Vaccination with mica / b alpha 3 domain for treatment of cancer

Abstract

To provide compositions and methods for treating cancer in a subject by eliciting an immune response against an MIC alpha 3-domain polypeptide.SOLUTION: A fusion protein is administered comprising a monomeric ferritin subunit protein joined to a MIC alpha 3-domain protein, where the monomeric ferritin subunit protein comprises a domain that allows the fusion protein to self-assemble into nanoparticles.SELECTED DRAWING: Figure 11

Description

[Technical field]

[0001] Related Applications This application claims priority to and benefit of U.S. Provisional Application No. 62 / 263,377, filed December 4, 2015, and No. 62 / 422,454, filed November 15, 2016, the contents of each of which are incorporated by reference in their entirety.

[0002] INCORPORATION BY REFERENCE TO SEQUENCE LISTING The contents of the text file entitled "DFCI-126-001WO-Sequence Listing.txt", created on December 2, 2016 and measuring 30.9 KB in size, are incorporated herein by reference in their entirety.

[0003] FIELD OF THEINVENTION The present invention relates generally to compositions and methods for inducing an anti-tumor immune response in a subject.

[0004] Government involvement This invention was made with Government support under R01CA173755 awarded by the National Institutes of Health. The Government has certain rights in this invention. [Background technology]

[0005] 2. Background of the Invention Recent advances in the field of cancer immunotherapy have demonstrated the ability of the immune system to eradicate even advanced cancers. These therapies are rapidly changing the face of cancer treatment. Unlike monoclonal antibody therapy, which requires repeated administration of antibodies to prevent tumor recurrence, vaccines can induce endogenous immune memory and therefore have the potential to provide long-term protection.

[0006] Selection of antigens for vaccine therapy requires a comprehensive understanding of the biological role of candidate antigens in tumor growth and their expression levels by tumor cells compared to normal tissues. MICA and the closely related MICB protein (abbreviated as MIC) are antigens that are expressed at very low levels or absent by normal cells, but are widely upregulated by a variety of different cancers secondary to genomic damage. MIC is an important ligand for the NKG2D receptor on cytotoxic lymphocytes, specifically NK cells, CD8 T cells and gamma-delta T cells. Expression of MIC targets such cells for elimination by the immune system. However, many tumors have been found to escape this important immune surveillance pathway by shedding MIC from the cell surface, a process in which the MIC alpha 3 domain is unfolded by the disulfide isomerase ERp5 and becomes sensitive to cleavage by matrix metalloproteases such as ADAM 10 and ADAM 17. Shedding MIC causes downregulation of the NKG2D receptor on NK cells and CD8 T cells. Thus, proteolytic cleavage converts an immunostimulatory protein into an immunosuppressant.

[0007] Thus, there is a need for compounds that inhibit MICA shedding. Summary of the Invention

[0008] In various aspects, the present invention provides a vaccine composition comprising as an immunogenic component an effective amount of a peptide comprising the MIC alpha 3-domain, where effective amount means an amount effective to induce an immune response against the MIC alpha 3-domain.

[0009] The MIC alpha 3-domain is a MICA or MICB alpha 3-domain. Optionally, the MIC alpha 3-domain is non-glycosylated. Preferably, the peptide comprises the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4. In various aspects, the vaccine composition comprises a plurality of peptides. In some aspects, the peptides are conjugated to a carrier protein.

[0010] In another aspect, the present invention provides a fusion protein having a monomeric ferritin subunit protein linked to a MIC alpha 3-domain protein. The monomeric ferritin subunit protein has a domain that allows the fusion protein to self-assemble into nanoparticles. In a preferred embodiment, the monomeric subunit is a Helicobacter pylori ferritin protein. Optionally, the fusion protein is further conjugated to a CpG oligonucleotide.

[0011] In yet another aspect, the present invention provides a nanoparticle comprising a fusion protein according to the present invention, the nanoparticle comprising a plurality of MIC alpha 3-domain peptides.

[0012] In yet another aspect, the present invention provides a vaccine composition comprising the nanoparticles according to the invention. The vaccine composition may further comprise GM-CSF.

[0013] In a further aspect, the present invention provides a method for treating cancer in a subject by administering to the subject a vaccine composition according to the present invention. Optionally, the vaccine composition contains GM-CSF. The subject has tested positive for shedding MIC in serum. The vaccine composition is administered as part of a therapeutic regimen. The therapeutic regimen includes, for example, radiation therapy, targeted therapy, immunotherapy, or chemotherapy. Optionally, the subject is further administered one or more vaccines specific for antigens other than MIC alpha 3-domain antigens.

[0014] In another aspect, the invention provides a method for treating cancer by administering to a subject a vaccine comprising cells expressing the MIC alpha-3 domain. In a further aspect, the invention provides a method for treating cancer in which an immune response against the MIC is induced by the use of replicating or non-replicating viruses.

[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Methods and materials similar or equivalent to those described herein can be used in the practice of this invention, but suitable methods and materials are described below.All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety.In case of conflict, the present specification, including definitions, will take precedence.In addition, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting.

[0016] [The present invention 1001] A vaccine composition comprising as an immunogenic component an effective amount of a peptide comprising or consisting of the MIC alpha 3-domain, A vaccine composition, wherein the effective amount is an amount effective to induce an immune response against the MIC alpha 3-domain. [The present invention 1002] 1001. The vaccine composition of the present invention, wherein the MIC alpha 3-domain is a MICA or MICB alpha 3-domain. [The present invention 1003] The vaccine composition of claim 1001 or 1002, wherein the MIC alpha 3-domain is non-glycosylated. [The present invention 1004] The vaccine composition of any of claims 1001 to 1003, wherein the peptide comprises or consists of the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4. [The present invention 1005] The vaccine composition according to any one of claims 1001 to 1004, comprising a plurality of peptides. [The present invention 1006] The vaccine composition of any of claims 1001 to 1005, wherein the peptide is conjugated to a carrier protein. [The present invention 1007] The vaccine composition of the present invention 1001 further comprising GM-CSF. [The present invention 1008] 1. A fusion protein comprising a monomeric ferritin subunit protein linked to a MIC alpha 3-domain protein, A fusion protein in which a monomeric ferritin subunit protein contains a domain that enables the fusion protein to self-assemble into a nanoparticle. [The present invention 1009] The fusion protein of the present invention, wherein the monomeric subunit is a monomeric subunit of Helicobacter pylori ferritin protein. [The present invention 1010] The fusion protein of any one of claims 1008 to 1009, further comprising a cytosine-guanosine (CpG) oligonucleotide sequence. [The present invention 1011] A nanoparticle comprising any one of the fusion proteins of the present inventions 1008 to 1010. [The present invention 1012] A nanoparticle comprising a plurality of MIC alpha 3-domain peptides. [The present invention 1013] A vaccine composition comprising the nanoparticles of the present invention. [The present invention 1014] The vaccine composition of the present invention 1013, further comprising GM-CSF. [The present invention 1015] A method for treating cancer in a subject, comprising the step of administering to the subject any one of the vaccine compositions of the present inventions 1001 to 1007 or 1013 to 1014. [The present invention 1016] The method of claim 1015, further comprising administering GM-CSF. [The present invention 1017] The method of claim 1015 or 1016, wherein the vaccine composition is administered as part of a therapeutic regimen. [The present invention 1018] The method of claim 1017, wherein the therapeutic regimen is radiation therapy, targeted therapy, immunotherapy, or chemotherapy. [The present invention 1019] The method of any of claims 1015 to 1018, wherein said subject has tested positive for shedding MIC in serum. [The present invention 1020] The method of any of claims 1015 to 1019, comprising the step of administering to the subject one or more vaccines specific for an antigen other than a MIC alpha 3-domain antigen. [The present invention 1021] A method for treating cancer comprising administering to said subject a vaccine comprising cells expressing a MIC alpha-3 domain. [The present invention 1022] A method for treating cancer in which an immune response against a MIC is induced through the use of replicating or non-replicating viruses. Other features and advantages of the invention will be apparent from and are encompassed by the following detailed description and claims. [Brief description of the drawings]

[0017] [Figure 1A] Schematic diagram showing the interaction between NKG2D homodimer and MICA. The MICA-alpha3 domain is identified for reference. From Nat Immunol. 2001 May;2(5):443-51. Complex structure of the activating immunoreceptor NKG2D and its MHC class I-like ligand MICA. [Figure 1B] FIG. 1 is a schematic illustrating one mechanism by which tumors evade immune surveillance through the shedding of MICs from the tumor cell surface. [Figure 1C] FIG. 1 is a schematic depicting a ferritin particle. [Figure 1D] FIG. 1 is a schematic depicting maps showing cellular and humoral immune responses to human and mouse ferritin. [Figure 2A] FIG. 1 is a schematic diagram of the MICA alpha 3 ferritin fusion gene construct. [Figure 2B] Size-exclusion chromatogram of MICA alpha 3-ferritin using an XK16 / 60 Superdex200 column (flow rate: 2 ml / min; running buffer: 50 mM Tris, 150 mM NaCl pH 7.5) (left). Figure 2B further depicts an SDS gel under reducing conditions containing samples taken from 22-27 min at the protein peak (right). [Figure 2C] FIG. 1 is a schematic diagram of the deglycosylated MICA alpha 3 construct. [Figure 2D] Size-exclusion chromatogram of MICA alpha 3 using a Superdex200 column (flow rate: 1 ml / min; running buffer: 50 mM Tris, 150 mM NaCl pH 7.5) (left). Figure 2D further depicts an SDS gel under reducing conditions containing samples taken from 16-20 min at the peak protein (right). [Diagram 3] Schematic diagram depicting the use of mesoporous silica rod (MSR) vaccines for subcutaneous injection and the resulting induction of strong immune responses. See Kim, J & Aileen, WL et al. Nature Biotech. 2015. [Figure 4]Figure 4A-B: A series of graphs depicting the efficacy of MICα3 domain vaccine in a lung metastasis model. The data shown in Figure 4A was obtained as follows: B6 mice were immunized with 200 μg MICα3 protein, 1 μg GM-CSF and 100 μg CpG-ODN either as a scaffold-free bolus (bolus) or within a mesoporous silica rod (MSR) scaffold (MSR vaccine). Mice received one booster injection on day 28. Three weeks later, mice were challenged with an iv injection of 5X105 B16-MIC tumor cells. The number of lung metastases was quantified on day 14 after tumor cell injection. The data obtained in Figure 4B was obtained as follows: Shed MIC was quantified by ELISA on days 0, 5, and 13 after tumor cell injection. Previous experiments had shown that MICα3 domain-specific antibodies did not interfere with the ELISA used to detect shed MIC. [Diagram 5] Figure 5A-B: A series of plots and graphs demonstrating that vaccination with MICA-ferritin fusion protein induces high titers of MICA-specific antibodies. Figure 5A depicts FACS plots of MICAα3-specific antibodies in the serum of immunized mice against full-length MICA expressed on the surface of B16F10 mouse melanoma cells. B16F10 melanoma cells were transfected with human MICA cDNA (allele 009) and then labeled with an isotype control antibody (negative control) or a saturating concentration of MICA-specific mouse mAb (6D4, positive control). This system was then used to test serum from mice vaccinated with MICAα3-ferritin or control antigen (OVA). A PE-labeled anti-mouse IgG secondary antibody was used to detect antibody bound to the cell surface. Fluorescence was quantified by FACS. Strong staining was detected even in 1 μl of serum from mice on days 14-42 post-vaccination. FIG. 5B depicts a bar graph of mean fluorescence intensity (MFI) showing + / - SD of three replicates of binding of MICAα3-specific antibodies in the serum of immunized mice to full-length MICA expressed on the surface of B16F10 mouse melanoma cells. [Figure 6]1 is a series of graphs depicting tested sera from MICA-ferritin immunized mice assayed by ELISA to determine the various subclasses of IgG induced upon vaccination. [Figure 7] FIG. 13 is a graph showing that serum antibodies in the MICA-ferritin immunized group prevent MICA shedding from the tumor cell surface. [Figure 8] Figure 8A-B: A series of graphs depicting the therapeutic activity of the MICA-ferritin vaccine. C57BL / 6 mice were immunized with MICAα3-ferritin or ovalbumin and received a booster injection on day 28. Mice were challenged by intravenous injection of 5x105 B16-MICA tumor cells, which formed lung metastases. The number of lung metastases was counted on day 14 (Figure 8A) and shed MICA was quantified in serum (Figure 8B). The MICAα3 domain vaccine substantially reduced the number of lung metastases, while the control vaccine had no effect. Furthermore, shed MICA became undetectable in the serum of mice that received the MICAα3-ferritin vaccine, while shed MICA levels were very high in both control groups. Figure 8A demonstrates that immunization with MICA alpha3-ferritin prevents metastases compared to naive controls or animals that received OVA-protein injections. FIG. 8B is a graph depicting the results obtained by ELISA showing that vaccinated mice had undetectable levels of sMICA (shedding MICA) in the serum. [Figure 9] Figures 9A-C: A series of graphs depicting the titers of antibodies induced by the MICA-ferritin vaccine (Figure 9A), as well as the effect of vaccine dosage on the number of lung nodules (Figure 9B) and the amount of sMICA (Figure 9C). [Figure 10A] FIG. 10A is a series of graphs depicting the effect of a deglycosylated form of the MICAα3 vaccine (not linked to ferritin nanoparticles) on the binding of MICAα3-specific antibodies in the serum of immunized mice to full-length MICA expressed on the surface of B16F10 mouse melanoma cells, as examined by flow cytometry. [Figure 10B]FIG. 10B is a graph depicting the results from an ELISA assay used to determine the different subclasses of IgG induced by vaccination. [Figure 11] Figures 11A-B: A series of graphs depicting that the MICAα3 vaccine alone (without ferritin fusion) has significant therapeutic utility in vivo. Figure 11A is a graph depicting the number of lung metastases after vaccination with the MICAα vaccine alone. Figure 11B is a graph depicting the amount of sMICA in serum at days 0, 5, and 13 after vaccination with the MICAα vaccine alone. [Figure 12] Figure 12A-B: MICA-ferritin vaccine slows tumor growth in B16F10 subcutaneous melanoma model. In Figure 12A, 7-week-old C57BL / 6 female mice (n=8) were immunized with MICA-ferritin vaccine and boosted on day 12. On day 25 after the first vaccination, mice were challenged with 0.5x106 MICA-expressing B16F10 cells subcutaneously and tumor volumes were measured every other day. Tumor growth in the MICA-ferritin immunized group was found to be significantly slower (open squares) compared to the naive, untreated age-matched control group (closed circles). In Figure 12B, sMICA levels were undetectable in the serum of mice immunized with MICA-ferritin vaccine (open triangles), whereas high levels of sMICA were detected in the serum of non-immunized control group (closed triangles) within 2 weeks after tumor challenge. [Figure 13]Figure 13A-B: Depletion of CD8 T cells accelerates tumor growth in a MICA-ferritin vaccinated B16F10 subcutaneous melanoma model. In Figure 13A, 7-week-old C57BL / 6 female mice were immunized with MICA-ferritin vaccine (n=16) or OVA control vaccine (n=8) and boosted on day 14. On day 21 after the first vaccination, mice were challenged with a subcutaneous injection of 0.5x106 MICA-expressing B16F10 cells. Mice received intravenous injections of 200μg of anti-CD8 antibody (n=8) or isotype control antibody (n=8) at a dose of 100μg per mouse two days prior to tumor challenge and twice weekly thereafter until the study endpoint. Tumor volumes were measured every other day. Mice were euthanized when tumors reached ≥250mm2. Tumors reached maximum volume by day 12 in OVA protein vaccinated control mice treated with CD8 antibody (open triangles) and by day 14 in naive, untreated, non-depleted controls (filled circles). CD8 depletion accelerated tumor growth in the MICA-vaccinated group (filled triangles) compared to the MICA-vaccinated group that received isotype antibody (open squares). In FIG. 13B, survival analysis of the CD8 depletion experiment showing age-matched naive, untreated, non-depleted control group (thick solid line), OVA protein vaccinated group (thin dotted line), MICA-ferritin vaccinated, CD8 depleted (thick dotted line), and MICA-ferritin vaccinated, isotype antibody injected mice (thin solid line). [Figure 14]Figure 14A-B: NK cells contribute to the therapeutic effect of MICA-ferritin vaccine in B16F10 subcutaneous melanoma model. In Figure 14A, 7-week-old C57BL / 6 female mice were immunized with MICA-ferritin vaccine (n=16) and boosted on day 14. On day 21 after the first vaccination, mice were challenged with subcutaneous injections of 0.5x106 MICA-expressing B16F10 cells. Mice received intravenous injections of 200μg of anti-NK1.1 antibody (n=8) or isotype control antibody (n=8) at a dose of 100μg per mouse 2 days prior to tumor challenge and twice weekly thereafter until the study endpoint. Tumor volumes were measured every other day. Mice were euthanized when tumors reached ≥250mm2. Tumors reached maximum volume by day 14 in the naive, untreated, non-removed control group (black circles). NK cell depletion accelerated tumor growth in the MICA-vaccinated group (open triangles) compared to the MICA-vaccinated group that received isotype antibody (closed squares). In Figure 14B, survival analysis of the NK cell depletion experiment showing age-matched naive, untreated, non-depleted control group (thick solid line); MICA-ferritin vaccinated, NK cell depleted (dotted line), and MICA-ferritin vaccinated, isotype antibody injected mice (thin solid line). [Figure 15]Figure 15A-B: Serum polyclonal antibodies raised in response to MICA-ferritin vaccine prevent lung metastasis of B16F10-MICA tumor cells. In Figure 15A, 8-week-old Ighmtm1Cgn / J female mice (n=12) were challenged with intravenous injection of 0.5x106 MICA-expressing B16F10 melanoma cells. Mice were randomized into three cohorts, each containing 4 mice. On days 1, 2, 4 and 6 after tumor challenge, mice were injected (intraperitoneal route) with 100μl of end-point serum from naive, OVA-protein or MICA-ferritin immunized C57BL / 6 mice. Mice were euthanized 14 days after tumor challenge; lungs were removed and fixed in 10% neutral buffered formalin, and the number of lung metastases was quantified. Mice injected with serum from the MICA-ferritin vaccinated group (open squares) had significantly fewer lung metastases compared with untreated, age-matched controls (closed circles) and with serum from the OVA-protein immunized group. In Figure 15B, sMICA levels were lower in mice that received serum from the MICA-ferritin vaccinated group (open squares) compared with mice that received serum from the naive or OVA-protein immunized groups. [Figure 16] Figure 16A-B: MICA-ferritin vaccine also controls B16F10-MICB005 subcutaneous tumor growth. In Figure 16A, 7-week-old C57BL / 6 female mice (n=4) were immunized with MICA-ferritin vaccine and boosted on day 14. On day 21 after the first vaccination, mice were challenged with 0.5x106 MICB-expressing B16F10 cells subcutaneously and tumor volumes were measured every other day. B16F10-MICB tumor growth in the MICA-ferritin immunized group was found to be significantly slower (open squares) compared to the OVA-protein immunized control group (filled circles). In Figure 16B, sMICB levels were nearly undetectable in the serum of mice immunized with MICA-ferritin vaccine (open squares), while high levels of sMICB were detected in the serum of the OVA-protein immunized control group (filled circles) within 2 weeks after tumor challenge. [Figure 17]A series of graphs showing staining of MICA expressing B16 cell line with serum from mice immunized with MICA-ferritin vaccine formulated with mesoporous silica rods (MSR) (dashed line) or without MSR and direct conjugation of CpG to MICA-ferritin (thin solid line). These data illustrate that vaccination with CpG directly conjugated to MICA-ferritin peptide induces stronger immune response against MICA alpha 3 domain compared to vaccine formulated with MSR scaffold. For MSR vaccine, 5mg MSR+200ug protein+100ug CpG+1ug GM-CSF; immunize on day 0; boost on day 14; serum from day 28. For direct conjugation, 200ug protein conjugated to approximately 5ug CpG (primary immunization); boost (100ug protein conjugated to approximately 5ug CpG + AddaVax (100ul) + GM-CSF (1ug); immunize on day 0; boost on day 21; serum from day 28. [Figure 18] Figure 18A: Electron micrograph of purified MICAα3-ferritin nanoparticles. Figure 18B: SDS-PAGE of vaccine protein after affinity and gel filtration chromatography. [Figure 19] Figure 1 shows that polyclonal antibodies induced by MICAα3 domain vaccines inhibit MICA shedding by human tumor cells. MICA shedding by the human A375 melanoma cell line was quantified using a sandwich ELISA. Addition of small amounts of serum (1-10 μl) from mice vaccinated with MICAα3-ferritin strongly inhibited shedding, whereas addition of serum from control mice had little effect. [Figure 20]Figure 20A-C: A series of graphs showing that MICA-ferritin vaccine induces secondary T cell responses against neoantigens. We investigated whether MICBα3 domain vaccine induces secondary responses against tumor neoantigens. Lymph node T cells were labeled with CFSE and cultured for 3 days with four different neoantigen peptides previously identified as CD4 T cell epitopes for B16F10 tumors. CD4 T cell responses were confirmed for three of the four peptides based on intracellular IFNγ staining in proliferating cells (CFSElow). We hypothesize that MICA antibodies trigger Fc receptor-mediated uptake of apoptotic tumor fragments by dendritic cells, thereby promoting T cell responses against neoantigens. In Figure 20A-20B, B6 mice were immunized with MICBα3-ferritin or OVA (n=5 / group) and injected with B16F10-MICB tumor cells. Ten days after tumor implantation, T cells were isolated from tumor-draining lymph nodes and labeled with CFSE. T cells were cultured with CD11c+ spleen cells for 3 days in the presence of four different CD4 neoantigen peptides (10 μg / ml) previously identified for B16F10 tumors. Intracellular IFNγ staining was performed to quantify proliferating T cells positive for intracellular IFNγ (CFSE-low). T cell responses to neoantigen peptides were compared between mice immunized with OVA control antigen (Figure 20A) or MICBα3 domain (Figure 20B). Both T cell populations were incubated in vitro with M30 neoantigen. In Figure 20C, a summary of T cell responses to three neoantigens (M30, M44 and M48) where enhanced T cell responses were observed in MICB-immunized mice. [Figure 21]21A-B: Graphs showing that immunization with MICA-ferritin nanoparticles conjugated with CpG induces high titer antibodies. In FIG. 21A, macaque MICA / B-ferritin was conjugated to CpG ODN 1826 by CLICK chemistry (Protein-Oligo Conjugation Kit, Solulink). Briefly, S-HyNic (succinimidyl-6-hydrazino-nicotinamide) linker was conjugated to the protein through the primary amine on lysine, and S-4B (succinimidyl-4-formylbenzamide) linker was added to the CpG oligo. The modified protein and oligo were incubated in a catalytic conjugation reaction. Following this reaction, excess unconjugated CpG was removed by size exclusion chromatography. The formed protein-oligo conjugate bond (stable bis-arylhydrazone bond) is UV traceable at 350 nm (see graph). In Figure 21B, CpG-conjugated proteins were used to immunize C57BL / 6 mice. MICA / B-specific antibodies in serum were analyzed on day 14 by labeling of B16-MICA cells. CpG-linked proteins induced higher titers of antibodies (thin solid line) compared to MICA-ferritin protein formulated with the scaffold (dotted line; thick solid line indicates background staining levels). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Detailed Description of the Invention The present invention provides a vaccine for cancer. More specifically, the present invention provides a MIC alpha 3-domain vaccine that can induce immune responses against the MIC alpha 3-domain. Importantly, the vaccine induces antibodies against the MIC alpha 3 domain, but does not induce antibodies against the alpha 1-alpha 2 domain of MIC, so as not to interfere with the binding of the alpha 1-alpha 2 domain to the NKG2D receptor on NK cells.

[0019] The goal of the vaccine is to induce polyclonal antibodies that bind to the membrane-proximal Ig domain of MICA and inhibit the proteolytic shedding of this protein from tumor cells. The MICA alpha 3 domain was expressed on the surface of nanoparticles. Specifically, the MICA alpha 3 domain coding sequence was fused to a ferritin sequence (from H. pylori), considering that ferritin spontaneously forms nanoparticles. The vaccine was formulated either with an immunization scaffold (mesoporous silica rod) using CpG as an adjuvant and GM-CSF to recruit dendritic cells to the injection site, or by directly conjugating CpG to GM-CSF and the MICA alpha 3 domain-ferritin fusion protein. Injection of these vaccines was found to induce high titer antibodies directed against the MICA alpha 3 domain. Surprisingly, the MICA alpha 3 domain-ferritin fusion protein with CpG directly conjugated achieved higher antibody titers.

[0020] These antibodies induced by the vaccine composition of the present invention bound to multiple MICA alleles and stained tumor cells that were MICA positive. Importantly, these polyclonal antibodies inhibited the shedding of MICA by tumor cells. The in vivo efficacy of the vaccine was tested in a metastatic mouse model of melanoma. B16F10 melanoma cells were genetically modified to express MICA and injected intravenously after two vaccinations of mice. The vaccine provided a high level of protection, while control mice had numerous lung metastases (approximately 150-200).

[0021] The vaccine of the present invention is conceptually different from conventional cancer vaccines, which attempt to induce immune responses that eliminate all cancer cells expressing a particular antigen. In contrast, the goal of the vaccine of the present invention is to prevent tumors from escaping from important immune surveillance pathways. The vaccine is considered safe based on patient studies with MICA antibodies and the fact that MIC expression flags cells for elimination by cytotoxic lymphocytes. The advantages of the vaccine approach are: low-cost vaccine, long-term protection against escape from immune surveillance, induction of polyclonal antibodies that inhibit shedding and rapidly remove shed MIC by forming immune complexes, and induction of T cell responses against other tumor antigens by enhanced uptake of apoptotic tumor fragments by dendritic cells.

[0022] Further provided by the present invention are self-assembling ferritin-based nanoparticles that display an immunogenic portion of MICA alpha 3 domain on their surface. Optionally, the nanoparticles further comprise CpG oligonucleotides. For example, CpG oligonucleotides are covalently attached to MICA alpha 3 domain-ferritin fusion proteins. Such nanoparticles are useful for vaccinating individuals. Thus, the present invention also relates to fusion proteins for producing such nanoparticles, and nucleic acid molecules encoding such proteins. Furthermore, the present invention relates to methods for producing the nanoparticles of the present invention, and methods for using such nanoparticles to vaccinate individuals.

[0023] Further provided by the present invention is a vaccine composition comprising a MIC alpha 3-domain peptide linked to a CpG oligonucleotide.

[0024] Vaccines against MIC alpha 3 domain proteins The present invention provides a vaccine composition suitable for administration to humans, comprising at least one MIC alpha 3-domain peptide as an immunogenic component. The MIC alpha 3-domain peptide comprises or consists of the full-length alpha 3 domain of MICA or MICB, which domain corresponds to amino acids 181-274 of SEQ ID NO: 1 or SEQ ID NO: 2. Optionally, the peptide comprises one or more flanking amino acids. In the present context, the term "flanking amino acids" refers to amino acids adjacent to the MIC alpha 3-domain sequence in the full-length reference sequence [SEQ ID NO: 1 for MICA or SEQ ID NO: 2 for MICB]. In some embodiments, the peptide comprises 2, 4, 6, 8, or 10 flanking amino acids on either its N-terminus or C-terminus, or on both termini. In some embodiments, the vaccine peptide is non-glycosylated. TIFF2024177207000002.tif114130

[0025] In a preferred embodiment, the vaccine comprises a peptide having the following amino acid sequence: TIFF2024177207000003.tif41161.

[0026] In another embodiment, the vaccine composition comprises a nucleic acid encoding a MIC alpha 3-domain sequence. The nucleic acid may be in the form of an expression vector, such as a plasmid or viral vector, or the nucleic acid may be packaged into a nanoparticle. In one embodiment, the nucleic acid is delivered to the subject by injection. In one embodiment, the nucleic acid is injected as purified DNA or in the form of nanoparticles. In one embodiment, modified immune cells are injected that have been modified to express the nucleic acid. In one embodiment, the immune cells are modified by in vitro infection or transfection with a vector containing the nucleic acid.

[0027] The peptides forming or incorporated into the vaccine compositions of the present invention, if chemically synthesized, are preferably purified from contaminating chemical precursors or substantially free of cellular material from the cell or tissue source from which they are derived. In specific embodiments, the peptides are 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% free of contaminating chemical precursors, proteins, lipids or nucleic acids. In preferred embodiments, the peptides are substantially free of contaminating viruses. Preferably, each composition for administration to a subject is at least 95%, at least 97%, or at least 99% free of contaminating viruses.

[0028] In one embodiment, the MIC alpha 3-domain peptide of the vaccine composition of the present invention comprises or consists of one or more peptides that are at least 90%, at least 95%, at least 98%, or at least 99% identical to a peptide comprising amino acids 181-274 of SEQ ID NO: 1 or SEQ ID NO: 2. In the present context, the term "similar" refers to amino acid sequence similarity defined according to the number of conservative and non-conservative amino acid changes in a query sequence compared to a reference sequence. Conservative and non-conservative amino acid changes are known in the art. See, for example, WR Taylor, The Classification of Amino Acid Conservation, J. Theor. Biol. 1986 119:205-218, and D. Bordo and P. Argos, Suggestions for "Safe" Residue Substitutions in Site-Directed Mutagensis, 1991 J. Mol. Biol. 217:721-729. In general, conservative amino acid change refers to the substitution of one amino acid with another amino acid that has substantially similar chemical properties, particularly with respect to amino acid side chain.Non-conservative change refers to the substitution of one amino acid with another amino acid that has substantially different chemical properties.In general, conservative substitution is recognized in the art as being unlikely to affect the overall structure or biological function of polypeptide, while non-conservative change is recognized as being more likely to affect structure and function.

[0029] Non-limiting examples of conservative amino acid changes include substitutions of amino acids within the following groups: aliphatic, aromatic, polar, non-polar, acidic, basic, phosphorylatable hydrophobic, hydrophilic, slightly non-polar, slightly polar, highly non-polar, and highly polar. Non-limiting examples of non-conservative amino acid changes include substitutions of amino acids between the aforementioned groups.

[0030] In one embodiment, conservative amino acid changes are those substitutions in which the substitution matrix for a pair of residues has a positive value. Examples of amino acid substitution matrices are known in the art, such as the BLOSUM50 matrix or the PAM250 matrix (see WA Pearson, Rapid and Sensitive Sequence Comparison with FASTP and FASTA, Meth. Enzymology, 1990 183:63-98, ed. R. Doolittle, Academic Press, San Diego). For further examples of scoring matrices and comparisons between them, see MS Johnson and JP Overington, 1993, A Structural Basis for Sequence Comparisons: An Evaluation of Scoring Methodologies, J. Mol. Biol. 233:716-738.

[0031] In preferred embodiments, a conservative amino acid change is the substitution of one amino acid with another amino acid within the same chemical group, where the group is selected from neutral polar amino acids (Ser, Thr, Pro, Ala, Gly, Asn, Gln), negatively charged polar amino acids (Asp, Glu), positively charged polar amino acids (His, Arg, Lys), nonpolar amino acids lacking a ring structure (Met, Ile, Leu, Val), nonpolar amino acids having a ring structure (Phe, Tyr, Trp), and cysteine.

[0032] In various embodiments, the peptide is conjugated to a CpG oligonucleotide sequence.

[0033] In other embodiments, the peptide is conjugated to a carrier protein. The term "carrier protein" is intended to include both small peptides and large polypeptides (>10 kDa). The carrier protein can be any peptide or protein. It can contain one or more T-helper epitopes. Carrier proteins can be: tetanus toxoid (TT), tetanus toxoid fragment C, non-toxic mutants of tetanus toxin [note that all such mutants of TT are considered to be the same type of carrier protein for the purposes of the present invention], polypeptides containing tetanus toxin T cell epitopes, e.g., N19 (WO2006 / 067632), diphtheria toxoid (DT), CRM197, other non-toxic mutants of diphtheria toxin, e.g., CRM176, CRM 197, CRM228, CRM 45 (Uchida et al J. Biol. Chem. 218; 3838-3844, 1973); CRM 9, CRM 45, CRM102, CRM as described in Nicholls and Youle, Genetically Engineered Toxins, Ed: Frankel, Maecel Dekker Inc, 1992. 103 and CRM107 and other mutations; deletions or mutations of Glu-148 to Asp, Gln or Ser and / or Ala 158 to Gly and other mutations disclosed in U.S. Pat. No. 4,709,017 or U.S. Pat. No. 4,950,740; mutations of at least one or more residues Lys 516, Lys 526, Phe 530 and / or Lys 534 and other mutations disclosed in U.S. Pat. No. 5,917,017 or U.S. Pat. No. 6,455,673; or fragments disclosed in U.S. Pat. No. 5,843,711] (note that all such variants of DT are considered to be the same type of carrier protein for the purposes of the present invention), pneumococcal pneumolysin (Kuo et al (1995) Infect Immun 63; 2706-13), OMPC (meningococcal outer membrane protein - Usually, meningococcal serogroup B (N.meningitidis serogroup B) - EP0372501), synthetic peptides (EP0378881, EP0427347), heat shock proteins (WO 93 / 17712, WO 94 / 03208), pertussis proteins (WO 98 / 58668, EP0471177), cytokines, lymphokines, growth factors or hormones (WO 91 / 01146), artificial proteins containing multiple human CD4+ T cell epitopes from antigens derived from various pathogens (Falugi et al (2001) Eur J Immunol 31; 3816-3824), e.g. the N19 protein (Baraldoi et al (2004) Infect Immun 72; 4884-7), the pneumococcal surface protein PspA (WO 02 / 091998), iron uptake protein (WO 01 / 72337), toxins A or B of C. difficile (WO 00 / 61761), H. influenzae protein D (EP594610 and WO 00 / 56360), Streptococcus pneumoniae PhtA (WO 98 / 18930, also called Sp36), Streptococcus pneumoniae PhtD (disclosed in WO 00 / 37105, also called Sp036D), Streptococcus pneumoniae PhtB (disclosed in WO 00 / 37105, also called Sp036B), or PhtE (disclosed in WO00 / 30299, called BVH-3).

[0034] In one embodiment, the carrier protein may be selected from the group consisting of: tetanus toxoid (TT), fragment C of tetanus toxoid, diphtheria toxoid (DT), CRM197, pneumolysin (Ply), protein D, PhtD, PhtDE and N19. In one embodiment, the carrier protein is CRM197.

[0035] Vaccine containing HA-ferritin fusion protein The inventors have also discovered that fusions of MIC alpha 3-domain peptides with ferritin proteins (MIC alpha 3-ferritin fusion proteins) result in vaccines that induce robust immune responses against cancer. Such MIC alpha 3-ferritin fusion proteins self-assemble into nanoparticles that display immunogenic portions of MIC alpha 3-domain peptides on their surface. These nanoparticles are useful for vaccinating individuals with MIC alpha 3-domain. Thus, one aspect of the present invention is a MIC alpha 3-ferritin fusion protein that includes a monomeric ferritin subunit as disclosed herein linked to a MIC alpha 3-domain peptide as disclosed herein. The MIC alpha 3-ferritin fusion protein can self-assemble into nanoparticles. In various aspects, the fusion protein further comprises a CpG oligonucleotide sequence. The CpG oligonucleotide sequence can be covalently attached to the MIC alpha 3-ferritin fusion protein.

[0036] Ferritin is a globular protein found in all animals, bacteria and plants that acts primarily to control the rate and location of polynuclear Fe(III)2O3 formation through the transport of hydrated iron ions and protons to and from the mineralized core. The globular form of ferritin is composed of monomeric subunit proteins (also called monomeric ferritin subunits), which are polypeptides with molecular weights of approximately 17-20 kDa. Each monomeric ferritin subunit has a helical bundle topology, which contains four antiparallel helical motifs, with a fifth, shorter helix (the c-terminal helix) lying approximately perpendicular to the long axis of the four-helix bundle. By convention, the helices are designated `A, B, C and D and E`, respectively, from the N-terminus. The N-terminal sequence lies adjacent to the capsid triad axis and extends to the surface, while the E helices pack together at the tetraad axis and the C-terminus extends into the particle core. This packing results in the creation of two pores on the capsid surface. One or both of these pores are predicted to represent points of diffusion of hydrated iron into and out of the capsid. Following production, these monomeric ferritin subunit proteins self-assemble into globular ferritin proteins. Thus, the globular form of ferritin contains 24 monomeric ferritin subunit proteins and has a capsid-like structure with 432 symmetry.

[0037] According to the present invention, the monomeric ferritin subunit of the present invention is a full-length single polypeptide of a ferritin protein, or any portion thereof, that can direct the self-assembly of the monomeric ferritin subunit into a globular form of the protein. The amino acid sequence from the monomeric ferritin subunit of any known ferritin protein can be used to generate the fusion protein of the present invention, so long as the monomeric ferritin subunit is capable of self-assembly into a nanoparticle that displays the MIC alpha 3-domain on its surface. In one embodiment, the monomeric subunit is derived from a ferritin protein selected from the group consisting of bacterial ferritin protein, plant ferritin protein, algal ferritin protein, insect ferritin protein, fungal ferritin protein and mammalian ferritin protein. In one embodiment, the ferritin protein is derived from Helicobacter pylori.

[0038] The MIC alpha 3-ferritin fusion proteins of the present invention need not contain the full length sequence of a monomeric subunit polypeptide of a ferritin protein. A portion or region of a monomeric ferritin subunit protein can be utilized as long as the portion contains an amino acid sequence that directs self-assembly of the monomeric ferritin subunit into a globular form of the protein. One example of such a region is located at amino acids 5-167 of the Helicobacter pylori ferritin protein. More specific regions are described in Zhang, Y. Self-Assembly in the Ferritin Nano-Cage Protein Super Family. 2011, Int. J. Mol. Sci., 12, 5406-5421, which are incorporated herein by reference in their entirety.

[0039] One embodiment of the invention is a MIC alpha 3-ferritin fusion protein comprising a MIC alpha 3-domain protein of the invention linked to at least 25 contiguous amino acids, at least 50 contiguous amino acids, at least 75 contiguous amino acids, at least 100 contiguous amino acids, or at least 150 contiguous amino acids from a monomeric ferritin subunit, wherein the MIC alpha 3-ferritin fusion protein is capable of self-assembly into nanoparticles. One embodiment of the invention is a MIC alpha 3-ferritin fusion protein comprising a MIC alpha 3-domain protein of the invention linked to at least 25 contiguous amino acids, at least 50 contiguous amino acids, at least 75 contiguous amino acids, at least 100 contiguous amino acids, or at least 150 contiguous amino acids from a region of a ferritin protein that corresponds to an amino acid sequence of a Helicobacter pylori ferritin monomeric subunit that directs self-assembly of the monomeric subunit into a globular form of the ferritin protein, wherein the MIC alpha 3-ferritin fusion protein is capable of self-assembly into nanoparticles.

[0040] It is well known in the art that some mutations can be made in the amino acid sequence of a protein without affecting the activity of the protein. Such mutations include insertion of an amino acid residue, deletion of an amino acid residue, and substitution of an amino acid residue. Thus, in one embodiment, the sequence of a monomeric ferritin subunit is sufficiently different from the sequence of a ferritin subunit naturally found in a mammal, so that when the mutant monomeric ferritin subunit is introduced into a mammal, it does not cause the production of antibodies that react with the natural ferritin protein of the mammal. According to the present invention, such a monomeric subunit is called immunogenically neutral. One aspect of the invention is a MIC alpha 3-ferritin fusion protein comprising a MIC alpha 3-domain protein of the invention linked to an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, and at least 97% identical to the amino acid sequence of a monomeric ferritin subunit responsible for directing the self-assembly of the monomeric ferritin subunit into a globular form of the protein, wherein the MIC alpha 3-ferritin fusion protein is capable of self-assembly into nanoparticles. In one embodiment, the HA-ferritin fusion protein comprises a polypeptide sequence that is identical in sequence to a monomeric ferritin subunit. One aspect of the invention is a MIC alpha 3-ferritin fusion protein comprising a MIC alpha 3-domain protein of the invention linked to an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, and at least 97% identical to the amino acid sequence of a monomeric ferritin subunit from Helicobacter pylori, wherein the MIC alpha 3-ferritin fusion protein is capable of self-assembly into nanoparticles.

[0041] In some embodiments, it may be useful to engineer mutations into the amino acid sequence of the protein of the present invention. For example, it may be useful to modify sites such as enzyme recognition sites or glycosylation sites in the monomeric ferritin subunit, trimerization domain or linker sequence to give the fusion protein advantageous properties (e.g., solubility, half-life, masking part of the protein from immune surveillance). In this regard, it is known that the monomeric subunit of ferritin is not naturally glycosylated. However, when expressed as a secretory protein in mammalian cells or yeast cells, it may be glycosylated. Thus, in one embodiment, a potential N-linked glycosylation site in the amino acid sequence from the monomeric ferritin subunit is mutated so that the mutated ferritin subunit sequence is no longer glycosylated at the mutated site.

[0042] The proteins of the present invention are encoded by the nucleic acid molecules of the present invention. Moreover, they are expressed by the nucleic acid constructs of the present invention. As used herein, a nucleic acid construct is a recombinant expression vector, i.e., a vector linked to a nucleic acid molecule encoding a protein, such that the nucleic acid molecule can express the protein when the nucleic acid construct is administered, for example, to a subject or an organ, tissue, or cell. A vector also allows the transport of a nucleic acid molecule to a cell in an environment (for example, but not limited to, an organism, tissue, or cell culture). The nucleic acid constructs of the present disclosure are generated by human intervention. The nucleic acid construct can be DNA, RNA, or variants thereof. The vector can be a DNA plasmid, a viral vector, or other vector. In one embodiment, the vector can be a cytomegalovirus (CMV), retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, poliovirus, Sindbis virus, or any other DNA or RNA viral vector. In one embodiment, the vector can be a pseudotyped lentivirus or retrovirus vector. In one embodiment, the vector can be a DNA plasmid. In one embodiment, the vector can be a DNA plasmid that includes a plasmid component and a viral component to enable nucleic acid molecule delivery and expression. Methods for constructing the nucleic acid construct of the present disclosure are well known. See, for example, Molecular Cloning: a Laboratory Manual, 3rd edition, Sambrook et al. 2001 Cold Spring Harbor Laboratory Press, and Current Protocols in Molecular Biology, Ausubel et al. eds., John Wiley & Sons, 1994. In one embodiment, the vector is a DNA plasmid, such as a CMV / R plasmid, such as CMV / R or CMV / R 8 KB (also referred to herein as CMV / R 8 kb). Examples of CMV / R and CMV / R 8 kb are provided herein.CMV / R is also described in U.S. Patent No. 7,094,598 B2, issued August 22, 2006.

[0043] As used herein, a nucleic acid molecule includes a nucleic acid sequence that encodes a MIC alpha 3-domain peptide immunogen, a ferritin monomer subunit, and / or a MIC alpha 3-ferritin fusion protein of the present invention. A nucleic acid molecule can be produced recombinantly, synthetically, or by a combination of recombinant and synthetic procedures. A nucleic acid molecule of the present disclosure can have a wild-type nucleic acid sequence or a codon-modified nucleic acid sequence, for example, to incorporate a codon that is better recognized by the human translation system. In one embodiment, a nucleic acid molecule can be engineered to introduce or eliminate a codon that encodes a different amino acid, for example, to introduce a codon that encodes an N-linked glycosylation site. Methods for generating a nucleic acid molecule of the present disclosure are known in the art, especially once the nucleic acid sequence is known. It should be recognized that a nucleic acid construct can include one nucleic acid molecule or more than one nucleic acid molecule. It should also be recognized that a nucleic acid molecule can encode one protein or more than one protein.

[0044] In one embodiment, the ferritin monomeric subunit is derived from the ferritin protein of Helicobacter pylori.

[0045] Further embodied in the present invention are nucleic acid sequences that are variants of the nucleic acid sequences encoding the proteins of the present invention. Such variants include nucleotide insertions, deletions, and substitutions, so long as they do not affect the ability of the fusion proteins of the present invention to self-assemble into nanoparticles or significantly affect the ability of the MIC alpha 3-domain portion of the fusion protein to elicit an immune response against the MIC alpha 3-domain protein.

[0046] Further encompassed by the present invention is an expression system for producing the fusion protein of the present invention. In one embodiment, the nucleic acid molecule of the present invention is operably linked to a promoter. As used herein, "operably linked" means that the protein encoded by the linked nucleic acid molecule can be expressed when the linked promoter is activated. Promoters useful for carrying out the present invention are known to those skilled in the art. One embodiment of the present invention is a recombinant cell comprising the nucleic acid molecule of the present invention. One embodiment of the present invention is a recombinant virus comprising the nucleic acid molecule of the present invention.

[0047] As indicated above, recombinant production of the ferritin fusion protein of the present invention can be performed using any suitable conventional recombinant technique currently known in the art. For example, molecular cloning of a fusion protein, such as ferritin and a suitable protein (e.g., recombinant MIC alpha 3-domain protein), can be performed by expression in E. coli using a suitable monomeric subunit protein, such as Helicobacter pylori ferritin monomeric subunit. The construct can then be transformed into a protein-expressing cell, grown to a suitable size, and induced to produce the fusion protein.

[0048] As described, the MIC alpha 3-ferritin fusion proteins of the present invention contain monomeric subunits of ferritin, so that they can self-assemble. According to the present invention, the supramolecules resulting from such self-assembly are called MIC alpha 3-expressing ferritin-based nanoparticles. For ease of discussion, MIC alpha 3-expressing ferritin-based nanoparticles are simply called nanoparticles (np). The nanoparticles of the present invention have the same structural characteristics as the aforementioned ferritin protein. That is, they contain 24 subunits and have 432 symmetry. In the case of the nanoparticles of the present invention, the subunits are fusion proteins comprising ferritin monomeric subunits linked to MIC alpha 3-domain proteins. Such nanoparticles display at least a portion of the MIC alpha 3-domain proteins on their surface. Thus, one aspect of the present invention is a nanoparticle comprising a MIC alpha 3-ferritin fusion protein, where the fusion protein comprises a monomeric ferritin subunit linked to a MIC alpha 3-domain protein. In one aspect, the nanoparticle is octahedral.

[0049] Since the MIC alpha 3-ferritin fusion proteins and nanoparticles of the present invention can induce an immune response against MIC alpha 3-domain proteins, they can be used as vaccines to treat cancer. According to the present invention, the vaccine can be a MIC alpha 3-domain peptide immunogen, a MIC alpha 3-ferritin fusion protein, or a nanoparticle of the present invention. The vaccine of the present invention can also contain other components, such as adjuvants, buffers, etc. Although any adjuvant can be used, preferred embodiments can contain the following: chemical adjuvants, such as aluminum phosphate, benzalkonium chloride, ubenimex, and QS21; genetic adjuvants, such as the IL-2 gene or fragments thereof, the granulocyte macrophage colony stimulating factor (GM-CSF) gene or fragments thereof, the IL-18 gene or fragments thereof, the chemokine (C--C motif) ligand 21 (CCL21) gene or fragments thereof, the IL-6 gene or fragments thereof, CpG, LPS, TLR agonists, and other immune stimulatory genes; protein adjuvants, such as IL-2, or fragments thereof, granulocyte-macrophage colony-stimulating factor (GM-CSF) or fragments thereof, IL-18 or fragments thereof, chemokine (C--C motif) ligand 21 (CCL21) or fragments thereof, IL-6 or fragments thereof, CpG, LPS, TLR agonists, and other immune stimulating cytokines or fragments thereof; lipid adjuvants, such as cationic liposomes, N3 (cationic lipid), monophosphoryl lipid A (MPL1); other adjuvants, such as cholera toxin, enterotoxin, Fms-like tyrosine kinase-3 ligand (Flt-3L), bupivacaine, marcaine, and levamisole.

[0050] Mesoporous Silica The vaccine composition according to the present invention may further comprise an immunization scaffold. In one embodiment, the immunization scaffold is a mesoporous silica nanoparticle (MSR). The MSR may be any shape or form, for example, a rod, a sphere, a wire, a cube, or a polyhedron. The shape or form of the MSR is typically the result of specific reaction conditions. For example, mesoporous silica nanoparticles can be synthesized by any method known in the art, for example, by reacting tetraethyl orthosilicate with a template made of micellar rods. As a result, nano-sized spheres or rods filled with regularly arranged pores are collected. The template can then be removed by washing with a solvent adjusted to an appropriate pH. In another technique, mesoporous particles could be synthesized using a simple sol-gel method or spray drying method. Tetraethyl orthosilicate is also used with an additional polymer monomer (as a template). Other methods include those described in U.S. Patent Application Publication Nos. 20150072009, 20120264599, and 20120256336, which are incorporated by reference herein.

[0051] Granulocyte-macrophage colony-stimulating factor (GM-CSF) Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a protein secreted by macrophages, T cells, mast cells, endothelial cells, and fibroblasts. Specifically, GM-CSF is a cytokine that functions as a white blood cell growth factor. GM-CSF stimulates stem cells to produce granulocytes and monocytes. Monocytes leave the bloodstream and migrate into tissues, where they then mature into macrophages.

[0052] The scaffold device described herein contains and releases GM-CSF polypeptides, attracting host DCs to the device. The intended GM-CSF polypeptides are isolated from endogenous sources or synthesized in vivo or in vitro. Endogenous GM-CSF polypeptides are isolated from healthy human tissues. Synthetic GM-CSF polypeptides are synthesized in vivo after transfection or transformation of template DNA into a host organism or host cell, such as a mammal or cultured human cell line. Alternatively, synthetic GM-CSF polypeptides are synthesized in vitro by polymerase chain reaction (PCR) or other art-recognized methods (e.g., Sambrook, J., Fritsch, EF, and Maniatis, T., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY, Vol. 1, 2, 3 (1989), incorporated herein by reference).

[0053] The GM-CSF polypeptide is modified to increase the protein stability in vivo. Alternatively, the GM-CSF polypeptide is engineered to be more or less immunogenic. Endogenous mature human GM-CSF polypeptide is reportedly glycosylated at amino acid residues 23 (leucine), 27 (asparagine), and 39 (glutamic acid) (see U.S. Pat. No. 5,073,627). The GM-CSF polypeptide of the present invention is modified at one or more of these amino acid residues for glycosylation status.

[0054] The GM-CSF polypeptide is recombinant. Alternatively, the GM-CSF polypeptide is a humanized derivative of a mammalian GM-CSF polypeptide. Exemplary mammalian species from which the GM-CSF polypeptide is derived include, but are not limited to, mouse, rat, hamster, guinea pig, ferret, cat, dog, monkey, or primate. In a preferred embodiment, the GM-CSF is a recombinant human protein (PeproTech, Catalog No. 300-03). Alternatively, the GM-CSF is a recombinant murine (mouse) protein (PeproTech, Catalog No. 315-03). Finally, the GM-CSF is a humanized derivative of a recombinant mouse protein.

[0055] Human recombinant GM-CSF (PeproTech, catalog number 300-03) is encoded by the following polypeptide sequence: TIFF2024177207000004.tif19145.

[0056] Mouse recombinant GM-CSF (PeproTech, Cat. No. 315-03) is encoded by the following polypeptide sequence: TIFF2024177207000005.tif19142.

[0057] Human endogenous GM-CSF is encoded by the following mRNA sequence (NCBI Accession No. NM — 000758 and SEQ ID NO: 28): TIFF2024177207000006.tif81161.

[0058] Human endogenous GM-CSF is encoded by the following amino acid sequence (NCBI Accession No. NP000749.2 and SEQ ID NO: 29): TIFF2024177207000007.tif19157.

[0059] Cytosine-guanosine (CpG) oligonucleotide (CpG-ODN) sequences CpG sites are regions of deoxyribonucleic acid (DNA) in which a cysteine ​​nucleotide occurs next to a guanine nucleotide in the linear sequence of bases along its length (the "p" indicates a phosphate bond between them, distinguishing them from cytosine-guanine complementary base pairing). CpG sites have a crucial role in DNA methylation, one of several endogenous mechanisms used by cells to silence gene expression. Methylation of CpG sites in promoter elements can cause gene silencing. It is known that in the case of cancer, tumor suppressor genes are often silenced and oncogenes or oncogenes are expressed. CpG sites in the promoter regions of tumor suppressor genes (which inhibit cancer formation) have been shown to be methylated, whereas CpG sites in the promoter regions of oncogenes are hypomethylated or unmethylated in certain cancers. The TLR-9 receptor binds to unmethylated CpG sites in DNA.

[0060] The vaccine composition described herein comprises CpG oligonucleotides. The CpG oligonucleotides are isolated from endogenous sources or are synthesized in vivo or in vitro. Exemplary sources of endogenous CpG oligonucleotides include, but are not limited to, microorganisms, bacteria, fungi, protozoa, viruses, molds, or parasites. Alternatively, endogenous CpG oligonucleotides are isolated from mammalian benign or malignant neoplastic tumors. Synthetic CpG oligonucleotides are synthesized in vivo after transfection or transformation of template DNA into a host organism. Alternatively, synthetic CpG oligonucleotides are synthesized in vitro by polymerase chain reaction (PCR) or other art-recognized methods (Sambrook, J., Fritsch, EF, and Maniatis, T., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY, Vol. 1, 2, 3 (1989), incorporated herein by reference).

[0061] CpG oligonucleotides are presented for cellular uptake by dendritic cells. For example, naked CpG oligonucleotides are used. The term "naked" is used to describe isolated endogenous or synthetic polynucleotides (or oligonucleotides) without additional substituents. In another embodiment, CpG oligonucleotides are bound to one or more compounds to increase cellular uptake efficiency. Alternatively or additionally, CpG oligonucleotides are bound to one or more compounds to increase the stability of oligonucleotides in scaffolds and / or dendritic cells. CpG oligonucleotides are optionally concentrated before cellular uptake. For example, CpG oligonucleotides are concentrated with cationic polymer polyethylimine (PEI), which increases cellular uptake efficiency into dendritic cells.

[0062] CpG oligonucleotides can be divided into several classes. For example, the exemplary CpG-ODNs encompassed by the compositions, methods and devices of the present invention are stimulatory, neutral or inhibitory. The term "stimulatory" describes the class of CpG-ODN sequences that activate TLR9. The term "neutral" describes the class of CpG-ODN sequences that do not activate TLR9. The term "inhibitory" describes the class of CpG-ODN sequences that inhibit TLR9. The term "activate TLR9" describes the process by which TLR9 initiates intracellular signaling.

[0063] Stimulatory CpG-ODNs can be further divided into three types, A, B, and C, which differ in their immunostimulatory activity. Type A stimulatory CpG ODNs are characterized by a phosphodiester central CpG-containing palindromic motif and a phosphorothioate 3' poly-G string. Following TLR9 activation, these CpG ODNs induce high levels of IFN-alpha production from plasmacytoid dendritic cells (pDCs). Type A CpG ODNs weakly stimulate TLR9-dependent NF-kappa B signaling.

[0064] Type B stimulatory CpG ODN comprises a complete phosphorothioate backbone with one or more CpG dinucleotides. After TLR9 activation, these CpG-ODNs strongly activate B cells. In contrast to type A CpG-ODN, type B CpG-ODNs weakly stimulate IFN-alpha secretion.

[0065] C-type stimulatory CpG ODN contains the characteristics of A-type and B-type. C-type CpG-ODN contains a complete phosphorothioate backbone and a CpG-containing palindromic motif. Like A-type CpG ODN, C-type CpG ODN induces strong IFN-alpha production from pDC. Like B-type CpG ODN, C-type CpG ODN induces strong B cell stimulation.

[0066] Exemplary stimulatory CpG ODNs include, but are not limited to, ODN 1585, ODN 1668, ODN 1826, ODN 2006, ODN 2006-G5, ODN 2216, ODN 2336, ODN 2395, ODN M362 (all from InvivoGen). The present invention also encompasses any humanized version of the aforementioned CpG ODNs. In one preferred embodiment, the compositions, methods, and devices of the present invention include ODN 1826 (the 5' to 3' sequence is tccatgacgttcctgacgtt (SEQ ID NO: 30), where the CpG element is in bold).

[0067] Neutral or control CpG ODNs that do not stimulate TLR9 are encompassed by the present invention. These ODNs contain the same sequences as their stimulatory counterparts, but contain GpC dinucleotides instead of CpG dinucleotides.

[0068] Exemplary neutral or control CpG ODNs encompassed by the present invention include, but are not limited to, ODN 1585 control, ODN 1668 control, ODN 1826 control, ODN 2006 control, ODN 2216 control, ODN 2336 control, ODN 2395 control, ODN M362 control (all from InvivoGen).The present invention also encompasses any humanized version of the aforementioned CpG ODNs.

[0069] Treatment and Administration Methods The vaccine composition of the present invention is useful for the prevention and treatment of cancer. Thus, the present invention provides a method for preventing cancer in a subject at risk of developing cancer, and a method for treating cancer in a subject in need of such treatment. In one embodiment, the cancer is selected from the group consisting of prostate cancer, multiple myeloma, glioblastoma multiforme, and melanoma. In one embodiment, the cancer is melanoma.

[0070] In one embodiment, the vaccine composition of the present invention is administered to a subject having a cancer associated with overexpression of MICA. Overexpression of MICA can be determined using any method known in the art for measuring the expression level of a protein or corresponding nucleic acid. Such methods include, but are not limited to, Western blot, Northern blot, Southern blot, ELISA, immunoprecipitation, immunofluorescence, flow cytometry, immunocytochemistry, nucleic acid hybridization techniques, nucleic acid reverse transcription, and nucleic acid amplification. In one embodiment, the cancer is selected from the group consisting of melanoma, lung cancer, breast cancer, kidney cancer, ovarian cancer, prostate cancer, pancreatic cancer, gastric cancer, and colon cancer, lymphoma, or leukemia. In one embodiment, the cancer is melanoma. In one embodiment, the cancer is a plasma cell malignancy, such as multiple myeloma (MM) or a pre-malignant condition of plasma cells. In some embodiments, the subject has been diagnosed with cancer or has been diagnosed with a predisposition to develop cancer.

[0071] The vaccine compositions of the invention may be administered separately or as part of a therapeutic regimen or combination therapy, as described below. The vaccine compositions of the invention may also be administered alone or in multiple doses, for example in a prime-boost strategy. In the present context, the term "prime-boost" refers to the use of two different immunogens in succession. The two different immunogens are typically administered consecutively, after a certain period of time, such as 10-30 days or 10-60 days. In one embodiment, the certain period of time is 2-4 weeks. Thus, for example, in one embodiment, the vaccine composition of the invention is administered at time zero and a second vaccine composition of the invention (comprising a different immunogen) is administered after a certain period of time, for example 10-30 days, 10-60 days, or 2-4 weeks.

[0072] The first and second vaccine compositions can be, but need not be, the same composition. Thus, in one embodiment of the invention, the step of administering a vaccine comprises administering a first vaccine composition and then later administering a second vaccine composition.

[0073] In one embodiment, one or more different vaccine compositions of the invention are administered to a subject at multiple sites as described in U.S. Patent No. 8,110,196. Preferably, each site drains to a lymph node or group of lymph nodes. In one embodiment, the vaccine composition of the invention is administered to multiple sites that drain to two or more lymph nodes selected from the group consisting of head and neck lymph nodes, axillary lymph nodes, tracheobronchial lymph nodes, abdominal wall lymph nodes, gastric lymph nodes, ileocolic lymph nodes, and inguinal and infrainguinal lymph nodes. In another embodiment, the site is selected from the group consisting of right arm, left arm, right thigh, left thigh, right shoulder, left shoulder, right breast, left breast, abdomen, right buttock, and left buttock. In one embodiment, the site is or drains to a nonencapsulated cluster of lymphoid tissue selected from the group consisting of tonsils, adenoids, appendix, and Peyer's patches. In one embodiment, the vaccine composition of the invention is administered to the site that drains the spleen.

[0074] In one embodiment, each vaccine composition is administered by a route independently selected from the group consisting of intradermal, subcutaneous, transdermal, intramuscular, oral, rectal, vaginal, inhalation, and combinations thereof. In one embodiment, at least one composition is injected directly into an anatomically distinct lymph node, lymph node cluster, or non-encapsulated cluster of lymphoid tissue.

[0075] Any suitable route of administration, such as intradermal, subcutaneous, intravenous, intramuscular, or mucosal, is encompassed by the method of the present invention. Mucosal administration routes include, but are not limited to, oral, rectal, vaginal, and intranasal administration. In a preferred embodiment, at least one composition is administered transdermally, intradermally, subcutaneously, orally, rectally, vaginally, or by inhalation. Any route approved by the Food and Drug Administration (FDA) can be used for the vaccine composition of the present invention. Exemplary administration methods are described in the FDA's CDER Data Standards Manual, version number 004 (available at fda.give / cder / dsm / DRG / drg00301.htm).

[0076] Preferably, the route of administration is selected to target the composition to a particular site, for example, by direct injection into a lymph node or lymph node cluster, by oral administration to target gastric lymph nodes, by anal administration to target rectal lymph nodes, by inhalation or aerosol to target pulmonary lymph nodes, or by any other suitable route of administration.

[0077] When the method of the invention involves administering vaccine compositions to multiple sites, each composition is preferably administered substantially simultaneously, e.g., within 1-8 hours or during the same physician's visit, hi one embodiment, each composition is administered within 1-2 hours, 1-3 hours, 1-4 hours, or 1-5 hours.

[0078] When the vaccine composition is in the form of a scaffold, the method of vaccinating a subject includes implanting the scaffold composition into the subject, preferably subcutaneously. In some embodiments, the method of vaccinating a subject can include implanting or injecting the scaffold vaccine composition into two or more regions of the subject's anatomy.

[0079] In one embodiment, the method of the invention further comprises administering to the subject antigen-presenting cells sensitized with at least one MIC peptide. In a preferred embodiment, the antigen-presenting cells are dendritic cells.

[0080] In one embodiment, the method further comprises administering one or more adjuvants to the subject. In one embodiment, the one or more adjuvants are selected from the group consisting of oil-based adjuvants, CpG DNA adjuvants, polyinosinic acid:polycytidylic acid (usually abbreviated as poly(I:C)), mineral salt adjuvants, mineral salt gel adjuvants, particulate adjuvants, microparticulate adjuvants, mucosal adjuvants, and cytokines. Such adjuvants can be formulated with the composition of the present invention, or can be administered separately from the composition, for example, before, at the same time, or after the composition is administered to the subject. One or more adjuvants can be covalently bound to the peptide or fusion protein of the present invention. For example, a CpG DNA adjuvant is covalently bound to the peptide or fusion protein of the present invention.

[0081] The methods disclosed herein can be applied to a wide range of species, including humans, non-human primates (e.g., monkeys), horses, cows, pigs, sheep, deer, elk, goats, dogs, cats, weasels, rabbits, guinea pigs, hamsters, rats, and mice.

[0082] The term "treat" or "treating" as used herein refers to partially or completely alleviating, suppressing, improving, and / or relieving the disease or condition suffered by the subject. In some cases, treatment can result in the permanent disappearance of the disease or condition suffered by the subject.

[0083] Generally, the method includes selecting a subject who is at risk of or suffers from a condition or disease.In some cases, the disease or condition of the subject can be treated with the pharmaceutical composition disclosed herein.For example, in some cases, the method includes selecting a subject who has cancer, for example, where the cancer of the subject can be treated by targeting one or both of MICA.

[0084] In some cases, the treatment method can include single administration, multiple administration, and repeated administration as necessary for the prevention or treatment of the disease or condition that the subject suffers from.In some cases, the treatment method can include evaluating the level of disease in the subject before, during, and / or after treatment.In some cases, treatment can be continued until a reduction in the level of disease in the subject is detected.

[0085] The terms "administer", "administering" or "administration" as used herein refer to implanting, absorbing, ingesting, injecting or inhaling the peptide of the present invention, regardless of the form. In some cases, one or more peptides disclosed herein can be administered topically (e.g., intranasally) and / or orally to a subject. For example, the methods described herein include administering an effective amount of a compound or composition of a compound to achieve a desired or stated effect. The specific dosage and treatment regimen for a particular patient will depend on a variety of factors, including, for example, the activity of the particular compound utilized, age, weight, general health, sex, diet, time of administration, rate of excretion, drug combination, severity and course of the disease, condition or symptom, the patient's predisposition to the disease, and the judgment of the treating physician.

[0086] After administration, the subject may be evaluated to detect, assess, or determine the level of disease. In some cases, treatment may be continued until a change (e.g., a decrease) in the level of disease in the subject is detected.

[0087] Upon improvement of the patient's condition (e.g., a change (e.g., a decrease) in the level of disease in the subject), a maintenance dose of the compound, composition or combination of the present invention can be administered as needed. Thereafter, depending on the symptoms, the dosage or frequency of administration, or both, can be reduced until the improved condition is maintained. However, the patient may require intermittent treatment on a long-term basis upon recurrence of disease symptoms.

[0088] In some cases, the present disclosure provides a method for detecting immune cells, such as B cells and / or memory B cells, from a human subject. Such methods can be used to monitor the level of immune cells, such as B cells and / or memory B cells, in a human subject, for example, after an event. Exemplary events include, but are not limited to, detection of disease, infection; administration of a therapeutic composition disclosed herein, administration of a therapeutic agent or treatment regimen, administration of a vaccine, induction of an immune response, etc. Such methods can be used clinically and / or for research.

[0089] Effective Amounts and Dosages In one embodiment, an effective amount of a vaccine composition of the invention is an amount sufficient to reduce the severity of cancer in a patient having cancer, or to reduce or ameliorate the severity of one or more symptoms thereof, to prevent the progression of cancer, to prevent further metastasis of cancer, to cause clinical regression of cancer, or to enhance or improve the therapeutic effect of another therapy or therapeutic agent administered simultaneously with, before or after the vaccine composition of the invention.

[0090] Symptoms of cancer are well known to those of skill in the art and include, but are not limited to, changes in the appearance of a mole, including unusual mole shape, asymmetry, border, color, and / or diameter, newly pigmented skin areas, abnormal moles, dark areas under fingernails, breast lumps, nipple changes, breast cysts, breast pain, death, weight loss, weakness, extreme fatigue, difficulty eating, loss of appetite, chronic cough, worsening shortness of breath, hemoptysis, blood in the urine, blood in the stool, nausea, vomiting, liver metastases, lung metastases, bone metastases, abdominal bloating, swelling, fluid in the abdominal cavity, vaginal bleeding, constipation, abdominal distension, colon perforation, acute peritonitis (infection, fever, pain), pain, vomiting of blood, excessive sweating, fever, high blood pressure, anemia, diarrhea, jaundice, dizziness, chills, muscle cramps, colon metastases, lung metastases, bladder metastases, liver metastases, bone metastases, kidney metastases, and pancreatic metastases, difficulty swallowing, and the like.

[0091] In one embodiment, the effective amount of the vaccine composition of the present invention is sufficient to generate an antibody-secreting B cell or cytotoxic T cell-mediated immune response directed against one or more peptides of the vaccine composition of the present invention.In one embodiment, the effective amount of the vaccine composition of the present invention is sufficient to generate an antibody-secreting B cell or cytotoxic T cell-mediated immune response directed against cancer cells.The ability of the vaccine composition of the present invention to induce an immune response can be determined using any conventional method available to those skilled in the art.In one embodiment, the effective amount of each composition is sufficient to generate a cytotoxic T cell response in a subject, for example, as measured by mixed lymphocyte T cell assay.

[0092] In one embodiment, an effective amount of the vaccine composition administered to a subject or to a particular site in a subject is an amount that delivers 1-1000 micrograms of one or more peptides of the composition. In one embodiment, the amount of peptide is 1-100 micrograms, 1-200 micrograms, 1-300 micrograms, 1-400 micrograms, 1-500 micrograms, 1-600 micrograms, 1-700 micrograms, 1-800 micrograms, or 1-900 micrograms. In another embodiment, the amount of peptide is 1-10 micrograms, 1-20 micrograms, 1-30 micrograms, 1-40 micrograms, 1-50 micrograms, 1-60 micrograms, 1-70 micrograms, 1-80 micrograms, or 1-90 micrograms. In one embodiment, the total amount of peptide administered to a subject does not exceed 5 milligrams. In one embodiment, the total amount of peptide administered to a subject does not exceed 2 milligrams.

[0093] Combination therapy The present invention also provides a method for treating or preventing cancer, comprising administering the vaccine composition of the present invention together with one or more additional therapeutic agents or treatment regimens to a subject in need thereof.In one embodiment, the vaccine composition of the present invention is administered as part of a treatment regimen that includes surgery, chemotherapy, or radiation therapy, immunotherapy, or any combination thereof.

[0094] In one embodiment, the treatment regimen comprises or further comprises one or more immune stimulants. In one embodiment, the one or more immune stimulants are selected from the group consisting of an anti-CTLA-4 antibody or peptide, an anti-PD-1 antibody or peptide, an anti-PDL-1 antibody or peptide, an anti-OX40 (also known as CD134, TNFRSF4, ACT35 and / or TXGP1L) antibody or peptide, an anti-GITR (also known as TNFRSF18, AITR and / or CD357) antibody or peptide, an anti-LAG-3 antibody or peptide, and / or an anti-TIM-3 antibody or peptide.

[0095] In one embodiment, the one or more immunostimulatory agents are selected from the anti-MICA antibodies described in WO 2013 / 049517 or WO 2008 / 036981. In one embodiment, the one or more immunostimulatory agents are selected from CM33322 Ab4, CM33322 Ab28, and CM33322 Ab29, which are described in U.S. Provisional Application Nos. 61 / 792,034 and 61 / 913,198, and U.S. Application No. 14 / 025,573.

[0096] In one embodiment, the treatment regimen comprises or further comprises one or more cytokines. In one embodiment, the vaccine composition of the present invention comprises one or more cytokines. In one embodiment, at least one cytokine is an interleukin or an interferon. In one embodiment, at least one cytokine is an interleukin selected from the group consisting of IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-13, IL-15, and IL-18. In another embodiment, at least one cytokine is an interferon selected from IFN alpha, IFN beta, and IFN gamma.

[0097] In one embodiment, the vaccine composition of the present invention is administered as part of a treatment regimen that includes administering to the subject at least one chemotherapeutic agent selected from the group consisting of histone deacetylase inhibitors ("HDAC") inhibitors, proteasome inhibitors, alkylating agents, and topoisomerase inhibitors.

[0098] In one embodiment, the chemotherapeutic agent is an HDAC inhibitor selected from the group consisting of: hydroxamic acids, vorinostat (Zolinza), suberoylanilide hydroxamic acid (SAHA) (Merck), trichostatin A (TSA), LAQ824 (Novartis), panobinostat (LBH589) (Novartis), belinostat (PXD101) (CuraGen), ITF2357 Italfarmaco SpA (Cinisello), cyclic tetrapeptides, depsipeptides (romidepsin, FK228) (Gloucester Pharmaceuticals), benzamides, entinostat (SNDX-275 / MS-275) (Syndax Pharmaceuticals), MGCD0103 (Celgene), short chain aliphatic acids, valproic acid, phenylbutyric acid, AN-9, pivanex (Titan Pharmaceutical), CHR-3996 (Chroma Therapeutics), and CHR-2845 (Chroma Therapeutics).

[0099] In one embodiment, the chemotherapeutic agent is a proteasome inhibitor selected from the group consisting of bortezomib (Millennium Pharmaceuticals), NPI-0052 (Nereus Pharmaceuticals), carfilzomib (PR-171) (Onyx Pharmaceuticals), CEP 18770, and MLN9708.

[0100] In one embodiment, the chemotherapeutic agent is an alkylating agent, such as mephalan.

[0101] In one embodiment, the chemotherapeutic agent is a topoisomerase inhibitor, such as adriamycin (doxorubicin).

[0102] In one embodiment, the treatment regimen includes or further includes one or more of chemotherapy, radiation therapy, cytokines, chemokines and other biological signaling molecules, tumor-specific vaccines, cellular cancer vaccines (e.g., GM-CSF transduced cancer cells), tumor-specific monoclonal antibodies, autologous and allogeneic stem cell rescue (e.g., to enhance graft-versus-tumor effects), other therapeutic antibodies, molecular targeted therapy, anti-angiogenic therapy, infectious agents for therapeutic purposes (e.g., tumor-localized bacteria), and gene therapy.

[0103] kit The present invention provides a pharmaceutical pack or kit for carrying out the method or treatment regimen of the present invention.In one embodiment, the kit comprises the vaccine composition of the present invention in lyophilized form.In one embodiment, the kit comprises the vaccine composition of the present invention in the form of a protein scaffold.

[0104] In another embodiment, the kit further comprises a cytokine or adjuvant in one or more additional containers.

[0105] The composition in each container may be in the form of a pharma- ceutically acceptable solution, e.g., in combination with sterile saline, dextrose solution, or buffer, or other pharma- ceutically acceptable, sterile liquid. Alternatively, the composition may be lyophilized or dehydrated; in this case, the kit optionally further comprises, in a separate container, a pharma- ceutically acceptable solution, preferably sterile (e.g., saline, dextrose solution, etc.), for reconstituting the composition to make a solution for injection.

[0106] In another embodiment, the kit further comprises one or more reusable or disposable administration devices (e.g., syringes, needles, dispensing pens), preferably packaged in a sterile form, and / or packaged alcohol pads. Optionally, instructions may be included for administration of the composition by the clinician or by the patient. The kit may also comprise other materials, such as metal or plastic foils, such as blister packs.

[0107] In some embodiments, the present disclosure provides methods for using any one or more of the vaccine compositions disclosed herein (shown below as "X") in the following methods:

[0108] Substance X for use as a medicament in treating one or more diseases or conditions disclosed herein (e.g., cancer, referred to in the examples below as "Y"); use of substance X for the manufacture of a medicament for the treatment of Y; and substance X for use in the treatment of Y.

[0109] In some cases, the therapeutic compositions disclosed herein may be formulated for sale in the United States, import into the United States, and / or export from the United States.

[0110] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Methods and materials are described herein for use in the present invention; other suitable methods and materials known in the art can also be used.Materials, methods, and examples are illustrative only and are not intended to be limiting.All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety.In case of conflict, the present specification, including definitions, will control. EXAMPLES

[0111] Example 1: General Method Vector construction Multivalent vaccines induce substantially higher titer antibody responses compared to monovalent proteins. Multivalent display was used herein, where the MICA alpha 3 domain is fused to Helicobacter pylori (H. pylori) ferritin. Ferritin-based nanoparticles have recently been shown to induce high titer antibodies for influenza and EBV vaccines. Ferritin is found in most organisms as an iron storage protein. Ferritin is a self-assembling particle that forms spherical particles with octahedral symmetry, consisting of 24 subunits. See Figures 1C and 1D for a schematic of ferritin particles and maps showing the cellular and humoral immune responses to human and mouse ferritin. The MICA alpha 3 ferritin fusion gene (abbreviated as MICA-ferritin) was generated by fusing the gene encoding the alpha 3 domain of MICA to H. pylori ferritin using a Gly-Ser-Gly linker (Figure 2A). A point mutation (Asn19Gln) was introduced into H. pylori ferritin to remove a potential N-glycosylation site. To determine the antibody response of MICAα3 alone (without ferritin), a deglycosylated version of the MICAα3 gene was generated by mutating 7 of the 8 potential N-glycosylation sites to Asp or Gln. A C-terminal HA tag was introduced for downstream protein purification purposes. The gene was synthesized using the GeneArt® Gene Synthesis platform and codon-optimized for insect cell expression. The synthesized gene was cloned into the pAcDB3 baculovirus expression vector. (See FIG. 2C).

[0112] Assays demonstrating the generation of a MIC alpha 3 domain vaccine and a deglycosylated MICA alpha 3 vaccine are shown in Figures 2B and 2D, respectively.

[0113] Protein Biosynthesis and Purification MICAα3 and MICA-ferritin fusion proteins were expressed in Sf9 (Spodoptera frugiperda) insect cells by infecting these cells with recombinant baculoviruses at a multiplicity of infection of 10. Cells were grown in Sf900 serum-free expression medium (Life Technologies) and culture supernatants were harvested 3 days after transfection. Supernatants were concentrated and then exchanged into Tris buffer (50 mM Tris, 150 mM NaCl, pH 7.5 buffer). Proteins were purified by HA affinity chromatography and aggregates were removed by size exclusion chromatography using a Superose 6 column (GE Healthcare). Purified proteins were buffer exchanged into PBS using a PD-10 desalting column (GE) and concentrated to 1 mg / ml using an Amicon Ultra 4 ml centrifugal filter. Protein purity and size were confirmed by SDS-PAGE.

[0114] MPS vaccine preparation and immunization The scaffold vaccine described herein was recently reported (Kim et al. Nat. Biotechnol.2015, 33, 64-72). Mesoporous silica rods (MSRs) injected with a needle spontaneously organize in vivo to form macroporous structures resembling haystacks, which provide a 3D cellular microenvironment for dendritic cells. This biodegradable scaffold recruits and educates dendritic cells, which then migrate to lymph nodes, where they induce immune responses. 5 mg of MSRs were loaded with 1 μg of GM-CSF (to recruit dendritic cells), 100 μg of CpG oligonucleotides (to induce dendritic cell activation), and 200 μg of MICA-ferritin fusion or control protein (ovalbumin) for 12 hours at room temperature. The particles were lyophilized, resuspended in PBS, and injected subcutaneously into the flank of C57BL / 6 mice. Mice received a boost on days 14 or 21 after the initial immunization. Age-matched non-immunized mice (naive) and mice immunized with ovalbumin were used as control groups for the MICA-ferritin immunization experiments. As an additional control in the MICAα3 experiments, mice were immunized with all vaccine components but without the MSR scaffold (bolus).

[0115] Pulmonary metastasis experiments in MICAα3- and MICA-ferritin-immunized mice C57Bl / 6J mice were immunized with MICAα3 or MICAα-ferritin vaccine. Three weeks after the boost, 0.5x10 6 Mice were challenged with intravenous (iv) injection of MICA-expressing B16F10 melanoma cells. Serum was collected prior to tumor challenge and at weekly intervals and analyzed for shed MICA levels. Mice were euthanized 14 days after tumor challenge; lungs were removed and fixed in 10% neutral buffered formalin, and the number of lung metastases was quantified.

[0116] Flow cytometric analysis and ELISA to determine MICA antibody titers in immunized mice MICA-specific antibody titers were tested by ELISA using the full-length extracellular domain of MICA. Full-length MICA protein (0.2 μg) was coated onto 96-well ELISA plates overnight at 4° C. Plates were blocked with PBS / 2% BSA for 1 h at room temperature. Plates were washed and incubated with serial dilutions of sera taken at weekly intervals from each experimental group. Goat anti-mouse HRP was used as detection antibody. Flow cytometry analysis was used to assess binding of serum antibodies to full-length expressed on the surface of tumor cells. Briefly, 1×10 5 1x10 tumor cells were incubated with 1 μl of serum for 2 hours at 4°C. 5 Cells were stained with 1 μl of serum from non-immunized mice (naive), mice immunized with vaccine components without MSR scaffold (bolus) or MICA-ferritin vaccine (vaccinated) in 100 μl of PBS for 2 hours. Commercially available monoclonal antibody 6D4, which binds to the alpha1-alpha2 domain of MICA, was used as a positive control (10 μg). PE-conjugated anti-mouse IgG was used as the secondary antibody.

[0117] Example 2: Scaffold vaccine for induction of potent immune responses The MICα3 domain was expressed as a recombinant protein in the baculovirus system; the protein was displayed in a multivalent form on H. pylori ferritin, an iron storage protein with 24 identical subunits. Kim et al. Nat. Biotechnol.Herein, we use a vaccination approach using mesoporous silica rods (MSRs), first described in 2015, 33, 64-72, which is incorporated herein by reference in its entirety (see FIG. 3). MSRs, injected subcutaneously with a needle, spontaneously organize in vivo into macroporous structures, which provide a 3D cellular microenvironment for host immune cells. This system recruits large numbers of immune cells, exposes them to relevant antigens, and provides the appropriate molecular cues for the induction of a strong immune response. MICα3 domain proteins were absorbed into the MSRs together with GM-CSF (for dendritic cell recruitment) and CpG oligonucleotides (an adjuvant that activates dendritic cells). This vaccination approach allowed the induction of high titer antibodies specific for the MICα3 domain. These antibodies stained tumor cells expressing MIC and inhibited the shedding of MIC by tumor cells.

[0118] To test the antitumor activity of this vaccine, we utilized B16 melanoma cells transfected with human MIC. When these tumor cells are injected intravenously into non-immunized mice, they form numerous lung metastases (approximately 200 metastases / mouse). The MSR scaffold vaccine provided strong protection from such metastatic outcomes. When the vaccine components were injected as a bolus without the MSR scaffold, partial protection was observed, but the biological effect was significantly weaker. This result indicates that local recruitment of immune cells to the MSR scaffold greatly enhances the activity of this vaccine. (See Figure 4).

[0119] Example 3: Vaccination with MICA-ferritin fusion protein induces high titers of MICA-specific antibodies The binding of MICAα3-specific antibodies in the serum of immunized mice to full-length MICA expressed on the surface of B16F10 mouse melanoma cells was examined by flow cytometry. 5Cells were stained with 1 μl of serum from non-immunized mice (naive), mice immunized with control vaccine (OVA-protein) or MICA-ferritin vaccine (vaccinated) from days 14, 28 and 42 in 100 μl of PBS for 2 hours. Commercially available monoclonal antibody 6D4, which binds to the α1-α2 domain of MICA, was used as a positive control (10 μg). PE-conjugated anti-mouse IgG was used as a secondary antibody. MICAα3-specific antibodies (histograms - green (d14), blue (d28), red (d42)) in the serum of vaccinated mice showed significant binding to MICA expressed on the tumor cell surface (Figures 5A and 5B). The results of these assays demonstrate that MICA-ferritin fusion protein vaccination induces high titers of MICA-specific antibodies.

[0120] Example 4: Vaccination with MICA-ferritin fusion protein generates high levels of IGG1, IGG2A and IGG3 MICAA3-specific polyclonal antibody responses Sera from MICA-ferritin immunized mice were tested in ELISA to determine the different subclasses of IgG induced by vaccination. Sera from mice immunized with OVA-protein (bolus) or non-immunized mice (naive) were used as control groups. Full-length MICA was used as capture antigen and 1 / 1000 serum dilution was used in each well. HRP-conjugated anti-mouse IgG1, IgG2a, IgG2b or IgG3 were used for detection. Immunization with MICA-ferritin (vaccination) was found to induce high levels of all IgG subclasses tested (see Figure 6).

[0121] Example 5: Polyclonal antibodies raised in response to a MICA-ferritin vaccine prevent MICA shedding from the surface of human metastatic melanoma cell lines Induction of MICA antibodies in melanoma patients treated with autologous tumor vaccine (GVAX) and ipilimumab correlated with decreased serum soluble MICA (sMICA) levels. The extracellular portion of MICA / B contains two MHC class I-like domains (α1 and α2) and a membrane-proximal immunoglobulin domain (α3). It was shown that disulfide isomerase ERp5 cleaves the structural disulfide bond in the MICAα3 domain, and the resulting unfolding of this domain allows proteolytic cleavage by ADAM 10, ADAM 17 and MMP-14. The purpose of this assay was to determine whether polyclonal antibodies raised in response to MICA-ferritin vaccines would prevent MICA shedding from the human melanoma tumor cell line A375.

[0122] For these assays, 4x10 5 A375 melanoma cells were plated in 96-well plates with 200 μl of medium. The cells were incubated for 24 hours without serum or with serum from naive, OVA-protein immunized, or MICA-ferritin vaccinated mice (FIG. 7, bars with inverted triangles). sMICA in the supernatants was analyzed using a MICA ELISA kit that utilizes MICAα1-α2 domain antibodies for capture and detection. Lower levels of sMICA were detected in the supernatants of cells incubated with serum from MICA-ferritin vaccinated mice (FIG. 7, bars with inverted triangles) compared to cells incubated without serum and with serum from naive (FIG. 7, bars with circles and squares) or OVA-protein immunized mice (FIG. 7, bars with triangles), thus indicating that MICAα3-specific antibodies can inhibit the shedding of MICA from the tumor cell surface.

[0123] Example 6: Therapeutic Activity of the MICA-Ferritin Vaccine The therapeutic activity of the MICA-ferritin vaccine was tested using an aggressive B16F10 melanoma tumor model. B16F10 melanoma tumor cells were genetically modified to express human MICA. MICA is bound by the mouse NKG2D receptor, and therefore is a suitable model system. C57BL / 6 mice immunized with MICA-ferritin vaccine, OVA-protein vaccine (control antigen), and non-immunized control mice (age- and sex-matched) were challenged with iv injections of B16F10-MICA tumor cells. Serum was collected before tumor challenge, on days 7 and 13. Mice were euthanized 14 days after tumor challenge, and the number of lung metastases was quantified (see Figure 8A).

[0124] For these assays, 8-week-old C57BL / 6 female mice were immunized with MICA alpha 3-ferritin or ova-protein followed by a boost on day 28. Three weeks later, 5x10 5 Mice were challenged iv with 1000 MICA-expressing B16F10 melanoma cells. Mice were euthanized 14 days after tumor challenge and the number of lung metastases was quantified. Serum shed MICA (sMICA) levels were monitored by ELISA. These experiments demonstrated that MICA-ferritin vaccinated mice were nearly tumor-free. In contrast, non-immunized age-matched control groups (naive) and mice vaccinated with the control antigen - ovalbumin had numerous lung metastases (average of approximately 150 lung metastases / mouse) (Figure 8A). Importantly, sMICA was undetectable in the serum of mice immunized with the MICA-ferritin vaccine (triangles), whereas high levels of sMICA were detected in the serum of ovalbumin immunized mice (squares) and non-immunized control groups (circles) within 2 weeks of tumor challenge (Figure 8B).

[0125] Example 7: Determination of Effective Dosage of MICA-Ferritin Vaccine and Kinetics of Polyclonal Antibody Response In Vivo For these studies, mice received two injections of vaccine before tumor cell challenge. However, nearly maximal antibody levels are already achieved 2 weeks after primary immunization. To determine the optimal vaccination dosage and the kinetics of polyclonal antibody responses at different doses, C57Bl / 6J mice were immunized with different doses of MICA-ferritin protein (50-200 μg) absorbed into MSR. On day 17, mice received a boost. Serum was collected at weekly intervals by retro-orbital bleeding and MICA antibody titers were determined by ELISA. On day 25 after primary immunization, mice were challenged with an iv injection of MICA-expressing B16F10 melanoma cells. Serum was collected before tumor challenge and at weekly intervals to analyze shed MICA levels. Mice were euthanized 14 days after tumor challenge; lungs were removed and fixed in 10% neutral buffered formalin, and the number of lung metastases was quantified.

[0126] For these studies, 8-week-old C57BL / 6 female mice were immunized with MICA alpha 3-ferritin vaccine at various doses (50 μg, 100 μg, or 200 μg) and boosted on day 17. End-point antibody titers were determined by serially diluting sera and testing their binding to full-length MICA protein by ELISA. MICA-ferritin immunized mice elicited high levels of antibody titers by day 14 at all doses tested (10 5 ELISA endpoint titers), which increased approximately 1000-fold after boosting on day 17. Naive, untreated age-matched mice were used as a control group (see FIG. 9A).

[0127] On the 25th day after the first immunization, 0.5x10 6Mice were challenged with iv injection of 100 μg of MICA-expressing B16F10 melanoma cells. Serum was collected prior to tumor challenge and at weekly intervals to analyze shed MICA levels. Mice were euthanized 14 days after tumor challenge; lungs were removed and fixed in 10% neutral buffered formalin, and the number of lung metastases was quantified. Mice immunized with 100 μg and 200 μg were nearly tumor-free compared to mice immunized with 50 μg of vaccine (approximately 2-12 lung metastases). sMICA was undetectable in the serum of mice immunized with various doses of MICA-ferritin vaccine (50 μg - squares, 100 μg - up-pointing triangles, 200 μg - down-pointing triangles), whereas high levels of sMICA were detected in the serum of non-immunized control groups (open circles) within 2 weeks of tumor challenge (see Figures 9B and 9C).

[0128] Example 8: MICAA3 vaccine alone induces high titers of MICA-specific antibodies To determine the effect of MICAα3 vaccine alone (without ferritin) in generating MICA-specific polyclonal antibody responses, a deglycosylated version of the MICAα3 gene was generated by mutating seven of the eight potential N-glycosylation sites to Asp or Gln. Following protein production and purification as described in the Methods section, the MICAα3 vaccine was prepared by loading 5 mg of MSR with 1 μg GM-CSF, 100 μg CpG-ODN and 150 μg of deglycosylated MICAα3 protein (abbreviated as MICAα3 vaccine). The particles were then lyophilized, resuspended in cold PBS (150 μl) and injected subcutaneously into the flank of female C57Bl / 6J mice. Mice immunized with all vaccine components (bolus) except without the MSR scaffold, and age-matched untreated mice were used as control groups. Serum was collected at weekly intervals by retro-orbital bleeding. Mice received a boost on day 28 after the initial immunization.

[0129] For these studies, 1x10 5MICA009-expressing B16F10 melanoma cells were stained with 1 μl of serum from non-immunized mice (naive), mice immunized with MICAα3 without MSR (bolus) or MICAα3 vaccine (vaccinated) in 100 μl of PBS for 2 hours. Commercially available monoclonal antibody 6D4, which binds to the α1-α2 domain of MICA, was used as a positive control (10 μg). PE-conjugated anti-mouse IgG was used as a secondary antibody. MICAα3-specific antibodies in the serum of vaccinated mice and the bolus group showed significant binding to MICA expressed on the tumor cell surface after boosting at levels similar to those of the positive control group (see FIG. 10A).

[0130] Sera from MICAα3-immunized mice were tested in ELISA to determine the different subclasses of IgG induced by vaccination. Sera from non-immunized mice were used as control groups. Full-length MICA was used as capture antigen, and 1 / 1000 serum dilutions were used in each well. HRP-conjugated anti-mouse IgG1, IgG2a, IgG2b or IgG3 were used for detection. It was found that immunization with MICAα3 vaccine and bolus vaccine induced the production of all IgG subclasses tested, with IgG1 levels being higher than with MICA-ferritin vaccine (see FIG. 10B).

[0131] Example 8: MICAA3 vaccine alone (without ferritin fusion) shows significant therapeutic benefit in vivo For these studies, 8-week-old C57Bl / 6J female mice were immunized with a bolus of either the MICAα3 vaccine or all vaccine components except for the MSR scaffold. Naive, age-matched C57Bl / 6J female mice served as controls. Three weeks after the boost, 0.5x10 6Mice were challenged with an iv injection of 100 MICA-expressing B16F10 melanoma cells. Mice were euthanized 14 days after tumor challenge; lungs were removed and fixed in 10% neutral buffered formalin, and the number of lung metastases was quantified. MICAα3-vaccinated mice were nearly tumor-free compared to naive, age-matched controls. The number of lung metastases was significantly lower in the bolus group (approximately 100-125) compared to the non-immunized group (approximately 200-250) (see Figure 11A).

[0132] sMICA was undetectable in the serum of mice immunized with the MICAα3 vaccine (triangles), while elevated levels of sMICA were seen in the untreated control group (circles) within 2 weeks after tumor challenge. The bolus group had relatively lower levels of sMICA in serum compared to the control group (squares). (See FIG. 11B). The increased number of lung metastases and sMICA levels in mice immunized with the MICAα3 bolus compared to the vaccinated group are most likely due to the reduced levels of MICA-specific antibody titers observed by day 62 after the first immunization compared to the vaccinated group (data not shown).

[0133] Example 9: Determination of Cytotoxic Lymphocyte Populations Required for Vaccine Efficacy By depleting CD8 T cells or NK cells with mAb, we found that both CD8 T cells and NK cells contribute to the therapeutic effect of the vaccine (FIGS. 13A-13B and 14A-14B).

[0134] Example 10: ELISA assay for quantification of MIC antibodies An ELISA assay is used for quantification of MIC antibodies induced by the vaccine (Figure 6).

[0135] Example 11: Further studies Below are upcoming studies that will be conducted to further evaluate the vaccine performance. 1. Optimize the vaccine formulation by testing optimal amounts of antigen and comparing two adjuvants, CpG oligonucleotides and poly(I:C). 2. Vaccine efficacy is tested in multiple tumor models, specifically B16-MIC melanoma model (both subcutaneous and metastatic models) and orthotopic TRAMP-MIC model of prostate cancer. These studies involve measuring vaccine efficacy by evaluating inhibition of tumor growth and reduction of serum shedding MIC. 3. Serum is transferred from immunized mice to non-immunized recipients to determine whether the MIC-specific antibodies induced are sufficient for the protection conferred by the vaccine. 4. Determine whether the vaccine provides protection against secondary challenge with tumor cells that lack MIC expression due to induction of CD8 T cell responses against other tumor antigens. Mice surviving the B16-MIC metastasis model are challenged by iv injection of high doses of B16 tumor cells that either express or do not express the MIC.

[0136] Further investigations into biomarkers that reflect the mechanistic activity of the induced antibodies will be performed. These include the following approaches: 1. ELISA assay for shedding MIC in serum; assays are available and will be rigorously tested on serum samples from patients with advanced cancer. 2. Testing the functional activity of induced MIC α3 domain antibodies. We will investigate which human tumor cell lines are optimal for assays evaluating antibody-mediated inhibition of MIC shedding (a panel of cell lines is available). 3. Flow cytometric analysis of immune cells in peripheral blood and tumor biopsies. Of particular importance is quantification of surface NKG2D levels by CD8 T cells and NK cells; antibodies are available and panels are being optimized.

[0137] Example 12: Baculovirus expression of MICA002 alpha3 fused to ferritin (H. pylori) Objective: Insect cell expression of MICA(002) alpha 3 fused to ferritin nanoparticles General design: signal peptide, 6 his tag, linker, N-terminal HA peptide, MICA alpha 3 domain (*002:01), GSG linker, H. pylori ferritin, stop codon TIFF2024177207000008.tif36147

[0138] Strategy: Cloning into the pacDB3 vector between the SmaI and BamHI sites Strategy: Cloning into the pacDB3 vector Translation of DNAMAN18(1-945) Universal genetic code Total amino acids: 314, MW=35668 Max ORF:1-942, 314 AA, MW=35668 TIFF2024177207000009.tif187149

[0139] SEQ ID NO: 945 bp; Composition 254 A; 252 C; 241 G; 198 T; 0 Other Percentages: 26.9% A; 26.7% C; 25.5% G; 21.0% T; 0.0% Other Molecular weight (kDa):ssDNA: 291.81 dsDNA: 582.6 origin TIFF2024177207000010.tif127144

[0140] Step 1. Amplify the template for PCR 1 (signal peptide, 6 his, linker, HA, MICA alpha 3) from the C1347 construct using the following primers: Forward primer # Ferritin_baculo_Smalif TIFF2024177207000011.tif3128 Internal reverse primer:# ferritinbaculo_IRev TIFF2024177207000012.tif3147

[0141] Step 2. Amplify template for PCR 2 (ferritin) from C1347 using the following: Internal forward primer:# ferritin baculo_IF TIFF2024177207000013.tif4148Reverse primer:# ferritin baculo_BamHIRev TIFF2024177207000014.tif4137

[0142] Step 3: Fusion PCR using primers. # Ferritin_baculo_SmaIfor and # Ferritin baculo_BamHIRev

[0143] Restriction analysis of DNAMAN18 Methylation: dam-No dcm-No 117 enzymes were screened and 18 sites were found. TIFF2024177207000015.tif175128

[0144] List by part TIFF2024177207000016.tif35133

[0145] non-cleaving enzyme TIFF2024177207000017.tif67142

[0146] Ferritin from H. pylori TIFF2024177207000018.tif12143N19 position has been changed to Q (to eliminate an N-linked glycosylation site), starting at position 5 (underlined).

[0147] Ferritin [Helicobacter pylori] TIFF2024177207000019.tif191147

[0148] Example 11: Expression of deglycosylated MICA 002 protein in insect cells Objective: Baculovirus expression of deglycosylated MICA alpha 3 (*002:01) General design: Signal peptide, N-terminal HA peptide, MICA alpha 3 domain (*002:01), stop codon TIFF2024177207000020.tif20159

[0149] Strategy: Cloning into pAcDB3 BglII-EcoRI sites SEQ ID NO:432 bp; Composition 96 A; 125 C; 122 G; 89 T; 0 Other Percentages: 22.2% A; 28.9% C; 28.2% G; 20.6% T; 0.0% Other Molecular weight (kDa): ssDNA: 133.37 dsDNA: 266.4 origin TIFF2024177207000021.tif32153

[0150] Translation of DNAMAN1(1-432) Universal genetic code Total amino acids: 143, MW=15928 Max ORF:1-429, 143 AA, MW=15928 TIFF2024177207000022.tif92149

[0151] Restriction analysis of DNAMAN1 Methylation: dam-No dcm-No 117 enzymes were screened and 5 sites were found. TIFF2024177207000023.tif47128

[0152] List by part TIFF2024177207000024.tif7152

[0153] non-cleaving enzyme TIFF2024177207000025.tif75144

[0154] 4099: MICA002_baculo_BglIIfor TIFF2024177207000026.tif3130

[0155] 4100: MICA002_baculo_EcoRIRev TIFF2024177207000027.tif3128

[0156] Other Aspects Although the present invention has been described in conjunction with its detailed description, the foregoing description is intended to be illustrative of the invention and is not intended to limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0157] Sequence information SEQUENCE LISTING <110> Dana-Farber Cancer Institute, Inc. <120> VACCINATION WITH MICA / B ALPHA 3 DOMAIN FOR THE TREATMENT OF CANCER <150> US 62 / 263,377 <151> 2015-12-04 <150> US 62 / 422,454 <151> 2016-11-15 <160> 30 <170> PatentIn version 3.5 <210> 1 <211> 360 <212> PRT <213> Homo sapiens <400> 1 His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly Ser Val 1 5 10 15 Gln Ser Gly Phe Leu Ala Glu Val His Leu Asp Gly Gln Pro Phe Leu 20 25 30 Arg Tyr Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln Trp Ala 35 40 45 Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg Asp Leu 50 55 60 Thr Gly Asn Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile Lys Asp 65 70 75 80 Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys Glu Ile 85 90 95 His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr Asp Gly 100 105 110 Glu Leu Phe Leu Ser Gln Asn Val Glu Thr Glu Glu Trp Thr Val Pro 115 120 125 Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn Phe Leu 130 135 140 Lys Glu Asp Ala Met Lys Thr Lys Thr His Tyr His Ala Met His Ala 145 150 155 160 Asp Cys Leu Gln Glu Leu Arg Arg Tyr Leu Glu Ser Ser Val Val Leu 165 170 175 Arg Arg Thr Val Pro Pro Met Val Asn Val Thr Arg Ser Glu Ala Ser 180 185 190 Glu Gly Asn Ile Thr Val Thr Cys Arg Ala Ser Ser Phe Tyr Pro Arg 195 200 205 Asn Ile Thr Leu Thr Trp Arg Gln Asp Gly Val Ser Leu Ser His Asp 210 215 220 Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Asn Gly Thr Tyr Gln 225 230 235 240 Thr Trp Val Ala Thr Arg Ile Cys Gln Gly Glu Glu Gln Arg Phe Thr 245 250 255 Cys Tyr Met Glu His Ser Gly Asn His Ser Thr His Pro Val Pro Ser 260 265 270 Gly Lys Val Leu Val Leu Gln Ser His Trp Gln Thr Phe His Val Ser 275 280 285 Ala Val Ala Ala Ala Ala Ala Ala Ile Phe Val Ile Ile Ile Phe Tyr 290 295 300 Val Arg Cys Cys Lys Lys Lys Thr Ser Ala Ala Glu Gly Pro Glu Leu 305 310 315 320 Val Ser Leu Gln Val Leu Asp Gln His Pro Val Gly Thr Ser Asp His 325 330 335 Arg Asp Ala Thr Gln Leu Gly Phe Gln Pro Leu Met Ser Ala Leu Gly 340 345 350 Ser Thr Gly Ser Thr Glu Gly Ala 355 360 <210> 2 <211> 358 <212> PRT <213> Homo sapiens <400> 2 Pro His Ser Leu Arg Tyr Asn Leu Met Val Leu Ser Gln Asp Gly Ser 1 5 10 15 Val Gln Ser Gly Phe Leu Ala Glu Gly His Leu Asp Gly Gln Pro Phe 20 25 30 Leu Arg Tyr Asp Arg Gln Lys Arg Arg Ala Lys Pro Gln Gly Gln Trp 35 40 45 Ala Glu Asp Val Leu Gly Ala Lys Thr Trp Asp Thr Glu Thr Glu Asp 50 55 60 Leu Thr Glu Asn Gly Gln Asp Leu Arg Arg Thr Leu Thr His Ile Lys 65 70 75 80 Asp Gln Lys Gly Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys Glu 85 90 95 Ile His Glu Asp Ser Ser Thr Arg Gly Ser Arg His Phe Tyr Tyr Asp 100 105 110 Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Gln Glu Ser Thr Val 115 120 125 Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Thr Asn Phe 130 135 140 Trp Lys Glu Asp Ala Met Lys Thr Lys Thr His Tyr Arg Ala Met Gln 145 150 155 160 Ala Asp Cys Leu Gln Lys Leu Gln Arg Tyr Leu Lys Ser Gly Val Ala 165 170 175 Ile Arg Arg Thr Val Pro Pro Met Val Asn Val Thr Cys Ser Glu Val 180 185 190 Ser Glu Gly Asn Ile Thr Val Thr Cys Arg Ala Ser Ser Phe Tyr Pro 195 200 205 Arg Asn Ile Thr Leu Thr Trp Arg Gln Asp Gly Val Ser Leu Ser His 210 215 220 Asn Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Gly Thr Tyr Gln 225 230 235 240 Thr Trp Val Ala Thr Arg Ile Arg Gln Gly Glu Glu Gln Arg Phe Thr 245 250 255 Cys Tyr Met Glu His Ser Gly Asn His Gly Thr His Pro Val Pro Ser 260 265 270 Gly Lys Ala Leu Val Leu Gln Ser Gln Arg Thr Asp Phe Pro Tyr Val 275 280 285 Ser Ala Ala Met Pro Cys Phe Val Ile Ile Ile Ile Leu Cys Val Pro 290 295 300 Cys Cys Lys Lys Lys Thr Ser Ala Ala Glu Gly Pro Glu Leu Val Ser 305 310 315 320 Leu Gln Val Leu Asp Gln His Pro Val Gly Thr Gly Asp His Arg Asp 325 330 335 Ala Ala Gln Leu Gly Phe Gln Pro Leu Met Ser Ala Thr Gly Ser Thr 340 345 350 Gly Ser Thr Glu Gly Ala 355 <210> 3 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> Artificial polypeptide <400> 3 Arg Thr Val Pro Pro Met Val Asn Val Thr Arg Ser Glu Ala Ser Glu 1 5 10 15 Gly Asn Ile Thr Val Thr Cys Arg Ala Ser Gly Phe Tyr Pro Trp Asn 20 25 30 Ile Thr Leu Ser Trp Arg Gln Asp Gly Val Ser Leu Ser His Asp Thr 35 40 45 Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Asn Gly Thr Tyr Gln Thr 50 55 60 Trp Val Ala Thr Arg Ile Ser Gln Gly Glu Glu Gln Arg Phe Thr Cys 65 70 75 80 Tyr Met Glu His Ser Gly Asn His Ser Thr His Pro Val Pro Ser Gly 85 90 95 Lys Val Leu Val Leu Gln Ser His Trp Gln Thr Phe His 100 105 <210> 4 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> Artificial polypeptide <400> 4 Arg Thr Val Pro Pro Met Val Gln Val Thr Arg Ser Glu Ala Ser Glu 1 5 10 15 Gly Gln Ile Thr Val Thr Cys Arg Ala Ser Gly Phe Tyr Pro Trp Asn 20 25 30 Ile Asn Leu Ser Trp Arg Gln Asp Gly Val Ser Leu Ser His Asp Thr 35 40 45 Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Asn Gly Thr Tyr Gln Thr 50 55 60 Trp Val Ala Thr Arg Ile Ser Gln Gly Glu Glu Gln Arg Phe Thr Cys 65 70 75 80 Tyr Met Glu His Ser Gly Gln His Ser Thr His Pro Val Pro Ser Gly 85 90 95 Lys Val Leu Val Leu Gln Ser His Trp Gln Thr Phe His 100 105 <210> 5 <211> 314 <212> PRT <213> Artificial Sequence <220> <223> Artificial polypeptide <400> 5 Met Val Pro Cys Thr Leu Leu Leu Leu Leu Ala Ala Ala Leu Ala Pro 1 5 10 15 Thr Gln Thr Arg Ala His His His His His His Ser Lys Ser Tyr Pro 20 25 30 Tyr Asp Val Pro Asp Tyr Ala Arg Thr Val Pro Pro Met Val Asn Val 35 40 45 Thr Arg Ser Glu Ala Ser Glu Gly Asn Ile Thr Val Thr Cys Arg Ala 50 55 60 Ser Gly Phe Tyr Pro Trp Asn Ile Thr Leu Ser Trp Arg Gln Asp Gly 65 70 75 80 Val Ser Leu Ser His Asp Thr Gln Gln Trp Gly Asp Val Leu Pro Asp 85 90 95 Gly Asn Gly Thr Tyr Gln Thr Trp Val Ala Thr Arg Ile Ser Gln Gly 100 105 110 Glu Glu Gln Arg Phe Thr Cys Tyr Met Glu His Ser Gly Asn His Ser 115 120 125 Thr His Pro Val Pro Ser Gly Lys Val Leu Val Leu Gln Ser His Trp 130 135 140 Gln Thr Phe His Gly Ser Gly Asp Ile Ile Lys Leu Leu Asn Glu Gln 145 150 155 160 Val Asn Lys Glu Met Gln Ser Ser Asn Leu Tyr Met Ser Met Ser Ser 165 170 175 Trp Cys Tyr Thr His Ser Leu Asp Gly Ala Gly Leu Phe Leu Phe Asp 180 185 190 His Ala Ala Glu Glu Tyr Glu His Ala Lys Lys Leu Ile Ile Phe Leu 195 200 205 Asn Glu Asn Asn Val Pro Val Gln Leu Thr Ser Ile Ser Ala Pro Glu 210 215 220 His Lys Phe Glu Gly Leu Thr Gln Ile Phe Gln Lys Ala Tyr Glu His 225 230 235 240 Glu Gln His Ile Ser Glu Ser Ile Asn Asn Ile Val Asp His Ala Ile 245 250 255 Lys Ser Lys Asp His Ala Thr Phe Asn Phe Leu Gln Trp Tyr Val Ala 260 265 270 Glu Gln His Glu Glu Glu Val Leu Phe Lys Asp Ile Leu Asp Lys Ile 275 280 285 Glu Leu Ile Gly Asn Glu Asn His Gly Leu Tyr Leu Ala Asp Gln Tyr 290 295 300 Val Lys Gly Ile Ala Lys Ser Arg Lys Ser 305 310 <210> 6 <211> 945 <212> DNA <213> Artificial Sequence <220> <223> Artificial polynucleotide <400> 6 atggtcccct gtaccctgct gctgctgctg gctgctgcac tggcacctac tcagactcgg 60 gcccaccatc atcaccatca ctcaaaagt tacccctacg atgtccccga ctacgccagg 120 accgtgcccc ctatggtgaa cgtcacacgc tcagaagcta gcgagggcaa tatcaccgtg 180 acatgccgag catctgggtt ctatccttgg aacattacac tgagttggag gcaggacggg 240 gtgtccctgt ctcacgatac tcagcagtgg ggcgacgtgc tgccagatgg caatgggacc 300 taccagacat gggtggctac tcggatctcc cagggggagg aacagagatt cacctgctat 360 atggagcata gtggaacca ctcaacacat cctgtgccat ctggcaaggt gctggtcctg 420 cagagtcact ggcagacatt tcatggatca ggcgatatca ttaagctgct gaacgaacag 480 gtgaacaagg agatgcagtc tagtaacctg tacatgagca tgtcaagctg gtgttataca 540 cactccctgg acggagccgg cctgttcctg tttgatcacg ccgctgagga atacgaacat 600 gtaagaaac tgatcatttt cctgaatgag aacaatgtgc cagtccagct gactagcatt 660 tccgcacccg aacacaagtt cgagggcctg acccagatct ttcagaaagc ctacgaacac 720 gagcagcata tctctgaaag tatcacaac atcgtggacc acgcaatca gagacaagat 780 catgccacct tcactttct gcagtgtac gtggccgagc agcacgagga agaggtcctg 840 tttaggaca ttctggataa aatcgaactg attggcaatg agaatcacgg gctgtacctg 900 gcagatcagt atgtcaaggg catcgcaag tcaggaat catga 945 <210> 7 <211> 314 <212> PRT <213> Artificial Sequence <220> <223> Artificial polypeptide <400> 7 Met Val Pro Cys Thr Leo Leo Leo Leo Leo Wing Wing Leu Wing Pro 1 5 10 15 Thr Gln Thr Arg Ala His His His His His Ser Lys Ser Tyr Pro 20 25 30 Tyr Asp Val Pro Asp Tyr Ala Arg Thr Val Pro Met Val Asn Val 35 40 45 Thr Arg Ser Glu Ala Serves as Glu Gly Asn With Thr Val Thr Cys Arg Ala 50 55 60 Ser Gly Phe Tyr Pro Trp Asn Ile Thr Leu Ser Trp Arg Gln Asp Gly 65 70 75 80 Val Ser Leu Ser His Asp Thr Gln Gln Trp Gly Asp Val Leu Pro Asp 85 90 95 Gly Asn Gly Thr Tyr Gln Thr Trp Val Ala Thr Arg Ile Ser Gln Gly 100 105 110 Glu Glu Gln Arg Phe Thr Cys Tyr Met Glu His Ser Gly Asn His Ser 115 120 125 Thr His Pro Val Pro Ser Gly Lys Val Leu Val Leu Gln Ser His Trp 130 135 140 Gln Thr Phe His Gly Ser Gly Asp Ile Ile Lys Leu Leu Asn Glu Gln 145 150 155 160 Val Asn Lys Glu Met Gln Ser Ser Asn Leu Tyr Met Ser Met Ser Ser 165 170 175 Trp Cys Tyr Thr His Ser Leu Asp Gly Ala Gly Leu Phe Leu Phe Asp 180 185 190 His Ala Ala Glu Glu Tyr Glu His Ala Lys Lys Leu Ile Ile Phe Leu 195 200 205 Asn Glu Asn Asn Val Pro Val Gln Leu Thr Ser Ile Ser Ala Pro Glu 210 215 220 His Lys Phe Glu Gly Leu Thr Gln Ile Phe Gln Lys Ala Tyr Glu His 225 230 235 240 Glu Gln His Protein Ser Glu Ser Protein Asn Protein Val Asp Protein Protein 245 250 255 Lys Ser Lys Asp His Ala Thr Phe Asn Phe Leu Gln Trp Tyr Val Ala 260 265 270 Glu Gln His Glu Glu Glu Val Leu Phe Lys Asp Ile Leu Asp Lys Ile 275 280 285 Leu Glu With Gly Asn Glu Asn His Gly Leu Tyr Leu Asp Gln Tyr 290,295,300 Val Lys Gly Ile Ala Lys Ser Arg Lys Ser 305 310 <210> 8 <211> 945 <212> DNA <213> Artificial Sequence <220> <223> Artificial polynucleotide <400> 8 atggtcccct gtaccctgct gctgctgctg gctgctgcc tgcacctc tcagactcgg 60 gcccaccatc atcaccatca ctcaaaagt tacccctacg atgtccccga ctacgccagg 120 accgtgcccc ctatggtgaa cgtcacacgc tcagaagcta gcgagggcaa tatcaccgtg 180 acatgccgag catctgggtt ctatccttgg aacattacac tgagttggag gcaggacggg 240 gtgtccctgt ctcacgatac tcagcagtgg ggcgacgtgc tgccagatgg caatgggacc 300 taccagacat gggtggctac tcggatctcc cagggggagg aacagagatt cacctgctat 360 atggagcata gtggaacca ctcaacacat cctgtgccat ctggcaaggt gctggtcctg 420 cagagtcact ggcagacatt tcatggatca ggcgatatca ttaagctgct gaacgaacag 480 gtgaacaagg agatgcagtc tagtaacctg tacatgagca tgtcaagctg gtgttataca 540 cactccctgg acggagccgg cctgttcctg tttgatcacg ccgctgagga atacgaacat 600 gtaagaaac tgatcatttt cctgaatgag aacaatgtgc cagtccagct gactagcatt 660 tccgcacccg aacacaagtt cgagggcctg acccagatct ttcagaaagc ctacgaacac 720 gagcagcata tctctgaaag tatcaacaac atcgtggacc acgcaatcaa gagcaaagat 780 catgccacct tcaactttct gcagtggtac gtggccgagc agcacgagga agaggtcctg 840 900 gcagatcagt atgtcaaggg catcgcaaag tcaaggaat catga 945 <210> 9 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Artificial polynucleotide <400> 9 aaaaaacccg ggatggtccc ctgtaccctg ctgctgctgc 40 <210> 10 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Artificial polynucleotide <400> 10 gttcgttcag cagcttaatg atatcgcctg atccatgaaa tgtctgccag 50 <210> 11 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Artificial polynucleotide <400> 11 ctggcagaca tttcatggat caggcgatat cattaagctg ctgaacgaac 50 <210> 12 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Artificial polynucleotide <400> 12 aaaaaaggat cctcatgatt tccttgactt tgcgatgccc ttg 43 <210> 13 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> Artificial polynucleotide <220> <221> misc_feature <222> (7)..(10) <223> n is a, c, g, or t <400> 13 acctgcnnnn 10 <210> 14 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> Artificial polynucleotide <220> <221> misc_feature <222> (4)..(8) <223> n is a, c, g, or t <400> 14 gacnnnnngt c 11 <210> 15 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> Artificial polynucleotide <220> <221> misc_feature <222> (4)..(8) <223> n is a, c, g, or t <400> 15 cctnnnnnag g 11 <210> 16 <211> 167 <212> PRT <213> Helicobacter pylori <400> 16 Met Leu Ser Lys Asp Ile Ile Lys Leu Leu Asn Glu Gln Val Asn Lys 1 5 10 15 Glu Met Gln Ser Ser Asn Leu Tyr Met Ser Met Ser Ser Trp Cys Tyr 20 25 30 Thr His Ser Leu Asp Gly Ala Gly Leu Phe Leu Phe Asp His Ala Ala 35 40 45 Glu Glu Tyr Glu His Ala Lys Lys Leu Ile Ile Phe Leu Asn Glu Asn 50 55 60 Asn Val Pro Val Gln Leu Thr Ser Ile Ser Ala Pro Glu His Lys Phe 65 70 75 80 Glu Gly Leu Thr Gln Ile Phe Gln Lys Ala Tyr Glu His Glu Gln His 85 90 95 Ile Ser Glu Ser Ile Asn Asn Ile Val Asp His Ala Ile Lys Ser Lys 100 105 110 Asp His Ala Thr Phe Asn Phe Leu Gln Trp Tyr Val Ala Glu Gln His 115 120 125 Glu Glu Glu Val Leu Phe Lys Asp Ile Leu Asp Lys Ile Glu Leu Ile 130 135 140 Gly Asn Glu Asn His Gly Leu Tyr Leu Ala Asp Gln Tyr Val Lys Gly 145 150 155 160 Ile Ala Lys Ser Arg Lys Ser 165 <210> 17 <211> 167 <212> PRT <213> Helicobacter pylori <400> 17 Met Leu Ser Lys Asp Ile Ile Lys Leu Leu Asn Glu Gln Val Asn Lys 1 5 10 15 Glu Met Asn Ser Ser Asn Leu Tyr Met Ser Met Ser Ser Trp Cys Tyr 20 25 30 Thr His Ser Leu Asp Gly Ala Gly Leu Phe Leu Phe Asp His Ala Ala 35 40 45 Glu Glu Tyr Glu His Ala Lys Lys Leu Ile Ile Phe Leu Asn Glu Asn 50 55 60 Asn Val Pro Val Gln Leu Thr Ser Ile Ser Ala Pro Glu His Lys Phe 65 70 75 80 Glu Gly Leu Thr Gln Ile Phe Gln Lys Ala Tyr Glu His Glu Gln His 85 90 95 Is Glu Is Asn Isn Is Val Asp His Ala Is Lys Is Lys 100 105 110 Asp His Ala Thr Phe Asn Phe Leu Gln Trp Tyr Val Ala Glu Gln His 115 120 125 Glu Glu Glu Val Leu Phe Lys Asp Ile Leu Asp Lys Ile Glu Leu Ile 130 135 140 Gly Asn Glu Asn His Gly Leu Tyr Leu Ala Asp Gln Tyr Val Lys Gly 145 150 155 160 Ile Only Lys Ser Arg Lys Ser 165 <210> 18 <211> 429 <212> DNA <213> Artificial Sequence <220> <223> Artificial polynucleotide <400> 18 atggtcccct gtaccctgct gctgctgctg gctgctgcc tgcacctc tcagactcgg 60 gcctcaaaaa gttaccccta cgatgtcccc gactacccca ggaccgtgcc ccctatggtg 120 caggtcacac gctcagaagc tagcgaggg CAatcaccg tgacatgccg agcatctggg 180 ttctatcctt ggaacattaa cctgagttgg agcaggacg gggtgtccct gtctcacgat 240 actcagcagt ggggcgacgt gctgccagat ggcaatggga cctaccagac atgggtggct 300 actcggatct cccaggggga ggaacagaga ttcacctgct atatggagca tagtggacag 360 cactcaacac atcctgtgcc atctggcaag gtgctggtcc tgcagagtca ctggcagaca 420 tttcattga 429 <210> 19 <211> 429 <212> DNA <213> Artificial Sequence <220> <223> Artificial polynucleotide <400> 19 atggtcccct gtaccctgct gctgctgctg gctgctgcac tggcacctac tcagactcgg 60 gcctcaaaaa gttaccccta cgatgtcccc gactacgcca ggaccgtgcc ccctatggtg 120 caggtcacac gctcagaagc tagcgagggc caaatcaccg tgacatgccg agcatctggg 180 ttctatcctt ggaacattaa cctgagttgg aggcaggacg gggtgtccct gtctcacgat 240 actcagcagt ggggcgacgt gctgccagat ggcaatggga cctaccagac atgggtggct 300 actcggatct cccaggggga ggaacagaga ttcacctgct atatggagca tagtggacag 360 cactcaacac atcctgtgcc atctggcaag gtgctggtcc tgcagagtca ctggcagaca 420 tttcattga 429 <210> 20 <211> 142 <212> PRT <213> Artificial Sequence <220> <223> Artificial polypeptide <400> 20 Met Val Pro Cys Thr Leu Leu Leu Leu Leu Ala Ala Ala Leu Ala Pro 1 5 10 15 Thr Gln Thr Arg Ala Ser Lys Ser Tyr Pro Tyr Asp Val Pro Asp Tyr 20 25 30 Ala Arg Thr Val Pro Pro Met Val Gln Val Thr Arg Ser Glu Ala Ser 35 40 45 Glu Gly Gln Ile Thr Val Thr Cys Arg Ala Ser Gly Phe Tyr Pro Trp 50 55 60 Asn Ile Asn Leu Ser Trp Arg Gln Asp Gly Val Ser Leu Ser His Asp 65 70 75 80 Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Asn Gly Thr Tyr Gln 85 90 95 Thr Trp Val Ala Thr Arg Ile Ser Gln Gly Glu Glu Gln Arg Phe Thr 100 105 110 Cys Tyr Met Glu His Ser Gly Gln His Ser Thr His Pro Val Pro Ser 115 120 125 Gly Lys Val Leu Val Leu Gln Ser His Trp Gln Thr Phe His 130 135 140 <210> 21 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> Artificial polynucleotide <220> <221> misc_feature <222> (7)..(10) <223> n is a, c, g, or t <400> 21 acctgcnnnn 10 <210> 22 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> Artificial polynucleotide <220> <221> misc_feature <222> (4)..(8) <223> n is a, c, g, or t <400> 22 gacnnnnngt c 11 <210> 23 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Artificial polynucleotide <400> 23 aaaaaaagat ctatggtccc ctgtaccctg ctgctgctgc 40 <210> 24 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Artificial polynucleotide <400> 24 aaaaaagaat tctcaatgaa atgtctgcca gtgactctgc 40 <210> 25 <211> 142 <212> PRT <213> Artificial Sequence <220> <223> Artificial polypeptide <400> 25 Put Val Pro Cys Thr Leu Leu Leu Leu Leu Wing Wing Wing Slow Wing Pro 1 5 10 15 Thr Gln Thr Arg Ala Ser Lys Ser Tyr Pro Tyr Asp Val Pro Asp Tyr 20 25 30 Ala Arg Thr Val Pro Pro Met Val Gln Val Thr Arg Ser Glu Ala Ser 35 40 45 Glu Gly Gln Ile Thr Val Thr Cys Arg Ala Ser Gly Phe Tyr Pro Trp 50 55 60 Asn Ile Asn Leu Ser Trp Arg Gln Asp Gly Val Ser Leu Ser His Asp 65 70 75 80 Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Asn Gly Thr Tyr Gln 85 90 95 Thr Trp Val Ala Thr Arg Ile Ser Gln Gly Glu Glu Gln Arg Phe Thr 100 105 110 Cys Tyr Met Glu His Ser Gly Gln His Ser Thr His Pro Val Pro Ser 115 120 125 Gly Lys Val Leu Val Leu Gln Ser His Trp Gln Thr Phe His 130 135 140 <210> 26 <211> 128 <212> PRT <213> Artificial Sequence <220> <223> Artificial polypeptide <400> 26 Met Ala Pro Ala Arg Ser Pro Ser Pro Ser Thr Gln Pro Trp Glu His 1 5 10 15 Val Asn Ala Ile Gln Glu Ala Arg Arg Leu Leu Asn Leu Ser Arg Asp 20 25 30 Thr Ala Ala Glu Met Asn Glu Thr Val Glu Val Ile Ser Glu Met Phe 35 40 45 Asp Leu Gln Glu Pro Thr Cys Leu Gln Thr Arg Leu Glu Leu Tyr Lys 50 55 60 Gln Gly Leu Arg Gly Ser Leu Thr Lys Leu Lys Gly Pro Leu Thr Met 65 70 75 80 Met Ala Ser His Tyr Lys Gln His Cys Pro Pro Thr Pro Glu Thr Ser 85 90 95 Cys Ala Thr Gln Ile Ile Thr Phe Glu Ser Phe Lys Glu Asn Leu Lys 100 105 110 Asp Phe Leu Leu Val Ile Pro Phe Asp Cys Trp Glu Pro Val Gln Glu 115 120 125 <210> 27 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Artificial polypeptide <400> 27 Met Ala Pro Thr Arg Ser Pro Ile Thr Val Thr Arg Pro Trp Lys His 1 5 10 15 Val Glu Ala Ile Lys Glu Ala Leu Asn Leu Leu Asp Asp Met Pro Val 20 25 30 Thr Leu Asn Glu Glu Val Glu Val Val Ser Asn Glu Phe Ser Phe Lys 35 40 45 Lys Leu Thr Cys Val Gln Thr Arg Leu Lys Ile Phe Glu Gln Gly Leu 50 55 60 Arg Gly Asn Phe Thr Lys Leu Lys Gly Ala Leu Asn Met Thr Ala Ser 65 70 75 80 Tyr Tyr Gln Thr Tyr Cys Pro Pro Thr Pro Glu Thr Asp Cys Glu Thr 85 90 95 Gln Val Thr Thr Tyr Ala Asp Phe Ile Asp Ser Leu Lys Thr Phe Leu 100 105 110 Thr Asp Ile Pro Phe Glu Cys Lys Lys Pro Val Gln Lys 115 120 125 <210> 28 <211> 781 <212> DNA <213> Homo sapiens <400> 28 acacagagag aaaggctaaa gttctctgga ggatgtggct gcagagcctg ctgctcttgg 60 gcactgtggc ctgcagcatc tctgcaccg cccgctcgcc cagccccagc acgcagccct 120 gggagcatgt gaatgccatc caggaggccc ggcgtctcct gaacctgagt agagacactg 180 ctgctgagat gaatgaaaca gtagaagtca tctcagaaat gtttgacctc caggagccga 240 cctgcctaca gacccgcctg gagctgtaca agcagggcct gcggggcagc ctcaccaagc 300 tcaagggccc cttgaccatg atggccagcc actacaagca gcactgccct ccaaccccgg 360 aaacttcctg tgcaacccag attatcacct ttgaaagtttt caaagagaac ctgaaggact 420 ttctgcttgt catccccttt gactgctggg agccagtcca ggagtgagac cggccagatg 480 aggctggcca agccggggag ctgctctctc atgaaacaag agctagaaac tcaggatggt 540 catcttggag ggaccaaggg gtgggccaca gccatggtgg gagtggcctg gacctgccct 600 gggccacact gaccctgata caggcatggc agaagaatgg gaatatttta tactgacaga 660 aatcagtaat atttatatat ttatattttt aaaatattta tttatttatt tatttaagtt 720 catattccat atttattcaa gatgttttac cgtaataatt attattaaaa atatgcttct 780 at 781 <210> 29 <211> 144 <212> PRT <213> Homo sapiens <400> 29 Met Trp Leu Gln Ser Leu Leu Leu Leu Gly Thr Val Ala Cys Ser Ile 1 5 10 15 Ser Ala Pro Ala Arg Ser Pro Ser Pro Ser Thr Gln Pro Trp Glu His 20 25 30 Val Asn Ala Ile Gln Glu Ala Arg Arg Leu Leu Asn Leu Ser Arg Asp 35 40 45 Thr Ala Ala Glu Met Asn Glu Thr Val Glu Val Ile Ser Glu Met Phe 50 55 60 Asp Leu Gln Glu Pro Thr Cys Leu Gln Thr Arg Leu Glu Leu Tyr Lys 65 70 75 80 Gln Gly Leu Arg Gly Ser Leu Thr Lys Leu Lys Gly Pro Leu Thr Met 85 90 95 Met Ala Ser His Tyr Lys Gln His Cys Pro Pro Thr Pro Glu Thr Ser 100 105 110 Cys Ala Thr Gln Ile Ile Thr Phe Glu Ser Phe Lys Glu Asn Leu Lys 115 120 125 Asp Phe Leu Leu Val Ile Pro Phe Asp Cys Trp Glu Pro Val Gln Glu 130 135 140 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Artificial polynucleotide <400> 30 tccatgacgt tcctgacgtt 20

Claims

1. A vaccine composition comprising a peptide containing an effective amount of a MIC alpha 3-domain peptide as an immunogenic component, wherein the effective amount is an amount effective to induce an immune response against the MIC alpha 3-domain peptide, the peptide contains the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4, a vaccine composition.

2. The vaccine composition according to claim 1, wherein the MIC alpha 3-domain is of the non-glycosylated type.

3. The vaccine composition according to claim 1, wherein the peptide is conjugated to a carrier protein.

4. The vaccine composition according to claim 1, further comprising a GM-CSF polypeptide or a fragment thereof, polyinosinic acid:polycytidylic acid (poly(I:C)), cholera toxin, enterotoxin, Fms-like tyrosine kinase-3 ligand, bupivacaine, marcaine, or levamisole.

5. The vaccine composition according to claim 1, further comprising an immunization scaffold.

6. The vaccine composition according to claim 5, wherein the immunization scaffold contains mesoporous silica.

7. The vaccine composition according to claim 1, further comprising a chemical adjuvant, genetic adjuvant, protein adjuvant, lipid adjuvant, oily adjuvant, inorganic salt adjuvant, inorganic salt gel adjuvant, particulate adjuvant, microparticulate adjuvant, mucosal adjuvant, or cytokine.

8. The vaccine composition according to claim 7, wherein the chemical adjuvant contains aluminum phosphate, benzalkonium chloride, ubenimex, or QS21.

9. The vaccine composition according to claim 7, wherein the genetic adjuvant contains an IL-2 gene or a fragment thereof, granulocyte macrophage colony-stimulating factor gene or a fragment thereof, IL-18 gene or a fragment thereof, chemokine ligand 21 gene or a fragment thereof, IL-6 gene or a fragment thereof, CpG oligonucleotide or a fragment thereof, LPS, TLR agonist, or immunostimulatory gene. **Claim 10**: The vaccine composition according to claim 7, wherein the protein adjuvant comprises IL-2 or a fragment thereof, granulocyte macrophage colony-stimulating factor or a fragment thereof, IL-18 or a fragment thereof, chemokine ligand 21 or a fragment thereof, IL-6 or a fragment thereof, CpG, LPS, a TLR agonist, or an immunostimulatory cytokine or a fragment thereof. **Claim 11**: The vaccine composition according to claim 7, wherein the lipid adjuvant comprises cationic liposomes, cationic lipids, or monophosphoryl lipid A (MPL1). **Claim 12**: A vaccine composition comprising a peptide comprising the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4 as an immunogenic component, further comprising mesoporous silica, a GM-CSF polypeptide or a fragment thereof, and a CpG oligonucleotide or a fragment thereof, the vaccine composition. **Claim 13**: A synthetic nucleic acid encoding an MIC alpha 3-domain peptide, wherein the peptide comprises the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4, the synthetic nucleic acid. **Claim 14**: A vector comprising the synthetic nucleic acid according to claim 13. **Claim 15**: A cell comprising the vector according to claim 14. **Claim 16**: A vaccine composition comprising the synthetic nucleic acid according to claim 13. **Claim 17**: The vaccine composition according to claim 1, for treating cancer in a subject. **Claim 18**: The vaccine composition according to claim 17, wherein the subject was positive in a test for shed MIC in serum. **Claim 19**: The vaccine composition according to claim 17, wherein the cancer is associated with overexpression of MICA. **Claim 20**: The vaccine composition according to claim 17, administered as part of a treatment regimen. **Claim 21**: The vaccine composition according to claim 20, wherein the treatment regimen is radiotherapy, targeted therapy, immunotherapy, or chemotherapy. **Claim 22**: The vaccine composition according to claim 17, administered to a subject in combination with one or more vaccines specific for antigens other than the MIC alpha 3-domain antigen. **Claim 23**: The vaccine composition according to claim 1, for preventing the progression of cancer in a subject. **Claim 24**: The vaccine composition according to claim 23, wherein preventing the progression of cancer comprises preventing metastasis of cancer or delaying tumor growth.

25. The vaccine composition according to claim 23, wherein the subject was positive in the test for shed MIC in serum.

26. The vaccine composition according to claim 23, wherein the cancer is associated with overexpression of MICA.

27. The vaccine composition according to claim 1, for inducing clinical regression of cancer in a subject.

28. The vaccine composition according to claim 27, wherein the subject was positive in the test for shed MIC in serum.

29. The vaccine composition according to claim 27, wherein the cancer is associated with overexpression of MICA.

30. The vaccine composition according to claim 1, for inducing an immune response against the MIC alpha 3-domain in a subject.

31. The vaccine composition according to claim 30, which induces an immune response against the MIC alpha 3-domain, but does not induce an immune response against either the MIC alpha 1-domain or the MIC alpha 2-domain.

32. The vaccine composition according to claim 30, wherein the subject was positive in the test for shed MIC in serum.

33. The vaccine composition according to claim 30, wherein the cancer is associated with overexpression of MICA.

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