Vaccine adjuvants and formulations

Abstract

Compositions comprising an antigen, a carbohydrate, and a metabolizable oil, methods of administering such compositions to a subject, methods of making such compounds, and related compositions, methods, and uses.

Description

[0001] Related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 806,422, filed February 15, 2019, which is incorporated herein by reference in its entirety for all purposes.

[0003] Sequence Listing

[0004] This specification references a sequence listing (submitted electronically as a .txt file named "CCI_005_Seq_Listing.txt" on February 13, 2020). The .txt file was generated on February 13, 2020 and is 6 kb in size. The entire contents of the sequence listing are incorporated herein by reference. Background Art

[0005] Vaccines generally contain at least two main components: an immunogen, which serves as the target of the adaptive immune response, and an adjuvant, which enhances the adaptive immune response. Complete Freund's adjuvant (CFA) is a suspension of dead mycobacteria in a liquid prepared from non-metabolizable oils. CFA is widely considered the "gold standard" against which all other adjuvants are compared, as its effectiveness in inducing adaptive immunity has been demonstrated for over 70 years. However, CFA cannot be used as an adjuvant in human vaccinations because its toxic effects are primarily related to the induction of non-resolving granulomas and abscesses at the inoculation site.

[0006] Therefore, there is a need for adjuvants suitable for human vaccination that can also promote the induction of robust immune responses.

[0007] Breast cancer is the second most common cause of cancer-related death in women. There are several different genetic subtypes of breast cancer, and treatments are often tailored to specific subtypes. For example, hormone therapy and drugs that target the estrogen receptor (ER) are designed to treat ER-positive cancers. Triple-negative breast cancer (TNBC) is the most aggressive and deadliest form of breast cancer and is notoriously difficult to treat. TNBC cancer cells are negative for estrogen receptors (ER), progesterone receptors (PR), and HER2, making drugs designed to treat those receptors ineffective in TNBC.

[0008] Breast cancer treatments that are effective for refractory breast cancer subtypes, including TNBC, are needed. Summary of the Invention

[0009] Recent clinical vaccine formulations induce responses from proinflammatory T-helper type 1 cells that produce gamma-interferon (IFNy), but rarely, if ever, from T-helper type 17 cells that produce interleukin-17 (IL-17). The present invention encompasses the insight that many current clinical vaccine formulations are ineffective because they fail to elicit both type 1 and type 17 immune responses. In accordance with the present invention, compositions are provided that induce immune responses including both type 1 and type 17 T cells, while inducing limited toxicity or no toxicity.

[0010] The present invention also encompasses the development of vaccine formulations that can induce adaptive immune responses that effectively suppress and / or prevent the growth of breast cancer. The presently disclosed compositions comprise an alpha-lactalbumin polypeptide antigen and an adjuvant component as further described herein. Alpha-lactalbumin is constitutively overexpressed in most TNBCs and in a lower percentage of other forms of breast tumors. Thus, the presently disclosed vaccine formulations, compositions, and methods can be useful for treating and / or preventing the most aggressive forms of breast cancer.

[0011] In one aspect, compositions are provided comprising: a carbohydrate and a metabolizable oil, wherein (i) the composition further comprises an antigen, or (ii) the carbohydrate comprises a polysaccharide, and the composition comprises a mixture of at least two polysaccharides.

[0012] In some embodiments, the antigen is a tumor-associated antigen.

[0013] In some embodiments, the carbohydrate comprises a polysaccharide, and the composition comprises a mixture of at least two polysaccharides.

[0014] In some embodiments, the composition comprises a tumor-associated antigen, the carbohydrate comprises a polysaccharide, and the composition comprises a mixture of at least two polysaccharides.

[0015] In some embodiments, the composition (i) comprising an antigen or (ii) further comprising an antigen, when administered to a subject, is capable of inducing an antigen-specific T cell immune response including type 1 and type 17 proinflammatory T cell responses.

[0016] In some embodiments, the carbohydrate binds to a pattern recognition receptor. For example, the pattern recognition receptor can be TLR2 or dectin-1.

[0017] In some embodiments, the mixture of polysaccharides comprises at least three polysaccharides.

[0018] In some embodiments, the polysaccharide or each polysaccharide in the mixture is selected from the group consisting of chitin, dextran, dextran, lentanan, mannan, and combinations thereof.

[0019] In some embodiments, the polysaccharide or mixture of polysaccharides includes a glucan, such as a β-glucan (e.g., 1-3 β-glucan). For example, the mixture of polysaccharides can include a mixture of chitosan, glucan, and mannan. In some embodiments, at least 50% of the carbohydrates in the composition are β-glucans.

[0020] In some embodiments, the composition comprises zymosan.

[0021] In some embodiments, the metabolizable oil comprises a purified oil. For example, the purified oil can be a mineral oil, such as DRAKEOL TM 6VR.

[0022] In some embodiments, the metabolizable oil comprises a biodegradable oil. For example, the biodegradable oil can be isopropyl myristate, squalene oil, squalane oil, vegetable oil, or a combination thereof. In some embodiments, the biodegradable oil is a vegetable oil, such as, for example, a vegetable oil selected from the group consisting of almond oil, castor oil, celery seed oil, coconut oil, corn oil, cottonseed oil, olive oil, peanut oil, peach kernel oil, safflower oil, and soybean oil.

[0023] In some embodiments, the metabolizable oil is a pharmaceutical grade oil.

[0024] In some embodiments, the composition further comprises a surfactant, such as anhydrous mannitol monooleate, isoanhydrous mannitol monooleate, or a combination thereof. In some embodiments, the surfactant comprises anhydrous mannitol monooleate. For example, the composition may comprise MONTANIDE TM , such as MONTANIDE TM ISA 51 VG.

[0025] In some embodiments, the composition is an emulsion of water and oil, eg, a water-in-oil emulsion.

[0026] In some embodiments, the antigen comprises a polypeptide antigen. In some embodiments, the polypeptide antigen is a retired autologous antigen.

[0027] In some embodiments, the polypeptide antigen comprises an α-lactalbumin polypeptide. For example, the α-lactalbumin polypeptide may have an amino acid sequence comprising at least 8 consecutive amino acids of SEQ ID NO:5.

[0028] In some embodiments, the antigen and carbohydrate (or mixture of polysaccharides) are present in a ratio of about 10:1 to about 1:10 (w / w). In some embodiments, the antigen and carbohydrate (or mixture of polysaccharides) are present in a ratio of about 1:1.

[0029] In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

[0030] In some embodiments, the composition further comprises an antibiotic.

[0031] In one aspect, methods are provided comprising administering to a subject a therapeutically effective amount of a composition as disclosed herein, e.g., a composition comprising an antigen, a carbohydrate, and a metabolizable oil.

[0032] In some embodiments, the subject is a mammal, e.g., a human.

[0033] In some embodiments, the subject is a non-lactating female subject.

[0034] In some embodiments, the subject has cancer or is at risk of developing cancer.

[0035] In some embodiments, the subject has not been diagnosed with cancer.

[0036] In some embodiments, the cancer is breast cancer, e.g., metastatic breast cancer, primary breast cancer, and / or triple negative breast cancer.

[0037] In some embodiments, the cancer comprises cells that overexpress a-lactalbumin.

[0038] In some embodiments, the therapeutically effective amount comprises more than one dose, e.g., three or more doses. In some embodiments, the therapeutically effective amount comprises no more than three doses.

[0039] In some embodiments, the interval between each dose administration is one week or more, e.g., at least four weeks. In some embodiments, the interval between each dose administration is about four weeks.

[0040] In some embodiments, each dose contains about the same amount of antigen. In some embodiments, each dose contains about the same amount of antigen and the same amount of carbohydrate.

[0041] In some embodiments, each dose contains between about 1 pg and about 5 mg of antigen, e.g., between about 50 pg and about 2 mg of antigen, or between about 100 pg and about 1 mg of antigen.

[0042] In some embodiments, the composition is administered by subcutaneous, intradermal, subdermal, or intramuscular injection.

[0043] In some embodiments, administration of the composition induces an antigen-specific T cell immune response. In some embodiments, the T cell immune response includes CD4+ T cells, CD8+ T cells, or both. In some embodiments, the T cell immune response includes a type 1 or type 17 proinflammatory T cell response. In some embodiments, the T cell immune response includes both type 1 and type 17 proinflammatory T cell responses.

[0044] In some embodiments, administration results in a reduction in granuloma formation relative to a reference level, e.g., a level of granuloma formation observed in a subject administered a composition comprising Freund's complete adjuvant.

[0045] In some embodiments, the subject has been administered, will be administered, or is concurrently administered an additional anti-cancer therapy, e.g., an anti-cancer therapy comprising an anti-cancer agent. In some embodiments, the additional anti-cancer agent is selected from the group consisting of bevacizumab, bleomycin, carboplatin, cisplatin, cyclophosphamide, docetaxel, doxorubicin, etoposide, gemcitabine, letrozole, olaparib, tamoxifen, topotecan, trabectedin, a CTLA4 antibody, a PD-1 antibody, a PD-L1 antibody, and a TGFβ antibody.

[0046] In some embodiments, a composition is provided comprising an alpha-lactalbumin polypeptide, zymosan, and MONTANIDE® ISA 201 VG TM wherein the alpha-lactalbumin polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the alpha-lactalbumin polypeptide comprises an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 5.

[0047] In one aspect, a formulation is provided comprising an alpha-lactalbumin polypeptide, zymosan, and MONTANIDE® ISA 201 VG TM wherein the alpha-lactalbumin polypeptide and zymosan are present in the formulation at a ratio of between about 1 :5 (w / w) and 5: 1 (w / w), and wherein the alpha-lactalbumin polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the alpha-lactalbumin polypeptide comprises an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 5.

[0048] In one aspect, a method of making a composition or formulation disclosed herein is provided, comprising the step of mixing an aqueous solution comprising an antigen with an emulsion comprising a carbohydrate and a metabolizable oil. In some embodiments, the ratio of the aqueous solution to the emulsion is between about 1 :2 to about 2: 1 (v / v), e.g., about 1 : 1 (v / v).

[0049] In one aspect, there is provided use of a composition or formulation as disclosed herein for the manufacture of a medicament for preventing, treating or ameliorating cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 Figure 1 shows the induction of proinflammatory T cell immunity by vaccination with an α-lactalbumin / complete Freund's adjuvant (CFA) emulsion. Six- to eight-week-old BALB / c female mice (n=3) were inoculated with 200 μl of a water-in-oil emulsion containing 100 μg of recombinant mouse α-lactalbumin and 200 μg of Mycobacterium tuberculosis strain H37RA in complete Freund's adjuvant (CFA). The mean splenocyte frequency of proinflammatory T cells producing IFNγ (type 1) and IL-17 (type 17) was determined four weeks after vaccination. Error bars represent ±SD.

[0051] Figure 2 Shown is a comparison of the efficacy of several adjuvants and CFA in inducing type 1 / 17 T cell immunity. 4 weeks after 200 μl of an emulsion containing 100 μg of recombinant mouse α-lactalbumin and a conventional dose of various adjuvants including CFA were inoculated into 6-8 week old BALB / cJ female mice, the splenocyte frequency of type 1 and type 17 proinflammatory T cells was measured. Data are expressed as the average α-lactalbumin-specific type 1 and type 17 spot-forming units (SFU) obtained using each test adjuvant divided by the SFU obtained using CFA as a "gold standard" adjuvant on the same day. The horizontal black dotted line indicates that the frequency is equivalent to the frequency obtained using CFA as an adjuvant (SFU=1 induced using CFA). Some immunizations are performed twice, two weeks apart, and indicated with x2.

[0052] Figures 3A-3H Shown are 4T1 mouse mammary tumor growth using different α-lactalbumin / adjuvant combinations. Figure 3A Female BALB / cJ mice were subcutaneously inoculated with 200 μL of emulsion containing 100 μg recombinant mouse α-lactalbumin and 200 μg CFA at 6-8 weeks of age. Control mice were inoculated with 200 μg CFA alone. Two weeks after vaccination, mice were subcutaneously inoculated with 2 × 10 4 4T1 mouse mammary tumor cells were used and tumor growth was measured every other day using a vernier caliper. This same protocol was subsequently used to determine the Figure 2 Tumor growth was measured using various other adjuvants as shown in the Table 1 using the doses recommended by each manufacturer, including ( Figure 3B )GPI-0100x2、( Figure 3C )Sigma lipid A, ( Figure 3D )AS02B lipid A, ( Figure 3E )CpG DNA x2, ( Figure 3F)CpG DNA+α-Gal-Cer x2, ( Figure 3G )β-glucan peptides in IFA and ( Figure 3H ) Zymosan in IFA. Some vaccinations were performed twice, two weeks apart, and are indicated by x2. Asterisks indicate significant differences between experimentally vaccinated and control-vaccinated mice (P < 0.05).

[0053] Figure 4 The results of Zymosan / IFA and Zymosan / MONTANIDE are shown. TM Comparison of the induction of type 1 / type 17 pro-inflammatory T cells Female BALB / cJ mice aged 6-8 weeks were inoculated with 100 μL IFA or 100 μL MONTANIDE. TM An emulsion of 100 μg of aqueous recombinant mouse α-lactalbumin emulsified with 200 μg of zymosan in 1% vaccinia virus (VV) was administered. Four weeks after vaccination, splenocyte frequencies of proinflammatory type 1 (IFNγ) and type 17 (IL-17) T cells were determined by ELISPOT assay. Data show the mean spot-forming units (SFU) of the recall response to 50 μg / mL recombinant mouse α-lactalbumin minus the mean background response of cultures without recall antigen (mean background <5 SFU per assay). Error bars represent ±SE.

[0054] Figure 5 Results of a vaccine dose study are shown. Eight-week-old female BALB / cJ mice were inoculated subcutaneously on the ventral side with 200 μL of an emulsion containing equal amounts of 100-1000 μg each of α-lactalbumin and zymosan. Recombinant mouse α-lactalbumin (FLAG-N-mαlac-C-HIS) was solubilized in sterile USP-grade water, and zymosan was suspended in MONTANIDE TM ISA51 VG. Groups of three mice received one, two, or three vaccinations, four weeks apart. Four weeks after the final vaccination, splenocytes were analyzed by ELISPOT using capture / antibody pairs specific for mouse IFNγ, IL-5, and IL-17 to assess the frequency of splenocytes expressing type 1, type 2, and type 17 T cell lineages, respectively.

[0055] Figure 6-8 The results of single vaccination of group A mice ( Figure 6 ), double-inoculated group B mice ( Figure 7 ) and mice in group C vaccinated three times ( Figure 8) Body weight change from the time of first inoculation. Body weight change was normalized to the body weight obtained on day 0 (set as 100%) and plotted as the percentage increase or decrease relative to this initial starting point. Body weight was recorded for each mouse at the same time each day. Arrows indicate one or more inoculation days. Error bars represent ±SE.

[0056] Figure 9-11 The results of the single vaccination of group A mice ( Figure 9 ), double vaccination group B mice ( Figure 10 ) and mice in group C vaccinated three times ( Figure 11 ) Change in body temperature from the time of the first vaccination. Changes in body temperature were normalized to the body temperature obtained on day 0 (set as 100%) and plotted as the percentage increase or decrease relative to this initial starting point. Body temperature was recorded for each mouse at the same time each day. Arrows indicate one or more vaccination days. Error bars represent ±SE.

[0057] Figure 12-14 The results of the single vaccination of group A mice ( Figure 12 ), double vaccination group B mice ( Figure 13 ) and mice in group C vaccinated three times ( Figure 15 ) Spleen, liver, and kidney weights were recorded at necropsy. Weights were expressed as a percentage of total body weight, and the mean percentages for each mouse subgroup were plotted (as shown in Table 2 in Example 5). Error bars represent ± SE.

[0058] Figure 15 Subcutaneous administration of Zymosan / MONTANIDE is shown as further described in Example 5. TM Representative appearance of immunization sites in mice treated with α-lactalbumin emulsion. Figure 15 The following are photos of mice taken about two weeks after the second injection and about six weeks after the first injection. The blue arrow indicates the first injection site, and the red arrow indicates the second injection site. Figure 15 As shown, the first injection site showed improvement in appearance compared to the second injection site, indicating that the granuloma resolved over time.

[0059] Figure 16 Shown is a subcutaneous injection of Zymosan / MONTANIDE as further described in Example 5. TMEndpoint mean clinical scores for granulomas at three consecutive injection sites in BALB / c mice treated with α-lactalbumin emulsion. Oily granulomas were typically observed at the injection site and were graded according to the following criteria: 0, normal; 1, minimal; 2, mild; 3, moderate; 4, severe. For each injection site, the mean value for all mice in all treatment groups (n=25) was calculated. Error bars represent ±SE.

[0060] Figure 17 A summary of the severity of focal liver inflammation in all groups in the toxicology studies described in Example 5 is presented.

[0061] Figure 18 The study protocol for the clinical trial described in Example 8 is shown. The protocol indicates the timeline for dosing, toxicity assessments, and immune monitoring blood draws. DETAILED DESCRIPTION

[0062] The T cells that mediate adaptive immune response are divided into subgroups according to their cytokine profiles. Type 1 proinflammatory T cells produce IFNγ and mediate immunity to viral and bacterial infections, while type 2 regulatory T cells produce interleukin (IL) -4, IL-5 and IL-13 and mediate humoral immunity to parasitic infections. Recent studies have determined that the 17 types of proinflammatory T cells that produce IL-17 are unique subtypes, which also play a prominent role in inflammation. Type 1 and type 17 T cell lineages are needed to induce optimized tissue damage for self-protein (Steinman et al., (2007) Nat Med 13:139-145; Luger et al., (2008) J Exp Med 205:799-810).

[0063] Anti-cancer vaccines are designed to stimulate the immune system to attack cancer cells. These vaccines typically contain antigens that are preferentially expressed by cancer cells ("tumor-associated antigens"). Most current clinical vaccine formulations induce pro-inflammatory type 1 immunity, but rarely, if ever, induce type 17 immunity. The present invention encompasses the view that many current clinical vaccine formulations are ineffective because they fail to elicit type 1 and type 17 immune responses. According to the present invention, the compositions provided induce an immune response that includes type 1 and type 17 pro-inflammatory T cells. In addition, the compositions disclosed herein induce limited or no toxicity when injected into animal models, indicating that they are suitable for clinical use in humans.

[0064] The present invention also encompasses the concept that vaccine formulations comprising α-lactalbumin polypeptides and adjuvant components, as disclosed herein, can prevent and / or ameliorate breast cancer. α-lactalbumin is constitutively overexpressed in most TNBCs (the most aggressive and lethal form of breast cancer). Therefore, the vaccine formulations, compositions, and methods disclosed herein may be useful for treating and preventing the most lethal form of breast cancer.

[0065] General Description

[0066] Provided herein are methods and compositions for treating and / or preventing breast cancer by inducing an immune response against α-lactalbumin. As described herein, the present disclosure relates to immunogen / adjuvant combinations that induce adaptive immune responses (e.g., type 1 and type 17 T cells) for inhibiting the growth of breast cancer. In some aspects, the composition comprises an α-lactalbumin polypeptide and zymosan. In some aspects, the composition comprises an α-lactalbumin polypeptide and a MONTANIDE. TM In some aspects, the composition comprises α-lactalbumin polypeptide, zymosan, and MONTANIDE TM α-lactalbumin with zymosan and / or MONTANIDE of the present disclosure TM The combination of α-lactalbumin and zymosan and / or MONTANIDE induced high frequencies of type 1 / type 17 T cells associated with effective tumor immunity without inducing the non-resolving granulomas associated with CFA (the "gold standard" adjuvant). TM Combined vaccination provides a unique approach for providing safe and effective immunity against the growth of human breast cancer.

[0067] I. Definition

[0068] For convenience, certain terms used in the specification, examples, and appended claims are collected here.

[0069] The articles "a" and "an" are used herein to refer to one or to more than one (ie, to at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

[0070] As used herein, "adjuvant" means a substance that, when administered before, simultaneously with, or after administration of an antigen, accelerates, prolongs, and / or enhances the quality and / or intensity of the immune response to an antigen as compared to the response elicited by administration of the antigen alone.

[0071] As used herein, "anti-cancer therapy" means a therapy intended to treat, improve, and / or reduce the risk or progression of cancer or a cancerous condition. In some embodiments, the anti-cancer therapy comprises an anti-cancer agent, i.e., an agent used to treat, improve, and / or reduce the risk or progression of cancer or a cancerous condition.

[0072] As used herein, the term "antigen" has its ordinary meaning in the art and refers to any molecule or portion of a molecule that can generate an immune response, such as an antibody and / or T cell response, by itself or in combination with an adjuvant and / or a pharmaceutically acceptable carrier.

[0073] As used herein, the term "administering" means providing an agent or composition to a subject, and includes, but is not limited to, administration by a healthcare professional and self-administration.

[0074] As used herein, "biodegradable" when used with respect to a material means a material that, when introduced into a cell, can be decomposed by cellular machinery (e.g., enzymatic degradation) or by hydrolysis into components that the cell can reuse or dispose of without producing a significant toxic effect on the cell. In some embodiments, the components produced by the decomposition of the biodegradable material do not induce inflammation and / or other adverse effects in the body. In some embodiments, the biodegradable material is enzymatically decomposable. Alternatively or additionally, in some embodiments, the biodegradable material is decomposed by hydrolysis.

[0075] The term "immune response" refers to any response of the immune system to an antigen or antigenic determinant in this article. Exemplary immune responses include humoral immune responses (e.g., the production of antigen-specific antibodies (neutralizing or other antibodies)) and cell-mediated immune responses (e.g., lymphocyte proliferation). Type 1 proinflammatory immune responses are characterized by the production of IFNγ. Type 2 regulatory immune responses are characterized by the expression of IL-4 or IL-5. Type 17 proinflammatory immune responses are characterized by the expression of IL-17. In some cases, a mixed immune response can be produced. For example, in some cases, a mixed type 1 / type 17 inflammatory immune response is generated, characterized by the expression of IFNγ and IL-17.

[0076] As used herein, the phrase "metabolizable oil" means an oil that, when introduced into an organism, (1) can be broken down or eliminated from the organism to a greater extent; (2) can be broken down or eliminated from the organism more rapidly; and / or (3) results in a reduction in granuloma formation compared to a reference level (such as the level of granuloma formation in a subject administered complete Freund's adjuvant or the level of granuloma formation in a subject administered incomplete Freund's adjuvant). Thus, the phrase "metabolizable oil" as used herein does not need to be fully metabolizable. "Reduction in granuloma formation" can be characterized by, for example, one or more of: fewer granulomas formed, reduced severity of granulomas, more rapid reduction in granuloma severity, and more rapid resolution (partial or complete) of granulomas.

[0077] As used herein, the terms "polypeptide" and "protein" are used interchangeably and generally have their art-recognized meanings of polymers of at least three amino acids. The term "polypeptide" may refer to a polypeptide in a neutral (uncharged) form or a salt form, as well as unmodified or modified (e.g., by glycosylation, side chain oxidation, or phosphorylation). The term "polypeptide" may also be used to refer to a specific functional class of polypeptides. When used to refer to a functional class of a polypeptide, the term is intended to include functional fragments, variants (e.g., allelic variants), and derivatives of a reference polypeptide, as well as the full-length, wild-type form of the reference polypeptide. In some embodiments, a polypeptide of a certain functional class shares at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 97.5% sequence identity with the full-length form of the reference polypeptide at the amino acid level. For example, as used herein, "α-lactalbumin polypeptide" includes α-lactalbumin and polypeptides having an amino acid sequence having sufficient sequence identity to the amino acid sequence of α-lactalbumin (or a portion thereof) to elicit an α-lactalbumin-specific immune response.

[0078] As used herein, "percent identity" between amino acid sequences is synonymous with "percent homology," which can be determined using the algorithm of Kailin and Altschul (Proc. Natl. Acad. Sci. USA 87, 2264-2268, 1990), as modified by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90, 5873-5877, 1993). The algorithm described is incorporated into the NBLAST and XBLAST programs of Altschul et al. (J. Mol. Biol. 215, 403-410, 1990). BLAST nucleotide searches are performed with the NBLAST program, score = 100, word length = 12 to obtain nucleotide sequences homologous to the polynucleotides described herein. BLAST protein searches are performed with the XBLAST program, score = 50, word length = 3 to obtain amino acid sequences homologous to a reference polypeptide. To obtain blank alignments for comparison purposes, Gapped BLAST was used as described in Altschul et al. (Nucleic Acids Res. 25, 3389-3402, 1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) were used.

[0079] As used herein, the phrase "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, thickener, solvent, or encapsulating material, that participates in carrying or delivering the subject compound from one organ or part of the body to another. The term "carrier" encompasses carriers that are not covalently attached to the compound or composition being delivered by the carrier as well as carriers that are covalently attached to them.

[0080] As used herein, the term "purified" means the enrichment of a molecule, compound, or composition relative to other components normally associated with the molecule, compound, or composition in its native environment. The term "purified" does not necessarily indicate that complete purity of a molecule, compound, or composition has been achieved. In some embodiments, a "purified" molecule, compound, or composition is at least 90%, at least 95%, or at least 97.5% free of other components.

[0081] As used herein, the term "tumor-associated antigen" has its recognized meaning in the art and refers to an antigen whose expression is highly correlated with tumor cells. Tumor-associated antigens may or may not be expressed in normal cells. In some embodiments, tumor-associated antigens are overexpressed in tumor cells. In some embodiments, expression of tumor-associated antigens is associated with a specific subtype or multiple specific subtypes of tumor cells.

[0082] As used herein, the terms "subject" and "patient" are interchangeable and refer to an organism that receives a treatment or vaccine (e.g., by administering a composition or formulation disclosed herein). Examples of subjects and patients include mammals, such as humans or non-human animals.

[0083] As used herein, the phrases "therapeutically effective amount" and "effective amount" mean an amount of an agent effective to produce the desired therapeutic effect in at least one subpopulation of cells in a subject, at a reasonable benefit / risk ratio applicable to any medical treatment.

[0084] "Treating" a disease in a subject or "treating" a subject with a disease means subjecting the subject to drug therapy, eg, administration of a drug, such that at least one symptom of the disease is alleviated or prevented from worsening.

[0085] The term "reference" refers to any sample, standard, or level used for comparison purposes. The phrases "reference standard" and "reference level" are used interchangeably and refer to a value or number derived from a reference sample or subject. In some embodiments, the subject or sample from which the reference level is derived is matched to the subject sample according to at least one of the following criteria: age, weight, disease stage, and overall health.

[0086] For example, in some embodiments, the reference level is a clinical grade or score or an average clinical grade or score.

[0087] The term "retired autologous protein" refers to an autologous protein that is no longer expressed at autoimmunogenic levels in normal aging tissue. The term "retired autologous antigen" refers to an antigen derived from a retired autologous protein. In some embodiments, the retired autologous antigen comprises a fragment of a retired autologous protein. In some embodiments, the retired autologous antigen comprises a full-length version of a retired autologous protein.

[0088] As used herein, the term "surfactant" has its art-recognized meaning and refers to a substance that tends to reduce the surface tension between two liquids, between a gas and a liquid, or between a liquid and a solid. In some embodiments, the surfactant is an emulsifier, i.e., a substance that stabilizes an emulsion.

[0089] II. Composition

[0090] In one aspect, a composition is provided comprising: a carbohydrate and a metabolizable oil, wherein (i) the composition further comprises an antigen, or (ii) the carbohydrate comprises a polysaccharide and the composition comprises a mixture of at least two polysaccharides.

[0091] In some embodiments, the antigen is a tumor-associated antigen.

[0092] In some embodiments, the carbohydrate comprises a polysaccharide, and the composition comprises a mixture of at least two polysaccharides.

[0093] In some embodiments, the composition comprises a tumor-associated antigen, the carbohydrate comprises a polysaccharide, and the composition comprises a mixture of at least two polysaccharides.

[0094] In some embodiments, the composition, when administered to a subject, elicits an antigen-specific T cell immune response comprising at least one of (i) a type 1 proinflammatory response and (ii) a type 17 proinflammatory T cell response.

[0095] In some embodiments, the composition comprising an antigen or further comprising an antigen, when administered to a subject, induces an antigen-specific T cell immune response that includes both type 1 and type 17 proinflammatory T cell responses.

[0096] As further described herein, the composition may further comprise a surfactant.

[0097] A.Carbohydrates

[0098] In some embodiments, the carbohydrate comprises a polysaccharide, for example, a polysaccharide selected from the group consisting of chitin, dextran, glucan, lentanan, mannan, and combinations thereof.

[0099] In some embodiments, the composition comprises a mixture of polysaccharides, for example, a mixture comprising at least three polysaccharides.

[0100] In some embodiments, each polysaccharide in the mixture is selected from the group consisting of chitin, dextran, glucan, lentanan, mannan, and combinations thereof.

[0101] In some embodiments, the polysaccharide or mixture of polysaccharides includes a glucan, for example a β-glucan, such as, but not limited to, 1-3 β-glucan. In some embodiments, at least 50% of the carbohydrates in the composition are β-glucans.

[0102] In some embodiments, the mixture of polysaccharides includes a mixture of chitin, glucan, and mannan.

[0103] In some embodiments, the carbohydrate binds to a pattern recognition receptor, such as TLR2 and / or dectin-1.

[0104] For example, in some embodiments, the composition comprises zymosan. Zymosan is a mixture of crude cell wall components of baker's yeast extract from Saccharomyces cerevisiae, primarily composed of β-glucans (50-57%), mannans, and chitin. The U.S. Food and Drug Administration (FDA) gives these yeast extract-derived β-glucans a GRAS ("Generally Recognized as Safe") rating. Yeast zymosan serves as a rich source of β (1,3) glucans. Yeast-derived β (1,3) glucans appear to stimulate the immune system in part by activating the innate immune system as part of the body's basic defense against fungal infections (Huang et al., (2013) Clin Vaccine Immunol 20: 1585-1591). Yeast β(1,3) glucan is a polysaccharide composed primarily of β(1-3)-linked glucose molecules with periodic β(1-3) branches connected by β(1-6) linkages and is more formally known as poly(1-6)-β-glucopyranosyl-(1-3)-β-D-glucopyranose.

[0105] B. Metabolizable oil

[0106] As used herein, the phrase "metabolizable oil" means an oil that, when introduced into an organism, (1) can be broken down or eliminated from the organism to a greater extent; (2) can be broken down or eliminated from the organism more rapidly; and / or (3) results in reduced granuloma formation compared to incomplete Freund's adjuvant. Thus, the phrase "metabolizable oil" as used herein is not limited to being fully metabolizable. "Reduced granuloma formation" can be characterized by, for example, one or more of the following: fewer granulomas formed, reduced severity of granulomas, and more rapid resolution of granulomas.

[0107] In some embodiments, the metabolizable oil comprises mineral oil.

[0108] In some embodiments, the metabolizable oil comprises a purified oil, for example, a purified mineral oil (such as, but not limited to, DRAKEOL TM 6VR).

[0109] In some embodiments, metabolizable oil comprises biodegradable oil.The limiting examples of biodegradable oil comprises isopropyl myristate, squalene oil (for example, MF59), squalane oil, vegetable oil or its combination.In some embodiments, biodegradable oil is vegetable oil, such as, for example almond oil, castor oil, cyperus rotundus oil, coconut oil, corn oil, cottonseed oil, olive oil, peanut oil, peach kernel oil, safflower oil, soybean oil or its combination.

[0110] In some embodiments, the metabolizable oil comprises fish oil.

[0111] In certain embodiments, the metabolizable oil is a pharmaceutical grade oil.

[0112] C. Surfactants / emulsions

[0113] In some embodiments, provided compositions comprise one or more surfactants. Non-limiting examples of suitable surfactants include anhydrous mannitol monooleate, isoanhydrous mannitol monooleate, and combinations thereof. In some embodiments, the composition comprises anhydrous mannitol monooleate.

[0114] In some embodiments, provided compositions comprise MONTANIDE TM , such as Montanide TM The ISA series of adjuvants contain metabolisable oils.

[0115] MONTANIDE TMISA (ISA = Incomplete Seppic Adjuvant) adjuvants (Seppic SA, Paris, France) are a group of oil / surfactant based adjuvants in which different surfactants are combined with non-metabolizable mineral oil, metabolizable oil or a mixture of both. They are usually prepared as emulsions with aqueous antigen solutions. Various MONTANIDE TM The adjuvants for the ISA group were used as water-in-oil emulsions, oil-in-water emulsions, or water-in-oil-in-water emulsions.

[0116] In some embodiments, the composition comprises MONTANIDE TM ISA 51, MONTANIDE TM ISA 51VG or any bioequivalent adjuvant derived therefrom (e.g., by replacing oleic acid isolated from olives with oleic acid isolated from another source or synthetically derived). MONTANIDE TM ISA 51 is a highly purified mineral oil (DRAKEOL TM A mixture of 6VR) and a surfactant (dehydrated mannitol monooleate). TM ISA 51 VG is a similar composition, in which the oleic acid is obtained from olives rather than from animal sources.

[0117] In some embodiments, provided compositions are emulsions of water and oil, such as water-in-oil emulsions. Methods for preparing water-in-oil (w / o) emulsions are well known in the art. Water-in-oil emulsions can be obtained by any of a variety of approaches, such as using any of a variety of devices, such as high shear mixers, vortex mixers, and syringes with or without connectors (e.g., T- or I-connectors). In some embodiments, provided compositions comprise an adjuvant that produces a depot effect (such as MONTANIDE TM Adjuvants) are agents that cause the antigen in the same composition to be slowly released throughout the body, thereby prolonging the exposure of immune cells to the antigen.

[0118] D.Antigen

[0119] Generally, any molecule or portion of a molecule that resists the desired immune response can be used as an antigen. Antigens can include, but are not limited to, any of the following: peptides, polypeptides, proteins, cells (or components thereof), live attenuated pathogens (or components thereof), and heat-inactivated pathogens (or components thereof).

[0120] In some embodiments, the antigens are non-self antigens, ie, they are foreign to the organism to which the composition comprising the antigen is intended to be administered.

[0121] In some embodiments, the antigens are autoantigens because they are or were expressed in at least some cells of the organism to which the composition comprising the antigen is intended to be administered. In some embodiments, the antigens are retired self-proteins because they were once expressed in the organism but are no longer expressed at autoimmunogenic levels in non-malignant mature cells.

[0122] In some embodiments, the antigen is a tumor-associated antigen.

[0123] In some embodiments, provided compositions or formulations comprise a mixture of different antigens.

[0124] The antigen may comprise one or more modifications. For example, one or more modifications that affect the processing, cellular uptake, immunogenicity and / or stability (e.g., within a peptide / MHC complex) of the antigen or fragment thereof may be used.

[0125] In some embodiments, the antigen comprises a polypeptide antigen. Polypeptide antigens can be any of a variety of lengths, and their sequences can be consistent or inconsistent with the sequences of naturally occurring proteins. For example, in some embodiments, full-length or nearly full-length proteins can be used as polypeptide antigens. In some embodiments, the antigen or antigen mixture comprises one or more fragments or variants of a protein.

[0126] α-lactalbumin peptide

[0127] In some embodiments, the antigen comprises an α-lactalbumin polypeptide or an immunogenic fragment thereof. In some embodiments, the antigen comprises a plurality (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, or 10) of different α-lactalbumin polypeptides or fragments. In some embodiments, provided compositions comprise a nucleic acid encoding an α-lactalbumin polypeptide in place of or in addition to an α-lactalbumin polypeptide.

[0128] The LALBA gene encodes α-lactalbumin, the major milk protein. α-lactalbumin forms the regulatory subunit of the lactose synthase (LS) heterodimer, and β1,4-galactosyltransferase (β4Gal-T1) forms the catalytic component. Together, these proteins enable LS to produce lactose by transferring a galactose moiety to glucose. As a monomer, α-lactalbumin strongly binds calcium and zinc ions and may possess bactericidal or antitumor activities. The human LALBA gene contains five exons.

[0129] The human α-lactalbumin precursor protein has 142 amino acids and a molecular weight of 14,178 Da, while human α-lactalbumin has 123 amino acids. In some embodiments, the α-lactalbumin polypeptide has 123 amino acids. The term "α-lactalbumin polypeptide" is intended to include fragments, variants (e.g., allelic variants) and derivatives thereof. Representative human α-lactalbumin cDNAs and human α-lactalbumin protein sequences are well known in the art and are publicly available from the National Center for Biotechnology Information (NCBI). For example, at least one human UBE2D3 subtype is known. The human UBE2D3 subtype (NP_002280.1) can be encoded by a transcript variant (NM_002289.2). The nucleic acid and polypeptide sequences of α-lactalbumin homologs in organisms other than humans are well known and include, for example, chimpanzee α-lactalbumin (XM_016924811.2 and XP_016780300.1), monkey α-lactalbumin (XM_001102116.2 and XP_001102116.1), canine α-lactalbumin (NM_001003129.1 and NP_001003129.1), bovine α-lactalbumin (NM_174378.2 and NP_776803.1), mouse α-lactalbumin (NM_010679.1 and NP_034809.1), and rat α-lactalbumin (NM_012594.1 and NP_036726.1). Each of the above mRNA and protein sequences is incorporated herein by reference. Representative sequences of α-lactalbumin homologs are listed in Table 1 below.

[0130] Table 1

[0131] SEQ ID NO: 1 Human LALBA amino acid precursor sequence (NP_002280.1)

[0132]

[0133] SEQ ID NO: 2 Human LALBA cDNA sequence (NM_002289.2; CDS: 27-455)

[0134]

[0135]

[0136] SEQ ID NO: 3 Mouse LALBA amino acid sequence (NP_034809.1)

[0137]

[0138] SEQ ID NO: 4 Mouse LALBA cDNA sequence (NM_010679.1; CDS: 13-444)

[0139]

[0140] SEQ ID NO:5 human LALBA amino acid sequence

[0141]

[0142] * Included in Table 1 are RNA nucleic acid molecules (e.g., thymine is replaced with uridine), nucleic acid molecules encoding LALBA homologs, and DNA or RNA nucleic acid sequences comprising a nucleic acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identical over its entire length to the nucleic acid sequence of any SEQ ID NO listed in Table 1, or a portion thereof. Such nucleic acid molecules can function as the full-length nucleic acids as further described herein.

[0143] * Table 1 includes homologs of LALBA and polypeptide molecules comprising an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identical over its entire length to the amino acid sequence of any SEQ ID NO listed in Table 1, or a portion thereof. Such polypeptides may have the functions of the full-length polypeptides as further described herein.

[0144] In some embodiments, provided herein are α-lactalbumin polypeptides and / or nucleic acids encoding α-lactalbumin polypeptides. α-lactalbumin polypeptides are polypeptides comprising an amino acid sequence having sufficient sequence identity to the amino acid sequence of α-lactalbumin or a portion thereof to induce an α-lactalbumin-specific immune response.

[0145] In some embodiments, a fusion polypeptide comprising α-lactalbumin and a heterologous polypeptide is provided.

[0146] In certain embodiments, the alpha-lactalbumin polypeptide has an amino acid sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, or 140 consecutive amino acids of the alpha-lactalbumin amino acid sequence listed in Table 1 (e.g., SEQ ID NO: 1, 3, or 5). In some embodiments, the consecutive amino acids are identical to the alpha-lactalbumin amino acid sequence listed in Table 1.

[0147] In certain embodiments, the alpha-lactalbumin polypeptide has an amino acid sequence consisting essentially of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, or 140 consecutive amino acids of the alpha-lactalbumin amino acid sequence listed in Table 1. In some embodiments, the consecutive amino acids are identical to the amino acid sequence of the alpha-lactalbumin amino acid sequence listed in Table 1.

[0148] In certain embodiments, the alpha-lactalbumin polypeptide has an amino acid sequence consisting of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, or 140 consecutive amino acids of the alpha-lactalbumin amino acid sequence. In some embodiments, the consecutive amino acids are identical to the alpha-lactalbumin amino acid sequence listed in Table 1.

[0149] In some embodiments, the alpha-lactalbumin polypeptide has an amino acid sequence comprising 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, or 140 consecutive amino acids that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the alpha-lactalbumin amino acid sequence listed in Table 1. In some embodiments, the contiguous amino acids are identical to the α-lactalbumin amino acid sequence listed in Table 1.

[0150] In some embodiments, the α-lactalbumin polypeptide has an amino acid sequence comprising at least 8 contiguous amino acids of SEQ ID NO:5.

[0151] In some embodiments, the alpha-lactalbumin polypeptide has an amino acid sequence consisting essentially of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, or 140 contiguous amino acids that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an alpha-lactalbumin amino acid sequence set forth in Table 1. In some embodiments, the contiguous amino acids are identical to an alpha-lactalbumin amino acid sequence set forth in Table 1.

[0152] In some embodiments, the alpha-lactalbumin polypeptide has an amino acid sequence consisting essentially of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, or 140 contiguous amino acids that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an alpha-lactalbumin amino acid sequence set forth in Table 1. In some embodiments, the contiguous amino acids are identical to an alpha-lactalbumin amino acid sequence set forth in Table 1.

[0153] As is well known to those skilled in the art, polypeptides having substantial sequence similarity can elicit identical or very similar immune responses in a host organism. Thus, in some embodiments, alpha-lactalbumin polypeptides that are derivatives, equivalents, variants, fragments, or mutants of alpha-lactalbumin can also be suitable for use in the methods and compositions provided herein.

[0154] In some embodiments, the alpha-lactalbumin polypeptides provided are functional equivalents in that they have an amino acid sequence that is altered (e.g., by conservative substitution) relative to an alpha-lactalbumin polypeptide but still can elicit an immune response. As used herein, the term "conservative substitution" denotes the replacement of an amino acid residue by another of a biologically similar residue. It is well understood that amino acids within the same conservative group can often be substituted for one another without significantly altering the function or immunogenicity of the protein.

[0155] In some embodiments, nucleic acids are provided herein, such as DNA molecules encoding the α-lactalbumin polypeptides described herein. In some embodiments, compositions are provided that include an expression vector comprising an open reading frame encoding the α-lactalbumin polypeptide. In some embodiments, the α-lactalbumin nucleic acid includes regulatory elements that promote expression of the open reading frame. Such elements may include, for example, one or more of a promoter, a start codon, a stop codon, and a polyadenylation signal. In addition, one or more enhancers may be included. These elements may be operably linked to a sequence encoding the α-lactalbumin polypeptide.

[0156] Examples of promoters include, but are not limited to, the simian virus 40 (SV40) promoter, the mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV) (such as the HIV long terminal repeat (LTR) promoter), Moloney virus, cytomegalovirus (CMV) (such as the CMV immediate early promoter), Epstein-Barr virus (EBV), Rous sarcoma virus (RSV), and promoters from human genes (such as human actin, human myosin, human hemoglobin, human muscle creatine, and human metallothionein). Examples of suitable polyadenylation signals include, but are not limited to, the SV40 polyadenylation signal and the LTR polyadenylation signal.

[0157] Non-limiting examples of enhancers or enhancer / promoters include, for example, enhancers from human actin, human myosin, human hemoglobin, human muscle creatine, and viral enhancers such as CMV, RSV, and EBV.

[0158] In some embodiments, the nucleic acid provided is incorporated into a vector or delivery vehicle. Useful delivery vehicles include, but are not limited to, biodegradable microcapsules, immunostimulating complexes (ISCOMs), liposomes, and genetically engineered attenuated live vectors (such as viruses or bacteria).

[0159] In some embodiments, the vector is a viral vector, non-limiting examples of which include lentivirus, retrovirus, herpes virus, adenovirus, adeno-associated virus, vaccinia virus, baculovirus, fowlpox virus, AV pox virus, modified vaccinia Ankara (MVA) virus and other recombinant viruses. For example, a vaccinia virus vector can be used to infect dendritic virus.

[0160] F. Formulations and Pharmaceutical Compositions

[0161] In some embodiments, the antigen and carbohydrate are present in a ratio of from about 10:1 to about 1:10 (w / w), e.g., from about 5:1 to about 1:5 (w / w), from about 4:1 to 1:4 (w / w), from about 3:1 to about 1:3 (w / w), or from about 1:2 to about 2:1 (w / w). In some embodiments, the antigen and carbohydrate are present in a ratio of about 1:1 (w / w).

[0162] In some embodiments, provided compositions comprise an antigen, zymosan, and MONTANIDE TM In some embodiments, the antigen is a polypeptide antigen.

[0163] For example, a composition that may be suitable for treating and / or preventing breast cancer may include α-lactalbumin polypeptide, zymosan, and MONTANIDE TM , wherein the α-lactalbumin polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the α-lactalbumin polypeptide comprises an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 5.

[0164] In some embodiments, provided compositions are formulated as emulsions of water and oil, such as water-in-oil emulsions.

[0165] In some embodiments, a method comprising an antigen, zymosan, and MONTANIDE is provided. TM In some embodiments, the antigen is a polypeptide antigen.

[0166] For example, in some embodiments, the formulation includes α-lactalbumin polypeptide, zymosan, and MONTANIDE TM The invention also provides a water-in-oil emulsion of the present invention, wherein the α-lactalbumin polypeptide and zymosan are present in the formulation at a ratio of between about 1:5 (w / w) and 5:1 (w / w), and wherein the α-lactalbumin polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of SEQ ID NO: 5. In some such embodiments, the α-lactalbumin polypeptide comprises an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 5.

[0167] In some aspects, provided herein are pharmaceutical compositions (eg, vaccine compositions). For example, in some embodiments, provided compositions further comprise a pharmaceutically acceptable carrier.

[0168] In some embodiments, the composition further comprises an antibiotic.

[0169] The pharmaceutical compositions disclosed herein can be specifically formulated for administration in solid or liquid form, including forms suitable for: (1) oral administration, such as drenches (aqueous or non-aqueous solutions or suspensions), tablets (e.g., those for buccal, sublingual, or systemic absorption), boluses, powders, granules, or pastes (e.g., for application to the tongue); or (2) parenteral administration, such as, for example, subcutaneous, intramuscular, intravenous, or epidural injection. Non-limiting examples of formulations suitable for parenteral administration include sterile solutions, sterile suspensions, and sustained-release formulations.

[0170] The method for preparing these preparations or compositions may include the step of combining the antigen with a carbohydrate, a metabolizable oil, a pharmaceutically acceptable carrier, and optionally one or more auxiliary ingredients. Generally speaking, the preparations can be prepared by uniformly and intimately combining one or more of the composition ingredients described herein with a pharmaceutically acceptable liquid carrier, a pharmaceutically acceptable carrier in finely divided solid form, or both, and then shaping the product if necessary.

[0171] The pharmaceutical composition that is suitable for parenteral administration can be provided as pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solution, dispersion, suspension or emulsion.Alternatively or additionally, the pharmaceutical composition that is used for parenteral administration can be provided as sterile powder, and this sterile powder can be reconstituted into sterile injectable solution or dispersion before use.This type of injectable solution may contain one or more agents, one or more suspending agents and / or one or more thickening agents that make the preparation isotonic with the blood of the expected recipient.For example, the injectable solution can comprise one or more in sugar, alcohol, antioxidant, buffer, antibacterial agent and solute.

[0172] Examples of suitable aqueous and non-aqueous pharmaceutically acceptable carriers include, but are not limited to, water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof; vegetable oils, such as olive oil; and injectable organic esters, such as ethyl oleate. Suitable fluidity can be maintained, for example, by using coating materials (such as lecithin), by maintaining the desired particle size in the case of dispersions, and by using surfactants.

[0173] Pharmaceutical compositions disclosed herein can be formulated as emulsions. For example, there is provided a vaccine composition formulated as an emulsion, which provides a substitute for vaccines based on aluminum. Emulsion formulations can be prepared by emulsifying antigens dissolved in aqueous buffer with oil (such as any metabolizable oil), as further described herein. Emulsion formulations can form short-term reservoirs to promote the vaccine phagocytosis of innate immune cells, which leads to an immune response (Leenaars, Koedam et al., 1998). The oil used in such emulsions can be used for unique immunostimulation, and leads to an immune response stronger than that resulting from the immune response of vaccines comprising aluminum adjuvants (De Gregorio, Caproni et al. 2013).

[0174] In some embodiments, the present disclosure provides a pharmaceutical composition comprising an emulsion (e.g., a water-in-oil emulsion) comprising an antigen and a metabolizable oil as described herein. For example, in some embodiments, the present disclosure provides a pharmaceutical composition comprising an α-lactalbumin polypeptide, zymosan, and a metabolizable oil.

[0175] In certain embodiments, the present disclosure provides pharmaceutical compositions comprising about 40-60% v / v aqueous antigen emulsified with about 40-60% v / v metabolizable oil (optionally, mixed with a carbohydrate as further described herein). For example, the pharmaceutical composition may comprise about 0.1-25 mg / mL (e.g., 0.5-5 mg / mL) of the antigen in about 50% v / v metabolizable oil / carbohydrate composition.

[0176] In some embodiments, the emulsion of the pharmaceutical composition disclosed herein is formed by mixing an aqueous antigen with a metabolizable oil in a ratio of about 1.5:1 to about 1:1.5, such as about 1.5:1, about 1.4:1, about 1.3:1, about 1.2:1, about 1.1:1, about 1:1, about 1:1.1, about 1:1.2, about 1:1.3, about 1:1.4, or about 1:1.5, or any value therebetween. In some embodiments, the carbohydrate mixture is suspended in the metabolizable oil prior to forming the emulsion. In some embodiments, the zymosan is suspended in the metabolizable oil prior to forming the emulsion.

[0177] The pharmaceutical compositions disclosed herein can be formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

[0178] The metabolizable oils and / or carbohydrates in provided pharmaceutical compositions can, in some embodiments, act as adjuvants that increase the immunogenicity of the pharmaceutical compositions.

[0179] In some embodiments, additional physiologically acceptable adjuvants are employed. Such additional adjuvants can be used or incorporated in any of a number of ways, including but not limited to: (i) after reconstitution of an antigen (e.g., a polypeptide antigen) and optionally emulsification with a metabolizable oil as defined above, mixed with other components in a pharmaceutical composition provided herein, (ii) a portion of a recovered antigen-containing composition as provided herein, (iii) physically connected to one or more antigens to be reconstituted; and (iv) administered separately to a subject. Additional adjuvants can, for example, slow down the release of an antigen (e.g., an additional adjuvant can be a liposome) and / or it can be an adjuvant that is immunogenic in itself, thereby synergizing with an antigen (i.e., an antigen present in the composition provided).

[0180] For example, the additional adjuvant can be a known adjuvant or other substance that promotes antigen uptake, recruits immune system cells to the site of administration, and / or promotes immune activation of responding lymphoid cells. Examples of suitable additional adjuvants include, but are not limited to, immunomodulatory molecules (e.g., cytokines), oil and water emulsions, aluminum hydroxide, dextran, dextran sulfate, iron oxide, sodium alginate, Bacto adjuvant, synthetic polymers (such as polyamino acids and amino acid copolymers), saponin, paraffin oil, and muramyl dipeptide. In some embodiments, the additional adjuvant is Adjuvant 65, α-GalCer, aluminum phosphate, aluminum hydroxide, calcium phosphate, β-glucan peptide, CpG DNA, GM-CSF, GPI-0100, IFA, IFN-γ, IL-17, lipid A, lipopolysaccharide, Lipovant, MONTANIDE TM , N-acetyl-muramyl-L-alanyl-D-isoglutamine, Pam3CSK4, quil A, trehalose dimycolate or yeast polysan. In some embodiments, the additional adjuvant induces a mixed type 1 / type 17 immune response.

[0181] In some embodiments, the additional adjuvant is an immunomodulatory molecule that enhances the immune response. For example, the immunomodulatory molecule can be a cytokine, a chemokine, or an immunostimulatory agent, a recombinant form of any of the foregoing, or a nucleic acid encoding any of the foregoing.

[0182] Examples of immunomodulatory cytokines include, but are not limited to, interferons (such as IFNα, IFNβ, and IFNγ), interleukins (such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-17, and IL-20), tumor necrosis factors (such as TNFα and TNFβ), erythropoietin (EPO), FLT-3 ligand, gIp10, TCA-3, MCP-1, MIF, MIP-1α, MIP-1β, Rantes, macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), and granulocyte-macrophage colony stimulating factor (GM-CSF), as well as functional fragments of any of the foregoing.

[0183] In some embodiments, provided compositions comprise immunomodulatory chemokines that bind to chemokine receptors (e.g., CXC, CC, C, or CX3C chemokine receptors). Examples of chemokines include, but are not limited to, Mip1α, Mip-1β, Mip-3α (Larc), Mip-3β, Rantes, Hcc-1, Mpif-1, Mpif-2, Mcp-1, Mcp-2, Mcp-3, Mcp-4, Mcp-5, eotaxin, Tarc, Elc, I309, IL-8, Gcp-2Gro-α, Gro-β, Gro-γ, Nap-2, Ena-78, Gcp-2, Ip-10, Mig, I-Tac, Sdf-1, and Bca-1 (Blc), as well as functional fragments of any of the foregoing substances.

[0184] G. Additional doses

[0185] In certain embodiments, the compositions provided herein further comprise one or more additional agents, such as, but not limited to, anti-cancer agents (eg, chemotherapeutic agents), immunotherapeutic agents, immunomodulatory agents, and / or anti-angiogenic agents.

[0186] In some embodiments, the composition comprises an additional anticancer agent. In some embodiments, the anticancer agent is selected from the group consisting of bevacizumab, bleomycin, carboplatin, cisplatin, cyclophosphamide, docetaxel, doxorubicin, etoposide, gemcitabine, letrozole, olaparib, tamoxifen, topotecan, trabectedin, CTLA4 antibody, PD-1 antibody, PD-L1 antibody, and TGFβ antibody.

[0187] In some embodiments, the additional agent is a naturally occurring or synthetic anticancer agent, such as described in "Cancer Chemotherapeutic Agents," American Chemical Society, 1995, WO Foye Ed.

[0188] In some embodiments, the anticancer agent comprises a small molecule.

[0189] In some embodiments, the anticancer agent is a receptor antagonist or blocker. In some embodiments, the chemotherapeutic agent is selected from the group consisting of a VEGF receptor antagonist (such as, for example, vatalanib (PTK-787 / ZK222584), SU-5416, SU-6668, SU-11248, SU-14813, AZD-6474, AZD-2171, CP-547632, CEP-7055, AG-013736, IM-842, or GW-786034), a VEGFtrap, an EGFR and / or HER2 antagonist (such as, for example, gefitinib, erlotinib, CI-1033, GW-2016, Herceptin, Iressa (ZD-1839), Tarceva (OSI-774), PKI-166, EKB-569, or HKI-272), an integrin receptor antagonist, and a protein kinase receptor antagonist (e.g., atrasentan). In some embodiments, the chemotherapeutic agent inhibits expression of HER2.

[0190] In some embodiments, the anticancer agent comprises an antagonist of a protein kinase, such as an antagonist of a mitogen-activated protein kinase (eg, BAY-43-9006 or BAY-57-9006) or imatinib.

[0191] In some embodiments, the anti-cancer agent comprises a tubulin-binding agent.

[0192] In some embodiments, the anti-cancer agent comprises an antibody. For example, chemotherapeutic antibodies include, but are not limited to, antibodies against cytokines (e.g., TGF ), antibodies targeting cancer cell surface molecules, and antibodies targeting growth factors or their receptors. Non-limiting examples of antibody chemotherapeutic agents include alemtuzumab, apolizumab, bevacizumab, daclizumab, cetuximab, ibritumomab, mitumomab, matuzumab, oregovomab, rituximab, vitaxin (chorionic gonadotropin receptor antibody), DC101 (VEGFR2 antibody), ID09C3 (MHC class II monoclonal antibody), and IMC-1C11 (kinase insert domain receptor antibody).

[0193] In some embodiments, the anti-cancer agent comprises a cell cycle inhibitor.

[0194] In some embodiments, the anti-cancer agent comprises a cytokine inhibitor.

[0195] In some embodiments, the anti-cancer agent comprises a hypoxia-selective cytotoxin.

[0196] In some embodiments, the anti-cancer agent comprises a TNF inhibitor, such as etanercept.

[0197] In some embodiments, the anti-cancer agent comprises an interferon, such as interferon beta.

[0198] In some embodiments, the anti-cancer agent comprises an interleukin, such as IL-10 or IL-12.

[0199] In some embodiments, the anti-cancer agent comprises an immunomodulatory agent, such as lenalidomide or thalidomide.

[0200] In some embodiments, the anticancer agent comprises an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is an inhibitor of CTLA4, such as a CTLA4 antibody (e.g., ipilimumab (BMS), tremelimumab (AstraZeneca) and / or KAHR-102 (KAHR Medical)). In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-1, such as a PD-1 antibody (e.g., nivolumab (BMS), pembrolizumab / lambrolizumab (Merck), pidilizumab (Curetech), AMP-224 (GSK), AMP-514 (AstraZeneca), STI-A1110 (Sorrento), and / or TSR-042 (Tesaro). In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-L1 and / or PD-L2, such as a PD-L1 and / or PD-L2 antibody (e.g., RG-7446 (Roche), BMS-936559 (BMS), MEDI-4736 (AstraZeneca), MSB-0020718C (Merck), AUR-012 (Pierre Fabre Med), STI-A1010 (Sorrento)). In some embodiments, the anticancer agent comprises a leukotriene antagonist.

[0201] In some embodiments, the anticancer agent includes a DNA alkylating agent such as, for example, a nitrogen mustard or a derivative thereof (e.g., bendamustine, chlorambucil, chlormethine (mechlorethamine), an oxazolidinone (e.g., cyclophosphamide, ifosfamide, and trofosfamide), melphalan, nitromin, uramustine), a nitrosourea (e.g., carmustine, lomustine, or streptozotocin), an alkyl sulfonate (e.g., busulfan), an ethyleneimine (aziridine) (e.g., thiotepa or altretinoin), a metal salt (e.g., carboplatin, cisplatin, or oxaliplatin), or a hydrazine (e.g., altretinoin, procarbazine, dacarbazine, or temozolomide).

[0202] In some embodiments, the anticancer agent comprises a platinum compound, such as, for example, cisplatin, oxaliplatin, carboplatin, satraplatin, tetraplatin, or ipriboplatin.

[0203] In some embodiments, the anticancer agent includes a DNA intercalator, for example, an anthracycline, such as daunorubicin, doxorubicin (Adriamycin), liposomal doxorubicin (doxil), epirubicin, or idarubicin.

[0204] In some embodiments, the anticancer agent comprises a DNA minor groove binding compound.

[0205] In some embodiments, the anti-cancer agent comprises a DNA cross-linking agent.

[0206] In some embodiments, the anticancer agent includes an antimetabolite, such as, for example, a pyrimidine or purine analog or antagonist or a nucleoside diphosphate reductase inhibitor. Non-limiting examples of antimetabolites include cytarabine, 5-fluorouracil (5-FU), pemetrexed, tegafur / uracil, uracil mustard, floxuridine, fludarabine, gemcitabine, capecitabine, mercaptopurine, cladribine, thioguanine, methotrexate, pentostatin or hydroxyurea.

[0207] In some embodiments, anticancer agents include inhibitors of DNA transcription, RNA translation, or protein expression. Non-limiting examples of DNA transcription inhibitors include topoisomerase I or II inhibitors (e.g., camptothecin, irinotecan, topotecan, epipodophyllotoxin, etoposide, teniposide, or tricyclic carboxamide-based agents) and transcription factor complex inhibitors (such as, for example, ESX / DRIP130 / Sur-2 complex inhibitors).

[0208] In some embodiments, the anticancer agent includes a proteasome inhibitor, such as, for example, bortezomib.

[0209] In some embodiments, the anticancer agent comprises an enzyme, such as asparaginase or pegylated asparaginase (pegaspargase).

[0210] In some embodiments, the anticancer agent comprises an oligonucleotide or a polynucleotide.

[0211] In some embodiments, the anticancer agent comprises a histone deacetylase inhibitor, such as, for example, SAHA, MD-275, trichostatin A, CBHA, LAQ824, or valproic acid.

[0212] In some embodiments, the chemotherapeutic agent comprises a chemoradiosensitizer or protectant.

[0213] In some embodiments, anticancer agents include oncogene inhibitors, such as P53 or Rb inhibitors.

[0214] In some embodiments, the anticancer agent includes a plant-derived agent such as a taxane (e.g., paclitaxel or docetaxel), a vinca alkaloid (e.g., navelbine, vinblastine, vincristine, vindesine, or vinorelbine), or a tropical alkaloid (e.g., colchicine or a derivative thereof).

[0215] In some embodiments, the anticancer agent comprises a quinazoline or a derivative thereof, such as, for example, afatanib, erlotinib, gefitinib, or lapatinib.

[0216] In some embodiments, the anticancer agent includes an antimitotic agent, such as an antimitotic peptide (eg, phomopsin and dolastatin), an antimitotic carbamate derivative (eg, combretastatin (A4) or amphetinile).

[0217] In some embodiments, the anticancer agent comprises steganacin.

[0218] In some embodiments, anticancer agents include hormone blockers, such as antiandrogens, antiestrogens, gonadotropin-releasing hormone (GNrH) antagonists (e.g., abarelix), GNrH analogs, and aromatase inhibitors. Non-limiting examples of such anti-androgens include nilutamide (anandron), bicalutamide, casodex, cyproterone acetate, flutamide, mitotane, and nilutamide (nilutamide). Non-limiting examples of antiestrogens include droloxifene, raloxifene, tamoxifen, trioxifene, and zindoxifene. Non-limiting examples of GNrH analogs include leuprolide (leuprolide), buserelin, goserelin, and triptorelin. Non-limiting examples of aromatase inhibitors include aminogluthetimide, anastrozole, fadrozole, formestane, or letrozole, and testolactone. Additional examples of hormone blockers include finasteride.

[0219] In some embodiments, the anticancer agent is a hormone or a derivative thereof, such as an estrogen (e.g., estramustine (T-66), 17-β-estradiol (including derivatives ICI 164,384 or ICI 182,780), a gestagen, or a progestogen (e.g., megestrol acetate).

[0220] In some embodiments, the anticancer agent includes a piperazine derivative, such as piprobroman.

[0221] In some embodiments, the anticancer agent comprises a glutathione analog, such as TLK-286.

[0222] In some embodiments, the anticancer agent comprises a biological response modifier, such as aldesleukin or denileukin.

[0223] In some embodiments, the anticancer agent includes a matrix metalloproteinase inhibitor, such as marimastat, TIMP-1, or TIMP-2.

[0224] In some embodiments, the anticancer agent includes a complex of a rare earth element, such as a lanthanide complex.

[0225] In some embodiments, the anticancer agent includes a metal with anticancer effects, such as zinc.

[0226] In some embodiments, the anticancer agent comprises a photochemically activated drug, such as a porfimer, a photoporphyrin, a benzoporphyrin derivative, a pheophorbide derivative, merocyanine 540 (MC-540), or tin etioporpurin.

[0227] In some embodiments, anticancer agents include agents used in photochemotherapy, such as psoralen used with ultraviolet therapy.

[0228] In some embodiments, the anticancer agent comprises a nitroaromatic compound, such as RSU- 1069, RB-6145, or CB- 1954. In some embodiments, the chemotherapeutic agent comprises a nitroxyl group or an N-oxide, such as, for example, SR-4233.

[0229] In some embodiments, the anticancer agent comprises antisense RNA or DNA, such as oblimersen.

[0230] In some embodiments, the anticancer agent comprises a halogenated pyrimidine analog, such as bromodeoxyuridine or iododeoxyuridine.

[0231] In some embodiments, the additional agent includes an angiogenesis inhibitor, such as, for example, DC-101, novavastatin, tetrathiomolybdate, a thymidine phosphorylase inhibitor, or TNP-470.

[0232] In some embodiments, additional agents include antibiotics (including macrolides), antifungals, or antiparasitics that may or may not have anticancer effects. Non-limiting examples of antibiotics that can be used as additional agents include acridine, actinomycin, amsacrine, ansamitocin, anthramycin, bleomycin, chloramphenicol, dactinomycin, distamycin, duocarmycin, geldanamycin, ketoconazole, liblomycin, maytansine, mithramycin, mitomycin, mitoxantrone, fusobactin, nitroimidazoles (e.g., benznidazole, metronidazole, misonidazole, nimorazole, NLA-1, NLP-1), nitroacridines, nitroquinolines, nitropyrazolacridines, olivomycin, phleomycin, phthalanilides (e.g., propamidine or stipemidine), dibenzimidazoles, prinomycin, rifamycin, rhizoxin, squalamine, tanespimycin (17-allylaminogeldanamycin), or a derivative or salt of any of the foregoing.

[0233] In some embodiments, the additional agent includes an aziridoquinone (eg, mitomycin C, BMY-42355, AZQ, or EO-9).

[0234] In some embodiments, the additional agent includes a 2-nitroimidazole, such as misonidazole, NLP-1 or NLA-1, a nitroacridine, a nitroquinoline, a nitropyrazoleacridine.

[0235] In some embodiments, the additional agent comprises an anti-inflammatory agent, such as, for example, a steroid or a nonsteroidal anti-inflammatory drug. Non-limiting examples of steroids include prednisone, prednisolone, methylprednisolone, dexamethasone, budesonide, fluocortolone, or triamcinolone. Non-limiting examples of additional anti-inflammatory agents include acetylsalicylic acid, mesalamine, ibuprofen, naproxen, flurbiprofen, fenoprofen, fenbufen, ketoprofen, indoprofen, pirprofen, carprofen, oxaprozin, pranoprofen, miprofen, thioxaprofen, suprofen, alminoprofen, tiaprofenic acid, fluprofen, indomethacin, sulindac, tolmetin, zomepirac, nabumetone, diclofenac, fenclofenac, alclofenac, bromfenac, ibufenac, aceclofenac, acemetacin, fentiazac, clidanac, etodolac, oxpinac, mefenamic acid, meclofenamic acid, flufenamic acid, niflumic acid, tolfenamic acid, diflunisal, flufenalil, piroxicam, tenoxicam, lomoxicam, nimesulide, meloxicam, celecoxib, and rofecoxib.

[0236] In some embodiments, the additional agent includes a bisphosphonate or a derivative thereof, such as, for example, minodronic acid or a derivative thereof (YM-529, Ono-5920, YH-529), zoledronic acid monohydrate, ibandronate sodium hydrate, or clodronate disodium.

[0237] In some embodiments, the additional agent is in the form of a pharmaceutically acceptable salt, hydrate, and / or solvate. In some embodiments, the chemotherapeutic agent is in the form of a single optical isomer, a mixture of single enantiomers, or a racemate thereof.

[0238] E. Nucleic acid

[0239] In some embodiments, the composition comprises a nucleic acid (e.g., a DNA or RNA molecule) encoding a polypeptide antigen described herein, such as an α-lactalbumin polypeptide. In such embodiments, the composition may comprise a nucleic acid substituted for the antigen or an adjunct antigen. In some embodiments, the composition comprises an expression vector comprising an open reading frame encoding a polypeptide (e.g., an α-lactalbumin polypeptide).

[0240] When taken up by cells (e.g., muscle cells, antigen presenting cells (APCs) such as dendritic cells, macrophages, etc.), the DNA molecule can be present in the cell as an extrachromosomal molecule and / or can be integrated into the chromosome. DNA can be introduced into the cell in the form of a plasmid, which can be retained as a separate genetic material. Alternatively, linear DNA that can be integrated into the chromosome can be introduced into the cell. Optionally, when the DNA is introduced into the cell, a reagent that promotes the integration of the DNA into the chromosome can be added.

[0241] II. Treatment Methods

[0242] In one aspect, methods are provided comprising administering to a subject a therapeutically effective amount of a composition as disclosed herein.

[0243] In some embodiments, provided herein are methods for treating or preventing cancer and / or inducing an immune response against cancer (eg, breast cancer).

[0244] Subjects

[0245] The methods described herein can be used to treat any subject in need thereof.

[0246] Generally, the subject to which the presently disclosed compositions or formulations are administered has an adaptive immune system. In some embodiments, the subject is a mammal. Examples of subjects include, but are not limited to, humans, livestock, dogs, cats, mice, rats, and transgenic species thereof. In some embodiments, the subject is a human.

[0247] In some embodiments, the subject has cancer or is at risk of developing cancer. For example, the subject may be diagnosed with cancer. The cancer may be a primary cancer or a metastatic cancer. The subject may have any stage of cancer, for example, Phase I, Phase II, Phase III, or Phase IV, with or without lymph node involvement and with or without metastasis. The compositions provided can prevent or reduce further growth of cancer and / or otherwise ameliorate cancer (e.g., prevent or reduce metastasis).

[0248] In some embodiments, the subject does not have cancer but is determined to be at risk for developing cancer, e.g., because of the presence of one or more risk factors, such as environmental exposures, the presence of one or more genetic mutations or variations, family history, etc.

[0249] In some embodiments, the subject has not been diagnosed with cancer. For example, provided compositions and formulations can be used as prophylactic vaccines, e.g., in individuals identified as at risk, in one or more subpopulations for which prophylaxis may be particularly effective, etc. For example, in the case of a vaccine against breast cancer, the subject may be, for example, a non-lactating woman.

[0250] In some embodiments, the cancer is breast cancer (e.g., primary breast cancer, metastatic breast cancer). In some embodiments, the breast cancer is triple-negative breast cancer (negative for estrogen receptor (ER), progesterone receptor (PR), and HER2) or comprises triple-negative cells. In some embodiments, the breast cancer is positive for at least one of ER, PR, and HER2, or comprises cells that are positive for at least one of ER, PR, and HER2.

[0251] In some embodiments, the subject has undergone surgery to remove at least part of a breast tumor. In some embodiments, the subject has a genetic predisposition to breast cancer due to a mutation associated with such risk (e.g., a BRCA1 or BRCA2 gene mutation). In some embodiments, the subject has a family history of breast cancer.

[0252] In some embodiments, the cancer expresses or overexpresses a polypeptide, or a polypeptide fragment and / or variant thereof, for use as an antigen in a provided composition or formulation. For example, in some embodiments, the cancer (e.g., breast cancer) expresses or overexpresses alpha-lactalbumin.

[0253] In some embodiments, the subject has been administered, will be administered, or is concurrently administered with an additional therapy. Additional therapies may include, for example, surgical resection, radiotherapy, chemotherapy, and / or other immunotherapy modalities. In some embodiments, additional therapies include additional agents as described herein.

[0254] For example, in some embodiments, the subject has been administered, will be administered, or is concurrently administered an anti-cancer therapy comprising an anti-cancer agent as described herein.

[0255] In some embodiments, administration is timed relative to the additional therapy in a manner to avoid interfering with the immunogenicity of the compositions as described herein.

[0256] In some embodiments, the subject has been administered an additional therapy, and as a result of the additional therapy, the subject does not exhibit clinical symptoms of the disease for which the subject is being treated, e.g., there is no clinically measurable tumor. However, in some embodiments, the subject is determined to have a risk of disease recurrence or progression. For example, when the disease is cancer, the subject may be determined in some embodiments to have a risk of cancer recurrence or progression, e.g., near the original tumor site and / or at the metastatic site. Such subjects may be further classified as high-risk and low-risk subjects. Classification may be based on, for example, features observed before and / or after treatment with additional therapy. These features are known in the clinical field and may be defined for each type of cancer. Typical features of high-risk subgroups include invasion of adjacent tissues and / or lymph node involvement. Therefore, for example, the pharmaceutical compositions described herein may be administered to the subject to elicit an anti-cancer response to prevent cancer recurrence or progression.

[0257] Route of administration

[0258] The compositions disclosed herein (including pharmaceutical compositions) can be administered by any appropriate route of administration, including oral, parenteral, and other routes of administration discussed in the "Formulations and Pharmaceutical Compositions" subsection of the "Compositions" section. In some embodiments, a therapeutically effective amount of the composition is administered by a systemic route of administration (e.g., by oral or parenteral administration). In some embodiments, a therapeutically effective amount of the composition is administered topically. In some embodiments, a therapeutically effective amount of the composition is administered by subcutaneous, intradermal, subdermal, or intramuscular injection.

[0259] dose

[0260] In certain embodiments, the therapeutically effective amount comprises more than one dose, for example, at least two doses or at least three doses. In some embodiments, the therapeutically effective amount comprises no more than three doses, for example, exactly three doses. In some embodiments, each dose is administered one or more weeks apart, for example, at least two or more weeks apart, at least three or more weeks apart, or at least four weeks apart. In some embodiments, each dose is administered about four weeks apart.

[0261] In some embodiments, each dose contains about the same amount of antigen.In some embodiments, each dose contains about the same amount of antigen and the same amount of carbohydrate.

[0262] In some embodiments, an initial dose is administered and the immunological and / or clinical response of the subject is monitored. Suitable immunological monitoring methods include using the patient's peripheral blood mononuclear cells (PBMC) as responders, neoplastic cells or antigens as stimulants to determine memory or recall responses. Immune responses can also be determined by the presence of delayed inflammatory responses at the application site. After the initial dose, one or more doses may be administered, as appropriate, for example, monthly, half-monthly or weekly, until the desired effect is achieved. Thereafter, additional booster doses or maintenance doses may be given as needed, especially when the immunological or clinical benefit appears to subside.

[0263] Appropriate dosage can be determined, for example, by reference to the resulting plasma concentration of a subject to which the dosage is administered. For example, the maximum plasma concentration (Cmax) and the area under the plasma concentration-time curve from time 0 to infinity (AUC(0-4)) can be used. Dosage includes dosages that produce certain Cmax and AUC(0-4) expected values.

[0264] The dosage may depend on a variety of factors, such as, for example, the activity of the particular antigen or composition; the route of administration; the time of administration; the rate of excretion or metabolism of the components of the particular composition used; the duration of treatment; other drugs, compounds and / or materials used in combination with the particular antigen composition; the age, sex, weight, condition, general health and previous medical history of the subject; and similar factors well known in the medical arts.

[0265] In general, a "therapeutically effective amount" of a composition described herein will be the lowest amount effective to produce the desired immune, prophylactic, or therapeutic effect. For example, in some embodiments, a therapeutically effective amount is an amount capable of inducing an effective humoral or cellular T cell response in a subject to be treated, or in certain embodiments, an effective systemic immune response. Such an effective amount will generally depend on certain factors, such as those described above.

[0266] In some embodiments, each dose contains between about 1 μg to about 20 mg of antigen, for example, between about 1 μg to about 5 mg, between about 50 μg to about 2 mg, or between about 100 μg to about 1 mg of antigen. For example, in some embodiments, each dose contains about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 10 μg, about 15 μg, about 20 μg, about 30 μg, about 40 μg, about 50 μg, about 60 μg, about 70 μg, about 80 μg, about 90 μg, about 100 μg, about 150 μg, about 200 μg, about 250 μg, about 300 μg, about 350 μg, about 400 μg, about 450 μg, about 500 μg, about 550 μg, about 600 μg, about 650 μg, about 700 μg, about 750 μg, about 800 μg, about 850 μg, about 900 μg, about 950 μg, about 1 mg, about 1.5 mg, about 2 mg, about 2.5 mg, about 3 mg, about 3.5 mg, about 4 mg, about 4.5 mg, about 5.5 mg, about 6 mg, about 6.5 mg, about 7 mg, about 7.5 mg, about 8 mg, about 8.5 mg, about 9 mg, about 9.5 mg, about 10 mg, about 15 mg, about 20 mg, or any value therebetween.

[0267] In some embodiments, each dose contains between about 1 μg to about 20 mg of carbohydrate, for example, between about 10 μg to about 10 mg, about 50 μg to about 5 mg, about 100 μg to about 2 mg, or about 100 μg to 1 mg of carbohydrate. For example, in some embodiments, each dose contains about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 10 μg, about 15 μg, about 20 μg, about 30 μg, about 40 μg, about 50 μg, about 60 μg, about 70 μg, about 80 μg, about 90 μg, about 100 μg, about 150 μg, about 200 μg, about 250 μg, about 300 μg, about 350 μg, about 400 μg, about 450 μg, about 500 μg, about 550 μg, about 600 μg, about 650 μg, about 700 μg, about 800 μg, about 900 μg, about 1000 μg, about 1500 μg, about 2000 μg, about 2500 μg, about 3000 μg, about 3500 μg, about 4000 μg, about 4500 μg, about 5000 μg, about 5500 μg, about 6000 μg, about 6500 μg, about or about 20 mg, or any value therebetween, of carbohydrates.

[0268] answer

[0269] In some embodiments, administering the composition induces an immune response.

[0270] Generally, the immune response can include a humoral immune response, a cell-mediated immune response, or both.

[0271] The humoral response can be measured, for example, by standard immunoassays for antibody levels in serum samples from subjects who have received the pharmaceutical composition.

[0272] A cellular immune response is a response that generally involves T cells and can be measured in vitro or in vivo. For example, a general cellular immune response can be determined as T cell proliferation activity in cells (e.g., peripheral blood leukocytes (PBL)) sampled from a subject at an appropriate time after administration of a pharmaceutically acceptable composition. For example, after incubating PBMCs with a stimulator for an appropriate time, [ 3 [H] thymidine incorporation. The percentage of proliferating T cells can be determined using flow cytometry. Another way to measure cellular immunity involves measuring the frequency of circulating T cells that secrete proinflammatory type 1 and / or type 17 cytokines in response to antigen.

[0273] In some embodiments, the immune response comprises an antigen-specific T cell immune response, which may include, for example, CD4+ T cells, CD8+ T cells, or both. In some embodiments, the T cell immune response comprises a type 1 or type 17 proinflammatory T cell response. In some embodiments, the T cell immune response comprises both type 1 and type 17 proinflammatory T cell responses.

[0274] When the antigen is expressed on a cell, administration of the composition can elicit an immune response to the cell. For example, when the antigen is a tumor-associated antigen, administration of the composition can elicit an immune response to tumor cells expressing the antigen.

[0275] In some embodiments, administration results in a decrease in granuloma formation in the subject compared to a reference level. For example, a reference level can be the level of granuloma formation observed in subjects administered a composition comprising complete Freund's adjuvant. In some embodiments, a reference level is the level of granuloma formation observed in subjects administered a composition comprising incomplete Freund's adjuvant. "Reduction in granuloma formation" can be characterized, for example, by one or more of the following: fewer granulomas formed, reduced granuloma severity, a more rapid decrease in granuloma severity, and more rapid regression (partial or complete) of granulomas.

[0276] Cell therapy

[0277] In some embodiments, methods are provided that include administering to a subject cells (e.g., antigen-presenting cells or precursors thereof) that have been contacted in vitro with a composition as disclosed herein, or cells produced from such cells (such as antigen-sensitized antigen-presenting cells or antigen-specific lymphocytes).

[0278] IV. Production Method

[0279] In one aspect, methods for making compositions or preparations as disclosed herein are provided. Generally, such methods include the step of mixing an aqueous solution comprising an antigen with an emulsion comprising a carbohydrate and a metabolizable oil. Suitable antigens, carbohydrates and metabolizable oils, and suitable ratios between two or more components include those described herein, for example, in the "compositions" chapters and sections.

[0280] For example, in some embodiments, the ratio of aqueous solution to emulsion is between about 1:2 and about 2:1 (v / v). In some embodiments, the ratio of aqueous solution to emulsion is about 1:1 (v / v).

[0281] V. Application

[0282] In one aspect, methods of using a composition or formulation as disclosed herein for the manufacture of a medicament for preventing, treating, or ameliorating a disease or condition (eg, cancer) are provided.

[0283] The present invention can be further illustrated by the following examples, which should not be construed as limiting. The contents of all references, patents, and published patent applications cited throughout this application, as well as the figures, are incorporated herein by reference.

[0284] Example

[0285] Example 1: A single vaccination with α-lactalbumin in complete Freund's adjuvant (CFA) induces 1 Type 1 and 17 pro-inflammatory T cell immune responses

[0286] Previously published studies have shown that vaccination with recombinant mouse α-lactalbumin in mice inhibits the growth of both bulk and transplantable breast tumors when used in either a preventive or therapeutic setting. In those experiments where tumor growth inhibition was observed, CFA was used as an adjuvant.

[0287] In this example, the type of immune response elicited by α-lactalbumin in CFA was characterized by examining the splenocyte frequencies of type 1 (IFNγ-producing) and type 17 (IL-17-producing) T cells in mice administered the α-lactalbumin / CFA composition.

[0288] Mice were given a single subcutaneous injection of 100 μg of recombinant mouse α-lactalbumin in CFA (containing 200 μg of H37Ra Mycobacterium tuberculosis). Figure 1 As shown in Figure 2, the average splenocyte frequencies of α-lactalbumin-specific T cells producing IFNγ (type 1) and IL-17 (type 17) pro-inflammatory T cells reached levels of 1 / 6,700 and 1 / 12,700, respectively ( Figure 1 ).

[0289] Thus, a single injection of the α-lactalbumin / CFA composition was sufficient to elicit a proinflammatory immune response involving both type 1 and type 17 T cells.

[0290] Example 2: Development of alternative adjuvants to elicit type 1 and type 17 T cell responses

[0291] As shown in Example 1, antigen / CFA compositions can elicit both type 1 and type 17 T cell responses, indicating that CFA is a potent immunostimulatory adjuvant.

[0292] Unfortunately, CFA cannot be used as an adjuvant in human vaccination due to its toxic effects, which primarily involve the induction of nonresolving granulomas and abscesses at the vaccination site. To develop an alternative nontoxic adjuvant that mimics CFA by inducing similarly high frequencies of α-lactalbumin-specific type 1 and type 17 T cells, a series of adjuvants were tested with the goal of “reverse engineering” the optimized type 1 / type 17 adaptive immune response induced by CFA. Adjuvants tested included: 1) CFA, 200 μg per vaccination, 2) GPI-0100, a triterpene glycoside, 200 μg per vaccination, 3) Sigma lipid A (Ribi adjuvant), 50 μg per vaccination, 4) ASO2B lipid A, 50 μg per vaccination, 5) non-methylated CpG DNA, 100 μg per vaccination, 6) α-galactosylceramide (α-Gal-Cer), 10 μg per vaccination, 7) β-glucan peptide in IFA, 200 μg per vaccination, and 8) zymosan in IFA, 200 μg per vaccination. Each vaccine dose contained 100 μg of recombinant mouse α-lactalbumin protein in 100 μL of an aqueous solution emulsified with 100 μL of an adjuvant (if available) prepared according to the manufacturer's recommendations. In the absence of such recommendations, adjuvants were prepared according to the instructions provided in the references that demonstrated the induction of a substantial immune response. Among the adjuvants tested with recombinant mouse α-lactalbumin, β-glucan peptide / IFA and zymosan / IFA were the only adjuvants that induced type 1 and type 17 T cell frequencies that were associated with tumor immunity and similar to those observed with CFA as an adjuvant ( Figure 2 ).

[0293] In subsequent preclinical studies, in vivo inhibition of breast tumor growth was provided, consistent with that observed with α-lactalbumin / CFA ( Figure 3AThe only immunogen / adjuvant combination that showed a significant inhibitory effect on 4T1 mouse mammary tumor growth comparable to that of zymosan in IFA ( Figure 3H ; P < 0.02). All other adjuvants tested did not provide any in vivo inhibitory effect on breast tumor growth ( Figure 3B -G).

[0294] These results indicate that the combination of zymosan and IFA is an effective adjuvant in vaccine compositions. Furthermore, an anticancer vaccine comprising zymosan, IFA, and an antigen expressed on cancer cells successfully inhibited tumor growth in vivo.

[0295] Example 3: Development of a non-toxic adjuvant that elicits both type 1 and type 17 T cell responses

[0296] A large part of the toxic effects induced by CFA is likely due to the IFA used to suspend dead Mycobacterium tuberculosis. IFA is often used as a stand-alone adjuvant to induce the production of type 2 regulatory T cells and antibodies. IFA is prepared from non-metabolizable oils, including paraffin oil. These non-metabolizable oils are retained for a long time and promote antigen presentation. However, this retention may actually lead to the unresolved granulomas and abscesses at the inoculation site observed in the case of CFA. Therefore, in order to develop a non-toxic alternative adjuvant suitable for human vaccination that can trigger a high frequency of antigen-specific type 1 and type 17 pro-inflammatory T cells together with the antigen, an alternative to IFA is being sought. This alternative can then be used as a vaccine adjuvant together with yeast polysaccharide.

[0297] Montanide TM ISA 51 VG (Seppic, Paris, France) was tested as a potential alternative to IFA. TM ISA 51 VG is a high purity mineral oil (DRAKEOL TM MONTANIDE is a GMP-grade mixture of a surfactant (6VR) and a surfactant (dehydrated mannitol monoglyceride). When mixed with an aqueous immunogen in a 50 / 50 ratio, a water-in-oil emulsion is obtained. Similar to IFA, this water-in-oil emulsion acts as a stand-alone vaccine for generating an enhanced immune response. TM ISA51 VG as a Montanide TM Alternative to ISA 51. The difference between these two grades is the source of oleic acid used to make the surfactant (mannitol monooleate). Used in the preparation of MONTANIDE TM Oleic acid in ISA 51 is of animal origin. Due to concerns about bovine spongiform encephalopathy (BSE) and other transmissible spongiform encephalopathies, MONTANIDE TMThe oleic acid used in ISA 51 VG is of vegetable origin. Since 2006, MONTANIDE TM ISA 51 VG (hereinafter referred to as "MONTANIDE TM ”) has been used in more than 150 human clinical trials worldwide involving over 10,000 patients. Detailed composition, manufacturing process, analytical controls, and stability data are described in the Drug Master File (DMF) or Common Technical Document (CTD) registered in different countries (DMF Type IV, Nos. 9756 and 10870 in the United States; BBMF Nos. 12130 and 14167 in the United States).

[0298] To test Montanide TM As an adjuvant component, female BALB / cJ mice aged 6-8 weeks were administered a single dose of an emulsion containing either 100 μL of IFA or 100 μL of MONTANIDE. TM Four weeks after vaccination, splenocyte frequencies of proinflammatory type 1 (IFNγ) and type 17 (IL-17) T cells were determined by ELISPOT assay. Figure 4 The data in show the mean spot forming units (SFU) of the recall response to 50 μg / mL recombinant mouse α-lactalbumin minus the mean background response of cultures without recall antigen (mean background < 5 SFU per assay).

[0299] like Figure 4 As shown in TM When used as an adjuvant, the production of both type 1 and type 17 pro-inflammatory T cells was induced. However, the level of induction was much lower than that achieved using zymosan as an adjuvant in IFA. Therefore, multiple doses containing zymosan / MONTANIDE may be required. TM Vaccines as adjuvants to achieve high T cell frequencies associated with tumor growth inhibition.

[0300] These results indicate that adjuvants containing yeast polysaccharide in a metabolizable oil can support antigen-induced type 1 and type 17 immune responses.

[0301] Example 4: α-lactalbumin / zymosan / MONTANIDE TM Effective dose of vaccine

[0302] To determine the use of Zymosan / Montanide TMThe effective dose or doses of the adjuvant combination required to provide high frequencies of α-lactalbumin-specific type 1 / type 17 T cells are administered to female BALB / c mice in one or more doses of 200 μL of an emulsion containing equal amounts (by weight) of α-lactalbumin and zymosan, with each dose containing 100-1000 μg of each of α-lactalbumin and zymosan.

[0303] Recombinant mouse α-lactalbumin (FLAG-N-mαlac-C-HIS) was solubilized in sterile USP grade water and zymosan was suspended in MONTANIDE TM Four weeks after the last vaccination, splenocytes were subjected to ELISPOT analysis using capture / antibody pairs specific for mouse IFNγ, IL-5, and IL-17 to assess the frequencies of splenocytes of type 1, type 2, and type 17 T cell lineages generated, respectively.

[0304] Three mice per group received one, two, or three doses, four weeks apart. Four weeks after the last dose, splenocyte frequencies of type 1 proinflammatory T cells, type 17 proinflammatory T cells, and type 2 regulatory T cells were measured by ELISPOT assay using capture / antibody pairs specific for mouse IFNγ, IL-17, and IL-5, respectively.

[0305] like Figure 5 As shown, at all doses tested (100 μg, 250 μg, 500 μg, and 1000 μg each of α-lactalbumin and zymosan), both type 1 and type 17 responses were elicited after the third dose. Three doses of an emulsion containing 1 mg α-lactalbumin / 1 mg zymosan consistently generated high frequencies of type 1 / type 17 T cells ( Figure 5 ).

[0306] Furthermore, no abscesses were observed at any dose, and all granulomas that developed at the vaccination site resolved completely within 1-2 weeks after vaccination. No long-term adverse effects were observed. TM It is a potent immunogen / adjuvant combination used to induce α-lactalbumin-specific type 1 and type 17 T cells at frequencies generally sufficient to inhibit breast tumor growth without inducing non-resolving granulomas and abscesses at the vaccination site.

[0307] In summary, the results described in Examples 2-3 indicate that vaccination with α-lactalbumin / zymosan / IFA can induce type 1 / type 17 T cell immunity and inhibit breast tumor growth similar to vaccination with α-lactalbumin / CFA. TMThe resulting water-in-oil emulsion, when used with appropriate antigens, induced antigen-specific type 1 / type 17 proinflammatory T cells, but at a lower frequency than that observed with IFA. TM The emulsion induced type 1 / type 17 T cell frequencies that were associated with inhibition of breast tumor growth without inducing abscesses or non-resolving granulomas. TM The emulsion induces type 1 / type 17 immunity that is associated with potent breast tumor growth inhibition.

[0308] Example 5: α-lactalbumin / zymosan / MONTANIDE TM Toxicological characteristics of the vaccine

[0309] For evaluation included in MONTANIDE TM Is the adjuvant zymosan in the α-lactalbumin / zymosan / MONTANIDE vaccine related to toxicity? TM The mice were examined histopathologically and bioassay-wise.

[0310] Study Design

[0311] Three groups of 25 mice each (Groups A, B, and C) received one, two, or three doses, respectively. Each group was further divided into five subgroups of five mice each, each receiving: 1) control vaccine; 2) vaccine containing 100 μg of recombinant mouse α-lactalbumin (FLAG-N-mαlac-C-HIS variant); 3) vaccine containing 1000 μg of recombinant mouse α-lactalbumin (FLAG-N-mαlac-C-HIS variant); 4) vaccine containing 100 μg of recombinant human α-lactalbumin (HISTEV-N-hαlac-COOH variant); and 5) vaccine containing 1000 μg of recombinant human α-lactalbumin (HISTEV-N-hαlac-COOH variant). All vaccines were produced at MONTANIDE TM ISA 51 VG is a water-in-oil emulsion with equal volumes of recombinant α-lactalbumin as target antigen in the aqueous phase and zymosan as adjuvant in the oily phase.

[0312] Mice in group A received a single dose, mice in group B received two doses with a one-month interval, and mice in group C received three doses with a one-month interval, as shown in Table 2.

[0313] Table 2. Study design

[0314]

[0315]

[0316] Materials and methods

[0317] Recombinant alpha-lactalbumin

[0318] The open reading frame cDNA nucleotide sequences of mouse alpha-lactalbumin (NCBI Reference Sequence: NM_010679.1) and human alpha-lactalbumin (NCBI Reference Sequence: NM_002289.2) were modified by replacing mammalian codons with more efficient prokaryotic sequences that encode the same amino acid (Dapcel, Cleveland, OH) to ensure optimal protein folding and production in prokaryotic expression systems. Optimized DNA sequences were synthesized de novo (GeneArt, Regensburg, Germany). Mouse alpha-lactalbumin DNA was inserted into a pET3a expression vector (GeneArt) to provide recombinant mouse alpha-lactalbumin containing an N-terminal FLAG-tag and a C-terminal 6xHis-tag (FLAG-N-malac-C-HIS). Human alpha-lactalbumin DNA was also inserted into a pET3a expression vector (GeneArt) to provide recombinant human alpha-lactalbumin containing a 6xHis-tag linked to the N-terminus and a tobacco etch virus nuclear inclusion a endopeptidase (TEV protease) as a cleavage site for removal of the 6xHis-tag from the recombinant human alpha-lactalbumin protein (HIS TEV-N-halac-COOH). Plasmids containing these inserts were transformed into E. coli strain BL21 Star (Invitrogen, Carlsbad, CA). High-level expressing colonies were selected after induction with isopropyl beta-D-l-thiogalactopyranoside (IPTG; Amresco, Solon, OH) and sequenced to confirm proper orientation and alignment. 6xHis-tagged proteins were purified under denaturing and reducing conditions using nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography (Qiagen Sciences, Germantown, MD). Proteins were purified by reverse phase high performance liquid chromatography (HPLC) to obtain endotoxin-free proteins prior to use in vitro.

[0319] Water

[0320] Sterile USP grade water was obtained commercially (Corning) and used to solubilize recombinant alpha-lactalbumin protein in the aqueous phase of the emulsion.

[0321] Zymosan

[0322] Zymosan A was obtained commercially (Sigma-Aldrich) and used as a vaccine adjuvant.

[0323] MONTANIDE TM MONTANIDE TM ISA 51 VG was commercially available (Seppic) and used as solvent for suspending zymosan and for maintaining and stabilizing the emulsion phase.

[0324] Vaccine preparation

[0325] The recombinant α-lactalbumin protein solution was adjusted to a concentration of 10 mg / ml in sterile USP grade water to prepare a stock solution. Zymosan was suspended in MONTANIDE at a concentration of 10 mg / ml. TM To prepare a stock solution, add ISA 51 VG. To inoculate with a high dose of 1000 μg recombinant α-lactalbumin + 1000 μg zymosan, load a 3.0 mL syringe with the aqueous recombinant α-lactalbumin solution and lock it into a double female luerlock connector. Load another syringe with an equal volume of the oily zymosan suspension and lock it into the other end of the connector.

[0326] Emulsification consists of two stages: pre-emulsification at a very slow speed and final emulsification at a high speed. The plunger of the syringe containing aqueous recombinant α-lactalbumin is fully pushed so that both phases are in one syringe. During the slow pre-emulsification stage, the entire formulation is slowly and repeatedly passed from one syringe to another through a connector for a total of 30 cycles, with each plunger advancement taking 4 seconds (each cycle taking 8 seconds to complete). At the end of this pre-emulsification stage, the speed is increased sharply so that 80 additional complete cycles are performed as quickly as possible. The entire emulsion is then inserted into a syringe and a sterile 26-gauge needle is attached.

[0327] Before vaccination, the formation of a stable emulsion was confirmed by the drop test: if a drop of emulsion maintained its structural integrity for at least 5 minutes when placed in a beaker of water, the emulsion was considered stable. A high-dose vaccine (1000 μg recombinant α-lactalbumin + 1000 μg zymosan) was administered subcutaneously to the dorsal neck area with 200 μL of the emulsion. To prepare an emulsion containing a low-dose vaccine (100 μg recombinant α-lactalbumin + 100 μg zymosan), the stock reagent was diluted to 10% of its original concentration with an appropriate solvent before emulsion preparation.

[0328] Mice and vaccine administration

[0329] Female BALB / cJ mice were purchased commercially at 6-7 weeks of age (Jackson Laboratory, Bar Harbor, ME) and vaccinated at 8-10 weeks of age.

[0330] Each dose of 200 μl of the emulsion was injected subcutaneously in the dorsal neck region of the mice. All vaccinations were performed dorsally, starting near the back of the scalp and continuing in a caudal direction approximately 1.5 cm from each previous vaccination site for subsequent vaccinations.

[0331] To facilitate precise injections, mice were immobilized under isoflurane anesthesia prior to each dose. Following each dose, mice were housed and maintained in microisolator cages with free access to sterile chow and water. Mice were observed daily for any changes in behavior, including altered social activity, isolation, changes in appearance or grooming behavior, excessive attention to the injection site, and excessive scratching.

[0332] Biometrics

[0333] Baseline body weight and temperature were obtained immediately before each administration and every three days thereafter. Body weight and temperature changes during the experiment were plotted relative to the percentage of baseline deviation, with body weight on day 0 being taken as 100%. Body temperature was measured using an Optris LSIR thermometer (Micro-Epsilon, Raleigh, NC), with a temperature probe placed at the same distance from the xiphoid process during each measurement. The weight of the liver, spleen, and kidneys was measured during autopsy and calculated as the percentage of each mouse's total body weight.

[0334] autopsy

[0335] Each group of mice was housed in a single cage, with each mouse individually distinguished by a different permanent ear punch and secondarily by a temporary tail mark. Mice were euthanized 14-16 days after their last vaccination.

[0336] On the day of necropsy, place one cage at a time in a chemical fume hood where the necropsy will be performed. Observe the mice's grooming patterns and injection site condition.

[0337] Body temperature and body weight were recorded, and the mice were then placed in a chamber connected to an isoflurane vaporizer (providing 97.5% pure oxygen and 2.5% isoflurane). After deep anesthesia, the mice were placed ventrally upwards and the chest area was wiped with a 70% isopropyl alcohol preparation pad so that cardiac puncture could be performed with a 1.0 mL syringe and a 22-gauge needle. After collecting approximately 0.5-0.8 mL of whole blood, the mice were dislocated from the cervical vertebrae and a midline ventral incision was made in the abdominal wall from the lower abdomen to the xiphoid process with surgical scissors to expose the contents of the abdominal cavity. Then, an incision was made through the diaphragm, cutting through the thorax. The vena cava was opened to achieve perfusion outlets, and the 26-gauge needle on a 30 mL syringe equipped with cold sterile PBS was inserted into the left ventricle for perfusion. The mice were slowly perfused with a total volume of 30 mL of cold PBS. The spleen, liver, and kidneys were taken out and weighed, and then placed in 10% phosphate-buffered formalin. After this, the remaining tissue to be collected was carefully removed and placed in a fixative. After 24 hours, the tissues were removed, washed with PBS, and placed in 70% ethanol until histopathological processing. The brains of mice in group C were fixed for a longer period and then stored in 70% ethanol.

[0338] Histopathology

[0339] The following tissues were collected for histopathological examination: kidney, brain, large intestine, liver, lung, ovary, spleen, heart, uterus, skin (injection site), stomach, bladder, mammary gland, and small intestine. Beginning with Groups B and C, skin samples were collected from each injection site to evaluate the healing process of the injection site over time. For Group C, additional tissues were collected and analyzed, including the thymus, mesenteric lymph nodes, and mandibular salivary glands. Tissue processing for Group C was also modified to include: 1) filling the bladder and lungs with formalin as part of the collection process; and 2) formalin fixation of the brain in the skull, where the calvaria was removed for five days, rather than fixation for 24 hours. All tissues were fixed overnight in 10% phosphate-buffered formalin, washed with PBS, and stored in 70% ethanol until processing. Tissues were paraffin-embedded, sectioned, mounted on slides, and stained with hematoxylin and eosin. Tissue sections were analyzed by a veterinary pathologist.

[0340] result

[0341] Figure 6-8 Shown are changes in body weight during the observation period. Figure 9-11 The changes in body temperature for each group and subgroup are shown. Figure 12-14 The weights of the spleen, liver, and kidneys recorded at necropsy are shown and expressed as a percentage of total body weight.

[0342] There were no apparent mortality or severe morbidity issues throughout the study. Morbidity appeared to be limited to the injection site, with incomplete healing or return of hair growth at the injection site during the 14-15 days post first dose or during the 45 day or 75 day observation periods in mice receiving two or three doses, respectively. Overall, mice exhibited normal behavior in terms of eating, drinking, and socializing. Generally, mice gained weight over the course of the study, with the exception of a transient, slight weight loss immediately following each vaccination, which was quickly recovered by the mice. Figure 6-8

[0343] Mice receiving the highest dose vaccines (M1000 and H1000) exhibited substantial interest in their injection sites 6 days post-injection. The injection sites typically became hairless and had a granulomatous appearance by day 9 and began to crust over by day 12. By the time of euthanasia on days 14-15 (Group A), the crusts had resolved and the injection sites were almost completely healed, but remained hairless. This pattern of irritation, hair loss, crusting, and healing resolution was also observed in the M1000 and H1000 subgroups in the case of the second and third vaccinations. For example, by the time of the second vaccination, Group B mice exhibited apparent healing from the first vaccination. The initial injection sites of mice receiving multiple doses exhibited continued improvement throughout the study, but did not exhibit complete healing or full return of hair growth by the 45 day observation period (for mice receiving two doses) or the 75 day observation period (for mice receiving three doses).

[0344] However, the degree of healing generally correlated with the age of the injection site, with the oldest injection sites appearing the healthiest. Conversely, the area of hair loss around the older injection sites was typically greater. It is not currently known why this occurs, but it is possible that the mice find it easier to scratch at the initial injection site (closest to their heads).

[0345] Mice receiving three doses of human alpha-lactalbumin (H100) at a low dose, but not mouse alpha-lactalbumin (M100), exhibited the same pattern of irritation, hair loss, crusting, and healing resolution as mice injected with the highest vaccine dose. This aggressive response was limited to mice inoculated with the human alpha-lactalbumin protein and only occurred after the third inoculation. Without wishing to be bound by any particular theory, the aggressive response of these mice can be related to the enhanced immunogenicity of the heterologous antigen and the high level of immunity achieved against three doses of the heterologous antigen.

[0346] Figure 15 and 16 shows resolution of granulomas at the injection site over time. Figure 15 shows photographs of mice taken approximately two weeks after the second injection and approximately six weeks after the first injection. The blue arrow indicates the first injection site and the red arrow indicates the second injection site. As​ Figure 15 As shown, the first injection site exhibited improved appearance compared to the second injection site, indicating resolution of granulomas over time.

[0347] Figure 16 The average grade of oil granulomas at the injection sites, assessed using the following grading system: 0, normal; 1, very mild; 2, mild; 3, moderate; 4, severe, is shown. For each injection site, the average grade was calculated for all mice (n=25) in all treatment groups. Error bars represent ± SE. On average, all injection sites initially exhibited a moderate oil granuloma. However, these granulomas resolved significantly over time, with significant improvements at the end of the experiment between the first and third immunizations (P<0.001) and between the second and third immunizations (P<0.05).

[0348] Results of the histopathological analysis of the mice are shown in Tables 3A and 3B (Group A; single dose), Tables 4A and 4B (Group B; two doses), and Tables 5A-5C (Group C; three doses). The following symbols were used in Tables 3A, 3B, 4A, 4B, 5A, 5B, and 5C:

[0349] n = normal (no lesion)

[0350] BI = biliary inflammation

[0351] FI = focal inflammation

[0352] OG = oil granuloma (injection site)

[0353] Ab = abscess

[0354] At = atrophy

[0355] GCH = germinal center hyperplasia

[0356] LPH = lamina propria hyperplasia

[0357] MH = myeloid hyperplasia

[0358] em = epicardial mineralization

[0359] NP = nephropathy

[0360] FC = fatty change

[0361] FN = focal necrosis

[0362] u = ulcer

[0363] n / s = no section

[0364] ns = not found

[0365] np = not present

[0366] grade:

[0367] 1 = Very mild

[0368] 2 = Mild

[0369] 3 = Moderate

[0370] 4 = Severe

[0371] In group A (single dose) (Tables 3A and 3B), no adverse effects on organs were found in mice vaccinated with human or mouse α-lactalbumin (M and H subgroups). Lesions were found at the injection site; these lesions were oily granulomas containing the injected material, macrophages, and some neutrophils. In some mice, abscesses were found at the injection site. Abscesses indicate that the injected material was contaminated with bacteria or that bacterial contamination occurred during the injection procedure. Most of the other lesions found were considered incidental events because they were very mild to mild and were also found in the control group. Inflammatory cell lesions in the liver were observed in many mice, which may be related to the injection site lesions.

[0372] Table 3A. Histopathological analysis of mice in group A

[0373]

[0374]

[0375]

[0376] Table 3B. Histopathological analysis of mice in group A

[0377]

[0378]

[0379] In Group B (two doses) (Tables 4A and 4B), liver inflammatory cell lesions were found in many mice. The liver lesions were random and bilious and mainly composed of lymphocytes, sometimes with neutrophils and macrophages. Some liver lesions were myeloid lesions (hepatic myeloid hyperplasia). The liver lesions included mild biliary inflammation and were probably not associated with adverse effects on hepatocytes and liver function.

[0380] Splenic myeloproliferative lesions observed in some mice may represent a response to injection site lesions. Splenic germinal center hyperplasia observed in some mice may represent a response to injection site lesions and / or a response to antigen.

[0381] Breast tissue is often present in skin biopsies but not in breast biopsies, which often contain only muscle.

[0382] Abscesses composed of neutrophils were observed at the injection site in some mice, which may indicate sterile abscesses that were either free of bacteria or contained bacteria. Bacteria were generally absent from the lesions.

[0383] Table 4 Histopathological analysis of mice in group A and B

[0384]

[0385]

[0386]

[0387] Table 4B. Histopathological analysis of mice in group B

[0388]

[0389]

[0390]

[0391] In Group C (three doses) (Tables 5A-5C), histopathological findings were similar to those in Groups A and B. The most severe skin lesions occurred at the third (most recent) injection site. No organ-specific toxic lesions were observed. The majority of the dose-independent lesions observed were incidental.

[0392] Liver lesions were also most severe in mice in the high-dose group. These lesions resembled those caused by Helicobacter hepatica, a bacterium commonly found in immunodeficient mice. To evaluate the cause of these liver lesions, the livers of three mice with numerous foci of liver inflammation containing an infiltrate composed primarily of lymphocytes and some macrophages, with occasional necrosis, were stained with Steiner stein. Positive control sections of human stomachs containing bacteria were also included. None of the livers of the mice with inflammatory lesions had lesions that stained positive for bacteria. Therefore, the liver lesions observed in those mice were unlikely to be due to infection with Helicobacter hepatica.

[0393] Table 5 Histopathological analysis of mice in group A and C

[0394]

[0395]

[0396]

[0397] Table 5 Histopathological analysis of mice in group B.C

[0398]

[0399]

[0400] Table 5C Histopathological analysis of mice in group C

[0401]

[0402]

[0403]

[0404] Figure 17 A summary of the severity of focal liver inflammation in all groups is presented.

[0405] In summary, these results suggest that the liver lesions were random, focal, bilious, and predominantly mild and benign, with the most severe lesions occurring in high-dose mice. The liver lesions likely had no adverse effects on hepatocytes or liver function.

[0406] Example 6: Using α-lactalbumin / zymosan / MONTANIDE TM Immunization against breast cancer growth Preventive suppression

[0407] MMTV-neu mice can be used as a mouse model for metastatic breast cancer. MMTV-neu mice express the neu protooncogene under the control of the mouse mammary tumor virus (MMTV) long terminal repeat and develop spontaneous mammary tumors with a 50% incidence by 205 days of age. (See, for example, Guy CT et al. Expression of the neu protooncogene in the mammary epithelium of transgenic mice induces metastatic disease. Proc Natl Acad Sci USA. 1992;89:10578–10582.)

[0408] To test α-lactalbumin / zymosan / MONTANIDE TM To test the ability of a vaccine to inhibit breast cancer growth, MMTV-neu mice are administered three or more doses of a formulation containing α-lactalbumin in zymosan and Montanide, for example, at a dose of 1000 μg each of zymosan and Montanide. Additional groups of mice can be used as controls or for comparison and can include mice receiving: 1) α-lactalbumin in CFA (positive control); 2) α-lactalbumin in zymosan and IFA; or 3) α-lactalbumin in MONTANIDE without antigen. TM Zymosan in (negative control).

[0409] To evaluate the effect of α-lactalbumin / zymosan / MONTANIDETM The ability of the vaccine to inhibit cancer growth before it appears can be tested by administering the first, second, and third doses to mice before tumors are expected to appear, for example, at 6-10 weeks. Tumor incidence and / or size can be examined and compared between groups.

[0410] Example 7: Effect of immunization with α-lactalbumin / zymosan / Montanide on the growth of established breast cancer The inhibitory effect

[0411] For the detection of α-lactalbumin / zymosan / MONTANIDE TM The ability of the vaccine to inhibit further growth of established breast cancer is tested by administering three or more doses of a vaccine containing zymosan and MONTANIDE to mice from one or more appropriate cancer models. TM α-lactalbumin in pharmaceutical agents, such as zymosan and montanide TM Each dose of 1000 μg is administered. The experiments and mouse groupings are similar to those described in Example 4, except that the timing of dosing may be different, depending on the cancer model. For example, at least one, two, or three doses are administered to mice of a certain age at which, based on known characteristics of the mouse model, a significant proportion of mice in the cancer model have established cancer. Alternatively, at least one, two, or three doses are administered after the mice exhibit at least one sign that they have developed at least one tumor (e.g., the mice have at least one palpable tumor).

[0412] Any of a variety of suitable cancer models can be used to test the effect on established mammary tumors. By way of non-limiting example, MMTV-PyVT transgenic mice constitutively express an activated form of the neu oncogene in mammary tissue and develop invasive, palpable mammary tumors by 5 weeks of age. (See, e.g., Guy CT, Cardiff RD, Muller WJ. Induction of mammary tumors by expression of polyomavirus middle Toncogene: a transgenic mouse model for metastatic disease. Mol Cell Biol. 1992; 12: 954–961.) Therefore, MMTV-PyVT mice can be used to evaluate the effect on bulk tumor growth.

[0413] Transplantable breast cancer models may also be suitable. For example, mice can be inoculated by injection with tumor cells from a breast cancer cell line such as 4T1.

[0414] Tumor size is examined at one or more different time points (eg, time after immunization, time after tumor cell inoculation, etc.) and compared between groups.

[0415] Example 8: Phase I clinical trial of α-lactalbumin vaccine in patients with non-metastatic triple-negative breast cancer at high risk of recurrence Bed Test

[0416] Treatment of triple-negative breast cancer is inadequate and hampered by the lack of actionable therapeutic targets. The results described in Examples 2-4 and other preclinical studies indicate that a combination of α-lactalbumin (antigen) and zymosan (in MONTANIDE TM The vaccine (in a solvent) can induce an immune response consistent with effective breast tumor prevention and treatment. In addition, the vaccine has great potential for treating human triple-negative breast cancer.

[0417] An open-label Phase I clinical trial is planned to determine the dose and schedule for use of the vaccine in future trials. This trial will include an initial dose escalation phase to determine the efficacy of α-lactalbumin / zymosan / MONTANIDE TM The maximum tolerated dose (MTD) of the vaccine in patients with non-metastatic triple-negative breast cancer (TNBC). Subsequently, cohort expansion was used to explore the relationship between vaccine dose and immune response in order to select the dose for the Phase II trial.

[0418] Figure 18 The study protocol is shown, indicating the timeline for dosing, toxicity assessments, and blood sampling for immunological monitoring.

[0419] Purpose and destination

[0420] Primary objective: To determine the effect of α-lactalbumin / zymosan / MONTANIDE TM The maximum tolerated dose (MTD) of the vaccine in patients with non-metastatic TNBC.

[0421] Secondary objective: To measure the immune response to the vaccine, focusing on the ability to induce proinflammatory T cell responses consistent with tumor protection. This assessment was performed using ELISPOT assays to determine the peripheral blood frequency of T cells that produce gamma interferon (IFNγ; type 1) and IL-17 (type 17) in response to recombinant human α-lactalbumin. Based on these immunological assays, the lowest dose that produces an immune effect—the minimum immune dose (LID)—was identified.

[0422] Immune response was defined as the response to α-lactalbumin / zymosan / MONTANIDE TM The response to vaccination was induced by 1 / 30,000 IFNγ-secreting (type 1) and / or IL-17-secreting (type 17) T cells induced by peripheral blood mononuclear cells after treatment. The LID was defined as the lowest tolerated dose at which an immune response was observed in >1 / 10 patients.

[0423] Exploratory objectives: Determine the optimal immunization dose (OID) of alpha-lactalbumin vaccine in the patient population with operable TNBC based on the ELISPOT assay. The OID is defined as the dose at which the majority of patients develop an immune response as defined above. If more than one dose level has the same number of immune responders, the lowest dose among these dose levels is defined as the OID. The OID must be < MTD.

[0424] Correlation objectives:

[0425] • Examine the cellular response to alpha-lactalbumin / Zymosan / MONTANIDE TM vaccination in the patient population with operable TNBC using the ELISPOT assay for IFNy and IL-17 production in response to alpha-lactalbumin stimulation.

[0426] • Examine the humoral response to alpha-lactalbumin / Zymosan / MONTANIDE TM vaccination in the patient population with operable TNBC using the ELISA assay.

[0427] Safety objectives: Determine the incidence of adverse events (drug-related and emergent treatments).

[0428] Subjects

[0429] Forty to sixty subjects who meet the following inclusion criteria but are not excluded by any of the following exclusion criteria are enrolled. These subjects represent a high-risk population with operable TNBC. Subjects are enrolled after completion of all standard therapy, which can include chemotherapy, surgery, or radiation therapy. Chemotherapy can be given pre- or post-operatively.

[0430] Inclusion criteria: This trial is open to men and women, and to members of all racial and ethnic groups. Potential subjects must meet all of the following inclusion criteria to be eligible for enrollment:

[0431] • Histologically proven invasive breast cancer

[0432] • Primary tumor is estrogen receptor (ER) negative (<1% of cells ER), progesterone receptor (PR) negative (<1% of cells PR), and HER2 negative (0-1+, IHC or FISH ratio <2.0, signal number <6 / cell) ("triple negative breast cancer").

[0433] • High risk, defined as any of the following:

[0434] • Pathological stage IIA, IIB, IIIA, IIIB, or IIIC by AJCC 6, or

[0435] Residual invasive cancer in the breast or regional nodules after preoperative chemotherapy

[0436] Six months or less since the last active therapy (chemotherapy, radiotherapy, or surgery) and <12 months since the start of current cancer treatment.

[0437] Treatment prior to enrollment must be consistent with the current NCCN guidelines, which can be found on the website at "https: / / www.nccn.org / "

[0438] Age > 18 years old

[0439] ECOG (Eastern Cooperative Oncology Group) performance status 0-1. (See, e.g., Oken M, Creech R, Tormey D, et al. Toxicity and response criteria of the Eastern Cooperative Oncology Group. Am J Clin Oncol. 1982; 5: 649-655; or available on the website at “https: / / ecog-acrin.org / resources / ecog-performance-status”).

[0440] Adequate major organ function, defined as white blood cell count >3,000 / mcL; hemoglobin >10.0 g / dL; platelets >100,000 / mcL; total bilirubin within normal limits; ALT / AST <2.5 × upper limit of normal (ULN); and serum creatinine <1.5 × ULN

[0441] Serum prolactin level must be < upper limit of normal (ULN)

[0442] Able to understand and willing to sign and provide written informed consent

[0443] Archival tissue may be used for potential correlative studies (e.g., assays for α-lactalbumin expression or tumor-infiltrating lymphocytes), but tumors do not need to overexpress α-lactalbumin for inclusion.

[0444] The subjects agreed not to use alternative treatments from the time of signing the informed consent until 30 days after the last vaccination.

[0445] Exclusion Criteria. Any of the following characteristics will exclude a potential subject from this study:

[0446] Received cytotoxic chemotherapy within 4 weeks before entering the study

[0447] Received radiotherapy within 4 weeks before entering the study

[0448] Toxicity from prior therapy failed to resolve to National Cancer Institute's Common Terminology Criteria for Adverse Events (CTCAE) grade 0-1, except for alopecia and grade 2 neuropathy.

[0449] Unless systemic corticosteroids are required (defined as physiologic replacement of prednisone 10 mg / day or equivalent).

[0450] Requirement for immunosuppression (eg, history of organ transplant)

[0451] Known HIV infection

[0452] Active or planned breastfeeding or pregnancy

[0453] Currently taking or planning to take oral contraceptives

[0454] Refusing to use an effective non-hormonal method of contraception. Acceptable methods of contraception include, but are not limited to, barrier contraception (diaphragm or condom), non-hormonal intrauterine devices, and vasectomy for the male partner.

[0455] · Received any other investigational agents within the past 4 weeks.

[0456] Any known recurrence or metastasis

[0457] History of another active invasive malignancy within 5 years before study entry

[0458] History of allergic reaction to α-lactalbumin, human milk (excluding lactose intolerance), zymosan, or other agents used in this study

[0459] Uncontrolled concurrent illness, including but not limited to ongoing or active infection, symptomatic congestive heart failure, unstable angina, arrhythmias, or psychiatric / social conditions that may limit compliance with study requirements

[0460] Known hyperprolactinemia

[0461] Currently receiving medications known to cause hyperprolactinemia

[0462] Known allergy to penicillin

[0463] Subject screening

[0464] Subjects were screened by review of medical records, history, and physical examination. Pathology materials were also reviewed to determine study eligibility. Screening was performed within 4 weeks prior to administration of the first vaccination.

[0465] Potentially eligible subjects who agreed to participate in the study underwent laboratory tests, including a complete blood count (CBC) with differential, prolactin level, and comprehensive metabolic profile (total protein, albumin, calcium, total bilirubin, alkaline phosphatase, AST, glucose, BUN, creatinine, sodium, potassium, chloride, bicarbonate, and ALT), and a pregnancy test.

[0466] Subjects are not required to undergo scans to rule out metastatic disease, but will be managed according to American Society of Clinical Oncology (ASCO) and National Comprehensive Cancer Network (NCCN) guidelines. Potential study subjects with signs or symptoms of metastatic disease or unexplained abnormal alkaline phosphatase or liver function tests will undergo standard of care imaging to rule out the presence of metastases.

[0467] Vaccine preparations

[0468] cGMP-grade Zymosan A (Sigma-Aldrich Fine Chemicals, Buchs, Switzerland) was suspended in GMP-grade MONTANIDE TM ISA 51 VG (Seppic, Fairfield, NJ) was used to prepare Zymosan / MONTANIDE TM MONTANIDE TM ISA 51 VG is a light mineral oil / surfactant solvent that facilitates the formation of water-in-oil emulsions and has a much higher metabolic capacity than the incomplete Freund's adjuvant (IFA) oil used to formulate CFA. GMP-grade recombinant human α-lactalbumin (rhαlac) (List Biologicals, Campbell, CA) in USP-grade HO was combined with zymosan / MONTANIDE TM The suspensions were mixed to create a water-in-oil emulsion. Each emulsion product was prepared by a clinical pharmacist on the day of use and administered to each patient within 2 hours of dispensing.

[0469] Dosing and administration

[0470] Subjects were administered α-lactalbumin / zymosan / MONTANIDE by subcutaneous administration at alternating sites (left thigh, right thigh, and abdomen). TM Vaccine. Subjects received three doses, four weeks apart. In the initial dose escalation phase, patients were sequentially enrolled in one of six different dose levels (as shown in Table 6), with each dose level consisting of a cohort of 1-6 patients until the MTD was identified. Dose level 1 was 1% of the dose routinely given to mice in previous preclinical studies. Intra-patient dose escalation was not allowed.

[0471] Table 6. Dose levels in the dose escalation study

[0472]

[0473]

[0474] mcg = microgram

[0475] MONTANIDE TM was used as the solvent at all dose levels.

[0476] Dose escalation continued within each cohort according to the accelerated titration dose escalation schedule summarized in Table 7. Dose-limiting toxicities (DLTs) generally corresponded to CTCAE 5th edition grade 2 or higher toxicities. (The CTCAE 5th edition can be found on the website at https: / / ctep.cancer.gov / protocolDevelopment / electronic_applications / docs / CTCAE_v5_Quick_Reference_8.5x11.pdf). Once grade 1 toxicity was documented, the dose escalation schedule listed in Table 8 was used.

[0477] Table 7. Accelerated titration dose escalation schedule

[0478]

[0479] Table 8. Dose escalation after grade 1 toxicity was documented

[0480]

[0481] Once the MTD is identified, the dose level expansion phase will begin. The MTD will be expanded to 10 subjects, and immunology-related studies will be performed in all 10 subjects. Table 9 summarizes the regimen for the dose expansion phase.

[0482] Table 9. Dose Expansion Phase Schedule (Once MTD is identified and expanded to 10 subjects)

[0483]

[0484] After the MTD was expanded, lower doses were sequentially expanded to 10 patients until the lowest dose level associated with an immune response was expanded.

[0485] Evaluate

[0486] Subjects were evaluated according to the study calendar listed in Table 10. Subjects underwent a medical history and physical examination (including breast examination) at Screening, Day 0, every 4 weeks, and at the Day 84 visit.

[0487] Evaluate subjects for toxicity for 4 weeks after the last vaccination or until all toxicities resolve to Grade 0-1, whichever occurs later. Contact all patients or observe them in long-term follow-up for long-term monitoring of toxicity, relapse, and survival.

[0488] Any subject who received at least one vaccination as part of this trial was considered evaluable for toxicity. To allow for safe dose escalation, subjects who withdrew from treatment before receiving three doses of the investigational vaccine in the absence of dose-limiting toxicity (DLT) were replaced. Subjects who experienced any DLT after any dose were considered fully evaluable and were not replaced, even if no relevant immunological studies were performed.

[0489] Table 10. Study calendar

[0490]

[0491]

[0492] 1 The screening visit occurred within 28 days before the Day 0 visit. No scans were required to exclude metastatic disease.

[0493] 2 All visit days are ±3 working days.

[0494] 3 The CMP (comprehensive metabolic profile) includes total protein, albumin, calcium, total bilirubin, alkaline phosphatase, AST, glucose, BUN, creatinine, sodium, potassium, chloride, bicarbonate, anion gap, and ALT.

[0495] 4 ELISPOT frequencies of α-lactalbumin- and ovalbumin-specific T cells; percentages of culture supernatant levels of IFNγ and IL-17 determined by flow cytometry of central versus effector memory T cells; and direct ELISA measurements of α-lactalbumin antibody titers.

[0496] 5 Allow ≥2 hours for vaccine preparation

[0497] 6 After vaccine administration, vital signs were measured every 15 minutes for 60 minutes and again at 120 minutes. The patients were observed for 120 minutes.

[0498] Example 9: Vaccine formulation comprising zymosan suspended in MONTANIDE TM IMMUNE®

[0499] Other vaccine formulations containing adjuvants as disclosed herein can be generated and tested according to the present invention. For example, any target antigen, such as a polypeptide antigen, can be mixed with a vaccine suspended in MONTANIDE. TM The target antigen may be expressed, for example, on tumor cells and / or pathogenic organisms against which the vaccine formulation is designed.

[0500] The vaccine formulations are then tested by any of the various methods disclosed in this disclosure or as described below.

[0501] Antigen-specific recall response

[0502] To evaluate antigen-specific recall responses, mice are administered one or more doses of the vaccine formulation. Ten days after the last dose, lymph node cells are collected from the mice. Lymph node cells are incubated in the presence of serial dilutions of the antigen (the antigen used in the vaccine formulation or an irrelevant antigen as a negative control). The cells are subjected to a proliferation assay. For example, cell cultures can be pulsed with labeled thymidine, and a "stimulation index" can be calculated by dividing the (labeled) counts of the culture containing the antigen used in the vaccine formulation by the counts of the culture containing the irrelevant antigen. Enhanced proliferation in cultures containing the antigen used in the vaccine indicates an antigen-specific recall response.

[0503] Response characteristics

[0504] Lymph node cells from mice administered one or more doses of the vaccine formulation can be analyzed for cell surface markers (e.g., CD4+ and CD8+) (e.g., by flow cytometry) and / or by cytokine release (e.g., by ELISA or ELISPOT assays). The cytokine release profile may indicate the type of immune response (e.g., type 1, type 2, or type 17). A cytokine release profile that exhibits both type 1 and type 17 responses indicates that the vaccine formulation is potentially effective.

[0505] Tumor growth inhibition

[0506] Antitumor vaccine formulations contain target antigens expressed in tumor cells. These formulations can be tested for their ability to inhibit tumor growth in appropriate animal tumor models. Controls may include one or more of the following: adjuvants (zymosan and montanide TM ), formulation alone (as a negative control) or formulations containing the target antigen and different commonly used adjuvants.

[0507] Drug administration studies

[0508] To determine the effective dose of a vaccine formulation, a range of amounts and / or dose times can be used in each dose (e.g., within a range of amounts of target antigen and / or zymosan). Output indicators can include one or more of the following: antigen-specific recall response, response characteristics, or tumor growth inhibition (in the case of anti-cancer vaccine formulations).

[0509] Example 10: Emulsification solution

[0510] To obtain a mixture comprising an antigen (eg, a polypeptide antigen) and MONTANIDE TM For emulsions of ISA 51, two silicone-free syringes connected by a connector (I or T-connector) can be used to create high shear conditions to embed the aqueous droplets into the surrounding oil phase. The antigen can be dissolved in an aqueous solution, such as water or saline solution (most commonly PBS or NaCl 0.9% saline buffer), and one volume of this aqueous antigen solution can be mixed with one volume of MONTANIDE TM ISA 51 mixing. The mixture can then be loaded into a device comprising two silicone-free syringes connected, for example, by an I-connector, which is used to perform a pre-emulsification step comprising a plurality (e.g., about 20) of slow cycles (e.g., cycles lasting about 4 seconds), followed by an emulsification step comprising a plurality (e.g., about 40) of rapid cycles. A cycle is defined as transferring the entire solution (aqueous phase and adjuvant) from the first syringe to the other syringe and then transferring the entire solution back to its originating syringe.

[0511] Incorporated by reference

[0512] All publications, patents, and patent applications mentioned herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event of a conflict, the present application, including any definitions herein, will control.

[0513] Equivalent plan

[0514] 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. Such equivalents are intended to be encompassed by the following claims. Sequence Listing <110> Cleveland Clinic Foundation <120> Vaccine adjuvants and formulations <130> CCI-005 WO <150> 62 / 806,422 <151> 2019-02-15 <160> 5 <170> PatentIn version 3.5 <210> 1 <211> 142 <212> PRT <213> 智人(Homo sapiens) <400> 1 Met Arg Phe Phe Val Pro Leu Phe Leu Val Gly Ile Leu Phe Pro Ala 1 5 10 15 Ile Leu Ala Lys Gln Phe Thr Lys Cys Glu Leu Ser Gln Leu Leu Lys 20 25 30 Asp Ile Asp Gly Tyr Gly Gly Ile Ala Leu Pro Glu Leu Ile Cys Thr 35 40 45 Met Phe His Thr Ser Gly Tyr Asp Thr Gln Ala Ile Val Glu Asn Asn 50 55 60 Glu Ser Thr Glu Tyr Gly Leu Phe Gln Ile Ser Asn Lys Leu Trp Cys 65 70 75 80 Lys Ser Ser Gln Val Pro Gln Ser Arg Asn Ile Cys Asp Ile Ser Cys 85 90 95 Asp Lys Phe Leu Asp Asp Asp Ile Thr Asp Asp Ile Met Cys Ala Lys 100 105 110 Lys Ile Leu Asp Ile Lys Gly Ile Asp Tyr Trp Leu Ala His Lys Ala 115 120 125 Leu Cys Thr Glu Lys Leu Glu Gln Trp Leu Cys Glu Lys Leu 130 135 140 <210> 2 <211> 742 <212> DNA <213>智人(Homo sapiens) <400> 2 atttcaggtt cttgggggta gccaaaatga ggttctttgt ccctctgttc ctggtgggca 60 tcctgttccc tgccatcctg gccaagcaat tcacaaaatg tgagctgtcc cagctgctga 120 aagacataga tggttatgga ggcatcgctt tgcctgaatt gatctgtacc atgtttcaca 180 ccagtggtta tgacacacaa gccatagttg aaaacaatga aagcacggaa tatggactct 240 tccagatcag tataagctt tggtgcaaga gcagccaggt ccctcagtca aggaacatct 300 gtgacatctc ctgtgacaag ttcctggatg atgacattac tgatgacata atgtgtgcca 360 agaagatcct ggatattaaa ggaattgact actggttggc ccataaagcc ctctgcactg 420 agaagctgga acagtggctt tgtgagaagt tgtgagtgtc tgctgtcctt ggcacccctg 480 cccactccac actcctggaa tacctcttcc ctaatgccac ctcagtttgt ttctttctgt 540 tccccaag cttatctgtc tctgagcctt gggccctgta gtgacatcac cgaattcttg 600 aagactattt tccagggatg cctgagtggt gcactgagct ctagaccctt actcagtgcc 660 ttcgatggca cttcactac agcacagatt tcacctgt cttgataaa ggtcccactt 720 tgagtcaaaaaaaaaaa 742 <210> 3 <211> 143 <212> PRT <213>小家鼠(Muscles) <400> 3 Met Met His Phe Val Pro Leu Phe Leu Val Cys Ile Leu Ser Leu Pro 1 5 10 15 Phe Gln Ala Thr Glu Leu Thr Lys Cys Lys Val Ser His Ala Ile 20 25 30 Lys Asp With Asp Gly Tyr Gln Gly Serves Less Glu Than Ala Cys 35 40 45 Val Leu Phe His Thr Ser Gly Tyr Asp Thr Gln Ala Val Val Asn Asp 50 55 60 Asn Gly Ser Thr Glu Tyr Gly Leu Phe Gln Ile Ser Asp Arg Phe Trp 65 70 75 80 Cys Lys Ser Ser Glu Phe Pro Glu Ser Glu Asn Ile Cys Gly Ile Ser 85 90 95 Cys Asp Lys Leu Leu Asp Asp Glu Leu Asp Asp Asp Ile Ala Cys Ala 100 105 110 Lys Lys Ile Leu Ala Ile Lys Gly Ile Asp Tyr Trp Lys Ala Tyr Lys 115 120 125 Pro Met Cys Ser Glu Lys Leu Glu Gln Trp Arg Cys Glu Lys Pro 130 135 140 <210> 4 <211> 733 <212> DNA <213>小家鼠(Mus musculus) <400> 4 ggagcagtca aaatgatgca tttcgttcct ttgttcctgg tgtgtatttt gtcgttgcct 60 gcctttcaag ccacagagct tacaaaatgc aaggtgtccc atgccattaa agacatagat 120 ggctatcaag gcatctcttt gcttgaatgg gcctgtgttt tatttcatac cagtggctac 180 gacacacaag ctgttgtcaa cgacaacggc agcacagagt acggactctt ccagatcagt 240 gacagattt ggtgtaaaag tagtgagttc cccgagtcgg agaacatctg tggcatctcc 300 tgtgacaagt tattggatga cgagttggat gatgacatag cgtgtgccaa gaagatcctg 360 gctatcaaag gaatcgacta ctggaaagcc tacaagccca tgtgctctga gaagcttgaa 420 cagtggcgtt gtgagaagcc ctgagccccc ccccccccc ccccgtcct tgctgctcct 480 gccccgtggt caggaatgcc tcttccctaa ggctacctca gcttggctct tgctattcct 540 gtgaagatga tctgcctctg agccttgtac cctgtagtga caccaccgga ctctagagga 600 cttttttttc cctatgggag tgtgactggc gcactggact gcaaaccctt gcttagtgac 660 ggcgagggtc tcgatggggg ttttacaaaa tcgagagagc cctctcctgt cccaaataaa 720 gggccagact is 733 <210> 5 <211> 123 <212> PRT <213>智人(Homo sapiens) <400> 5 Lys Gln Phe Thr Lys Cys Glu Leu Ser Gln Leu Leu Lys Asp Ile Asp 1 5 10 15 Gly Tyr Gly Gly Ile Ala Leu Pro Glu Leu Ile Cys Thr Met Phe His 20 25 30 Thr Ser Gly Tyr Asp Thr Gln Ala Ile Val Glu Asn Asn Glu Ser Thr 35 40 45 Glu Tyr Gly Leu Phe Gln Ile Ser Asn Lys Leu Trp Cys Lys Ser Ser 50 55 60 Gln Val Pro Gln Ser Arg Asn Ile Cys Asp Ile Ser Cys Asp Lys Phe 65 70 75 80 Leu Asp Asp Asp Ile Thr Asp Asp Ile Met Cys Ala Lys Lys Ile Leu 85 90 95 Asp Ile Lys Gly Ile Asp Tyr Trp Leu Ala His Lys Ala Leu Cys Thr 100 105 110 Glu Lys Leu Glu Gln Trp Leu Cys Glu Lys Leu 115 120

Claims

1. A composition comprising: α-lactalbumin polypeptide, Zymosan, Dehydrated mannitol monooleate, and Purified mineral oil.

2. The composition of claim 1, wherein when the composition is administered to a subject, the composition is capable of inducing an antigen-specific T cell immune response comprising type 1 and type 17 proinflammatory T cell responses.

3. The composition of claim 1, wherein the zymosan binds to a pattern recognition receptor. The composition of claim 3 , wherein the pattern recognition receptor is TLR2 or dectin-1.

5. The composition of claim 1, wherein the purified mineral oil is DRAKEOL TM 6VR.

6. The composition of claim 1, wherein the composition comprises MONTANIDE TM ISA 51VG.

7. The composition of claim 6, wherein the composition is a water and oil emulsion.

8. The composition of claim 7, wherein the composition is a water-in-oil emulsion.

9. The composition of claim 1, wherein the α-lactalbumin polypeptide has an amino acid sequence comprising at least 8 consecutive amino acids of SEQ ID NO:

5.

10. The composition of claim 1, wherein the α-lactalbumin polypeptide and the zymosan are present in a ratio of 10:1 to 1:10 (w / w). The composition of claim 10 , wherein the α-lactalbumin polypeptide and the zymosan are present in a ratio of 1:1 (w / w).

12. The composition of claim 1, further comprising a pharmaceutically acceptable carrier.

13. The composition of claim 1, further comprising an antibiotic.

14. Use of the composition according to any one of claims 1 to 13 in the preparation of a medicament for treating breast cancer in a human subject.

15. The use according to claim 14, wherein the subject is a non-lactating female subject.

16. The use according to claim 14, wherein the subject has cancer or is at risk of developing cancer.

17. The use according to claim 14, wherein the subject has not been diagnosed with cancer.

18. The use according to claim 14, wherein the breast cancer is metastatic breast cancer. The use according to claim 14 , wherein the breast cancer is primary breast cancer.

20. The use according to claim 14, wherein the breast cancer is triple-negative breast cancer.

21. The use according to claim 14, wherein the breast cancer comprises cells that overexpress α-lactalbumin.

22. A composition comprising α-lactalbumin polypeptide, zymosan and MONTANIDE TM ISA 51VG, wherein the α-lactalbumin polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to the amino acid sequence of SEQ ID NO:

5.

23. The composition of claim 22, wherein the α-lactalbumin polypeptide comprises an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO:

5.

24. A formulation comprising a water-in-oil emulsion comprising an α-lactalbumin polypeptide, zymosan, anhydrous mannitol monooleate, and purified mineral oil, wherein the α-lactalbumin polypeptide and zymosan are present in the formulation in a ratio of between 1:5 (w / w) and 5:1 (w / w), and wherein the α-lactalbumin polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to the amino acid sequence of SEQ ID NO:

5.

25. A preparation comprising α-lactalbumin polypeptide, zymosan and MONTANIDE TM A water-in-oil emulsion of ISA 51VG, wherein the α-lactalbumin polypeptide and zymosan are present in the formulation in a ratio of between 1:5 (w / w) and 5:1 (w / w), and wherein the α-lactalbumin polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to the amino acid sequence of SEQ ID NO:

5.

26. The formulation of claim 25, wherein the α-lactalbumin polypeptide comprises an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 5.

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