WO2017079303A1 - Sequentially orchestrated immune checkpoint therapy for the treatment and prevention of cancer - Google Patents

Abstract

Provided herein are methods and kits for the treatment and/or prevention of cancer through the induction or enhancement of an immune response in a subject using one or more immune checkpoint inhibitors, alone or in combination, with targeted cancer vaccines.

Description

SEQUENTIALL Y ORCHESTRA TED IMMUNE CHECKPOINT THERAPY FOR THE TREATMENT AND PREVENTION OF CANCER RELATED APPLICATIONS

This application claims the benefit of priority to U.S. Provisional Patent Application No. 62/249,593, filed November 2, 2015, which is hereby incorporated by reference in its entirety. BACKGROUND

Subjects with triple negative breast cancer (TNBC) have the poorest prognosis of all breast cancer subtypes with respect to five year overall survival. TNBC tumors do not express any of the three common growth factor receptors (i.e., estrogen receptors, progesterone receptors, and HER2 receptors for epidermal growth factor) that can be targeted with specific therapeutic antagonists. Current standard of care for TNBC involves surgery, radiation therapy, and chemotherapy with no available receptor-targeted therapy.

Surgical intervention has an immediate effect on reducing tumor load. Likewise, chemotherapy and radiation therapy typically induce rapid measurable tumor shrinkage. These interventions may result in enduring beneficial effects in extending overall survival, but all too often the effects of these therapies have limited durability beyond the treatment period. Hormone therapy does not typically induce rapid measureable effects on tumor growth, and the long-term beneficial effects of hormone therapy require extended administration of drugs that block the tumor growth effects of estrogen and progesterone. In all cases, these conventional therapies are accompanied by deleterious side effects that are often debilitating and frequently preclude subject compliance with additional or extended treatments.

Thus, there is a great need for new and effective targeted therapies to improve the overall survival of subjects with TNBC. SUMMARY

In certain aspects, provided herein are methods for the treatment and/or prevention of TNBC through the induction or enhancement of an immune response using sequential treatment with a first immune checkpoint inhibitor and second immune checkpoint inhibitor, singly or in combination. In addition to TNBC, such methods may be useful in the treatment and/or prevention of breast, ovarian, prostate, or other cancers. Said treatments may combine one or more immune checkpoint inhibitors with targeted cancer vaccines comprising polypeptides selected e.g., from a-lactalbumin, aS l casein, β-casein, κ-casein, or anti-Mullerian Hormone Receptor, Type II (AMHR2), to induce or enhance the immune response in a subject.

In certain aspects, provided herein is a method of inducing or enhancing an immune response in a subject ( e.g., a human subject) in need thereof, the method comprising the steps of (a) administering an effective amount of a first immune checkpoint inhibitor ( e.g., an inhibitor of CTLA4) to a subject to prime the immune response in the subject; and/or (b) administering an effective amount of a second immune checkpoint inhibitor ( e.g., an inhibitor of PD-1, PD-Ll, or PD-L2) to the subject to enhance the primed immune response in the subject. In some embodiments, the subject has triple negative breast cancer (T BC), breast cancer, ovarian cancer, or prostate cancer. In some embodiments, the method further comprises administering a targeted cancer vaccine prior to, subsequently, or conjointly with the first and/or second immune checkpoint inhibitor. In some embodiments, the targeted cancer vaccine is an immunogenic composition comprising an immunogenic polypeptide selected from the group consisting of human a-lactalbumin (e.g., at least a portion of SEQ ID NO: 1), aS l casein (e.g., at least a portion of SEQ ID NO: 2), β-casein (e.g., at least a portion of SEQ ID NO: 3), κ-casein (e.g., at least a portion of SEQ ID NO: 4), and anti- Mullerian Hormone Receptor, Type II (AMHR2) (e.g., at least a portion of SEQ ID NO: 5). In some embodiments, administration of the first immune checkpoint inhibitor, second immune checkpoint inhibitor, or targeted cancer vaccine induces an immune response against the ovarian cancer tumor in the subject (e.g., a T cell immune response, such as a type 1 and/or type 17 immune response and/or a B cell immune response, such as an IgG response). In some embodiments, the subject is administered multiple doses of the first immune checkpoint, second immune checkpoint, and/or targeted cancer vaccine (e.g., at least 2, 3, 4, 5 or 6 doses). Said doses may be given at periodic intervals occurring daily, weekly, biweekly, and monthly. In some embodiments, the methods further comprise using the first immune checkpoint inhibitor or second immune checkpoint inhibitor in

combination with a chemotherapeutic agent (e.g., sunitinib or sorafenib) or a corticosteroid treatment.

In certain aspects, provided herein is a method of preventing cancer in a subject in need thereof (e.g., a human subject), the method comprising the steps of (a) administering an effective amount of a first immune checkpoint inhibitor ( e.g., an inhibitor of CTLA4) to a subject to prime the immune response in the subject; and/or (b) administering an effective amount of a second immune checkpoint inhibitor ( e.g., inhibitor of PD-1, PD-L1, or PD-L) to the subject to enhance the primed immune response in the subject. In some embodiments, the subject has triple negative breast cancer (TNBC), breast cancer, ovarian cancer, or prostate cancer. In some embodiments, the method further comprise administering a targeted cancer vaccine prior to, subsequently, or conjointly with the first and/or second immune checkpoint inhibitor. In some embodiments, the targeted cancer vaccine is an immunogenic composition comprising an immunogenic polypeptide selected from the group consisting of human a-lactalbumin (e.g., at least a portion of SEQ ID NO: 1), aSl casein (e.g., at least a portion of SEQ ID NO: 2), β-casein (e.g., at least a portion of SEQ ID NO: 3), K-casein (e.g., at least a portion of SEQ ID NO: 4), and anti-Mullerian Hormone Receptor, Type II (AMHR2) (e.g., at least a portion of SEQ ID NO: 5). In some

embodiments, administration of the first immune checkpoint inhibitor, second immune checkpoint inhibitor, or targeted cancer vaccine induces an immune response against the ovarian cancer tumor in the subject (e.g., a T cell immune response, such as a type 1 and/or type 17 immune response and/or a B cell immune response, such as an IgG response). In some embodiments, the subject is administered multiple doses of the first immune checkpoint, second immune checkpoint, and/or targeted cancer vaccine (e.g., at least 2, 3, 4, 5 or 6 doses). Said doses may be given at periodic intervals occurring daily, weekly, biweekly, monthly. In some embodiments, the methods further comprise using the first immune checkpoint inhibitor or second immune checkpoint inhibitor in combination with a chemotherapeutic agent (e.g., sunitinib or sorafenib) or a corticosteroid treatment.

In some embodiments of the methods provided herein, the targeted cancer vaccine comprises an adjuvant. In some embodiments, the adjuvant is Adjuvant 65, a-GalCer, aluminum phosphate, aluminum hydroxide, calcium phosphate, β-Glucan Peptide, CpG DNA, GM-CSF, GPI-0100, IF A, IFN-γ, IL-17, lipid A, lipopoly saccharide, Lipovant, Montanide, N-acetyl-muramyl-L-alanyl-D-isoglutamine, Pam3CSK4, poly-IC, quil A, trehalose dimycolate, or zymosan. In some embodiments, the adjuvant is one that induces a mixed type 1/type 17 immune response.

In some embodiments of the methods provided herein, the method further comprises the step of determining whether the administration of the sequential treatment with a first and second immune checkpoint inhibitor induces an immune response against the cancer tumor. In some embodiments, the immune response is a T cell immune response (e.g., a mixed type 1/type 17 immune response). In some embodiments, the immune response is detected by detecting the presence of cytokines (e.g., IFN-γ and/or IL-17) in a subject sample (e.g., a subject serum sample). In some embodiments, the cytokines are detected by contacting the sample with, directly or indirectly, detectably labeled antibodies specific for the cytokines to be detected. In some embodiments, the cytokines are detected by performing an ELISA assay. In some embodiments, the immune response is a B cell immune response. In some embodiments, the B cell immune response is detected in a subject's sample (e.g., a serum sample).

In certain embodiments, the methods provided herein further comprise the step of administering an additional agent to the subject. In some embodiments, the additional agent is an anti-cancer agent. In some embodiments, the anti-cancer agent is paclitaxel, cisplatin, topotecan, gemcitabine, bleomycin, etoposide, carboplatin, docetaxel, doxorubicin, topotecan, cyclophosphamide, trabectedin, olaparib, tamoxifen, letrozole or bevacizumab. In some embodiments, the anti-cancer agent is an immune checkpoint inhibitor.

In some embodiments, the first 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 second 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-A1 1 10 (Sorrento) and/or TSR-042 (Tesaro). In some embodiments, the second immune checkpoint inhibitor is an inhibitor of PD-L1 and/or PD-L2, such as an anti-PD-Ll and/or an 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 certain embodiments of the methods provided herein, the human subject is predisposed to breast or ovarian cancer. In some embodiments, the genome of the subject comprises a BRCAl or BRCA2 mutation that predisposes the subject to breast or ovarian cancer. In some embodiments, the subject has a family history of ovarian cancer. In some embodiments, the subject is a post-menopausal human female.

In certain aspects, provided herein is a kit comprising a first immune checkpoint inhibitor, a second immune checkpoint inhibitor, and/or a targeted cancer vaccine for preventing and/or treating cancer (e.g., breast cancer, TNBC, ovarian, prostate, etc.) in a subject (e.g., a human subject), the kit or composition comprising a first immune checkpoint inhibitor ( e.g., an inhibitor of CTLA4), a second immune checkpoint inhibitor ( e.g., an inhibitor of PD-1), and a targeted cancer vaccine which is an immunogenic composition comprising an immunogenic polypeptide selected from the group consisting of human a-lactalbumin (e.g., at least a portion of SEQ ID NO: 1), aS l casein (e.g., at least a portion of SEQ ID NO: 2), β-casein (e.g., at least a portion of SEQ ID NO: 3), κ-casein (e.g., at least a portion of SEQ ID NO: 4), and anti-Mullerian Hormone Receptor, Type II (AMHR2) (e.g., at least a portion of SEQ ID NO: 5). In some embodiments, the kit comprises multiple doses of the first immune checkpoint inhibitor, a second immune checkpoint inhibitor, and/or a targeted cancer vaccine. In some embodiments, the kit further comprises instructions for use.

In certain embodiments of the kits and compositions provided herein, the kit or composition further comprises an adjuvant. In some embodiments, the adjuvant is Adjuvant 65, a-GalCer, aluminum phosphate, aluminum hydroxide, calcium phosphate, β-Glucan Peptide, CpG DNA, GM-CSF, GPI-0100, IF A, IFN-γ, IL-17, lipid A, lipopoly saccharide, Lipovant, Montanide, N-acetyl-muramyl-L-alanyl-D-isoglutamine, Pam3CSK4, quil A, trehalose dimycolate or zymosan. In some embodiments, adjuvant is one that induces a mixed type 1/type 17 immune response.

In some embodiments of the kits or compositions provided herein, the kit or composition further comprises an additional agent (e.g., an anti-cancer agent). In some embodiments, the additional agent is an anti-cancer agent selected from the group consisting of paclitaxel, cisplatin, topotecan, gemcitabine, bleomycin, etoposide, carboplatin, docetaxel, doxorubicin, topotecan, cyclophosphamide, trabectedin, olaparib, tamoxifen, letrozole and bevacizumab.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and

modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is shows the frequencies of IFNy-producing T cells in eight week old C57BL/6 female mice vaccinated with a single subcutaneous injection of 100 μg of recombinant mouse AMHR2-ED in complete Freund's adjuvant and subsequently injected intraperitoneally with 100 μg of checkpoint inhibitor antibody (BioXCell, West Lebanon, H) or control during the early priming phase on days 0, 5, 10, and 15 and/or during the late effector phase on days 18, 23, 28, and 33, as indicated. Control mice were injected with PBS during early and late phases. Error bars show +SE. Asterisk indicates significance. DETAILED DESCRIPTION

General

Provided herein are methods and kits for the treatment and/or prevention of cancer (e.g., breast cancer, TNBC, ovarian, prostate, etc.) through the induction or enhancement of an immune response in a subject using sequential treatments with a first immune checkpoint inhibitor and second immune checkpoint inhibitor, singly or in combination. Said methods may be combined with targeted cancer vaccines (e.g., vaccines directed to a- lactalbumin, aSl casein, β-casein, κ-casein, and anti-Mullerian Hormone Receptor, Type II (AMHR2)) administered prior to, subsequently, or conjointly with the first and/or second immune checkpoint inhibitor.

Definitions

For convenience, certain terms employed in the specification, examples, and appended claims are collected here.

The articles "a" and "a«" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

As used herein, the term "administering" means providing a pharmaceutical agent or composition to a subject, and includes, but is not limited to, administering by a medical professional and self-administering.

As used herein, the phrase "Anti-Mullerian Hormone Receptor, Type IF, abbreviated (AMHR2), is a serine/threonine kinase receptor homologous to type II receptors of the transforming growth factor beta (TGFP) family. The human AMHR2 gene contains 11 exons with seven known alternatively spliced variants producing three known coded proteins, one additional variant with protein coding features, and three non-coding transcripts with no open reading frames. In adult women, the longest human protein coding transcript for a 573 amino acid long protein (SEQ ID NO: 5) is normally expressed only in the ovary and comprises a 127 amino acid extracellular domain (AMHR2-ED), a 26 amino acid transmembrane domain, and a 403 amino acid cytoplasmic domain (AMHR2-CD). AMHR2 signaling causes regression of the Miillerian ducts during male development and regulates oocyte development and follicle production in adult females, thereby providing substantial control of ovarian reserve and fertility. AMHR2 is expressed in the vast majority of human EOCs, including 90% of primary EOCs, 78% of borderline malignancies, 77-86%) of non-EOC ovarian tumors, and 56%> of malignant ascites from grade III-IV ovarian cancers. In normal tissues, AMHR2-CD expression is predominantly confined to the ovaries. While some AMHR2-CD expression also occurs in a small number of additional human tissues, AMHR2-ED is expressed exclusively in the ovary. What is more, ovarian expression of both AMHR2-CD and AMHR2-ED is reduced in post-menopausal ovaries. The mRNA sequences of the three protein-coding isoforms of AMHR2 are provided at NCBI reference numbers NM_020547.2, NM_001164690.1 and

NM 001164690.1, which encode for proteins having amino acid sequences provided at NCBI reference numbers NP_065434.1 , NP_001158162.1 and NP_001158163.1 , respectively. Each of the above mRNA and protein sequences are hereby incorporated by reference.

The term "immune response" refers herein to any response to an antigen or antigenic determinant by the immune system. Exemplary immune responses include humoral immune responses {e.g. production of antigen-specific antibodies (neutralizing or otherwise)) and cell-mediated immune responses {e.g. lymphocyte proliferation). Type-1 inflammatory immune responses are characterized by the production of type-1 cytokines, such as IFNy. Type-2 inflammatory immune responses are characterized by expression of type-2 cytokines, such as IL-4 or IL-5. Type-17 inflammatory immune responses are characterized by expression of type-17 cytokines, and particularly IL-17. In some instances, a mixed immune response can be generated. For example, in some instances a mixed type- l/type-17 inflammatory immune response is generated that is characterized by the expression of both IFNy and IL-17.

As used herein, "percent identity^'' between amino acid sequences is synonymous with percent homology.' " which can be determined using the algorithm of Karlin and Altschul (Proa Nail. Acad. Set USA 87, 2264-2268, 1990), modified by Karlin and Altschul (Proc. Natl. Acad. Set. USA 90, 5873-5877, 1993). The noted algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (J. Mol. Biol. 2 5, 403-410, 1990). BLAST nucleotide searches are performed with the NBLAST program, score=TG0, wordlength=12, to obtain nucleotide sequences homologous to a polynucleotide described herein. BLAST protein searches are performed with the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to a reference polypeptide. To obtain gapped alignments for comparison purposes, Gapped BLAST is utilized 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) are used.

The phrase "pharmaceutically-acceptable carrier" as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.

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

The phrases "therapeutically-effective amount" and "effective amount" as used herein means the amount of an agent which is effective for producing the desired therapeutic effect in at least a sub-population of cells in a subject at a reasonable

benefit/risk ratio applicable to any medical treatment. The induction of an effective immune response involves clonal expansion of high affinity T cells to frequency levels that can produce a clinically relevant immune response. It also involves production of high titers of high affinity anti-tumor antibodies that may participate effectively in the ultimate demise of the tumor. The completion of this immune process often requires multiple booster vaccinations and typically takes several months to develop and reach maturity. In some embodiments, the efficacy is dramatically improved if the treatment and methods provided herein are administered prior to any tumor taken root or any tumor which has acquired mutations capable of maintaining the adaptive plasticity and immortality of the tumor.

"Treating" a disease in a subject or "treating" a subject having a disease refers to subjecting the subject to a pharmaceutical treatment, e.g., the administration of a drug, such that at least one symptom of the disease is decreased or prevented from worsening. Immune Checkpoint Inhibitors

In some embodiments, immune checkpoint inhibitors may be directed to immune checkpoint regulators, including but not limited to, CTLA4, PD-1, PD-L1, PD-L2, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, 2B4, ICOS, HVEM, PD-L2, CD160, gp49B, PIR- B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, BTLA, SIRPalpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, LAG-3, BTLA, A2aR and many more.

In some embodiments, immune checkpoint inhibitors may comprise agents that inhibit immune checkpoint nucleic acids and/or proteins. Inhibition of one or more immune checkpoints can block or otherwise neutralize inhibitory signaling to promote

immunomodulation. Exemplary agents useful for inhibiting immune checkpoints include antibodies, small molecules, peptides, peptidomimetics, natural ligands, and derivatives of natural ligands, that can either bind and/or inactivate or inhibit immune checkpoint proteins, or fragments thereof; as well as RNA interference, antisense, nucleic acid aptamers, etc. that can downregulate the expression and/or activity of immune checkpoint nucleic acids, or fragments thereof. Exemplary agents for upregulating an immune response include antibodies against one or more immune checkpoint proteins block the interaction between the proteins and its natural receptor(s); a non-activating form of one or more immune checkpoint proteins (e.g., a dominant negative polypeptide); small molecules or peptides that block the interaction between one or more immune checkpoint proteins and its natural receptor(s); fusion proteins (e.g. the extracellular portion of an immune checkpoint inhibition protein fused to the Fc portion of an antibody or immunoglobulin) that bind to its natural receptor(s); nucleic acid molecules that block immune checkpoint nucleic acid transcription or translation; and the like. Such agents can directly block the interaction between the one or more immune checkpoints and its natural receptor(s) (e.g., antibodies) to prevent inhibitory signaling and upregulate an immune response. Alternatively, agents can indirectly block the interaction between one or more immune checkpoint proteins and its natural receptor(s) to prevent inhibitory signaling and upregulate an immune response. For example, a soluble version of an immune checkpoint protein ligand such as a stabilized extracellular domain can binding to its receptor to indirectly reduce the effective

concentration of the receptor to bind to an appropriate ligand.

In some embodiments, the first 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 second 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 second immune checkpoint inhibitor is an inhibitor of PD-L1 and/or PD-L2, such as an anti-PD-Ll and/or an 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)).

Targeted cancer vaccines

Provided herein are methods and kits for the treatment and/or prevention of cancer

(e.g., breast cancer, T BC, ovarian, prostate, etc.) through the induction or enhancement of an immune response in a subject using sequential treatments with a first and second immune checkpoint inhibitor. Said methods may be combined with targeted cancer vaccines (e.g., vaccines directed to a-lactalbumin, aSl casein, β-casein, κ-casein, and anti- Mullerian Hormone Receptor, Type II (AMHR2)). Said targeted cancer vaccines may comprise the vaccines described in US patent no. 9,125,848, US publication 20140178418, or WO2013/158553. Each of the aforementioned patents or patent publications are hereby incorporated by reference.

In some embodiments, the targeted cancer vaccine is a human breast cancer vaccine comprising an immunogenic polypeptide. The immunogenic polypeptide comprises human a-lactalbumin according to the amino acid sequence:

KQFTKCELSQ LLKDIDGYGG IALPELICTM FHTSGYDTQA IVENNESTEY GLFQIS KLW CKSSQVPQSR NICDISCDKF LDDDITDDIM CAKKILDIKG IDYWLAHKAL CTEKLEQWLC EKL (SEQ ID NO: 1).

It is appreciated that human α-lactalbumin is processed in vivo by proteases to smaller peptide fragments, which are able to bind to MHC class I and/or MHC class II molecules on antigen presenting cells. Subsequently, T-cell receptors recognize and bind to the MHC molecule to which the peptide is bound, forming the primary signal that initiates an immune response.

In some embodiments, the targeted cancer vaccine is a human breast cancer vaccine comprising an immunogenic polypeptide. The immunogenic polypeptide comprises human aSl casein according to the amino acid sequence: RPKLP LRYPERLQ P SESSEPIPLE SREEYMNGMN RQRNILREKQ

TDEIKDTR E STQNCVVAEP EKMESSISSS SEEMSLSKCA EQFCRL EYN

QLQLQAAHAQEQIRRMNENS HVQVPFQQLN QLAAYPYAVW YYPQIMQYVP

FPPFSDIS P TAHENYEKNNVMLQW (SEQ ID NO: 2).

In some embodiments, the targeted cancer vaccine is a human breast cancer vaccine comprising an immunogenic polypeptide. The immunogenic polypeptide comprises human β-casein according to the amino acid sequence:

ALALARETIE SLSSSEESIT EYKQKVEKVK HEDQQQGEDE HQDKIYPSFQ

PQPLIYPFVE PIPYGFLPQN ILPLAQPAVV LPVPQPEIME VPKAKDTVYT KGRVMPVLKS PTIPFFDPQI PKLTDLENLH LPLPLLQPLM QQVPQPIPQT

LALPPQPLWS VPQPKVLPIP QQVVPYPQRA VPVQALLLNQ ELLLNPTHQI

YPVTQPLAPV HNPISV (SEQ ID NO: 3).

In some embodiments, the targeted cancer vaccine is a human breast cancer vaccine comprising an immunogenic polypeptide. The immunogenic polypeptide comprises human K-casein according to the amino acid sequence:

EVQNQKQPAC HENDERPFYQ KTAPYVPMYY VPNSYPYYGT

NLYQRRPAIA INNPYVPRTY YANPAVVRPH AQIPQRQYLP NSHPPTVVRR

PNLHPSFIAI PPKKIQDKII IPTINTIATV EPTPAPATEP TVDSVVTPEA FSESIITSTP

ETTTVAVTPP TA (SEQ ID NO: 4).

In some embodiments, the targeted cancer vaccine is a human ovarian cancer vaccine comprising an immunogenic polypeptide. The immunogenic polypeptide comprises

Human AMHR2 according to the amino acid sequence:

MLGSLGLWALLPTAVEAPPNRRTCVFFEAPGVRGSTKTLGELLDTGTELPR

AIRCLYSRCCFGIWNLTQDRAQVEMQGCRDSDEPGCESLHCDPSPRAHPSPGSTLFT CSCGTDFCNANYSHLPPPGSPGTPGSQGPQAAPGESIWMALVLLGLFLLLLLLLGSII

L ALLQRKN YRVRGEP VPEPRPD S GRD W S VELQELPELCF S Q VIREGGH A V VW AGQ

LQGKLVAIKAFPPRSVAQFQAERALYELPGLQHDHIVRFITASRGGPGRLLSGPLLV

LELHPKGSLCHYLTQYTSDWGSSLRMALSLAQGLAFLHEERWQNGQYKPGIAHRD

LSSQNVLIREDGSCAIGDLGLALVLPGLTQPPAWTPTQPQGPAAIMEAGTQRYMAP ELLDKTLDLQDWGM ALRRADI YSL ALLLWEIL SRCPDLRPD S SPPPFQL AYE AELG

NTPTSDELWALAVQERRRPYIPSTWRCFATDPDGLRELLEDCWDADPEARLTAEC

VQQRLAALAHPQESHPFPESCPRGCPPLCPEDCTSIPAPTILPCRPQRSACHFSVQQG

PCSRNPQPACTLSPV (SEQ ID NO: 5). In some embodiments, the targeted cancer vaccine further comprises an adjuvant and a pharmaceutically acceptable carrier. As used herein, the term "adjuvant" refers to an agent that stimulates the immune system and increases the response to a vaccine. Vaccine adjuvants are well-known to those of skill in the art. As used herein, the term "carrier" refers to an ingredient other than the active component(s) in a formulation. The choice of carrier will to a large extent depend on factors such as the particular mode of administration or application, the effect of the carrier on solubility and stability, and the nature of the dosage form. Pharmaceutically acceptable carriers for polypeptide antigens are well known in the art.

In some embodiments, the vaccine is administered prophylactically to prevent cancer (e.g., breast cancer, TNBC, ovarian, prostate, etc.).

In some embodiments, the targeted cancer vaccine is administered to inhibit tumor cell expansion. The vaccine may be administered prior to or after the detection of tumor cells in a subject. Inhibition of tumor cell expansion is understood to refer to preventing, stopping, slowing the growth, or killing of tumor cells.

In some embodiments, T cells of the human immune system are activated, enhanced, or induced after sequential administration of an effective amount of a first immune checkpoint inhibitor followed by administration of an effective amount of a second immune checkpoint inhibitor. In some embodiments, T cells of the human immune system are activated, enhanced, or induced after sequential administration of an effective amount of a first immune checkpoint inhibitor conjointly administered with an effective amount of a targeted cancer vaccine, followed by administration of an effective amount of a second immune checkpoint inhibitor. In some embodiments, T cells of the human immune system are activated, enhanced, or induced after sequential administration of an effective amount of a first immune checkpoint inhibitor, followed by an effective amount of a second immune checkpoint inhibitor conjointly administered with an effective amount of a targeted cancer vaccine. The activated T cells may be CD4⁺ and/or CD8⁺.

In some embodiments, after sequential administration of an effective amount of a first immune checkpoint inhibitor followed by administration of an effective amount of a second immune checkpoint inhibitor, with or without administration of an effective amount of a targeted cancer vaccine, a proinflammatory response is induced by subsequent encounter of immune cells with immunogenic composition. The proinflammatory immune response comprises production of proinflammatory cytokines and/or chemokines, for example, interferon gamma (IFNy) and/or interleukin 2 (IL-2). Proinflammatory cytokines and chemokines are well known in the art.

In one embodiment, a method of treating cancer in a human subject is disclosed. The method comprises the step of administering to the subject a composition comprising human a-lactalbumin, an adjuvant, and a pharmaceutically acceptable carrier, in an amount effective to induce a breast tissue specific inflammatory response in the human subject. In one embodiment, the adjuvant is GPI-0100.

In one embodiment, a method of treating cancer in a human subject is disclosed. The method comprises the step of administering to the subject a composition, the composition comprising isolated human dendritic cells that have been loaded with human a-lactalbumin, in an amount effective to induce a breast tissue specific inflammatory response in the human subject.

An effective amount of a first immune checkpoint inhibitor, second immune checkpoint inhibitor, or targeted cancer vaccine, or combination thereof refers to an amount that is sufficient to be taken up by antigen presenting cells and/or activate T cells to elicit an immune response.

According to various embodiments for treatment or prevention of cancer (e.g., breast cancer, T BC, ovarian, prostate, etc.), one or more booster injections of the vaccine are administered.

T cells recognize discrete peptides of protein antigens presented in the context of antigen presenting molecules that are typically expressed on macrophages and dendritic cells of the immune system. Peptide recognition typically occurs following phagocytic processing of the antigen by antigen-presenting cells and loading of small peptide fragments onto Major Histocompatibility Complex (MHC) class I and/or class II molecules. After CD4⁺ T cells recognize peptides presented on MHC class II molecules, they proliferate rapidly and become effector T cells that may activate other immune effector cells.

CD8⁺ T cells are believed to recognize peptides presented by MHC class I molecules, upon which they develop into cytotoxic effector cells capable of lysing and eliminating cells that express a particular protein. CD4 and CD8 molecules serve as co- receptors because their interactions with MHC molecules. They are believed to be required for an effective T cell mediated immune response. The ability of human a-lactalbumin to be an effective polypeptide antigen in a vaccine against breast cancer depends on whether human a-lactalbumin is sufficiently immunogenic in humans to generate a proinflammatory immune response. The immunogenicity of a particular protein, such as human α-lactalbumin, is highly unpredictable, and depends in part upon the particular amino acid sequence of the protein, its uptake and processing by antigen presenting cells into smaller peptide fragments, the availability of appropriate MHC binding sites for the processed peptide fragments, and the availability of appropriately responsive T cells with specific receptor sequences that can recognize and bind the peptide in the context of the MHC binding pocket.

In some embodiments, the targeted cancer vaccine is a multivalent cancer vaccine comprising: a polypeptide comprising a 15 amino acid fragment of α-lactalbumin (SEQ ID NO: 1) or a polypeptide, at least 20 amino acids in length, having 90% sequence identity with an amino acid sequence contained in SEQ ID NO: 1 and a polypeptide comprising a 15 amino acid fragment of aSl casein (SEQ ID NO: 2) or a polypeptide, at least 20 amino acids in length, having 90% sequence identity with an amino acid sequence contained in SEQ ID NO: 2.

In some embodiments, the targeted cancer vaccine is a multivalent cancer vaccine comprising: a polypeptide comprising a 15 amino acid fragment of α-lactalbumin (SEQ ID NO: 1) or a polypeptide, at least 20 amino acids in length, having 90% sequence identity with an amino acid sequence contained in SEQ ID NO: 1 and a polypeptide comprising a 15 amino acid fragment of β-casein (SEQ ID NO: 3) or a polypeptide, at least 20 amino acids in length, having 90% sequence identity with an amino acid sequence contained in SEQ ID NO: 3.

In some embodiments, the targeted cancer vaccine is a multivalent cancer vaccine comprising: a polypeptide comprising a 15 amino acid fragment of α-lactalbumin (SEQ ID NO: 1) or a polypeptide, at least 20 amino acids in length, having 90% sequence identity with an amino acid sequence contained in SEQ ID NO: 1 and a polypeptide comprising a 15 amino acid fragment of κ-casein (SEQ ID NO: 4)or a polypeptide, at least 20 amino acids in length, having 90% sequence identity with an amino acid sequence contained in SEQ ID NO: 4.

In some embodiments, the targeted cancer vaccine is a multivalent cancer vaccine comprising: a polypeptide comprising a 15 amino acid fragment of α-lactalbumin (SEQ ID NO: 1) or a polypeptide, at least 20 amino acids in length, having 90% sequence identity with an amino acid sequence contained in SEQ ID NO: 1; a polypeptide comprising a 15 amino acid fragment of aSl casein (SEQ ID NO: 2) or a polypeptide, at least 20 amino acids in length, having 90% sequence identity with an amino acid sequence contained in SEQ ID NO: 2; and a polypeptide comprising a 15 amino acid fragment of β-casein (SEQ ID NO: 3) or a polypeptide, at least 20 amino acids in length, having 90% sequence identity with an amino acid sequence contained in SEQ ID NO: 3.

In some embodiments, the targeted cancer vaccine is a multivalent cancer vaccine comprising: a polypeptide comprising a 15 amino acid fragment of a-lactalbumin (SEQ ID NO: 1) or a polypeptide, at least 20 amino acids in length, having 90% sequence identity with an amino acid sequence contained in SEQ ID NO: 1; a polypeptide comprising a 15 amino acid fragment of aSl casein (SEQ ID NO: 2) or a polypeptide, at least 20 amino acids in length, having 90% sequence identity with an amino acid sequence contained in SEQ ID NO: 2; and a polypeptide comprising a 15 amino acid fragment of κ-casein (SEQ ID NO: 4)or a polypeptide, at least 20 amino acids in length, having 90% sequence identity with an amino acid sequence contained in SEQ ID NO: 4.

In some embodiments, the targeted cancer vaccine is a multivalent cancer vaccine comprising: a polypeptide comprising a 15 amino acid fragment of a-lactalbumin (SEQ ID NO: 1) or a polypeptide, at least 20 amino acids in length, having 90% sequence identity with an amino acid sequence contained in SEQ ID NO: 1; a polypeptide comprising a 15 amino acid fragment of aSl casein (SEQ ID NO: 2) or a polypeptide, at least 20 amino acids in length, having 90% sequence identity with an amino acid sequence contained in SEQ ID NO: 2; a polypeptide comprising a 15 amino acid fragment of β-casein (SEQ ID NO: 3) or a polypeptide, at least 20 amino acids in length, having 90% sequence identity with an amino acid sequence contained in SEQ ID NO: 3; and a polypeptide comprising a 15 amino acid fragment of κ-casein (SEQ ID NO: 4) or a polypeptide, at least 20 amino acids in length, having 90% sequence identity with an amino acid sequence contained in SEQ ID NO: 4.

In some embodiments, the targeted cancer vaccine is a multivalent cancer vaccine comprising a polypeptide of SEQ ID NO: 1; and two or more polypeptides selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4.

In some embodiments, the targeted cancer vaccine is a multivalent cancer vaccine comprising one of said polypeptides covalently linked to an immune-enhancing cytokine selected from the group consisting of granulocyte-macrophage colony stimulating factor, interleukin-2 and interleukin-4.

In some embodiments, the targeted cancer vaccine is a multivalent cancer vaccine comprising: a polypeptide, at least 20 amino acids in length, having 95% sequence identity with an amino acid sequence contained in SEQ ID NO: 1 ; a polypeptide, at least 20 amino acids in length, having 95% sequence identity with an amino acid sequence contained in SEQ ID NO: 2; a polypeptide, at least 20 amino acids in length, having 95% sequence identity with an amino acid sequence contained in SEQ ID NO: 3; and a polypeptide, at least 20 amino acids in length, having 95% sequence identity with an amino acid sequence contained in SEQ ID NO: 4.

In some embodiments, the targeted cancer vaccine is a multivalent cancer vaccine comprising said polypeptides linked to one another in a linear chain via an amino acid linker.

In some embodiments, the method comprises the steps of identifying cancer subjects having tumors that fail to express estrogen receptors, progesterone receptors, and human epidermal growth factor receptor 2; selecting those identified breast cancer subjects and administering a multivalent composition comprising a polypeptide of SEQ ID NO: 1, or a 15 amino acid fragment of SEQ ID NO: 1 ; and a polypeptides selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, or a 15 amino acid fragment of SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

In certain embodiments, the targeted cancer vaccine is a prostate cancer vaccine such as PROSTVAC, a poxvirus-based vaccine against prostate-specific antigen (PSA).

In other embodiments, the targeted breast cancer vaccine is Neuvax™, autologous dendritic cells (DC) against peptides derived from HER2, peptides derived from HER2, insulin-like growth factor binding protein 2 (IGFBP2), and insulin-like growth factor 1 receptor (IGFIR) overexpressed in ductal carcinoma in situ (DCIS), glycosylated MUCl, DCs targeted to notch antagonist delta-like 1 homologue (DLK1), or vaccines targeting folate receptor alpha protein (FOLR1), MAGE-A and NY-ESO-1, mammaglobin-A

(SCGB2A2; secretoglobin, family 2A, member 2), or heat shock protein 90 kDa beta, member 1 (gp96 or HSP90B 1).

In some embodiments, the targeted cancer vaccine is a AMHR2 polypeptide (e.g., a polypeptide containing the AMHR2-ED or an immunogenic fragment thereof, or the AMHR2-CD or an immunogenic fragment thereof) or a nucleic acid encoding an AMHR2 polypeptide. In some embodiments, the AMHR2 polypeptides are polypeptides that include an amino acid sequence that corresponds to the amino acid sequence of an AMHR2 protein, the AMHR2-ED, the AMHR2-CD, and/or a portion of the AMHR2 amino acid sequence of sufficient length to elicit an AMHR2-specific immune response. In certain embodiments, the AMHR2 polypeptide also includes amino acids that do not correspond to the amino acid sequence (e.g., a fusion protein comprising an AMHR2 amino acid sequence and an amino acid sequence corresponding to a non-AMHR2 protein or polypeptide). In some

embodiments, the AMHR2 polypeptide only includes amino acid sequence corresponding to an AMHR2 protein or fragment thereof. In some embodiments, the amino acid sequence is in the cytoplasmic domain of an AMHR2 protein. In some embodiments, the amino acid sequence is in the extracellular domain of an AMHR2 protein. In some embodiments of the methods, compositions and kits provided herein, the AMHR2 polypeptide does not comprise an amino acid sequence identical to the extracellular domain of AMHR2. In some embodiments, the AMHR2 polypeptide does not comprise an amino acid sequence identical to the transmembrane domain of AMHR2. In some embodiments, the AMHR2 polypeptide does not comprise an amino acid sequence identical to the cytoplasmic domain of AMHR2.

In some embodiments of the methods provided herein, the targeted cancer vaccines comprise polypeptides comprising amino acid sequences at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NOs: 1, 2, 3, 4, or 5. In some embodiments of the methods provided herein, the targeted cancer vaccines has an amino acid sequence that consists essentially of the amino acid sequence set forth in SEQ ID NOs: 1, 2, 3, 4, or 5.

In some embodiments of the methods, compositions and kits provided herein, the AMHR2 polypeptide has an amino acid sequence that consists of 8, 9, 10, 1 1, 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, 1 10, 1 15, 120, 125, 130, 140, 150, 160, 170, 180, 190 or 200 consecutive amino acids that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 77%, 98% or 99% identical to an amino acid sequence in an AMHR2 protein.

As is well-known to those skilled in the art, polypeptides having substantial sequence similarities can cause identical or very simi lar immune reaction in a host animal . Accordingly, in some embodiments, a derivative, equivalent, variant, fragment, or mutant of the targeted cancer vaccine or fragment thereof can also suitable for the methods, compositions and kits provided herein.

In some embodiments, the altered polypeptide may have an altered amino acid sequence, for example by conservative substitution, yet still elicits immune responses which react with the unaltered protein antigen, and are considered functional equivalents. As used herein, the term "conservative substitutioi denotes the replacement of an amino acid residue by another, biologically similar residue. It is well known in the art that the amino acids within the same conservative group can typically substitute for one another without substantially affecting the function of a protein. According to certain embodiments, the derivative, equivalents, variants, or mutants of the ligand-binding domain of a targeted cancer vaccine are at least 85% homologous to a sequence set forth in SEQ ID NOs: 1, 2, 3, 4, or 5. In some embodiments, the homology is at least 90%, at least 95%, or at least 98%.

In some embodiments, provided herein is a nucleic acid encoding an AMHR2 polypeptide described herein, such as a DNA molecule encoding a targeted cancer vaccine. In some embodiments the composition comprises an expression vector comprising an open reading frame encoding a targeted cancer vaccine. In some embodiments, the targeted cancer vaccine nucleic acid includes regulatory elements necessary for expression of the open reading frame. Such elements can include, for example, a promoter, an initiation codon, a stop codon, and a polyadenylation signal. In addition, enhancers can be included. These elements can be operably linked to a sequence that encodes the targeted cancer vaccine polypeptide.

Examples of promoters include but are not limited to promoters from Simian Virus 40 (SV40), 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) as well as promoters from human genes such as human actin, human myosin, human hemoglobin, human muscle creatine, and human

metalothionein. Examples of suitable polyadenylation signals include but are not limited to SV40 polyadenylation signals and LTR polyadenylation signals.

In addition to the regulatory elements required for expression, other elements may also be included in the nucleic acid molecule. Such additional elements include enhancers. Enhancers include the promoters described hereinabove. Preferred enhancers/promoters include, for example, human actin, human myosin, human hemoglobin, human muscle creatine and viral enhancers such as those from CMV, RSV and EB V.

In some embodiments, the nucleic acid can be operably incorporated in a carrier or delivery vector. Useful delivery vectors include but are not limited to biodegradable microcapsules, immuno-stimulating complexes (ISCOMs) or liposomes, and genetically engineered attenuated live carriers such as viruses or bacteria.

In some embodiments, the vector is a viral vector, such as lentiviruses, retroviruses, herpes viruses, adenoviruses, adeno-associated viruses, vaccinia viruses, baculoviruses, Fowl pox, AV-pox, modified vaccinia Ankara (MVA) and other recombinant viruses. For example, a vaccinia virus vector can be used to infect dendritic cells (DC).

In some embodiments, the identification of immunogenic breast tumor-specific neoantigens that may be highly specific for each tumor can lead to the development of personalized immunotherapies involving tumor-specific customized vaccines. For example, the identification of a group of neoantigens common to many breast tumors that could form the basis for developing a multivalent vaccine for treatment and perhaps prophylaxis against defined subtypes of breast cancer. Customized immunotherapies may include active and DC personalized vaccines targeted against individual breast tumor neoantigens or overexpressed self-proteins as well as passively transferred tumor-specific immunity in the form of genetically modified cloned T cells.

Pharmaceutical Compositions

in certain aspects, provided herein are pharmaceutical compositions comprising a first immune checkpoint inhibitor, a second immune checkpoint inhibitor, and/or targeted cancer vaccine. In some embodiments, the composition comprises a pharmaceutically acceptable carrier. In some embodiments, the composition includes a combination of multiple ( e.g., two or more) a first immune checkpoint inhibitor, a second immune checkpoint inhibitor, and/or targeted cancer vaccine.

The pharmaceutical compositions disclosed herein may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; or (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation.

Methods of preparing these formulations or compositions include the step of bringing into association any of the first immune checkpoint inhibitor, second immune checkpoint inhibitor, and/or targeted cancer vaccine described herein with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association an agent described herein with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

Pharmaceutical compositions suitable for parenteral administration comprise any of the first immune checkpoint inhibitor, second immune checkpoint inhibitor, and/or targeted cancer vaccine described herein in combination with one or more pharmaceutically- acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain sugars, alcohols, antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

Regardless of the route of administration selected, the agents provided herein, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions disclosed herein, are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art.

In some embodiments, the pharmaceutical composition described, when

administered to a subject, can elicit an immune response against a cell that expresses human a-lactalbumin (e.g., at least a portion of SEQ ID NO: 1), aSl casein (e.g., at least a portion of SEQ ID NO: 2), β-casein (e.g., at least a portion of SEQ ID NO: 3), κ-casein (e.g., at least a portion of SEQ ID NO: 4), or anti-Mullerian Hormone Receptor, Type II (AMHR2) (e.g., at least a portion of SEQ ID NO: 5). Such pharmaceutical compositions can be useful as vaccine compositions for prophylactic and/or therapeutic treatment of cancer (e.g., T BC, breast, ovarian, prostate, etc.).

In some embodiments, the pharmaceutical composition further comprises a physiologically acceptable adjuvant. In some embodiments, the adjuvant employed provides for increased immunogenicity of the pharmaceutical composition. The adjuvant can be one that provides for slow release of antigen (e.g., the adjuvant can be a liposome), or it can be an adjuvant that is immunogenic in its own right thereby functioning synergistically with antigens. For example, the adjuvant can be a known adjuvant or other substance that promotes antigen uptake, recruits immune system cells to the site of administration, or facilitates the immune activation of responding lymphoid cells.

Adjuvants include, but are not limited to, immunomodulatory molecules (e.g., cytokines), oil and water emulsions, aluminum hydroxide, glucan, dextran sulfate, iron oxide, sodium alginate, Bacto- Adjuvant, synthetic polymers such as poly amino acids and co-polymers of amino acids, saponin, paraffin oil, and muramyl dipeptide. In some embodiments, the adjuvant is Adjuvant 65, a-GalCer, aluminum phosphate, aluminum hydroxide, calcium phosphate, β-Glucan Peptide, CpG DNA, GM-CSF, GPI-OlOO, IF A, IFN-γ, IL-17, lipid A, lipopolysaccharide, Lipovant, Montanide, N-acetyl-muramyl-L-alanyl-D-isoglutamine, Pam3CSK4, quil A, trehalose dimycolate or zymosan. In some embodiments, adjuvant is one that induces a mixed type 1/type 17 immune response.

In some embodiments, the adjuvant is an immunomodulatory molecule. For example, the immunomodulatory molecule can be a recombinant protein cytokine, chemokine, or immunostimulatory agent or nucleic acid encoding cytokines, chemokines, or immunostimulatory agents designed to enhance the immunologic response.

Examples of immunomodulatory cytokines include interferons (e.g., IFNa, IFNPand IFNy), interleukins (e.g., 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 (e.g., TNFa and TNFP), erythropoietin (EPO), FLT-3 ligand, glplO, TCA-3, MCP-1, MIF, MIP-1. alpha., ΜΙΡ-Ιβ, 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.

In some embodiments, an immunomodulatory chemokine that binds to a chemokine receptor, i.e., a CXC, CC, C, or CX3C chemokine receptor, also can be included in the compositions provided here. Examples of chemokines include, but are not limited to, Mipla, Mip-ΐβ, Mip-3a (Larc), Μίρ-3β, Rantes, Hcc-1, Mpif-1, Mpif-2, Mcp-1, Mcp-2, Mcp-3, Mcp-4, Mcp-5, Eotaxin, Tare, Elc, 1309, IL-8, Gcp-2 Gro-a, 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.

In certain embodiments, compositions provided herein also comprise one or more other agents such as, but not limited to, chemotherapeutic, immunotherapeutic,

immunomodulatory and/or anti-angiogenic agents.

In some embodiments, the one or more other agents can be a chemotherapeutic agent, naturally occurring or synthetic, for example as described in "Cancer

Chemotherapeutic Agents", American Chemical Society, 1995, W. O. Foye Ed.

In one embodiment, the chemotherapeutic agent is selected from the group consisting of a small molecule receptor antagonists such as vatalanib, SU 11248 or AZD- 6474, EGFR or HER2 antagonists such as gefitinib, erlotinib, CI-1033 or Herceptin, antibodies such as bevacizumab, cetuximab, rituximab, DNA alkylating drugs such as cisplatin, oxaliplatin or carboplatin, anthracyclines such as doxorubicin or epirubicin, an antimetabolite such as 5-FU, pemetrexed, gemcitabine or capecitabine, a camptothecin such as irinotecan or topotecan, an anti-cancer drug such as paclitaxel or docetaxel, an epipodophyllotoxin such as etoposide or teniposide, a proteasome inhibitor such as bortezomib or anti-inflammatory drugs such as celecoxib or rofecoxib, optionally in form of the pharmaceutically acceptable salts, in form of the hydrates and/or solvates and optionally in the form of the individual optical isomers, mixtures of the individual enantiomers or racemates thereof.

In another 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-1033 or GW-2016, an EGFR antagonist such as iressa (ZD- 1839), tarceva (OSI-774), PKI-166, EKB-569, HKI-272 or herceptin, an antagonist of the mitogen-activated protein kinase such as BAY-43-9006 or BAY-57-9006, a quinazoline derivative such as 4-[(3-chloro-4-fluorophenypamino]-6-{[4-(N,N-dimethylamino)-l-oxo- 2-bute-- n-l-yl]amino}-7-((S)-tetrahydrofuran-3-yloxy)quinazoline or 4-[(3-chloro-4- fluoro-phenyl)amino]-6-{[4-(homomorpholin-4-yl)-l-oxo-2-bu- -ten-l-yl]amino}-7-[(S)- (tetrahydrofuran-3-yl)oxy] -quinazoline, or a pharmaceutically acceptable salt thereof, a protein kinase receptor antagonist which is not classified under the synthetic small molecules such as atrasentan, rituximab, cetuximab, Avastin.TM. (bevacizumab), EVIC- 1C11, erbitux (C-225), DC-101, EMD-72000, vitaxin, imatinib, a protein tyrosine kinase inhibitor which is a fusion protein such as VEGFtrap, an alkylating agent or a platinum compound such as melphalan, cyclophosphamide, an oxazaphosphorine, cisplatin, carboplatin, oxaliplatin, satraplatin, tetraplatin, iproplatin, mitomycin, streptozocin, carmustine (BCNU), lomustine (CCNU), busulfan, ifosfamide, streptozocin, thiotepa, chlorambucil, a nitrogen mustard such as mechlorethamine, an ethyleneimine compound, an alkylsulphonate, daunorubicin, doxorubicin (adriamycin), liposomal doxorubicin (doxil), epirubicin, idarubicin, mitoxantrone, amsacrine, dactinomycin, distamycin or a derivative thereof, netropsin, pibenzimol, mitomycin, CC-1065, a duocarmycin, mithramycin, chromomycin, olivomycin, a phtalanilide such as propamidine or stilbamidine, an anthramycin, an aziridine, a nitrosourea or a derivative thereof, a pyrimidine or purine analogue or antagonist or an inhibitor of the nucleoside diphosphate reductase such as cytarabine, 5-fluorouracile (5-FU), pemetrexed, tegafur/uracil, uracil mustard, fludarabine, gemcitabine, capecitabine, mercaptopurine, cladribine, thioguanine, methotrexate, pentostatin, hydroxyurea, or folic acid, a phleomycin, a bleomycin or a derivative or salt thereof, CHPP, BZPP, MTPP, BAPP, liblomycin, an acridine or a derivative thereof, a rifamycin, an actinomycin, adramycin, a camptothecin such as irinotecan (camptosar) or topotecan, an amsacrine or analogue thereof, a tricyclic carboxamide, an histonedeacetylase inhibitor such as SAHA, MD-275, trichostatin A, CBHA, LAQ824, or valproic acid, an anti-cancer drug from plants such as paclitaxel (taxol), docetaxel or taxotere, a vinca alkaloid such as navelbine, vinblastin, vincristin, vindesine or vinorelbine, a tropolone alkaloid such as colchicine or a derivative thereof, a macrolide such as maytansine, an ansamitocin or rhizoxin, an antimitotic peptide such as phomopsin or dolastatin, an epipodophyllotoxin or a derivative of podophyllotoxin such as etoposide or teniposide, a steganacin, an antimitotic carbamate derivative such as combretastatin or amphetinile, procarbazine, a proteasome inhibitor such as bortezomib, an enzyme such as asparaginase, pegylated asparaginase (pegaspargase) or a thymidine-phosphorylase inhibitor, a gestagen or an estrogen such as estramustine (T-66) or megestrol, an anti-androgen such as flutamide, casodex, anandron or cyproterone acetate, an aromatase inhibitor such as aminogluthetimide, anastrozole, formestan or letrozole, a GNrH analogue such as leuprorelin, buserelin, goserelin or triptorelin, an anti-estrogen such as tamoxifen or its citrate salt, droloxifene, trioxifene, raloxifene or zindoxifene, a derivative of 17.beta.- estradiol such as ICI 164,384 or ICI 182,780, aminoglutethimide, formestane, fadrozole, finasteride, ketoconazole, a LH-RH antagonist such as leuprolide, a steroid such as prednisone, prednisolone, methylprednisolone, dexamethasone, budenoside, fluocortolone or triamcinolone, an interferon such as interferon .beta., an interleukin such as IL-10 or IL- 12, an anti-T F. alpha, antibody such as etanercept, an immunomodulatory drug such as thalidomide, its R- and S-enantiomers and its derivatives, or revimid (CC-5013), a leukotrien antagonist, mitomycin C, an aziridoquinone such as BMY-42355, AZQ or EO-9, a 2-nitroimidazole such as misonidazole, LP-1 or LA-1, a nitroacridine, a

nitroquinoline, a nitropyrazoloacridine, a "dual-function" nitro aromatic such as RSU-1069 or RB-6145, CB-1954, a N-oxide of nitrogen mustard such as nitromin, a metal complex of a nitrogen mustard, an anti-CD3 or anti-CD25 antibody, a tolerance induction agent, a biphosphonate or derivative thereof such as minodronic acid or its derivatives (YM-529, Ono-5920, YH-529), zoledronic acid monohydrate, ibandronate sodium hydrate or clodronate disodium, a nitroimidazole such as metronidazole, misonidazole, benznidazole or nimorazole, a nitroaryl compound such as RSU-1069, a nitroxyl or N-oxide such as SR- 4233, an halogenated pyrimidine analogue such as bromodeoxyuridine, iododeoxyuridine, a thiophosphate such as WR-272 1, a photo-chemically activated drug such as porfimer, photofrin, a benzoporphyrin derivative, a pheophorbide derivative, merocyanin 540 (MC- 540) or tin etioporpurin, an ant-template or an anti-sense RNA or DNA such as oblimersen, a non-steroidal inflammatory drug such as acetylsalicyclic acid, mesalazin, ibuprofen, naproxen, flurbiprofen, fenoprofen, fenbufen, ketoprofen, indoprofen, pirprofen, carprofen, oxaprozin, pranoprofen, miroprofen, tioxaprofen, 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, nifluminic acid, tolfenamic acid, diflunisal, flufenisal, piroxicam, tenoxicam, lomoxicam, nimesulide, meloxicam, celecoxib, rofecoxib, or a pharmaceutically acceptable salt of a non-steroidal inflammatory drug, a cytotoxic antibiotic, an antibody targeting the surface molecules of cancer cells such as apolizumab or 1D09C3, an inhibitor of metalloproteinases such as

TEVIP-l or TIMP-2, Zinc, an inhibitor of oncogenes such as P53 and Rb, a complex of rare earth elements such as the heterocyclic complexes of lanthanides, a photo- chemotherapeutic 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 modulator geldanamycin and its derivative 17-allylaminogeldanamycin or 17-AAG, or a therapeutic agent selected from IM-842, tetrathiomolybdate, squalamine, combrestatin A4, T P-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 antagonist 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, an EGFR antagonist such as iressa (gefitinib, ZD-1839), tarceva (erlotinib, OSI-774), PKI-166, EKB-569, HKI-272 or herceptin, an antagonist of the mitogen-activated protein kinase such as BAY-43-9006 or BAY-57-9006, atrasentan, rituximab, cetuximab, Avastin.TM. (bevacizumab), IMC-1C11, erbitux (C-225), DC-101, EMD-72000, vitaxin, imatinib, an alkylating agent or a platinum compound such as melphalan, cyclophosphamide, cisplatin, carboplatin, oxaliplatin, satraplatin, daunorubicin, doxorubicin (adriamycin), liposomal doxorubicin (doxil), epirubicin, idarubicin, a pyrimidine or purine analogue or antagonist or an inhibitor of the nucleoside diphosphate reductase such as cytarabine, 5-fluorouracile (5-FU), pemetrexed, tegafur/uracil, gemcitabine, capecitabine, mercaptopurine, methotrexate, an anti-cancer drug such as paclitaxel (taxol) or docetaxel, a vinca alkaloid such as navelbine, vinblastin, vincristin, vindesine or vinorelbine, an antimitotic peptide such as dolastatin, an

epipodophyllotoxin or a derivative of podophyllotoxin such as etoposide or teniposide, a non-steroidal inflammatory drug such as meloxicam, celecoxib, rofecoxib, an antibody targeting the surface molecules of cancer cells such as apolizumab or ID09C3 or the heat shock protein HSP90 modulator geldanamycin and its derivative 17- allylaminogeldanamycin or 17-AAG.

In another embodiment, the chemotherapeutic agent is selected from the group consisting of compounds interacting with or binding tubulin, synthetic small molecule VEGF receptor antagonists, small molecule growth factor receptor antagonists, inhibitors of the EGF receptor and/or VEGF receptor and/or integrin receptors or any other protein tyrosine kinase receptors which are not classified under the synthetic small-molecules, inhibitors directed to EGF receptor and/or VEGF receptor and/or integrin receptors or any other protein tyrosine kinase receptors, which are fusion proteins, compounds which interact with nucleic acids, and which are classified as alkylating agents or platinum compounds, compounds which interact with nucleic acids and which are classified as anthracyclines, as DNA intercalators or as DNA cross-linking agents, including DNA minor-groove binding compounds, anti-metabolites, naturally occurring, semi-synthetic or synthetic bleomycin type antibiotics, inhibitors of DNA transcribing enzymes, and especially the topoisomerase I or topoisomerase II inhibitors, chromatin modifying agents, mitosis inhibitors, anti-mitotic 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 directed against cytokines, oral and parenteral tolerance induction agents, supportive agents, chemical radiation sensitizers and protectors, photo-chemically activated drugs, synthetic poly- or oligonucleotides, optionally modified or conjugated, non-steroidal anti-inflammatory drugs, cytotoxic antibiotics, antibodies targeting the surface molecules of cancer cells, antibodies targeting growth factors or their receptors, inhibitors of

metalloproteinases, metals, inhibitors of oncogenes, inhibitors of gene transcription or of RNA translation or protein expression, complexes of rare earth elements, and photo- chemotherapeutic agents.

In other embodiments, the chemotherapeutic agent is selected from the group consisting of sunitinib, sorafenib, paclitaxel (taxol), docetaxel, a vinca alkaloid such as navelbine, vinblastin, vincristin, vindesine or vinorelbine, an alkylating agent or a platinum compound such as melphalan, cyclophosphamide, an oxazaphosphorine, cisplatin, carboplatin, oxaliplatin, satraplatin, tetraplatin, iproplatin, mitomycin, streptozocin, carmustine (BCNU), lomustine (CCNU), busulfan, ifosfamide, streptozocin, thiotepa, chlorambucil, a nitrogen mustard such as mechlorethamine, an immunomodulatory drug such as thalidomide, its R- and S-enantiomers and its derivatives, or revimid (CC-5013)), an ethyleneimine compound, an alkylsulphonate, daunorubicin, doxorubicin (adriamycin), liposomal doxorubicin (doxil), epirubicin, idarubicin, mitoxantrone, amsacrine,

dactinomycin, distamycin or a derivative thereof, netropsin, pibenzimol, mitomycin, CC- 1065, a duocarmycin, mithramycin, chromomycin, olivomycin, a phtalanilide such as propamidine or stilbamidine, an anthramycin, an aziridine, a nitrosourea or a derivative thereof, a pyrimidine or purine analogue or antagonist or an inhibitor of the nucleoside diphosphate reductase such as cytarabine, 5-fluorouracile (5-FU), uracil mustard, fludarabine, gemcitabine, capecitabine, mercaptopurine, cladribine, thioguanine, methotrexate, pentostatin, hydroxyurea, or folic acid, an acridine or a derivative thereof, a rifamycin, an actinomycin, adramycin, a camptothecin such as irinotecan (camptosar) or topotecan, an amsacrine or analogue thereof, a tricyclic carboxamide, an histonedeacetylase inhibitor such as SAHA, MD-275, trichostatin A, CBHA, LAQ824, or valproic acid, a proteasome inhibitor such as bortezomib, a small molecule VEGF receptor antagonist such as vatalanib (PTK-787/ZK222584), SU-5416, SU-6668, SU-1 1248, SU-14813, AZD-6474, AZD-2171, CP-547632, CEP-7055, AG-013736, IM-842 or GW-786034, an antagonist of the mitogen-activated protein kinase such as BAY-43-9006 or BAY-57-9006, a dual EGFR/HER2 antagonist such as gefitinib, erlotinib, CI-1033 or GW-2016, an EGFR antagonist such as iressa (ZD-1839), tarceva (OSI-774), PKI-166, EKB-569, HKI-272 or herceptin, a quinazoline derivative such as 4-[(3-chloro-4-fluorophenyl)amino]-6-{ [-4- (Ν,Ν-dimethylamino)- 1 -oxo-2-but- -en- 1 -yl]amino}-7-((S)-tetrahydrofuran-3-yloxy)- quinazoline or 4-[(3-chloro-4-fluoro-phenyl)amino]-6-{ [4-(homomorpholin-4-yl)-l-oxo-2- bu- -ten-l-yl]amino}-7-[(S)-(tetrahydrofuran-3-yl)oxy]-quinazoline, or a pharmaceutically acceptable salt thereof, an inhibitor of the transcription factor complex ESX/DRIP130/Sur- 2, an inhibitor of HER-2 expression, such as the heat shock protein HSP90 modulator geldanamycin and its derivative 17-allylaminogeldanamycin or 17-AAG, a protein kinase receptor antagonist which is not classified under the synthetic small molecules such as atrasentan, rituximab, cetuximab, Avastin.TM. (bevacizumab), IMC-1C1 1, erbitux (C-225), DC-101, EMD-72000, vitaxin, imatinib, and an antibody targeting the surface molecules of cancer cells such as apolizumab or 1D09C3.

In some embodiments, the composition comprises a nucleic acid encoding an AMHR2 polypeptide described herein, such as a DNA molecule encoding an AMHR2 polypeptide. In some embodiments the composition comprises an expression vector comprising an open reading frame encoding an AMHR2 polypeptide.

When taken up by a cell (e.g., muscle cell, an antigen-presenting cell (APC) such as a dendritic cell, macrophage, etc.), a DNA molecule can be present in the cell as an extrachromosomal molecule and/or can integrate into the chromosome. DNA can be introduced into cells in the form of a plasmid which can remain as separate genetic material. Alternatively, linear DNAs that can integrate into the chromosome can be introduced into the cell. Optionally, when introducing DNA into a cell, reagents which promote DNA integration into chromosomes can be added. Therapeutic Methods

Immune checkpoint inhibitors that target the CTLA-4 immune inhibitory pathway (e.g. , ipilimumab) may impact the priming phase of T cell activation whereas those acting on the PD-1 inhibitory pathway (e.g., pembrolizumab, nivolumab, pidlizumab, MK-3475) or PD-Ll inhibitory pathway (e.g., BMS-936559, MPDL3280A) may impact the activity of T cells that are already primed. Treatment involving both forms of checkpoint inhibitors may provide both enhanced immune priming against the tumor, as well as, enhancement of any established tumor immunity already in place. The synergy that may occur when both pathways are inhibited simultaneously may allow for effective treatment regimens involving lower doses, shorter time courses, and diminished toxicities. Combination therapies may also involve co-treatment with a targeted cancer vaccine plus a first immune checkpoint inhibitors that targets the CTLA-4 pathway during the priming phase of vaccination. Combination therapies may also combine targeted cancer vaccination in combination with first immune checkpoint inhibitors that target the CTLA-4 pathway during the priming phase, followed by treatment with a second immune checkpoint inhibitor that targets the PD-1, PD-Ll, or PD-L2 pathway during the post-priming effector stage of the immune response. Such sequentially orchestrated treatment regimens may provide an enhanced response to the vaccine in the first phase of combination therapy with one first immune checkpoint inhibitor followed by the induction of an enhanced response to the tumor with another second immune checkpoint inhibitor during a subsequent second phase of treatment. This aggressive combination therapy may be particularly useful and effective against tumors like T BC that appear to be only modestly immunogenic and are known to be aggressive and notoriously resistant to currently available treatments.

In certain aspects, provided herein is a method of inducing or enhancing an immune response in a subject ( e.g., a human subject) in need thereof, the method comprising the steps of (a) administering an effective amount of a first immune checkpoint inhibitor ( e.g., an inhibitor of CTLA4) to a subject to prime the immune response in the subject; and (b) administering an effective amount of a second immune checkpoint inhibitor ( e.g., an inhibitor of PD-1) to the subject to enhance the primed immune response in the subject. In some embodiments, the subject has triple negative breast cancer (TNBC) subject, breast cancer, ovarian cancer, or prostate cancer. In some embodiments, the method further comprise administering a targeted cancer vaccine prior to, subsequently, or conjointly with the first or second immune checkpoint inhibitor. In some embodiments, the targeted cancer vaccine is an immunogenic composition comprising an immunogenic polypeptide selected from the group consisting of human a-lactalbumin (e.g., at least a portion of SEQ ID NO: 1), aS l casein (e.g., at least a portion of SEQ ID NO: 2), β-casein (e.g., at least a portion of SEQ ID NO: 3), κ-casein (e.g., at least a portion of SEQ ID NO: 4), and anti-Mullerian Hormone Receptor, Type II (AMHR2) (e.g., at least a portion of SEQ ID NO: 5). In some embodiments, administration of the first immune checkpoint inhibitor, second immune checkpoint inhibitor, or targeted cancer vaccine induces an immune response against the ovarian cancer tumor in the subject (e.g., a T cell immune response, such as a type 1 and/or type 17 immune response and/or a B cell immune response, such as an IgG response). In some embodiments, the subject is administered multiple doses of the first immune checkpoint, second immune checkpoint, and/or targeted cancer vaccine (e.g., at least 2, 3, 4, 5 or 6 doses). Said doses may be given at periodic intervals occurring daily, weekly, biweekly, monthly. In some embodiments, the methods further comprise using the first immune checkpoint inhibitor or second immune checkpoint inhibitor in combination with a chemotherapeutic agent (e.g., sunitinib or sorafenib) or a corticosteroid treatment.

In certain aspects, provided herein is a method of preventing cancer in a subject comprising the steps of ( e.g., a human subject) in need thereof, the method comprising the steps of (a) administering an effective amount of a first immune checkpoint inhibitor ( e.g., an inhibitor of CTLA4) to a subject to prime the immune response in the subject; and (b) administering an effective amount of a second immune checkpoint inhibitor ( e.g., an inhibitor of PD-1) to the subject to enhance the primed immune response in the subject. In some embodiments, the subject has triple negative breast cancer (TNBC) subject, breast cancer, ovarian cancer, or prostate cancer. In some embodiments, the method further comprise administering a targeted cancer vaccine prior to, subsequently, or conjointly with the first or second immune checkpoint inhibitor. In some embodiments, the targeted cancer vaccine is an immunogenic composition comprising an immunogenic polypeptide selected from the group consisting of human a-lactalbumin (e.g., at least a portion of SEQ ID NO: 1), aS l casein (e.g., at least a portion of SEQ ID NO: 2), β-casein (e.g., at least a portion of SEQ ID NO: 3), κ-casein (e.g., at least a portion of SEQ ID NO: 4), and anti-Mullerian Hormone Receptor, Type II (AMHR2) (e.g., at least a portion of SEQ ID NO: 5). In some embodiments, administration of the first immune checkpoint inhibitor, second immune checkpoint inhibitor, or targeted cancer vaccine induces an immune response against the ovarian cancer tumor in the subject (e.g., a T cell immune response, such as a type 1 and/or type 17 immune response and/or a B cell immune response, such as an IgG response). In some embodiments, the subject is administered multiple doses of the first immune checkpoint, second immune checkpoint, and/or targeted cancer vaccine (e.g., at least 2, 3, 4, 5 or 6 doses). Said doses may be given at periodic intervals occurring daily, weekly, biweekly, monthly. In some embodiments, the methods further comprise using the first immune checkpoint inhibitor or second immune checkpoint inhibitor in combination with a chemotherapeutic agent (e.g., sunitinib or sorafenib) or a corticosteroid treatment.

In certain embodiments, the prevention and treatment methods provided herein may be combined with corticosteroid treatment to counteract any side effects from immune checkpoint inhibitor therapy. Known common side effects include a number of

autoimmune-related inflammatory events like hypophysitis, pancreatitis, hepatitis, dermatitis, colitis, uveitis, pulmonary abnormalities, renal insufficiency, and a variety of hematologic and neurologic abnormalities. Thyroid dysfunction including Hashimoto's thyroiditis and Graves' disease are also common side effects. Any array of autoimmune inflammatory complications that occur following treatment with checkpoint inhibitors may be controlled effectively by prompt and aggressive corticosteroid treatment along with hormone supplement for treatment of endocrinopathies.

The methods described herein can be used to treat any subject in need thereof. As used herein, a "subject in need thereof includes any subject who has ovarian, breast, or other cancer, who has had ovarian or breast cancer and/or who is predisposed to ovarian or breast cancer. For example, in some embodiments, the subject has an ovarian cancer tumor {e.g., an ovarian cancer tumor expressing AMHR2). In some embodiments, the subject has undergone surgery to remove at least part of an ovarian or breast cancer tumor. In some embodiments, the subject is predisposed to ovarian or breast cancer due to having a BRCA1 or BRCA2 mutation in her genome that predisposes the subject to ovarian cancer. In some embodiments, the subject has a family history of ovarian or breast cancer.

The pharmaceutical compositions disclosed herein may be delivered by any suitable route of administration, including orally and parenterally. In certain embodiments the pharmaceutical compositions are delivered generally {e.g., via oral or parenteral

administration).

The dosage of the subject agent may be determined by reference to the plasma concentrations of the agent. For example, the maximum plasma concentration (Cmax) and the area under the plasma concentration-time curve from time 0 to infinity (AUC (0-4)) may be used. Dosages include those that produce the above values for Cmax and AUC (0- 4) and other dosages resulting in larger or smaller values for those parameters.

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

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

A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could prescribe and/or administer doses of the agents employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

In general, a suitable daily dose of an agent described herein will be that amount of the agent which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.

In one aspect, provided herein is a method of eliciting in a subject an immune response to a cell that expresses the immunogenic compositions of the targeted cancer vaccine. The method comprises: administering to the subject a pharmaceutical composition described herein, wherein the pharmaceutically acceptable composition, when administered to the subject, elicits an immune response to the cell that expresses the immunogenic compositions of the targeted cancer vaccine.

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

A humoral response can be determined by a standard immunoassay for antibody levels in a serum sample from the subject receiving the pharmaceutical composition. A cellular immune response is a response that involves T cells and can be determined in vitro or in vivo. For example, a general cellular immune response can be determined as the T cell proliferative activity in cells (e.g., peripheral blood leukocytes (PBLs)) sampled from the subject at a suitable time following the administering of a pharmaceutically acceptable composition. Following incubation of e.g., PBMCs with a stimulator for an appropriate period, [³H]thymidine incorporation can be determined. The subset of T cells that is proliferating can be determined using flow cytometry.

In certain aspects, the methods provided herein include administering to both human and non-human mammals. Veterinary applications also are contemplated. In some embodiments, the subject can be any living female organism in which an immune response can be elicited. Examples of subjects include, without limitation, humans, livestock, dogs, cats, mice, rats, and transgenic species thereof.

In certain embodiments, the subject has a history of ovarian cancer and has been administered another mode of therapy. The other therapy may have included e.g., surgical resection, radiotherapy, chemotherapy, and other modes of immunotherapy whereby as a result of the other therapy, the subject presents no clinically measurable tumor. However, the subject can be one determined to be at risk for recurrence or progression of the cancer, either near the original tumor site, or by metastases. Such subjects can be further categorized as high-risk and low-risk subjects. The subdivision can be made on the basis of features observed before or after the initial treatment. These features are known in the clinical arts, and are suitably defined for each different cancer. Features typical of high risk subgroups are those in which the tumor has invaded neighboring tissues, or which show involvement of lymph nodes. Thus, for example, a pharmaceutical composition described herein can be administered to the subject to elicit an anti-cancer response primarily as a prophylactic measure against recurrence.

In some embodiments, the pharmaceutical composition can be administered at any time that is appropriate. For example, the administering can be conducted before or during traditional therapy of a subject having an ovarian cancer tumor, and continued after the tumor becomes clinically undetectable. The administering also can be continued in a subject showing signs of recurrence.

In some embodiments, the pharmaceutical composition can be administered in a therapeutically or a prophylactically effective amount. Administering the pharmaceutical composition to the subject can be carried out using known procedures, and at dosages and for periods of time sufficient to achieve a desired effect. In some embodiments, the pharmaceutical composition can be administered to the subject at any suitable site, for example a site that is distal to or proximal to a primary tumor. The route of administering can be parenteral, intramuscular, subcutaneous, intradermal, intraperitoneal, intranasal, intravenous (including via an indwelling catheter), via an afferent lymph vessel, or by any other route suitable in view of the neoplastic disease being treated and the subject's condition. Preferably, the dose will be administered in an amount and for a period of time effective in bringing about a desired response, be it eliciting the immune response or the prophylactic or therapeutic treatment of the neoplastic disease and/or symptoms associated therewith.

The pharmaceutically acceptable composition can be given subsequent to, preceding, or contemporaneously with other therapies including therapies that also elicit an immune response in the subject. For example, the subject may previously or concurrently be treated by chemotherapy, radiation therapy, and other forms of immunotherapy, such other therapies preferably provided in such a way so as not to interfere with the

immunogenicity of the compositions described herein.

Administering can be properly timed by the care giver (e.g., physician,

veterinarian), and can depend on the clinical condition of the subject, the objectives of administering, and/or other therapies also being contemplated or administered. In some embodiments, an initial dose can be administered, and the subject monitored for an immunological and/or clinical response. Suitable means of immunological monitoring include using subject's peripheral blood lymphocyte (PBL) as responders and neoplastic cells as stimulators. An immunological reaction also can be determined by a delayed inflammatory response at the site of administering. One or more doses subsequent to the initial dose can be given as appropriate, typically on a monthly, semimonthly, or preferably a weekly basis, until the desired effect is achieved. Thereafter, additional booster or maintenance doses can be given as required, particularly when the immunological or clinical benefit appears to subside.

EXAMPLE

As described herein, checkpoint inhibitors that target the CTLA-4 immune inhibitory pathway (e.g., ipilimumab) appear to have their predominant impact on the priming phase of T cell activation whereas those acting on the PD-1 inhibitory pathway (e.g., pembrolizumab, nivolumab, pidlizumab, MK-3475) or PD-Ll inhibitory pathway (e.g., BMS-936559, MPDL3280A) have their predominant impact on the activity of T cells that are already primed and in the effector phase. We therefore tested whether sequential treatment involving both forms of checkpoint inhibitors provide enhanced immune priming against targeted tumor antigens while avoiding the severe toxicities particularly associated with extended use of CTLA-4 targeted therapies using a vaccine effective in prevention and treatment of murine epithelial ovarian carcinoma (EOC). Eight week old C57BL/6 female mice were vaccinated with a single subcutaneous injection of 100 μg of recombinant mouse AMHR2-ED in complete Freund's adjuvant. Each mouse was subsequently injected intraperitoneally with 100 μg of checkpoint inhibitor antibody (BioXCell, West Lebanon, H) during the early priming phase on days 0, 5, 10, and 15 and/or during the late effector phase on days 18, 23, 28, and 33. Mice were euthanized 3 days after the last treatment, and splenocyte frequencies of IFNy-producing T cells were determined by ELISPOT analysis using recall responses to 50 μg/ml of AMHR2-ED minus recall responses to 50 μg/ml of ovalbumin. Control mice were injected with PBS during early and late phases. Vaccination of C57BL/6 mice with the extracellular domain of anti-Mullerian hormone receptor II

(AMHR2-ED) provided significant inhibition of murine EOC tumor growth and significant enhancement of overall survival. As seen in Figure 1, mice were treated with anti-CTLA-4 but not anti-PD-Ll during the priming phase immediately following AMHR2-ED vaccination exhibited enhanced frequencies of type-1 interferon-gamma (IFNy)-producing T cells. This the frequencies of type-1 interferon-gamma (IFNy)-producing T cells was further enhanced in mice treated with anti-CTLA-4 only during the priming phase were subsequently treated with anti-PD-Ll during the effector phase (Figure 1). There was no detectable increase in frequencies of IFNy-producing T cells when vaccinated mice were treated with anti-PD-Ll during either the priming phase or the effector phase (Figure 1). These results indicate that combination treatment of ant-CTLA-4 during the priming phase of tumor immune development followed by anti-PD-Ll treatment during the effector phase of tumor immune development provides enhanced tumor immunity while avoiding the substantial toxicities associated with extended use of anti-CTLA-4 therapy.

Incorporation by Reference

All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

Equivalents

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.

Claims

What is claimed is:

1. A method of inducing or enhancing an immune response in a subject in need thereof, the method comprising the steps of:

(a) administering an effective amount of a first immune checkpoint inhibitor to a subject to prime the immune response in the subject; and

(b) administering an effective amount of a second immune checkpoint inhibitor to the subject to enhance the primed immune response in the subject.

2. The method of claim 1, wherein the subject has triple negative breast cancer (TNBC) subject, breast cancer, ovarian cancer, or prostate cancer.

3. The method of any one of claims 1 to 2, wherein said method further comprises coadministering a targeted cancer vaccine.

4. The method of claim 3, wherein the targeted cancer vaccine is administered prior to, subsequently, or conjointly with the first or second immune checkpoint inhibitor.

5. The method of claim 4, wherein the targeted cancer vaccine is conjointly

administered with the first immune checkpoint inhibitor.

6. The method of claim 5, wherein the targeted cancer vaccine is selected from the group consisting of a breast cancer vaccine, prostate cancer vaccine, ovarian cancer vaccine, and a multivalent breast cancer vaccine.

7. The method of any one of claims 1 to 6, wherein the first immune checkpoint inhibitor is an inhibitor of CTLA4.

8. The method of claim 7, wherein the inhibitor of CTLA4 is selected from the group consisting of ipilimumab, tremelimumab, and KAHR-102.

9. The method of any one of claims 1 to 6, wherein the second immune checkpoint inhibitor is an inhibitor of PD-1.

10. The method of claim 9, wherein the inhibitor of PD-1 is an anti-PD-1 antibody.

11. The method of claim 10, wherein the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab/lambrolizumab, pidilizumab, AMP -224, AMP- 514, STI-A1110, MK-3475, and TSR-042.

12. The method of any one of claims 1 to 6, wherein the second immune checkpoint inhibitor is an inhibitor of PD-L1 and/or PD-L2 antibody.

13. The method of claim 12, wherein the inhibitor of PD-L1 and/or PD-L2 antibody is selected from the group consisting of MPDL3280A, RG-7446, BMS-936559, MEDI-4736, MSB-0020718C, AUR-012, and STI-A1010.

14. The method of any one of claims 1 to 13, wherein the subject is human.

15. The method of claim 14, wherein the subject is cancer-free or healthy.

16. The method of claim 14, wherein the subject has a high-risk of developing breast cancer or ovarian cancer.

17. The method of claim 6, wherein the targeted cancer vaccine is an immunogenic composition comprising an immunogenic polypeptide selected from the group consisting of human a-lactalbumin, aSl casein, β-casein, κ-casein, and anti -Mullen an Hormone

Receptor, Type II (AMHR2).

18. The method of claim 17, wherein the immunogenic composition further comprises an adjuvant, a pharmaceutically acceptable carrier, or both.

19. The method of claim 18, wherein the adjuvant is selected from the group consisting of Adjuvant 65, a-GalCer, aluminum phosphate, aluminum hydroxide, calcium phosphate, β-Glucan Peptide, CpG DNA, GM-CSF, GPI-0100, IF A, IFN-γ, IL-17, lipid A,

lipopolysaccharide, Lipovant, Montanide, N-acetyl-muramyl-L-alanyl-D-isoglutamine, Pam3CSK4, poly-IC, quil A, trehalose, dimycolate, and zymosan.

20. The method of claim 18 or 19, wherein the adjuvant is one that induces a mixed type 1/type 17 immune response.

21. The method of any one of claims 17 to 19, wherein the immunogenic polypeptide is human a-lactalbumin, said polypeptide consists essentially of a human a-lactalbumin sequence as set forth in SEQ ID NO: 1.

22. The method of claim 6, wherein the targeted cancer vaccine is a multivalent breast cancer vaccine.

23. The method of claim 22, wherein the multivalent cancer vaccine comprises: two or more immunogenic polypeptides selected from the group consisting of: a polypeptide comprising a 15 amino acid fragment of human α-lactalbumin (SEQ ID NO: 1), a polypeptide comprising a 15 amino acid fragment of human aSl casein (SEQ ID NO: 2), a polypeptide comprising a 15 amino acid fragment of human β-casein (SEQ ID NO: 3), a polypeptide comprising a 15 amino acid fragment of human κ-casein (SEQ ID NO: 4), a polypeptide, at least 20 amino acids in length, having 90% sequence identity with an amino acid sequence contained in SEQ ID NO: 1, a polypeptide, at least 20 amino acids in length, having 90% sequence identity with an amino acid sequence contained in SEQ ID NO: 2, a polypeptide, at least 20 amino acids in length, having 90% sequence identity with an amino acid sequence contained in SEQ ID NO: 3, and a polypeptide, at least 20 amino acids in length, having 90% sequence identity with an amino acid sequence contained in SEQ ID NO: 4, or combination thereof.

24. The method of claim 23, wherein each of said polypeptides are linked to one another as one fusion antigen containing all hydrophilic domains of each of said human a- lactalbumin, human aSl casein, human β-casein, and human κ-casein polypeptides.

25. The method of any one of claims 1 to 24, wherein said method further comprises administering chemotherapy and/or radiotherapy and/or immunotherapy to said subject.

26. The method of any one of claims 1 to 25, wherein the immune response is a proinflammatory immune response, said immune response is induced by subsequent encounter of immune cells with a-lactalbumin, aSl casein, β-casein or κ-casein.

27. The method of any one of claims 1 to 26, further comprising the step of contacting the T cells with a composition comprising isolated human dendritic cells previously exposed to the targeted cancer vaccine of claim 6.

28. The method of claim 27, wherein the dendritic cells are autologous or allogeneic.

29. The method any one of claims 1 to 28, wherein T cells are activated after administration of the first immune checkpoint inhibitor to the subject.

30. The method of claim 29, wherein the T cells are CD4⁺.

31. The method of claim 29, wherein the T cells are CD8⁺.

32. The method of claim 26, wherein the proinflammatory immune response comprises production of IFNy by T cells.

33. The method of any one of claims 1 to 28, wherein B cells are activated after administration of the first immune checkpoint inhibitor to the subject.

34. The method of claim 26, wherein the proinflammatory immune response is breast tissue specific, ovarian tissue specific, or prostate tissue specific.

35. The method of any one of claims 1-34, wherein the subject has a breast tumor that overexpresses a-lactalbumin.

36. The method of any one of claims 1-35, wherein said method further comprises administering a corticosteroid against autoimmune-related inflammation, pancreatitis, hepatitis, dermatitis, colitis, uveitis, pulmonary abnormalities, renal insufficiency, hematologic and neurologic abnormalities, thyroid dysfunction, Hashimoto's thyroiditis, and Graves' disease.

37. The method of any one of claims 1-35, wherein said method further comprises administering a chemotherapeutic agent.

38. The method of claim 37, wherein the chemotherapeutic agent is selected from the group consisting of sunitinib or sorafenib

39. The method of any one of claims 1 to 38, further comprising the step of determining whether the administration of the first immune checkpoint inhibitor, second immune checkpoint inhibitor, and/or targeted cancer vaccine induces an immune response in the subject.

40. The method of any one of claims 1 to 39, wherein the subject is administered multiple doses of the checkpoint inhibitor or targeted cancer vaccine.

41. The method of claim 6, wherein the targeted cancer vaccine is a ovarian cancer vaccine.

42. The method of claim 41, wherein the ovarian cancer vaccine is an immunogenic composition comprising an Anti-Mullerian Hormone Receptor, Type II (AMHR2) polypeptide, wherein the AMHR2 polypeptide has an amino acid sequence that comprises at least 8 consecutive amino acids of SEQ ID NO: 5.

43. The method of claim 42, wherein administration of the immunogenic composition induces expression of anti-AMHR2 IgG antibodies by the subject.

44. The method of any one of claims 1 to 43, further comprising the step of

administering an additional anti-cancer agent to the subject.

45. The method of claim 44, wherein the additional anti-cancer agent is selected from the group consisting of paclitaxel, cisplatin, topotecan, gemcitabine, bleomycin, etoposide, carboplatin, docetaxel, doxorubicin, topotecan, cyclophosphamide, trabectedin, olaparib, tamoxifen, letrozole, bevacizumab,.

46. The method of any one of claims 1 to 29, wherein the subject has undergone surgery to remove at least part of the ovarian cancer tumor.

47. A method of preventing cancer in a subject comprising the steps of:

(a) administering an effective amount of a first immune checkpoint inhibitor to a subject to prime the immune response in the subject; and

(b) administering an effective amount of a second immune checkpoint inhibitor to the subject to enhance the primed immune response in the subject.

48. The method of claim 47, wherein the cancer is triple negative breast cancer (TNBC) subject, breast cancer, ovarian cancer, or prostate cancer.

49. The method of any one of claims 47 to 48, wherein said method further comprises co-administering a targeted cancer vaccine.

50. The method of claim 49, wherein the targeted cancer vaccine is administered prior to, subsequently, or conjointly with the first or second immune checkpoint inhibitor.

51. The method of any one of claims 49 to 50, wherein the targeted cancer vaccine is a conjointly administered with the first immune checkpoint inhibitor.

52. The method of claim 51, wherein the targeted cancer vaccine is selected from the group consisting of a breast cancer vaccine, prostate cancer vaccine, ovarian cancer vaccine, and a multivalent breast cancer vaccine.

53. The method of any one of claims 47 to 52, wherein the first immune checkpoint inhibitor is an inhibitor of CTLA4.

54. The method of claim 53, wherein the inhibitor of CTLA4 is selected from the group consisting of ipilimumab, tremelimumab, and KAHR-102.

55. The method of any one of claims 47 to 52, wherein the second immune checkpoint inhibitor is an inhibitor of PD-1.

56. The method of claim 55, wherein the inhibitor of PD-1 is an anti-PD-1 antibody.

57. The method of claim 56, wherein the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab/lambrolizumab, pidilizumab, AMP -224, AMP- 514, STI-A1110, MK-3475, and TSR-042.

58. The method of any one of claims 47 to 52, wherein the second immune checkpoint inhibitor is an inhibitor of PD-L1 and/or PD-L2 antibody.

59. The method of claim 58, wherein the inhibitor of PD-L1 and/or PD-L2 antibody is selected from the group consisting of MPDL3280A, RG-7446, BMS-936559, MEDI-4736, MSB-0020718C, AUR-012, and STI-A1010.

60. The method of any one of claims 47 to 59, wherein the subject is human.

61. The method of claim 60, wherein the subject is cancer-free or healthy.

62. The method of claim 62, wherein the subject has a high-risk of developing breast cancer or ovarian cancer.

63. The method of claim 52, wherein the targeted cancer vaccine is an immunogenic composition comprising an immunogenic polypeptide selected from the group consisting of human a-lactalbumin, aSl casein, β-casein, κ-casein, and anti-Mullerian Hormone

Receptor, Type II (AMHR2).

64. The method of claim 63, wherein the immunogenic composition further comprises an adjuvant, a pharmaceutically acceptable carrier, or both.

65. The method of claim 64, wherein the adjuvant is selected from the group consisting of Adjuvant 65, a-GalCer, aluminum phosphate, aluminum hydroxide, calcium phosphate, β-Glucan Peptide, CpG DNA, GM-CSF, GPI-0100, IF A, IFN-γ, IL-17, lipid A, lipopolysaccharide, Lipovant, Montanide, N-acetyl-muramyl-L-alanyl-D-isoglutamine, Pam3CSK4, poly-IC, quil A, trehalose, dimycolate, and zymosan.

66. The method of claim 64 or 65, wherein the adjuvant is one that induces a mixed type 1/type 17 immune response.

67. The method of any one of claims 63 to 66, wherein the immunogenic polypeptide is human a-lactalbumin, said polypeptide consists essentially of a human a-lactalbumin sequence as set forth in SEQ ID NO: 1.

68. The method of claim 52, wherein the targeted cancer vaccine is a multivalent breast cancer vaccine.

69. The method of claim 68, wherein the multivalent cancer vaccine comprises: two or more immunogenic polypeptides selected from the group consisting of: a polypeptide comprising a 15 amino acid fragment of human α-lactalbumin (SEQ ID NO: 1), a polypeptide comprising a 15 amino acid fragment of human aSl casein (SEQ ID NO: 2), a polypeptide comprising a 15 amino acid fragment of human β-casein (SEQ ID NO: 3), a polypeptide comprising a 15 amino acid fragment of human κ-casein (SEQ ID NO: 4), a polypeptide, at least 20 amino acids in length, having 90% sequence identity with an amino acid sequence contained in SEQ ID NO: 1, a polypeptide, at least 20 amino acids in length, having 90% sequence identity with an amino acid sequence contained in SEQ ID NO: 2, a polypeptide, at least 20 amino acids in length, having 90% sequence identity with an amino acid sequence contained in SEQ ID NO: 3, and a polypeptide, at least 20 amino acids in length, having 90% sequence identity with an amino acid sequence contained in SEQ ID NO: 4, or combination thereof.

70. The method of claim 69, wherein each of said polypeptides are linked to one another as one fusion antigen containing all hydrophilic domains of each of said human a- lactalbumin, human aSl casein, human β-casein, and human κ-casein polypeptides.

71. The method of any one of claims 47 to 70, wherein said method further comprises administering chemotherapy and/or radiotherapy and/or immunotherapy to said subject.

72. The method of any one of claims 47 to 71, wherein the immune response is a proinflammatory immune response, said immune response is induced by subsequent encounter of immune cells with α-lactalbumin, aSl casein, β-casein or κ-casein.

73. The method of any one of claims 47 to 72, further comprising the step of contacting the T cells with a composition comprising isolated human dendritic cells previously exposed to the targeted cancer vaccine of claim 52.

74. The method of claim 73, wherein the dendritic cells are autologous or allogeneic.

75. The method any one of claims 47 to 74, wherein T cells are activated after administration of the first immune checkpoint inhibitor to the subject.

76. The method of claim 75, wherein the T cells are CD4⁺.

77. The method of claim 75, wherein the T cells are CD8⁺.

78. The method of claim 72, wherein the proinflammatory immune response comprises production of IFNy by T cells.

79. The method of any one of claims 47 to 74, wherein B cells are activated after administration of the first immune checkpoint inhibitor to the subject.

80. The method of claim 72, wherein the proinflammatory immune response is breast tissue specific, ovarian tissue specific, or prostate tissue specific.

81. The method of any one of claims 47 to 80, wherein said method further comprises administering a corticosteroid against autoimmune-related inflammation, pancreatitis, hepatitis, dermatitis, colitis, uveitis, pulmonary abnormalities, renal insufficiency, hematologic and neurologic abnormalities, thyroid dysfunction, Hashimoto's thyroiditis, and Graves' disease.

82. The method of any one of claims 47 to 81, wherein said method further comprises administering a chemotherapeutic agent.

83. The method of claim 82, wherein the chemotherapeutic agent is selected from the group consisting of sunitinib or sorafenib.

84. The method of claim 83, wherein the chemotherapeutic agent is sunitinib.

85. The method of any one of claims 47 to 84, further comprising the step of determining whether the administration of the first immune checkpoint inhibitor, second checkpoint inhibitor, or targeted cancer vaccine induces an immune response in the subject.

86. The method of any one of claims 47 to 85, wherein the subject is administered multiple doses of the first immune checkpoint inhibitor, second immune checkpoint inhibitor, or targeted cancer vaccine.

87. The method of claim 52, wherein the targeted cancer vaccine is a ovarian cancer vaccine.

88. The method of claim 87, wherein the ovarian cancer vaccine is an immunogenic composition comprising an AMHR2 polypeptide, wherein the AMHR2 polypeptide has an amino acid sequence that comprises at least 8 consecutive amino acids of SEQ ID NO: 5.

89. The method of claim 88, wherein administration of the immunogenic composition induces expression of anti-AMHR2 IgG antibodies by the subject.

90. The method of any one of claims 47 to 89, further comprising the step of administering an additional anti-cancer agent to the subject.

91. The method of claim 90, wherein the additional anti-cancer agent is selected from the group consisting of paclitaxel, cisplatin, topotecan, gemcitabine, bleomycin, etoposide, carboplatin, docetaxel, doxorubicin, topotecan, cyclophosphamide, trabectedin, olaparib, tamoxifen, letrozole, bevacizumab.

92. The method of any one of claims 47 to 91, wherein the subject is predisposed to ovarian or breast cancer.

93. The method of claim 92, wherein the genome of the subject comprises a BRCA1 or BRCA2 mutation that predisposes the subject to ovarian or breast cancer.

94. The method of claim 92 or 93, wherein the subject has a family history of ovarian or cancer.

95. The method of any one of claims 47 to 94, wherein the subject is a post-menopausal human female.