AMHR2-ed cancer vaccine formulation

Abstract

Provided herein are compositions, systems, kits, and methods of using compositions comprising at least a portion of an anti-Mullerian hormone receptor type II extracellular domain (AMHR-ED) and an adjuvant comprising i) a squalene oil, ii) a nonionic surfactant (e.g., Tween 80), iii) an emulsifier (e.g., sorbitan trioleate), and iv) a buffer (e.g., sorbitan trioleate). A citrate buffer). In certain embodiments, such compositions are administered to a female subject to treat or prevent ovarian cancer or endometrial cancer (e.g., by inducing the subject to express an anti-AMHR2-ED IgG antibody in vivo).

Description

[0001] Related Applications

[0002] This application claims priority to U.S. Provisional Application Serial No. 63 / 310,731, filed on February 16, 2022, which is incorporated herein by reference in its entirety.

[0003] Sequence Listing

[0004] The text of the computer-readable sequence listing entitled "40282-601_SEQUENCE_LISTING" with a file size of 10,200 bytes, created on February 15, 2023 and filed with this application, is incorporated herein by reference in its entirety. Technical Field

[0005] Provided herein are compositions, systems, kits, and methods of using compositions comprising at least a portion of an anti-Mullerian hormone receptor type II extracellular domain (AMHR-ED) and an adjuvant comprising: i) squalene oil, ii) a nonionic surfactant (e.g., Tween 80), iii) an emulsifier (e.g., sorbitan trioleate), and iv) a buffer (e.g., citrate buffer). In certain embodiments, such compositions are administered to females to treat or prevent ovarian or endometrial cancer (e.g., by inducing expression of anti-AMHR2-ED IgG antibodies in vivo in a subject). Background Art

[0006] Epithelial ovarian cancer (EOC) is the most lethal of all gynecological malignancies in postmenopausal women, accounting for more than 75% of all cases [1,2]. The constellation of non-definitive symptoms associated with the onset of EOC and the lack of effective biomarkers for early detection often lead to late diagnosis at advanced disease stages, accompanied by high rates of disease recurrence and poor prognosis after current standards of care [1-7]. Therefore, more effective approaches to control this disease remain needed. Inducing ovarian tumor immunity through vaccination is a promising approach, supported by the increased overall survival observed in patients whose ovarian tumors are infiltrated by T cells [8].

[0007] Studies have shown that anti-Müllerian hormone receptor type II (AMHR2) is expressed in approximately 92% of primary EOC, 78% of borderline malignancies, 77%–86% of non-EOC ovarian tumors, and 56% of malignant ascites from grade III-IV ovarian cancer [9-12]. AMHR2 is a serine / threonine kinase receptor homologous to the type II receptor of the transforming growth factor-β (TGFβ) superfamily

[13] . Anti-Müllerian hormone (AMH) is the cognate ligand of AMHR2, and binding of AMH to the extracellular domain of AMHR2 (AMHR2-ED) signals cell cycle arrest and programmed cell death, leading to regression of the Müllerian ducts during male fetal development, as well as regulation of oocyte development and control of ovarian reserve and fertility in adult females [14,15]. In adult women, the longest AMHR2 transcript encodes a 573 amino acid protein that is expressed exclusively in the ovary and consists of a 403 amino acid cytoplasmic domain (AMHR2-CD) with kinase activity and a 26 amino acid hydrophobic transmembrane domain, both of which show extraovarian expression

[16] . The long ovary-specific transcript also encodes a 127 amino acid ligand-binding extracellular domain (AMHR2-ED) that is expressed exclusively in the human premenopausal ovary

[16] , and this expression decreases to non-immunogenic levels in the postmenopausal ovary

[17] . Summary of the invention

[0008] Provided herein are compositions, systems, kits, and methods of using compositions comprising at least a portion of an anti-Mullerian hormone receptor type II extracellular domain (AMHR-ED) and an adjuvant comprising: i) squalene oil, ii) a nonionic surfactant (e.g., Tween 80), iii) an emulsifier (e.g., sorbitan trioleate), and iv) a buffer (e.g., citrate buffer). In certain embodiments, such compositions are administered to females to treat or prevent ovarian or endometrial cancer (e.g., by inducing expression of anti-AMHR2-ED IgG antibodies in vivo in a subject).

[0009] In some embodiments, compositions are provided herein, comprising: a) a polypeptide comprising at least a portion of an anti-Mullerian hormone receptor type II extracellular domain (AMHR-ED); and b) an adjuvant comprising: i) squalene oil, ii) a nonionic surfactant, iii) an emulsifier, and iv) a buffer. In some embodiments, the polypeptide is configured to activate CD4+T cells when administered to a subject (e.g., a human subject, such as a human female subject). In certain embodiments, the polypeptide is configured to induce the production of AMHR2-ED specific IgG in vivo when administered to a subject (e.g., a human subject, such as a human female subject).

[0010] In other embodiments, provided herein is a system or kit comprising: a) a polypeptide comprising at least a portion of an anti-Mullerian hormone receptor type II extracellular domain (AMHR-ED) (e.g., from humans); and b) an adjuvant comprising: i) squalene oil, ii) a nonionic surfactant, iii) an emulsifier, and iv) a buffer. In some embodiments, the polypeptide is configured to activate CD4+T cells when administered to a subject (e.g., a human subject, such as a human female subject). In certain embodiments, the polypeptide is configured to induce the production of AMHR2-ED specific IgG in vivo when administered to a subject (e.g., a human subject, such as a human female subject).

[0011] In some embodiments, provided herein is a method for treating ovarian cancer or endometrial cancer tumors in female subjects, the method comprising: administering the above and herein described compositions to female subjects (e.g., human females). In other embodiments, provided herein is a method for preventing ovarian cancer and / or endometrial cancer in subjects (e.g., human female subjects), the method comprising: administering the above and herein described compositions to subjects. In some embodiments, the subject does not suffer from detectable ovarian cancer and / or endometrial cancer.

[0012] In certain embodiments, the ovarian cancer tumor is a primary ovarian cancer tumor. In certain embodiments, the ovarian cancer tumor is a metastatic tumor. In certain embodiments, the ovarian cancer tumor is an epithelial ovarian cancer (EOC) tumor.

[0013] In some embodiments, administration of the composition induces an immune response against ovarian and / or endometrial cancer tumors in the subject. In some embodiments, administration of the composition induces expression of anti-AMHR2-ED IgG antibodies in the subject.

[0014] In some embodiments, ovarian cancer and / or endometrial cancer tumors express AMHR2-ED. In some embodiments, the method further comprises the step of determining whether ovarian and / or endometrial cancer tumors express AMHR2-ED. In other embodiments, the method further comprises the step of determining whether administration of the composition induces an immune response against ovarian or endometrial cancer tumors in the subject. In other embodiments, the method further comprises repeatedly administering the composition to the subject (e.g., administering once, twice, three times, etc.). In a specific embodiment, the adjuvant comprises ADDAVAX TM (InvivoGen) or MF59 (Novartis, Siena), which are commercially available.

[0015] In some embodiments, the method provided herein also includes the step of applying other anticancer agents to the subject. In a specific embodiment, other anticancer agents are selected from the group consisting of: paclitaxel, cisplatin, topotecan, gemcitabine, bleomycin, etoposide, carboplatin, docetaxel, doxorubicin, topotecan, cyclophosphamide, trabectedin, olaparib, tamoxifen, letrozole, bevacizumab, anti-CTLA4 antibodies, anti-PD-1 antibodies and anti-PD-L1 antibodies. In some embodiments, other agents are anticancer agents. In some embodiments, the anticancer agent is paclitaxel, cisplatin, topotecan, gemcitabine, bleomycin, etoposide, carboplatin, docetaxel, doxorubicin, topotecan, cyclophosphamide, trabectedin, olaparib, tamoxifen, letrozole or bevacizumab. In some embodiments, the anticancer agent is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is an inhibitor of CT LA4, such as an anti-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 an anti-PD-1 antibody (e.g., nivolumab (BMS), pembrolizumab / lambruzumab (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 an anti-PD-L1 and / or anti-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 subject has undergone surgery to remove at least a portion of an ovarian theca and / or endometrial cancer tumor.

[0016] In some embodiments, the emulsifier includes sorbitan trioleate. In further embodiments, the nonionic surfactant includes polysorbate 80, polyglycerol alkyl ethers, glucosyl dialkyl ethers, crown ethers, ester-linked surfactants, polyoxyethylene alkyl ethers, Brij, Spans (sorbitan esters) and Tweens (polysorbates). In other embodiments, the buffer includes citrate buffer or phosphate buffered saline. In other embodiments, the pH of the composition is between 5.5 and 6.9.

[0017] In certain embodiments, the composition is in the form of an emulsion. In a specific embodiment, the emulsion is a nanoemulsion of a first and a second component, wherein the first component comprises an emulsifier and squalene oil, and the second component comprises a nonionic surfactant and a buffer. In other embodiments, the emulsifier is present in the first component at about 0.4-0.6 weight / volume (e.g., 0.4%, 0.5% or 0.6%). In other embodiments, squalene oil is present in the first component at about 4-6% weight / volume (e.g., 0.4%, 0.5% or 0.6%). In some embodiments, a nonionic surfactant is present in the second component at about 0.4-0.6 weight / volume (e.g., 0.4%, 0.5% or 0.6%). In other embodiments, a buffer is present in the second component at about 4-6% weight / volume (e.g., 4%, 5% or 6%). In a specific embodiment, the nanoemulsion is composed of particles having an average size of about 150-170nm.

[0018] In additional embodiments, the adjuvant further comprises at least one of: a saponin, cholesterol, a phospholipid, a phosphatidylcholine, and a second buffer. In some embodiments, the second buffer comprises phosphate buffered saline.

[0019] In some embodiments, the composition comprises an oil phase and an aqueous phase. In other embodiments, squalene is present at 4.0-5.5% (e.g., 4...4.3...4.6...4.9...5.2...5.5%) of the oil phase. In other embodiments, nonionic surfactants are present at about 0.4-0.6% (e.g., 0.4%, 0.5% or 0.6%) of the aqueous phase. In other embodiments, emulsifiers are present at about 0.4-0.6% (e.g., 0.4%, 0.5% or 0.6%) of the aqueous phase.

[0020] In some embodiments, the adjuvant further comprises alpha-tocopherol. In certain embodiments, the adjuvant further comprises monophosphoryl lipid A. In specific embodiments, the adjuvant further comprises an immunomodulator. In further embodiments, the immunomodulator comprises synthetic trehalose diphtherate.

[0021] In certain embodiments, the polypeptide comprises an amino acid sequence in SEQ ID NO:7, 8, 9 or 10. In some embodiments, the polypeptide comprises a complete human AMHR-ED or a nearly complete human AMHR2-ED. In some embodiments, the polypeptide comprises at least 20 consecutive amino acids from SEQ ID NO:7. In other embodiments, the polypeptide comprises at least 40 consecutive amino acids from SEQ ID NO:7. In certain embodiments, the polypeptide comprises at least 60 consecutive amino acids from SEQ ID NO:7. In other embodiments, the polypeptide comprises at least 100 consecutive amino acids from SEQ ID NO:7. In some embodiments, the polypeptide comprises at least 115 consecutive amino acids from SEQ ID NO:7. In other embodiments, the polypeptide comprises at least 122 consecutive amino acids from SEQ ID NO:7. In other embodiments, the polypeptide has an amino acid sequence comprising 100 consecutive amino acids, which are at least 80% identical to the amino acid sequence in the extracellular domain of AMHR2. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 AMHR2 gene expression in mouse EOC is shown. qRT-PCR analysis of ID8 and MOVCAR EOC cell lines showed that both cell lines expressed the AMHR2-ED and AMHR2-CD domains, but the expression of each AMHR2 domain in MOVCAR cells was consistently approximately twofold higher than the expression levels measured in ID8 cells. Error bars represent ±SD.

[0023] Figure 2 Shown using ADDAVAX TM The AMHR2-ED vaccine was formulated to induce a high IgG response. ELISA analysis showed that when immunizing C57BL / 6 female mice, ADDAVAX TM As an adjuvant resulted in (A) antigen-specific IgG responses to the AMHR2-ED immunogen, and (B) antigen-specific IgG isotype responses similar to those obtained using CFA as an adjuvant. Sera were analyzed 8 weeks after immunization using a 1:10,000 dilution. Error bars represent ±SD.

[0024] Figure 3 shows the use of ADDAVAX TM Vaccination with AMHR2-ED as an adjuvant was highly effective in inhibiting the growth of EOC in mice. Compared with vaccination with CFA alone or vaccination against AMHR2-ED in CFA, the use of ADDAVAX TMProphylactic AMHR2-ED vaccination as an adjuvant resulted in (A) significant inhibition of MOVCAR growth and (B) significantly increased overall survival in TgMISIIR-Tag(low) female mice. TM AMHR2-ED vaccination also effectively inhibited the growth of (C) MOVCAR and (D) ID8 tumors. Error bars = ± SD, and asterisks indicate significance.

[0025] Figure 4 shows the use of ADDAVAX TM Adjuvanted AMHR2-ED vaccination resulted in T cell infiltration of murine EOC. TM Corresponding EOC tissues of vaccinated mice (arrows, upper row) were compared with those of mice vaccinated with ADDAVAX. TM AMHR2-ED vaccination results in infiltration of CD3+ T cells (arrows, lower row, left), CD4+ T cells (arrows, lower row, center), and CD8+ T cells (arrows, lower row, right). Representative images of several tissues examined from several different mice. Tissue magnification = 20X.

[0026] Figure 5 Exemplary sequences are shown. Picture A shows an exemplary human AMHR2-ED amino acid sequence (SEQ ID NO:7). Picture B shows an exemplary N-terminally truncated human AMHR2-ED amino acid sequence (SEQ ID NO:8). Picture C shows an exemplary C-terminally truncated human AMHR2-ED amino acid sequence (SEQ ID NO:9). Picture D shows an exemplary N-terminally and C-terminally truncated human AMHR2-ED amino acid sequence (SEQ ID NO:10).

[0027] definition

[0028] For convenience, certain terms used in the specification, examples, and appended claims are collected here.

[0029] The articles "a" and "an" are used herein to refer to one or more than one (ie, at least one) of the grammatical object of the article. For example, "element" means one element or more than one element.

[0030] As used herein, the term "administering" means providing an agent or composition to a subject (eg, intravenously), and includes, but is not limited to, administration by a medical professional and self-administration.

[0031] 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 or solvent encapsulating material, that participates in carrying or transporting the subject compound from one organ or part of the body to another organ or part of the body. In certain embodiments, the compositions herein contain a pharmaceutically acceptable carrier.

[0032] As used herein, the term "subject" means a human or non-human animal selected for treatment or therapy.

[0033] 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 a subpopulation of cells in a subject, at a reasonable benefit / risk ratio applicable to any medical treatment.

[0034] "Treating" a disease in a subject or "treating" a subject having a disease means subjecting the subject to medical treatment, such as administration of a vaccine, such that at least one symptom of the disease is alleviated or prevented from worsening. DETAILED DESCRIPTION

[0035] Provided herein are compositions, systems, kits, and methods of using compositions comprising at least a portion of an anti-Mullerian hormone receptor type II extracellular domain (AMHR-ED) and an adjuvant comprising: i) squalene oil, ii) a nonionic surfactant (e.g., Tween 80), iii) an emulsifier (e.g., sorbitan trioleate), and iv) a buffer (e.g., a citrate buffer). In certain embodiments, such compositions are administered to female subjects (e.g., humans) to treat or prevent ovarian cancer or endometrial cancer (e.g., by inducing the subject to express anti-AMHR2-ED IgG antibodies in vivo).

[0036] In certain embodiments, the adjuvant employed herein is ADDAVAX TM (InvivoGen) and / or chemically related to ADDAVAX TM Adjuvants that are identical to and / or highly similar to those marketed. ADDAVAX TM It is a nanoemulsion of 2 components: i) sorbitan trioleate (0.5% w / v) in squalene oil (5% v / v), and ii) Tween 80 (0.5% w / v) in sodium citrate buffer (10 mM, pH 6.5), wherein the nanoemulsion is produced using a microfluidizer and filtered through a 0.22-μm filter to remove large droplets and sterilize the final product. The particle size is about 160 nm.

[0037] In certain embodiments, the adjuvant employed herein is MF59 (Novartis, Siena) and / or an adjuvant that is chemically identical and / or highly similar to an adjuvant sold as MF59. MF59 is an approximately 160 nm particle emulsion comprising: i) squalene: 9.75 mg; ii) polysorbate 80: 1.175 mg; iii) sorbitan trioleate: 1.175 mg; iv) sodium citrate: 0.66 mg; and v) citric acid: 0.04 mg.

[0038] In certain embodiments, the adjuvant employed herein is Abisco-100 (referred to as Matrix M when prepared according to GMP standards) and / or an adjuvant that is chemically identical and / or highly similar to an adjuvant sold as Abisco-100. Abisco-100 has the following chemical content: purified saponins obtained from a crude extract of the plant soap tree (Quillaja Saponaria Molina); cholesterol from lanolin and phosphatidylcholine (phospholipids) from fresh egg yolk; in a suspension of nanoscale (40nm) caged particles composed of the above ingredients in PBS. Matrix M (or Abisco-100) is composed of a mixture of Matrix A and Matrix C in a ratio of 80:20 to 95:5 (preferably 85:15). Matrix A causes T cell induction and has low toxicity, and Matrix C induces antibodies and has some toxicity. Matrix C contains the C fraction separated by QS, which corresponds to QS21. Fraction A (in Matrix A) corresponds to QS7.

[0039] In some embodiments, the adjuvant employed herein is AS03 adjuvant and / or an adjuvant that is chemically identical and / or highly similar to an adjuvant sold as AS03 adjuvant. AS03 adjuvant is an emulsion adjuvant consisting of an oil phase (10.69 mg squalene, 11.86 mg DL-α-tocopherol) and an aqueous phase (4.86 mg polysorbate 80) per 0.5-mL adult dose. ASO3 has PBS.

[0040] In certain embodiments, the adjuvant employed herein is Ribi adjuvant and / or an adjuvant that is chemically identical and / or highly similar to an adjuvant sold as Ribi adjuvant. Ribi adjuvant is an oil-in-water emulsion containing 2% squalene-Tween80-water, 0.5 mg monophosphoryl lipid A (a low toxicity derivative of the lipid A region of lipopolysaccharide (LPS)) and 0.5 mg synthetic trehalose diphtheriae ester (an immunomodulator). The adjuvant is in PBS.

[0041] In certain embodiments, the compositions provided herein include an AMHR2-ED polypeptide and / or an immunologically active fragment of an AMHR2-ED polypeptide. Polypeptides with substantial sequence similarity can induce the same or very similar immune responses in host animals. Therefore, in some embodiments, derivatives, equivalents, variants, fragments or mutants of AMHR2-ED proteins (e.g., as shown in SEQ ID NO: 7) or fragments thereof may also be applicable to the methods, compositions and kits provided herein.

[0042] In some embodiments, variants or derivatives of AMHR2-ED polypeptides (e.g., SEQ ID NO: 7-10) are provided herein. The altered polypeptide may have an altered amino acid sequence, such as by conservative substitution, but still induces an immune response that reacts with an unchanged protein antigen, and is considered to be a functional equivalent. As used herein, the term "conservative substitution" means that an amino acid residue is replaced by another biologically similar residue. It is well known in the art that amino acids within the same conservative group can generally be substituted with each other without having a substantial effect on the function of the protein. According to certain embodiments, a derivative, equivalent, variant or mutant of the ligand binding domain of an AMHR2-ED polypeptide is a polypeptide that is at least 85% homologous to the sequence of an AMHR2-ED protein or a fragment thereof. In some embodiments, the homology is at least 90%, at least 95% or at least 98% (e.g., compared to SEQ ID NO: 7-10).

[0043] In certain aspects, provided herein are pharmaceutical compositions (e.g., vaccine compositions) comprising the AMHR2-ED polypeptides described herein and the adjuvants described herein. In some embodiments, the compositions comprise a pharmaceutically acceptable carrier. In some embodiments, the compositions include a combination of multiple (e.g., two or more) AMHR2-ED polypeptides or nucleic acids described herein.

[0044] The pharmaceutical compositions disclosed herein can be specifically formulated for administration in solid or liquid form, including those suitable for: (1) oral administration, e.g., drenches (aqueous or non-aqueous solutions or suspensions); tablets, e.g., tablets targeted for oral, sublingual, and systemic absorption; pills; powders; granules; pastes for the tongue; or (2) parenteral administration, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection, as, e.g., sterile solutions or suspensions or sustained release formulations.

[0045] The method for preparing these preparations or compositions comprises the step of associating the AMHR2-ED polypeptide and adjuvant described herein with a carrier and optionally one or more auxiliary ingredients. In general, the preparations are prepared by uniformly and intimately associating the medicaments described herein with a liquid carrier or a finely divided solid carrier or both and then shaping the product if necessary.

[0046] Pharmaceutical compositions suitable for parenteral administration comprise an AMHR2-ED polypeptide described herein and an adjuvant in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders to be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain sugars, alcohols, antioxidants, buffers, bacteriostats, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.

[0047] Examples of suitable aqueous and non-aqueous carriers (which may be used in pharmaceutical compositions) include 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 may be maintained, for example, by the use of coatings (such as lecithin), by maintaining the desired particle size (in the case of dispersions), and by the use of surfactants.

[0048] Regardless of which route of administration is chosen, the agents provided herein and / or the pharmaceutical compositions disclosed herein, which can be used in a suitable hydrated form, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

[0049] In some embodiments, the pharmaceutical composition can elicit an immune response against cells expressing AMHR2-ED when administered to a subject. Such pharmaceutical compositions can be used as vaccine compositions for the preventive and / or therapeutic treatment of ovarian cancer or endometrial cancer.

[0050] In certain embodiments, the compositions provided herein also include one or more other agents, such as, but not limited to, chemotherapeutic agents, immunotherapeutic agents, immunomodulators and / or anti-angiogenic agents. In some embodiments, one or more other agents can be chemotherapeutic agents, naturally occurring or synthetic, for example, as described in "Cancer Chemotherapeutic Agents", American Chemical Society, 1995, WO Foye Ed.

[0051] In one embodiment, the chemotherapeutic agent is selected from the group consisting of: a small molecule receptor antagonist such as vatalanib, SU 11248 or AZD-6474; an EGFR or HER2 antagonist such as gefitinib, erlotinib, CI-1033 or herceptin; an antibody such as bevacizumab, cetuximab, rituximab; a DNA alkylating drug such as cisplatin, oxaliplatin or carboplatin; an anthracycline such as doxorubicin or epirubicin; an antimetabolite such as 5-FU, pemetrexed, gemcitabine or capecitabine. citabine); camptothecins such as irinotecan or topotecan; anticancer drugs such as paclitaxel or docetaxel; epipodophyllotoxins such as etoposide or teniposide; proteasome inhibitors such as bortezomib; or anti-inflammatory drugs such as celecoxib or rofecoxib, optionally in the form of a pharmaceutically acceptable salt, hydrate and / or solvate and optionally in the form of an individual optical isomer, an individual enantiomer or a mixture of its racemates.

[0052] In one embodiment, the chemotherapeutic agent is selected from the group consisting of: a small molecule VEGF receptor antagonist such as 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 dual EGFR / HER2 antagonist such as gefitinib, erlotinib, CI-1 033 or GW-2016; EGFR antagonists such as Iressa (ZD-1839), Tarceva (OSI-774), PKI-166, EKB-569, HKI-272 or Herceptin; antagonists of mitogen-activated protein kinases such as BAY-43-9006 or BAY-57-9006; quinazoline derivatives such as 4-[(3-chloro-4-fluorophenylamino]-6-{[4-(N,N-dimethylamino)-1-oxo-2-buten-1-yl] amino}-7-((S)-tetrahydrofuran-3-yloxy)quinazoline or 4-[(3-chloro-4-fluorophenyl)amino]-6-{[4-(homomorpholin-4-yl)-1-oxo-2-buten-1-yl]amino}-7-[(S)-(tetrahydrofuran-3-yl)oxy]-quinazoline, or a pharmaceutically acceptable salt thereof; protein kinase receptor antagonists that are not synthetic small molecules, such as atrasentan, rituximab, cetuximab, Avastin.TM. (bevacizumab), IMC -1C11, erbitux (C-225), DC-101, EMD-72000, vitaxin, imatinib; protein tyrosine kinase inhibitors as fusion proteins, such as VEGFtrap; alkylating agents or platinum compounds, such as melphalan, cyclophosphamide, oxazaphosphorine, cisplatin, carboplatin, oxaliplatin, satraplatin, tetraplatin aplatin, iproplatin, mitomycin, streptozocin, carmustine (BCNU), lomustine (CCNU), busulfan, ifosfamide, streptozocin, thiotepa, chlorambucil; nitrogen mustards such as mechloretha mine; ethyleneimine compounds; alkyl sulfonates;daunorubicin; doxorubicin (adriamycin); liposomal doxorubicin (doxil); epirubicin; idarubicin; mitoxantrone; amsacrine; dactinomycin; distamycin or its derivatives; netropsin; pibenzimol; mitomycin; CC-1065; duocarmycin; mithramycin; chromomycin; olivomycin; phtalanilides, such as propamidine or stilbamidine; anthramycin in); aziridine; nitrosourea or its derivatives; pyrimidine or purine analogs or antagonists, or nucleoside diphosphate reductase inhibitors, such as cytarabine, 5-fluorouracil (5-FU), pemetrexed, tegafur / uracil, uracil mustard, fludarabine, gemcitabine, capecitabine, mercaptopurine, cladribine , thioguanine, methotrexate, pentostatin, hydroxyurea or folic acid; phleomycin; bleomycin or its derivatives or salts thereof; CHPP; BZPP; MTPP; BAPP; liblomycin; acridine or its derivatives; rifamycin; actinomycin; adrenaline amycin); camptothecins such as irinotecan (camptosar) or topotecan; amsacrine or its analogs; tricyclic carboxamides; histone deacetylase inhibitors such as SAHA, MD-275, trichostatin A, CBHA, LAQ824, or valproic acid; plant-derived anticancer drugs such as paclitaxel (taxol), docetaxel, or taxotere;Vinca alkaloids such as navelbine, vinblastin, vincristin, vindesine or vino relbine; tropolone alkaloids such as colchicine or a derivative thereof; macrolides such as maytansine; ansamitocin or rhizoxin; antimitotic peptides such as phomopsin or dolastatin; epipodophyllotoxin or a podophyllotoxin derivative such as etoposide or teniposide; steganacin; antimitotic carbamate derivatives such as combretastatin. atin or amphetamine; procarbazine; proteasome inhibitors such as bortezomib; enzymes such as asparaginase, pegylated asparaginase (pegaspargase), or thymidine phosphorylase inhibitors; progestins or estrogens such as estramustine (T-66) or megestrol; antiandrogens such as flutamide, casodex, anandron, or cyproterone acetate; acetate; aromatase inhibitors such as aminogluthetimide, anastrozole, formestan or letrozole; GNrH analogs such as leuprorelin, buserelin, goserelin or triptorelin; antiestrogens such as tamoxifen or its citrate, droloxifene, trioxifene, raloxifene or zindoxifene; 17β-estradiol derivatives such as ICI 164,384 or ICI 182,780, aminoglutethimide or phentermine; de), formestan, fadrozole, finasteride, ketoconazole; LH-RH antagonists such as leuprolide acetate;steroids, such as prednisone, prednisolone, methylprednisolone, dexamethasone, budesonide, fluocortolone or triamcinolone; interferons, such as interferon beta; interleukins, such as IL-10 or IL-12; anti-TNFα antibodies, such as etanercept; immunomodulatory drugs, such as thalidomide, its R-enantiomer and S-enantiomer, and derivatives thereof, or revimid (CC-5013); leukotriene antagonists; mitomycin C; aziridoquinones, such as BMY-42355, AZQ or EO-9; 2-nitroimidazoles, such as misonidazole (misonidazole); dazole), NLP-1 or NLA-1; nitroacridine; nitroquinoline; nitropyrazoloacridine; "dual-function" nitroaromatics, such as RSU-1069 or RB-6145, CB-1954; N-oxides of nitrogen mustards, such as nitromin; metal complexes of nitrogen mustards; anti-CD3 or anti-CD25 antibodies; tolerance inducing agents; bisphosphonates or derivatives thereof, such as minodronic acid or its derivatives (YM-529, Ono-5920, YH-529), zoledronic acid monohydrate, ibandronate sodium hydrate or clodronate disodium; nitroimidazoles, such as metronidazole, misonidazole, and chloranilamide. le), benznidazole or nimorazole; nitroaryl compounds such as RSU-1069; nitroxyl or N-oxides such as SR-4233; halogenated pyrimidine analogs such as bromodeoxyuridine, iododeoxyuridine; thiophosphates such as WR-272 1; photochemically active drugs such as porfim er, photofrin; benzoporphyrin derivatives; pheophorbide derivatives, merocyanin 540 (MC-540) or tin etioporpurin;anti-template or antisense RNA or DNA, such as oblimersen; nonsteroidal anti-inflammatory drugs, such as acetylsalicyclic acid, mesalazine, ibuprofen, naproxen, flurbiprofen, fenoprofen, fenbufen, ketoprofen, ind oprofen, pirprofen, carprofen, oxaprozin, pranoprofen, miroprofen, tioxaprofen, suprofen, alminoprofen, tiaprofenic acid d), fluprofen, indomethacin, sulindac, tolmetin, zomepirac, nabumetone, diclofenac, fenclofenac, alclofenac, bromfenac, ibufenac, aceclofenac, acemetacin, fentiazac, clidanac, etodol ac, oxpinac, mefenamic acid, meclofenamic acid, flufenamic acid, nifluminic acid, tolfenamic acid, diflunisal, flufenis a1), piroxicam, tenoxicam, lornoxicam, nimesulide, meloxicam, celecoxib, rofecoxib or a pharmaceutically acceptable salt of a nonsteroidal anti-inflammatory drug; cytotoxic antibiotics; antibodies targeting surface molecules of cancer cells, such as apolizumab or 1D09C3; metalloproteinase inhibitors, such as TIMP-1 or TIMP-2, zinc; oncogene inhibitors, such as P53 and Rb; rare earth element complexes, such as lanthanide heterocyclic complexes;a photochemotherapeutic agent such as PUVA; an inhibitor of the transcription factor complex ESX / DRIP130 / Sur-2; an inhibitor of HER-2 expression, such as the heat shock protein HSP90 regulator geldanamycin, and its derivatives 17-allylaminogeldanamycin or 17-AAG; or an agent selected from IM-842, tetrathiomolybdate, squalamine, combrestatin A4, TNP-470, marimastat, neovastat, bicalutamide, abarelix, oregovomab, mitumomab, TLK-286, alemtuzumab, ibritumomab, temozolomide, denileukin diftitox, aldesleukin, dacarbazine, floxuridine, plicamycin, mitotane, pipobroman, plicamycin, tamoxifen, and testolactone. Preferred compounds include small molecule VEGF receptor antagonists such as vatalanib (PTK-787 / ZK222584), SU-5416, SU-6668, SU-11248, SU-14813, AZD-6474; EGFR / HER2 antagonists such as CI-1033 or GW-2016; EGFR antagonists such as Iressa (gefitinib, ZD-1839), Tarceva (erlotinib, OSI-774), PKI-166, EKB-569, HKI-272 or Herceptin; mitogen activated protein Antagonists of leukokinase, such as BAY-43-9006 or BAY-57-9006; atrasentan, rituximab, cetuximab, Avastin.TM. (bevacizumab), IMC-1C11, Erbitux (C-225), DC-101, EMD-72000, Vitasin, imatinib; alkylating agents or platinum compounds, such as melphalan, cyclophosphamide, cisplatin, carboplatin, oxaliplatin, satraplatin; daunorubicin; doxorubicin (adriamycin); liposomal doxorubicin (liposomal doxorubicin hydrochloride); epirubicin; idarubicin;Pyrimidine or purine analogs or antagonists, or nucleoside diphosphate reductase inhibitors, such as cytarabine, 5-fluorouracil (5-FU), pemetrexed, tegafur / uracil, gemcitabine, capecitabine, mercaptopurine, methotrexate; anticancer drugs, such as paclitaxel (Taxol) or docetaxel; vinca alkaloids, such as navelbine, vinblastine, vincristine, vindesine or vinorelbine; antimitotic peptides, such as dolastatin; epipodophyllotoxin or podophyllotoxin derivatives, such as etoposide or teniposide; nonsteroidal anti-inflammatory drugs, such as meloxicam, celecoxib, rofecoxib; antibodies targeting cancer cell surface molecules, such as apolizumab or ID09C3; and the heat shock protein HSP90 modulator geldanamycin, and its derivative 17-allylaminogeldanamycin or 17-AAG. ;

[0053] In another embodiment, the chemotherapeutic agent is selected from the group consisting of: a compound that interacts with or binds to tubulin, a synthetic small molecule VEGF receptor antagonist, a small molecule growth factor receptor antagonist, an inhibitor of the EGF receptor and / or VEGF receptor and / or integrin receptor or any other protein tyrosine kinase receptor that is not a synthetic small molecule, an inhibitor of the EGF receptor and / or VEGF receptor and / or integrin receptor or any other protein tyrosine kinase receptor that is a fusion protein, a compound that interacts with nucleic acids and is classified as an alkylating agent or a platinum compound, a compound that interacts with nucleic acids and is classified as an anthracycline, a DNA intercalator or a DNA cross-linker (including DNA minor groove binding compounds), an antimetabolite, a naturally occurring, semisynthetic or synthetic bleomycin-type antibiotic, an inhibitor of DNA transcriptase (and and in particular topoisomerase I or topoisomerase II inhibitors), chromatin modifiers, mitotic inhibitors, antimitotic agents, cell cycle inhibitors, proteasome inhibitors, enzymes, hormones, hormone antagonists, hormone inhibitors, inhibitors of steroid biosynthesis, steroids, cytokines, hypoxia-selective cytotoxins, inhibitors of cytokines, lymphokines, antibodies against cytokines, oral and parenteral tolerance inducers, adjuvants, chemoradiation sensitizers and protectants, photochemically activated drugs, synthetic polynucleotides or oligonucleotides (optionally modified or conjugated), non-steroidal anti-inflammatory drugs, cytotoxic antibiotics, antibodies targeting cancer cell surface molecules, antibodies targeting growth factors or their receptors, inhibitors of metalloproteinases, metals, inhibitors of oncogenes, inhibitors of gene transcription or RNA translation or protein expression, rare earth element complexes and photochemotherapeutic agents.

[0054] In other embodiments, the chemotherapeutic agent is selected from the group consisting of: paclitaxel (Taxol), docetaxel; vinca alkaloids such as navelbine, vinblastine, vincristine, vindesine, or vinorelbine; alkylating agents or platinum compounds such as melphalan, cyclophosphamide, oxazaphosphorin, cisplatin, carboplatin, oxaliplatin, satraplatin, tetraplatin, iproplatin, mitomycin, streptozocin, carmustine (BCNU), lomustine (CCNU), busulfan, ifosfamide, streptozocin, thiotepa, chlorambucil; nitrogen mustards such as dichloromethane; immunomodulatory drugs such as thalidomide, its R-enantiomer and S-enantiomer, and derivatives thereof, or revitra. mede (CC-5013); ethyleneimine compounds; alkyl sulfonates; daunorubicin; doxorubicin (adriamycin); liposome doxorubicin (liposome doxorubicin hydrochloride); epirubicin; idarubicin; mitoxantrone; amsacrine; actinomycin D; distamycin or its derivatives; spindles; pibendzimol; mitomycin; CC-1065; duokamycin; mithramycin; chromomycin; olivetomycin; phthalimide, such as propamidine or distilamidine; anthramycin; aziridine; nitrosourea or its derivatives; pyrimidine or purine analogs or antagonists, or nucleoside diphosphate reductase inhibitors, such as cytarabine, 5-fluorouracil (5-FU), uracil mustard, Fludarabine, gemcitabine, capecitabine, mercaptopurine, cladribine, thioguanine, methotrexate, pentostatin, hydroxyurea or folic acid; acridine or its derivatives; rifamycin; actinomycin; methacycline; camptothecin, such as irinotecan (Capto) or topotecan; amsacrine or its analogs; tricyclic carboxamide; histone deacetylase inhibitors, such as SAHA, MD-275, trichostatin A, CBHA, LAQ824 or valproic acid; proteasome inhibitors, such as botilimab; small molecule VEGF receptor antagonists, such as 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; antagonists of mitogen-activated protein kinases, such as BAY-43-9006 or BAY-57-9006; dual EGFR / HER2 antagonists, such as gefitinib, erlotinib, CI-1033 or GW-2016; EGFR antagonists, such as Iressa (ZD-1839), Tarceva (OSI-774), PKI-166, EKB-569, HKI-272 or Herceptin;Quinazoline derivatives, such as 4-[(3-chloro-4-fluorophenyl)amino]-6-{[-4-(N,N-dimethylamino)-1-oxo-2-buten-1-yl]amino}-7-((S)-tetrahydrofuran-3-yloxy)-quinazoline or 4-[(3-chloro-4-fluorophenyl)amino]-6-{[4-(homomorpholin-4-yl)-1-oxo-2-buten-1-yl]amino}-7-[(S)-(tetrahydrofuran-3-yl)oxy]-quinazoline, or pharmaceutically acceptable salts thereof; transcription factor complex ESX / DRIP130 / Su r-2 inhibitors; HER-2 expression inhibitors, such as heat shock protein HSP90 regulator geldanamycin, and its derivatives 17-allylaminogeldanamycin or 17-AAG; protein kinase receptor antagonists that are not synthetic small molecules, such as atrasentan, rituximab, cetuximab, Avastin.TM. (bevacizumab), IMC-1C11, Erbitux (C-225), DC-101, EMD-72000, Vitasin, imatinib; and antibodies targeting cancer cell surface molecules, such as apolizumab or 1D09C3. ;

[0055] In some embodiments, the anticancer agent is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is an inhibitor of CTLA4, such as an anti-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 an anti-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 an anti-PD-L1 and / or anti-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)).

[0056] In certain aspects, provided herein are methods for treating or preventing ovarian cancer or endometrial cancer and / or for inducing an immune response to ovarian cancer or endometrial cancer tumors expressing AMHR2-ED. In certain embodiments, the method comprises administering a pharmaceutical composition as described herein to a subject. In some embodiments, the ovarian cancer or endometrial cancer tumor is a primary tumor. In some embodiments, the ovarian cancer or endometrial cancer tumor is a metastatic tumor. In some embodiments, the ovarian cancer tumor is an epithelial ovarian cancer (EOC) tumor. In some embodiments, the ovarian cancer tumor expresses AMHR2-ED.

[0057] The methods described herein can be used to treat any subject in need. As used herein, "subject in need" includes any subject suffering from ovarian cancer or endometrial cancer, having suffered from ovarian cancer or endometrial cancer and / or being susceptible to ovarian cancer or endometrial cancer. For example, in some embodiments, the subject suffers from ovarian cancer or endometrial cancer tumor (e.g., ovarian cancer tumor expressing AMHR2-ED). In some embodiments, the subject has undergone surgery to remove at least a portion of the ovarian membrane or endometrial cancer tumor. In some embodiments, the subject is susceptible to ovarian cancer due to having a BRCA1 or BRCA2 gene mutation that makes the subject susceptible to ovarian cancer in its genome. In some embodiments, the subject is susceptible to ovarian cancer due to having a TP53 gene mutation, a CHEK2 gene mutation, a RAD51 gene mutation, a BRIP1 gene mutation and / or a PALB2 gene mutation. In some embodiments, the subject has a family history of ovarian cancer.

[0058] The pharmaceutical compositions disclosed herein can be delivered by any suitable route of administration, including oral and parenteral. In certain embodiments, the pharmaceutical composition is usually delivered (e.g., by oral or parenteral administration). In some embodiments, the pharmaceutical composition is delivered intravenously. In some embodiments, the pharmaceutical composition is delivered intramuscularly. In some embodiments, the pharmaceutical composition is delivered subcutaneously.

[0059] The dosage of the drug of the present invention can be determined with reference to the plasma concentration of the drug. 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. The dosage includes those dosages that produce the above-mentioned Cmax and AUC(0-4) values, as well as other dosages that produce greater or lesser values ​​of these parameters.

[0060] Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration without being toxic to the patient.

[0061] The selected dosage level will depend upon a variety of factors, including the activity of the specific agent employed; the route of administration; the time of administration; the rate of excretion or metabolism of the specific compound employed; the duration of the treatment; other drugs, compounds and / or materials used in combination with the specific compound employed; the age, sex, weight, condition, general health and prior medical history of the patient being treated; and like factors well known in the medical arts.

[0062] A doctor or veterinarian with ordinary skills in the art can easily determine and prescribe the effective amount of the desired pharmaceutical composition. For example, a doctor or veterinarian can prescribe and / or administer a dosage of the medicament employed in the pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved.

[0063] Generally, a suitable daily dose of an agent described herein will be the amount of the agent that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend on the factors described above.

[0064] In one aspect, the present invention provides a method for inducing an immune response to cells expressing AMHR2-ED (e.g., ovarian cancer tumor cells) in a subject. The method includes: administering a pharmaceutical composition as described herein to a subject, wherein the pharmaceutically acceptable composition induces an immune response to cells expressing AMHR2-ED when administered to the subject. Typically, the immune response can include a humoral immune response, a cell-mediated immune response, or both.

[0065] The humoral response can be determined by standard immunoassays for antibody levels in serum samples from subjects receiving the pharmaceutical composition. A cellular immune response is a response involving T cells and can be determined 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. After incubating, for example, PBMCs with a stimulant for an appropriate period of time, [ 3 H] Thymidine incorporation. Proliferating T cell subsets can be determined using flow cytometry.

[0066] In some aspects, the methods provided herein include administration to humans and non-human mammals. Veterinary applications are also contemplated. In some embodiments, the subject can be any living female organism in which an immune response can be elicited. Examples of subjects include, but are not limited to, humans, livestock, dogs, cats, mice, rats, and transgenic species thereof.

[0067] In certain embodiments, the subject has a history of ovarian cancer or endometrial cancer and has been administered another treatment mode. Other therapies may include, for example, surgical resection, radiotherapy, chemotherapy, and other immunotherapy modes, whereby, as a result of other therapies, the subject does not present a clinically measurable tumor. However, the subject may be a person who is determined to be at risk of cancer recurrence or progression near the original tumor site or due to metastasis. Such subjects may be further classified into high-risk subjects and low-risk subjects. It may be subdivided according to the features observed before or after the initial treatment. These features are known in the clinical field and are appropriately defined for each different cancer. The typical features of the high-risk subgroup are those features in which the tumor has invaded adjacent tissues or shows lymph node involvement. Therefore, for example, a pharmaceutical composition as described herein may be applied to a subject to induce an anticancer response primarily as a preventive measure for recurrence.

[0068] In some embodiments, the pharmaceutical composition can be administered at any time as appropriate. For example, administration can be performed before or during conventional therapy for a subject with an ovarian or endometrial cancer tumor and continued after the tumor becomes clinically undetectable. Administration can also be continued in a subject showing signs of recurrence.

[0069] In some embodiments, the pharmaceutical composition can be administered in a therapeutically or prophylactically effective amount. The pharmaceutical composition can be administered to a subject using known procedures, at dosages and for periods of time sufficient to achieve the desired effect.

[0070] In some embodiments, the pharmaceutical composition can be administered to the subject at any suitable site (e.g., at a site distal to or proximal to the primary tumor). The route of administration can be parenteral, intramuscular, subcutaneous, intradermal, intraperitoneal, intranasal, intravenous (including via an indwelling catheter), via an afferent lymphatic vessel, or by any other suitable route depending on the neoplastic disease being treated and the condition of the subject. Preferably, the dose will be administered in an amount and time period effective to produce a desired response, which is a preventive or therapeutic treatment that induces an immune response or a neoplastic disease and / or symptoms associated therewith.

[0071] Pharmaceutically acceptable compositions can be administered after, before or simultaneously with other therapies, including therapies that also induce an immune response in the subject. For example, the subject may be treated with chemotherapy, radiotherapy and other forms of immunotherapy in advance or simultaneously, such other therapies are preferably provided in a manner that does not interfere with the immunogenicity of the compositions described herein.

[0072] The administration time can be appropriately determined by a caregiver (e.g., a doctor, a veterinarian), and can depend on the clinical condition of the subject, the purpose of administration, and / or other therapies being considered or administered. In some embodiments, an initial dose can be administered, and the immune response and / or clinical response of the subject can be monitored. Suitable immune monitoring means include using the patient's peripheral blood lymphocytes (PBL) as responders, and using tumor cells as stimulants. The immune response can also be determined by the delayed inflammatory response of the application site. One or more doses after the initial dose can be given as appropriate, usually on a monthly, semi-monthly or preferably weekly basis, until the desired effect is achieved. Thereafter, additional booster or maintenance doses can be given as needed, particularly when the immunological or clinical benefits seem to subside.

[0073] Example

[0074] Example 1

[0075] Using ADDAVAX TM Ovarian cancer vaccine formulation as adjuvant induces significant inhibition of epithelial ovarian cancer growth in mice

[0076] Epithelial ovarian cancer (EOC) is the most lethal of all human gynecological malignancies. Previous studies have demonstrated that a single vaccination of female C57BL / 6 mice with the extracellular domain of anti-Müllerian hormone receptor II (AMHR2-ED) in an emulsion containing complete Freund's adjuvant (CFA) provides significant prevention and therapy against murine EOC. Tumor immunity was found to be mediated by AMHR2-ED-specific IgG that significantly induced EOC cell death via a caspase-3-dependent apoptotic signaling cascade. However, despite the recognized usefulness of CFA as a "gold standard" adjuvant in proof-of-principle animal studies, its use in human clinical vaccination has been precluded by its propensity to induce non-resolving granulomas that are often accompanied by painful abscess formation. Due to AD DAVAX TM Adjuvants have been used safely and successfully in multiple human clinical trials to generate high-titer antibodies, so we examined the potential of using ADDAVAX TM We found that AMHR2-ED in ADDAVAX can induce effective tumor immunity in female C57BL / 6 mice. TM The formulation induced high serum titers of IgG and highly significant tumor immunity similar to that obtained using CFA as an adjuvant. Our results suggest that in ADDAVAX TM Vaccination against AMHR2-ED should provide a novel and effective approach to control human EOC.

[0077] 2. Materials and Methods

[0078] 2.1. Generation of recombinant mouse AMHR2-ED. As previously described, the complete 125 amino acid DNA coding sequence of mature native AMHR2-ED lacking the signal peptide was used to generate expression constructs

[17] (NCBI reference sequence: NM_144547.2; Uniprot Q8K592). To optimize protein folding and increase overall yield, substitutions of the native codon sequence were performed (Dapcel, Cleveland, OH), and optimized DNA was synthesized de novo to include an N-terminal methionine followed by a FLAG tag, and a C-terminal 6xHis tag. 6xHis-tagged AMHR2-ED was purified using nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography (Qiagen, Valencia, CA) under denaturing conditions. 6xHis-tagged AMHR2-ED was further purified by reverse-phase high-performance liquid chromatography (HPLC) prior to in vitro use. Endotoxin levels were determined to be <0.05 endotoxin units (<5 pg) / mg recombinant protein as previously described

[17] .

[0079] 2.2. Mice and murine EOC cell lines. TgMISIIR-Tag(DR26) and TgMISIIR-Tag(low) transgenic mice were generously provided by Dr. Denise C. Connolly (Developmental Therapeutics Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania). Female TgMISIIR-Tag(DR 26) transgenic mice develop bilateral spontaneous EOC due to expression of the large T antigen (Tag) of SV40 under the control of the AMHR2 promoter

[27] . TgMISIIR-Tag(low) transgenic female mice do not develop spontaneous ovarian cancer due to erroneous transgene insertion, but these mice are immunologically tolerant to SV40-TAg and effectively serve as histocompatibility receptors for transplantable mouse ovarian carcinoma (MOVCAR) cells derived from the ascites of TgMISIIR-Tag(DR26) mice

[28] . MOVCAR cells were cultured in DMEM (Media Preparation Core, Cleveland Clinic, Cle veland, OH) supplemented with 5% fetal bovine serum (HyClone, Logan, UT), 5% HEPES buffer (Sigma-Aldrich, St. Louis, MO), 2mM L-glutamine (Thermo Fisher Scientific, Waltham, MA) and 1% penicillin / streptomycin (Invitrogen, Carlsbad, CA). Colonies of transgenic mice were established and maintained by breeding male transgenic mice with wild-type isogenic C57BL / 6 female mice (Jackson Laboratory, Bar Harbor, ME). MOVCAR cells were subcutaneously injected into tissue-compatible TgMISIIR-Tag (low) transgenic female mice for the development of EOC. ID8 mouse ovarian surface epithelial cells (MOSEC) were purchased commercially (Sigma-Aldrich) and cultured in DMEM (Media Preparation Core) containing 4% fetal bovine serum (HyClone), 2 mM L-glutamine (Thermo Fisher Scientific), 1% penicillin / streptomycin (Invitrogen), and insulin-transferrin-sodium selenite medium supplement (Sigma-Aldrich). When injected subcutaneously into C57BL / 6 female mice, ID8 cells developed EOC

[29] .

[0080] 2.3. Transplantable ovarian tumors and treatment. MOVCAR cells (4x10 6 cells) and ID8 cells (5x10 6100 μL of recombinant mouse AMHR2-ED in 100 μL of sterile double-distilled deionized water (DDDH2O) and 100 μL of CFA (Difco, Detroit, MI) containing 200 μg of M. tuberculosis H37Ra were used to immunize mice by a single subcutaneous injection in the right flank. ADDAVAX TM The emulsion was purchased from Invivogen (San Diego, CA; Catalog No.: vac-adx-10), and 100 μL was suspended with 100 μL of sterile DDDH2O containing 100 μg of recombinant mouse AMHR2-ED, so that a final emulsion volume of 200 μL was injected into the abdomen of female TgMISIIR-Tag (low) or female C57BL / 6 mice. For prevention, mice were vaccinated 15 days before tumor inoculation. For therapeutic intervention, mice were vaccinated with AMHR2-ED when tumors became palpable. All experiments were started when mice were 6-7 weeks old. Tumor growth was assessed regularly using a vernier caliper, and the endpoints of all experiments involving transplantable tumors were defined by tumor measurements of 17 mm in any direction.

[0081] 2.4. Immunohistochemistry. Briefly, CD3+ T cells were immunostained in 5 μm sections of formalin-fixed paraffin-embedded ID8 tumor tissue using a primary CD3-specific rabbit monoclonal IgG antibody (Abcam, Cambridge, UK; catalog number: ab16669) at a dilution of 1:100, followed by immunostaining with a horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody (Abcam; catalog number: ab214880). CD4+ and CD8+ T cells were immunostained in 5 μm sections of ovarian tumors by incubation with a primary CD4-specific rabbit monoclonal IgG antibody (Abcam; catalog number: ab183685) at a dilution of 1:1000 or a primary CD8-specific rabbit monoclonal IgG antibody (Abcam; catalog number: ab217344) at a dilution of 1:500, followed by incubation with a HRP-conjugated goat anti-rabbit IgG secondary antibody (Abcam; catalog # ab214880). Visualization was achieved using a DAB substrate kit (BD Pharmingen, San Diego, CA; catalog number: 550880), followed by counterstaining with hematoxylin and mounting the sections in mounting medium (Richard-Allan Scientific, Kalama zoo, MI; catalog number: 4112) for light microscopy.

[0082] 2.5. Real-time quantitative RT-PCR (qRT-PCR) Tissues were excised and stored frozen in RN Alater stabilization solution (Invitrogen; catalog number: AM7021). RNA was extracted from each tissue by homogenization in TRIZO L reagent (Invitrogen; catalog number: 15596026). qRT-PCR was performed using SYBR Green PCR mix (Applied Biosystems, Carlsbad, CA; catalog number: 4309155) and gene-specific primer pairs (Invitrogen) including the following: mouse AMHR2-CD primer: forward-CTGAGCCGCTGTTCC GATTTGA (SEQ ID NO: 1); reverse-ATGTTGGGGCGCTTCCTCTCC T (SEQ ID NO: 2); mouse AMHR2-ED primer: forward-GCGGGGAAGCACAAAGACACT (SEQ ID NO: 3); reverse-CCGGCCATGGGTAAGA TTCC (SEQ ID NO: 4); mouse β-actin primer: forward-GGT CAT CA C TAT TGG CAA CG (SEQ ID NO: 5); reverse-ACG GAT GTC AAC GTCACA CT (SEQ ID NO: 6). Relative gene expression was determined by normalizing each gene of interest to β-actin expression in each sample.

[0083] 2.6. Enzyme-linked immunosorbent assays (ELISAs). ELISAs were performed as described previously

[29] . Briefly, serum samples were taken from vaccinated mice eight weeks after vaccination and added to triplicate wells of 96-well plates pre-coated with AMH R2-ED or with the control antigen ovalbumin (Sigma-Aldrich; Catalog No. A7641-250MG) and recombinant mouse β-casein (a protein produced in Escherichia coli in our laboratory in a manner similar to that of AMHR2-ED) after diluting each serum sample 1:10,000

[17] . After each step of incubation, the absorbance at 405 nm was determined as described previously according to the manufacturer's instructions

[17] . Isotype-specific serum antibody responses to AMHR2-ED were determined at a dilution of 1:10,000 using a mouse typing kit (Bio-Rad, Hercules, CA; Catalog No. 1722051) according to the manufacturer's instructions.

[0084] 2.7. Biostatistical analysis. Student's t test and two-way ANOVA were used to compare the differences between mRNA expression levels. Two-way ANOVA was used to compare the differences between tumor growth curves. The differences in mouse survival were determined using log-rank and chi-square using related samples. All experiments were repeated at least three times independently.

[0085] 3. Results

[0086] 3.1. Elevated AMHR2 expression in mouse ovarian tumor cell lines. We determined the relative gene expression of AMHR2-ED and AMHR2-CD in ID8 and MOVCAR murine EOC cells. We found that, although both MOVCAR and ID8 cells expressed high levels of AMHR2 domains, the expression level of each domain in MOVCAR cells was consistently approximately twofold that in ID8 cells ( Figure 1 ).

[0087] 3.2. Using ADDAVAX TM Adjuvanted AMHR2-ED vaccination induces robust IgG responses in female C57BL / 6 mice. TM As an adjuvant to induce a robust IgG response to AMHR2-ED, we used CFA and ADDAVAX TM C57BL / 6 female mice aged 6-7 weeks were immunized with 100 μg of recombinant mouse AMHR2-ED as an adjuvant. Eight weeks after immunization, serum IgG responses at a dilution of 1:10,000 were assessed in response to AMHR2-ED and in response to the irrelevant antigens ovalbumin and recombinant mouse β-casein. We found that the use of ADDAVAX TM The absorbance obtained as an adjuvant was similar to that obtained using CFA as an adjuvant ( Figure 2 a). We also found that using ADDAVAX TM The IgG isotype distribution generated as an adjuvant was also similar to that obtained using CFA as an adjuvant ( Figure 2 b).

[0088] 3.3. Using ADDAVAX TM We next examined the inhibition of murine EOC tumor growth induced by AMHR2-ED vaccination with ADDAVAX compared to that induced when AMHR2-ED vaccination was incorporated with CFA as an adjuvant. TMWe found that vaccination with ADDAVAX 15 days before inoculation of MOVCAR cells into TgMISIIR-Tag(low) female mice was beneficial compared with vaccination with CFA alone or with AMHR2-ED vaccination with CFA as an adjuvant. TM Prophylactic vaccination against AMHR2-ED with adjuvant significantly inhibited the growth of MOVCAR-derived tumors ( Figure 3 a; P < 0.0001 for each). In addition, compared with vaccination with CFA alone ( Figure 3 b; P < 0.009), or compared with vaccination against AMHR2-ED using CFA as an adjuvant ( Figure 3 b; P < 0.006), using ADDAVAX TM Prophylactic vaccination against AMHR2-ED as an adjuvant provided a statistically significant increase in overall survival. We also found that vaccination with CFA or ADDAVAX was associated with a statistically significant increase in overall survival compared with vaccination with CFA alone. TM As an adjuvant, therapeutic vaccination against AMHR2-ED had similar effects when MOVCAR tumors became palpable ( Figure 3 c; P < 0.0001, respectively). However, when female C57BL / 6 mice were treated with ADDAVAX alone after the inoculated ID8 EOC cells developed palpable tumors, TM Or use ADDAVAX TM Perhaps most clearly demonstrated when administering vaccination with AMHR2-ED as an adjuvant TM The efficacy ( Figure 3 d; P<0.0001).

[0089] 3.4. Using ADDAVAX TM Vaccination with AMHR2-ED as an adjuvant resulted in T cell infiltration of EOC in mice. We next performed 175 days after tumor inoculation and with ADDAVAX TM We examined immune cell infiltration of ID8 tumors 168 days after vaccination with AMHR2-ED as an adjuvant. We observed a large infiltration of CD3+, CD4+, and CD8+ T cells ( Figure 4 The observed CD8+ T cell infiltration into ID8 tumors was quite surprising, as we had previously observed no CD8+ T cell infiltration in C57BL / 6 female mice vaccinated against AMHR2-ED when CFA was used as an adjuvant

[17] .

[0090] The current example shows that using ADDAVAX TMOur AMH R2-ED vaccine formulated as an adjuvant induced robust IgG responses and inhibited the growth of mouse EOC in a similar (and often superior) manner compared to using CFA as an adjuvant. In fact, by some comparisons, ADDAVAX TM It appears to be unexpectedly superior to CFA, most notably inducing significantly enhanced overall survival in the transplantable MOVCAR EOC mouse model and inducing T cell infiltration of CD8+ T cells in the ID8-derived EOC tumor bed, which was not seen in previous studies using CFA as an adjuvant

[17] . Therefore, our data support the use of ADDAVAX TM Instead of CFA as an adjuvant, it can be used to prevent EOC in women at high genetic risk. Studies have shown that 39%–44% of women with deleterious BRCA1 variants and 11%–17% of women with deleterious BRCA2 variants develop ovarian cancer by the age of 70–80 years [30–32]. These high-risk groups of women have a high incidence of EOC and the greatest need for an EOC preventive vaccine, so it is important to treat these women with the adjuvant and AMHR2-ED peptide described in this article.

[0091] References

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[0128] Incorporated by Reference

[0129] All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. A composition comprising: a) a polypeptide comprising at least a portion of an anti-Müllerian hormone receptor type II extracellular domain (AMHR-ED); and b) an adjuvant, said adjuvant comprising: i) Squalene oil, ii) nonionic surfactants, iii) an emulsifier, and iv) Buffering agents.

2. The composition of claim 1, wherein the emulsifier comprises sorbitan trioleate, and / or the nonionic surfactant comprises polysorbate 80.

3. A composition as claimed in claim 1 or claim 2, wherein the polypeptide is from human AMHR-ED.

4. The composition of any one of claims 1 to 3, wherein the buffer comprises citrate buffer or phosphate buffered saline.

5. The composition of any one of claims 1 to 4, wherein the pH of the composition is between 5.5 and 6.

9.

6. The composition of any one of claims 1 to 5, wherein the composition is in the form of an emulsion.

7. The composition of claim 6, wherein the emulsion is a nanoemulsion of a first and a second component, wherein the first component comprises the emulsifier and squalene oil, and the second component comprises the nonionic surfactant and the buffer.

8. The composition of claim 7, wherein the emulsifier is present in the first component at about 0.4-0.6 weight / volume.

9. A composition as claimed in claim 7 or claim 8, wherein the squalene oil is present in the first component at about 4-6% weight / volume.

10. The composition of any one of claims 7 to 9, wherein the nonionic surfactant is present in the second component at about 0.4-0.6 weight / volume.

11. The composition of any one of claims 7 to 10, wherein the buffer is present in the second component at about 4-6% weight / volume.

12. The composition of any one of claims 7 to 11, wherein the nanoemulsion is composed of particles having an average size of about 150-170 nm.

13. The composition of any one of claims 1 to 12, wherein the adjuvant further comprises at least one of: a saponin, cholesterol, a phospholipid, a phosphatidylcholine, and a second buffer.

14. The composition of any one of claims 1 to 13, wherein the adjuvant further comprises alpha-tocopherol.

15. The composition of any one of claims 1 to 14, wherein the adjuvant further comprises monophosphoryl lipid A.

16. The composition of any one of claims 1 to 15, wherein the adjuvant further comprises an immunomodulator.

17. The composition of claim 16, wherein the immunomodulator comprises synthetic trehalose diphtherate.

18. The composition of any one of claims 1 to 17, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 7, 8, 9 or 10.

19. The composition of any one of claims 1 to 17, wherein the polypeptide comprises complete human AMHR-ED or nearly complete human AMHR2-ED.

20. The composition of any one of claims 1 to 17, wherein the polypeptide comprises at least 20 consecutive amino acids from SEQ ID NO:

7.

21. The composition of any one of claims 1 to 17, wherein the polypeptide comprises at least 40 consecutive amino acids from SEQ ID NO:

7.

22. The composition of any one of claims 1 to 17, wherein the polypeptide comprises at least 60 consecutive amino acids from SEQ ID NO:

7.

23. The composition of any one of claims 1 to 17, wherein the polypeptide comprises at least 100 consecutive amino acids from SEQ ID NO:

7.

24. The composition of any one of claims 1 to 17, wherein the polypeptide comprises at least 115 consecutive amino acids from SEQ ID NO:

7.

25. The composition of any one of claims 1 to 17, wherein the polypeptide comprises at least 122 consecutive amino acids from SEQ ID NO:

7.

26. The composition of any one of claims 1 to 17, wherein the polypeptide has an amino acid sequence comprising 100 consecutive amino acids that are at least 80% identical to an amino acid sequence in the extracellular domain of AMHR2.

27. The composition of any one of claims 1 to 26, wherein administration of the composition to a subject activates CD4+T cells in the subject.

28. The composition of any one of claims 1 to 27, wherein administration of the composition to a subject induces the production of AMHR2 ED-specific IgG in the subject.

29. The composition of claim 27 or 28, wherein the subject is a human female.

30. A method of treating an ovarian cancer or endometrial cancer tumor in a female subject, the method comprising: The composition of any one of claims 1 to 29 is administered to the female subject.

31. The method of claim 30, wherein the subject has an ovarian cancer tumor, and the ovarian cancer tumor is a primary ovarian cancer tumor or a metastatic tumor.

32. The method of claim 30 or 31, wherein the female subject is a human.

33. The method of any one of claims 30 to 32, wherein the subject has an ovarian cancer tumor and the ovarian cancer tumor is an epithelial ovarian cancer (EOC) tumor.

34. The method of any one of claims 30 to 33, wherein administration of the composition induces an immune response in the subject against the ovarian cancer and / or endometrial cancer tumor.

35. The method of any one of claims 30 to 34, wherein the ovarian and / or endometrial cancer tumor expresses AMHR2-ED.

36. The method of any one of claims 30 to 35, wherein the method further comprises the step of determining whether the ovarian cancer and / or endometrial cancer tumor expresses AMHR2-ED.

37. The method of any one of claims 30 to 36, further comprising the step of determining whether administration of the composition induces an immune response in the subject against the ovarian or endometrial cancer tumor.

38. The method of any one of claims 30 to 37, further comprising repeating the administration of the composition to the subject.

39. The method of any one of claims 30 to 38, wherein administration of the composition induces the subject to express anti-AMHR2-ED IgG antibodies.

40. The method of any one of claims 30 to 39, further comprising the step of administering to the subject an additional anti-cancer agent.

41. The method of claim 40, wherein the additional anticancer agent is paclitaxel, cisplatin, topotecan, gemcitabine, bleomycin, etoposide, carboplatin, docetaxel, doxorubicin, topotecan, cyclophosphamide, trabectedin, olaparib, tamoxifen, letrozole, bevacizumab, anti-CTLA4 antibody, anti-PD-1 antibody, or anti-PD-L1 antibody.

42. The method of any one of claims 30 to 41, wherein the subject has undergone surgery to remove at least a portion of the ovarian theca and / or endometrial cancer tumor.

43. A method for preventing ovarian cancer and / or endometrial cancer, the method comprising: The composition of any one of claims 1 to 29 is administered to a female subject.

44. The method of claim 43, wherein the subject does not have detectable ovarian cancer and / or endometrial cancer.

45. The method of claim 43 or 44, wherein the subject has an increased risk of developing ovarian cancer and / or endometrial cancer.

46. ​​A system or kit, comprising: a) a polypeptide comprising at least a portion of an anti-Müllerian hormone receptor type II extracellular domain (AMHR-ED); as well as b) an adjuvant, the adjuvant comprising: i) Squalene oil, ii) nonionic surfactants, iii) an emulsifier, and iv) Buffering agents.

47. The system or kit of claim 46, wherein the polypeptide is from human AMHR-ED.

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