WO2022034523A1 - Phagocytosis-inducing compounds and methods of use - Google Patents

Abstract

The invention relates to a peptide for inducing phagocytosis by an antigen presenting cell without inflammation and related method of facilitating the uptake of an immunotherapy composition by contacting an antigen presenting cell with the peptide and with an immunotherapy composition.

Description

PHAGOCYTOSIS-INDUCING COMPOUNDS AND METHODS OF USE

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63/065,308, filed August 13, 2020. The entire disclosure of U.S. Provisional Patent Application No. 63/065,308 is incorporated herein by reference.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing submitted as an electronic text file named “8774-9-PCT_Seq_Listing_ST25.txt”, having a size in bytes of 2,366 bytes, and created on August 11, 2021. The information contained in this electronic file is hereby incorporated by reference in its entirety pursuant to 37 CFR § 1.52(e)(5).

BACKGROUND

Peptides are short linear chains of amino acids. They are usually <50 amino acids in length and are often stabilized by disulfide bonds (Hayashi MA, et al. Natural Peptides with Potential Applications in Drug Development, Diagnosis, and/or Biotechnology. Int J Pept. 2012;2012:757838). They can be designed by rational methods with high specificity to bind and modulate a protein interaction of interest. Many sequences, structures and pattern interactions of oncogenic proteins are available; as such peptides can be designed specifically as an inhibitor of these interactions (Bidwell GL and Raucher D. Therapeutic peptides for cancer therapy. Part I - peptide inhibitors of signal transduction cascades. Expert Opin Drug Deliv. 2009;6(10): 1033-47) - for example, if an interaction of two proteins is known, a peptide can inhibit this interaction provided the sequence of the binding site is known (Draeger LJ and Mullen GP. Interaction of the bHLH-zip domain of c-Myc with Hl-type peptides. Characterization of helicity in the Hl peptides by NMR. J Biol Chem. 1994;269(3): 1785-93). If a protein-protein interaction site is unknown, a series of overlapping peptides of the desired protein may be synthesized and can be tested for their capability to bind and inhibit this target interaction (Chen IT, et al. Characterization of p21Cipl/Wafl peptide domains required for cyclin E/Cdk2 and PCNA interaction. Oncogene. 1996;12(3):595-607). The peptide sequence can also be modulated easily, due to their ease of synthesis either by chemical or molecular biological techniques (Bidwell GL., et al. 2009).

The peptide RP426 having a sequence of KARKAAKRAF (SEQ ID NO: 1) is composed of alternating hydrophobic and hydrophilic amino acids. RP426 has been referred to as an anti-inflammatory peptide (U.S. Publication No. 2016/0101150). RP426 has been shown to have low binding affinity for CD206.

Therapeutic peptides, such as RP426, have several important advantages over proteins or antibodies: they are small in size, easy to synthesize and have the ability to penetrate cell membranes. They also have high activity, specificity and affinity; minimal drug-drug interaction; and biological and chemical diversity. An added benefit of using peptides as a treatment is that they do not accumulate in specific organs (e.g. kidney or liver), which can help to minimize their toxic side effects (Ali R, et al. New Peptide Based Therapeutic Approaches. In: Ghulam Md A, Ishfaq Ahmed S, editors. Advances in Protein Chemistry. Jeddah: OMICS Group eBooks; 2013). They can also be rapidly synthesized and easily modified (Boohaker RJ, et al. The use of therapeutic peptides to target and to kill cancer cells. Curr Med Chem. 2012;19(22):3794-804) and are less immunogenic than recombinant antibodies or proteins (McGregor DP. Discovering and improving novel peptide therapeutics. Curr Opin Pharmacol. 2008;8(5):616-9). Therapeutic peptides show great potential in the treatment of many diseases. In the case of cancer, these peptides can be used in a variety of ways, including carrying cytotoxic drugs, vaccines, hormones and radionuclides (Thundimadathil J. Cancer Treatment Using Peptides: Current Therapies and Future Prospects. J Amino Acids. 2012;2012:Article ID 967347).

Phagocytosis is a process by which antigen presenting cells take up antigens from the environment as a preliminary step to instigating an immune response against the antigen. While many substances are known in the prior art to enhance phagocytosis, and thus enhance antigen presentation (e.g., bacteria or bacterial proteins), these prior art substances simultaneously enhance inflammation. RP426 is one of a series of short peptides that mimic certain bacterial surface molecules. Surprisingly and advantageously as disclosed herein, it has been found that RP426 and related peptides enhance phagocytosis by antigen presenting cells without also upregulating inflammation. This makes RP426 and related peptides useful in various methods and compositions.

SUMMARY

One embodiment is a peptide composed of three domains: a striapathic domain with the sequence KARKAAKRAF (SEQ ID NO: 1), an amidated cysteine, and a polyethylene glycol. The polyethylene glycol can be located at the carboxy terminus of the striapathic domain, bound to the amidation of the amidated cysteine, and/or bound to the R-group amine of the carboxy-terminal lysine of the striapathic domain. In some embodiments, the peptide has a molecular weight no more than 5 kDa. The polyethylene glycol can be tetraethylene glycol.

In some embodiments, the peptide is conjugated to a particle. The peptide can be conjugated to the particle via, for example, a maleimide linker. The particle can be a virion, including but not limited to an adenovirus. The particle can be a polymer bead, including but not limited to a polystyrene bead.

Another embodiment is a method of facilitating uptake of an immunotherapy composition by an antigen presenting cell (APC). The method includes contacting the APC with a peptide of the invention and contacting the APC with the immunotherapy composition. In some embodiments, the peptide binds to CD206 on the APC surface.

The immunotherapy composition can be of many different forms. The immunotherapy composition can comprise a virion, a cancer associated antigen, an infectious disease associated antigen, a viral vector encoding at least one antigen, a yeast immunotherapy composition comprising at least one antigen, and/or other immunotherapy compositions. In some embodiments the immunotherapy composition is a replication defective adenovirus vector comprising an E2b deletion and a nucleic acid sequence encoding an antigen, and the immunotherapy composition can further comprise a replication defective adenovirus vector comprising an El deletion, an E3 deletion, an E4 deletion, or a combination thereof. In some embodiments the immunotherapy composition comprises a yeast immunotherapy composition, and the yeast immunotherapy composition can comprise an intact, heat-inactivated yeast cell comprising at least one antigen.

In some embodiments, the antigen presenting cell is a dendritic cell, a macrophage, or a B lymphocyte.

In some embodiments, contacting the APC with the peptide occurs before contacting the APC with the immunotherapy composition. For example, contacting the APC with the peptide can occur from about 1 to about 2 hours before contacting the APC with the immunotherapy composition.

DESCIRPTION OF THE DRAWINGS

Figs. 1A-1C: NLP06 monocyte-derived dendritic cells (moDCs) treated with pegylated peptides then infected with an adenovirus vector containing a constitutive green fluorescent protein expression vector (AdV-GFP). Multiplicity of infection (MOI) 10,000.

Figs: 1D-1F: NLP06 moDCs treated with pegylated peptides then infected with

AdV-GFP. MOI 1000. Figs. 2A-2C: NLP04 moDCs treated with functionalized RP426 then infected with AdV-GFP. MOI 1000.

Figs. 2D-2F: NLP04 GMDCs (GM-CSF-induced dendritic cells) treated with functionalized RP426 then infected with AdV-GFP. MOI 10,000

Fig. 3: Viability of NLP04 moDCs treated with functionalized RP182 and RP426 then infected with AdV-GFP. MOI 1000.

Figs. 4A-4C: NLP04 GMDCs treated with functionalized RP426 then infected with AdV-GFP. MOI 1000.

Fig. 5: Binding affinity of RP182 and RP426 variants by surface plasmon resonance (SPR).

Figs. 6A-6H: NLP04 moDCs treated with RP182 and RP426 variants and infected with AdV-null O/N.

Fig. 7: RP426 particle functionalization scheme.

Figs. 8A-8F : NLP04 G/4-DCs treated with RP426 nanoparticles infected with AdV- GFP. MOI 1000.

DETAILED DESCRIPTION

Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings that are commonly understood by those of ordinary skill in the art. Generally, nomenclature used in connection with, and techniques of, chemistry, molecular biology, cell and cancer biology, immunology, microbiology, pharmacology, and protein and nucleic acid chemistry are those well-known and commonly used in the art.

All publications, patents, and published patent applications referred to in this application are specifically incorporated by reference herein. In case of conflict, the present specification, including its specific definitions, will control.

The use of RP426 and related peptides to facilitate antigen uptake or phagocytosis while causing little or no inflammation is disclosed herein.

Certain cell types phagocytose various particles, such as bacteria or other particles, and degrade the particles. Antigen presenting cells (APCs) are capable of then displaying, or presenting, fragments of the degraded particles on the surface of the cell. APCs are important in the normal function of immune system, and APCs are also useful as immunotherapeutic compositions. APCs can be exposed to antigens, phagocytose and display the antigens, and facilitate elimination of particles that contain the displayed antigen and other antigens. This is particularly useful in cancer immunotherapy, wherein an APC can be used to display a cancer associated antigen and contribute to the elimination of cells that contain the displayed cancer associated antigen and other antigens.

Disclosed herein is the surprising finding that RP426 peptide and related peptides increase phagocytosis without enhancing inflammation. It has been found that when antigen presenting cells (APCs) are pre-treated with a peptide, such as RP426, prior to exposure to adenovirus encoding green fluorescent protein (GFP), the APCs show increased phagocytosis of the adenovirus encoding GFP relative to control cells.

Striapathic peptides

One embodiment relates to peptides comprising the sequence KARKAAKRAF (SEQ ID NO: 1) and to other related sequences. Sequences can be at least about 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, 60%, 50% identical or similar to KARKAAKRAF (SEQ ID NO: 1). Sequence identity and/or similarity can be determined for a portion of an amino acid sequence. For example, a sequence longer than ten amino acids can contain a portion of that sequence that is 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, 60%, 50% identical or similar to KARKAAKRAF (SEQ ID NO: 1).

The terms "peptide" and "polypeptide" are used synonymously herein to refer to polymers constructed from amino acid residues.

The term "amino acid residue " as used herein refers to any naturally occurring amino acid (L or D form), non-naturally occurring amino acid, or amino acid mimetic (such as peptiod monomer).

The "length" of a polypeptide is the number of amino acid residues linked end-to- end that constitute the polypeptide, excluding any non-peptide linkers and/or modifications that the polypeptide may contain.

The term "striapathic," as used herein, refers to a peptide having an alternating sequence of hydrophobic and hydrophilic modules. A "hydrophobic module" is made up of a peptide sequence consisting of one to five hydrophobic amino acid residues. Likewise, a hydrophilic module is made up of a peptide sequence consisting of one to five hydrophilic amino acid residues.

Hydrophobic amino acid residues are characterized by a functional group ("side chain") that has predominantly non-polar chemical properties. Such hydrophobic amino acid residues can be naturally occurring (L or D form) or non-naturally occurring. Alternatively, hydrophobic amino acid residues can be amino acid mimetics characterized by a functional group ("side chain") that has predominantly non-polar chemical properties. Conversely, hydrophilic amino acid residues are characterized by a functional group ("side chain") that has predominantly polar (charged or uncharged) chemical properties. Such hydrophilic amino acid residues can be naturally occurring (L or D form) or non-naturally occurring. Alternatively, hydrophilic amino acid residues can be amino acid mimetics characterized by a functional group ("side chain") that has predominantly polar (charged or uncharged) chemical properties. Examples of hydrophilic and hydrophobic amino acid residues are shown in Table 1, below. Suitable non-naturally occurring amino acid residues and amino acid mimetics are known in the art. See, e.g., Liang et al. (2013), "An Index for Characterization of Natural and Non-Natural Amino Acids for Peptidomimetics," PLoS ONE 8(7):e67844.

Table 1. Hydrophobic and Hydrophilic Amino Acid Residues

Although most amino acid residues can be considered as either hydrophobic or hydrophilic, a few, depending on their context, can behave as either hydrophobic or hydrophilic. For example, due to their relatively weak non-polar characteristics, glycine, proline, and/or cysteine can sometimes function as hydrophilic amino acid residues. Conversely, due to their bulky, slightly hydrophobic side chains, histidine and arginine can sometimes function as hydrophobic amino acid residues.

The term “phagocytosis” refers to the process by which a cell, such as an antigen presenting cell, takes up or engulfs a large particle or particles, such as in the present invention, an immunotherapy composition.

The term "phagocytosis-inducing property," as used herein, refers to any property of a polypeptide that can be evaluated in silico, in vitro, and/or in vivo, that facilitates or increases, or would be expected to facilitate or increase, phagocytosis.

One embodiment of the invention is a striapathic peptide having a phagocytosisinducing property. Such peptides of the invention can have the following characteristics: a length of 3 to 24 amino acid residues; a striapathic region that comprises at least 25% of the length of the polypeptide; and at least one phagocytosis-inducing property.

The phagocytosis-inducing peptide and/or its striapathic region can have a length that is greater than 3 amino acid residues and/or less than 24 amino acid residues. Thus, the requisite length of the polypeptide can be, for example, 3 to 20, 3 to 18, 3 to 16, 3 to 14, 3 to 12, 4 to 20, 4 to 18, 4 to 16, 4 to 14, 4 to 12, 5 to 20, 5 to 18, 5 to 16, 5 to 14, 5 to 12, 6 to 20, 6 to 18, 6 to 16, 6 to 14, 6 to 12, 7 to 20, 7 to 18, 7 to 16, 7 to 14, or in certain embodiments 7 to 12 amino acid residues. For a phagocytosis-inducing peptide that is longer than 12 amino acid residues, it can be advantageous to design a kink in the secondary structure (e.g., such as produced by a proline residue) such that the polypeptide has a striapathic region that is 12 or fewer amino acid residues in length. The striapathic region of a phagocytosis-inducing peptide can comprise at least 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the length of the polypeptide.

A phagocytosis-inducing peptide can have a striapathic region that includes at least two hydrophobic modules and one or more (e.g., two or three) hydrophilic modules. Alternatively, a phagocytosis-inducing peptide can have a striapathic region that includes at least three hydrophobic modules and two or more (e.g., three or four) hydrophilic modules; a striapathic region that includes at least two hydrophilic modules and one or more (e.g., two or three) hydrophilic modules; or a striapathic region that includes at least three hydrophilic modules and two or more (e.g., three or four) hydrophobic modules.

Antigen presenting cells The term “antigen-presenting cell” or “APC” as referred to herein, refers to a cell that displays antigens on its surface in the process known as antigen presentation. T-cells can recognize these complexes using their T-cell receptors (TCRs), so APCs may process antigens and present them to T-cells. Examples of APCs include, but are not limited to, dendritic cells (DCs), monocytes, macrophages, certain B-cells, and certain activated epithelial cells.

Dendritic cells are antigen presenting cells that are part of the mammalian immune system. Dendritic cells recognize pathogens and present antigens from those pathogens on the dendritic cell surface for other cells in the immune system. Immature dendritic cells constantly sample foreign antigens from the environment in order to detect pathogens such as viruses and bacteria. This is accomplished by pattern recognition receptors (PRRs), such as CD206. PRRs recognize distinctive chemical moieties that appear in some groups of pathogens, and once they come into contact with pathogens, they become activated mature dendritic cells, and begin to migrate to the lymph nodes. Immature dendritic cells digest pathogens via phagocytosis, break down proteins, and then display their fragments on the cell surface using major histocompatibility complex (MHC). At the same time, they increase the ability to activate T cells by increasing the amount of cell surface receptors such as CD80, CD86 and CD40, which are used as co-receptors in T cell activation. They also induce the migration of dendritic cells into the spleen through the blood vessel or into the lymph node through the lymphatic system by increasing the expression of CCR7. Dendritic cells are therein used as antigen presenting cells to present the antigen of pathogens to helper T cells, cytotoxic T cells (killer T cells), and B cells or activate the cells via non-antigen specific co-stimulatory signals.

Dendritic cells can function in the immune prevention and response to cancer by presenting cancer associated antigens. For example, cancer-associated dendritic cells, such as CD 103 -positive dendritic cells, play an important role in the T cell-cancer immune response by transporting cancer antigens to the draining lymph node and cross-presenting the cancer antigen to cytotoxic T cells.

Immunotherapy compositions

Immunotherapy compositions help the immune system recognize, inhibit growth of, and/or eliminate cancer and organisms that cause disease. In some embodiments an immunotherapy composition comprises at least one antigen. In some embodiments, an immunotherapy composition comprises at least one delivery vehicle. Antigens of the present invention include but are not limited to antigens derived from any of a variety of infectious agents or cancer cells.

Antigens

According to the present invention, the general use herein of the term "antigen" refers: to any portion of a protein (peptide, partial protein, full-length protein), wherein the protein is naturally occurring or synthetically derived, to a cellular composition (whole cell, cell lysate or disrupted cells), to an organism (whole organism, lysate or disrupted cells) or to a carbohydrate, or other molecule, or a portion thereof. An antigen may elicit an antigenspecific immune response (e.g., a humoral and/or a cell-mediated immune response) against the same or similar antigens that are encountered by an element of the immune system (e.g., T cells, antibodies).

The methods of the disclosure may include the administration of an antigen as part of an immunotherapy composition. In some embodiments, the antigen is a cancer associated antigen, i.e. an antigen associated with a metaplastic, dysplastic, or neoplastic cell. A cancer associated antigen may comprise a tumor associated antigen, which is enriched in tumor cells as compared to non-tumor cells. A cancer associated antigen may be a tumor specific antigen, which is found in, on, or associated with tumor cells but not non-tumor cells. In further embodiments, the antigen is enriched in or specific to a patient's cancer, as compared to the patient’s non-tumor cells. In some embodiments, the antigen is chemically or recombinantly synthesized. In some embodiments, the antigen comprises one or more of a cancer associated antigen, tumor associated antigen, tumor specific antigen, tumor cell lysate, inactivated tumor cell, apoptotic tumor cell, or tumor-derived nucleic acids, including mRNA. In some embodiments, the antigen is cell-free. The antigen may be one known in the art or described herein.

Antigens of the present invention include but are not limited to antigens derived from a variety of tumor proteins. Illustrative tumor proteins useful in the present invention include, but are not limited to any one or more of, WT1, HPV E6, HPV E7, p53, MAGE-A 1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, BAGE, DAM-6, -10, GAGE-1, -2, -8, GAGE-3, -4, -5, -6, -7B, NA88-A, NY-ESO-1, MART-1, MC1R, GplOO, PSA, PSM, Tyrosinase, TRP-1, TRP-2, ART-4, CAMEL, CEA, Cyp-B, Her2/neu, BRCA1, hTERT, hTRT, iCE, MUC1, MUC2, PRAME, P15, RU1, RU2, SART- 1, S ART-3, WT1, AFP, p- catenin/m, Caspase-8/m, CEA, CDK-4/m, ELF2M, GnT-V, G250, HSP70-2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, Myosin/m, RAGE, SART-2, TRP- 2/INT2, 707-AP, Annexin II, CDC27/m, TPl/mbcr-abl, ETV6/AML, LDLR/FUT, Pml/RARa, and TEL/ AML 1. Further non-limiting examples of cancer antigens include antigenic fragments and polypeptides from VEGFR-2, MMPs, Survivin, TEM8, PMSA, CA125, folate binding protein (FBP), HER2/neu, MUC1, NYESO 1, PSA, Carcinoembryonic antigen (CEA), a-fetoprotein (AFP), heat shock proteins (e.g., hsp70 or hsp90 proteins) from a particular type of tumor, MICAS ligands of NKG2D, epithelial cell adhesion molecule (Ep-CAM/TACSTDl), mesothelin, tumor-associated glycoprotein 72 (TAG-72), gplOO, Melan-A, MART-1, KDR, RCAS1, MDA7, cancer-associated viral vaccines (e.g., human papillomavirus antigens), prostate specific antigen (PSA, PSMA), RAGE (renal antigen), CAMEL (CTL-recognized antigen on melanoma), CT antigens (such as MAGE-B5, -B6, -C2, -C3, and D; Mage-12; CT10; NY-ESO-1, SSX-2, GAGE, BAGE, MAGE, and SAGE), mucin antigens (e.g., MUC1, mucin-CA125, etc.), cancer-associated ganglioside antigens, tyrosinase, gp75, C-myc, Marti, MelanA, MUM-1, MUM-2, MUM- 3, HLA-B7, Ep-CAM, tumor-derived heat shock proteins, and the like (see also, e.g., Acres et al., Curr Opin Mol Ther 2004 February, 6:40-7; Taylor-Papadimitriou et al., Biochim Biophys Acta. 1999 Oct. 8; 1455(2-3):301-13; Emens et al., Cancer Biol Ther. 2003 July- August; 2(4 Suppl 1):5161-8; and Ohshima et al., Int J Cancer. 2001 Jul. 1; 93(l):91-6). Other exemplary cancer antigen targets include CA 195 tumor-associated antigen-like antigen (see, e.g., U.S. Pat. No. 5,324,822) and female urine squamous cell carcinoma-like antigens (see, e.g., U.S. Pat. No. 5,306,811), and the breast cell cancer antigens described in U.S. Pat. No. 4, 960, 716. These and other tumor proteins are known to the skilled artisan.

The cancer antigen may be any type of cancer antigen. The cancer antigen may be an epithelial cancer antigen, (e.g., breast, gastrointestinal, lung), a prostate specific cancer antigen (PSA) or prostate specific membrane antigen (PSMA), a bladder cancer antigen, a skin (melanoma) cancer antigen, a lung (e.g., small cell lung) cancer antigen, a colon cancer antigen, an ovarian cancer antigen, a brain cancer antigen, a gastric cancer antigen, a renal cell carcinoma antigen, a pancreatic cancer antigen, a liver cancer antigen, an esophageal cancer antigen, a head and neck cancer antigen, or a colorectal cancer antigen. A cancer antigen can also be an antigen specifically expressed by the patient's cancer or an antigen known to be specifically expressed by the patient's cancer.

As used herein, an "infectious agent" is any living organism capable of infecting a host. In some embodiments, the antigen is an infectious disease associated antigen, i.e. an antigen derived from an infectious agent. Infectious agents include, for example, bacteria, any variety of viruses, such as, single stranded RNA viruses, single stranded DNA viruses, fungi, parasites, and protozoa. Examples of infectious agents include, but are not limited to, Actinobacillus spp., Actinomyces spp., Adenovirus (types 1, 2, 3, 4, 5 et 7), Adenovirus (types 40 and 41), Aerococcus spp., Aeromonas hydrophila, Ancylostoma duodenale, Angiostrongylus cantonensis, Ascaris lumbricoides, Ascaris spp., Aspergillus spp., Babesia spp, B. microti, Bacillus anthracis, Bacillus cereus, Bacteroides spp., Balantidium coli, Bartonella bacilliformis, Blastomyces dermatitidis, Bluetongue virus, Bordetella bronchiseptica, Bordetella pertussis, Borrelia afzelii, Borrelia burgdorferi, Borrelia garinii, Branhamella catarrhalis, Brucella spp. (B. abortus, B. canis, B. melitensis, B. suis), Brugia spp., Burkholderia, (Pseudomonas) mallei, Burkholderia (Pseudomonas) pseudomallei, California serogroup, Campylobacter fetus subsp. Fetus, Campylobacter jejuni, C. coli, C. fetus subsp. Jejuni, Candida albicans, Capnocytophaga spp., Chikungunya virus, Chlamydia psittaci, Chlamydia trachomatis, Citrobacter spp., Clonorchis sinensis, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Clostridium spp. (with the exception of those species listed above), Coccidioides immitis, Colorado tick fever virus, Cory neb acterium diphtheriae, Coxiella burnetii, Coxsackievirus, Creutzfeldt- Jakob agent, Kuru agent, Crimean-Congo hemorrhagic fever virus, Cryptococcus neoformans, Cryptosporidium parvum, Cytomegalovirus, Cyclospora cayatanesis, Dengue virus (1, 2, 3, 4), Diphtheroids, Eastern (Western) equine encephalitis virus, Ebola virus, Echinococcus granulosus, Echinococcus multilocularis, Echovirus, Edwardsiella tarda, Entamoeba histolytica, Enterobacter spp., Enterovirus 70, Epidermophyton floccosum, Ehrlichia spp, Ehrlichia sennetsu, Microsporum spp. Trichophyton spp., Epstein-Barr virus, Escherichia coli, enterohemorrhagic, Escherichia coli, enteroinvasive, Escherichia coli, enteropathogenic, Escherichia coli, enterotoxigenic, Fasciola hepatica, Francisella tularensis, Fusobacterium spp., Gemella haemolysans, Giardia lamblia, Guanarito virus, Haemophilus ducreyi, Haemophilus influenzae (group b), Hantavirus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Herpes simplex virus, Herpesvirus simiae, Histoplasma capsulatum, Human coronavirus, Human immunodeficiency virus, Human papillomavirus, Human rotavirus, Human T- lymphotrophic virus, Influenza virus including H5N1, Junin virus / Machupo virus, Klebsiella spp., Kyasanur Forest disease virus, Lactobacillus spp., Lassa virus, Legionella pneumophila, Leishmania major, Leishmania infantum, Leishmania spp., Leptospira interrogans, Listeria monocytogenes, Lymphocytic choriomeningitis virus, Machupo virus, Marburg virus, Measles virus, Micrococcus spp., Moraxella spp., Mycobacterium spp. (other than M. bovis, M. tuberculosis, M. avium,, M. leprae), Mycobacterium tuberculosis, M. bovis, Mycoplasma hominis, M. orale, M. salivarium, M. fermentans, Mycoplasma pneumoniae, Naegleria fowleri, Necator americanus, Neisseria gonorrhoeae, Neisseria meningitides, Neisseria spp. (other than N. gonorrhoeae and N. meningitidis), Nocardia spp., Norwalk virus, Omsk hemorrhagic fever virus, Onchocerca volvulus, Opisthorchis spp., Parvovirus B19, Pasteurella spp., Peptococcus spp., Peptostreptococcus spp., Plasmodium falciparum, Plasmodium vivax, Plasmodium spp., Plesiomonas shigelloides, Powassan encephalitis virus, Proteus spp., Pseudomonas spp. (other than P. mallei, P. pseudomallei), Rabies virus, Respiratory syncytial virus, Rhinovirus, Rickettsia akari, Rickettsia prowazekii, R. Canada, Rickettsia rickettsii, Rift Valley virus, Ross river virus / O'Nyong-Nyong virus, Rubella virus, Salmonella choleraesuis, Salmonella paratyphi, Salmonella typhi, Salmonella spp. (with the exception of those species listed above), Schistosoma spp., Scrapie agent, Serratia spp., Shigella spp., Sindbis virus, Sporothrix schenckii, St. Louis encephalitis virus, Murray Valley encephalitis virus, Staphylococcus aureus, Streptobacillus moniliformis, Streptococcus agalactiae, Streptococcus faecalis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus salivarius, Taenia saginata, Taenia solium, Toxocara canis, T. cati, T. cruzi, Toxoplasma gondii, Treponema pallidum, Trichinella spp., Trichomonas vaginalis, Trichuris trichiura, Trypanosoma brucei, Trypanosoma cruzi, Ureaplasma urealyticum, Vaccinia virus, Varicella-zoster virus,, eastern equine encephalitis virus (EEEV), severe acute respiratory virus (SARS), Venezuelan equine encephalitis virus (VEEV), Vesicular stomatitis virus, Vibrio cholerae, serovar 01, Vibrio parahaemolyticus, West Nile virus, Wuchereria bancrofti, Yellow fever virus, Yersinia enterocolitica, Yersinia pseudotuberculosis, and Yersinia pestis.

Examples of infectious agents associated with human malignancies include Epstein- Barr virus, Helicobacter pylori, Hepatitis B virus, Hepatitis C virus, Human heresvirus-8, Human immunodeficiency virus, Human papillomavirus, Human T cell leukemia virus, liver flukes, and Schistosoma haematobium.

Antigens may include proteins, or variants or fragments thereof, produced by any of the infectious organisms described herein, such as, but not limited to, viral coat proteins, i.e., influenza neuraminidase and hemagglutinin, HIV gpl60 or derivatives thereof, HIV Gag, HIV Nef, HIV Pol, SARS coat proteins, herpes virion proteins, WNV proteins, etc. Target antigens may also include bacterial surface proteins including pneumococcal PsaA, PspA, LytA, surface or virulence associated proteins of bacterial pathogens such as Nisseria gonnorhea, outer membrane proteins or surface proteases. Antigens may also include proteins, or variants or fragments thereof, of infectious agents associated with human malignancies such as the human papillomavirus (HPV) oncoproteins E6 and E7.

Delivery vehicles

Some embodiments of the immunotherapy compositions of the present invention comprise a delivery vehicle. The delivery vehicle may comprise a virion comprising nucleic acid surrounded by a capsid, a yeast, or another delivery vehicle. The virion can be an adenovirus.

The term "adenovirus" or "Ad" refers to a group of non-enveloped DNA viruses from the family Adenoviridae. In addition to human hosts, these viruses can be found in, but are not limited to, avian, bovine, porcine and canine species. The present invention contemplates the use of any adenovirus from any of the four genera of the family Adenoviridae (e.g., Aviadenovirus, Mastadenovirus, Atadenovirus and Siadenovirus) , along with any of the serotypes of each species. Ad also pertains to genetic derivatives of any of these viral serotypes, including but not limited to, genetic mutation, deletion or transposition of homologous or heterologous DNA sequences.

Adenoviruses can be engineered to take many forms. For example, an adenovirus can be a non-replicating Ad that does not contain any heterologous nucleic acid sequences for expression, that has the early region 1 (El) deleted (removed), that has the nonessential early region 3 (E3) deleted, that has all viral coding regions deleted, or that has all or parts of the El, E2, E3, and/or E4 DNA gene sequences deleted from the virus. Adenoviruses also can contain deletions in the DNA polymerase gene (pol) and/or deletions of the preterminal protein (pTP).

Thus, the present invention contemplates the use of E2b deleted adenovirus vectors, such as those described in U.S. Patent Nos. 6,063,622; 6,451,596; 6,057,158: and 6,083,750.

Adenoviruses (Ad) are non-enveloped double stranded DNA viruses, approximately 60-110 nm in diameter. In humans, adenoviruses can cause relatively mild, self-limiting diseases of the upper respiratory tract, gastroenteritis, or conjunctivitis. However, most are asymptomatic in immunocompetent individuals. Importantly, adenoviruses have not been associated with any neoplastic disease in humans. There are currently about 50 known serotypes of human Ad, the most extensively characterized serotypes being serotype 2 (Ad2) and serotype 5 (Ad5). The adenovirus genome is a double stranded, linear DNA molecule, approximately 36 kb in length. The genome is flanked by cis-acting inverted terminal repeats (ITRs), which are required for viral DNA replication in cis. A cis-acting packaging signal ( ), required for the encapsidation of the Ad genome, is located near the left ITR (relative to the conventional map of Ad) (Figs. 1A-1F). The Ad genome comprises two set of genes: early region genes, El A, E1B, E2, E3, and E4, which are transcribed before DNA replication; and late region genes, LI to L5, which are transcribed and expressed at high levels after the initiation of DNA replication. The early region genes are necessary for activating transcription of other viral regions, altering the host cellular environment to enhance virus replication, and replication of the viral DNA. The E1A transcription unit encodes two major El A proteins that are involved in transcriptional regulation of the virus. The two major E1B proteins are involved in stimulation of viral mRNA transport, blocking El A-induced apoptosis and blocking host mRNA transport. The E2b gene encodes the viral polymerase and terminal protein precursor, and is necessary for viral replication.

Several modified adenoviruses have been produced that rendered the virus replication defective and capable of evading host immunity. For example, AD5 has been produced that lack the El and E2b genes, resulting in adenoviruses vector that are unable to replicate with the use of helper-cells. The production of adenoviruses is well known in the art.

An immunotherapy composition may comprise a yeast vehicle and one or more antigens. In conjunction with a yeast vehicle, antigens may be expressed as recombinant proteins by the yeast vehicle (e.g., by an intact yeast or yeast spheroplast, which can optionally be further processed to a yeast cytoplast, yeast ghost, or yeast membrane extract or fraction thereof), or one or more antigens may be loaded into a yeast vehicle or otherwise complexed with, attached to, mixed with or administered with a yeast vehicle as described herein to form a composition of the present invention.

According to the present invention, reference to a "heterologous" protein or "heterologous" antigen, including a heterologous fusion protein, in connection with an adenovirus, yeast, or other type of vehicle of the invention, means that the protein or antigen is not a protein or antigen that is naturally expressed by the adenovirus, yeast, or other type of vehicle, although a fusion protein that includes heterologous antigen or heterologous protein may also include sequences or proteins or portions thereof that are also naturally expressed by adenovirus, yeast, or other type of vehicle.

Functionalization

PEGylation PEGylation, the attachment of one or more polyethylene glycol moieties to a composition, can accomplish any of several functions, in particular to act as a spacer or linker or to improve the pharmacodynamic properties of the PEGylated composition.

PEG is a well-known, water soluble polymer that is commercially available or can be prepared by ring-opening polymerization of ethylene glycol according to methods well known in the art (Sandler and Karo, Polymer Synthesis, Academic Press, New York, Vol. 3, pages 138-161). The term “PEG” is used broadly to encompass any polyethylene glycol molecule, without regard to size or to modification at an end of the PEG, and can be represented by the formula (1)

H-(O-CH₂-CH₂)n-OH, (1) where n is at least 2.

Polyethylene glycol can be many different molecular weights, dependent on how many ethylene glycol units are incorporated into the polyethylene glycol. One skilled in the art can select a suitable molecular mass for the PEG, e.g., based on how the pegylated peptide will be used therapeutically, the desired dosage, circulation time, resistance to proteolysis, immunogenicity, and other considerations. For a discussion of PEG and its use to enhance the properties of proteins, see N. V. Katre, Advanced Drug Delivery Reviews 10: 91-114 (1993). Practical molecular masses include from about 80 Daltons (Da) to 100,000 Da (n is 2 to 2300).

Poleyethylene glycol (PEG) can have relatively few ethylene glycol units. In that case, the polyethylene glycol can be described using the number of ethylene glycol units. Such polyethylene glycol is exemplified by PEG-3, PEG-4, PEG-5, PEG-6, PEG-7, PEG- 8, PEG-9, PEG- 10, wherein the number refers to the number of ethylene glycol units in the polyethylene glycol. For example, PEG-4 is tetraethylene glycol. Relatively large polyethylene glycol can be described using the approximate or average molecular weight of the polyethylene glycol. For example, polyethylene glycol can be PEG of about 5kDA, PEG of about lOkDA, PEG of about 20kDA, PEG of about 30kDA, PEG of about 40kDA, PEG of about 50kDA, and/or PEG of about lOOkDA.

It is preferred that the combined molecular mass of PEG on an activated linker is suitable for pharmaceutical use. Thus, the combined molecular mass of the PEG molecules should not exceed 100,000 Da. For example, if three PEG molecules are attached to a linker, where each PEG molecule has the same molecular mass of 12,000 Da (each n is about 270), then the total molecular mass of PEG on the linker is about 36,000 Da (total n is about 820). The molecular masses of the PEG attached to the linker can also be different, e.g., of three molecules on a linker two PEG molecules can be 5,000 Da each (each n is about 110) and one PEG molecule can be 12,000 Da (n is about 270).

The chemical PEGylation of a peptide can result in a protein preparation comprising the peptide PEGylated at one or more sites. For example, a peptide may be PEGylated at one or more s-amino groups of lysine residues and/or at the N-terminal amino group. A peptide can be PEGylated at the R-group amine of a C-terminal lysine. Selective PEGylation at the N-terminal amino acid can also be performed, for example according to Felix, A. M., et al., ACS Symp. Ser. 680 (Poly(ethylene glycol)) (1997) 218-238. Selective N-terminal PEGylation can be achieved, for example, during solid-phase synthesis by coupling of aNa- PEGylated amino acid derivative to the N-l terminal amino acid of the peptide chain. C- terminal PEGylation can also be accomplished. Amino acids can be amidated and PEGylated, for example a peptide can contain an amidated cysteine at its C-terminus and be PEGylated at the amidated cysteine. PEGylation can also be accomplished at other sites, including at disulfide bonds or at specific amino acids. Side chain PEGylation can be performed, for example, during solid-phase synthesis by coupling of Ns-PEGylated lysine derivatives to the growing chain. Combined N-terminal and side chain PEGylation is feasible, for example, either as described above within solid-phase synthesis or by solution phase synthesis by applying activated PEG reagents to an amino deprotected peptide.

PEGylation can be accomplished with activated PEG molecules. A wide variety of PEG derivatives suitable for use in the preparation of PEG-protein and PEG-peptide conjugates are available. Activated PEG derivatives are known in the art and are described in, for example, Morpurgo, M., et al., J. Bioconjug. Chem. 7 (1996) 363-368, for PEG- vinylsulfone. Linear chain and branched chain PEG species are suitable for the preparation of PEGylated peptides.

PEGylation can be used as a spacer and/or linker when it is desirable to link two or more compositions. For example, a peptide can be conjugated to a first end of a PEG-4 linker, with the second end of the linker being conjugated to a solid support, such as a polystyrene bead. A PEG linker can provide space to prevent steric hindrance, for example to prevent steric hindrance by a solid support from negatively impacting the intended function of a peptide that is conjugated to the solid support. A PEG linker can also provide flexibility and maneuverability, for example by allowing a peptide that is conjugated to a solid support from being held rigidly in a particular orientation relative to the solid support. Such flexibility can allow a composition to rotate or move freely, for example allowing a peptide that is conjugated to a solid support to rotate or move freely relative to the solid support. Such free movement can be important, for example to ensure that surfaces of a composition that need to be exposed for proper function of the composition are not obscured or buried, which could affect the function of the composition.

PEGylation can improve the pharmacological properties of peptides. For example, PEGylation can increase the in vitro or in vivo half-life peptides. In some embodiments, a PEGylated peptide has an in vitro or in vivo half-life of at least about 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, or 170 hours. In some embodiments, a PEGylated peptide has an in vitro or in vivo half-life of or greater than about 2 days, 2.5 days, 3 days, 3.5 days, 4 days, 4.5 days, 5 days, 5.5 days, 6 days, 6.5 days, 7 days, 7.5 days, 8 days, 8.5 days, 9 days, 9.5 days, 10 days, 11 days, 12 days, 13 days, or 14, days. In some embodiments, a PEGylated peptide has an in vitro or in vivo half-life ranging between about 0.5 and 14 days, 0.5 and 10 days, between 1 day and 10 days, between 1 day and 9 days, between 1 day and 8 days, between 1 day and 7 days, between 1 day and 6 days, between 1 day and 5 days, between 1 day and 4 days, between 1 day and 3 days, between 2 days and 10 days, between 2 days and 9 days, between 2 days and 8 days, between 2 days and 7 days, between 2 days and 6 days, between 2 days and 5 days, between 2 days and 4 days, between 2 day and 3 days, between 2.5 days and 10 days, between 2.5 days and 9 days, between 2.5 days and 8 days, between 2.5 days and 7 days, between 2.5 days and 6 days, between 2.5 days and 5 days, between 2.5 days and 4 days, between 3 days and 10 days, between 3 days and 9 days, between 3 days and 8 days, between 3 days and 7 days, between 3 days and 6 days, between 3 days and 5 days, between 3 days and 4 days, between

3.5 days and 10 days, between 3.5 days and 9 days, between 3.5 days and 8 days, between

3.5 days and 7 days, between 3.5 days and 6 days, between 3.5 days and 5 days, between

3.5 days and 4 days, between 4 days and 10 days, between 4 days and 9 days, between 4 days and 8 days, between 4 days and 7 days, between 4 days and 6 days, between 4 days and 5 days, between 4.5 days and 10 days, between 4.5 days and 9 days, between 4.5 days and 8 days, between 4.5 days and 7 days, between 4.5 days and 6 days, between 4.5 days and 5 days, between 5 days and 10 days, between 5 days and 9 days, between 5 days and 8 days, between 5 days and 7 days, between 5 days and 6 days, between 5.5 days and 10 days, between 5.5 days and 9 days, between 5.5 days and 8 days, between 5.5 days and 7 days, between 5.5 days and 6 days, between 6 days and 10 days, between 7 days and 10 days, between 8 days and 10 days, between 9 days and 10 days, between 10 days and 11 days, between 11 days and 12 days, between 12 days and 13 days, between 13 days and 14 days. Solid supports The inventors have made the surprising discovery that RP426 more effectively stimulates phagocytosis when it is linked to a solid support. For example, see Figure 7C. Solid supports for peptides and other therapeutics can serve many functions and take many forms, such as polymer beads. Solid supports can provide a physical platform to stabilize support-associated compositions, including peptides and immunotherapy compositions of the present invention, for example by sterically hindering object degradation or altering the dynamics of compositions within the body of a patient. Solid supports can also provide a means to separate support-associated compositions from those not associated with the support. Solid supports can be in various forms, including but not limited to surfaces (e.g. of microtiter plate wells or capillaries), membranes (e.g. nitrocellulose membrane), porous materials (e.g. monoliths, as in Liu J, Chen CF, Chang CW, DeVoe DL. Flow-through immunosensors using antibody-immobilized polymer monoliths. Biosens Bioelectron. 2010;26(l): 182— 188. doi: 10.1016/j.bios.2010.06.007), and/or beads (e.g. polymer beads). Polymer beads can also take many forms, for example agarose, cross-linked agarose (e.g. Sepharose®), acrylamide or polyacrylamide, or polystyrene beads. Compositions can be conjugated to solid supports, such as beads. For example, peptides can be conjugated to beads. Conjugation can be accomplished by various means, for example via a maleimide linker.

Inflammation

Inflammation is a complex protective response to pathogens, tissue damage, and exposure to irritants, involving alteration in blood vessels, mobilization of immune cells, and release of a variety of chemical and peptide mediators. While inflammation serves to remove the initial cause of cell injury and eliminate necrotic cells from damaged tissue, the inflammatory response can itself be damaging. For example, inflammation resulting from acute processes, such as cancer treatment or viral or bacterial infection, can result in pain, tissue damage, and septic shock.

Controlling inflammation is especially important with certain methods of cancer treatment, such as certain targeted immunological therapies. For example, some patients given certain immune therapies experience a dangerous and sometimes life-threatening side effect called cytokine release syndrome (CRS), a systemic inflammatory response in which there is a rapid and massive release of cytokines into the bloodstream, leading to dangerously low blood pressure, high fever and shivering. In severe cases of CRS, patients experience a cytokine storm (a.k.a. cytokine cascade or hypercytokinemia), in which there is a positive feedback loop between cytokines and white blood cells with highly elevated levels of cytokines. In a cytokine storm, numerous proinflammatory cytokines, such as interleukin-1 (IL-1), IL-6, g-interferon (g-IFN), and tumor necrosis factor-a (TNFa), are released, resulting in hypotension, hemorrhage, and, ultimately, multiorgan failure. This can lead to potentially life-threatening complications including cardiac dysfunction, adult respiratory distress syndrome, neurologic toxicity, renal and/or hepatic failure, pulmonary edema and disseminated intravascular coagulation.

Non-inflammatory treatments for cancer and other conditions are thus desirable to avoid the deleterious effects of inflammation. The peptides of the present invention can be non-inflammatory, as demonstrated in Example 3. Inflammation in vitro or in vivo can be measured by many methods, for example by measuring the levels or release of cytokines. Such cytokines can include IFNa, IL-12p70, IL-ip, MCP-1, IL-8, IL-10, IL-6, and TNFa. Inflammation can also be measured by testing for C-reactive protein (CRP), white blood cell count, erythrocyte sedimentation rate (ESR), procalcitonin (PCT), or various other markers of inflammation. The term “non-inflammatory” refers to a method or composition that does not cause an increase in inflammation of more than about 5%, about 10%, about 20%, about 30%, about 50%, about 75%, or about 100%, for which inflammation can be quantified by one or more of the abovementioned methods.

Treatment

The compositions provided herein are useful for a variety of clinical applications in which it is desirable to facilitate the uptake by an APC of an immunotherapy composition being administered to a subject for treatment of a disease or condition. The compositions of the invention may be administered for the treatment of various diseases or conditions or used in the manufacture of a medicament for the treatment of various diseases or conditions. As used herein, the terms "treat," "treating," and similar words shall mean stabilizing and/or reducing the symptoms of a disease or condition. In some aspects, the compositions of the invention can be used to prevent the occurrence of a disease or condition or curing a medical condition or disease.

The terms "patient" and "individual" are used interchangeably and refer to either a human or a non-human animal. These terms include mammals such as humans, primates, livestock animals (e.g., bovines, porcines), companion animals (e.g., canines, felines) and rodents (e.g., mice and rats).

The term "pharmaceutically acceptable carrier" refers to a non-toxic carrier that may be administered to a patient, together with compositions of this invention, and which does not destroy the pharmacological activity of the active agents within the composition. The term "excipient" refers to an additive in a formulation or composition that is not a pharmaceutically active ingredient.

The term "pharmaceutically effective amount" refers to an amount effective to treat a patient, e.g., effecting a beneficial and/or desirable alteration in the general health of a patient suffering from a disease (including but not limited to cancer or viral or other infection). The skilled worker will recognize that treating includes, but is not limited to, killing cells (such as cancer cells), preventing the growth of new cancer cells, causing tumor regression (a decrease in tumor size), causing a decrease in metastasis, improving vital functions of a patient, improving the well-being of the patient, decreasing pain, improving appetite, improving the patient's weight, and any combination thereof. A "pharmaceutically effective amount" also refers to the amount required to improve the clinical symptoms of a patient.

Methods are also provided for treating or ameliorating the symptoms of any of the infectious diseases or cancers as described herein. The methods of treatment comprise administering the compositions of the invention one or more times to individuals suffering from or at risk from suffering from an infectious disease or cancer as described herein. As such, the present invention provides methods for vaccinating against infectious diseases or cancers in individuals who are at risk of developing such a disease. Individuals at risk may be individuals who may be exposed to an infectious agent at some time or have been previously exposed but do not yet have symptoms of infection or individuals having a genetic predisposition to developing a cancer or being particularly susceptible to an infectious agent.

Routes and frequency of administration of the therapeutic compositions described herein, as well as dosage, will vary from individual to individual, and from disease to disease, and may be readily established using standard techniques. In general, the pharmaceutical compositions and vaccines may be administered by injection (e.g., intracutaneous, intramuscular, intravenous or subcutaneous), intranasally (e.g., by aspiration), in pill form (e.g. swallowing, suppository for vaginal or rectal delivery).

As described herein, the compositions of the invention are suitable for parenteral administration. These compositions may be administered, for example, intraperitoneally, intravenously, or intrathecally, parenterally, orthotopically, subcutaneously, topically, nasally, orally, sublingually, intraocularly, by means of an implantable depot, using nanoparticle-based delivery systems, microneedle patch, microspheres, beads, osmotic or mechanical pumps, and/or other mechanical means. One of skill in the art would appreciate that a method of administering the composition of the invention would depend on factors such as the age, weight, and physical condition of the patient being treated, and the disease or condition being treated. The skilled worker would, thus, be able to select a method of administration optimal for a patient on a case-by-case basis.

In conjunction with any of the foregoing methods, the compositions can be administered in combination with another drug. In each case, the composition of the invention can be administered prior to, at the same time as, or after the administration of the other drug. For the treatment of cancer, the injectable compositions of the invention can be administered in combination with a chemotherapeutic agent selected from the group consisting of steroids, anthracyclines, thyroid hormone replacement drugs, thymidylate- targeted drugs, Chimeric Antigen Receptor/T cell therapies, and other cell therapies. Specific chemotherapeutic agents include, for example, Gemcitabine, Docetaxel, Bleomycin, Erlotinib, Gefitinib, Lapatinib, Imatinib, Dasatinib, Nilotinib, Bosutinib, Crizotinib, Ceritinib, Trametinib, Bevacizumab, Sunitinib, Sorafenib, Trastuzumab, Ado- trastuzumab emtansine, Rituximab, Ipilimumab, Rapamycin, Temsirolimus, Everolimus, Methotrexate, Doxorubicin, Abraxane, Folfirinox, Cisplatin, Carboplatin, 5-fluorouracil, Teysumo, Paclitaxel, Prednisone, Levothyroxine, and Pemetrexed.

Alternatively, for the methods of treating cancer, the compositions of the invention can be administered in combination with radiation therapy or can be administered prior to, or after the administration of the radiation therapy.

In some embodiments, it may be beneficial to include one or more excipients in a composition of the invention. One of skill in the art would appreciate that the choice of any one excipient may influence the choice of any other excipient. For example, the choice of a particular excipient may preclude the use of one or more additional excipients because the combination of excipients would produce undesirable effects. One of skill in the art would be able to empirically determine which excipients, if any, to include in the formulations or compositions of the invention. Excipients of the invention may include, but are not limited to, co-solvents, solubilizing agents, buffers, pH adjusting agents, bulking agents, surfactants, encapsulating agents, tonicity-adjusting agents, stabilizing agents, protectants, and viscosity modifiers. In some embodiments, it may be beneficial to include a pharmaceutically acceptable carrier in the compositions of the invention.

In some embodiments, it may be beneficial to include a solubilizing agent in the compositions of the invention. Solubilizing agents may be useful for increasing the solubility of any of the components of the formulation or composition, including peptides of the invention or an excipient. The solubilizing agents described herein are not intended to constitute an exhaustive list but are provided merely as exemplary solubilizing agents that may be used in the formulations or compositions of the invention. In certain embodiments, solubilizing agents include, but are not limited to, ethyl alcohol, tert- butyl alcohol, polyethylene glycol, glycerol, methylparaben, propylparaben, polyethylene glycol, polyvinyl pyrrolidone, and any pharmaceutically acceptable salts and/or combinations thereof.

The pH of the compositions of the invention may be any pH that provides desirable properties for the composition. Desirable properties may include, for example, peptide (such as RP426) stability, increased peptide retention as compared to compositions at other pHs, and improved filtration efficiency.

In some embodiments, it may be beneficial to include a tonicity-adjusting agent in the compositions of the invention. The tonicity of a liquid composition is an important consideration when administering the composition to a patient, for example, by parenteral administration. Tonicity-adjusting agents, thus, may be used to help make a composition suitable for administration. Tonicity-adjusting agents are well known in the art. Accordingly, the tonicity-adjusting agents described herein are not intended to constitute an exhaustive list but are provided merely as exemplary tonicity-adjusting agents that may be used in the formulations or compositions of the invention. Tonicity-adjusting agents may be ionic or non- ionic and include, but are not limited to, inorganic salts, amino acids, carbohydrates, sugars, sugar alcohols, and carbohydrates. Exemplary inorganic salts may include sodium chloride, potassium chloride, sodium sulfate, and potassium sulfate. An exemplary amino acid is glycine. Exemplary sugars may include sugar alcohols such as glycerol, propylene glycol, glucose, sucrose, lactose, and mannitol.

In some embodiments, it may be beneficial to include a stabilizing agent in the compositions of the invention. Stabilizing agents help increase the stability of peptides (such as RP426) in compositions of the invention.

In some embodiments, it may be beneficial to include a protectant in the compositions of the invention. Protectants are agents that protect a pharmaceutically active ingredient (e.g., RP426) from an undesirable condition (e.g., instability caused by freezing or lyophilization, or oxidation). Protectants can include, for example, cryoprotectants, lyoprotectants, and antioxidants. Cryoprotectants are useful in preventing loss of potency of an active pharmaceutical ingredient (e.g., RP426) when a formulation is exposed to a temperature below its freezing point. For example, a cryoprotectant could be included in a reconstituted lyophilized formulation of the invention so that the formulation could be frozen before dilution for intravenous (IV) administration. Cryoprotectants are well known in the art. Accordingly, the cryoprotectants described herein are not intended to constitute an exhaustive list but are provided merely as exemplary cryoprotectants that may be used in the formulations or compositions of the invention. Cryoprotectants include, but are not limited to, solvents, surfactants, encapsulating agents, stabilizing agents, viscosity modifiers, and combinations thereof. Cryoprotectants may include, for example, disaccharides (e.g., sucrose, lactose, maltose, and trehalose), polyols (e.g., glycerol, mannitol, sorbitol, and dulcitol), glycols (e.g., ethylene glycol, polyethylene glycol, propylene glycol).

Lyoprotectants are useful in stabilizing the components of a lyophilized formulation or composition. For example, a peptide such as RP426 could be lyophilized with a lyoprotectant prior to reconstitution. Lyoprotectants are well known in the art. Accordingly, the lyoprotectants described herein are not intended to constitute an exhaustive list but are provided merely as exemplary lyoprotectants that may be used in the formulations or compositions of the invention. Lyoprotectacts include, but are not limited to, solvents, surfactants, encapsulating agents, stabilizing agents, viscosity modifiers, and combinations thereof. Exemplary lyoprotectants may be, for example, sugars and polyols, Trehalose, sucrose, dextran, and hydroxypropyl-beta-cyclodextrin are non-limiting examples of lyoprotectants.

Antioxidants are useful in preventing oxidation of the components of a composition. Oxidation may result in aggregation of a drug product or other detrimental effects to the purity of the drug product or its potency. Antioxidants are well known in the art. Accordingly, the antioxidants described herein are not intended to constitute an exhaustive list but are provided merely as exemplary antioxidants that may be used in the formulations or compositions of the invention. Antioxidants may be, for example, sodium ascorbate, citrate, thiols, metabisulfite, and combinations thereof.

Throughout this specification, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer (or components) or group of integers (or components), but not the exclusion of any other integer (or components) or group of integers (or components).

The singular forms "a," "an," and "the" include the plurals unless the context clearly dictates otherwise. The term "including" is used to mean "including but not limited to." "Including" and "including but not limited to" are used interchangeably.

EXAMPLES

The following examples are put forth to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the embodiments and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, and temperature is in degrees Celsius. Standard abbreviations are used.

Example 1. Uptake by dendritic cells

This example illustrates that peptides of the present invention comprising a striapathic domain, an amidated cysteine, and polyethylene glycol more effectively stimulate uptake of a viral vector encoding the test protein GFP.

Test Articles. Table 2 contains the peptide test articles by name and amino acid sequence. The peptides were custom synthesized by third party vendors (Polypeptide Group, Innopep). Table 2. Peptides used as test articles

Dendritic cell AdV uptake assay. Peripheral blood mononuclear cells (PBMCs) were isolated from leukopaks derived from unique donors using Ficoll-paque density gradient (VWR) then monocytes were isolated using plastic adherence from a portion of the isolated PBMCs. The remainder of the PBMCs are frozen in liquid nitrogen. Dendritic cells (monocyte-derived dendritic cells, moDCs) were differentiated from the monocytes by culturing with granulocyte-macrophage colony-stimulating factor (GM-CSF) (200U/ml, Peprotech) and IL-4 (lOOU/ml, Peprotech) for five to seven days in complete RPMI with 10% fetal calf serum (Omega Scientific). At the end of culturing, the moDCs are harvested and frozen. On the day of the experiment, moDCs are thawed and placed in culture. After a one-hour recovery time, the moDCs are treated with the test articles and infected with an adenovirus vector containing a constitutive green fluorescent protein expression vector (AdV-GFP). Following a 16 to 18 hour incubation time the infected moDCs are harvested with trypsin-EDTA and fixed with 2% PF A, then the fixed cells are processed using flow cytometry for GFP expression. The data were analyzed by percent GFP positive cells compared to uninfected control cells and the geometric mean fluorescence intensity (gMFI) of the GFP positive cells. GFP score is the product of the percent GFP positive and the GFP gMFI.

The data in Figs. 1A-1F show that PEGylation of RP426 (RP426 Peg4Cys-NH2) more effectively stimulates AdV-GFP uptake than does unmodified RP182 or PEGylated RP182 (RP182Peg4Cys-NH2).

The data in Figs. 2A-2F show that functionalized RP426 (RP426C-NH2 and RP182Peg4Cys-NH2) more effectively stimulate AdV-GFP uptake than does native RP182.

The data in Figure 3 show RP426 does not affect toxicity (measured as viability), as compared to RP182, and that the indicated modifications to RP426 do not make it more toxic.

The data in Figs. 4A-4C show that RP426C-NH2 and RP426Peg4Cys-NH2 most effectively stimulate AdV-GFP uptake, as compared to the indicated peptides.

This example illustrates that peptides of the present invention comprising a striapathic domain, an amidated cysteine, and polyethylene glycol more effectively bind to CD206.

Peptide/receptor affinity. Bio-layer interferometry (BLI) was used to determine the affinity of the test articles to human CD206 (huCD206) and mouse CD206 (muCD206). Recombinant huCD206 and muCD206 were purchased (R&D Systems), and biotinylation was performed using EZ-link NHS-Peg4-Biotin (Thermo Fisher) following the manufacturer’s instruction with three-fold molar excess of NHS-biotin solution. After biotinylation, free biotin molecules were removed using Centri-Sep Spin desalting column (Fisher Scientific). BLI experiments were performed on an Octet Red96e (Molecular Devices) instrument at 25 °C. All experiments used an assay buffer composed of 10 mM HEPES, pH 7.4, 150mM NaCl, 0.02% tween 20, 0.1% BSA. Streptavidin coated (SA) biosensors were loaded with biotinylated huCD206 and muCD206. A concentration series (1000, 500, 250, 125, 62.5, 0 nM) of the test articles were used to obtain kinetic data, and the biosensors were regenerated 3 times with 10 mM glycine, pH1.5 between assays. All data were evaluated using Octet data analysis software and the steady-state fits were used to determine KD.

The data in Figure 5 show that modification of RP426 with C-NH2 (RP426C-NH2 and RP426Peg4Cys-NH2) enables RP426 binding to CD206.

Example 3. Inflammation

This example illustrates that peptides of the present invention comprising a striapathic domain, an amidated cysteine, and polyethylene glycol do not induce inflammation.

Cytometric bead array. Cytometry bead array (CBA) was used to characterize the cytokine secretion profile of infected moDCs. Culture supernatants from moDCs treated with the test articles and infected with an empty AdV vector (AdV-null) for 16 to 18 hours were harvested. Protein levels of IFNa, IL-12p70, IL-ip, MCP-1, IL-8, IL-10, IL-6, and TNFa were determined using the corresponding BD CBA Flex Set (BD Bioscience) with a modified version of the manufacturer’s recommended protocol. Briefly, the supernatants were incubated with 0.5 ml of cytokine specific beads and 0.5 ml of secondary antibody for two hours. Following incubation, the beads are centrifuged and washed two times, then acquired using flow cytometry. A standard curve of known quantities for each cytokine is included for quantification.

The data in Figs. 6A-6H show that RP426Peg4Cys-NH2 and RP426C-NH2 do not have a substantial effect on the cytokine profile of treated cells, as compared to native RP182.

This example illustrates that peptides of the present invention comprising a striapathic domain, an amidated cysteine, and polyethylene glycol can be linked to be solid supports, in this case beads, without diminishing the peptides’ phagocytosis-inducing activity.

RP426 nanoparticles. Amino-functionalized 0.25 pm polystyrene particles (Spherotech) were washed twice with phosphate buffered saline (PBS) to remove sodium azide by centrifugation at 15,000 x g. The particles with then resuspended in a 50 mg/mL solution of maleimide-Peg6-succinmidyl ester (Sigma- Aldrich) in PBS, so as to provide a 10-fold molar excess relative to the number of amines present on the particles’ surface. This was allowed to react while mixing for 30 minutes. The particles were then washed again with PBS and immediately resuspended in a 10 mg/ml solution of the RP426-Peg4Cys-NH2 peptide, making a two-fold molar excess relative to the particles’ reactive sites. This reaction was mixed for two hours, then the particles were washed twice with PBS before being resuspended in PBS prior to addition to cell cultures.

Figure 7 depicts a scheme for attachment of RP426 to a solid support of polystyrene beads.

The data in Figs. 8A-8C show that linking RP426Peg4Cys to polystyrene beads does not have a substantial effect on AdV-GFP uptake, as compared to free RP426Peg4Cys-NH2.

The data in Figs. 8D-8F show that RP426Peg4Cys linked to polystyrene beads more effectively stimulates AdV-GFP uptake than does free RP426Peg4Cys-NH2.

Unless otherwise specified, it is to be understood that each embodiment of the invention may be used alone or in combination with any one or more other embodiments of the invention.

Variations, modifications, and other implementations of what is described herein will occur to those of ordinary skill without departing from the spirit and the scope of the invention. Accordingly, the invention is not to be limited only to the preceding illustrative description.

Each of the embodiments of the invention may be combined individually or in combination with one or more other embodiments of the invention.

Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, numerous equivalents to the compounds, compositions, and methods of use thereof described herein. Such equivalents are considered to be within the scope of the invention.

The contents of all references, patents and published patent applications cited throughout this Application, as well as their associated figures are hereby incorporated by reference in their entirety. In case of conflict, the present specification, including its specific definitions, will control.

Claims

What is Claimed is:

1. A peptide composed of three domains: a. a striapathic domain consisting of SEQ ID NO: 1 ; b. an amidated cysteine; and c. a polyethylene glycol.

2. The peptide of claim 1, wherein the polyethylene glycol is located at the carboxy terminus of the striapathic domain.

3. The peptide of claim 2, wherein the polyethylene glycol is bound to the amidation of the amidated cysteine.

4. The peptide of any one of the previous claims, wherein the polyethylene glycol is bound to the R-group amine of the carboxy-terminal lysine of the striapathic domain.

5. The peptide of any one of the previous claims, wherein the peptide has a molecular weight no more than 5 kDa.

6. The peptide of claim 5, wherein the polyethylene glycol is a tetraethylene glycol.

7. The peptide of any one of the previous claims, wherein the peptide is conjugated to a particle.

8. The peptide of claim 7, wherein the peptide is conjugated to the particle via a maleimide linker.

9. The peptide of claim 7 or claim 8, wherein the particle is a virion.

10. The peptide of claim 9, wherein the virion is an adenovirus.

11. The peptide of claim 7 or 8, wherein the particle is a polymer bead.

12. The peptide of claim 11, wherein the polymer is polystyrene.

13. A method of facilitating uptake of an immunotherapy composition by an antigen presenting cell (APC), the method comprising: a) contacting the APC with the peptide of any one of the previous claims; and b) contacting the APC with the immunotherapy composition.

14. The method of claim 13, wherein the peptide binds to CD206 on the APC surface.

15. The method of claim 13, wherein the immunotherapy composition is the virion.

16. The method of claim 13, wherein the immunotherapy composition comprises an antigen selected from the group consisting of a cancer associated antigen and an infectious disease associated antigen.

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17. The method of claim 13, wherein the immunotherapy composition is selected from the group consisting of a viral vector encoding at least one antigen and a yeast immunotherapy composition comprising at least one antigen.

18. The method of claim 17, wherein the immunotherapy composition is a replication defective adenovirus vector comprising an E2b deletion and a nucleic acid sequence encoding an antigen.

19. The method of claim 18, wherein the replication defective adenovirus vector further comprises an El deletion, an E3 deletion, an E4 deletion, or a combination thereof.

20. The method of claim 17, wherein the immunotherapy composition is a yeast immunotherapy composition.

21. The method of claim 20, wherein the yeast immunotherapy composition comprises an intact, heat-inactivated yeast cell comprising at least one antigen.

22. The method of claim 13, wherein the APC is selected from the group consisting of a dendritic cell, a macrophage, and a B lymphocyte.

23. The method of claim 19, wherein the APC is a dendritic cell.

24. The method of any one of the claims 13-23, wherein contacting the APC with the peptide occurs before contacting the APC with the immunotherapy composition.

25. The method of claim 24, wherein contacting the APC with the peptide occurs from about 1 to about 2 hours before contacting the APC with the immunotherapy composition.

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