Th2 vaccine-based obesity inflammation prevention and treatment

Abstract

Overexpressed proteins associated with inflammatory adipocytes have been demonstrated in TNF-a inflammatory adipocyte lines derived from humans and mice as well as in visceral fats derived from mice fed with an inflammatory high fat, high sucrose diet. The adipocyte-associated proteins have immunogenicity in humans and mice and can be used as vaccines for driving type II T cells into inflammatory adipose tissue. Compositions and methods are provided for the prevention and treatment of diseases associated with metabolic obesity, including cancers such as breast cancer and non-alcoholic fatty liver disease (NAFLD).

Description

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 371,999, filed on August 19, 2022, the entire contents of which are incorporated by reference into this application.

[0002] Reference to a sequence listing

[0003] The contents of the XML file of the sequence listing named "UW78_Seq" (41kb in size, created on August 3, 2023, and submitted electronically with this application) are incorporated herein by reference in their entirety.

[0004] Thank you for the government's support

[0005] This invention was made with government support under Grant No. W81XWH-11-1-0760 awarded by the U.S. Army Medical Research and Materiel Command. The government has certain rights in this invention. Background Art

[0006] Obesity is a significant risk factor for breast cancer, especially for postmenopausal women. Obesity that develops after menopause increases the risk of developing estrogen receptor-positive breast cancer by 20-40% compared with women of normal weight. The dose-response relationship between body mass index (BMI) and postmenopausal cancer consists of a 12% increased risk for every 5 kg / m2 increase. 1 Obesity is associated with the risk of triple-negative breast cancer (TNBC) in premenopausal women. 2 Compared with women with a BMI ≤ 25 kg / m2, individuals with a BMI ≥ 30 kg / m2 showed an 82% increased risk of triple-negative breast cancer (TNBC) (95% CI 1.32-2.51). Women with metabolic syndrome, the triad of obesity, type 2 diabetes, and hypertension may be at the highest risk. 3,4 BMI is not an accurate measure of obesity. The term “metabolic obesity” is used to describe individuals who are not obese based on height and weight, but who have hyperinsulinemia, insulin resistance, and are at increased risk for type 2 diabetes, hypertriglyceridemia, premature coronary heart disease+, and breast cancer, as do overtly obese individuals. 6-8

[0007] There remains a need to treat those with metabolic disorders that significantly increase their risk of breast cancer, as well as to effectively target obesity-related conditions, including prediabetes, cancer, and inflammation. Summary of the invention

[0008] Materials described herein meet these and other needs by providing vaccines that drive type II T cells into inflammatory adipose tissue. Overexpressed proteins associated with inflammatory adipocytes are identified herein and are immunogenic in humans and mice. Newly identified antigens DUSP1, FABP4, PAI-1, and ATGL and their epitopes, as well as epitopes of IGF-1R and HIF1a identified herein, can be used in such vaccines. Therefore, in particular, a nucleic acid molecule is provided comprising a nucleic acid sequence encoding a polypeptide, wherein the polypeptide comprises at least two epitopes selected from the group consisting of: (a) amino acids 53-67 of FABP4; (b) amino acids 29-43 of DUSP1; (c) amino acids 94-108, 120-134, 158-172, 186-200, 270-284 and / or 288-302 of PAI-1; (d) amino acids 93-107, 213-227 and / or 374-388 of ATGL; (e) amino acids 276-295 of HIF1a; (f) amino acids 388-402 and / or 545-558 of IGF-1R; and (g) an amino acid sequence having at least 90% identity with any one of the preceding items. In some embodiments, the polypeptide is selected from the group consisting of: (a) amino acids 53-67 of FABP4; (b) amino acids 29-43 of DUSP1; (c) amino acids 94-108, 120-134, 158-172, 186-200, 270-284 and / or 288-302 of PAI-1; (d) amino acids 93-107, 213-227 and / or 374-388 of ATGL; (e) amino acids 276-295 of HIF1a; (f) amino acids 388-402 and / or 545-558 of IGF-1R; and (g) an amino acid sequence that is at least 90% identical to any of the foregoing items. In some embodiments, the polypeptide comprises each of the following: (a) amino acids 53-67 of FABP4; (b) amino acids 29-43 of DUSP1; (c) amino acids 94-108 and 158-172 of PAI-1; (d) amino acids 213-227 and 374-388 of ATGL; (e) amino acids 276-295 of HIF1a; and (f) amino acids 388-402 and 545-558 of IGF-1R. The amino acid sequences of these epitopes are listed in Table 1.

[0009] In some embodiments, the polypeptide is selected from amino acids 53-67 of FABP4, amino acids 29-43 of DUSP1, amino acids 276-295 of HIF1a, and combinations thereof.These epitopes are associated with more IL-10 secreting T cells.

[0010] In some embodiments, the polypeptide comprises amino acids 94-108 and / or 158-172 of PAI-1, amino acids 213-227 and / or 374-388 of ATGL, amino acids 388-402 and / or 545-558 of IGF-1R, or a combination thereof. These epitopes are associated with significantly more antigen-specific IL-10 responses.

[0011] In some embodiments, the polypeptide comprises one or more epitopes of (a), (b), (c), and (d) as listed above. In some embodiments, the polypeptide comprises at least three epitopes selected from (a), (b), (c), and (d). In some embodiments, the polypeptide comprises at least one of each of (a), (b), (c), and (d).

[0012] In some embodiments, the polypeptide comprises at least one of each of (a), (b), (c), (d), (e), and (f), and optionally further comprises one or more linker sequences disposed between the epitopes. In some embodiments, the linker sequence is GPGPG or GGGS.

[0013] In some embodiments, the nucleic acid sequence includes a promoter sequence. In some embodiments, the nucleic acid sequence includes a heterologous sequence. In some embodiments, the heterologous sequence encodes a promoter, a transcription start site, a translation start site, a detectable marker, an mRNA processing splice site, a polyadenylation sequence, and / or a regulatory element.

[0014] Also described herein are vectors comprising nucleic acid molecules as described herein, wherein the vector is capable of directing the expression of the encoded polypeptide. In some embodiments, the vector is a DNA plasmid, messenger RNA (mRNA) or a viral vector. In some embodiments, the viral vector is a lentiviral vector, an adenoviral vector or a poxvirus vector. In some embodiments, the vector is mRNA.

[0015] Additionally provided herein is a polypeptide comprising at least two epitopes selected from the group consisting of epitopes listed in Table 1. In some embodiments, the selected epitopes include an amino acid sequence having at least 90% identity to any one of the amino acid sequences listed in Table 1. In some embodiments, the polypeptide comprises a combination of at least three, four, five, six or more epitopes listed in Table 1. In some embodiments, the polypeptide comprises at least one of each of (a), (b), (c), (d), (e) and (f). In some embodiments, the polypeptide further comprises one or more linker sequences disposed between the epitopes.

[0016] Compositions comprising the polypeptides described herein are also provided.In some embodiments, the composition further comprises an adjuvant.

[0017] In addition, a method for causing type 2 immune responses for inflammation related to obesity, for preventing or treating inflammation in obesity, and / or for preventing or treating a disease related to metabolic obesity in a subject is described herein. In some embodiments, the method includes administering a composition comprising nucleic acid molecules, polypeptides or compositions as described herein to the subject. In some embodiments, the disease related to metabolic obesity is cancer. In some embodiments, cancer is breast cancer. In some embodiments, the disease related to metabolic obesity is non-alcoholic fatty liver disease (NAFLD), metabolic-associated fatty liver disease (MAFLD) or prediabetes.

[0018] In some embodiments, the subject is a human.In some embodiments, the subject is suspected of having or has been diagnosed with obesity, a disease associated with metabolic obesity, cancer, such as prediabetes / insulin insensitivity, breast cancer, MAFLD, or NAFLD. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1A-Figure 1D IL-10 selective epitopes can be identified from proteins abnormally expressed in obese adipose tissue. Th ratios when stimulated with the indicated epitopes from (1A) FABP4, (1B) DUSP1 (1C) ATGL, or (1D) PAI1. The bars to the right of "0" represent Th-2 selective epitopes, while the bars to the left of "0" represent Th1 selective epitopes. n = 10 healthy donors.

[0020] Figure 2A-2D .Overexpressed antigens identified in human obese adipose tissue are also overexpressed in adipose tissue of obese mice. (2A) Representative Western blots of the indicated proteins in unstimulated differentiated mouse adipocytes or TNFa-stimulated differentiated mouse adipocytes, ***<0.001, ****p<0.0001. (2B) Relative pixel density (±SD) of the indicated proteins as measured by densitometry. (2C) Representative Western blots of cell lysates of the indicated antigens from mice fed a high-fat diet (HFD) or a normal diet (NCD). α / β tubulin was used as a loading control. (2D) Relative pixel density (±SEM) as measured by densitometry, normalized to expression observed in mouse tissue on NCD; n=5 mice; **p<0.05, **p<0.01, **p<0.001.

[0021] Figure 3A-Figure 3FIL-10 secretory epitopes identified by in vitro screening generate Th2-selective responses after immunization with each single antigen. Mean (±SEM) of IL-10 corrected points / well (CSPW) in splenocytes from mice vaccinated with peptides or peptide pools and stimulated with the indicated epitopes from (3A) FABP4, (3B) DUSP1, (3C) HIF1a, (3D) PAI1, (3E) ATGL, and (3F) IGF-IR are presented as box plots, showing all points per mouse. n = 8-12 mice / group; **p<0.01, ***p<0.001, ****p<0.0001.

[0022] Figure 4A-4C .AdVac immunization generates IL-10-dominated T cell responses to all antigens in most mice. (4A) Corrected IL-10 dots / well (cSPW) in splenocytes from mice immunized with AdVac and stimulated with the indicated antigens are presented as box and whisker plots, with the line at the median and whiskers from smallest to largest, showing all dots. (4B) Percentage of mice that responded to the indicated antigens. (4C) Percentage of mice that responded to zero antigens (bottom bar), three antigens (white bar, second from the bottom), five antigens (middle bar), or six antigens (top bar), n=10 mice / group; *p<0.05, **p<0.01.

[0023] Figure 5 Experimental design for both the male C57BL / 6 diet-induced obesity model and the female TgMMTV-neu mouse breast cancer model. wks = age in weeks.

[0024] Figure 6A-6D .AdVac increases glucose sensitivity and reduces insulin resistance in obese mice. (6A) Glucose tolerance test (GTT) of lean mice (triangles) or obese mice immunized with adjuvant alone (control; filled circles) or AdVac (open squares). (6B) Area under the curve (AUC) of GTT. (6C) Insulin tolerance test (ITT) of lean mice (triangles) or obese mice immunized with control (filled circles) or AdVac (open squares). (6D) AUC of ITT. n = 20 mice / group; **p < 0.01, ***p < 0.001, ****p < 0.0001 (compared with control obese).

[0025] Figure 7A-7D.AdVac reduced CD8+T cell levels in adipose tissue of obese mice and increased T regulatory cell levels. Percentage of CD8+T cells in all surviving lymphocytes from (7A) adipose tissue or (7B) spleen of lean mice or obese mice immunized with adjuvant (alum) or AdVac vaccine alone. Percentage of CD4+ / FOXP3+T regulatory cells in (7C) adipose tissue or (7D) spleen of lean mice or obese mice immunized with alum or AdVac. All data are presented in box and whisker plots, with horizontal lines at the median and whiskers from minimum to maximum, showing all points; n = 17-20 mice / group; *p, 0.05, **p<0.01, ****p<0.0001.

[0026] Figure 8A-8F .ADVac metabolically reprograms the adipose tissue microenvironment. (8A) Heat map of adipose tissue metabolites in obese mice immunized with adjuvant (control) or ADVac alone. (8B) Four major metabolite pathways associated with significant decreases in metabolite levels after ADVac immunization. Normalized metabolite levels of (8C) glutamine, (8D) nicotinamide, (8E) glucose-1-phosphate, and (8F) glucose-6-phosphate in obese mice immunized with adjuvant (control) or ADVac alone or lean untreated mice are presented as box plots and whisker plots, showing all points, with the horizontal line at the median and whiskers from minimum to maximum. n = 7-10 mice / group; **p<0.01, ***p<0.001, ****p<0.0001.

[0027] Figure 9A-9B Obese TgMMTV-neu mice developed a greater number of tumors and grew faster than lean TgMMTV-neu mice. (9A) Kaplan-Meier curve showing the percentage of tumor-free in control obese mice (lower line) and obese mice immunized with AdVac (upper line). (9B) Tumor growth rate (mm3 / day) in lean or obese mice. n=15 mice / group; **p<0.01, ***p<0.001.

[0028] Figure 10A-10C.AdVac immunization reduces CD8+ T cells in mammary tissue, lowers serum leptin levels, and results in significant tumor suppression in obese Tg-MMTV-neu mice. (10A) Percentage of CD8+ T cells in surviving lymphocytes from mammary fat of the indicated treatment groups. (10B) Mean (±SEM) serum leptin (ng / ml) from mice immunized with the indicated vaccines. (10C) Survival curves show the percentage of tumor-free in control obese mice (lower line) and obese mice immunized with AdVac (upper line). n=7-15 mice / group; *p<0.05, **p<0.01, ***p<0.001. DETAILED DESCRIPTION

[0029] Invention as described herein is based on the discovery and identification of overexpressed proteins associated with inflammatory adipocytes. Overexpression of these antigens has been confirmed in the TNF-α inflammatory adipocyte system derived from humans and mice and in the visceral fat of mice derived from the high-fat, high-sucrose (HFHS) diet that generates inflammation in feeding. Adipocyte-related proteins have immunogenicity in humans and mice. This discovery provides help for the development of the compositions and methods for preventing and treating diseases associated with metabolic obesity, including cancer (such as breast cancer) and non-alcoholic fatty liver disease (NAFLD) .

[0030] definition

[0031] Unless otherwise specified, all scientific and technical terms used in this application have the meanings commonly used in the art. As used in this application, the following words or phrases have the designated meanings.

[0032] As used herein, the term "comprising" is intended to mean that compositions and methods include the described elements, but do not exclude other elements. As used herein, the transition phrase "consisting essentially of..." (and grammatical variations) should be interpreted as covering the described materials or steps "and those that do not substantially affect the basic and novel features of the described embodiments." Therefore, as used herein, the term "consisting essentially of..." should not be interpreted as being equivalent to "comprising." "Consisting of..." means excluding other elements and substantial method steps that exceed trace ingredients for applying the compositions disclosed herein. The aspects defined by each of these transition terms are within the scope of the disclosure herein.

[0033] As used herein, "linker" refers to an amino acid sequence that connects two proteins, polypeptides, peptides, domains, regions or motifs, and can provide a spacer function that is compatible with the interaction of the two sub-binding domains, so that the resulting polypeptide retains a specific binding affinity for the target molecule or retains signal transduction activity. In certain embodiments, the linker is composed of about two to about 35 amino acids, for example, or about four to about 20 amino acids or about eight to about 15 amino acids or about 15 to about 25 amino acids.

[0034] As used herein, the term "nucleic acid sequence" or "polynucleotide" refers to nucleotides of any length, which are deoxynucleotides (i.e., DNA) or derivatives thereof; ribonucleotides (i.e., RNA) or derivatives thereof; or peptide nucleic acids (PNA) or derivatives thereof. The term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, oligonucleotides (oligo), or other natural, synthetic, modified, mutated, or non-natural forms of DNA or RNA.

[0035] The term "vector" refers to, but is not limited to, a recombinant genetic construct or plasmid or expression construct or expression vector that retains the ability to infect and transduce non-dividing cells and / or slowly dividing cells and integrate into the genome of the target cell. The vector may be derived from or based on a wild-type virus. Aspects of the present disclosure relate to adeno-associated viral vectors, adenoviral vectors, and lentiviral vectors.

[0036] As used herein, the term "expression control element" refers to any sequence that regulates the expression of a coding sequence (such as a gene). Exemplary expression control elements include, but are not limited to, promoters, enhancers, microRNAs, post-transcriptional regulatory elements, polyadenylation signal sequences, and introns. Expression control elements can be, but are not limited to, constitutive, inducible, repressive, or tissue-specific. A "promoter" is a control sequence that is a region in a polynucleotide sequence that controls transcription initiation and rate. It may contain genetic elements that regulatory proteins and molecules can bind to, such as RNA polymerases and other transcription factors. In some embodiments, the expression controlled by the promoter is tissue-specific. An "enhancer" is a region of DNA that can bind to increase the probability or frequency of transcription by activating proteins. Non-limiting exemplary enhancers and post-transcriptional regulatory elements include CMV enhancers and WPRE.

[0037] The term "multicistronic" or "polycistronic" or "bicistronic" or "tricistronic" refers to an mRNA with multiple (i.e., two or three) coding regions or exons, and therefore will have the ability to express two or more or three or more or four or more proteins from the mRNA in a single construct. Polycistronic vectors express two or more separate proteins from the same mRNA at the same time. The two most widely used strategies for constructing a polycistronic configuration are by using 1) IRES or 2) 2A self-cleavage site. "IRES" refers to an internal ribosome entry site or portion thereof of a virus, prokaryotic or eukaryotic origin used in a polycistronic vector construct. In some embodiments, IRES is an RNA element that allows translation to be initiated in a cap-independent manner. The term "self-cleaving peptide" or "sequence encoding a self-cleaving peptide" or "2A self-cleavage site" refers to a linker sequence that is used in a vector construct to integrate a site to promote ribosome jumping and thereby produce two polypeptides from a single promoter, such self-cleaving peptides include but are not limited to T2A and P2A peptides or sequences encoding self-cleaving peptides.

[0038] "Homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing positions in each sequence that can be aligned for comparison purposes. When a position in the compared sequences is occupied by the same base or amino acid, then the molecules are homologous at that position. The degree of homology between sequences varies with the number of matching or homologous positions shared by the sequences. "Unrelated" or "non-homologous" sequences have less than 40% identity, or alternatively less than 25% identity, to one of the sequences disclosed herein.

[0039] The percentage similarity or complementarity of any of the disclosed sequences can be determined, for example, by comparing sequence information using the GAP computer program version 6.0 available from the University of Wisconsin Genetics Computer Group (UWGCG). The GAP program utilizes the alignment method of Needleman and Wunsch (1970). In short, the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids) that are similar divided by the total number of symbols in the shorter of the two sequences. Preferred default parameters for the GAP program include: (1) a unary comparison matrix for nucleotides (containing a value of 1 for identity and 0 for non-identity) and a weighted comparison matrix of Gribskov and Burgess (1986), (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap; and (3) no penalty for end gaps.

[0040] "Nucleotide sequence" refers to a heteropolymer of deoxyribonucleotides, ribonucleotides, or peptide nucleic acid sequences that can be assembled from smaller fragments, isolated from larger fragments, or chemically synthesized de novo or by combining shorter oligonucleotide linker moieties or from a series of oligonucleotides.

[0041] As used herein, the terms "protein", "peptide" and "polypeptide" refer to amino acid subunits, amino acid analogs or peptide mimetics. The subunits can be linked by peptide bonds. In another aspect, the subunits can be linked by other bonds, such as esters, ethers, etc. As used herein, the term "amino acid" refers to natural and / or non-natural or synthetic amino acids.

[0042] As used herein, the term "recombinant expression system" or "recombinant expression vector" refers to a genetic construct formed by recombination and used to express certain genetic materials.

[0043] The term "effective amount" or "therapeutically effective amount" or "prophylactically effective amount" refers to an amount of an active agent described herein that is effective to provide a desired / intended result and / or biological activity. Thus, for example, in various embodiments, an effective amount of a composition described herein is an amount effective to cause alleviation or slowing of disease progression and / or improvement or alleviation of symptoms of a disease and / or treatment of a disease.

[0044] When the disclosure herein relates to small molecules, polypeptides, proteins, polynucleotides, nucleic acids, oligonucleotides, antisense or miRNA, its equivalents or biological equivalents are intended to fall within the scope of the present disclosure. As used herein, when referring to a reference small molecule, polypeptide, protein, polynucleotide, nucleic acid, oligonucleotide, antisense or miRNA, the term "its biological equivalent" is intended to be synonymous with "its equivalent", even if these reference molecules have minimal homology, while still maintaining the desired structure or function. Unless explicitly stated herein, it is considered that any nucleic acid, polynucleotide, oligonucleotide, antisense, miRNA, polypeptide or protein mentioned herein also includes its equivalent. For example, an equivalent is intended to have at least about 70% homology or identity, or at least 80% homology or identity, and alternatively, or at least 85%, or alternatively at least about 90%, or alternatively at least about 95%, or alternatively 98% homology or identity, and exhibits a biological activity substantially equivalent to a reference protein, polypeptide or nucleic acid. Alternatively, when reference is made to a polynucleotide, its equivalent is a polynucleotide that hybridizes under stringent conditions to the reference polynucleotide or its complement.

[0045] In some embodiments disclosed herein, polypeptide and / or polynucleotide sequences are provided herein for use in gene and protein transfer and expression techniques described herein. Such sequences provided herein can be used to provide expression products and to produce substantially identical sequences of proteins with the same biological properties. These "biological equivalents" or "biologically active" or "equivalent" polypeptides are encoded by equivalent polynucleotides as described herein. When compared using a sequence identity method run under default conditions, they can have a primary amino acid sequence with at least 60%, or alternatively, at least 65%, or alternatively, at least 70%, or alternatively, at least 75%, or alternatively, at least 80%, or alternatively, at least 85%, or alternatively at least 90%, or alternatively at least 95% or alternatively at least 98% identity with a reference polypeptide. Specific polynucleotide or polypeptide sequences are provided as examples of specific embodiments. Amino acid sequences can be modified by using alternative amino acids with similar charges. Additionally, equivalent polynucleotides are polynucleotides that hybridize with reference polynucleotides or their complements under stringent conditions, or, with respect to polypeptides, polypeptides encoded by polynucleotides that hybridize with reference coding polynucleotides or their complementary chains under stringent conditions. Alternatively, an equivalent polypeptide or protein is a polypeptide or protein expressed by an equivalent polynucleotide.

[0046] "Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonds between nucleotide residue bases. Hydrogen bonds can occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner. The complex can contain two chains that form a double-stranded structure, three or more chains that form a multi-stranded complex, a single self-hybridizing chain, or any combination of these. The hybridization reaction can constitute a step in a broader process, such as the initiation of a PCR reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme.

[0047] Examples of stringent hybridization conditions include: incubation temperature of about 25°C to about 37°C; hybridization buffer concentration of about 6x SSC to about 10x SSC; formamide concentration of about 0% to about 25%; and washing solution of about 4x SSC to about 8x SSC. Examples of moderate hybridization conditions include: incubation temperature of about 40°C to about 50°C; buffer concentration of about 9x SSC to about 2x SSC; formamide concentration of about 30% to about 50%; and washing solution of about 5x SSC to about 2x SSC. Examples of high stringency conditions include: incubation temperature of about 55°C to about 68°C; buffer concentration of about 1x SSC to about 0.1x SSC; formamide concentration of about 55% to about 75%; and washing solution of about 1x SSC, 0.1x SSC or deionized water. In general, the hybridization incubation time is 5 minutes to 24 hours, with 1, 2 or more washing steps, and the washing incubation time is about 1, 2 or 15 minutes. SSC is 0.15M NaCl and 15mM citrate buffer. It will be appreciated that SSC equivalents using other buffer systems may be employed.

[0048] As used herein, "treating" or "treatment" of a disease in a subject refers to (1) preventing symptoms or disease from occurring in a subject susceptible to or not yet exhibiting symptoms of the disease; (2) inhibiting a disease or preventing its development; or (3) alleviating or causing regression of a disease or disease symptom. As understood in the art, "treatment" is a method for obtaining a beneficial or desired result, including a clinical result. For the purposes of the compositions, therapies, nucleic acid molecules and methods disclosed herein for inducing an immune response, a beneficial or desired result may include one or more of (but not limited to) alleviating or ameliorating one or more symptoms of a disease associated with metabolic obesity, alleviating its symptoms, stabilizing the state of the disease (including the disease) (i.e., not worsening), delaying or slowing the disease (including the disease), progression, improving or alleviating the state of a disease (including the disease), a disease associated with metabolic obesity (whether partial or complete), and alleviation, whether detectable or undetectable.

[0049] As used herein, the term "isolated" means that a naturally occurring DNA fragment, DNA molecule, coding sequence or oligonucleotide is removed from its natural environment, or is a synthetic molecule or cloned product. Preferably, the DNA fragment, DNA molecule, coding sequence or oligonucleotide is purified, i.e., substantially free of any other DNA fragment, DNA molecule, coding sequence or oligonucleotide and associated cellular products or other impurities.

[0050] As used herein, the term "cell" refers to a prokaryotic cell or a eukaryotic cell, which is optionally obtained from a subject or a commercial source. Cells treated, transfected, transformed, or otherwise contacted with the compositions and / or nucleic acid molecules disclosed herein include, but are not limited to, cells of humans, non-human animals, mammals, or non-human mammals, including, but not limited to, cells of murine, canine, or non-human primate species. Cells treated, transfected, transformed, or otherwise contacted with the compositions and / or nucleic acid molecules disclosed herein are, but are not limited to, T cells, antigen presenting cells, and other suitable host cells.

[0051] As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, horses, sheep, dogs, cows, pigs, chickens, and other veterinary subjects.

[0052] As used herein, "a" or "an" means at least one, unless specifically stated otherwise.

[0053] As used herein, "preventing" or "protecting against" a condition or disease means hindering, reducing, or delaying the occurrence or progression of the condition or disease.

[0054] When the term "encoding" is applied to a nucleic acid sequence, it means that if the polynucleotide in its native state or when manipulated by methods well known to those skilled in the art can be transcribed and / or translated to produce an effector RNA, mRNA, or mRNA that can encode a polypeptide and / or fragment thereof, then the polynucleotide "encodes" a polypeptide, mRNA or effector RNA. The antisense strand is the complementary strand of such a nucleic acid, and the coding sequence can be deduced therefrom.

[0055] As used herein, the term "expression" or "gene expression" refers to the process of transcribing a polynucleotide into mRNA and / or the process of subsequently translating the transcribed mRNA into a peptide, polypeptide or protein. If the polynucleotide is derived from genomic DNA, expression may include splicing of mRNA in a eukaryotic cell. The expression level of a gene can be determined by measuring the amount of mRNA or protein in a cell or tissue sample; in addition, the expression levels of multiple genes can be determined to establish an expression profile for a particular sample.

[0056] As used herein, the term "functional" may be applied to the modification of any molecular, biological or cellular material to direct it to achieve a specific, designated effect.

[0057] As used herein, the term "about" when referring to a measurable value (such as an amount, level or concentration), for example but not limited to, is meant to cover variations of 20%, 10%, 5%, 1%, 0.5% or even 0.1% of a specified amount, or a fold difference in a level that is quantifiably compared to a standard or control or reference material, such as 1-fold, 2-fold, 3-fold, 4-fold... 10-fold, 100-fold, etc., of a specified comparison level.

[0058] When the terms "acceptable," "effective," or "sufficient" are used to describe the selection of any component, range, dosage form, etc. disclosed herein, it is intended to mean that the component, range, dosage form, etc. is suitable for the disclosed purpose.

[0059] As used herein, the term "adeno-associated virus" or "AAV" refers to a member of the class of viruses associated with the name and belongs to the Parvoviridae family, genus dependoparvovirus. Multiple serotypes of the virus are known to be suitable for gene delivery; all known serotypes can infect cells of various tissue types. At least 11 or 12 serotypes numbered sequentially are disclosed in the prior art. Non-limiting exemplary serotypes that can be used in the methods disclosed herein include any of the 11 or 12 serotypes, for example, AAV2, AAV5 and AAV8, or variant serotypes, such as AAV-DJ. AAV structural particles are composed of 60 protein molecules consisting of VP1, VP2 and VP3. Each particle contains approximately 5 VP1 proteins, 5 VP2 proteins and 50 VP3 proteins, arranged in an icosahedral structure.

[0060] abbreviation

[0061]

[0062] Compositions and nucleic acid molecules

[0063] Compositions and / or nucleic acid molecules for preventing and treating inflammation associated with obesity or diseases associated with metabolic obesity in a subject are provided. A nucleic acid molecule is disclosed herein, comprising a nucleic acid sequence encoding a polypeptide comprising one or more epitopes described herein. In some embodiments, the polypeptide is selected from the group consisting of: (a) amino acids 53-67 of FABP4; (b) amino acids 29-43 of DUSP1; (c) amino acids 94-108, 120-134, 158-172, 186-200, 270-284 and / or 288-302 of PAI-1; (d) amino acids 93-107, 213-227 and / or 374-388 of ATGL; (e) amino acids 276-295 of HIF1a; (f) amino acids 388-402 and / or 545-558 of IGF-1R; and (g) an amino acid sequence having at least 90% identity to any of the foregoing items. In some embodiments, the polypeptide comprises each of the following: (a) amino acids 53-67 of FABP4; (b) amino acids 29-43 of DUSP1; (c) amino acids 94-108 and 158-172 of PAI-1; (d) amino acids 213-227 and 374-388 of ATGL; (e) amino acids 276-295 of HIF1a; and (f) amino acids 388-402 and 545-558 of IGF-1R. The amino acid sequences of these epitopes are listed in Table 1.

[0064] Table 1: Epitopes included in ADVac (designated) and candidate epitopes for inclusion in ADVac

[0065]

[0066]

[0067] *The mature IGF1R protein is cleaved 30 amino acids from the N-terminus. When referring to the proprotein rather than the mature protein sequence, these amino acid residues are numbered 575-588.

[0068] In some embodiments, the nucleic acid sequence encodes a polypeptide comprising a combination of one or more epitopes. In some embodiments, the encoded polypeptide comprises one or more epitopes listed in Table 1. In some embodiments, the polypeptide comprises the first nine epitopes listed in Table 1. In some embodiments, the polypeptide comprises one or more epitopes listed in the second half of Table 1 as candidates for inclusion in the ADVac vaccine. In some embodiments, the polypeptide comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more epitopes listed in Table 1.

[0069] In some embodiments, the nucleic acid sequence optionally further comprises one or more joint sequences arranged between the epitopes. In some embodiments, the joint sequence is glycine-serine. In some embodiments, the nucleic acid sequence comprises a promoter sequence. In some embodiments, the nucleic acid sequence comprises a heterologous sequence. In some embodiments, the heterologous sequence encodes a promoter, a transcription start site, a translation start site, a detectable marker, an mRNA processing splice site, a polyadenylation sequence and / or a regulatory element.

[0070] Such nucleic acid molecules can be delivered by viral or non-viral means. An example of viral delivery is adeno-associated virus (AAV). Other examples include retrovirus, slow virus and baculovirus delivery. An example of non-viral delivery method is cell penetrating peptide (CPP). Polynucleotide constructs can also be modified, such as by chemical modification, to improve its stability and / or delivery suitability. In some embodiments, oligonucleotides are modified by locked nucleic acid and / or thiophosphate bonds. In some embodiments, delivery systems are selected to improve bioavailability, such as, for example, PEGylated liposomes, lipidoids or biodegradable polymers.

[0071] Viral vectors

[0072] In some embodiments, the vector disclosed herein is a viral vector. In some embodiments, the vector is an adenoviral vector, an adeno-associated virus (AAV) vector or a lentiviral vector. In some embodiments, the vector is a retroviral vector, an adenovirus / retroviral chimera vector, a herpes simplex virus I or II vector, a parvovirus vector, an avian reticuloendotheliosis virus (reticuloendotheliosis viral) vector, a poliovirus vector, a papillomavirus vector, a vaccinia virus vector or any hybrid or chimeric vector integrating the advantages of two or more viral vectors. In some embodiments, the vector further comprises one or more expression control elements operably connected to the polynucleotide. In some embodiments, the vector further comprises one or more selectable markers.

[0073] In some embodiments, the vector disclosed herein is a low toxicity AAV vector. In some embodiments, the AAV vector will not integrate into the host genome, thereby causing a low possibility of insertional mutagenesis. In some embodiments, the AAV vector can encode a total polynucleotide ranging from 4.5 kb to 4.75 kb. In some embodiments, exemplary AAV vectors that can be used in any of the compositions, systems, methods, and kits described herein can include AAV1 vectors, modified AAV1 vectors, AAV2 vectors, modified AAV2 vectors, AAV3 vectors, modified AAV3 vectors, AAV4 vectors, modified AAV4 vectors, AAV5 vectors, modified AAV5 vectors, AAV6 vectors, modified AAV6 vectors, AAV7 vectors, modified AAV7 vectors, AAV8 vectors, AAV9 vectors, AAV.rh10 vectors, modified AAV.rh10 vectors, AAV.rh32 / 33 vectors, modified AAV.rh32 / 33 vectors, AAV.rh43 vectors, modified AAV.rh43 vectors, AAV.rh64R1 vectors, and modified AAV.rh64R1 vectors, and any combinations or equivalents thereof.

[0074] In some embodiments, the vector disclosed herein is a lentiviral vector. In one embodiment, the lentiviral vector is an integrase-functional lentiviral vector (ICLV). In some embodiments, a lentiviral vector may refer to a transgenic plasmid vector and a combination of a transgenic plasmid vector and a related plasmid (e.g., a packaging plasmid, a rev expression plasmid, an envelope plasmid), and a lentiviral-based particle capable of introducing exogenous nucleic acids into cells via a viral or virus-like entry mechanism. Lentiviral vectors are well known in the art. In some embodiments, exemplary lentiviral vectors may be used with any of the compositions, nucleic acid molecules, and / or methods described herein, and may include human immunodeficiency virus (HIV) 1 vectors, modified human immunodeficiency virus (HIV) 1 vectors, human immunodeficiency virus (HIV) 2 vectors, modified human immunodeficiency virus (HIV) 2 vectors, black and white mangabey immunodeficiency virus (SIV SM ) vector, modified black and white mangabey immunodeficiency virus (SIV SM ) vector, African green monkey simian immunodeficiency virus (SIV AGM ) vector, modified African green monkey simian immunodeficiency virus (SIV AGM) vector, equine infectious anemia virus (EIAV) vector, modified equine infectious anemia virus (EIAV) vector, feline immunodeficiency virus (FIV) vector, modified feline immunodeficiency virus (FIV) vector, Visna / maedi virus (VNV / VMV) vector, modified Visna / maedi virus (VNV / VMV) vector, caprine arthritis-encephalitis virus (CAEV) vector, modified caprine arthritis-encephalitis virus (CAEV) vector, bovine immunodeficiency virus (BIV) or modified bovine immunodeficiency virus (BIV).

[0075] In some embodiments of the compositions and / or nucleic acid molecules and / or methods of the present disclosure, the vector of the present disclosure is a viral vector. In some embodiments, the viral vector comprises a sequence separated from or derived from a retrovirus. In some embodiments, the viral vector comprises a sequence separated from or derived from a lentivirus. In some embodiments, the viral vector comprises a sequence separated from or derived from an adenovirus. In some embodiments, the viral vector comprises a sequence separated from or derived from an adeno-associated virus (AAV). In some embodiments, the viral vector is a replication-deficient type. In some embodiments, the viral vector is isolated or recombinant. In some embodiments, the viral vector is self-complementary.

[0076] In some embodiments of the compositions and / or nucleic acid molecules and / or methods of the present disclosure, the viral vector comprises a sequence separated from or derived from an adeno-associated virus (AAV). In some embodiments, the viral vector comprises an inverted terminal repeat or capsid sequence, which is separated from or derived from an AAV of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or AAV12, or the vector and / or component are derived from a synthetic AAV serotype, such as but not limited to Anc80 AAV (ancestor of AAV 1, 2, 6, 8 and 9). In some embodiments, the viral vector is a replication-deficient type. In some embodiments, the viral vector is isolated or recombinant (rAAV). In some embodiments, the viral vector is self-complementary (scAAV).

[0077] In some embodiments of the compositions and methods of the present disclosure, the vector of the present disclosure is a non-viral vector. In some embodiments, the vector comprises or consists of a nanoparticle, a micelle, a liposome or a lipoplex, a polymer vesicle, a polymer complex, or a dendrimer.

[0078] In some embodiments, the expression vectors or viral vectors disclosed herein are used to transfect, transform, or contact a cell that is a eukaryotic cell. In some embodiments, the cell is an animal cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a bovine cell, a mouse cell, a cat cell, a horse cell, a pig cell, a dog cell, a monkey cell, or a human cell.

[0079] In some embodiments, the cell is a packaging cell or a producer cell for producing viral particles.

[0080] In some embodiments, provided herein are viral particles comprising, consisting of, or consisting essentially of a vector comprising, consisting of, or consisting essentially of a polynucleotide sequence encoding one or more polypeptides as described herein.

[0081] In general, methods of packaging genetic material (such as RNA or DNA) into one or more vectors are well known in the art. For example, packaging vectors and cell lines can be used to package genetic material and introduced via traditional recombinant methods.

[0082] In some embodiments, packaging vectors may include but are not limited to retroviral vectors, lentiviral vectors, adenoviral vectors and adeno-associated viral vectors. Packaging vectors contain elements and sequences that contribute to the delivery of genetic material into cells. For example, a retroviral construct is a packaging plasmid comprising at least one retroviral auxiliary DNA sequence derived from a replication-defective retroviral genome, the retroviral genome trans-encoding all virion proteins required for packaging replication-defective retroviral vectors, and used to produce virion proteins capable of packaging replication-defective retroviral vectors in high titer without the need to produce replication-defective helper viruses. Retroviral DNA sequences lack the region of natural enhancers and / or promoters encoding viral 5'LTR, and lack the psi functional sequence responsible for packaging auxiliary genome and 3'LTR simultaneously, but encode external polyadenylation sites, for example, SV40 polyadenylation sites, and external enhancers and / or promoters that instruct efficient transcription in the cell type in which the virus is expected to be produced. The retrovirus is a leukemia virus, such as Moloney murine leukemia virus (MMLV), human immunodeficiency virus (HIV) or gibbon ape leukemia virus (GALV). The foreign enhancer and promoter can be the human cytomegalovirus (HCMV) immediate early (IE) enhancer and promoter, the enhancer and promoter (U3 region) of Moloney murine sarcoma virus (MMSV), the U3 region of Rous sarcoma (Rous Sarcoma) virus (RSV), the U3 region of spleen focus forming virus (SFFV), or the HCMV IE enhancer connected to the natural Moloney murine leukemia virus (MMLV) promoter.

[0083] A retroviral packaging plasmid can consist of two retroviral helper DNA sequences encoded by a plasmid-based expression vector, for example, wherein the first helper sequence contains cDNAs encoding the gag and pol proteins of an ecotropic MMLV or GALV and the second helper sequence contains a cDNA encoding the env protein. The host range determining Env gene may be derived from a gene encoding heterotropic, amphotropic, ecotropic, polytropic (mink focus forming) or 10A1 murine leukemia virus env protein, or a gibbon ape leukemia virus (GALV env protein, human immunodeficiency virus env (gp160) protein, vesicular stomatitis virus (VSV) G protein, human T cell leukemia (HTLV) type I and II env gene products, a chimeric envelope gene derived from a combination of one or more of the above env genes, or a chimeric envelope gene encoding cytoplasmic and transmembrane env gene products of the above, and monoclonal antibodies directed against specific surface molecules on desired target cells. Similar vector-based systems may employ other vectors, such as sleeping beauty vectors or transposon elements.

[0084] The resulting packaged expression system can then be introduced via an appropriate route of administration, as discussed in detail with respect to the method aspects disclosed herein.

[0085] Peptides

[0086] Also provided are polypeptides comprising one or more epitopes of MCPyV. In some embodiments, the polypeptide is selected from the group consisting of: (a) amino acids 53-67 of FABP4; (b) amino acids 29-43 of DUSP1; (c) amino acids 94-108, 120-134, 158-172, 186-200, 270-284 and / or 288-302 of PAI-1; (d) amino acids 93-107, 213-227 and / or 374-388 of ATGL; (e) amino acids 276-295 of HIF1a; (f) amino acids 388-402 and / or 545-558 of IGF-1R; and (g) an amino acid sequence having at least 90% identity to any of the foregoing items. The amino acid sequences of these epitopes are listed in Table 1. In some embodiments, the selected epitope comprises an amino acid sequence having at least 90% identity to any one of the amino acid sequences listed in Table 1. In some embodiments, the polypeptide comprises a combination of at least three, four, five, six or more epitopes listed in Table 1. In some embodiments, the polypeptide comprises at least one of each of (a), (b), (c), (d), (e) and (f). In some embodiments, the polypeptide further comprises one or more linker sequences disposed between the epitopes.

[0087] Pharmaceutical composition

[0088] The pharmaceutical compositions disclosed herein include one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may include buffers such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine, antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of the present disclosure may be formulated for administration as a vaccine, for example, including an adjuvant.

[0089] For example, in some embodiments, a composition comprising a polypeptide is provided, wherein the composition further comprises an adjuvant. In some embodiments, the adjuvant causes a T cell response. In some embodiments, the composition comprises a nucleic acid molecule disclosed herein.

[0090] method

[0091] Described herein are methods for causing type 2 immune responses to inflammation associated with obesity, for preventing or treating inflammation in obesity, and for preventing or treating diseases associated with metabolic obesity in a subject. Also provided are methods for reducing insulin resistance, reducing systemic leptin levels, and reducing tumors associated with obesity. Examples of diseases associated with metabolic obesity include, but are not limited to, diabetes, prediabetes, nonalcoholic fatty liver disease (NAFLD), metabolic-associated fatty liver disease (MAFLD), colitis, and cancer, including, for example, breast cancer. In typical embodiments, methods include administering compositions as described herein to a subject in need. Such compositions are formulated for delivering a therapeutically effective amount of epitopes, as described herein. The determination of amount and appropriate mode of administration is determined under the guidance of the attending physician, taking into account the condition of individual patients.

[0092] Administration and Dosage

[0093] The compositions and / or nucleic acid molecules disclosed herein are administered in any suitable manner, typically using a pharmaceutically acceptable carrier. Suitable methods for administering the compositions, compounds, molecules, nucleic acids and vectors in the context of the invention to a subject are available, and although more than one route may be used to administer a particular composition, a particular route can often provide a more direct and more effective response than another route.

[0094] In the context of the disclosure herein, the dose administered to a patient should be sufficient to achieve a beneficial therapeutic response in the patient over time, or to inhibit disease progression. Thus, the composition is administered to a subject in an amount sufficient to induce an effective response and / or alleviate, mitigate, cure, or at least partially inhibit the symptoms and / or complications of the disease. An amount sufficient to achieve this goal is defined as a "therapeutically effective dose."

[0095] The route of administration, sequence and / or frequency and dosage of the therapeutic compositions disclosed herein will vary from person to person and can be readily determined using standard techniques. In general, an appropriate dosage and treatment regimen provides an amount of active compound sufficient to provide therapeutic and / or preventive benefit. This response can be monitored by determining an improvement in clinical outcomes in treated patients compared to untreated patients.

[0096] Example

[0097] The following examples are provided to illustrate the invention and to aid one of ordinary skill in making and using the invention. The examples are not intended to otherwise limit the scope of the invention in any way.

[0098] Example 1: Type I adipose inflammation reflects an adaptive immune response that leads to metabolic dysregulation.

[0099] The inflammatory infiltrate found in adipose tissue of 'metabolic obese' individuals is an adaptive type I T cell-mediated response that drives metabolic dysfunction. 1 Infiltration of CD8 T cells into fat is an early event. 2 Type I cytokines secreted by CD8 T cells upregulate co-stimulatory molecules on enlarged adipocytes, which act as antigen-presenting cells, providing antigenic peptides to CD4 and CD8 T cells, further stimulating type I T cell activation. 3-5 Therefore, the inflamed adipocyte vaccine is directed against adipocyte-associated proteins. Type I CD4 T cells are essential for maintaining inflammation. 6,7 The resulting effector and memory T cells compete for glucose and fatty acids, leading to metabolic dysfunction in both adipose tissue and the T cells themselves. 8 T cells fail to maintain immune surveillance 9 And the secretion of adipose factors promotes epithelial to mesenchymal and malignant transformation. 10,11 Losing weight doesn’t solve the problem. Even when individuals return to normal weight, immune memory prevents inflammation from resolving. Studies of once-obese mice showed that serum cytokine expression and pro-inflammatory genes in the mammary fat pad were similar to those of currently obese mice and much higher than those of lean controls. 12,13 Furthermore, defects in T cell metabolism cannot be completely corrected by weight loss. 14Similar effects have been observed in humans. Although insulin sensitivity is restored in formerly obese individuals after bariatric surgery and weight loss, serum biomarkers of systemic inflammation and pro-inflammatory gene expression in adipose tissue persist. 15,16 , and T cell levels remained unchanged. 17 Obesogenic T cell memory would lead to long-term increased adipose inflammation in once-obese individuals. 18,19,20 Clinical strategies to reduce type I T cells or increase type II (anti-inflammatory) T cells in inflammatory fat would be beneficial in preventing breast cancer.

[0100] References

[0101] 1. Mathis, D, Cell Metab17, 851-859 (2013).

[0102] 2. Nishimura, S., et al. Nat Med 15, 914-920 (2009).

[0103] 3. Lumeng, CN, Bodzin, JL & Saltiel, ARJ Clin Invest 117, 175-184 (2007).

[0104] 4. Xiao, L., et al. IntJ Obes (L..ond) 40, 112-120 (2016).

[0105] 5. Missiou, A., et al. Thromb Haemost 103, 788-796 (2010).

[0106] 6. Deng, T., et al. Cell Metab 17, 411-422 (2013).

[0107] 7. Cho, KW, et al. Cell Rep 9, 605-617 (2014).

[0108] 8. Ringel, AE, et al. Cell 183, 1848-1866e1826 (2020).

[0109] 9, Zhang, C., et al. Cell Metab 31, 148-161 e145(2020).

[0110] 10. Avtanski, D., et al. Cytokine 120, 155-164 (2019).

[0111] 11.Tiwari,P.,et al.J Exp Med 216,1345-1358(2019).

[0112] 12.Rossi,EL,et al.Cancer Prev Res(Phila)9,339-348(2016).

[0113] 13.Zamarron,BF,et al.Diabetes 66,392-406(2017).

[0114] 14, Rebeles, J., et al. J Infect Dis 219, 1652-1661 (2019).

[0115] 15.Hagman,DK,et al.Metabolism 70,12-22(2017).

[0116] 16.Schmitz,J.,et al.Mol Metab 5,328-339(2016).

[0117] 17.Garcia-Rubio,J.,et al.Sci Rep 8,15203(2018).

[0118] 18.Zou,J.,et al.Cell Mol Immunol 15,630-639(2018).

[0119] 19.Sbierski-Kind,J.,et al.J Immunol 205,45-55(2020).

[0120] 20.Cecil,DL,et al.Breast Cancer Res Treat 139,657-665(201 3).

[0121] Example 2: Development of a vaccine that drives type II T cells into inflammatory adipose tissue.

[0122] This example demonstrates the development of a vaccine that can drive type II T cells into inflammatory adipose tissue. We explored the potential of comprehensive metabolomic profiling and applied artificial intelligence methods to a large set of plasma mass spectrometry data collected from 353 breast cancer cases and 141 controls at the MD Anderson Cancer Center to reveal signatures of lipids and other metabolites involved in distinguishing cancer cases from controls. Validation of the signature in an independent group of 85 cases and 170 matched controls resulted in an area under the curve (AUC) of 0.812 (CI 0.755-0.870). These data and our published data on breast cancer signatures in circulation justify a discovery strategy focused on obesity-related breast cancer risk markers.

[0123] Antigen identification. Antigens were identified by searching PubMed for the following keywords: [(“obese” OR “fat” OR “obesity”) AND (“inflammation” OR “inflammatory” OR “immune” OR “immunogenic”) AND (“overexpressed”) NOT (“macrophage” OR “neutrophil” OR “innate” OR “monocyte” OR “granulocyte” OR “myeloid” OR “dendritic”) NOT (“nervous system” OR “endocrine system” OR “muscular system” OR “liver”) NOT (“expressed in brown adipose tissue”), 19 candidate antigens were retrieved. Targets were given priority if they were associated with hypoxia and / or metabolic syndrome. The 10 candidates finally identified are listed in Table 3.

[0124] Human subjects. Sample collection was approved by the Human Subjects Department of the University of Washington (UW). Peripheral blood mononuclear cells (PBMCs) were collected from 10 volunteer controls (4 males and 6 females, median age: 49 years, range 18-79 years) and cryopreserved as previously described (1).

[0125] Analysis of peptide-specific T cell responses. Peptides predicted to bind to human MHCII hybrids were selected using a web-based algorithm as described previously (Table 3) (2). Peptides were constructed and purified by high-performance liquid chromatography (>90% purity; CPC Scientific). Antigen-specific IFN-gamma (γ) or IL-10 production by human PBMCs was assessed by ELISPOT as previously described using 10 μg / ml peptide (3). Antigen-specific cytokine secretion by mouse splenocytes was assessed by ELISPOT as previously published with the following modifications; splenocytes were incubated with antigen for 72 hours, and then spots were developed using the AEC substrate kit (BD Biosciences) (2). Positive reactions were defined by a statistically significant difference (p<0.05) between the mean number of spots in the experimental wells and the mean number of spots in the no-antigen control wells, and all experiments were performed in duplicate four times. Data are reported as the mean number of spots for each experimental antigen minus the mean number of spots detected in the no-antigen control wells ± SEM or SD (corrected spots per well: 2x10 5 PBMC or 3.5x10 5 The product ratio of the mean response amplitude and the response incidence rate was calculated by IFN-γ and IL-10 ELISPOT assay (IL-10 发生率X幅度 / IFN-γ 发生率X幅度 )(4). Donors with a ratio greater than 1.4 and ≤10% producing an IFN-γ response defined a Th2 selective epitope and a ratio less than 1 defined a Th1 selective epitope. To facilitate graphical representation of the ratio values ​​for Th1 epitopes, the ratio was inverted (IFN-γ / IL-10) and plotted in Figure 1.

[0126] Animal Model. Animal care and use were in accordance with institutional guidelines. Male C57BL / 6 (Jackson Laboratory) were used in this study. Mice were fed a high-fat diet (60% calories from fat; Bio-serve) or a normal diet starting at 10 weeks of age. Body weights of mice were measured weekly during the study. Ten-week-old TgMMTV-neu mice were fed a HFHS diet for 4 weeks and then randomly divided into 2 cohorts, one receiving adjuvant only (Alum) and one receiving ADVac. Mice were treated with Alum at 31 weeks of age or when tumors were >1000 mm 3 was executed.

[0127] Metabolic studies. Before conducting metabolic studies, mice were fasted for 4 h. For glucose tolerance test, mice were injected with 1 g glucose / kg body weight, and for insulin tolerance test mice, mice were injected with 0.75 U / kg body weight human insulin (Eli Lilly). Glucose concentrations in blood collected before and after injection at 30 min, 60 min, and 120 min were measured using a glucometer (Freestyle Freedom Lite).

[0128] Lipidomics. Ten milligrams of frozen fat samples were homogenized in 15% water, 60% methanol, and 25% dichloromethane. The mixture was vortexed for 5 seconds, and 25 μL of isotope-labeled internal standard mixture was added to the tube. The sample was left to incubate at room temperature for 30 minutes. Next, another portion of 55% water and 45% dichloromethane was added to the tube, followed by gentle vortexing for 5 seconds and centrifugation at 2500×g for 10 minutes at 15°C. The bottom organic layer was transferred to a new tube, and 900 μL of dichloromethane was added to the original tube for a second extraction. The combined extracts were concentrated under nitrogen and reconstituted in 250 μL of running solution (10 mM ammonium acetate in 50:50 methanol: dichloromethane). Quantitative lipidomics was performed with the Sciex Lipidyzer platform, which consists of a Shimadzu Nexera X2 LC-30AD pump, a Shimadzu Nexera X2SIL-30AC autosampler and an Sciex for Differential Mobility Spectroscopy (DMS) 5500 mass spectrometer. 1-Propanol was used as a chemical modifier for DMS. The sample was introduced into the mass spectrometer by flow injection analysis at 8uL / min. Each sample was injected twice, once with DMS turned on (PC / PE / LPC / LPE / SM) and once with DMS turned off (CE / CER / DAG / DCER / FFA / HCER / LCER / TAG). Multiple reaction monitoring (MRM) and positive / negative polarity switching were used to measure lipid molecular species. The positive ion mode detected lipid categories SM / DAG / CE / CER / DCER / HCER / DCER / TAG, and the negative ion mode detected lipid categories LPE / LPC / PC / PE / FFA. A total of 1070 lipids and fatty acids were analyzed. Analyst 1.6.3 and Lipidomics Workflow Manager1.0.5.0 were used to acquire and process data.

[0129] Aqueous metabolomics. Ten mg of frozen adipose tissue was homogenized in 80% methanol: 9% PBS: 11% H2O. After centrifugation, the aqueous extract was collected and dried in a Speed-Vac at 30°C. All samples were analyzed using an Agilent 7890GC instrument equipped with a 5977 mass selective detector (MSD) using an HP-5MS UI GC column. GCMS conditions were used according to Agilent's Fiehn library instructions. The retention times of individual metabolites were annotated according to known standards. Peak intensities were normalized to total ion counts (TIC) and protein concentrations and depicted as fold changes relative to controls. Data were acquired and processed using MetaboAnalyst 4.0.2.

[0130] Isolation and flow cytometry of stromal vascular cells. Epididymal adipose tissue was collected, weighed and placed in a C tube (Miltenyi) containing 5 ml of DMEM (supplemented with 10 mg / mL BSA, 0.03 mg / mL Liberase Blendzyme 3 (Worthington) and 50 U / mL DNase), and then incubated in a gentleMACs dissociator using the manufacturer's "mTDK1" program. The aqueous slurry was filtered and the red blood cells were lysed. The washed cells (1-2x10 6 ) were incubated with live / dead stain (FVS450; ThermoFisher), washed and incubated with FC receptor blocking antibodies to reduce nonspecific binding. After washing, cells were stained with fluorescent dye-coupled monoclonal antibodies for phenotypic analysis. Cells were surface stained with CD3 PE-Cy7, CD4 BV605, and CD8 APC-Cy7. After washing, cells were stained with FOXP3 Alex 488 according to the FOXP3 staining protocol of eBioscience. The % of CD8+ cells in surviving lymphocytes was analyzed. FOXP3-positive cells were analyzed in CD3+CD4+T cells. Only data for samples with counts>350 cells were included.

[0131] Vaccination. Mice were immunized subcutaneously using a 26 1 / 2 G needle. Each mouse was injected with 50 μl of Imject Alum Adjuvant (Thermo Fisher Scientific) peptide pool (100 μg each). In order to generate an effective immune response to the overexpressed self-antigen, four immunizations were performed at two-week intervals.

[0132] Cell culture and protein expression. 3T3-L1 preadipocytes were differentiated for 10 days with 0.5mM IBMX, 1ug / ml insulin, 0.25uM dexamethasone and 2uM rosiglitazone to become adipocytes. 94% of the cultures were positive for Oil Red-O. The cultures were stimulated with 2.5nM TNFa daily for 5 days and confirmed to be inflammatory (increased NFKb activation), insulin resistant (reduced IRS-1 expression) and to have reduced adiponectin expression. Adipose tissue was collected and homogenized using a mechanical ultrasonic generator and the lipid layer was removed. Cell lysates were separated by SDS / PAGE (4) and Western blotted. The antibodies used were rabbit anti-mouse IGF-IR (2 μg / mL; Genetex), rabbit anti-mouse ATGL (1 μg / mL; Genetex), rabbit anti-mouse PAI-1 (5 μg / mL; Genetex), goat anti-mouse FABP4 (1 μg / ml; Santa Cruz Biotech), rabbit anti-mouse HIF1a (2 μg / mL; abcam), HRP-conjugated goat anti-rabbit, and rabbit anti-goat (dilution 1:10,000; Invitrogen).

[0133] Serum ELISA. Serum amyloid A measurements were performed using the SAA mouse ELISA kit (Invitrogen). IL-6 and leptin measurements were performed using the corresponding mouse Duoset ELISA (Rand D Systems). Cholesterol was measured using the HDL and LDL / VLDL quantification kits, and triglycerides were measured using the TG quantification kit (LS Bio). All ELISAs were performed according to the manufacturer's instructions.

[0134] Statistical analysis. Data are expressed as mean + standard error of the mean (SEM). Statistical analysis was performed using Prism 7.0 or 8.0 software (GraphPad). Statistical comparisons between 2 groups were performed by unpaired two-tailed t-test. Statistical comparisons between 3 or more groups were performed by one-way ANOVA, followed by Tukey post hoc analysis to determine statistical significance. Values ​​of p < 0.05 were considered statistically significant.

[0135] Table 2: Genes associated with obesity, metabolic syndrome and / or type 2 diabetes

[0136]

[0137]

[0138] result

[0139] IL-10 selective epitopes can be identified from proteins that are abnormally expressed in obese adipose tissue. We sought to identify candidate antigens as targets for multi-epitope, multi-antigen anti-inflammatory vaccines. IFN-γ and IL-10 inducible epitopes were identified from the candidate antigens (Table 3). We have previously identified IL-10 selective epitopes for IGF-IR (p388-402 and p545-558) and HIF1a (p276-295) (4, 5). One of the six epitopes of FABP4 (p53--67; Figure 1A ), 1 / 5 epitopes in DUSP1 (p29-43; Figure 1B ), 2 / 5 epitopes of ATGL (p93-107, p213-227, and p374-388; Figure 1C ) and 6 / 8 epitopes of PAI-1 (p94-108, p120-134, p158-172, p186-200, p270-284, and p288-302) were identified as the major inducers of IL-10 in PBMCs ( Figure 1D ). No Th2 selective epitopes were identified in IGF-1, 11BHSDB1, SEMA3E, or SOCS3. These antigens were not considered further.

[0140] Table 3: Candidate antigens, recognition codes and epitope locations

[0141]

[0142]

[0143] We next questioned whether the candidate antigens were truly upregulated in inflammatory adipocytes. Protein expression of HIF1a, IGF-IR, PAI-1, and FABP4 was upregulated after rmTNF-a stimulation compared with resting control cells (all p<0.0001)( Figure 2A and Figure 2B ). Expression of ATGL was significantly reduced after stimulation compared to control (p<0.0001). We were unable to measure protein expression for DUSP1, but we observed a significant increase in gene expression after stimulation with TNFa compared to non-inflammatory controls (p<0.0001). In similar experiments, we questioned whether these proteins would also be upregulated in inflammatory fat derived from obese mice, providing us with a model for testing adipocytes against Th2 selective vaccines. Western blot analysis demonstrated that the majority of candidate antigens evaluated were overexpressed in inflammatory fat compared to controls ( Figure 2C and Figure 2DWestern blot analysis revealed that the protein expression of HIF1a (p = 0.018), PAI-1 (p < 0.0001), and FABP4 (p < 0.0001) was increased compared with the expression in adipose tissue of normal weight mice. The protein expression of IGF-IR and ATGL was not increased in obese adipose tissue ( Figure 2C and Figure 2D ).

[0144] The multi-antigen fat-directed vaccine generated a selective IL-10 response against all antigens in most mice. We initially validated that a single epitope for each antigen was highly homologous between mouse and human (Table 4) and could generate IL-10 and little IFN-γ response after immunization. For example, when mice vaccinated with the vaccine epitope FABP4-p53-67 were stimulated with the vaccine epitope compared with the negative control epitope HIVp52-66 (p < 0.0001; Figure 3A ), DUSP1-p29 vaccinated mice (p<0.0001; Figure 3B ) or HIF1a-p276-295 vaccinated mice ( p = 0.0004; Figure 3C ) splenocytes, significantly more IL-10 secreting T cells were observed. Similarly, as compared to controls, PAI-1-p94-108 and PAI-1-p158-172 epitope pools (all p < 0.001; Figure 3D ), ATGL-p213-227, and ATGL-p374-388 epitope pools (all p < 0.01; Figure 3E ) or IGF-IR-p545 and IGF-IR-p388 epitope pools (all p < 0.0001; Figure 3F ) vaccination generated significantly enhanced antigen-specific IL-10 responses to each epitope. No IFN-γ responses were detected for any epitope after vaccination.

[0145] Table 4: Homology of candidate antigens identified as vaccine targets

[0146] Candidate antigen % Mouse homology HIF1α 87％ FABP4 92％ IGF1R 96％ PAI1 79％ DUSP1 96％ ATGL 88％

[0147] Immunization with the multi-antigen fat-directed vaccine (which we call AdVac) generated significant IL-10 responses to each epitope as compared to the negative control epitope (all p < 0.01; Figure 4A There was no antigen competition with AdVac. The median incidence of response to any antigen was 80% (range 70-90%; Figure 4B ). Most mice responded to all antigens. Sixty percent of the mice induced a significant IL-10 response to all six antigens, although an additional 20% induced a response to a total of five antigens ( Figure 4C ).

[0148] AdVac reduced CD8+ T cell levels and increased T regulatory cell levels in adipose tissue of obese mice. Evaluation of the phenotype of adipose-infiltrating T cells in obese and lean individuals revealed that Th1 and CD8 T cells predominated in obese fat and were associated with insulin resistance. In addition, IL-10-secreting T regulatory cells (Tregs), which have potent anti-inflammatory effects, were present at very low levels (6). Therefore, we evaluated the CD8+ and Treg populations infiltrating in adipose tissue of immunoobese mice and compared these levels with those of normal weight mice. As compared with controls (11±1.1%; p=0.0011; Fig. 7A ), significantly fewer CD8+ T cells were observed in adipose tissue of obese mice immunized with AdVac compared to normal weight controls (mean; 7±0.8%). Although there were still significantly more CD8+ T cells in the fat of AdVac-immunized obese mice compared to normal weight controls (mean, 4.1±0.3% p=0.043), 40% of AdVac-immunized mice had CD8+ T cell levels within the mean and 2SD observed for normal weight mice. In contrast, none of the obese mice immunized in the control group had T cells in this range. The reduction in CD8+ T cells after vaccination was specific to adipose tissue, as no changes in this T cell population were observed in the matched spleen ( Figure 7B Although immunization with AdVac did not increase Tregs in adipose tissue of obese mice to the level detected in normal-weight mice (56±5.6%; p=0.0062), it did increase Tregs in adipose tissue of obese mice compared with control mice (16±1.5%; p=0.031; Figure 7C ) compared with the control group, the T cell population in the adipose tissue of mice immunized with AdVac was significantly increased (27±3.7%). No changes in Treg levels were observed after vaccination in matched spleens ( Fig.7D ).

[0149] AdVac increased glucose sensitivity and reduced insulin resistance in obese mice. AdVac did not alter serum cholesterol, triglyceride, or IL-6 levels compared to control immunized mice. However, AdVac increased glucose sensitivity. Blood glucose concentrations were significantly lower in AdVac-immunized obese mice compared to control obese mice at all time points assessed by GTT (all p<0.01; Fig. 6A ). Additionally, the AUC of AdVac-immunized mice was predicted to be lower than that of control-immunized obese mice (p<0.0001), but significantly higher than that of normal-weight mice (p<0.0001; Figure 6BSimilar results were observed in the insulin tolerance test (ITT). Blood glucose was lower in mice treated with AdVac at all time points during the study (all p < 0.001; Figure 6C ). As compared with control obese mice, the AUC of mice immunized with AdVac was significantly reduced (p<0.0001), but still higher than that measured in normal weight mice (p<0.0001; Fig.6D ).

[0150] AdVac reduced adipose tissue metabolites associated with glucose insensitivity and insulin resistance. Adipocytes have been shown to be regulators of glucose homeostasis and can regulate systemic energy balance by changing their own metabolism (Rosen, et al. Nature. 2006; 14; 444 (7121): 847-53). We next questioned whether immunization would induce changes in the adipose tissue metabolome or lipidome. Although only two lipids were significantly lower in the fat of immunized mice, the levels of 50 aqueous metabolites in the tissues of mice immunized with ADVac were significantly lower compared to controls ( Fig. 8A ). As compared with the control, the top four pathways regulated by vaccination (p>-log1.5; Figure 8B ) demonstrated a decrease in metabolites associated with glycolysis, an important pathway for cancer cell metabolism and growth. In the Warburg effect, cancer cells are observed not to produce energy normally through oxidative phosphorylation, but rather through aerobic glycolysis, taking up high levels of glucose followed by lactic acid fermentation. Pyruvate is the end product of glycolysis, and nicotinamide adenine dinucleotide is an important coenzyme that regulates glycolysis. Gluconeogenesis results in the production of glucose from non-carbohydrate carbon substrates and may help provide the high levels of glucose required for the abnormal metabolism of cancer cells. Increased glycolysis has been shown to be a key metabolic pathway that promotes breast cancer metastasis. Notably, after vaccination, four metabolites in the top pathway did not differ from levels observed in untreated lean mice ( Figure 8C-8F ).

[0151] Obese TgMMTV-neu mice developed a greater number of tumors, which grew at a faster rate, than lean TgMMTV-neu mice. Fig.9A is a survival curve showing the percentage of tumor-free mice at 30 weeks in obese mice (lower line) and lean mice (upper line). Fig. 9B Tumor growth rates (mm) in lean and obese mice are shown. 3 / sky).

[0152] AdVac restored insulin sensitivity, reduced systemic leptin levels, and resulted in lower tumor development in obese Tg-MMTV-neu mice. Similar to the diet-induced obesity (DIO) model, we observed increased glucose sensitivity in the AdVac immunized group compared to obese Tg-MMTV-neu mouse controls. GTT revealed significantly lower blood glucose levels before testing in the immunized group compared to controls (p<0.0001). The immunized group exhibited lower insulin resistance than controls, as significantly less glucose was detected in the blood at each measured time point (all p<0.0001). After immunization of obese mice, significantly fewer CD8+ cells were observed in mammary adipose tissue compared to control obese (p=0.001; Fig. 10A Significantly less leptin was detected in the serum of mice vaccinated with ADVac compared to controls (Alum; p=0.024; Fig. 10B ). The median age of tumor development was 25 weeks in the control cohort and 29 weeks in the ADVac immunization group (p = 0.009; Fig. 10C Sixty percent (9 / 15) of the vaccinated mice were tumor-free at the planned end of the study, while 100% of the control mice had developed tumors before the study endpoint.

[0153] References

[0154] 1. Disis ML, et al. J Immunol Methods. 2006; 308: 13-8.

[0155] 2.Park KH, et al.Cancer Res.2008;68:8400-9.

[0156] 3. Cecil DL, et al. Cancer Res. 2014;74:2710-8.

[0157] 4. Cecil DL, et al. Clin Cancer Res. 2017; 23: 3396-404.

[0158] 5. Cecil DL, et.al.J Immunother Cancer 2021 Mar;9(3):e002355

[0159] 6. McLaughlin T, et al. Arterioscler Thromb Vasc Biol, 2014;34:2637-43.

[0160] Example 3: Confirming the effectiveness and safety of breast cancer prevention vaccine targeting inflammatory adipocytes.

[0161] Two transgenic mouse models can be used to validate the efficacy of ADVac. First, the TgMMTV-neu mouse, a model of luminal B breast cancer, can be used. 7,8 Because the mice are born without lesions, the goal is to prevent primary breast cancer. The second model is TgC3(1)-Tag (C3T), which represents "basal-like" TNBC. 9 C3T mice are born with DCIS, so vaccination would represent an interception of breast cancer. We have shown that feeding the HFHS diet to TgMMTV-neu mice leads to insulin resistance and accelerated breast cancer development, with the median age of tumor development being 25 weeks in the HFHS cohort and 28.4 weeks in the normal diet cohort (p=0.0011). Mice fed the HSHS diet had faster tumor growth (average, 12.1 mm3 / day) compared to tumors in lean mice (average, 4.2 mm3 / day; p=0.004). 3 / day) and an increase in tumor multiplicity.

[0162] Table 5: Experimental groups

[0163]

[0164] In the TgMMTV-neu model, we will evaluate 5 experimental groups (Table 5). We included group 5 to document that antigen-specific IL-10 secreting T cells are maintained during active immunization and to evaluate the immunological efficacy of the vaccine booster. We will use the antigen-specific T cell lines from group 5 to determine the specific T cell receptors used to identify the antigens in ADVac. Defining the specific TCRVbeta(b) will allow us to track the distribution of vaccine-induced T cells. When mice become obese, mice receiving ADVac will begin immunization with 100ug of each epitope, 10-14 days apart, for a total of 4 times. Booster vaccines will be administered every 6 weeks until the end of the study. When mice have tumors reaching 1000mm 3Mice will be sacrificed at or if tumor-free at 52 weeks. We assume that we will be able to replicate a 60% protection rate. Secondary endpoints include the rate of tumor growth in mice developing tumors to ensure that ADVac does not accelerate tumor proliferation and the diversity of mammary tumor development. Spleens will be collected at the end of the experiment to assess ADVac antigen-specific immunity using IL-10 ELISPOT. Visceral and mammary adipose tissue and tumor samples will be collected and preserved. We will include 30 mice per group. Using a similar experimental design as described for the TgMMTV-neu model, we will test ADVac immunity in the C3T mouse model using a similar protocol for each group. C3T tumors develop earlier and grow faster than those of TgMMTV-neu, so we will start vaccination 1 week after the animals start feeding a specific diet. The expected primary outcome is that we will prevent tumors in 25% of the vaccinated mice. 10 Similar analyses as described for TgMMTV-neu will be performed.

[0165] Immune cells play a key role in the propagation of adipose inflammation and the generation of metabolic dysfunction. 11 We observed a significant difference in the incidence of palpable tumors in TgMMTV-neu mice immunized with ADVac at week 31. To this end, we will add 10 mice to the above TgMMTV-neu groups 1-4 to collect visceral and mammary fat and tumors at week 31 for the experiments described below. We expect to obtain samples from the following mouse cohorts; (1) lean mice bearing tumors, (2) obese mice bearing tumors, (3) obese mice bearing tumors injected with adjuvant control vaccine, (4) obese mice that are protected from tumor growth by ADVac immunization, and (5) obese mice that develop tumors despite vaccination.

[0166] We will use mass cytometry (CyTOF) to assess vaccine-induced changes in immune cell subsets and activation states. Using broad and unbiased cellular analysis, our goal will be to discern differences in cellular composition or phenotype between vaccine-protected and non-vaccine-protected mice. We will also use material from lean tumor-bearing mice and obese tumor-bearing mice as comparisons. The Newell lab has extensive experience using mass cytometry to study a variety of tissues and cell types, including multiple tumor types in both mice and humans. 12,13 To significantly reduce batch effects, normalized beads will be used 14 and antibody cocktail 15A frozen aliquot of the protected mice was prepared. We will use a multi-omic approach based on 10x Genomics single cell sequencing to more deeply compare the protein and gene expression profiles between protected and unprotected mice. For these experiments, cells will be stained with the CITE-seq antibody cocktail (Biolegend, containing all the wovens in the mass cytometry panel) prior to live cell FACS sorting and analysis using the 5'10x Genomics immunoassay platform.

[0167] As described, metabolic profiling of visceral and adipose tissues will be performed to identify significant differences between ADVac-protected and unprotected mice. A systems-level analysis of cellular metabolic function will be performed by real-time measurement of glucose and fatty acid energy metabolism using the Agilent Seahorse XF 96 Extracellular Flux Analyzer. Direct ex vivo samples of purified CD4 and CD8 T cells will be isolated from mammary fat, visceral fat, tumors, and spleens of obese mice immunized with alum vaccine or ADVac and compared to purified T cells from matched tissues isolated from unimmunized obese and lean control mice. The Kalia lab will measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) as readouts of mitochondrial respiration and glycolysis, respectively. The relative functional relevance of ECAR and OCR will be confirmed by forcing cellular respiration to shut down mitochondrial respiration using the glucose analog 2-deoxyglucose (2-DG) or oligomycin (ATP synthase inhibitor) and rotenone + antimycin A (complex I and II inhibitors). Trifluoromethoxycarbonyl cyanide phenylhydrazone will be used to measure the cellular spare respiratory capacity (SRC) relative to the basal OCR by collapsing the proton gradient, driving the respiratory chain to operate at maximum capacity. Etomoxir will be used to inhibit fatty acid oxidation to further confirm fatty acids as the main nutrient source. The metabolic function of T cells will be correlated with metabolites in the corresponding tissues.

[0168] References

[0169] 7. Guy, CT, et al. Proc Natl Acad Sci USA 89, 10578-10582 (1992).

[0170] 8. Herschkowitz, JI, et al. Genome Biol 8, R76 (2007).

[0171] 9. Maroulakou, IG, et al. Proc Natl Acad Sei USA 91, 11236-11240 (1994).

[0172] 10. Gad, E., et al. Breast Cancer Res Treat 148, 501-510 (2014).

[0173] 11. Turbitt, W Jet al. Immunol Rev 295, 203-219 (2020).

[0174] 12. Li, S., et al. Proc Natl Acad Sci USA 118 (2021).

[0175] 13. Cheng, Y., et al. Immunity 54, 1825-1840 e1827(2021).

[0176] 14. Finck, R., et al. Cytometry A 83, 483-494 (2013).

[0177] 15. Schulz, AR, et al. Cytometry A 95, 910-916 (2019).

[0178] Throughout this application, various publications are cited. The disclosures of these publications are hereby incorporated by reference into this application in their entireties in order to more fully describe the state of the art to which this invention pertains.

[0179] From the foregoing it will be appreciated that although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the invention is not to be limited except as by the appended claims.

Claims

1. A nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide comprising at least two epitopes selected from the group consisting of: (a) amino acids 53-67 of FABP4; (b) amino acids 29-43 of DUSP1; (c) amino acids 94-108, 120-134, 158-172, 186-200, 270-284 and / or 288-302 of PAI-1; (d) amino acids 93-107, 213-227 and / or 374-388 of ATGL; (e) amino acids 276-295 of HIF1a; (f) amino acids 388-402 and / or 545-558 of IGF-1R; and (g) an amino acid sequence that is at least 90% identical to any of the preceding items.

2. The nucleic acid molecule of claim 1, wherein the polypeptide is selected from amino acids 53-67 of FABP4, amino acids 29-43 of DUSP1, amino acids 276-295 of HIF1a, and combinations thereof.

3. The nucleic acid molecule of claim 1, wherein the polypeptide comprises amino acids 94-108 and / or 158-172 of PAI-1, amino acids 213-227 and / or 374-388 of ATGL, amino acids 388-402 and / or 545-558 of IGF-1R, or a combination thereof.

4. The nucleic acid molecule of claim 1, wherein the polypeptide comprises at least one of each of (a), (b), (c), and (d).

5. The nucleic acid molecule of claim 1, wherein the polypeptide comprises each of: (a) amino acids 53-67 of FABP4; (b) amino acids 29-43 of DUSP1; (c) amino acids 94-108 and 158-172 of PAI-1; (d) amino acids 213-227 and 374-388 of ATGL; (e) amino acids 276-295 of HIF1a; and (f) Amino acids 388-402 and 545-558 of IGF-1R.

6. A nucleic acid molecule as described in claim 1, wherein the polypeptide comprises at least one of each of (a), (b), (c), (d), (e) and (f), and optionally further comprises a linker sequence disposed between the epitopes.

7. The nucleic acid molecule of claim 6, wherein the linker sequence is selected from the group consisting of: GPGPG and GGGS.

8. The nucleic acid molecule of claim 1, wherein the nucleic acid sequence comprises a promoter sequence.

9. The nucleic acid molecule of claim 1, wherein the nucleic acid sequence comprises a heterologous sequence.

10. The nucleic acid molecule of claim 9, wherein the heterologous sequence encodes a promoter, a transcription start site, a translation start site, a detectable marker, an mRNA processing splice site, a polyadenylation sequence and / or a regulatory element.

11. A vector comprising the nucleic acid molecule of any one of claims 1 to 10, wherein the vector is capable of directing the expression of the encoded polypeptide.

12. The vector of claim 11, wherein the vector is a DNA plasmid, messenger RNA (mRNA) or a viral vector.

13. The vector of claim 12, wherein the viral vector is a lentiviral vector, an adenoviral vector or a poxviral vector.

14. The vector of claim 12, wherein the vector is mRNA.

15. A polypeptide comprising at least two epitopes selected from the group consisting of: (a) amino acids 53-67 of FABP4; (b) amino acids 29-43 of DUSP1; (c) amino acids 94-108, 120-134, 158-172, 186-200, 270-284 and / or 288-302 of PAI-1; (d) amino acids 93-107, 213-227 and / or 374-388 of ATGL; (e) amino acids 276-295 of HIF1a; (f) amino acids 388-402 and / or 545-558 of IGF-1R; and (g) an amino acid sequence that is at least 90% identical to any of the preceding items.

16. A composition comprising a polypeptide as described in claim 15, wherein the polypeptide comprises at least one of each of (a), (b), (c), (d), (e) and (f), and optionally further comprises one or more linker sequences disposed between the epitopes.

17. A composition comprising the polypeptide of claim 15, wherein the composition further comprises an adjuvant.

18. A method for eliciting a type 2 immune response to inflammation associated with obesity, for preventing or treating inflammation in obesity, and / or for preventing or treating a disease associated with metabolic obesity in a subject, the method comprising administering to the subject a composition comprising a nucleic acid molecule as described in claim 1 or a composition as described in claim 15.

19. The method of claim 18, wherein the disease associated with metabolic obesity is cancer.

20. The method of claim 19, wherein the cancer is breast cancer.

21. The method of claim 18, wherein the disease associated with metabolic obesity is non-alcoholic fatty liver disease (NAFLD).

22. The method of claim 18, wherein the disease associated with metabolic obesity is prediabetes.

23. The method of any one of claims 18 to 22, wherein the subject is a human.

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