 275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J 8:729 733) and immunoglobulins (Banerji et al. (1983) Cell 33:729 740; Queen and Baltimore (1983) Cell33:741 748), neuron specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle (1989) PNAS 86:5473 5477), pancreas specific promoters (Edlund et al. (1985) Science 230:912 916), and mammary gland specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264, 166).

Developmentally regulated promoters are also encompassed, e.g., the murine hox promoters (Kessel and Gruss (1990) Science 249:374 379) and the a-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev 3:537 546).

[0076] In an embodiment, the enhancer of RGMb interaction with PD-L2 is a soluble RGMb protein. A soluble RGMb protein may have a deletion or disruption of the GPI membrane anchoring domain. In an embodiment, the soluble RGMb protein is an RGMb.Fc fusion protein. Preferably, the Fc protein is human Fc.

[0077] The soluble RGMb protein and RGMb.Fc fusion protein may be generated by recombinant DNA methods known in the art. A nucleic acid sequence encoding soluble RGMb or Fc may be obtained from a DNA template using specifically designed

oligonucleotide primers and PCR methodologies. The nucleic acids encoding soluble RGMb and Fc may then be provided with appropriate linkers and ligated into an expression vector commonly available in the art (Wu et al, 1987, Methods in Enzymol 152:343-359, incorporated by reference herein in its entirety). For example, RGMb cDNA without its GPI anchor may be ligated into an expression vector alone or in frame with the Fc portion of human IgG. The vectors can be used to transform suitable hosts to produce the soluble RGMb or RGMb.Fc. fusion protein. [0078] In an embodiment of the methods of the present disclosure, the enhancer of RGMb interaction with PD-L2 is an RGMb protein comprising a mutation in a PD-L2 binding domain. The mutation may be a conservative or non-conservative substitution, a deletion, or an insertion. A conservative amino acid substitution is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Examples of groups of amino acids that have side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic- hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate, and (7) sulfur-containing side chains: cysteine and methionine. Preferably, the RGMb mutation increases its binding with PD-L2.

[0079] The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein. [0080] A host cell can be any prokaryotic or eukaryotic cell. For example, soluble proteins and fusion proteins of the present disclosure can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.

[0081] Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co precipitation, DEAE dextran mediated transfection, lipofection, or

electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals. [0082] For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Various selectable markers include those that confer resistance to drugs, such as G418, hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding the composition of the present disclosure or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).

[0083] A host cell, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) one or more proteins of the present disclosure. A host cell into which a recombinant expression vector encoding a protein of the present disclosure has been introduced must be cultured in a suitable medium such that the protein may be produced. The mutated RGMb and RGMb.Fc proteins may be recovered from the bacterial, mammalian, or other host cells, or from the culture medium by methods known in the art (Current Protocols in Immunology, vol. 2, chapter 8, Coligan et al. (ed.), John Wiley & Sons, Inc.; Pathogenic and Clinical Microbiology: A Laboratory Manual by Rowland et al., Little Brown & Co., June 1994; each of which is incorporated herein by reference in its entirety).

[0084] In an embodiment, the enhancer of RGMb interaction with PD-L2 is a nucleic acid that encodes RGMb or RGMb.Fc. Preferably, the nucleic acid encoding RGMb increases RGMb expression by at least 50%, 100%, 200%, 250%, 300%, 400%, 500%, or even 1000%.

[0085] Regulatory sequences operatively linked to a nucleic acid can be chosen that direct the continuous expression of the nucleic acid molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen that direct constitutive, tissue specific or cell type specific expression of nucleic acid. The expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. Nucleic Acid Formulations

[0086] Nucleic acid molecules can be formulated using one or more liposomes, lipoplexes, or lipid nanoparticles. In certain embodiments, pharmaceutical compositions of a nucleic acid molecule include liposomes. Liposomes may be used as a delivery vehicle for the administration of pharmaceutical formulations. Liposomes can be of different sizes, such as a multilamellar vesicle (MLV) that may be hundreds of nanometers in diameter and may contain a series of concentric bilayers separated by narrow aqueous compartments, a small unicellular vesicle (SUV) that may be smaller than 50 nm in diameter, and a large unilamellar vesicle (LUV) that may be between 50 and 500 nm in diameter. Liposome design may include opsonins or ligands in order to improve the attachment of liposomes to unhealthy tissue or to activate events such as endocytosis. Liposomes may contain a low or a high pH in order to improve the delivery of the pharmaceutical formulations.

[0087] The formation of liposomes may depend on the physicochemical characteristics such as the pharmaceutical formulation entrapped and the liposomal ingredients, the nature of the medium in which the lipid vesicles are dispersed, the effective concentration of the entrapped substance and its potential toxicity, any additional processes involved during the application and/or delivery of the vesicles, the optimization size, polydispersity and the shelf-life of the vesicles for the intended application, and the batch-to-batch reproducibility and possibility of large-scale production of safe and efficient liposomal products. [0088] For a thorough discussion of liposome, lipoplex and lipid nanoparticles constitution, see US20130244278; see also US20130245105 and US20130245107, each of which is incorporated by reference herein in its entirety.

[0089] Nucleic acid molecules can be formulated using natural and/or synthetic polymers. Examples of polymers which can be used for delivery include DYNAMIC

POLYCONJUGATE (Arrowhead Research Corp., Pasadena, CA) formulations from MIR US® Bio (Madison, Wl) and Roche Madison (Madison, Wl), PHASERX™ polymer formulations such as SMARTT POLYMER TECHNOLOGYT™ (Seattle, WA),

DMRI/DOPE, poloxamer, VAXFECTIN® adjuvant from Vical (San Diego, CA), chitosan, cyclodextrin from Calando Pharmaceuticals (Pasadena, CA), dendrimers and poly(lactic-co- gly colic acid) (PLGA) polymers, RONDEL™ (RNAi/Oligonucleotide Nanoparticle Delivery) polymers (Arrowhead Research Corporation, Pasadena, CA) and pH responsive co- block polymers such as, but not limited to, PHASERX™ (Seattle, WA).

[0090] An example of chitosan formulation includes a core of positively charged chitosan and an outer portion of negatively charged substrate (U.S. Pub. No. 20120258176). Chitosan includes N-trimethyl chitosan, mono-Ncarboxymethyl chitosan (MCC), N-palmitoyl chitosan (NPCS), EDTA-chitosan, low molecular weight chitosan, chitosan derivatives, or

combinations thereof.

[0091] The polymers used can undergo processing to reduce and/or inhibit the attachment of unwanted substances such as bacteria, to the surface of the polymer. The polymer may be processed by methods known and/or described in the art and/or described in WO2012150467.

[0092] An example of PLGA formulations include PLGA injectable depots (e.g.,

ELIGARD® formed by dissolving PLGA in 66% N-methyl-2-pyrrolidone (NMP) and the remainder being aqueous solvent and leuprolide. Once injected, the PLGA and leuprolide peptide precipitates into the subcutaneous space). [0093] Many polymer approaches have demonstrated efficacy in delivering oligonucleotides in vivo into the cell cytoplasm (deFougerolles Hum Gene Ther. 2008 19: 125-132). Two polymer approaches that have yielded robust in vivo delivery of nucleic acids, in this case with small interfering RNA (siRNA), are dynamic poly conjugates and cyclodextrin-based nanoparticles. The first of these delivery approaches uses dynamic poly conjugates and has been shown in vivo in mice to effectively deliver siRNA and silence endogenous target mRNA in hepatocytes (Rozema et al, Proc Natl Acad Sci USA. 2007 104: 12982-12887). This particular approach is a multicomponent polymer system of which key features include a membrane-active polymer to which nucleic acid, in this case siRNA, is covalently coupled via a disulfide bond and where both PEG (for charge masking) and N-acetylgalactosamine (for hepatocyte targeting) groups are linked via pH-sensitive bonds (Rozema et al, Proc Natl Acad Sci USA. 2007 104: 12982-12887). On binding to the hepatocyte and entry into the endosome, the polymer complex disassembles in the low-pH environment, with the polymer exposing its positive charge, leading to endosomal escape and cytoplasmic release of the siRNA from the polymer. Through replacement of the N acetylgalactosamine group with a mannose group, it was shown one could alter targeting from asialoglycoprotein receptor- expressing hepatocytes to sinusoidal endothelium and Kupffer cells. [0094] Another polymer approach involves using transferrin-targeted cyclodextrin containing poly cation nanoparticles. These nanoparticles have demonstrated targeted silencing of the EWS-FLI™ gene product in transferrin receptor-expressing Ewing's sarcoma tumor cells (Hu-Lieskovan et al. Cancer Res. 2005 65: 8984-8982) and siRNA formulated in these nanoparticles was well tolerated in non-human primates (Heidel et al, Proc Natl Acad Sci USA 2007 104:571521). Both of these delivery strategies incorporate rational approaches using both targeted delivery and endosomal escape mechanisms.

[0095] The polymer formulation can permit the sustained or delayed release of nucleic acid molecules (e.g., following intramuscular or subcutaneous injection). The altered release profile for the nucleic acid molecule can result in, for example, translation of an encoded protein over an extended period of time. The polymer formulation may also be used to increase the stability of the nucleic acid molecule. Biodegradable polymers have been previously used to protect nucleic acids from degradation and been shown to result in sustained release of payloads in vivo (Rozema et al, Proc Natl Acad Sci USA. 2007

104: 12982-12887; Sullivan et al, Expert Opin Drug Deliv. 2010 7: 1433-1446; Convertine et al., Biomacromolecules. 2010 Oct. 1; Chu et al, Acc Chem Res. 2012 Jan. 13; Manganiello et al, Biomaterials. 2012 33:2301-2309; Benoit et al, Biomacromolecules. 2011 12:2708- 2714; Singha et al., Nucleic Acid Ther. 2011 2: 133-147; deFougerolles Hum Gene Ther. 2008 19: 125-132; Schaffert and Wagner, Gene Ther. 2008 16: 1131-1138; Chaturvedi et al.,Expert Opin Drug Deliv. 2011 8: 1455-1468; Davis, Mol. Pharm. 2009 6:659-668; Davis, Nature 2010 464: 1067-1070).

[0096] The pharmaceutical compositions can be sustained release formulations. The sustained release formulations can be for subcutaneous delivery. Sustained release formulations may include PLGA microspheres, ethylene vinyl acetate (EVAc), poloxamer, GELSITE (Nanotherapeutics, Inc. Alachua, Ela.), HYLENEX® (Halozyme Therapeutics, San Diego, CA), surgical sealants such as fibrinogen polymers (Ethicon Inc. Cornelia, GA), TISSELL® (Baxter International, Inc Deerfield, IL), PEG-based sealants, and COSEAL® (Baxter International, Inc). Nucleic acids may be formulated in PLGA microspheres by preparing the PLGA microspheres with tunable release rates (e.g., days and weeks) and encapsulating the nucleic acid in the PLGA microspheres while maintaining the integrity of the nucleic acid during the encapsulation process. EVAc are non-biodegradeable, biocompatible polymers which are used extensively in pre-clinical sustained release implant applications. Poloxamer F-407 NF is a hydrophilic, nonionic surfactant triblock copolymer of poly oxyethylene-polyoxypropylenepolyoxy ethylene having a low viscosity at temperatures less than 5° C and forms a solid gel at temperatures greater than 15° C PEG-based surgical sealants comprise two synthetic PEG components mixed in a delivery device which can be prepared in one minute, seals in 3 minutes and is reabsorbed within 30 days. GELSITE® and natural polymers are capable of in-situ gelation at the site of administration. They have been shown to interact with protein and peptide therapeutic candidates through ionic interaction to provide a stabilizing effect.

[0097] Polymer formulations can also be selectively targeted through expression of different ligands as exemplified by folate, transferrin, and N-acetylgalactosamine (GalNAc) (Benoit et al., Biomacromolecules. 2011 12:2708-2714; Rozema et al, Proc Natl Acad Sci USA. 2007

104: 12982-12887; Davis, Mol. Pharm. 2009 6:659-668; Davis, Nature 2010 464: 1067-1070).

Nucleic acid molecules may be formulated with a PLGA-PEG block copolymer (see US Pub.

No. US20120004293 and U.S. Pat. No. 8,236,330) or PLGA-PEG-PLGA block copolymers (see U.S. Pat. No. 6,004,573). Nucleic acid molecules may be formulated with a diblock copolymer of PEG and PLA or PEG and PLGA (see U.S. Pat. No. 8,246,968).

[0098] A polyamine derivative may be used to deliver nucleic acid molecules or to treat and/or prevent a disease or to be included in an implantable or injectable device (U.S. Pub. No. 20100260817). A pharmaceutical composition may include the nucleic acid molecules and the polyamine derivative described in U.S. Pub. No. 20100260817. Nucleic acids may be delivered using a polyaminde polymer such as a polymer comprising a 1,3-dipolar addition polymer prepared by combining a carbohydrate diazide monomer with a dilkyne unite comprising oligoamines (U.S. Pat. No. 8,236,280).

[0099] Nucleic acids may be formulated with at least one polymer and/or derivatives thereof described in WO2011115862, WO2012082574 and WO2012068187 and U.S. Pub. No.

20120283427. Nucleic acids may be formulated with a polymer of formula Z as described in WO2011115862. Nucleic acids may be formulated with a polymer of formula Z, Z' or Z" as described in International Pub. Nos. WO2012082574 or WO2012068187. The polymers formulated with nucleic acids may be synthesized by the method described in International Pub. Nos. WO2012082574 or WO2012068187. [00100] Formulations of nucleic acid molecules may include at least one amine-containing polymer such as polylysine, polyethylene imine, poly(amidoamine) dendrimers or combinations thereof. Nucleic acid molecules may be formulated with at least one crosslinkable polyester. Crosslinkable polyesters include those known in the art and described in US Pub. No. 20120269761. The described polymers may be conjugated to a lipid- terminating PEG. PLGA may be conjugated to a lipid-terminating PEG forming PLGA- DSPE-PEG. PEG conjugates are described in International Publication No. W02008103276. The polymers may be conjugated using a ligand conjugate such as, but not limited to, the conjugates described in U.S. Pat. No. 8,273,363. [00101] Nucleic acid molecules may be conjugated with another compound, such as those described in U.S. Pat. Nos. 7,964,578 and 7,833,992. Nucleic acid molecules may be conjugated with conjugates of formula 1-122 as described in U.S. Pat. Nos. 7,964,578 and 7,833,992. Nucleic acid molecules may be conjugated with a metal such as gold. (See e.g., Giljohann et al. J. Amer. Chem. Soc. 2009 131(6): 2072-2073). In another example, nucleic acid molecules may be conjugated and/or encapsulated in gold-nanoparticles.

(WO201216269 and US20120302940). As described in US20100004313, a gene delivery composition may include a nucleotide sequence and a poloxamer. For example, nucleic acid molecules may be used in a gene delivery composition with the poloxamer described in US20100004313. [00102] Nucleic acid molecules may be formulated in a polyplex of one or more polymers (US20120237565 and US20120270927). In one embodiment, the polyplex comprises two or more cationic polymers. The cationic polymer may comprise a poly(ethylene imine) (PEI) such as linear PEI. Further details are provided in US20130244278.

[00103] In an embodiment of the present disclosure, the nucleic acid encoding RGMb or RGMb.Fc is conjugated to an antibody or a tumor cell-specific ligand in order to facilitate delivery to tumor cells. This may be accomplished by methods known in the art. For example, see Cuellar et al, Nucleic Acids Res. 2015 43: 1189-203; Schiffelers et al, Nucleic Acids. Res. 2004 32:el49; Liu et al, Nucleic Acids Res. 2014 42: 111805-11817.

[00104] Nucleic acid molecules can also be formulated as a nanoparticle using a combination of polymers, lipids, and/or other biodegradable agents, such as calcium phosphate.

Components may be combined in a core-shell, hybrid, and/or layer-by-layer architecture, to allow for fine-tuning of the nanoparticle so delivery of the nucleic acid molecule may be enhanced (Wang et al., Nat Mater. 2006 5:791-796; Fuller et al., Biomaterials. 2008 29: 1526- 1532; DeKoker et al, Adv Drug Deliv Rev. 2011 63:748-761 ; Endres et al, Biomaterials. 2011 32:7721-7731 ; Su et al. Mol Pharm. 2011 Jun. 6; 8(3):774-87). As a non-limiting example, the nanoparticle may comprise a plurality of polymers such as hydrophilic- hydrophobic polymers (e.g., PEG-PLGA), hydrophobic polymers (e.g., PEG) and/or hydrophilic polymers (WO20120225129). The composition of nanoparticles is thoroughly discussed in US20130244278 and US20150086612.

[00105] Biodegradable calcium phosphate nanoparticles in combination with lipids and/or polymers have been shown to deliver nucleic acid molecules in vivo. In one embodiment, a lipid coated calcium phosphate nanoparticle, which may also contain a targeting ligand such as anisamide, may be used to deliver the nucleic acid molecule. For example, to effectively deliver siRNA in a mouse metastatic lung model a lipid coated calcium phosphate nanoparticle was used (Li et al., J Contr Rel. 2010 142: 416-421 ; Li et al., J Contr Rel. 2012 158: 108-114; Yang et al, Mol Ther. 2012 20:609-615). This delivery system combines both a targeted nanoparticle and a component to enhance the endosomal escape, calcium phosphate, in order to improve delivery of the siRNA.

[00106] Calcium phosphate with a PEG-polyanion block copolymer may be used to deliver nucleic acid molecules (Kazikawa et al., J Contr Rel. 2004 97:345-356; Kazikawa et al, J Contr Rel. 2006 111 :368-370.

[00107] A PEG-charge-conversional polymer (Pitella et al., Biomaterials. 2011 32:31063114) may be used to form a nanoparticle to deliver nucleic acid molecules. The PEG-charge- conversional polymer may improve upon the PEG-polyanion block copolymers by being cleaved into a poly cation at acidic pH, thus enhancing endosomal escape. [00108] In certain embodiments, the formulation comprising nucleic acid molecules is a nanoparticle that may comprise at least one lipid. The lipid may be selected from DLin- DMA, DLin-K-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG and PEGylated lipids. In another aspect, the lipid may be a cationic lipid such as DLin-DMA, DLin-D-DMA, DLin- MC3-DMA, DLin-KC2-DMA and

DODMA. [00109] The lipid to nucleic acid molecule ratio in the formulation may be between 10: 1 and 30: 10. The mean size of the nanoparticle formulation may comprise the nucleic acid molecules between 60 and 225 nm. The PDI of the nanoparticle formulation comprising the modified mRNA is between 0.03 and 0.15. The zeta potential of the lipid may be from -10 to +10 at a pH of 7.4.

[00110] The formulations of nucleic acid molecules may comprise a fusogenic lipid, cholesterol and a PEG lipid. The formulation may have a molar ratio 50: 10:38.5: 1.5-3.0 (cationic lipid:fusogenic lipid:cholesterol:PEG lipid). The PEG lipid may be, for example, PEG-c-DOMG, PEG-DMG. The fusogenic lipid may be DSPC. [00111] The formulation of nucleic acid molecules may be a PLGA microsphere that may be between 4 and 20 μιτι. The nucleic acid molecules may be released from the formulation at less than 50% in a 48 hour time period. The PLGA microsphere formulation may be stable in serum.

[00112] Stability may be determined relative to unformulated modified mRNA in 90%. The loading weight percent of the nucleic acid molecule PLGA microsphere may be at least 0.05%, at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4% or at least 0.5%. The encapsulation efficiency of the nucleic acid molecules in the PLGA microsphere may be at least 50%, at least 70%, at least 90% or at least 97%.

[00113] A lipid nanoparticle of the present disclosure may be formulated in a sealant such as, but not limited to, a fibrin sealant.

[00114] The use of core-shell nanoparticles has additionally focused on a high-throughput approach to synthesize cationic cross-linked nanogel cores and various shells (Siegwart et al, Proc Natl Acad Sci USA. 2011 108: 12996-13001). The complexation, delivery, and internalization of the polymeric nanoparticles can be precisely controlled by altering the chemical composition in both the core and shell components of the nanoparticle. For example, the core-shell nanoparticles may efficiently deliver siRNA to mouse hepatocytes after they covalently attach cholesterol to the nanoparticle.

[00115] A hollow lipid core comprising a middle PLGA layer and an outer neutral lipid layer containing PEG may be used to delivery of nucleic acid molecules. In mice bearing a luciferase-expressing tumor, it was determined that the lipid-polymer-lipid hybrid nanoparticle significantly suppressed luciferase expression, as compared to a conventional lipoplex (Shi et al., Angew Chem Int Ed. 2011 50:7027-7031).

[00116] The lipid nanoparticles may comprise a core of the nucleic acid molecules and a polymer shell. The polymer shell may be any of the polymers known in the art. The polymer shell may be used to protect the modified nucleic acids in the core. Core-shell nanoparticles for use with the nucleic acid molecules are described and may be formed by the methods described in U.S. Pat. No. 8,313,777. The core-shell nanoparticles may comprise a core of the nucleic acid molecules and a polymer shell. The polymer shell may be any of the polymers described herein and are known in the art. Further details are provided in US20130244278. Generation of Anti-PD-L2 Antibodies

[00117] In an embodiment, the enhancer of RGMb interaction with PD-L2 is a PD-L2- specific antibody or a fragment thereof. To prepare polyclonal antibodies or fragments thereof, the antibody or fragment protein may be synthesized in bacterial, fungal, or mammalian cells by expression of corresponding DNA sequences in a suitable cloning vehicle. The protein can be purified, coupled to a carrier protein, mixed with Freund's adjuvant (to enhance stimulation of the antigenic response in an inoculated animal), and injected into rabbits or other laboratory animals. Following booster injections at bi-weekly intervals, the rabbits or other laboratory animals are then bled and the sera isolated. The sera can be used directly or can be purified prior to use by various methods, including affinity chromatography employing reagents such as Protein A-Sepharose, antigen-Sepharose, and anti-mous e-Ig- S epharos e.

[00118] For generation of anti- PD-L2 antibodies that mimic the interaction of RGMb with PD-L2, synthetic peptides can be made that correspond to the PD-L2 domain that binds RGMb and used to inoculate the animals. [00119] Alternatively, monoclonal antibodies may be prepared using PD-L2 proteins and standard hybridoma technology (see, e.g., Kohler a/., Nature 256:495, 1975; Kohler et al, Eur. J. Immunol. 6:511, 1976; Kohler et al, Eur. J. Immunol. 6:292, 1976; Hammerling et al, In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, New York, NY, 1981). Once produced, monoclonal antibodies may be tested for specific PD-L2 protein recognition by Western blot or immunoprecipitation analysis. [00120] In certain embodiments, the PD-L2 -specific antibody is a bispecific antibody that simultaneously binds PD-L2 and a tumor antigen. Functionally, a bispecific antibody binds one antigen on one of its two binding arms, and binds a different antigen on its second binding arm, a binding arm being a pair of a heavy chain and light chain. Methods for generating bispecific antibodies are known in the art. For example, see Milstein and Cuello, Nature 1983 305: 537-40; Staerz et al., Nature 1985 628:31 ; Brennan et al, Science 1985 229:81-3.

[00121] Various delivery systems are known and can be used to administer the proteins or a pharmaceutical composition containing the proteins of the present disclosure. The proteins and pharmaceutical composition of the present disclosure can be administered systemically by any suitable route including, intravenous or intramuscular injection, intraventricular or intrathecal injection (for central nervous system administration), orally, topically, subcutaneously, subconjunctivally, or via intranasal, intradermal, sublingual, vaginal, rectal or epidural routes. The preferred route of administration is intravenous administration. [00122] Alternatively, the proteins and pharmaceutical compositions may be administered directly to a tumor, such as by intra-tumor injection. Other delivery systems well known in the art can be used for delivery of the proteins and pharmaceutical compositions of the present disclosure, for example via aqueous solutions, encapsulation in microparticles, or microcapsules. [00123] Pharmaceutical compositions containing proteins of the present disclosure may be combined with a pharmaceutically acceptable carrier. The term carrier refers to diluents, adjuvants and/or excipients such as fillers, binders, disintegrating agents, lubricants, silica flow conditioner, stabilizing agents or vehicles with which the peptide, peptide derivative or peptidomimetic is administered. Such pharmaceutical carriers include sterile liquids such as water and oils including mineral oil, vegetable oil (e.g., peanut oil, soybean oil, sesame oil and canola oil), animal oil or oil of synthetic origin. Aqueous glycerol and dextrose solutions as well as saline solutions may also be employed as liquid carriers of the phamlaceutical compositions of the present disclosure. Of course, the choice of the carrier depends on the nature of the peptide, peptide derivative or peptidomimetic, its solubility and other physiological properties as well as the target site of delivery and application. Examples of suitable pharmaceutical carriers are described in Remington: The Science and Practice of Pharmacy by Alfonso R. Gennaro, 2003, 21st edition, Mack Publishing Company. [00124] Further pharmaceutically suitable materials that may be incorporated in

pharmaceutical preparations include absorption enhancers, pH regulators and buffers, osmolarity adjusters, preservatives, stabilizers, antioxidants, surfactants, thickeners, emollient, dispersing agents, flavoring agents, coloring agents and wetting agents. [00125] Examples of suitable pharmaceutical excipients include, water, glucose, sucrose, lactose, glycol, ethanol, glycerol monostearate, gelatin, rice, starch, flour, chalk, sodium stearate, malt, sodium chloride and the like. The pharmaceutical compositions can take the form of solutions, capsules, tablets, creams, gels, powders, sustained release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides (see Remington: The Science and Practice of Pharmacy by Alfonso R. Gennaro, 2003, 21st edition, Mack Publishing Company). Such compositions contain a therapeutically effective amount of the therapeutic composition, together with a suitable amount of carrier so as to provide the form for proper administration to the subject. The formulations are designed so as to suit the mode of administration and the target site of action (e.g., a particular organ or cell type).

[00126] Pharmaceutical compositions can be formulated as neutral or salt forms.

Pharmaceutically acceptable salts include those that form with free amino groups and those that react with free carboxyl groups. Non-toxic alkali metal, alkaline earth metal and ammonium salts commonly used in the pharmaceutical industry include sodium, potassium, lithium, calcium, magnesium, barium, ammonium, and protamine zinc salts, which are prepared by methods well known in the art. The term also includes non-toxic acid addition salts, which are generally prepared by reacting the compounds of the present disclosure with suitable organic or inorganic acid. Representative salts include the hydrobromide, hydrochloride, valerate, oxalate, oleate, laureate, borate, benzoate, sulfate, bisulfate, acetate, phosphate, tysolate, citrate, maleate, fumarate, tartrate, succinate, napsylate salts and the like.

[00127] Examples of fillers or binders that may be used in accordance with the present disclosure include acacia, alginic acid, calcium phosphate (dibasic), carboxymethylcellulose, carboxymethylcellulose sodium, hydroxy ethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, dextrin, dextrates, sucrose, tylose, pregelatinized starch, calcium sulfate, amylose, glycine, bentonite, maltose, sorbitol, ethylcellulose, disodium hydrogen phosphate, disodium phosphate, disodium pyrosulfite, polyvinyl alcohol, gelatin, glucose, guar gum, liquid glucose, compressible sugar, magnesium aluminum silicate, maltodextrin, polyethylene oxide, polymethacrylates, povidone, sodium alginate, tragacanth, microcrystalline cellulose, starch, and zein. Another most preferred filler or binder consists of microcrystalline cellulose.

[00128] Examples of disintegrating agents that may be used include alginic acid,

carboxymethylcellulose, carboxymethylcellulose sodium, hydroxypropylcellulose (low substituted), microcrystalline cellulose, powdered cellulose, colloidal silicon dioxide, sodium croscarmellose, crospovidone, methylcellulose, polacrilin potassium, povidone, sodium alginate, sodium starch glycolate, starch, disodium disulfite, disodium edathamil, disodium edetate, disodiumethylenediaminetetraacetate (EDTA) crosslinked polyvinylpyrollidines, pregelatinized starch, carboxy methyl starch, sodium carboxymethyl starch and

microcrystalline cellulose.

[00129] Examples of lubricants include calcium stearate, canola oil, glyceryl palmitostearate, hydrogenated vegetable oil (type I), magnesium oxide, magnesium stearate, mineral oil, poloxamer, polyethylene glycol, sodium lauryl sulfate, sodium stearate fumarate, stearic acid, talc, zinc stearate, glyceryl behapate, magnesium lauryl sulfate, boric acid, sodium benzoate, sodium acetate, sodium benzoate/sodium acetate (in combination) and DL leucine.

[00130] Examples of silica flow conditioners include colloidal silicon dioxide, magnesium aluminum silicate and guar gum. Another most preferred silica flow conditioner consists of silicon dioxide. [00131] Examples of stabilizing agents include acacia, albumin, polyvinyl alcohol, alginic acid, bentonite, dicalcium phosphate, carboxymethylcellulose, hydroxypropylcellulose, colloidal silicon dioxide, cyclodextrins, glyceryl monostearate, hydroxypropyl

methylcellulose, magnesium trisilicate, magnesium aluminum silicate, propylene glycol, propylene glycol alginate, sodium alginate, carnauba wax, xanthan gum, starch, stearate(s), stearic acid, stearic monoglyceride and stearyl alcohol.

Cancer Therapies

[00132] The methods of the present disclosure contemplate administration of enhancers of RGMb and PD-L2 interaction with one or more cancer therapies. Cancer therapies include, but are not limited to, immunotherapies, such as monoclonal antibodies, bispecific antibodies that target a tumor antigen and an immune cell-specific protein, immune checkpoint inhibitors, T cell receptor therapy, and chimeric antigen receptor therapy; hormone therapy; signal transduction inhibitors; gene expression modulators; apoptosis inducers; angiogenesis inhibitors; antibody -drug conjugates, cancer vaccines, and gene therapies.

[00133] Monoclonal antibody immunotherapy includes administration of monoclonal antibodies that bind antigens on cancer cells, inducing an immune response, e.g.

alumtuzumab; and antibodies that bind growth factors and growth factor receptors, inhibiting cancer cell proliferation, e.g. trastuzumab.

T Cell Receptor (TCR) Therapy

[00134] In an embodiment, TCR therapy is administered. The TCR therapy may be an isolated recombinant TCR. Alternatively, the TCR therapy comprises a cell expressing a recombinant TCR on the surface that specifically binds to a major histocompatibility complex (MHC) molecule complexed with one or more tumor antigens. Recombinant TCR-expressing cells may be generated by methods known in the art. Host T cells may be isolated from a subject having cancer or expressing tumor antigens and transfected or transduced with nucleic acid constructs encoding a recombinant TCR, then administered to the subject from whom they were isolated (Hombach, et al. 2001, Cancer Res. 61 : 1976-1982, incorporated by reference herein in its entirety).

[00135] An alternative approach is to infuse a subject with a polyclonal T cell product.

Monocytic cells or dendritic cells may be electroporated with mRNA encoding tumor antigens, and co-cultured in vivo with T cells to produce polyclonal T cells. Alternatively, the DCs with mRNA encoding tumor antigens can be administered to a subject directly as a vaccine composition.

Chimeric Antigen Receptor (CAR) Therapies

[00136] In an embodiment of the present disclosure, CAR therapy is administered. The CAR therapy may comprise a cell expressing a chimeric antigen receptor on the surface that specifically binds to a tumor antigen. CAR-expressing cells may be generated by methods known in the art, and may be administered to the subject from whom they were isolated.

Immune Checkpoint Protein Inhibitors

[00137] In certain embodiments, the methods of the present disclosure include administration of immune checkpoint protein inhibitors. Exemplary immune checkpoint protein inhibitors that may be administered include, without limitation, antagonistic antibodies, such as anti- PD-1, anti-PD-Ll, PD-L2, anti-cytotoxic T-lymphocyte-associated protein 4 (anti-CTLA-4), anti-T cell immunoglobulin and mucin protein 3 (anti-TIM-3), anti-lymphocyte-activation protein 3 (anti-LAG-3), anti-carcinoembryonic antigen-related cell adhesion molecule 1 (anti- CEACAM1), anti-glucocorticoid-induced tumor necrosis factor receptor-related protein (anti- GITR), or anti-tumor necrosis factor receptor superfamily, member 4 (anti-TNFRSF4). Or, the subject can also be administered an indoleamine-pyrrole 2,3 -di oxygenase (IDO)inhibitor, such as 4-amino-N-(3-chloro-4-fluorophenyl)-N'-hydroxy-l,2,5-oxadiazole-3- carboximidamide, norharmane, rosmarinic acid, a cyclooxygenase-2 (COX-2) inhibitor, or alpha-methyl tryptophan.

Antibody-Drug Conjugates

[00138] In an embodiment, a cancer therapy comprising an antibody conjugated to a chemotherapy drug or radioactive particle is employed. Antibody-drug conjugates may be designed by methods known in the art. For example, see Kovtun et al. 2006, Cancer Res. 66:3214; Goldenberg 2002 J Nucl Med 43:693-713.

Adjuvants

[00139] Certain embodiments of the methods disclosed herein comprise administration of an enhancer of RGMb interaction with PD-L2, such as a soluble, chimeric, fusion or pegylated RGMb protein with a cancer therapy, such as an immunotherapy or vaccine, and an adjuvant to enhance the immune response.

[00140] A variety of adjuvants may be employed, including, for example, systemic adjuvants and mucosal adjuvants. A systemic adjuvant is an adjuvant that can be delivered

parenterally. Systemic adjuvants include adjuvants that create a depot effect, adjuvants that stimulate the immune system, and adjuvants that do both. An adjuvant that creates a depot effect is an adjuvant that causes the antigen to be slowly released in the body, thus prolonging the exposure of immune cells to the antigen. This class of adjuvants includes alum (e.g., aluminum hydroxide, aluminum phosphate); or emulsion-based formulations including mineral oil, nonmineral oil, water-in-oil or oil-in-water-in oil emulsion, oil-in-water emulsions such as Seppic ISA series of Montanide adjuvants (e.g., Montanide ISA 720, AirLiquide, Paris, France); MF-59 (a squalene-in-water emulsion stabilized with Span 85 and Tween 80; Chiron Corporation, Emeryville, Calif; and PROVAX (an oil-in-water emulsion containing a stabilizing detergent and a micelle-forming agent; IDEC, Pharmaceuticals Corporation, San Diego, Calif).

[00141] Other adjuvants stimulate the immune system and result in, e.g. more pronounced immune cell secretion of cytokines or IgG or improved cytolytic potential. This class of adjuvants includes immunostimulatory nucleic acids, such as CpG oligonucleotides; saponins purified from the bark of the Q. saponaria tree, such as QS21 and QS7; STING ligands; TLR2 agonists; TLR5 agonists (e.g., flagellin); TLR7 agonists;

poly[di(carboxylatophenoxy)phosphazene (PCPP polymer; Virus Research Institute, USA); RNA mimetics such as polyinosinic:polycytidylic acid (poly I:C) or poly I:C stabilized with poly-lysine (poly-ICLC [Hiltonol®; Oncovir, Inc.]; derivatives of lipopoly saccharides (LPS) such as monophosphoryl lipid A (MPL; Ribi ImmunoChem Research, Inc., Hamilton, Mont.), muramyl dipeptide (MDP; Ribi) and threonylmuramyl dipeptide (t-MDP; Ribi); OM- 174 (a glucosamine disaccharide related to lipid A; OM Pharma SA, Meyrin, Switzerland); and Leishmania elongation factor (a purified Leishmania protein; Corixa Corporation, Seattle, Wash.).

[00142] Other systemic adjuvants are adjuvants that create a depot effect and stimulate the immune system. These compounds have both of the above-identified functions of systemic adjuvants. This class of adjuvants includes but is not limited to ISCOMs (Immunostimulating complexes which contain mixed saponins, lipids and form virus-sized particles with pores that can hold antigen; CSL, Melbourne, Australia); AS01 which is a liposome based formulation containing MPL and QS21 (GlaxoSmithKline, Belgium); AS02

(GlaxoSmithKline , which is an oil-in-water emulsion containing MPL and QS21 :

GlaxoSmithKline, Rixensart, Belgium); AS04 (GlaxoSmithKline, which contains alum and MPL; GSK, Belgium); AS 15 which is a liposome based formulation containing CpG oligonucleotides, MPL and QS21 (GlaxoSmithKline, Belgium); non-ionic block copolymers that form micelles such as CRL 1005 (these contain a linear chain of hydrophobic polyoxypropylene flanked by chains of polyoxyethylene; Vaxcel, Inc., Norcross, Ga.); and Syntex Adjuvant Formulation (SAF, an oil-in-water emulsion containing Tween 80 and a nonionic block copolymer; Syntex Chemicals, Inc., Boulder, Colo.). [00143] Useful mucosal adjuvants are capable of inducing a mucosal immune response in a subject when administered to a mucosal surface in conjunction with complexes of the present disclosure. Mucosal adjuvants include CpG nucleic acids (e.g. PCT published patent application WO 1999061056), bacterial toxins: e.g., Cholera toxin (CT), CT derivatives including but not limited to CT B subunit (CTB); CTD53 (Val to Asp); CTK97 (Val to Lys); CTK104 (Tyr to Lys); CTD53/K63 (Val to Asp, Ser to Lys); CTH54 (Arg to His); CTN107 (His to Asn); CTE114 (Ser to Glu); CTE112K (Glu to Lys); CTS61F (Ser to Phe); CTS 106 (Pro to Lys); and CTK63 (Ser to Lys), Zonula occludens toxin (zot), Escherichia coli heat- labile enterotoxin, Labile Toxin (LT), LT derivatives including but not limited to LT B subunit (LTB); LT7K (Arg to Lys); LT61F (Ser to Phe); LT112K (Glu to Lys); LT118E (Gly to Glu); LT146E (Arg to Glu); LT192G (Arg to Gly); LTK63 (Ser to Lys); and LTR72 (Ala to Arg), Pertussis toxin, PT. including PT-9K/129G; Toxin derivatives (see below); Lipid A derivatives (e.g., monophosphoryl lipid A, MPL); Muramyl Dipeptide (MDP) derivatives; bacterial outer membrane proteins (e.g., outer surface protein A (OspA) lipoprotein of Borrelia burgdorferi, outer membrane protein of Neisseria meningitidis); oil-in-water emulsions (e.g., MF59; aluminum salts (Isaka et al, 1998, 1999); and Saponins (e.g., QS21, Agenus Inc., Lexington, Mass.), ISCOMs, MF-59 (a squalene-in-water emulsion stabilized with Span 85 and Tween 80; Chiron Corporation, Emeryville, Calif); the Seppic ISA series of Montanide adjuvants (e.g., Montanide ISA 720; AirLiquide, Paris, France); PROVAX (an oil-in-water emulsion containing a stabilizing detergent and a micelle-forming agent; IDEC Pharmaceuticals Corporation, San Diego, Calif); Syntext Adjuvant Formulation (SAF;

Syntex Chemicals, Inc., Boulder, Colo.); poly[di(carboxylatophenoxy)]phosphazene (PCPP polymer; Virus Research Institute, USA) and Leishmania elongation factor (Corixa

Corporation, Seattle, Wash.)

[00144] Other useful adjuvants include: inflammasome inducers such as NLRP3

inflammasome inducers (e.g., alum crystals, ATP, chitosan, calcium pyrophosphate dihydrate crystals, hemozoin, monosodium urate crystals, nano-SiCK nigericin, and mincle agonists), AIM2 inflammasome inducers (e.g., poly(dA:dT)), NLRC4 inflammasome inducers (e.g., flagellin), NLRPl inflammasome inducers (e.g., muramyldipeptide), and noncanonical inflammasome inducers (e.g., P-l,3-glucan from A. faecalis, heat-killed C. albicans, PD- glucan from lichen Lasallia pustulata, and hot alkali treated zymosan); NODI agonists such as D-y-Glu-mDAP and L-Ala-y-D-Glu-mDAP; NOD2 agonists such as murabutide, muramyl dipeptide, muramyl tripeptide, muramyl tetrapeptide, and N-glycolylated muramyl dipeptide; NOD1/NOD2 agonists such as MurNAc-L-Ala-y-D-Glu-mDAP-PGN-like molecule and peptidoglycan; bryostatin-1, and toll-like receptor (TLR) agonists such as TLR2 ligands (e.g., heat-killed bacteria and cell-wall components), TLR3 ligands (e.g., poly(A:U) and poly(LC)), TLR4 ligands (e.g., lipopolysaccharides and monophosphoryl lipid A), TLR5 ligands (e.g., flagellin and heat killed Salmonella typhimurium), TLR7/8 ligands (e.g., single-stranded RNAs), TLR9 ligands (e.g., CpG oligodeoxynucleotides), and TLR13 ligands (e.g., 23S rRNA derived oligoribonucleotide). [00145] Adjuvants of the present disclosure may be administered prior to, during, or following administration of the cancer therapies. Administration of the adjuvant and immunotherapeutic or vaccine compositions can be at the same or different administration sites.

[00146] It is contemplated that the methods of the present disclosure are useful for treating a cancer in a subject in need. Cancer types that may be treated include, without limitation, breast cancer, colon cancer, non-small cell lung carcinoma, testicular cancer, ovarian cancer, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, cervical cancer, endometrial cancer, esophageal cancer, gallbladder cancer, kidney cancer, laryngeal cancer,

hypopharyngeal cancer, gastrointestinal cancer, liver cancer, lung cancer, oropharyngeal cancer, pancreatic cancer, penile cancer, prostate cancer, stomach cancer, thymus cancer, thyroid cancer, vaginal cancer, vulvar cancer, skin cancer, bone cancer, leukemia, lymphoma, neuronal cancer of the nervous system, and non-neuronal cancer of the nervous system.

[00147] In the methods of the present disclosure, the subject is a mammal. The mammal may be selected from rodents, primates, dogs, cats, camelids and ungulates. The term "rodent" refers to any species that is a member of the order rodentia including mice, rats, hamsters, gerbils and rabbits. The term "primate" refers to any species that is a member of the order primates, including monkeys, apes and humans. The term "camelids" refers to any species that is a member of the family camelidae including camels and llamas. The term "ungulates" refers to any species that is a member of the superorder ungulata including cattle, horses and camelids. In an embodiment, the subject is a human.

[00148] All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. [00149] Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are offered by way of example only, and the invention is to be limited only by the terms of the appended claims along with the full scope of equivalents to which such claims are entitled.

Claims

1. A method of reducing tolerization of T cells to one or more tumor antigens in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a soluble, chimeric, fusion or pegylated RGMb protein.

2. The method of claim 1, wherein the soluble, chimeric, fusion or pegylated RGMb protein is targeted to a cancer cell expressing the one or more tumor antigens.

3. The method of claim 1, wherein the soluble, chimeric, fusion or pegylated RGMb protein is a soluble RGMb protein.

4. The method of claim 3, wherein the soluble RGMb protein is RGMb.Fc.

5. The method of claim 4, wherein the Fc is human Fc.

6. The method of claim 5, wherein the soluble RGMb protein is targeted to a cancer cell expressing the one or more tumor antigens.

7. The method of claim 1, wherein the soluble, chimeric, fusion or pegylated RGMb protein is an RGMb protein comprising a mutation in a PD-L2 binding domain.

8. The method of claim 7, wherein the mutation increases RGMb binding to PD-L2.

9. The method of claim 8, wherein the mutated RGMb protein is targeted to a cancer cell expressing one of the one or more tumor antigens.

10. The method of claim 1, wherein the soluble, chimeric, fusion or pegylated RGMb protein is administered directly to a tumor in the subject.

11. The method of claim 10, wherein the soluble, chimeric, fusion or pegylated RGMb

protein is injected directly into a tumor in the subject.

12. The method of claim 1, wherein the soluble, chimeric, fusion or pegylated RGMb protein is administered systemically.

13. The method of claim 12, wherein the systemic administration is a route of administration selected from the group consisting of intravenous, intramuscular, intraventricular, intrathecal, oral, topical, subcutaneous, subconjunctival, intranasal, intradermal, sublingual, vaginal, rectal and epidural.

14. The method of claim 13, wherein the route of administration is intravenous or

subcutaneous.

15. The method of claim 1, wherein the soluble, chimeric, fusion or pegylated RGMb protein is administered with one or more cancer therapies.

16. The method of claim 15, wherein the cancer therapy is selected from the group consisting of an immunotherapy, a hormone therapy, a signal transduction inhibitor, a gene expression modulator, an apoptosis inducer, an angiogenesis inhibitor, and an antibody- drug conjugate, a cancer vaccine, and a gene therapy.

17. The method of claim 16, wherein the immunotherapy is a T cell receptor (TCR) therapy targeting one of the one or more tumor antigens.

18. The method of claim 17, wherein the TCR therapy is an isolated recombinant TCR, wherein the recombinant TCR specifically binds to a major histocompatibility complex (MHC) molecule complexed with one of the one or more tumor antigens.

19. The method of claim 18, wherein the TCR therapy is a cell expressing a recombinant TCR on the surface, wherein the TCR specifically binds to an MHC molecule complexed with one of the one or more tumor antigens.

20. The method of claim 19, wherein the immunotherapy is a chimeric antigen receptor (CAR) therapy targeting one of the one or more tumor antigens.

21. The method of claim 20, wherein the CAR therapy is a cell expressing a CAR on the surface, wherein the CAR specifically binds to one of the one or more tumor antigens.

22. The method of claim 16, wherein the immunotherapy is a monoclonal antibody targeting a tumor antigen.

23. The method of claim 22, wherein the monoclonal antibody is conjugated to a

chemotherapy drug.

24. The method of claim 22, wherein the monoclonal antibody is conjugated to a radioactive particle.

25. The method of claim 16, wherein the immunotherapy is an inhibitor of an immune checkpoint protein.

26. The method of claim 25, wherein the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), T cell immunoglobulin and mucin protein 3 (TIM-3), lymphocyte-activation protein 3 (LAG-3), carcinoembryonic antigen-related cell adhesion molecule 1

(CEACAM1), glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR), and tumor necrosis factor receptor superfamily, member 4 (TNFRSF4).

27. The method of claim 25, wherein the immune checkpoint protein is a member of the indoleamine-pyrrole 2,3-dioxygenase (IDO) pathway.

28. The method of claim 27, wherein the inhibitor is selected from the group consisting of norharmane, rosmarinic acid, a cyclooxygenase-2 (COX-2) inhibitor, and alpha-methyl tryptophan.

29. The method of claim 26, wherein the immune checkpoint inhibitor is a monoclonal antibody.

30. The method of claim 16, wherein the immunotherapy is a bispecific antibody targeting a tumor antigen and an immune cell-specific protein.

31. The method of claim 16, wherein the cancer therapy is administered with an adjuvant.

32. The method of claim 31, wherein the adjuvant is selected from the group consisting of Quillaj a saponin 21 (QS21), Quillaja saponin 7 (QS7), monophosphoryl lipid A, aluminum hydroxide, an oil-in-water emulsion, bryostatin-1, a toll-like receptor (TLR) agonist, and CpG oligodeoxynucleotides.

33. A method of treating a cancer in a subject in need thereof, the method comprising

administering to the subject a therapeutically effective amount of a soluble, chimeric, fusion or pegylated RGMb protein.

34. The method of claim 33, wherein the soluble, chimeric, fusion or pegylated RGMb protein is targeted to a cancer cell.

35. The method of claim 33, wherein the soluble, chimeric, fusion or pegylated RGMb

protein is a soluble RGMb protein.

36. The method of claim 35, wherein the soluble RGMb protein is RGMb.Fc.

37. The method of claim 36, wherein the Fc is human Fc.

38. The method of claim 36, wherein the soluble RGMb protein is targeted to a cancer cell.

39. The method of claim 33, wherein the soluble, chimeric, fusion or pegylated RGMb

protein is an RGMb protein comprising a mutation in a PD-L2 binding domain.

40. The method of claim 39, wherein the mutation increases RGMb binding to PD-L2.

41. The method of claim 40, wherein the mutated RGMb protein is targeted to a cancer cell.

42. The method of claim 33, wherein the soluble, chimeric, fusion or pegylated RGMb

protein is administered with one or more cancer therapies.

43. The method of claim 42, wherein the cancer therapy is selected from the group consisting of an immunotherapy, a hormone therapy, a signal transduction inhibitor, a gene expression modulator, an apoptosis inducer, an angiogenesis inhibitor, and an antibody- drug conjugate, a cancer vaccine, and a gene therapy.

44. The method of claim 43, wherein the immunotherapy is a T cell receptor (TCR) therapy targeting a tumor antigen associated with the cancer.

45. The method of claim 44, wherein the TCR therapy is an isolated recombinant TCR, wherein the TCR specifically binds to a major histocompatibility complex (MHC) molecule complexed with the tumor antigen.

46. The method of claim 44, wherein the TCR therapy is a cell expressing a recombinant TCR on the surface, wherein the recombinant TCR specifically binds to an MHC molecule complexed with the tumor antigen.

47. The method of claim 43, wherein the immunotherapy is a chimeric antigen receptor (CAR) therapy targeting the tumor antigen.

48. The method of claim 47, wherein the CAR therapy is a cell expressing a CAR on the surface, wherein the CAR specifically binds to the tumor antigen.

49. The method of claim 43, wherein the immunotherapy is a monoclonal antibody targeting the tumor antigen.

50. The method of claim 49, wherein the monoclonal antibody is conjugated to a

chemotherapy drug.

51. The method of claim 49, wherein the monoclonal antibody is conjugated to a radioactive particle.

52. The method of claim 43, wherein the immunotherapy is an inhibitor of an immune checkpoint protein.

53. The method of claim 52, wherein the immune checkpoint protein is selected from the group consisting of PD-1, PD-L1, PD-L2, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), T cell immunoglobulin and mucin protein 3 (TIM-3), lymphocyte-activation protein 3 (LAG-3), carcinoembryonic antigen-related cell adhesion molecule 1

(CEACAM1), glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR), and tumor necrosis factor receptor superfamily, member 4 (TNFRSF4).

54. The method of claim 52, wherein the immune checkpoint protein is a member of the indoleamine-pyrrole 2,3-dioxygenase (IDO) pathway.

55. The method of claim 54, wherein the inhibitor is selected from the group consisting of norharmane, rosmarinic acid, a cyclooxygenase-2 (COX-2) inhibitor, and alpha-methyl tryptophan.

56. The method of claim 52, wherein the inhibitor of an immune checkpoint protein is a monoclonal antibody.

57. The method of claim 43, wherein the immunotherapy is a bispecific antibody targeting a tumor antigen and an immune cell-specific protein.

58. The method of claim 42, wherein the cancer therapy is administered with an adjuvant.

59. The method of claim 58, wherein the adjuvant is selected from the group consisting of Quillaj a saponin 21 (QS21), Quillaja saponin 7 (QS7), monophosphoryl lipid A, aluminum hydroxide, an oil-in-water emulsion, bryostatin-1 , a toll-like receptor (TLR) agonist, and CpG oligodeoxynucleotides.

60. The method of claim 1 or 33, wherein the cancer is selected from the group consisting of breast cancer, colon cancer, non-small cell lung carcinoma, testicular cancer, ovarian cancer, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, cervical cancer, endometrial cancer, esophageal cancer, gallbladder cancer, kidney cancer, laryngeal cancer, hypopharyngeal cancer, gastrointestinal cancer, liver cancer, lung cancer, oropharyngeal cancer, pancreatic cancer, penile cancer, prostate cancer, stomach cancer, thymus cancer, thyroid cancer, vaginal cancer, vulvar cancer, skin cancer, bone cancer, leukemia, lymphoma, neuronal cancer of the nervous system, and non-neuronal cancer of the nervous system.

61. The method of claim 1 or 33, wherein the subject is a human.

62. The method of claim 33, wherein the soluble, chimeric, fusion or pegylated RGMb

protein is administered systemically.

63. The method of claim 62, wherein the systemic administration is a route of administration selected from the group consisting of intravenous, intramuscular, intraventricular, intrathecal, oral, topical, subcutaneous, subconjunctival, intranasal, intradermal, sublingual, vaginal, rectal and epidural.

64. The method of claim 63, wherein the route of administration is intravenous or

subcutaneous.