Albumin binding peptide conjugates and methods thereof

Abstract

To provide albumin binding peptide conjugates and methods thereof.SOLUTION: The present invention provides a conjugate comprising an albumin binding peptide and a cargo, compositions for directing cargos to the lymphatic system, and vaccines. The methods of the invention can be used to increase an immune response, or to treat cancer or an infectious disease. In one aspect, a conjugate is provided comprising (a) an albumin binding peptide and (b) an immunomodulatory molecule, where the albumin binding peptide is operatively coupled to the immunomodulatory molecule with or without a linker domain.SELECTED DRAWING: None

Description

[Technical field]

[0001] Paragraph of relevant information This application claims the benefit of the priority date of U.S. Provisional Application No. 62 / 543,163, filed August 9, 2017, the entire contents of which are incorporated herein by reference.

[0002] Government funding This invention was made with Government support under Grant No. R01 EB022433 awarded by the National Institutes of Health. The Government has certain rights in this invention. [Background technology]

[0003] Peptide vaccines to induce T cell responses have great potential for use against cancer and other diseases, but their efficacy is limited (Hacohen, N., Fritsch, EF, Carter, TA, Lander, ES & Wu, CJ Getting personal with neoantigen-based therapeutic cancer vaccines. Cancer Immunol Res 1, 11-15, doi:10.1158 / 2326-6066. CIR-13-0022 (2013); Melief, CJM & van der Burg, SI Immunotherapy of established (pre) malignant disease by synthetic long peptide vaccines. Nat Rev Cancer 8, 351-360 (2008)). One factor limiting the activity of current vaccines is insufficient targeting to lymph nodes, where the immune response is stimulated. Enhanced lymph node targeting can be achieved by appending diacyl lipids separated by a PEG spacer to the peptide antigen (Liu, H. et al., Structure-based programming of lymph-node targeting in molecular vaccines. Nature 507, 519-522, doi:10.1038 / nature12978 (2014)). The mechanism of action is thought to involve binding of endogenous albumin upon injection, allowing the peptide antigen to "hitchhike" to the draining lymph node, similar to the mechanism of action of clinically used sentinel lymph node mapping dyes (Lindner, V. & Heinle, H. Binding properties of circulating evans blue in rabbits as determined by disc electrophoresis. Atherosclerosis 43, 417-422, (1982)). However, there remains a need for improved compositions to enhance the antigenicity of peptide vaccines. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Hacohen, N. et al. Cancer Immunol Res (2013) 1, 11~15 [Non-Patent Document 2] Liu, H. et al., Nature (2014) 507, 519-522 Summary of the Invention [Means for solving the problem]

[0005] The present disclosure is based, at least in part, on the discovery that albumin-binding peptides covalently bound to immunomodulatory molecules (e.g., peptide antigens or molecular adjuvants) can be used to enhance the antigenicity of the immunomodulatory molecules. The present disclosure is also based, at least in part, on the discovery that albumin-binding peptides covalently bound to immunomodulatory molecules (e.g., peptide antigens or molecular adjuvants) can stimulate endogenous immune responses. Furthermore, the present disclosure is based, at least in part, on the discovery that albumin-binding peptides covalently bound to immunomodulatory molecules (e.g., peptide antigens or molecular adjuvants) can be used in methods for treating cancer or infectious diseases or for increasing immune responses in a subject. Thus, in one aspect, the present disclosure provides a conjugate comprising an albumin binding peptide and an immunomodulatory molecule, wherein the albumin binding peptide is operably linked to the immunomodulatory molecule with or without a linker domain.

[0006] In another aspect, the disclosure provides a composition for targeting an immunomodulatory molecule to the lymphatic system, the composition comprising a conjugate comprising an albumin binding peptide and an immunomodulatory molecule, wherein the albumin binding peptide is operably linked to the immunomodulatory molecule with or without a linker domain.

[0007] In certain embodiments, the albumin binding peptide is operably linked to an immunomodulatory molecule.

[0008] In some embodiments, the albumin binding peptide is operably linked to the immunomodulatory molecule via a linker domain. In some embodiments, the linker domain is (Gly4Ser) n In one embodiment, the linker domain comprises a Gly-Ser linker, such as a PEG linker. 2000 Linkers such as polyethylene glycol (PEG) linkers.

[0009] In some embodiments, the immunomodulatory molecule is a peptide antigen or a molecular adjuvant. In some embodiments, the peptide antigen is a cancer antigen. In some embodiments, the peptide antigen is a melanoma antigen. In some embodiments, the molecular adjuvant is CpG DNA.

[0010] In one embodiment, the albumin binding peptide comprises the amino acid sequence DICLPRWGCLW.

[0011] In some embodiments, the present invention provides a vaccine comprising a conjugate of the present disclosure. In some embodiments, the vaccine comprises a conjugate of the present disclosure and a pharma- ceutically acceptable carrier or excipient. In some embodiments, the vaccine comprises a conjugate of the present disclosure and an adjuvant.

[0012] In certain embodiments, the conjugate exhibits increased accumulation in lymph nodes when administered to a subject in vivo compared to administration of the antigen alone.

[0013] In certain embodiments, the present invention provides an immunogenic composition comprising a conjugate of the present disclosure.In certain embodiments, the present invention provides an immunogenic composition comprising a vaccine of the present disclosure.

[0014] In certain embodiments, the present disclosure provides a method for treating cancer or an infectious disease, comprising administering to a subject an amount of a conjugate of the present disclosure effective to reduce one or more symptoms of the cancer or infectious disease compared to a control.

[0015] In certain embodiments, the present disclosure provides a method for increasing an immune response in a subject, comprising administering to the subject an amount of a conjugate of the present disclosure effective to increase the immune response in the subject.

[0016] In some embodiments, the subject has cancer or an infectious disease.

[0017] In some embodiments, the invention provides a nucleic acid molecule encoding a conjugate of the present disclosure. In some embodiments, the invention provides a recombinant expression vector comprising a nucleic acid molecule encoding a conjugate of the present disclosure. In some embodiments, the invention provides a host cell transformed with the recombinant expression vector.

[0018] In certain embodiments, the invention provides a method of making a conjugate of the present disclosure, comprising providing a host cell comprising a nucleic acid sequence encoding the conjugate, and maintaining the host cell under conditions in which the conjugate is expressed. [Brief description of the drawings]

[0019] The patent or application contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0020] [Figure 1]1 is a schematic diagram illustrating an exemplary albumin-binding peptide-antigen construct. ABP-PEG2k-EGP long has the structure: an albumin-binding peptide (cyclized DICLPRWGCLW) operably linked to an EGP long peptide (AVGALEGPRNQDWLGVPRQL) via a PEG2k linker. ABP-G4S-EGP long has the structure: an albumin-binding peptide (cyclized DICLPRWGCLW) operably linked to an EGP long peptide (AVGALEGPRNQDWLGVPRQL) via a Gly4Ser linker.

[0021] [Diagram 2] Figure 2 is a schematic diagram illustrating the synthesis and purification of albumin-bound peptide vaccines. The constructs were synthesized as single units via automated solid-phase peptide synthesis. The constructs were then purified via reverse-phase HPLC and isolated in mg quantities.

[0022] [Diagram 3] Figure 3 is a schematic diagram illustrating the cyclization of albumin-bound peptide vaccines. The constructs were cyclized by allowing spontaneous disulfide formation. The buffer was removed via solid phase extraction.

[0023] [Figure 4] FIG. 4 illustrates the LC-MS analysis and characterization of albumin-bound peptide vaccines.

[0024] [Diagram 5] 5 is a schematic diagram illustrating the albumin-bound peptide vaccine immunization regiment. Mice were primed on day 0, boosted on day 14, and tetramer staining was performed on day 21 to assess T cell stimulation in peripheral blood.

[0025] [Figure 6]Figure 6 graphically illustrates the results of albumin-bound peptide vaccine immunization. The graph on the left illustrates the percentage of CD44hi tetramer+ among all live CD8+ T cells. The graph on the right illustrates representative flow plots for unmodified EGP long peptide, ABP-PEG2K-EGP long construct, and ABP-GGGS-EGP long construct. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] The present disclosure provides a novel method for enhancing the antigenicity of peptide antigens by tethering them to albumin binding peptides (ABPs).

[0027] Prior to the discovery of the present invention, albumin binding peptides have remained largely unexplored in the context of vaccinology. Albumin binding peptides have previously been used to extend the pharmacokinetics of protein therapeutics in several preclinical studies (Dennis, MS et al., Albumin Binding as a General Strategy for Improving the Pharmacokinetics of Proteins. Journal of Biological Chemistry 277, 35035-35043 (2002); Levy, OE et al., Novel Exenatide Analogs with Peptidic Albumin Binding Domains: Potent Anti-Diabetic Agents with Extended Duration of Action. PLoS ONE 9, e87704, doi:10.1371 / journal.pone.0087704 (2014)).

[0028] The present disclosure provides the novel discovery that albumin-binding peptides (ABPs) are useful for generating peptide vaccines. ABPs covalently attached to peptide antigens can direct these antigens to the lymphatic system via albumin hitchhiking, a novel application of these underutilized sequences. ABPs also offer several technical advantages over lipid-based albumin binders such as "all on resin" synthesis. Furthermore, libraries of albumin-binding peptides have been characterized, which may allow precise control over albumin binding affinity, which may be important for the resulting immune response.

[0029] The cyclized peptide DICLPRWGCLW has been identified as the minimal core sequence for achieving binding to serum albumin from multiple species, including mouse and human (Dennis, MS, et al., Albumin Binding as a General Strategy for Improving the Pharmacokinetics of Proteins. Journal of Biological Chemistry 277, 35035-35043 (2002)). This sequence was selected as the albumin binding peptide and used in the experiments described herein.

[0030] In some embodiments, the albumin-binding peptide conjugates of the present disclosure comprise an albumin-binding peptide operably linked to a cargo, e.g., an immunomodulatory molecule. In some embodiments, the immunomodulatory molecule is an antigen, such as a peptide antigen, a polypeptide antigen, an antigenic protein, etc. In some embodiments, the immunomodulatory molecule is a molecular adjuvant.

[0031] In some embodiments, the present disclosure provides albumin-binding peptide conjugates for use in immunogenic compositions. In certain embodiments, the albumin-binding peptide conjugates effectively target lymph nodes. The albumin-binding peptide conjugates can be transported through the lymph from the site of administration to the lymph nodes, where they can accumulate and activate immune cells.

[0032] Albumin-binding peptide conjugates typically comprise at least two domains: (1) an albumin-binding peptide, and (2) a cargo (e.g., a peptide antigen or a molecular adjuvant). In some embodiments, the albumin-binding peptide may be operably linked to the cargo via a linker domain. Thus, in some embodiments, the albumin-binding peptide conjugate comprises three domains. Thus, in some embodiments, the general structure of the conjugate is ABP-C, where "ABP" is the albumin-binding peptide, and "C" is the cargo, such as a peptide antigen or a molecular adjuvant. In some embodiments, the general structure of the conjugate is ABP-LC, where "ABP" is the albumin-binding peptide, "L" is the linker domain, and "C" is the cargo, such as a peptide antigen or a molecular adjuvant.

[0033] definition Terms used in the claims and specification are defined as set forth below unless otherwise specified.

[0034] "Albumin-binding peptide conjugate" or "conjugate" refers to an albumin-binding peptide (ABP) conjugated to a cargo. In some embodiments, the albumin-binding peptide is conjugated to the cargo via a linker domain. In some embodiments, the "cargo" is an immunomodulatory molecule. The term "albumin-binding peptide conjugate" or "conjugate" may be used interchangeably with the terms "albumin-binding peptide-antigen construct" and "albumin-binding peptide vaccine."

[0035] "Cargo" refers to an agent that is conjugated to an albumin binding peptide. In some embodiments, the cargo is conjugated to the albumin binding peptide via a linker domain. In some embodiments, the cargo is an immunomodulatory molecule. In some embodiments, the cargo is an antigen. In some embodiments, the cargo is a peptide antigen. In some embodiments, the cargo is a molecular adjuvant. In some embodiments, the cargo is another agent.

[0036] "Immunomodulatory molecule" refers to an agent that modulates (e.g., stimulates, induces, enhances, reduces or diminishes) an immune response. In some embodiments, an immunomodulatory molecule can be an antigen. For example, in some embodiments, an antigen can be a peptide antigen. In some embodiments, an immunomodulatory molecule can be a molecular adjuvant.

[0037] "Amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code and those that are later modified, e.g., hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

[0038] Amino acids may be referred to herein by either their commonly known three letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may be referred to by their commonly accepted single-letter codes.

[0039] An "amino acid substitution" refers to the replacement of at least one existing amino acid residue in a given amino acid sequence (the amino acid sequence of the starting polypeptide) with another, different, "substitution" amino acid residue. An "amino acid insertion" refers to the incorporation of at least one additional amino acid into a given amino acid sequence. An insertion will usually consist of the insertion of one or two amino acid residues, although larger "peptide insertions", e.g., insertions of about 3 to about 5 or up to about 10, 15 or 20 amino acid residues, can also be made. The inserted residues may be naturally occurring or non-naturally occurring, as disclosed above. An "amino acid deletion" refers to the removal of at least one amino acid residue from a given amino acid sequence.

[0040] "Polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of a corresponding naturally occurring amino acid, as well as to naturally occurring and non-naturally occurring amino acid polymers.

[0041] "Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof, in either single- or double-stranded form. Unless otherwise limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly stated. Specifically, degenerate codon substitutions can be achieved by creating sequences in which the third position of one or more selected (or all) codons is substituted with mixed base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081, 1991; Ohtsuka et al., Biol. Chem. 260:2605-2608, 1985; and Cassol et al., 1992; Rossolini et al., Mol. Cell. Probes 8:91-98, 1994). For arginine and leucine, modifications at the second base can also be conservative. The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.

[0042] A polynucleotide as used herein may be composed of any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. For example, a polynucleotide may be composed of single-stranded and double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, RNA that is a mixture of single-stranded and double-stranded regions, hybrid molecules containing DNA and RNA, which may be single-stranded or more typically double-stranded or a mixture of single-stranded and double-stranded regions. Additionally, a polynucleotide may be composed of triple-stranded regions containing RNA or DNA or both RNA and DNA. A polynucleotide may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA, and thus "polynucleotide" encompasses chemically, enzymatically, or metabolically modified forms. A polynucleotide may be a synthetic or isolated nucleic acid polymer containing multiple nucleotide subunits.

[0043] As used herein, the terms "linked," "conjugated," "linked," "fused," or "fusion" are used interchangeably to refer to the joining of two or more elements or components or domains by any means, including chemical conjugation or recombinant means. Methods of chemical conjugation (e.g., using heterobifunctional crosslinkers) are known in the art.

[0044] A peptide, polypeptide or amino acid sequence "derived from" a specified polypeptide or protein refers to the origin of the polypeptide. Preferably, a peptide, polypeptide or amino acid sequence derived from a particular sequence has an amino acid sequence that is essentially identical to that sequence or a portion thereof, said portion consisting of at least 5-15 amino acids, at least 10-20 amino acids, at least 20-30 amino acids, at least 30-50 amino acids, or otherwise identifiable by one of skill in the art as having its origin in the sequence. A polypeptide derived from another peptide may have one or more amino acid residues substituted with another amino acid residue or have one or more insertions or deletions of amino acid residues compared to the starting polypeptide.

[0045] A peptide or polypeptide may include an amino acid sequence that does not occur in nature. Such variants necessarily have less than 100% sequence identity or similarity with the starting molecule. In certain embodiments, the variants will have an amino acid sequence that is about 75%-100% or less, more preferably about 80%-100%, more preferably about 85%-100%, more preferably about 90%-100% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%), and most preferably about 95%-100% amino acid sequence identity or similarity with the amino acid sequence of the starting polypeptide, for example over the length of the variant molecule.

[0046] In certain embodiments, there is one amino acid difference between the starting polypeptide sequence and the sequence derived therefrom. Identity or similarity with respect to this sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical to the starting amino acid residues (i.e., identical residues) after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.

[0047] In certain embodiments, the peptide or polypeptide is encoded by a nucleotide sequence, which may be useful for a number of applications including cloning, gene therapy, protein expression and purification, mutagenesis, DNA vaccination of a host in need of DNA vaccination, antibody generation for passive immunization, PCR, primer and probe generation, etc.

[0048] The term "ameliorating" refers to any therapeutically beneficial outcome in the treatment of a disease state, e.g., cancer, such as prevention, reduction in severity or progression, remission, or cure of the disease state.

[0049] The term "in vivo" refers to a process that takes place in a living organism.

[0050] As used herein, the terms "mammal" or "subject" or "patient" include both humans and non-humans, including, but not limited to, humans, non-human primates, canines, felines, murines, bovines, equines, and porcines. The terms "mammal", "subject" and "patient" are used interchangeably herein.

[0051] The terms "mammal," "subject," and "patient" refer to any individual who is the target of treatment with the compositions of the present disclosure. A subject may be a vertebrate, e.g., a mammal. A subject may be a human. A subject may be symptomatic or asymptomatic. The term does not denote a particular age or sex. It is intended to encompass adult and newborn subjects, whether male or female. A subject may include a control subject or a test subject.

[0052] The term "percent identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are identical when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to those skilled in the art) or by visual inspection. Depending on the application, the "percent identity" can exist over a region of the sequences being compared, e.g., over a functional domain, or alternatively over the entire length of the two sequences being compared. For sequence comparison, typically one sequence serves as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, subsequence coordinates are designated, and sequence algorithm program parameters are designated, if necessary. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence compared to the reference sequence based on the designated program parameters.

[0053] Optimal alignment of sequences for comparison can be carried out, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (generally, Ausubel et al., see below).

[0054] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the website of the National Center for Biotechnology Information.

[0055] As used herein, the term "gly-ser polypeptide linker" refers to a peptide consisting of glycine and serine residues. An exemplary gly-ser polypeptide linker comprises the amino acid sequence Ser(Gly4Ser)n. In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3, i.e., Ser(Gly4Ser)3. In some embodiments, n=4, i.e., Ser(Gly4Ser)4. In some embodiments, n=5. In some embodiments, n=6. In some embodiments, n=7. In some embodiments, n=8. In some embodiments, n=9. In some embodiments, n=10. Another exemplary gly-ser polypeptide linker comprises the amino acid sequence (Gly4Ser)n. In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3. In some embodiments, n=4. In some embodiments, n=5. In some embodiments, n=6. Another exemplary gly-ser polypeptide linker comprises the amino acid sequence (Gly3Ser)n. In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3. In some embodiments, n=4. In some embodiments, n=5. In some embodiments, n=6.

[0056] A "therapeutic antibody" is an antibody, antibody fragment, or antibody-derived construct that can bind to a cell surface antigen on a target cell and produce a therapeutic effect. Such antibodies can be chimeric, humanized, or fully human. Methods for producing such antibodies are known in the art. Such antibodies include single-chain Fc fragments of antibodies, minibodies, and diabodies. Any therapeutic antibody known in the art to be useful for cancer treatment can be used in a combination therapy suitable for use in the methods disclosed herein. The therapeutic antibody can be a monoclonal or polyclonal antibody. In a preferred embodiment, the therapeutic antibody targets a cancer antigen.

[0057] As used herein, "cancer antigen" refers to (i) a tumor-specific antigen, (ii) a tumor-associated antigen, (iii) a cell expressing a tumor-specific antigen, (iv) a cell expressing a tumor-associated antigen, (v) an embryonic antigen on a tumor, (vi) an autologous tumor cell, (vii) a tumor-specific membrane antigen, (viii) a tumor-associated membrane antigen, (ix) a growth factor receptor, (x) a growth factor ligand, and (xi) any other type of antigen or antigen-presenting cell or substance associated with cancer.

[0058] As used herein, we may use the terms "cancer" (or "cancerous"), "hyperproliferative" and "neoplastic" to refer to cells capable of autonomous proliferation (i.e., an abnormal state or condition characterized by rapidly proliferating cell proliferation). Hyperproliferative and neoplastic disease states may be classified as pathological (i.e., characterizing or constituting a disease state) or as non-pathological (i.e., deviating from normal but not associated with a disease state). These terms are intended to include any type of cancerous growth or oncogenic process, metastatic tissue or malignantly transformed cells, tissues or organs, regardless of histopathological type or stage of invasiveness. "Pathological hyperproliferative" cells occur in disease states characterized by malignant tumor growth. Examples of non-pathological hyperproliferative cells include the proliferation of cells associated with wound repair.

[0059] The terms "cancer" or "neoplasm" are used to refer to malignant diseases of various organ systems, including adenocarcinomas which are generally considered to include those affecting the lung, breast, skin (e.g., melanoma), thyroid, lymphatic glands and tissues, gastrointestinal tract and genitourinary tract, as well as malignancies such as most colon cancers, renal cell carcinoma, prostate cancer and / or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus.

[0060] The term "carcinoma" is art-recognized and refers to malignant diseases of epithelial or endocrine tissues, including respiratory system carcinoma, gastrointestinal system carcinoma, genitourinary system carcinoma, testicular carcinoma, breast carcinoma, prostate carcinoma, endocrine system carcinoma, and melanoma. Albumin-binding peptide conjugates can be used to treat patients who have, are suspected of having, or may be at high risk of developing any type of cancer, including renal carcinoma or melanoma, or any viral disease. Exemplary carcinomas include carcinomas forming from tissues of the cervix, lung, prostate, breast, skin, head and neck, colon, and ovary. The term also includes carcinosarcomas, which include malignant tumors composed of carcinomatous and sarcomatous tissues. "Adenocarcinoma" refers to carcinomas derived from glandular tissue or in which the tumor cells form recognizable glandular structures.

[0061] As used herein, "synergy" or "synergistic effect" in reference to the effect produced by two or more individual components refers to the phenomenon whereby the total effect produced by those components, when used in combination, is greater than the sum of the individual effects of each component acting alone.

[0062] The term "sufficient amount" or "an amount sufficient to" means an amount sufficient to produce a desired effect, e.g., an amount sufficient to reduce tumor size.

[0063] The term "effective amount" or "therapeutically effective amount" is a dosage sufficient to provide treatment for the disorder, disease, or condition being treated, to induce or enhance an immune response, ameliorate symptoms of the disease, or otherwise provide the desired pharmacological and / or physiological effect. Since prevention can be considered treatment, a therapeutically effective amount can be a "prophylactically effective amount."

[0064] As used herein, "combination therapy" encompasses administration of each agent or therapy in a sequential manner in a regimen that provides the beneficial effect of the combination, as well as co-administration of these agents or therapies in a substantially simultaneous manner, such as in a single capsule having a fixed ratio of these active agents or in multiple separate capsules for each agent. Combination therapy also includes combinations in which the individual elements may be administered at different times and / or by different routes, but which act in combination to provide a beneficial effect from the co-action or pharmacokinetic and pharmacodynamic effects of each agent or tumor treatment approach of the combination therapy.

[0065] As used herein, "about" will be understood by one of ordinary skill in the art and can vary to some extent depending on the context in which it is used. If there are uses of the term that are not clear to a person of ordinary skill in the art given the context in which the term is used, "about" will mean up to plus or minus 10% of the particular value.

[0066] As used herein, "cancer vaccine" refers to a treatment that induces the immune system to attack cells that bear one or more tumor-associated antigens. The vaccine can treat existing cancer (e.g., therapeutic cancer vaccine) or prevent the onset of cancer in an individual (e.g., prophylactic cancer vaccine). The vaccine creates memory cells that can recognize antigen-bearing tumor cells, thus preventing tumor growth. In some embodiments, the cancer vaccine comprises an immunostimulatory oligonucleotide. In some embodiments, the cancer vaccine comprises an albumin-binding peptide conjugate.

[0067] As used herein, an "immunostimulatory oligonucleotide" is an oligonucleotide that can stimulate (eg, induce or enhance) an immune response.

[0068] As used herein, "CG oligodeoxynucleotides (CG ODNs), also designated "CpG ODNs," are short, single-stranded synthetic DNA molecules that contain a cytosine nucleotide (C) followed by a guanine nucleotide (G). In certain embodiments, the immunostimulatory oligonucleotide is a CG ODN.

[0069] As used herein, an "immune cell" is a cell of hematopoietic origin that plays a role in the immune response. Immune cells include lymphocytes (e.g., B cells and T cells), natural killer cells, and myeloid cells (e.g., monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes).

[0070] The term "T cells" refers to CD4+ T cells or CD8+ T cells. The term T cells encompasses TH1 cells, TH2 cells and TH17 cells.

[0071] The term "T cell cytotoxicity" includes any immune response mediated by CD8+ T cell activation. Exemplary immune responses include cytokine production, CD8+ T cell proliferation, granzyme or perforin production, and clearance of infectious agents.

[0072] As generally used herein, "pharmacologically acceptable" refers to compounds, materials, compositions and / or dosage forms that are suitable for use in contact with the tissues, organs and / or bodily fluids of human beings and animals, within the bounds of sound medical judgment, without excessive toxicity, irritation, allergic response or other problem or complication commensurate with a reasonable benefit / risk ratio.

[0073] It must be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates to the contrary.

[0074] Albumin-binding peptides In one aspect, the conjugates of the present invention comprise an albumin-binding peptide (ABP) operably linked to a cargo (e.g., an immunomodulatory molecule such as a peptide antigen or a molecular adjuvant). In one embodiment, the albumin-binding peptide conjugates can target the cargo to the lymphatic system via albumin hitchhiking. For example, the albumin-binding peptide domain of the conjugate can bind endogenous albumin, which can allow the cargo to "hitchhike" to the draining lymph nodes. In some embodiments, the present disclosure provides a novel method for enhancing the antigenicity of a cargo (e.g., an immunomodulatory molecule such as a peptide antigen) by tethering the cargo to an albumin-binding peptide (ABP). In some embodiments, the albumin-binding peptide conjugates of the present disclosure enhance the antigenicity of a peptide vaccine.

[0075] In some embodiments of the present disclosure, albumin binding peptides can be identified by their ability to bind a target, preferably a serum protein such as serum albumin, in a direct binding assay or by competing for target binding with a known ligand for the target. In some embodiments, albumin binding peptides that bind serum albumin include linear and cyclic peptides, such as a cyclic peptide compound having the following formula, or a peptide that competes with a peptide of the following formula for binding to serum albumin of a particular mammalian species: [ka]

[0076] In some embodiments of the present disclosure, the albumin binding peptide compounds include peptides of the general formula above, wherein Xaa is an amino acid, and x and z are integers greater than or equal to 0 (zero), generally less than 100, preferably less than 10, more preferably 0, 1, 2, 3, 4 or 5, more preferably 4 or 5, and Xaa1 is selected from the group consisting of Ile, Phe, Tyr and Val.

[0077] Additional albumin binding peptides that bind serum albumin may be identified as described herein in the context of the following general formula: [ka] wherein Xaa is an amino acid, and x and z are integers greater than or equal to zero, generally less than 100, preferably less than 10, more preferably 0, 1, 2, 3, 4 or 5, more preferably 4 or 5.

[0078] In some embodiments of the present disclosure, albumin binding peptides include the following: [ka]

[0079] In some embodiments of the present disclosure, albumin binding peptides of the present disclosure can be identified by their ability to bind human serum albumin and compete for binding to human serum albumin in an in vitro assay with a peptide ligand having the following general formula: [ka] wherein Xaa is an amino acid, x and z are preferably 4 or 5, and Xaa1 is selected from the group consisting of Ile, Phe, Tyr, and Val.

[0080] In some embodiments, the albumin binding peptides of the present invention will compete with any of the peptides represented by SEQ ID NOs: 8-21 described herein above.

[0081] As will be understood from the above, the terms "compete" and "ability to compete" are relative terms. Thus, when used to describe the albumin-binding peptides of the present invention, they refer to albumin-binding peptides that, in a standard competitive assay as described herein, produce 50% inhibition of binding of a reference peptide when present at 50 μM, preferably when present at 1 μM, more preferably when present at 100 nM, preferably when present at 1 nM or less. However, albumin-binding peptides that have an affinity for serum albumin of less than about 1 nM, preferably about 1 pM to 1 nM, may also be albumin-binding peptides within the context of the present invention.

[0082] With regard to an in vitro assay system for determining whether a peptide or other compound has the "ability" to compete with an albumin-binding peptide for binding to serum albumin, as described herein, one of skill in the art can utilize any of a number of standard competitive assays. Competitive binding assays rely on the ability of a labeled standard to compete with the test sample analyte for binding to a limited amount of ligand. The amount of analyte in the test sample is inversely proportional to the amount of standard that becomes bound to the ligand.

[0083] Thus, one of skill in the art may determine whether a peptide or other compound has the ability to compete with an albumin-binding peptide for binding to albumin using techniques including, but not limited to, competitive assay systems using techniques such as radioimmunoassays (RIA), enzyme immunoassays (EIA), preferably enzyme-linked immunosorbent assays (ELISA), "sandwich" immunoassays, immunoradiometric assays, fluorescent immunoassays, and immunoelectrophoretic assays, to name just a few.

[0084] For these purposes, selected albumin binding peptides can be labeled with a detectable moiety (detectably labeled peptide ligand, hereafter referred to as "tracer") and used in a competitive assay with a candidate compound for binding albumin. A large number of detectable labels are available, which can be preferably divided into the following categories: (a) 35 S, 14 C. 125 I, 3 H and 131 Radioisotopes such as I. Albumin-binding peptides can be labeled with radioisotopes, for example, using techniques described in Coligen et al., 1991, eds., Current Protocols in Immunology, Volumes 1 and 2, Wiley-Interscience, New York, NY. Radioactivity can be measured using scintillation counting. (b) Fluorescent labels such as rare earth chelates (europium chelates) or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, Lissamine, phycoerythrin and Texas Red can be used. Fluorescent labels can be conjugated to albumin-binding peptides, for example, using the techniques disclosed in Current Protocols in Immunology, supra. Fluorescence can be quantified using a fluorimeter. (c) A variety of enzyme-substrate labels are available, and U.S. Patent No. 4,275,149 provides a review of some of these. The enzyme preferably catalyzes a chemical change in a chromogenic substrate that can be measured using a variety of techniques. For example, the enzyme may catalyze a color change in the substrate that can be measured spectrophotometrically. Or, the enzyme may change the fluorescence or chemiluminescence of the substrate. Techniques for quantifying the change in fluorescence are described above. The chemiluminescent substrate becomes electronically excited by a chemical reaction and may then emit light that can be measured (e.g., using a chemiluminometer) or donate energy to a fluorescent acceptor. Examples of enzyme labels include luciferases (e.g., firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidases such as horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like.

[0085] Examples of enzyme-substrate combinations include, for example: (i) horseradish peroxidase (HRP), which uses hydrogen peroxidase as a substrate, which oxidizes a dye precursor (e.g., ABTS, orthophenylenediamine (OPD), or 3,3',5,5'-tetramethylbenzidine hydrochloride (TMB)); (ii) alkaline phosphatase (AP) using p-nitrophenyl phosphate as a chromogenic substrate; and (iii) β-D-galactosidase (β-D-Gal) using a chromogenic substrate (e.g., p-nitrophenyl-β-D-galactosidase) or a fluorogenic substrate, 4-methylumbelliferyl-β-D-galactosidase.

[0086] According to a particular assay, the tracer is incubated with the immobilized target in the presence of varying concentrations of unlabeled candidate compound. Increasing concentrations of successful candidate compound effectively compete for binding of the tracer to the immobilized target. The concentration of unlabeled candidate compound at which 50% of the maximally bound tracer is displaced is known as the "IC 50 " and reflects the IgG binding affinity of the candidate compound. Thus, an IC of 1 mM 50 Candidate compounds with IC of 1 μM 50 The compound exhibits a significantly weaker interaction with the target than the candidate compound having

[0087] Thus, the present disclosure provides albumin binding peptides that have the ability to "compete" for target molecule binding, such as human serum albumin, in an in vitro assay as described. Preferably, the albumin binding peptide has an IC of less than 1 μM for a target, such as human serum albumin. 50 Preferred among these compounds are those having an IC of less than about 100 nM, preferably less than about 10 nM, or less than about 1 nM. 50 In further preferred embodiments according to this aspect of the invention, the compounds have an IC of less than about 100 pM, more preferably less than about 10 pM, against a target molecule such as human serum albumin. 50 Shows.

[0088] In some embodiments of the present disclosure, exemplary albumin binding peptides include: [ka]

[0089] Examples of albumin binding peptides, methods of identifying albumin binding peptides, and methods of making albumin binding peptides are known in the art, see, for example, U.S. Patent Publication No. 2004 / 0001827, which is incorporated herein by reference.

[0090] Cargo molecules In some embodiments, the cargo molecule of the albumin-binding peptide conjugate of the present disclosure is an immunomodulatory molecule. In some embodiments, the immunomodulatory molecule is an antigen, e.g., a peptide antigen, a protein antigen, a polypeptide antigen. In some embodiments, the immunomodulatory molecule is a molecular adjuvant, e.g., an immunostimulatory oligonucleotide. In some embodiments, the cargo can also be other oligonucleotides, peptides, toll-like receptor agonists or other immunomodulatory compounds, immunostimulatory molecules, dyes, MRI contrast agents, fluorophores, or small molecule drugs that require efficient transport to lymph nodes.

[0091] In some embodiments, the cargo can be an adjuvant such as an immunostimulatory oligonucleotide, and in some embodiments, the cargo molecule can be an antigen. The antigen can be a peptide, a protein, a polysaccharide, a sugar, a lipid, a nucleic acid, or a combination thereof. The antigen can be derived from a virus, a bacterium, a parasite, a plant, a protozoan, a fungus, a tissue, or a transformed cell such as a cancer cell or a leukemia cell, and can be a whole cell or an immunogenic component thereof, such as a cell wall component or a molecular component thereof.

[0092] Suitable antigens are known in the art and are available from commercial, governmental and scientific sources. In one embodiment, the antigen is a cancer antigen peptide or molecular adjuvant. The antigen can be a purified or partially purified peptide or polypeptide derived from a tumor. In some embodiments, the antigen is a recombinant peptide or polypeptide produced by expressing DNA encoding a peptide or polypeptide antigen in a non-compatible expression system. The antigen can be DNA encoding all or part of an antigenic protein, polypeptide or peptide. The DNA can be in the form of a vector DNA, such as a plasmid DNA.

[0093] In some embodiments, antigens are provided as single antigens or in combination. In some embodiments, antigens are provided as a complex mixture of polypeptides, peptides or nucleic acids. Exemplary antigens are listed below.

[0094] Peptide antigen In certain embodiments, the albumin binding peptide (ABP) conjugates described herein may comprise an ABP operably linked to a peptide antigen, an antigenic protein or an antigenic polypeptide, such as a tumor-associated antigen or a fragment thereof.

[0095] In some embodiments, the peptide antigen can be 2-100 amino acids (aa), for example, 5 amino acids, 10 amino acids, 15 amino acids, 20 amino acids, 25 amino acids, 30 amino acids, 35 amino acids, 40 amino acids, 45 amino acids, or 50 amino acids. In some embodiments, the peptide antigen can be more than 50 amino acids. In some embodiments, the peptide antigen can be more than 100 amino acids.

[0096] In some embodiments, the protein / peptide antigen may be linear, branched or cyclic. The peptide antigen may comprise D-amino acids, L-amino acids or a combination thereof. The peptide, polypeptide or protein antigen may be conjugated to the ABP at the N-terminus or C-terminus of the peptide, polypeptide or protein antigen.

[0097] In some embodiments, the peptide, protein or polypeptide antigen can be any peptide, protein or polypeptide that is capable of inducing or increasing the ability of the immune system to generate antibody and T cell responses against the peptide, protein or polypeptide.

[0098] Cancer antigens In some embodiments, the peptide antigen can be generated from a cancer antigen. A cancer antigen is an antigen that is usually preferentially expressed by cancer cells (i.e., the cancer antigen is expressed at a higher level in cancer cells than on non-cancerous cells), and in some cases, is expressed exclusively by cancer cells. The cancer antigen can be expressed within the cancer cell or on the surface of the cancer cell.

[0099] For example, the peptide antigen can be generated from any of the following cancer antigens: MART-1 / Melan-A, gp100, adenosine deaminase binding protein (ADAbp), FAP, cyclophilin b, colorectal-related antigen (CRC)-C017-1A / GA733, carcinoembryonic antigen (CEA), CAP-1, CAP-2, etv6, AML1, prostate-specific antigen (PSA), PSA-1, PSA-2, PSA-3, prostate-specific membrane antigen (PSMA), T-cell receptor / CD3-zeta chain and CD20. Cancer antigens are MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE -A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9, BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2 / neu, p21ras, RCAS1, a-fetoprotein, E-cadherin, a-catenin, β-catenin, γ-catenin, p120ctn, gp100Pmell 17, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin, connexin 37, Ig-idiotype, p15, gp75, GM2 ganglioside, GD2 ganglioside, human papillomavirus protein, Smad family of tumor antigens, lmp-1, PI A, EBV-encoded nuclear antigen (EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1, and CT-7, CD20, or c-erbB-2. Additional cancer antigens include tumor antigens described herein.

[0100] In some embodiments, the peptide antigen is generated from a melanoma antigen. In some embodiments, the melanoma antigen is gp100. In some embodiments, the peptide antigen is a modified peptide ligand form of gp100, referred to as EGP. The amino acid sequence of EGP is AVGAL EGPRNQDWL GVPRQL, where the underlined moieties have been shown to have a higher affinity for MHC than the native form.

[0101] In some embodiments, the albumin-binding peptide conjugates stimulate an immune response against a specific target, such as a tumor-associated antigen.

[0102] In certain embodiments, tumor-associated antigens are determined by sequencing the patient's tumor cells and identifying mutated proteins found only in tumors. In some embodiments, tumor-associated antigens are determined by analyzing the patient's tumor cells and identifying modified proteins (e.g., glycosylation, phosphorylation) found only in tumors. These antigens are referred to as "neo-antigens." Once a neo-antigen is identified, it can be used as an antigen for an albumin-binding peptide conjugate or to derive a peptide antigen for an albumin-binding peptide conjugate. In some embodiments, the ABP conjugates described herein comprise an ABP operably linked to a neo-antigen, with or without a linker domain. In some embodiments, the ABP conjugates described herein comprise an ABP operably linked to a peptide antigen derived from a neo-antigen, with or without a linker domain.

[0103] ii. Viral antigens In some embodiments, the peptide antigens may be generated from viral antigens. The viral antigens may be isolated from any virus, including, but not limited to, viruses from any of the following virus families: Arenaviridae, Arterivirus, Astroviridae, Baculoviridae, Badnavirus, Barnaviridae, Birnaviridae, Bromoviridae, Bunyaviridae, Caliciviridae, Capillovirus, Carlavirus, Caulimovirus, Circoviridae, Closterovirus, and / or other viruses. ovirus, Comoviridae, Coronaviridae (e.g., Coronaviruses, such as Severe Acute Respiratory Syndrome (SARS) virus), Corticoviridae, Cystoviridae, Deltavirus, Dianthovirus, Enamovirus, Filoviridae (e.g., Marburg virus and Ebola virus (e.g., Zaire, Reston, Ivory Coast, or Sudan strains)), Flaviviridae (e.g., Hepatitis C virus, Dengue virus 1,1), Dengue virus 2, Dengue virus 3 and Dengue virus 4), Hepadnaviridae, Herpesviridae (e.g., Human herpesviruses 1, 3, 4, 5 and 6 and Cytomegalovirus), Hypoviridae, Iridoviridae, Leviviridae, Lipothrixviridae, Microviridae, Orthomyxoviridae (e.g., Influenzavirus A, Influenzavirus B, Influenzavirus C, Influenzavirus D, Influenzavirus E, Influenzavirus F, Influenzavirus G, Influenzavirus H, Influenzavirus I, Influenzavirus I, Influenzavirus I, Influenzavirus I, Influenzavirus I, Influenzavirus I, Influenzavirus M, Influenzavirus N ... Influenza A and B and influenza C), Papovaviridae, Paramyxoviridae (e.g. measles, mumps and human respiratory syncytial virus), Parvoviridae, Picornaviridae (e.g. poliovirus, rhinovirus, hepatovirus and aphthovirus), Poxviridae (e.g. vaccinia, Suitable viral antigens include dengue virus, dengue virus, dengue sarcoma and smallpox virus), Reoviridae (e.g., rotavirus), Retroviridae (e.g., lentiviruses such as human immunodeficiency virus (HIV) 1 and HIV2), Rhabdoviridae (e.g., rabies virus, measles virus, respiratory syncytial virus, etc.), Togaviridae (e.g., rubella virus, dengue virus, etc.) and Totiviridae. Suitable viral antigens also include all or part of dengue protein M, dengue protein E, dengue D1NS1, dengue D1NS2 and dengue D1NS3.

[0104] Viral antigens may be derived from specific strains of papillomavirus, herpesvirus, e.g., herpes simplex 1 and 2; hepatitis virus, e.g., hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis delta D virus (HDV), hepatitis E virus (HEV), and hepatitis G virus (HGV), tick-borne encephalitis virus; parainfluenza, varicella zoster, cytomeglavirus, Epstein-Barr, rotavirus, rhinovirus, adenovirus, Coxsackievirus, equine encephalitis, Japanese encephalitis, yellow fever, Rift Valley fever, and lymphocytic choriomeningitis.

[0105] iii. Bacterial antigens In some embodiments, the peptide antigens may be generated from bacterial antigens, such as Actinomyces, Anabaena, Bacillus, Bacteroides, Bdellovibrio, Bordetella, Borrelia, Campylobacter, Caulobacter, Chlamydia, Chlorobium, Chromatium, and the like. atium, Clostridium, Corynebacterium, Cytophaga, Deinococcus, Escherichia, Francisella, Halobacterium, Heliobacter, Haemophilus, Hemophilus influenza type BB) (HIB), Hyphomicrobium, Legionella, Leptospirosis, Listeria, Meningococcus A, B and C, Methanobacterium, Micrococcus, Myobacterium, Mycoplasma, Myxococcus, Neisseria, Nitrobacter, Oscillatoria, Prochloron, Proteus, Pseudomonas The bacterial strain may be of any bacterial origin, including, but not limited to, Bacillus subtilis, Bacillus anguinea, Bacillus subtilis ...

[0106] iv. Parasite antigens In other embodiments, the peptide antigens may be generated from parasite antigens. Parasitic antigens include Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia ricketsii, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydial psittaci, Chlamydial trachomatis, Plasmodium falciparum, Trypanosoma brucei, Entamoeba histolytica, and others. Antigens may be derived from parasites such as, but not limited to, antigens from T. histolytica, T. gondii, T. vaginalis and T. mansoni. These include sporozoan antigens, circumsporozoite proteins, sporozoite surface proteins, liver stage antigens, apical membrane associated proteins or all or part of merozoite surface proteins.

[0107] v. Allergens and Environmental Antigens In some embodiments, the peptide antigens can be generated from allergens or environmental antigens. Allergens or environmental antigens can be, for example, antigens derived from naturally occurring allergens such as pollen allergens (tree, herb, weed and grass pollen allergens), insect antigens (inhalant, saliva and venom antigens), animal hair and dander allergens and food allergens. Important pollen allergens from trees, grasses and herbs include, among others, birch (Betula), alder (Alnus), hazel (Corylus), hornbeam (Carpinus) and olive (Olea), cedar (Cryptomeria and Juniperus), plane tree (Plant allergen), among others. from the taxonomic orders Fagales, Oleales, Pinales and Platanaceae, including the plane tree (Platanus), from the orders Poales, including the grasses of the genera Lolium, Phleum, Poa, Cynodon, Dactylis, Holcus, Phalaris, Secale and Sorghum, from the orders Asterales and Urticales, including especially the herbaceous genera Ambrosia, Artemisia and Parietaria.Other allergen antigens that may be used include house dust mites of the genus Dermatophagoides and Euroglyphus, storage mites, and the like. allergens from mites, e.g., Lepidoglyphys, Glycyphagus and Tyrophagus; allergens from cockroaches, midges and fleas, e.g., Blatella, Periplaneta, Chironomus and Ctenocepphalides; allergens from mammals, such as cats, dogs and horses; birds; venom allergens, including those resulting from stinging or biting insects, such as those from the taxonomic order Hymenoptera, which includes bees (superfamily Apidae), wasps (superfamily Vespidea) and ants (superfamily Formicoidae). Still other allergen antigens that may be used include inhalant allergens derived from fungi such as Alternaria and Cladosporium.

[0108] B. Molecular adjuvants In certain embodiments, the albumin binding peptide (ABP) conjugates described herein may include an ABP operably linked to a molecular adjuvant. In some embodiments, the molecular adjuvant is an immunostimulatory oligonucleotide, such as a CpG-containing oligonucleotide.

[0109] In some embodiments, the molecular adjuvant is an immunostimulatory oligonucleotide (eg, a CpG-containing oligonucleotide) capable of binding a pattern recognition receptor.

[0110] In some embodiments, immunostimulatory oligonucleotides can act as ligands for pattern recognition receptors (PRRs). Examples of PRRs include the Toll-like family of signaling molecules, which play a role in the initiation of innate immune responses and also influence later, more antigen-specific, adaptive immune responses. Thus, oligonucleotides can act as ligands for Toll-like family signaling molecules, such as Toll-like receptor 9 (TLR9).

[0111] For example, unmethylated CpG sites can be detected in humans by TLR9 on plasmacytoid dendritic cells and B cells (Zaida et al., Infection and Immunity, 76(5):2123-2129, (2008)). Thus, the sequence of the oligonucleotide can contain one or more unmethylated cytosine-guanine (CG or CpG, used interchangeably) dinucleotide motifs. "p" represents the phosphodiester backbone of DNA, as described in more detail below, and some oligonucleotides that contain CG can have a modified backbone, e.g., a phosphorothioate (PS) backbone.

[0112] In some embodiments, immunostimulatory oligonucleotides can contain more than one CG dinucleotide, located adjacently or separated by intervening nucleotides. The CpG motif can be present within the oligonucleotide sequence. Numerous nucleotide sequences that vary in the number and location of CG dinucleotides and the exact base sequences flanking the CG dimer stimulate TLR9.

[0113] Typically, CG ODNs are classified based on their sequence, secondary structure and effect on human peripheral blood mononuclear cells (PBMCs). The five classes are class A (type D), class B (type K), class C, class P and class S (Vollmer, J & Krieg, AM, Advanced drug delivery reviews 61 (3): 195-204 (2009), incorporated herein by reference). CG ODNs stimulate the production of type I interferons (e.g., IFNα) and induce the maturation of dendritic cells (DCs). Some classes of ODNs are also potent activators of natural killer (NK) cells through indirect cytokine signaling. Several classes are potent stimulators of human B cell and monocyte maturation (Weiner, GL, PNAS USA 94(20):10833-7 (1997); Dalpke, AH, Immunology 106(1):102-12 (2002); Hartmann, G, J of Immun. 164(3):1617-2 (2000); each of which is incorporated herein by reference).

[0114] Other PRR Toll-like receptors include TLR3 and TLR7, which can recognize double-stranded RNA, single-stranded and short double-stranded RNA, respectively, and the retinoic acid-inducible gene I (RIG-I)-like receptors, i.e., RIG-I and melanoma differentiation-associated gene 5 (MDA5), which are best known as RNA-sensing receptors in the cytosol. Thus, in some embodiments, the oligonucleotide contains a functional ligand for TLR3, TLR7 or RIG-I-like receptors, or a combination thereof.

[0115] Examples of immunostimulatory oligonucleotides and methods for making immunostimulatory oligonucleotides are known in the art, see, e.g., Bodera, P. Recent Pat Inflamm Allergy Drug Discov. 5(1):87-93 (2011), incorporated herein by reference.

[0116] In some embodiments, the oligonucleotide cargo comprises two or more immunostimulatory sequences. The oligonucleotide can be 2-100 nucleotide bases long or more, such as, for example, 5 nucleotide bases long, 10 nucleotide bases long, 15 nucleotide bases long, 20 nucleotide bases long, 25 nucleotide bases long, 30 nucleotide bases long, 35 nucleotide bases long, 40 nucleotide bases long, 45 nucleotide bases long, 50 nucleotide bases long, 60 nucleotide bases long, 70 nucleotide bases long, 80 nucleotide bases long, 90 nucleotide bases long, 95 nucleotide bases long, 98 nucleotide bases long, 100 nucleotide bases long.

[0117] The 3' or 5' end of the oligonucleotide may be conjugated to an albumin binding peptide (ABP). In a preferred embodiment, the 5' end of the oligonucleotide is linked to the ABP.

[0118] Oligonucleotides can be DNA or RNA nucleotides that typically contain a heterocyclic base (nucleobase), a sugar moiety attached to the heterocyclic base, and a phosphate moiety that esterifies the hydroxyl (hydroxy 1) functional group of the sugar moiety. The majority of naturally occurring nucleotides contain uracil, thymine, cytosine, adenine, and guanine as heterocyclic bases and ribose or deoxyribose sugars linked by phosphodiester bonds.

[0119] In some embodiments, oligonucleotides are composed of nucleotide analogs that are chemically modified to improve stability, half-life, or specificity or affinity for target receptors compared to their DNA or RNA counterparts. Chemical modifications include chemical modifications of nucleobases, sugar moieties, nucleotide bonds, or combinations thereof. As used herein, "modified nucleotide" or "chemically modified nucleotide" defines a nucleotide that has one or more chemical modifications of heterocyclic base, sugar moiety, or phosphate moiety components. In some embodiments, the charge of the modified nucleotide is reduced compared to the DNA or RNA oligonucleotide of the same nucleobase sequence. For example, the oligonucleotide can have a low negative charge, no charge, or a positive charge.

[0120] Typically, nucleoside analogs bear bases capable of hydrogen bonding by Watson-Crick base pairing to standard polynucleotide bases, and the analog backbone presents the bases in a manner that permits such hydrogen bonding in a sequence-specific manner between the oligonucleotide analog molecule and the bases in a standard polynucleotide (e.g., single-stranded RNA or single-stranded DNA). In some embodiments, the analogs have a phosphorus-containing backbone that is substantially uncharged.

[0121] i. Heterocyclic bases The major naturally occurring nucleotides include uracil, thymine, cytosine, adenine and guanine as heterocyclic bases. Oligonucleotides can include chemical modifications to their nucleobase components. Chemical modifications of heterocyclic bases or heterocyclic base analogs can be effective to increase binding affinity or stability in binding target sequences. Chemically modified heterocyclic bases include, but are not limited to, inosine, 5-(1-propynyl)uracil (pU), 5-(1-propynyl)cytosine (pC), 5-methylcytosine, 8-oxo-adenine, pseudocytosine, pseudoisocytosine, 5 and 2-ammo-5-(2'-deoxy-β-D-ribofuranosyl)pyridine (2-aminopyridine) and various pyrrolo- and pyrazolopyrimidine derivatives. Cyclic dinucleotides known to trigger cytosolic danger sensors such as STING could be used.

[0122] ii. Sugar modification Oligonucleotides can also contain nucleotides with modified sugar moieties or sugar moiety analogs. Sugar moiety modifications include, but are not limited to, 2'-O-aminoethoxy, 2'-O-aminoethyl (2'-OAE), 2'-O-methoxy, 2'-O-methyl, 2-guanidoethyl (2'-OGE), 2'-O,4'-C-methylene (LNA), 2'-O-(methoxyethyl) (2'-OME), and 2'-O-methyl)acetamido (2'-OMA). The 2'-O-aminoethyl sugar moiety substitution is particularly preferred because it is protonated at neutral pH, thus reducing charge repulsion between the TFO and the target duplex. This modification stabilizes the C3'-endo conformation of the ribose or dexyribose and also forms a bridge with the i-1 phosphate in the purine strand of the duplex.

[0123] In some embodiments, the oligonucleotide is a morpholino oligonucleotide. A morpholino oligonucleotide is typically composed of two more morpholino monomers containing a purine or pyrimidine base pairing moiety effective for binding to a base in a polynucleotide by base-specific hydrogen bonding, which are linked by a phosphorus-containing bond of 1-3 atoms long that connects the morpholino nitrogen of one monomer to the 5' exocyclic carbon of an adjacent monomer. The purine or pyrimidine base pairing moiety is typically adenine, cytosine, guanine, uracil or thymine. The synthesis, structure and binding properties of morpholino oligomers are described in detail in U.S. Patent Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,521,063 and 5,506,337.

[0124] Important properties of morpholino-based subunits typically include the ability to be linked in oligomeric form by stable uncharged backbone linkages; the ability to support nucleotide bases (e.g., adenine, cytosine, guanine, thymidine, uracil, or inosine) such that the formed polymers are capable of hybridizing to complementary base target nucleic acids (including target RNA) with high Tm, even with oligomers as short as 10-14 bases; the ability of the oligomers to be actively transported into mammalian cells; and the ability of oligomer:RNA heteroduplexes to resist RNAse degradation.

[0125] In some embodiments, oligonucleotides utilize morpholino-based subunits bearing base-pairing moieties linked by uncharged bonds, as described above.

[0126] iii. Internucleotide bond An oligonucleotide connected by an internucleotide bond, which refers to a chemical bond between two nucleoside moieties. Modifications to the phosphate backbone of DNA or RNA oligonucleotides can increase the binding affinity or stability of the oligonucleotide or reduce the susceptibility of the oligonucleotide to nuclease digestion. Cationic modifications, including but not limited to diethyl-ethylenediamide (DEED) or dimethyl-aminopropylamine (DMAP), can be particularly useful to reduce electrostatic repulsion between the oligonucleotide and the target.

[0127] Modification of phosphate backbone can also include replacing one of the non-bridging oxygens in phosphodiester bond with sulfur atom. This replacement creates phosphorothioate internucleoside bond instead of phosphodiester bond. The oligonucleotide containing phosphorothioate internucleoside bond has been shown to be more stable in vivo.

[0128] Examples of modified nucleotides with reduced charge include modified internucleotide linkages such as phosphate analogs with achiral, uncharged intersubunit linkages (e.g., Sterchak, EP et al., Organic Chem., 52:4202, (1987)) and morpholino-based uncharged polymers with achiral intersubunit linkages (see, e.g., U.S. Patent No. 5;034,S06), as discussed above. Some internucleotide linkage analogs include morpholidate, acetal, and polyamide-linked heterocycles.

[0129] In another embodiment, the oligonucleotide is composed of locked nucleic acid.Locked nucleic acid (LNA) is a modified RNA nucleotide (see, for example, Braasch et al., Chem. Biol., 8(1):1-7 (2001)).LNA forms hybrids with DNA that are more stable than DNA / DNA hybrids, with properties similar to those of peptide nucleic acid (PNA) / DNA hybrids.

[0130] Therefore, LNA can be used in the same way as PNA molecules are used. In some embodiments, LNA binding efficiency can be increased by adding positive charge to LNA. To make LNA, a commercially available nucleic acid synthesizer and standard phosphoramidite chemistry are used.

[0131] In some embodiments, the oligonucleotide is composed of peptide nucleic acid. Peptide nucleic acid (PNA) is a synthetic DNA mimic in which the entire phosphate backbone of the oligonucleotide is replaced by repeating N-(2-aminoethyl)-glycine units, and the phosphodiester bond is typically replaced by a peptide bond. Various heterocyclic bases are linked to the backbone by methylene carbonyl bonds. PNA resembles a normal DNA oligonucleotide, but maintains the spacing of the heterocyclic bases, which are achiral and neutrally charged molecules. Peptide nucleic acid is composed of peptide nucleic acid monomers.

[0132] Other backbone modifications include peptide and amino acid mutations and modifications. Thus, the backbone components of oligonucleotides such as PNAs can be peptide bonds or non-peptide peptide bonds. Examples include acetyl caps, amino spacers such as 8-amino-3,6-dioxaoctanoic acid (referred to herein as O-linker), and amino acids such as lysine are particularly useful when a positive charge is desired in PNAs, etc. Methods for chemical assembly of PNAs are well known. See, for example, U.S. Patent Nos. 5,539,082, 5,527,675, 5,623,049, 5,714,331, 5,736,336, 5,773,571 and 5,786,571.

[0133] Oligonucleotides may optionally include one or more terminal residues or modifications at one or both ends to increase stability and / or affinity of the oligonucleotide to its target. Commonly used positively charged moieties include the amino acids lysine and arginine, but other positively charged moieties may also be useful. Oligonucleotides may further be modified to be capped at the ends with propylamine groups to prevent degradation. Methods for 3' or 5' capping oligonucleotides are well known in the art.

[0134] In some embodiments, the oligonucleotide is single-stranded DNA, single-stranded RNA, or double-stranded RNA.

[0135] c. Other cargo In general, cargoes that can be conjugated to ABPs can include immunomodulatory, therapeutic, prophylactic or diagnostic agents. For example, chemotherapeutic drugs are of interest for targeting tumors, since albumin is known to accumulate in tumors due to the EPR effect and also due to rapid metabolism in tumors.

[0136] In some embodiments, the ABP conjugates disclosed herein include a detection label, e.g., a fluorophore, such as fluorescein or rhodamine, Alexa Fluor dyes, DyLight Fluor dyes, Quasar and Cal Fluor dyes, cyanine dyes (Cy3, Cy5, Cy5.5, Cy7) or other fluorescent dyes. The label can be the cargo or can be added to the cargo.

[0137] Linker Domain In some embodiments, the albumin binding peptide conjugate comprises a linker domain. In some embodiments, the albumin binding peptide conjugate comprises multiple linker domains. In some embodiments, the linker domain is a polypeptide linker, an ethylene glycol linker, or an oligonucleotide linker. In certain aspects, it is desirable to utilize a linker to fuse the albumin binding peptide or a variant or fragment thereof with one or more cargoes, such as an immunomodulatory molecule (e.g., an antigen, a peptide antigen, or a molecular adjuvant) to form the albumin binding peptide conjugate.

[0138] For example, the linker of the present invention can be utilized to ensure that the ABP is juxtaposed to ensure proper folding and formation of a functional immunomodulator or variant or fragment thereof. Preferably, a linker compatible with the present invention will be relatively non-immunogenic and will not inhibit any non-covalent association between the monomeric subunits of the binding protein. Exemplary linker domains are disclosed in U.S. Patent No. 6,660,843, which is incorporated herein by reference.

[0139] In some embodiments, the linker may be a non-cleavable linker or a cleavable linker. Non-cleavable linkers may include amide or phosphate bonds, and cleavable linkers may include disulfide bonds, acid cleavable bonds, ester bonds, anhydride bonds, biodegradable bonds, or enzyme cleavable bonds.

[0140] A. Polypeptide linker In certain embodiments, the albumin-binding peptide conjugates of the present invention utilize a polypeptide linker to link any two or more domains in-frame in a single polypeptide chain. In one embodiment, the two or more domains can be independently selected from any of the albumin-binding peptides or variants or fragments thereof or cargo molecules discussed herein. For example, in certain embodiments, a polypeptide linker can be used to fuse the albumin-binding peptide to an immunomodulatory molecule (e.g., a peptide antigen or a molecular adjuvant).

[0141] In some embodiments, the polypeptide linker is synthetic. As used herein, the term "synthetic" with respect to a polypeptide linker includes a peptide (or polypeptide) that includes an amino acid sequence (which may or may not be naturally occurring) that is linked by a linear sequence of amino acids to a sequence (e.g., an ABP sequence) that is not naturally linked thereto (which may or may not be naturally occurring). For example, the polypeptide linker may be a modified form (e.g., including mutations such as additions, substitutions, or deletions) of a naturally occurring polypeptide, or may include a non-naturally occurring polypeptide that includes a first amino acid sequence (which may or may not be naturally occurring).

[0142] In some embodiments, the polypeptide linker comprises or consists of a gly-ser linker. As used herein, the term "gly-ser linker" refers to a peptide consisting of glycine and serine residues. An exemplary gly / ser linker has the formula (Gly4Ser): nwherein n is a positive integer (e.g., 1, 2, 3, 4, or 5). In some embodiments, the gly / ser linker is (Gly4Ser)1. In some embodiments, the gly / ser linker is (Gly4Ser)2. In some embodiments, the gly / ser linker is (Gly4Ser)3. In some embodiments, the gly / ser linker is (Gly4Ser)4. In some embodiments, the gly / ser linker is (Gly4Ser)5. In some embodiments, the gly-ser linker may be inserted between two other sequences of a polypeptide linker (e.g., any of the polypeptide linker sequences described herein). In other embodiments, the gly-ser linker is attached to one or both ends of another sequence of a polypeptide linker (e.g., any of the polypeptide linker sequences described herein). In still other embodiments, two or more gly-ser linkers are incorporated consecutively into the polypeptide linker.

[0143] Other linkers suitable for use in albumin binding peptide conjugates are known in the art, such as the serine-rich linkers disclosed in U.S. Pat. No. 5,525,491, the helix-forming peptide linkers (e.g., A(EAAAK)nA (n=2-5)) disclosed in Arai et al., Protein Eng 2001;14:529-32, and the stable linkers disclosed in Chen et al., Mol Pharm 2011;8:457-65, namely, the dipeptide linker LE, the thrombin-sensitive disulfide cyclopeptide linker, and the alpha-helix-forming linker LEA(EAAAK)4ALEA(EAAAK)4ALE (SEQ ID NO: 38).

[0144] Other exemplary linkers include the GS linker (i.e., (GS)n), the GGSG (SEQ ID NO:39) linker (i.e., (GGSG)n), the GSAT (SEQ ID NO:40) linker, the SEG linker, and the GGS linker (i.e., (GGSGGS)n), where n is a positive integer (e.g., 1, 2, 3, 4, or 5). Other suitable linkers for use in albumin binding peptide conjugates can be found using publicly available databases such as the Linker Database (ibi.vu.nl / programs / linkerdbwww). The Linker Database is a database of inter-domain linkers in multi-functional enzymes that serve as potential linkers in novel fusion proteins (see, e.g., George et al., Protein Engineering 2002;15:871-9).

[0145] It will be understood that variant forms of these exemplary polypeptide linkers can be made by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence encoding the polypeptide linker, such that one or more amino acid substitutions, additions or deletions are introduced into the polypeptide linker. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.

[0146] The polypeptide linker of the present invention is at least one amino acid long and can be of various lengths. In one embodiment, the polypeptide linker of the present invention is about 1 to about 50 amino acids long. When used in this context, the term "about" refers to + / - 2 amino acid residues. The length of the linker must be a positive integer, so a length of about 1 to about 50 amino acids long means a length of 1 to 48 to 52 amino acids long. In another embodiment, the polypeptide linker of the present invention is about 1 to 5 amino acids long. In another embodiment, the polypeptide linker of the present invention is about 5 to 10 amino acids long. In another embodiment, the polypeptide linker of the present invention is about 10 to 20 amino acids long. In another embodiment, the polypeptide linker of the present invention is about 15 to about 50 amino acids long.

[0147] In another embodiment, a polypeptide linker of the invention is about 20 to about 45 amino acids in length. In another embodiment, a polypeptide linker of the invention is about 15 to about 25 amino acids in length. In another embodiment, a polypeptide linker of the invention is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or 61 or more amino acids in length.

[0148] Polypeptide linkers can be introduced into the polypeptide sequence using techniques known in the art. Modifications can be confirmed by DNA sequence analysis. Plasmid DNA can be used to transform host cells for stable production of the produced polypeptide.

[0149] B. Ethylene glycol linker In some embodiments, the linker domain is one or more ethylene glycol (EG) units, more preferably two or more EG units (i.e., polyethylene glycol (PEG)). In some embodiments, the linker domain comprises or consists of a polyethylene glycol (PEG) linker. Polyethylene glycol or PEG refers to a chemical compound composed of repeating ethylene glycol units. Exemplary "PEG linkers" include compounds of the formula: H-(O-CH2-CH2)n-OH, where n is a positive integer (e.g., 1, 10, 20, 50, 100, 200, 300, 400, 500, 600). In some embodiments, the PEG linker is PEG1000. In some embodiments, the PEG linker is PEG2000. In some embodiments, the PEG linker is PEG3000.

[0150] In some embodiments, the albumin-binding peptide conjugate comprises an immunomodulatory molecule (eg, a peptide antigen) linked to a polyethylene glycol (PEG) molecule, which is in turn linked to the albumin-binding peptide.

[0151] The exact number of ethylene glycol (EG) units will depend on the albumin binding peptide and cargo, but typically the ethylene glycol linker can have about 1 to about 100, about 20 to about 80, about 30 to about 70, or about 40 to about 60 EG units. In some embodiments, the ethylene glycol linker has about 45 to 55 EG units. For example, in one embodiment, the ethylene glycol linker has 45 EG units. For example, in one embodiment, the ethylene glycol linker has 48 EG units.

[0152] c. Oligonucleotide linker In some embodiments, the linker is an oligonucleotide. The linker can have any sequence, for example, the sequence of the oligonucleotide can be a random sequence or a sequence specifically selected for its molecular or biochemical properties. In some embodiments, the linker comprises one or more series of consecutive adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), or analogs thereof. In some embodiments, the linker consists of a series of consecutive adenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), or analogs thereof.

[0153] In one embodiment, the linker is one or more guanines, for example, 1 to 10 guanines. In some embodiments, the linker in the ABP conjugate can contain 0, 1 or 2 guanines.

[0154] immunogenic composition In some embodiments, the albumin-binding peptide conjugates of the present disclosure may be used as components in immunogenic compositions or in vaccines. In some embodiments, the immunogenic compositions disclosed herein comprise an albumin-binding peptide conjugate. In some embodiments, the immunogenic compositions disclosed herein comprise a combination of an albumin-binding peptide conjugate and an adjuvant. The combination of an adjuvant and an albumin-binding peptide conjugate may be referred to as a vaccine. When administered in combination to a subject, the adjuvant and the albumin-binding peptide conjugate may be administered in separate pharmaceutical compositions or may be administered together in the same pharmaceutical composition.

[0155] The immunogenic compositions can include albumin-binding peptide conjugates, such as albumin-binding peptides linked to immunomodulatory molecules (eg, peptide antigens or molecular adjuvants) administered alone or in combination with an adjuvant.

[0156] Adjuvants include alum (e.g., aluminum hydroxide, aluminum phosphate); saponins purified from the bark of the Q. saponaria tree, such as QS21 (a glycolipid that elutes in the 21st peak in HPLC fractionation; Antigenics, Inc., Worcester, Mass.); poly[di(carboxylate phenoxy)phosphazene (PCPP polymer; Virus Research Institute, USA), Flt3 ligand, Leishmania elongation factor (a purified Leishmania protein; Corixa Corporation, Seattle, Wash.), ISCOMS (mixed saponin, lipid-containing immunostimulatory complexes that form virus-sized particles with pores capable of retaining antigens; CSL, Melbourne, Australia), Pam3Cys, SB-AS4 (a SmithKline antibody that contains alum and MPL), and SB-AS5 (a serotonin-containing peptide that is a serotonin ... Adjuvant systems may include, but are not limited to, Beecham adjuvant system #4; SBB, Belgium), micelle-forming non-ionic block copolymers such as CRL1005, which contain linear chains of hydrophobic polyoxypropylene flanked by chains of polyoxyethylene (Vaxcel, Inc., Norcross, Ga) and Montanide IMS (e.g., IMS1312, aqueous nanoparticles combined with a soluble immunostimulant (Seppic)).

[0157] The adjuvant may be a TLR ligand, such as those discussed herein. Adjuvants that act through TLR3 include, but are not limited to, double-stranded RNA. Adjuvants that act through TLR4 include monophosphoryl lipid A (MPL). Adjuvants that act through TLR5 include, but are not limited to, lipopolysaccharide derivatives such as OM-174 (glucosamine disaccharide associated with lipid A; OM Pharma SA, Meyrin, Switzerland). Adjuvants that act through TLR7 and / or TLR8 include synthetic low molecular weight compounds such as single-stranded RNA, oligoribonucleotides (ORN), imidazoquinoline amines (e.g., imiquimod (R-837), resiquimod (R-848)). Adjuvants that act through TLR9 include synthetic oligodeoxynucleotides (ODN), such as DNA of viral or bacterial origin or CpG ODN. Another class of adjuvants are phosphorothioate-containing molecules, such as phosphorothioate nucleotide analogues and nucleic acids containing phosphorothioate backbone linkages.

[0158] Adjuvants can also be oil emulsions (e.g., Freund's adjuvant); saponin preparations; virosomes and virus-like particles; bacterial and microbial derivatives; immunostimulatory oligonucleotides; ADP-ribosylating toxins and detoxified derivatives; alum; BCG; mineral-containing compositions (e.g., mineral salts such as aluminum and calcium salts, hydroxides, phosphates, sulfates, etc.); bioadhesives and / or mucoadhesives; microparticles; liposomes; polyoxyethylene ether and polyoxyethylene ester preparations; polyphosphazenes; muramyl peptides; imidazoquinolone compounds; and surfactants (e.g., lysolecithin, Pluronic® polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol).

[0159] Adjuvants can also include immune modulators such as cytokines, interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g., interferon gamma), macrophage colony-stimulating factor, and tumor necrosis factor.

[0160] Pharmaceutical Compositions and Modes of Administration In certain embodiments, the albumin-binding peptide conjugate is administered to a subject in need thereof.

[0161] In some embodiments, the present disclosure provides a pharmaceutical composition comprising an albumin-binding peptide conjugate. In certain embodiments, the present disclosure provides a pharmaceutical composition comprising an albumin-binding peptide conjugate and a pharma- ceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative, and / or adjuvant.

[0162] The pharmaceutical compositions can be for administration by parenteral (intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection), transdermal (passively or using iontophoresis or electroporation) or transmucosal (nasal, vaginal, rectal or sublingual) routes of administration, or using a bioerodible insert, and can be formulated in a dosage form suitable for each route of administration.

[0163] In some embodiments, the compositions are administered systemically, for example, by intravenous or intraperitoneal administration, in an amount effective for delivery of the composition to targeted cells. Other possible routes include transdermal or oral.

[0164] In certain embodiments, the composition is administered locally, e.g., by injection directly into the site to be treated. In some embodiments, the composition is injected or otherwise administered directly into one or more tumors. Typically, local injection results in an increased local concentration of the composition that is higher than can be achieved by systemic administration. In some embodiments, the composition is delivered locally to the appropriate cells by using a catheter or syringe. Other means of locally delivering such compositions to cells include using an infusion pump (e.g., available from Alza Corporation, Palo Alto, Calif.) or incorporating the composition into a polymeric implant that can effect sustained release of nanolipogels to the area adjacent to the implant (see, e.g., P. Johnson and JG Lloyd-Jones, eds., Drug Delivery Systems (Chichester, England: Ellis Horwood Ltd., 1987)).

[0165] In some embodiments, acceptable formulation materials are preferably non-toxic to recipients at the dosages and concentrations employed. In some embodiments, the formulation materials are for subcutaneous and / or intravenous administration. In some embodiments, pharmaceutical compositions can contain formulation materials to modify, maintain or preserve, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition.In certain embodiments, suitable formulation materials include amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrate, phosphate, or other organic acids); bulking agents. agents) (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, β-cyclodextrin or hydroxypropyl-β-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrin); proteins (such as serum albumin, gelatin or immunoglobulins); colours, flavours and diluents; emulsifiers, hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexyl Examples of suitable pharmaceutical agents include, but are not limited to, glycerin, sorbic acid, or hydrogen peroxide; solvents (such as glycerin, propylene glycol, or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as Pluronic®, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal, and the like); stability enhancers (such as sucrose or sorbitol); tonicity enhancers (such as alkyl metal halides, preferably sodium or potassium chloride, mannitol sorbitol, and the like); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants. (Remington's Pharmaceutical Sciences, 18th Edition, A.R. Gennaro, ed., Mack Publishing Company (1995). In one embodiment, the formulation is PBS; 20 mM NaOAc, pH 5.2, 50 mM. NaCl; and / or 10 mM NAOAC, pH 5.2, 9% sucrose. In certain embodiments, optimal pharmaceutical compositions will be determined by those skilled in the art depending, for example, on the intended route of administration, delivery format, and desired dosage. See, for example, Remington's Pharmaceutical Sciences, supra. In certain embodiments, such compositions can affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the albumin-binding peptide conjugate.

[0166] In certain embodiments, the primary vehicle or carrier in a pharmaceutical composition can be aqueous or non-aqueous in nature. For example, in certain embodiments, a suitable vehicle or carrier can be water for injection, physiological saline solution, or artificial cerebrospinal fluid, which can be optionally supplemented with other materials common in compositions for parenteral administration. In certain embodiments, the saline solution comprises isotonic phosphate buffered saline. In certain embodiments, neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. In certain embodiments, the pharmaceutical composition comprises a Tris buffer of about pH 7.0-8.5 or an acetate buffer of about pH 4.0-5.5, and can further comprise sorbitol or a suitable substitute for sorbitol. In certain embodiments, compositions comprising albumin-binding peptide conjugates can be prepared for storage by mixing a selected composition having the desired purity with optionally added formulation agents (Remington's Pharmaceutical Sciences, supra) in the form of a lyophilized cake or aqueous solution. Furthermore, in certain embodiments, compositions comprising albumin-binding peptide conjugates can be formulated as lyophilizates using suitable excipients such as sucrose.

[0167] In some embodiments, the pharmaceutical compositions can be selected for parenteral delivery. In some embodiments, the compositions can be selected for inhalation or for delivery through the digestive tract, such as orally. The preparation of such pharma- ceutically acceptable compositions is within the capabilities of those skilled in the art.

[0168] In certain embodiments, the formulation components are present in concentrations that are acceptable to the site of administration. In certain embodiments, a buffer is used to maintain the composition at physiological pH or slightly lower, typically within a pH range of about 5 to about 8.

[0169] In some embodiments, the albumin-binding peptide conjugates of the present disclosure are administered in an aqueous solution by parenteral injection, hi some embodiments, the composition comprises albumin or other serum proteins.

[0170] In some embodiments, the formulation may be in the form of a suspension or emulsion. In general, a pharmaceutical composition is provided that contains an effective amount of an albumin-binding peptide conjugate, which may optionally contain pharma- ceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants, and / or carriers. Such compositions may include diluents such as sterile water, various buffer contents (e.g., Tris-HCl, acetate, phosphate), buffered saline solutions of various pH and ionic strengths, and may optionally contain additives such as surfactants and solubilizers (e.g., TWEEN® 20, TWEEN® 80, also known as polysorbate 20 or 80), antioxidants (e.g., ascorbic acid, sodium metabisulfite), as well as preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol). Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate.The formulation can be lyophilized and redissolved / resuspended immediately before use.The formulation can be sterilized, for example, by filtration through a bacteria-retaining filter, by incorporating a sterilizing agent into the composition, by irradiating the composition, or by heating the composition.

[0171] In some embodiments, when parenteral administration is envisaged, the therapeutic composition can be in the form of a parenterally acceptable pyrogen-free aqueous solution containing the albumin-binding peptide conjugate in a pharma- ceutically acceptable vehicle. In some embodiments, the parenteral injection vehicle is sterile distilled water, and the albumin-binding peptide conjugate is formulated as a sterile isotonic solution that is appropriately preserved. In some embodiments, the preparation can include formulating the desired molecule with agents such as injectable microspheres, bioerodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads or liposomes that can provide controlled or sustained release of the product and then deliver it via depot injection. In some embodiments, hyaluronic acid can also be used, which can have the effect of promoting a sustained period in the circulation. In some embodiments, an implantable drug delivery device can be used to introduce the desired molecule.

[0172] In some embodiments, the albumin-binding peptide conjugates of the present disclosure can be applied locally. Topical administration can include application to the pulmonary (pulmonary), nasal, oral (sublingual, buccal), vaginal, or rectal mucosa. In some cases, the conjugates can be transcytosed on albumin across the mucosal barrier.

[0173] In some embodiments, the pharmaceutical composition can be formulated for inhalation. In some embodiments, the albumin-binding peptide conjugate can be formulated as a dry powder for inhalation. In some embodiments, an inhalation solution containing the albumin-binding peptide conjugate can be formulated with a propellant for aerosol delivery. In some embodiments, the solution can be nebulized. Pulmonary administration is further described in PCT Application No. PCT / US94 / 001875, which describes pulmonary delivery of chemically modified proteins.

[0174] In certain embodiments, when delivered as either an aerosol or spray-dried particles having an aerodynamic diameter of less than about 5 microns, the composition is delivered to the lungs while being inhaled and can traverse the lung epithelial lining to the bloodstream.

[0175] A wide variety of mechanical devices designed for pulmonary delivery of therapeutic products can be used, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are well known to those skilled in the art. Some specific examples of commercially available devices are the Ultravent® nebulizer (Mallinckrodt Inc., St. Louis, Mo.); Acorn® II nebulizer (Marquest Medical Products, Englewood, Colo.); Ventolin® metered dose inhaler (Glaxo Inc., Research Triangle Park, NC); and Spinhaler® powder inhaler (Fisons Corp., Bedford, Mass.). Nektar, Alkermes, and Mannkind all have inhalable insulin powder preparations approved or in clinical trials whose technology can be applied to the formulations described herein.

[0176] In some embodiments, it is envisioned that the formulation can be administered orally. In some embodiments, the albumin-binding peptide conjugate administered in this manner can be formulated with or without carriers commonly used in the formulation of solid dosage forms such as tablets and capsules. In some embodiments, the capsule can be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized. In some embodiments, at least one additional agent can be included to facilitate absorption of the albumin-binding peptide conjugate. In some embodiments, diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents and binders can also be utilized.

[0177] In some embodiments, formulations for administration to mucosa will typically be spray-dried drug particles that may be incorporated into tablets, gels, capsules, suspensions or emulsions. Standard pharmaceutical excipients are available from any formulator. Oral formulations may be in the form of chewing gum, gel strips, tablets, capsules or lozenges.

[0178] In some embodiments, transdermal formulations may also be prepared. These are typically ointments, lotions, sprays or patches, all of which may be prepared using standard techniques. Transdermal formulations may include penetration enhancers.

[0179] In some embodiments, the pharmaceutical composition may contain an effective amount of the albumin-binding peptide conjugate in a mixture with non-toxic excipients suitable for tablet manufacture. In some embodiments, the solution may be prepared in unit dose form by dissolving the tablet in sterile water or another suitable vehicle. In some embodiments, suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate or bicarbonate, lactose or calcium phosphate; or binders such as starch, gelatin or gum arabic; or lubricants such as magnesium stearate, stearic acid or talc.

[0180] Further pharmaceutical compositions will be apparent to those skilled in the art, such as formulations comprising albumin-binding peptide conjugates in sustained or controlled delivery formulations. In some embodiments, techniques for formulating various other sustained or controlled delivery means, such as liposome carriers, bioerodible microparticles or porous beads, and depot injections, are also known to those skilled in the art. See, for example, PCT Application No. PCT / US93 / 00829, which describes controlled release of porous polymeric microparticles for delivery of pharmaceutical compositions. In some embodiments, sustained release preparations can include semipermeable polymer matrices in the form of shaped articles, such as films or microcapsules. Sustained release matrices can include polyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919 and European Patent No. 058,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et al., Biopolymers, 22:547-556 (1983)), poly(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater. Res., 15:167-277 (1981) and Langer, Chem. Tech., 12:98-105 (1982)), ethylene vinyl acetate (Langer et al., supra), or poly-D(-)-3-hydroxybutyrate (European Patent No. 133,988). In certain embodiments, sustained release compositions can also include liposomes, which can be prepared by any of several methods known in the art. See, e.g., Eppstein et al., Proc. Natl. Acad. Sci. USA, 82:3688-3692 (1985); EP 036,676; EP 088,046 and EP 143,949.

[0181] The pharmaceutical compositions used for in vivo administration are typically sterile. In some embodiments, this can be achieved by filtration through a sterile filtration membrane. In some embodiments, when the composition is lyophilized, sterilization can be carried out using this method either before or after lyophilization and reconstitution. In some embodiments, the composition for parenteral administration can be stored in lyophilized form or in a solution. In some embodiments, parenteral compositions are generally placed in a container with a sterile access port, for example, an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic injection needle.

[0182] In certain embodiments, once the pharmaceutical compositions have been formulated, they may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. In certain embodiments, such formulations may be stored either in a ready-to-use form or in a form that is reconstituted prior to administration (e.g., lyophilized).

[0183] In some embodiments, kits for making single dose administration units are provided. In some embodiments, the kits can contain both a first container with a dried compound and a second container with an aqueous formulation. In some embodiments, kits containing single and multi-chamber pre-filled syringes (e.g., liquid syringes and lyosyringes) are included.

[0184] In certain embodiments, the effective amount of a pharmaceutical composition comprising an albumin-binding peptide conjugate to be therapeutically utilized will depend, for example, on the context and purpose of the treatment. Those skilled in the art will appreciate that, according to certain embodiments, appropriate dosage levels for treatment may therefore vary in part depending on the molecule being delivered, the indication for which the albumin-binding peptide conjugate is being used, the route of administration, and the size (weight, body surface area, or organ size) and / or condition (age and general health) of the patient. In certain embodiments, the clinician can titrate the dosage and modify the route of administration to obtain optimal therapeutic effect. In certain embodiments, typical dosages for albumin-binding peptide conjugates can range from about 0.1 μg / kg up to about 10 mg / kg or more, depending on the factors mentioned above. In certain embodiments, dosages can range from 0.1 μg / kg up to about 10 mg / kg, or from 0.1 μg / kg up to about 1 mg / kg, or from 0.1 μg / kg up to about 0.1 mg / kg, or from 0.1 μg / kg up to about 0.05 mg / kg; or from 0.1 μg / kg up to about 0.01 mg / kg.

[0185] In some embodiments, the frequency of dosing will take into account the pharmacokinetic parameters of the albumin-binding peptide conjugate in the formulation used. In some embodiments, the clinician will administer the composition until a dosage is reached that achieves the desired effect. In some embodiments, the composition can therefore be administered as a single dose, or as two or more doses over time (which may or may not contain the same amount of the desired molecule), or as a continuous infusion via an implanted device or catheter. Further refinement of the appropriate dosage is routinely performed by those skilled in the art and falls within the scope of routine work performed by those skilled in the art. In some embodiments, the appropriate dosage can be ascertained through the use of appropriate dose-response data.

[0186] In some embodiments, the route of administration of the pharmaceutical composition is according to known methods, for example, via injection orally, intravenously, intraperitoneally, intracerebrally (intracemally), intraventricularly, intramuscularly, subcutaneously, intraocularly, intraarterially, intraportally or intralesionally; by sustained release system or by implanted device. In some embodiments, the composition can be administered by bolus injection or continuously by infusion, or by implanted device. In some embodiments, the individual components of the combination therapy can be administered by different routes.

[0187] In certain embodiments, the composition can be administered locally via implantation of a membrane, sponge or another suitable material onto which the desired molecule is absorbed or encapsulated. In certain embodiments, when an implantation device is used, the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be via diffusion, timed bolus or continuous administration. In certain embodiments, it may be desirable to use pharmaceutical compositions comprising albumin-binding peptide conjugates in an ex vivo manner. In such cases, cells, tissues and / or organs removed from a patient are exposed to pharmaceutical compositions comprising albumin-binding peptide conjugates, followed by subsequent implantation of the cells, tissues and / or organs back into the patient.

[0188] In some embodiments, the albumin-binding peptide conjugates can be delivered by implanting certain cells that have been genetically engineered, using methods such as those described herein, to express and secrete the polypeptide. In some embodiments, such cells can be animal or human cells and can be autologous, heterologous or xenogeneic. In some embodiments, the cells can be immortalized. In some embodiments, to reduce the probability of an immunological response, the cells can be encapsulated to avoid infiltration of surrounding tissues. In some embodiments, the encapsulation material is typically a biocompatible semipermeable polymeric enclosure or membrane that allows release of the albumin-binding peptide conjugates but prevents destruction of the cells by the patient's immune system or other harmful factors from the surrounding tissues.

[0189] Methods for producing albumin-binding peptide conjugates In some embodiments, the albumin-binding peptide conjugates described herein can be produced in transformed host cells using recombinant DNA technology.To do so, recombinant DNA molecules encoding albumin-binding peptide conjugates are prepared.Methods for preparing such DNA molecules are well known in the art.For example, the sequence encoding albumin-binding peptide conjugates could be excised from DNA using appropriate restriction enzymes.Alternatively, DNA molecules could be synthesized using chemical synthesis techniques, such as phosphoramidate method.A combination of these techniques could also be used.

[0190] The method for producing an albumin-binding peptide conjugate also includes a vector capable of expressing the albumin-binding peptide conjugate in a suitable host. The vector includes a DNA molecule encoding the albumin-binding peptide conjugate operably linked to a suitable expression control sequence. Methods for affecting this operably linkage, either before or after the DNA molecule is inserted into the vector, are well known. Expression control sequences include promoters, activators, enhancers, operators, ribosomal nuclease domains, start signals, stop signals, cap signals, polyadenylation signals and other signals involved in regulating transcription or translation.

[0191] The resulting vector, having the DNA molecule thereon, is used to transform an appropriate host, which can be carried out using methods well known in the art.

[0192] Any of a number of available and well-known host cells may be suitable for use in the methods disclosed herein. The selection of a particular host depends on a number of factors recognized by the art. These include, for example, compatibility with the selected expression vector, toxicity of the peptide encoded by the DNA molecule, rate of transformation, ease of recovery of the peptide, expression characteristics, biosafety and cost. These factors must be balanced, with the understanding that not all hosts may be equally effective in expressing a particular DNA sequence. Within these general guidelines, useful microbial hosts include bacteria (such as Escherichia coli species), yeast (such as Saccharomyces species) and other fungi, insects, plants, mammalian (including human) cells in culture or other hosts known in the art.

[0193] The transformed host is then cultured and purified. The host cells can be cultured under conventional fermentation conditions so that the desired compound is expressed. Such fermentation conditions are well known in the art. Finally, the peptide is purified from the culture by methods well known in the art.

[0194] Compounds can also be made by synthetic methods. For example, solid phase synthesis techniques can be used. Suitable techniques are well known in the art and include those described in Merrifield (1973), Chem. Polypeptides, pp. 335-61 (Katsoyannis and Panayotis eds.); Merrifield (1963), J. Am. Chem. Soc. 85: 2149; Davis et al. (1985), Biochem. Intl. 10: 394-414; Stewart and Young (1969), Solid Phase Peptide Synthesis; U.S. Patent No. 3,941,763; Finn et al. (1976), The Proteins (3rd ed.) 2: 105-253; and Erickson et al. (1976), The Proteins (3rd ed.) 2: 257-527. In some embodiments, solid phase synthesis is the technique for making albumin-binding peptide conjugates. Compounds containing derivatized peptides or containing non-peptide groups can be synthesized by well-known organic chemistry techniques.

[0195] In some embodiments, the albumin-binding peptide conjugates may be produced via solid phase peptide synthesis. In some embodiments, an automated synthesizer or manual solid phase peptide synthesis techniques are used to synthesize the ABP conjugates. In certain embodiments, solution peptide synthesis may be used to synthesize the ABP conjugates.

[0196] In some embodiments, the domains of the albumin binding peptide conjugates are synthesized as separate units or domains. In some embodiments, the domains of the albumin binding peptide conjugates are synthesized as a single unit.

[0197] For example, in some embodiments, an albumin-binding peptide conjugate comprising an albumin-binding peptide linked to a molecular adjuvant, with or without a linker domain, can be synthesized in two separate units using automated solid-phase peptide synthesis for the ABP and DNA synthesis for the molecular adjuvant (e.g., CpG antigen). The two components can then be linked in solution via Cu(I)-catalyzed click chemistry. Other methods commonly known in the art for solid-phase peptide synthesis techniques can be utilized to synthesize the ABP components. Other synthetic techniques commonly known in the art could be used to synthesize the adjuvant molecules (e.g., variations of DNA synthesis commonly known in the art for CpG domains and other DNA adjuvants, which vary with the nature of the molecular antigen). Bioconjugation strategies commonly known in the art (e.g., (amide / NHS ester, sulfhydryl / maleimide, azide / DBCO, and others) could be used to link the two components of the ABP conjugate.

[0198] In some embodiments, an albumin-binding peptide conjugate comprising an albumin-binding peptide linked to an antigen (eg, a peptide antigen), with or without a linker domain, may be synthesized as a single unit.

[0199] Other methods of molecular expression / synthesis are generally known to those of skill in the art.

[0200] Expression of Polypeptides The nucleic acid molecules can be contained, for example, within a vector capable of directing expression of the nucleic acid molecule in cells transduced with the vector.Thus, expression vectors containing nucleic acid molecules encoding albumin binding peptide conjugates and cells transfected with these vectors are among the embodiments.

[0201] Suitable vectors for use include T7-based vectors for use in bacteria (see, e.g., Rosenberg et al., Gene 56:125, 1987), pMSXND expression vector for use in mammalian cells (Lee and Nathans, J. Biol. Chem. 263:3521, 1988), and baculovirus-derived vectors for use in insect cells (e.g., expression vector pBacPAKS from Clontech, Palo Alto, Calif.). The nucleic acid insert encoding the polypeptide of interest in such vectors can be operably linked to a promoter selected, for example, based on the type of cell in which expression is desired. For example, the T7 promoter can be used in bacteria, the polyhedrin promoter can be used in insect cells, and the cytomegalovirus or metallothionein promoters can be used in mammals. Also, in the case of higher eukaryotes, tissue-specific and cell type-specific promoters are widely available. These promoters are so named because of their ability to direct the expression of nucleic acid molecules in a given tissue or cell type in the body. Those of skill in the art are well aware of the numerous promoters and other control elements that can be used to direct expression of a nucleic acid.

[0202] In addition to sequences that facilitate transcription of the inserted nucleic acid molecule, vectors can contain other genes that encode replication origins and selectable markers. For example, neomycin resistance (neomycin resistance) and / or cytoplasmic reductase (cytoplasmic reductase) are also contemplated. r The ) gene confers G418 resistance to cells in which it is expressed, thus allowing phenotypic selection of transfected cells. One of skill in the art can readily determine whether a given control element or selectable marker is appropriate for use in a particular experimental context.

[0203] Viral vectors suitable for use include, for example, retroviral vectors, adenoviral vectors and adeno-associated vectors, herpes virus, simian virus 40 (SV40), and bovine papilloma virus vectors (see, e.g., Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, NY).

[0204] Prokaryotic or eukaryotic cells that contain and express a nucleic acid molecule encoding an albumin-binding peptide conjugate are also suitable for use. The cell is a transfected cell, i.e., a cell into which a nucleic acid molecule, e.g., a nucleic acid molecule encoding an albumin-binding peptide conjugate, has been introduced by recombinant DNA techniques. The progeny of such cells are also considered suitable for use in the methods disclosed herein.

[0205] The exact components of the expression system are not critical. For example, albumin binding peptide conjugates can be produced in prokaryotic hosts such as the bacterium Escherichia coli, or in eukaryotic hosts such as insect cells (e.g., Sf21 cells) or mammalian cells (e.g., COS cells, NIH3T3 cells, or Hela cells). These cells are available from many sources, including the American Type Culture Collection (Manassas, Va.). When selecting an expression system, it is only important that the components are compatible with each other. Those skilled in the art are able to make such a decision. Furthermore, those skilled in the art may consult Ausubel et al. (Current Protocols in Molecular Biology, John Wiley and Sons, New York, NY, 1993) and Pouwels et al. (Cloning Vectors: A Laboratory Manual, 1985 Suppl. 1987) if guidance is needed in selecting an expression system.

[0206] The expressed albumin binding peptide conjugates can be purified from the expression system using conventional biochemical techniques and can be used as therapeutic agents, for example, as described herein.

[0207] How to use a. Methods for delivering albumin-binding peptide conjugates i. Lymph node targeting In some embodiments, the present disclosure provides that conjugation of cargo, such as immune-modulating molecules (e.g., antigenic peptides), to albumin-binding peptides (ABPs) can increase delivery and accumulation of the cargo to lymph nodes. Lymph nodes are oval-shaped organs of the immune system that are widely distributed throughout the body, including the axilla and stomach, and are connected by lymphatic vessels. Lymph nodes are home to B, T and other immune cells. Lymph nodes act as filters or traps for foreign particles and are important in the proper functioning of the immune system. Lymph nodes are densely packed with lymphocytes and white blood cells called macrophages.

[0208] Lymph node-targeted conjugates can be transported from the injection site to secondary organs of the lymphatic system (e.g., lymph nodes) where they interact with immune cells. Albumin binding of ABP conjugates prevents them from quickly flowing into the bloodstream, redirecting them to lymphatic vessels and draining lymph nodes, where they are believed to be filtered, accumulate, and present their cargo (e.g., peptide antigens or immunomodulatory molecules such as molecular adjuvants) to immune cells.

[0209] As discussed above, the albumin-binding peptides can be conjugated, for example, to an immunomodulatory molecule (e.g., a peptide antigen or a molecular adjuvant), which increases the immunostimulatory effect of the immunomodulatory molecule (e.g., a peptide antigen or a molecular adjuvant) compared to administering the unconjugated immunomodulatory molecule (e.g., a peptide antigen or a molecular adjuvant). In some embodiments, conjugation of a cargo (e.g., an immunomodulatory molecule) to an albumin-binding peptide increases accumulation of the cargo by 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more compared to the unconjugated cargo.

[0210] ii. Tissue-specific targeting Albumin-binding peptide conjugates can be used to increase delivery and accumulation of cargo (e.g., antigenic peptides, molecular adjuvants) to tissues at or near the site of administration. In some embodiments, the albumin-binding peptide conjugates can accumulate at the site of injection. In some embodiments, the albumin-binding peptide conjugates can be targeted, for example, to mucosal tissues.

[0211] b. Methods for increasing immune responses Albumin-bound peptide conjugates containing immunomodulatory molecules (e.g., peptide antigens or molecular adjuvants) can be administered in an amount effective to induce, increase, or enhance an immune response. An "immune response" refers to a response that induces, increases, induces, or perpetuates the activation or efficiency of innate or adaptive immunity. Additionally, albumin-bound peptide conjugates administered in the absence of other adjuvants can be used to promote tolerance rather than immunity to, for example, allergens or autoimmune antigens.

[0212] ABP conjugates can be delivered parenterally (by subcutaneous, intradermal or intramuscular injection) through lymphatics, or by systemic administration through the circulatory system. Lymph nodes can filter the conjugates bound to albumin. Thus, in some embodiments, parenteral administration does not result in systemic distribution, since ABP conjugates can be preferentially filtered by the nearest lymph node. This tendency also reduces systemic toxicity, such as splenic swelling.

[0213] Thus, in some embodiments, the ABP conjugate is administered to a site adjacent to or leading to one or more lymph nodes that are near the site in need of an immune response (i.e., near the site of a tumor or infection). In some embodiments, the ABP conjugate is administered in multiple doses at various locations throughout the body. In some embodiments, the ABP conjugate can also be administered directly to the site in need of an immune response (e.g., the site of a tumor or infection).

[0214] An immune response may be induced, increased or enhanced by the ABP conjugate compared to a control, e.g., an immune response in a subject is induced, increased or enhanced by the cargo alone or by the cargo delivered using another delivery strategy such as liposomes. In some embodiments, the ABP conjugate reduces inactivation of T cells and / or prolongs activation of T cells (i.e., increases antigen-specific proliferation of T cells, enhances cytokine production by T cells, stimulates differentiation and effector function of T cells, and / or promotes survival of T cells) or overcomes T cell exhaustion and / or anergy.

[0215] ABP conjugates can be used to induce an immune response, for example, when administering cargo alone or cargo in combination with another delivery system is ineffective. ABP conjugates can also be used to enhance or improve immune responses compared to administering cargo alone. In some embodiments, ABP conjugates can reduce the dosage required to induce, increase, or enhance an immune response, or shorten the time required for the immune system to respond after administration.

[0216] The ABP conjugates can be administered as part of a prophylactic vaccine or immunogenic composition that confers resistance in a subject to subsequent exposure to an infectious agent, or as part of a therapeutic vaccine that can be used to initiate or enhance a subject's immune response to a pre-existing antigen, such as a viral antigen, in a subject infected with a virus or with cancer.

[0217] The desired outcome of a prophylactic or therapeutic immune response may vary according to the disease or condition being treated or according to principles well known in the art. For example, an immune response to an infectious agent may completely prevent the establishment and replication of the infectious agent, affecting "sterile immunity" and the absence of any disease symptoms. However, a vaccine against an infectious agent may be considered effective if it reduces the number, severity, or duration of symptoms, reduces the number of symptomatic individuals in a population, or reduces the transmission of the infectious agent.

[0218] Similarly, an immune response to cancer, an allergen, or an infectious agent may treat the disease entirely, may alleviate symptoms, or may be one aspect in an overall therapeutic intervention against the disease.

[0219] The ABP conjugate may induce an improved effector cell response, such as a CD4 T cell immune response, against at least one of the component antigens or antigenic compositions, compared to the effector cell response obtained with the corresponding composition without the ABP conjugate. The term "improved effector cell response" refers to an effector cell response, such as a CD8 or CD4 response, obtained in a human patient after administration of the ABP conjugate that is higher than that obtained after administration of the same cargo without the ABP conjugate.

[0220] Improved effector cell responses can be obtained in patients who have not been immunologically stimulated, i.e., patients who are seronegative to the antigen. This seronegativity can be the result of the patient never having been confronted with the antigen (so-called "naive" patients) or of failure to respond to the antigen once encountered. In some embodiments, improved effector cell responses are obtained in immunocompromised subjects.

[0221] Improved effector cell responses can be assessed by measuring the number of cells producing any of the following cytokines: (1) cells producing at least two different cytokines (CD40L, IL-2, IFN-γ, TNF-α); (2) cells producing at least CD40L and another cytokine (IL-2, TNF-α, IFN-γ); (3) cells producing at least IL-2 and another cytokine (CD40L, TNF-α, IFN-γ); (4) cells producing at least IFN-γ and another cytokine (IL-2, TNF-α, CD40L); (5) and cells producing at least TNF-α and another cytokine (IL-2, CD40L, IFN-γ). Improved effector cell responses are present when the number of cells producing any of the above cytokines is higher after administration of the ABP conjugate composition compared to the control discussed above.

[0222] In some embodiments, the ABP conjugates increase the number of T cells that produce IFN-γ, TNF-α, or a combination thereof, or increase the production of IFN-γ, TNF-α, or a combination thereof in existing T cells.

[0223] In some embodiments, administration of the immunogenic composition alternatively or additionally induces an improved B memory cell response in the patient receiving the ABP conjugate compared to a control. Improved B memory cell response is intended to mean an increased frequency of peripheral blood B lymphocytes capable of differentiating into antibody-secreting plasma cells upon encountering an antigen, as measured by stimulation of in vitro differentiation.

[0224] In another embodiment, the immunogenic composition increases the primary immune response and the CD8 response. Administration of the ABP conjugate induces an improved CD4 T cell or CD8 T cell immune response to a specific antigen compared to a control. This method may allow for the induction of a CD4 T cell response that is more sustained in time.

[0225] In some embodiments, improved CD4 expression is obtained in unstimulated subjects. CD4 T cell immune responses, such as T cell immune responses, involve the induction of cross-reactive CD4 T helper responses. In particular, the amount of cross-reactive CD4 T cells is increased. The term "cross-reactive" CD4 response refers to CD4 T cells that target epitopes shared between influenza strains, for example.

[0226] It will be understood by those skilled in the art that the amount of albumin-binding peptide conjugate sufficient to stimulate or enhance an immune response, i.e., a therapeutically effective amount, will vary with the particular compound or composition selected, as well as the route of administration, the nature of the condition being treated, and the age and condition of the patient, and will ultimately be at the discretion of the patient's physician or pharmacist. The length of time over which the compounds used in the present methods are given will vary on an individual basis.

[0227] C. The disease being treated Cancer The albumin-binding peptide (ABP) conjugates of the present disclosure are useful for stimulating or enhancing immune response in treating cancer. In some embodiments, the albumin-binding peptide conjugates can be used as anti-cancer vaccines. Thus, for example, the compositions used herein that include albumin-binding peptide conjugates can be administered to patients with cancer.

[0228] Types of cancer that may be treated using the provided compositions and methods include, but are not limited to, the following: bladder, brain, breast, cervical, colorectal, esophageal, renal, liver, lung, nasopharyngeal, pancreatic, prostate, skin (e.g., melanoma), stomach, uterine, ovarian, testicular, and blood. Other non-limiting examples of cancers suitable for treatment using the compositions and methods of the present disclosure are described herein.

[0229] Malignant tumors that can be treated are classified herein according to the embryonic origin of the tissue from which they originate. Carcinomas are tumors that arise from endodermal or ectodermal tissues, such as the skin or the epithelial lining of internal organs and glands. Sarcomas, which occur less frequently, originate from mesodermal connective tissues, such as bone, fat and cartilage. Leukemias and lymphomas are malignant tumors of the hematopoietic cells of the bone marrow. Leukemias grow as single cells, whereas lymphomas tend to grow as tumor masses. Malignant tumors can appear in multiple organs or tissues of the body to establish cancer.

[0230] The ABP conjugates can be administered as immunogenic compositions or as part of a vaccine, such as a prophylactic or therapeutic vaccine, which can be used to initiate or enhance a subject's immune response to a pre-existing antigen, such as a tumor antigen, in a subject with cancer.

[0231] The desired outcome of preventive or therapeutic immune response can vary according to the disease, according to the principles well known in the art.Similarly, immune response against cancer can alleviate symptoms, or be one aspect of overall therapeutic intervention against disease.For example, administration of ABP conjugate can reduce tumor size or slow tumor growth compared to control.Stimulation of immune response against cancer can be combined with surgical, chemotherapy, radiation, hormonal and other immunological approaches to affect treatment.

[0232] ii. Infectious diseases In some embodiments, the ABP conjugates of the present disclosure are useful for treating acute or chronic infectious diseases. Since viral infections are primarily cleared by T cells, increasing T cell activity is therapeutically useful in situations where more rapid or complete clearance of infectious viral agents may be beneficial to an animal or human subject. Thus, ABP conjugate antagonists can be administered for the treatment of local or systemic viral infections, such as, but not limited to, immunodeficiency (e.g., HIV), papilloma (e.g., HPV), herpes (e.g., HSV), encephalitis, influenza (e.g., human influenza A virus) and cold (e.g., human rhinovirus) viral infections. For example, pharmaceutical formulations containing ABP conjugates can be administered topically to treat herpes lesions or viral skin diseases such as shingles or genital warts. ABP conjugates can also be administered to treat systemic viral diseases, including, but not limited to, AIDS, influenza, cold or encephalitis.

[0233] Representative infections that can be treated include Actinomyces, Anabaena, Bacillus, Bacteroides, Bdellovibrio, Bordetella, Borrelia, Campylobacter, Caulobacter, Chlamydia, Chlorobium, Chromatidium, and the like. Chromatium, Clostridium, Corynebacterium, Cytophaga, Deinococcus, Escherichia, Francisella, Halobacterium, Heliobacter, Haemophilus, Hemophilus influenza type BB) (HIB), Histoplasma, Hyphomicrobium, Legionella, Leishmania, Leptospirosis, Listeria, Meningococcus types A, B and C, Methanobacterium, Micrococcus, Myobacterium, Mycoplasma, Myxococcus, Neisseria, Nitrobacter, Oscillatoria, Prochloron, Proteus (P roteus, Pseudomonas, Phodospirillum, Rickettsia, Salmonella, Shigella, Spirillum, Spirochaeta, Staphylococcus, Streptococcus, Streptomyces, Sulfolobus, Thermoplasma, Thiobacillus and Treponema, Vibrio and Yersinia, Cryptococcus neoformans neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia ricketsii, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydial psittaciThese include, but are not limited to, infections caused by microorganisms including, but not limited to, Bacillus subtilis, Bacillus psittaci, Chlamydial trachomatis, Plasmodium falciparum, Plasmodium vivax, Trypanosoma brucei, Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalis, and Schistosoma mansoni.

[0234] In some embodiments, the type of disease to be treated or prevented is a chronic infectious disease caused by bacteria, viruses, protozoa, helminths, or other microbial pathogens that enter cells and are attacked, for example, by cytotoxic T lymphocytes.

[0235] In some embodiments, the infection being treated is a chronic infection caused by a hepatitis virus, human immunodeficiency virus (HIV), human T-lymphotropic virus (HTLV), herpes virus, Epstein-Barr virus, or human papilloma virus.

[0236] Combination therapy In some embodiments, the albumin-binding peptide conjugates of the present disclosure are administered in combination with one or more additional therapeutic agents. The therapeutic agents can be administered in the same pharmaceutical composition as the albumin-binding peptide conjugate, or the albumin-binding peptide conjugate and the additional therapeutic agent can be administered in separate pharmaceutical compositions.

[0237] In some embodiments, the albumin-binding peptide conjugate is administered in combination with a conventional therapeutic agent used for the treatment of the disease or condition being treated. Conventional therapeutic agents are known in the art and can be determined by the skilled artisan based on the disease or disorder being treated. For example, if the disease or condition is cancer, the albumin-binding peptide conjugate can be co-administered with a chemotherapeutic agent, or if the disease or condition is a bacterial infection, the albumin-binding peptide conjugate can be co-administered with an antibiotic. Other examples of therapeutic agents that can be co-administered with the albumin-binding peptide conjugate include anti-tumor antibodies and checkpoint inhibitors.

[0238] kit The kits can include the albumin binding peptide (ABP) conjugates disclosed herein and instructions for use. The kits can include the albumin binding peptide conjugates, one or more controls, as well as various buffers, reagents, enzymes and other standard components well known in the art in suitable containers.

[0239] The container may include at least one vial, well, test tube, flask, bottle, syringe or other container means into which the albumin-binding peptide conjugate may be placed, and in some cases may be appropriately aliquoted. If additional components are provided, the kit may contain additional containers into which the components may be placed. The kit may include a means for containing the albumin-binding peptide conjugate, and any other reagent containers sealed for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are retained. The container and / or kit may include a label having instructions for use and / or care. EXAMPLES

[0240] The following are examples of specific embodiments for carrying out the present invention. The examples are presented for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, temperatures, etc.), but of course, some experimental error and deviation should be taken into account.

[0241] Unless otherwise indicated, the practice of the present invention will employ conventional methods of protein chemistry, biochemistry, recombinant DNA technology, and pharmacology, which are within the skill of the art. Such techniques are explained fully in the literature, see, for example, TECreighton, Proteins: Structures and Molecular Properties (WH Freeman and Company, 1993); A. L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3 rd Ed. (Plenum Press) Vols A and B (l992).

[0242] Example 1 Exemplary ABP-Antigen Constructs This disclosure provides evidence that albumin-binding peptides can be used to enhance the antigenicity of peptide vaccines. Albumin-binding peptides have remained largely unexplored in the context of vaccinology. Albumin-binding peptides have previously been used to extend the pharmacokinetics of protein therapeutics in some preclinical studies. This disclosure provides the novel discovery that albumin-binding peptides can be used in the preparation of peptide vaccines. To investigate whether ABPs covalently linked to peptide antigens can target these antigens to the lymphatic system, exemplary ABP-antigen constructs were designed, synthesized, purified and characterized.

[0243] Various embodiments of the ABP-antigen constructs of the present disclosure are shown in FIG.

[0244] ABP-PEG2k-EGP-long has the following configuration: an albumin binding peptide (cyclized DICLPRWGCLW) operably linked to an EGP-long peptide (AVGALEGPRNQDWLGVPRQL) via a PEG2k linker.

[0245] ABP-G4S-EGPlong has the following structure: an albumin binding peptide (cyclized DICLPRWGCLW) operably linked to an EGPlong peptide (AVGALEGPRNQDWLGVPRQL) via a Gly4Ser linker.

[0246] Example 2 Synthesis and purification of ABP-antigen constructs The ABP-antigen construct was synthesized as a single unit via automated solid-phase peptide synthesis. Equal volumes of protected amino acid monomers (0.4 M in DMF, Chem Impex) and HATU (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, Chem Impex) were combined. PyAOP ((7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate, P3Bio) (0.34 M in DMF) was combined with diisopropylethylamine (neat, 1 M final concentration; Sigma) at 90° C. to form an active ester and coupled onto RINK-amide PEG (PCS Biomatrix) resin running at 90° C. After DMF washing, the N-terminus of the coupled monomer was deprotected with piperidine (1 M final concentration in DMF; Sigma), the peptiod resin was washed with DMF, and the cycle was repeated. These iterative coupling reactions (less than 1 min each) were carried out in an automated synthesizer as described (Mijalis, AJ et al., A fully automated flow-based approach for accelerated peptide synthesis. Nat Chem Biol advance online publication, doi:10.1038 / nchembio.2318(2017)), yielding the ABP-antigen construct. PEG was purchased with a protected amine and a carboxylic acid group at the opposite end. 2000 Linkers (Creative PEGworks) were similarly coupled manually in batches at room temperature onto peptide resins containing the automatically synthesized antigenic peptides. The resins were then transferred back to the automated synthesizer to complete the constructs. The crude peptides were cleaved overnight at room temperature (82.5% trifluoroacetic acid, 5% water, 5% thioanisole, 5% phenol, and 2.5% ethanedithiol, by volume; each from Sigma), triturated, and purified via reverse-phase HPLC (Figures 1 and 2).

[0247] Example 3 Cyclization of the ABP moiety and characterization of the ABP-antigen construct The ABP moiety was cyclized by incubating the purified construct (0.1 mg / mL) in 0.1 M NaHCO3 (Sigma), pH 8.0, for 24 h to promote disulfide formation. The cyclized construct was desalted using solid-phase extraction and analyzed using LC-MS to confirm identity and purity (Figures 3 and 4).

[0248] Example 4 Immunization with ABP-antigen constructs To test whether these albumin-binding peptides could enhance vaccination, a model melanoma antigen called gp100 was used. Vaccination against gp100 has shown significant preclinical activity (van Stipdonk, MJB et al., Design of agonistic altered peptides for the robust induction of CTL directed towards H-2Db in complex with the melanoma-associated epitope gp100.Cancer research 69,7784-7792,doi:10.1158 / 0008-5472.CAN-09-1724(2009)), and has been used clinically (Schwartzentruber,DJ et al.,gp100 Peptide Vaccine and Interleukin-2 in Patients with Advanced Melanoma. New England Journal of Medicine 364, 2119-2127, doi:10.1056 / NEJMoa1012863 (2011)). A modified peptide ligand form of gp100 (sequence AVGAL EGPRNQDWL GVPRQL,H2-D bThe agonistic altered peptide (epitope underlined) was shown to have enhanced affinity for MHC over the native form (called EGS) (van Stipdonk, MJB et al. Design of agonistic altered peptides for the robust induction of CTL directed towards H-2Db in complex with the melanoma-associated epitope gp100. Cancer research 69, 7784-7792, doi:10.1158 / 0008-5472. CAN-09-1724 (2009)). This antigen was used for testing in vivo immunization.

[0249] To test the immunogenicity of the ABP-modified peptides, 5 nmol of ABP-G4S-EGP, ABP-PEG, 2000 C57BL6 / J mice (Jackson Laboratories) were immunized with either -EGP or unmodified EGP peptide. Vaccines were formulated with 25 μg c-di-GMP (Invivogen) for adjuvant in a total volume of 100 μL (with sterile saline as diluent). Mice were primed on day 0, boosted on day 14, and tetramer staining was performed on day 21 to assess T cell stimulation in peripheral blood (Figure 5). Briefly, blood was isolated from the retro-orbital cavity, subjected to lysis to remove red blood cells, and incubated with tetramer (H-2Db gp100, MBL) for 30 min at room temperature, followed by surface staining for CD8 and CD44. DAPI was included as a viability marker and cells were subjected to LSR Fortessa (BD Biosciences). CD44 was detected among all live CD8+ T cells. hi Responses were quantified as a percentage of tetramer+. The response to the unmodified peptide was 1.45%, which was not statistically different from the background of 0.82% in unimmunized mice. The ABP-G4S-EGP construct stimulated a mean response of 5.56%, i.e., approximately 4-fold higher than the unmodified peptide. ABP-PEG2000 The -EGP construct stimulated a response of 8.58%, approximately 6-fold higher than the unmodified peptide (Figure 6).

[0250] In summary, G4S or PEG 2000 Addition of albumin-binding peptides linked to peptide antigens via either spacers showed enhanced T cell stimulation against clinically relevant tumor sequences compared to unmodified peptides, providing evidence that ABP-antigen constructs may be a broad platform for application to enhance the immunogenicity of poorly immunogenic peptides. [Table 1-1] [Table 1-2]

[0251] [ka] [ka]

[0252] Equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein which equivalents are intended to be encompassed by the following claims. The present invention provides, for example, the following items. (Item 1) (a) an albumin-binding peptide; (b) an immune modulator molecule; A conjugate comprising: A conjugate wherein the albumin binding peptide is operably linked to the immunomodulatory molecule, with or without a linker domain. (Item 2) The conjugate of item 1, wherein the albumin binding peptide is covalently bound to the immunomodulatory molecule. (Item 3) 2. The conjugate of claim 1, wherein the albumin binding peptide is operably linked to the immunomodulatory molecule via a linker domain. (Item 4) 4. The conjugate according to item 3, wherein the linker domain comprises a Gly-Ser linker or a polyethylene glycol (PEG) linker. (Item 5) The Gly-Ser linker is (Gly4Ser) n 5. The conjugate according to item 4, which is a linker. (Item 6) The PEG linker is PEG 2000 5. The conjugate according to item 4, which is a linker. (Item 7) The conjugate according to item 1, wherein the immunomodulatory molecule is a peptide antigen or a molecular adjuvant. (Item 8) The conjugate according to item 7, wherein the peptide antigen is a cancer antigen. (Item 9) 8. The conjugate according to item 7, wherein the peptide antigen is a melanoma antigen. (Item 10) 8. The conjugate according to item 7, wherein the molecular adjuvant is CpG DNA. (Item 11) 2. The conjugate according to item 1, wherein the albumin binding peptide comprises the amino acid sequence DICLPRWGCLW. (Item 12) A vaccine comprising a conjugate according to any of the preceding paragraphs and a pharma- ceutically acceptable carrier or excipient. (Item 13) 13. The vaccine of item 12, further comprising an adjuvant. (Item 14) 1. A composition for targeting an immunomodulatory molecule to the lymphatic system, said composition comprising: (a) an albumin-binding peptide; (b) an immune modulator molecule; and a conjugate comprising The composition, wherein said albumin binding peptide is operably linked to said immunomodulatory molecule, with or without a linker domain. (Item 15) 15. The composition of claim 14, wherein the conjugate exhibits increased accumulation in lymph nodes when administered to a subject in vivo compared to administration of the antigen alone. (Item 16) An immunogenic composition comprising the conjugate according to item 1. (Item 17) 13. An immunogenic composition comprising the vaccine according to item 12. (Item 18) 18. A method for treating cancer or an infectious disease, comprising administering to a subject a composition according to any one of items 14 to 17 in an amount effective to reduce one or more symptoms of said cancer or said infectious disease compared to a control. (Item 19) 18. A method for increasing an immune response in a subject, comprising administering to the subject an effective amount of the composition according to any one of items 14 to 17 for increasing the immune response in the subject. (Item 20) 20. The method of claim 19, wherein the subject has cancer or an infectious disease. (Item 21) A nucleic acid molecule encoding the conjugate according to item 1. (Item 22) 22. A recombinant expression vector comprising the nucleic acid molecule of item 21. (Item 23) A host cell transformed with the recombinant expression vector according to item 22. (Item 24) A method for producing the conjugate according to item 1, comprising providing a host cell comprising a nucleic acid sequence encoding the conjugate, and maintaining the host cell under conditions in which the conjugate is expressed.

Claims

1. (a) an albumin binding peptide, comprising: (i) the albumin binding peptide has the formula (Xaa) Ｘ -Asp-Xaa-Cys-Leu-Pro-Xaa-Trp-Gly-Cys-Leu-Trp-(Xaa) Ｚ (SEQ ID NO:5), where Xaa is an amino acid; Ｘ and Ｚ is an integer greater than or equal to zero; (ii) the albumin binding peptide comprises the amino acid sequence of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17; (iii) the albumin-binding peptide competes with any of the peptides set forth in SEQ ID NOs: 9 to 17 for binding to human serum albumin in an in vitro assay; or (iv) the albumin binding peptide has a structure represented by the formula X in an in vitro assay Ｘ DXCLPXWGCLWX Ｚ (SEQ ID NO: 22), where X is an amino acid that competes for binding to human serum albumin. An albumin-binding peptide; (b) an immunomodulatory molecule, the immunomodulatory molecule being a peptide antigen or an immunostimulatory oligonucleotide, capable of inducing a T cell response; A conjugate comprising: A conjugate, wherein the albumin binding peptide is operably linked to the immunomodulatory molecule with a linker domain, and the conjugate induces an increased T cell response compared to the immunomodulatory molecule alone.

2. The conjugate of claim 1 , wherein the linker domain comprises a polypeptide linker, an ethylene glycol (PEG) linker, or an oligonucleotide linker.

3. The conjugate of claim 2 , wherein the linker domain comprises a Gly-Ser linker.

4. The Gly-Ser linker is (Gly ４ Ser) ｎ The conjugate of claim 3 which is a linker.

5. The PEG linker is ２０００ The conjugate of claim 2 which is a linker.

6. The conjugate according to any one of claims 1 to 5, wherein the immunomodulatory molecule is a peptide antigen.

7. The conjugate of claim 6 , wherein the peptide antigen is a cancer antigen.

8. The conjugate of claim 6 , wherein the peptide antigen is a melanoma antigen.

9. The conjugate of any one of claims 1 to 5, wherein the immunostimulatory oligonucleotide is CpG DNA.

10. The conjugate according to any one of claims 1 to 9, wherein the albumin binding peptide comprises the amino acid sequence DICLPRWGCLW.

11. A vaccine comprising the conjugate of any one of claims 1 to 10 and a pharma- ceutically acceptable carrier or excipient.

12. The vaccine of claim 11 further comprising an adjuvant.

13. 11. A composition for use in a method for increasing delivery of an immunomodulatory molecule to the lymphatic system, said composition comprising a conjugate according to any one of claims 1 to 10.

14. The composition of claim 13, wherein the conjugate exhibits increased accumulation in lymph nodes when administered to a subject in vivo compared to administration of the immunomodulatory molecule alone.

15. A composition comprising the conjugate of any one of claims 1 to 10.

16. A composition comprising the vaccine of claim 11.

17. 11. A composition comprising a conjugate according to any one of claims 1 to 10 for treating cancer or an infectious disease, characterized in that said composition is administered to a subject so as to reduce one or more symptoms of said cancer or said infectious disease compared to a control.

18. A composition comprising a conjugate according to any one of claims 1 to 10 for increasing a T cell response in a subject.

19. 20. The composition of claim 18, wherein the subject has cancer or an infectious disease.

20. A nucleic acid molecule encoding the conjugate according to any one of claims 6 to 8, wherein the albumin binding peptide is linked to the peptide antigen with a polypeptide linker.

21. 21. A recombinant expression vector comprising the nucleic acid molecule of claim 20.

22. A host cell transformed with the recombinant expression vector of claim 21.

23. 21. A method for making a conjugate according to any one of claims 6 to 8, comprising providing a host cell comprising the nucleic acid molecule according to claim 20, and maintaining said host cell under conditions in which said conjugate is expressed.

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