# Patent

## Title
Dendritic cell vaccines for asparaginyl-β-hydroxylase expressing tumors

## Abstract

A vaccine containing AAH-loaded mature dendritic cells for treatment of AAH-expressing tumors in mammalian subjects. A method of producing primed dendritic cells is carried out by contacting isolated dendritic cells with an antigen such as AAH. Following the antigen-contacting step, the dendritic cells are contacted with a combination of cytokines such as GM-CSF and IFN-γ.

## Background
RELATED APPLICATIONS
This application claims priority to U.S. provisional patent application 61/228,429, filed Jul. 24, 2009, U.S. provisional patent application 61/231,127, filed Aug. 4, 2009, U.S. provisional application 61/239,288, filed Sep. 2, 2009 and U.S. provisional application 61/240,745, filed Sep. 9, 2009, the entire contents of which are hereby incorporated by reference.
REFERENCE TO A “SEQUENCE LISTING”
The sequence listing material in the text file entitled “21486_593001US_Sequence_Listing_ST25.txt” (10,093 bytes), which was created on Nov. 18, 2010, is herein incorporated by reference in its entirety.


BACKGROUND OF THE INVENTION
Liver cancer including hepatocellular and cholangiocellular carcinoma is one of the most common cancers in the world. In the United States, liver cancer is the fifth common cause of cancer death in men and the ninth in women. In spite of the dramatic progress in diagnosis and local control of liver cancer, the mortality rate in the United States has been increasing constantly. One of the reasons for the increasing mortality might be that, unlike other cancers including breast and colorectal cancers, liver cancer is resistant to systemic treatment such as chemotherapy. Therefore, there is no effective treatment of liver cancer once it becomes a systemic disease.
SUMMARY OF THE INVENTION
The invention provides a solution to the longstanding problem of an alternative to chemotherapy for treatment of patients diagnosed with cancer. A method of reducing growth of an aspartyl (asparaginyl)-β-hydroxylase (AAH or ASPH)-expressing tumor in a subject involves administering to the subject an isolated mature AAH-loaded dendritic cell or population of cells. Growth of an AAH-expressing tumor is reduced after administration of such a dendritic cell vaccine. For example, tumor growth and tumor burden is reduced by 10%, 20%, 50%, 75%, 2-fold, 5-fold, 10-fold, or more. The methods are used to reduce and eliminate AAH-expressing tumors from mammalian subjects, such as human patients. The compositions and methods are also suitable for use in companion animals and livestock, e.g., human, canine, feline, equine, bovine, or porcine subjects.
AAH-expressing tumors include most tumor types such as tumors of gastrointestinal tissues (e.g., esophagus, stomach, colon), pancreas, liver (e.g., cholangiocellular carcinoma, hepatocellular carcinoma), breast, prostate, cervix, ovary, fallopian tube, larynx, lung, thyroid, gall bladder, kidney, bladder, and brain (e.g., glioblastoma) as well as numerous others described below. AAH-expressing tumors include primary tumors that express and increased level of AAH compared to normal tissue as well as tumors that arise by metastasis from such AAH-overexpressing primary tumors.
The dendritic cells used in the vaccination method are preferably activated ex vivo with a combination of cytokines comprising GM-CSF and IFN-γ prior administering them to the subject. The latter step yields a population of dendritic cells with improved anti-tumor activity. An improved method of producing primed dendritic cells is carried out by contacting isolated dendritic cells with an antigen such as AAH or a combination of tumor antigens such as AAH and α-fetoprotein (AFP) and treating the cells to yield a population of matured and activated antigen-presenting cells. Following the antigen-contacting step, the dendritic cells are contacted with the combination of cytokines. For example, the combination comprises GM-CSF and IFN-γ. In other examples, the combination further comprises IL-4. Optionally, the combination comprises CD40L. The dendritic cells are contacted with the combination of cytokines for at least 10 hours (e.g., 12, 24, 36, 40, 48 hours or more). The antigen is in a soluble form or bound to a solid support. For example, the solid support comprises a polystyrene bead such as a biodegradable bead or particle. Dendritic cells are obtained from a subject by known methods such as leukapheresis or cytopheresis.
The subject from which the cells are obtained is suffering from or at risk of developing cancer. For example, the patient has been diagnosed with a tumor such as an AAH-expressing tumor. Patients who are at risk of developing cancer such as AAH-bearing tumors include those with a family history of individuals who have been identified with such a cancer.
Also within the invention is a vaccine composition comprising primed dendritic cells produced as described above. The vaccine is useful to inhibit tumor growth, prevent tumor growth, and inhibit or reduce metastasis. A method of inhibiting tumor growth in a mammal is carried out by identifying a subject suffering from an AAH-bearing tumor, and administering to the subject autologous dendritic cells, which have been primed according to the method described above. A method of preventing development of a tumor in a mammal, comprises the step of identifying a subject at risk of developing an AAH-bearing tumor (such as one with a family history of cancer), and administering to the subject the antigen-primed and activated autologous dendritic cells. A method of preventing metastasis of an AAH-bearing tumor is carried out by identifying a subject suffering from an AAH-bearing tumor, and administering to the subject autologous dendritic cells, as described above.
The polypeptides and other compositions of the invention are purified. For example, a substantially pure AAH polypeptide variant thereof is preferably obtained by expression of a recombinant nucleic acid encoding the polypeptide or by chemically synthesizing the protein. A polypeptide or protein is substantially pure when it is separated from those contaminants which accompany it in its natural state (proteins and other naturally-occurring organic molecules). Typically, the polypeptide is substantially pure when it constitutes at least 60%, by weight, of the protein in the preparation. Preferably, the protein in the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, AAH. Purity is measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. Accordingly, substantially pure polypeptides include recombinant polypeptides derived from a eucaryote but produced in E. coli or another procaryote, or in a eucaryote other than that from which the polypeptide was originally derived.
Dendritic cells or other cells, e.g., immune cells such as macrophages, B cells, T cells, used in the methods are purified or isolated. With regard to cells, the term “isolated” means that the cell is substantially free of other cell types or cellular material with which it naturally occurs. For example, a sample of cells of a particular tissue type or phenotype is “substantially pure” when it is at least 60% of the cell population. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99% or 100%, of the cell population. Purity is measured by any appropriate standard method, for example, by fluorescence-activated cell sorting (FACS).
The methods of generated antigen-primed DCs has several advantages over earlier methods. The cells are not only antigen-loaded but the subsequent cytokine incubation yield superior activated antigen-primed antigen presenting cells with improved anti-tumor and anti-metastatic activity.
These and other capabilities of the invention, along with the invention itself, will be more fully understood after a review of the following figures, detailed description, and claims. All references cited herein are hereby incorporated by reference.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a bar graph showing IL-12 production by isolated DCs.
FIG. 2 is a series of line graphs showing expression of various surface DC markers on isolated DCs.
FIG. 3 is a bar graph showing mean fluorescence on surface DC markers.
FIG. 4 is a series of flow cytometric graphs and a bar graph showing an increased population of CD8+ DCs grown in the presence of cytokines.
FIG. 5 is a bar graph showing IFN-γ secretion.
FIG. 6 is a bar graph showing IL-4 secretion.
FIG. 7 is a line graph showing the results of a cytotoxicity assay.
FIG. 8 is a diagram of AAH as expressed by a cell.
FIG. 9 is a diagram of a method of generating mature antigen-loaded DCs.
FIG. 10 is a flow chart of a schedule of immunization with AAH-loaded DCs.
FIG. 11 is a diagram showing a protocol for determining the therapeutic effect of AAH vaccination on tumor growth in mice.
FIG. 12 is a line graph showing therapeutic effect of AAH vaccination on tumor growth in mice.



DETAILED DESCRIPTION
AAH is a protein, the expression of which is associated with a number of cancer types (see, e.g., U.S. Pat. No. 6,815,415; U.S. Pat. No. 6,812,206; U.S. Pat. No. 6,797,696; U.S. Pat. No. 6,783,758; U.S. Pat. No. 6,835,370; and U.S. Pat. No. 7,094,556). Over-expression of HAAH has been detected by immunohistochemical staining (IHC) in numerous cancers lung (e.g., adenocarcinoma, bronchioalveolar carcinoma, and other non-small cell lung cancers such as squamous cell carcinoma subtype (Luu et al., 2009, Hum. Pathol. 40:639-644), liver (e.g., hepatocellular carcinoma, cholangiocarcinoma, and cancer of biliary epithelial cells (Wang et al., 2010, Hepatology 52:164-173), gastrointestinal tissues (e.g., colon, stomach, esophagus), pancreas, prostate, ovary, bile duct, breast, kidney, bladder, and brain (e.g., glioblastoma and neuroblastoma. Another survey of tumor tissue samples and cell lines confirmed AAH expression in the following additional cancers (compared to non-cancer tissues): laryngeal carcinoma, cervical cancer, fallopian tube cancer, liver cancer (e.g., cholangiocarcinoma), kidney cancer, breast cancer, cervical cancer, ovarian cancer, Fallopian tube carcinoma, laryngeal cancer, lung cancer, thyroid cancer, pancreatic cancer, thymic carcinoma, prostate cancer, bladder cancer, esophageal cancer, gastric cancer, gallbladder cancer, colon cancer, and rectal cancer (Song et al., 2010, Chinese J. of Cell. and Mol. Immunol. 26: 141-144). HAAH is highly specific for cancer; it has been detected by immunohistochemistry in >99% of tumor specimens tested (n>1000) and is not present in adjacent non-affected tissue, or in tissue samples from normal individuals.
Immunotherapy using AAH-primed cells is administered to patients with tumors that express AAH on their cell surface. Such tumors include liver cancers such as hepatocellular carcinoma and cholangiocarcinoma, as well as adenocarcinomas of the breast and colon. Such a therapeutic strategy is useful for not only treatment but prevention of cancers.
Prior to the invention, there has been no effective systemic treatment of liver cancer, in spite of the advances in diagnosis and local control of the disease. Immunotherapy is used to treat systemic and localized liver cancer, because of its specificity.
Dendritic cell vaccines using AAH were found to cure established hepatocellular carcinoma in immunocompetent mice. AAH dendritic cell vaccines reduce growth of AAH-expressing tumors to decrease tumor burden and eradicate tumors in humans as well.
Immunotherapy
Immunotherapy of malignant tumors is attractive in terms of the specific response to tumor cells. A rationale for immunotherapy is that cancer cells are replete with potential antigens, which become immunogenic when presented by antigen-presenting cells to helper and cytotoxic T cells. Immunotherapy for liver cancer, whether systemic or not, is a viable clinical approach, because liver cancer cells express several tumor-associated proteins, which are not or only slightly expressed in normal tissues. For example, AFP, which is not expressed in adult tissues, is extensively expressed in hepatocellular carcinoma (HCC). Several studies have shown that AFP-specific immune reaction can be induced not only in mice but also in humans. However, a clinical trial of HCC immunotherapy that targets AFP did not show even partial tumor responses. Some possible reasons are: because AFP is not involved in cancer progression, cancer cells that do not express AFP dominated during APP-targeting immunotherapy. In order for HCC cells to be recognized by AFP-responsive T cells, AFP should be presented on the cell surface with major histocompatibility complex (MHC) class I molecule, because AFP is not distributed on the cell surface. However, as MHC class I molecules are often down-regulated in many cancer cells, AFP may not be appropriately recognized by the T cells. To overcome the problems of liver cancer immunotherapy, studies were undertaken to identify another antigen that is associated with liver cancer to be used to primed cells for cell-based therapy. Given the results using AFP as an antigen, the anti-tumor results with AAH were surprising.
AAH, also known as ASPH, is strongly expressed in hepatocellular and cholangiocellular carcinoma, but is not expressed in normal cell counterparts of those tissue types. AAH is therefore a tumor-associated protein. AAH is useful for immunotherapy of liver cancer, because of its unique nature. First, AAH confers cancer cells on motility, which is associated with cancer metastasis; thus, therapy targeting AAH-expressing cells is effective to suppress metastatic lesions as well as the primary tumor. Second, because AAH is membrane protein, most part of which is exposed to extracellular space, it is easier for immune cells to access to the protein even when class I molecules are down-regulated. Third, other than AAH, no antigenic protein specific to cholangiocellular carcinoma is known. Thus, AAH is uniquely qualified as a target molecule of immunotherapy against liver cancer.
An immunotherapy approach involves injection of dendritic cells (DCs) loaded with the protein of interest. DCs are specialized cells that capture, process, and present antigens to T cells to induce and control T cell-mediated immunity. DCs are widely used to immunize not only laboratory animals but also tumor-bearing patients. The methods described herein include a DC-based immunization for induction of immunity against AAH. The present methods improve upon a previously described method for generating immunogenic mouse DCs loaded with protein of interest (Gehring et al., 2008, J. Immunol. Meth. 332:18-30) in at least two ways. One is that to stimulate DCs, lipopolysaccharides (LPS) was previously used, a strong pyrogen that may be inappropriate for in vivo use. Second is that DC stimulation was relatively weak, judging by IL-12 secretion by DCs, and by the expression of various surface DC markers. Improvements were therefore made to the method for the generation of immunogenic DCs.
Dendritic Cell Vaccines
The most promising approach for HCC immunotherapy is the one using DCs, as they have a strong capacity of inducing T cell-mediated tumor immunity. DCs are derived from hematopoietic cells, and are specialized to capture and process antigens, converting proteins to peptides that are presented on MHC molecules and recognized by T cells. DCs strongly induce and control T-cell mediated immunity.
DC-based immunotherapy is based on the fact that tumors are replete with potential antigens, and these become immunogenic when presented by DCs. DCs are obtained from a subject, e.g. by cytopheresis. The cells, which are now ex vivo are loaded (i.e., contacted) with tumor antigens (e.g., tumor cell lysates; apoptotic or necrotic tumor cells; recombinant, synthetic, or purified tumor antigen peptide or protein; or nucleic acid such as RNA encoding tumor antigens), stimulated for maturation, and re-injected into patients to induce strong T-cell immunity.
AAH Tumor Antigen
AAH is type II membrane protein with a catalytic domain in a C-terminal region. Most parts of the protein are located at the outside of the cell membrane, allowing the access of immune cells to this protein. AAH protein is strongly expressed in HCCs, cholangiocarcinomas, and adenocarcinomas of breast or colon origin, and is barely detectable or completely undetectable in normal tissue counterparts. Thus, AAH is an ideal target molecule for DC-based immunotherapy of HCC and other AAH-bearing tumors.
Overexpressed AAH in tumor cells increases the cell motility, which confers malignant phenotype on tumor cells. A clinicopathological study on HCC revealed that AAH overexpression is associated with histological grade and intrahepatic metastasis. Therefore, targeting the AAH-expressing tumor cell population suppresses the development and progression of AAH-bearing tumors.







TABLE 1





Amino acid sequence of Human AAH

















MAQRKNAKSS GNSSSSGSGS GSTSAGSSSP GARRETKHGG HKNGRKGGLS GTSFFTWFMV


 


 61
IALLGVWTSV AVVWFDLVDY EEVLGKLGIY DADGDGDFDV DDAKVLLGLK ERSTSEPAVP


 


121
PEEAEPHTEP EEQVPVEAEP QNIEDEAKEQ IQSLLHEMVH AEHVEGEDLQ QEDGPTGEPQ


 


181
QEDDEFLMAT DVDDRFETLE PEVSHEETEH SYHVEETVSQ DCNQDMEEMM SEQENPDSSE


 


241
PVVEDERLHH DTDDVTYQVY EEQAVYEPLE NEGIEITEVT APPEDNPVED SQVIVEEVSI


 


301
FPVEEQQEVP PETNRKTDDP EQKAKVKKKK PKLLNKFDKT IKAELDAAEK LRKRGKIEEA


 


361
VNAFKELVRK YPQSPRARYG KAQCEDDLAE KRRSNEVLRG AIETYQEVAS LPDVPADLLK


 


421
LSLKRRSDRQ QFLGHMRGSL LTLQRLVQLF PNDTSLKNDL GVGYLLIGDN DNAKKVYEEV


 


481
LSVTPNDGFA KVHYGFILKA QNKIAESIPY LKEGIESGDP GTDDGRFYFH LGDAMQRVGN


 


541
KEAYKWYELG HKRGHFASVW QRSLYNVNGL KAQPWWTPKE TGYTELVKSL ERNWKLIRDE


 


601
GLAVMDKAKG LFLPEDENLR EKGDWSQFTL WQQGRRNENA CKGAPKTCTL LEKFPETTGC


 


661
RRGQIKYSIM HPGTHVWPHT GPTNCRLRMH LGLVIPKEGC KIRCANETRT WEEGKVLIFD


 


721
DSFEHEVWQD ASSFRLIFIV DVWHPELTPQ QRRSLPAI





(SEQ ID NO: 1; GENBANK Accession No. S83325; His motif is underlined; conserved sequences within the catalytic domain are designated by bold type; catalytic domain comprises residues 650-700 of SEQ ID NO: 1).













TABLE 2





Human AAH cDNA sequence

















cggaccgtgc aatggcccag cgtaagaatg ccaagagcag cggcaacagc agcagcagcg


 


  61
gctccggcag cggtagcacg agtgcgggca gcagcagccc cggggcccgg agagagacaa


 


 121
agcatggagg acacaagaat gggaggaaag gcggactctc gggaacttca ttcttcacgt


 


 181
ggtttatggt gattgcattg ctgggcgtct ggacatctgt agctgtcgtt tggtttgatc


 


 241
ttgttgacta tgaggaagtt ctaggaaaac taggaatcta tgatgctgat ggtgatggag


 


 301
attttgatgt ggatgatgcc aaagttttat taggacttaa agagagatct acttcagagc


 


 361
cagcagtccc gccagaagag gctgagccac acactgagcc cgaggagcag gttcctgtgg


 


 421
aggcagaacc ccagaatatc gaagatgaag caaaagaaca aattcagtcc cttctccatg


 


 481
aaatggtaca cgcagaacat gttgagggag aagacttgca acaagaagat ggacccacag


 


 541
gagaaccaca acaagaggat gatgagtttc ttatggcgac tgatgtagat gatagatttg


 


 601
agaccctgga acctgaagta tctcatgaag aaaccgagca tagttaccac gtggaagaga


 


 661
cagtttcaca agactgtaat caggatatgg aagagatgat gtctgagcag gaaaatccag


 


 721
attccagtga accagtagta gaagatgaaa gattgcacca tgatacagat gatgtaacat


 


 781
accaagtcta tgaggaacaa gcagtatatg aacctctaga aaatgaaggg atagaaatca


 


 841
cagaagtaac tgctccccct gaggataatc ctgtagaaga ttcacaggta attgtagaag


 


 901
aagtaagcat ttttcctgtg gaagaacagc aggaagtacc accagaaaca aatagaaaaa


 


 961
cagatgatcc agaacaaaaa gcaaaagtta agaaaaagaa gcctaaactt ttaaataaat


 


1021
ttgataagac tattaaagct gaacttgatg ctgcagaaaa actccgtaaa aggggaaaaa


 


1081
ttgaggaagc agtgaatgca tttaaagaac tagtacgcaa ataccctcag agtccacgag


 


1141
caagatatgg gaaggcgcag tgtgaggatg atttggctga gaagaggaga agtaatgagg


 


1201
tgctacgtgg agccatcgag acctaccaag aggtggccag cctacctgat gtccctgcag


 


1261
acctgctgaa gctgagtttg aagcgtcgct cagacaggca acaatttcta ggtcatatga


 


1321
gaggttccct gcttaccctg cagagattag ttcaactatt tcccaatgat acttccttaa


 


1381
aaaatgacct tggcgtggga tacctcttga taggagataa tgacaatgca aagaaagttt


 


1441
atgaagaggt gctgagtgtg acacctaatg atggctttgc taaagtccat tatggcttca


 


1501
tcctgaaggc acagaacaaa attgctgaga gcatcccata tttaaaggaa ggaatagaat


 


1561
ccggagatcc tggcactgat gatgggagat tttatttcca cctgggggat gccatgcaga


 


1621
gggttgggaa caaagaggca tataagtggt atgagcttgg gcacaagaga ggacactttg


 


1681
catctgtctg gcaacgctca ctctacaatg tgaatggact gaaagcacag ccttggtgga


 


1741
ccccaaaaga aacgggctac acagagttag taaagtcttt agaaagaaac tggaagttaa


 


1801
tccgagatga aggccttgca gtgatggata aagccaaagg tctcttcctg cctgaggatg


 


1861
aaaacctgag ggaaaaaggg gactggagcc agttcacgct gtggcagcaa ggaagaagaa


 


1921
atgaaaatgc ctgcaaagga gctcctaaaa cctgtacctt actagaaaag ttccccgaga


 


1981
caacaggatg cagaagagga cagatcaaat attccatcat gcaccccggg actcacgtgt


 


2041
ggccgcacac agggcccaca aactgcaggc tccgaatgca cctgggcttg gtgattccca


 


2101
aggaaggctg caagattcga tgtgccaacg agaccaggac ctgggaggaa ggcaaggtgc


 


2161
tcatctttga tgactccttt gagcacgagg tatggcagga tgcctcatct ttccggctga


 


2221
tattcatcgt ggatgtgtgg catccggaac tgacaccaca gcagagacgc agccttccag


 


2281
caatttagca tgaattcatg caagcttggg aaactctgga gaga





(SEQ ID NO: 2; GENBANK Accession No. S83325; codon encoding initiating methionine is underlined).






Clinical Use

Antigen-presenting cells (APCs) such as DCs are obtained from subjects, e.g., human patients suffering from or at risk of developing cancer, using the process of leukapheresis. Such as procedure is typically carried out at an apheresis center (Day 1). Dendritic cells are purified using known methods are contacted with antigen, such as AAH or AAH and AFP together. Following antigen contact, the cells are cultured with a mixture of cytokines (Day 2-3). The antigen-primed and activated DCs are then administered to the patient (Day 3-4). An exemplary course of therapy includes three administrations of DCs over a four week period.
The following materials and methods were used to generated the data described herein.
Animals
7-8-week-old female BALB/c (H-2d) mice were purchased from Harlan Sprague Dawley, Inc., and kept under specific pathogen-free conditions.
Preparation of Magnetic Microbeads
Immunomagnetic beads (1.3 μm; Calbiochem) were washed three times with 50 mM borate buffer, pH 8.5, and resuspended in 50 mM borate buffer, pH 9.0, with 0.1 mg/ml AAH or GFP protein. Then the suspension was incubated overnight at room temperature under constant agitation. The beads were pelleted, washed with PBS, and resuspended in PBS at a concentration of 30 mg solid content/ml.
Isolation of DCs
DC isolation was performed using known methods (e.g., (Gehring et al., 2008, J. Immunol. Meth. 332:18-30)). Cells are optionally obtained by leukapheresis. 10 μg pUMVC3-hFLex, an expression plasmid encoding Fms-like tyrosine kinase receptor-3 ligand (FLT3L), was injected into the mouse tail vein on day 0 and 6. Splenocytes were prepared from the FLT3L-injected mice, using NH4Cl red blood cell-lysis buffer, and 5×107 cells were incubated in serum-free DMEM with 10 μl magnetic microbeads for 4-6 hours. The cells were collected, passed through a magnetic field using MACS MS column (Miltenyi) to enrich the cells ingesting magnetic beads. Then the cells were subjected to density-gradient centrifugation using Lympholyte M (Cedarlane) to eliminate free beads and dead cells. The viable cells were collected, washed twice with Hanks' buffered salt solution (HBSS), and used for subsequent experiments. The viability of the isolated cells was >90%, and the percentage of the CD11 c-positive population in the isolated cells were 70-80%.
Cell Culture
DCs were cultured in HEPES-buffered RPMI1640 supplemented with 10% mouse serum (Equitech Bio), 2 mM L-glutamine, 50 μM 2-mercaptoethanol, 100 U/ml penicillin, 100 μg/ml streptomycin, 20 ng/ml GMCSF, 100 ng/ml IL-4, 20 ng/ml IFN-γ, and 1 μg/ml CD40L (all cytokines were purchased from Peprotech) on a 6-well ultra-low attachment plate (Corning) for 40 h. For cytokine release, splenocytes were grown in serum-free X-VIVO 15 (Lonza) supplemented with 2 mM L-glutamine, 50 nM 2-mercaptoethanol, 100 U/ml penicillin, and 100 μg/ml streptomycin. For the cytotoxicity assay, splenocytes were grown in the complete medium consisting of HEPES-buffered RPMI1640 supplemented with 10% fetal bovine serum (Atlantic Bio), 2 mM L-glutamine, 50 μM 2-mercaptoethanol, 100 U/ml penicillin, 100 μg/ml streptomycin, 1× non-essential amino acids (Lonza), 0.5× amino acid solution (Invitrogen), and 1 mM sodium pyruvate. SP2/0 cells were cultured in Dulbecco's minimum essential medium supplemented with 20% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin.
Enzyme-Linked Immunosorbent Assay (ELISA)
DCs or splenocytes were grown in the serum-free medium with or without AAH protein for 48 h. Then the culture supernatants were collected, centrifuged, and subjected to ELISA assays. ELISA for IL-12, p70, IFN-γ, and IL-4 was performed using ELISA Ready-SET-Go! kit (eBioscience) according to the manufacturer's instructions.
Flow Cytometry
The expression of cell surface markers was analyzed by flow cytometry as described previously (Gehring, et al., 2008). 3-6×105 cells were used for staining. The cells were washed with staining buffer (HBSS containing 5% FBS), incubated with 2.5 μg/ml Mouse BD Fc Block (BD Biosciences) for 5 min at 4° C., and further incubated with staining antibodies for 30 min at 4° C. The antibodies used were anti-CD11c (HL3; BD Biosciences), anti-CD40 (3/23; BD Biosciences), anti-CD54 (3E2; BD Biosciences), anti-CD80 (16-10A1; BD Biosciences), anti-CD 86 (GLI; BD Biosciences), anti-I-Ad (AMS-32.1; BD Biosciences), and anti-CD8a (53-6.7; eBioscience), all of which were conjugated with fluorochrome. Hamster, rat, and mouse immunoglobulin G isotype-matched controls were also used for staining controls. After washing twice, the cells were resuspended in staining buffer, and were subjected to flow cytometry using FACSCalibur (BD Biosciences). Dead cells were eliminated from analysis by staining the cells with 7-amino-actinomycin D (eBioscience). Data were processed with CellQuest software (BD Biosciences) followed by FlowJo software (Tree Star).
Vaccination
After the culture with cytokines for 40 h, 2.5×105 cells were injected subcutaneously into the right and left flanks (5×105 cells per mouse) on day 0 and 14. On day 28, the immunized mice were used for further experiments including cytokine release and cytotoxicity assay.
Cytotoxicity Assay
Splenocytes were prepared from immunized mice, and 5×107 splenocytes were grown in complete medium with 0.5 μg/ml AAH protein for 2 days and further grown for 2 days after addition of 10 ng/ml IL-2. Two×104 target SP2/0 cells were mixed with 6-60×104 effector splenocytes in the serum-free medium, centrifuged at 200×g for 5 min, and incubated in CO2 incubator for 4 h. Culture supernatants were collected, and released lactate dehydrogenase activity in the supernatants were measured by using LDH Cytotoxicity Detection Kit (Roche) in accordance with manufacturer's instruction. % target cell lysis was calculated using the following formula:

((Aeffector+target−Aeffector−Atarget) divided by (Atarget+TritonX−Atarget))×100,

where A indicates absorbance at 490 nm.

IL-12 Secretion by the Isolated DCs Stimulated by Various Cytokines In Vitro

The protocol previously used includes in vivo expansion of DCs, ingestion of magnetic microbeads on which an antigen and stimulants of DCs including LPS and anti-CD40 antibody are coated, and isolation of DCs by passing through a magnetic column. Instead of stimulating DCs with the stimulants coated on the beads, the improved method involved stimulating isolated DCs by growing for two days in the presence of various cytokines, because stimulants should interact with the corresponding receptors on the cell surface, but the stimulants on the beads that were ingested by DCs cannot. There exist some cytokines that stimulate and promote maturation of DCs including IL-4, IFN-γ, and CD40L. Therefore, the combinatorial effect of these cytokines on the maturation of DCs was examined. Expansion, ingestion, and isolation of DCs were performed. Isolated cells were cultured for 40 hours in the presence of GM-CSF and/or IL-4, and/or IFN-γ, and/or CD40L. GM-CSF was always added because it was necessary for survival of DCs. Mature, but not immature, DCs vigorously produce IL-12, which is essential for polarizing helper and cytotoxic T cells. Therefore, IL-12 levels were used as an indicator of DC stimulation. Supernatants of the culture was collected, the concentration of IL-12 p70 was measured by ELISA. The results indicated that IL-12 p70 was the highest when all cytokines were present in the DC culture (FIG. 1). Each of the cytokines are used for stimulation alone or in combination. Preferably, all the cytokines are used to stimulate DCs. However, CD40L is optional.
Expression of Mature DC Markers on the Surface of Stimulated DCs
To investigate the effect of the cytokine stimulation on the expression of mature DC markers, the marker expression of DCs grown for 40 h in the presence of the cytokines to that of DCs without culture was measured. The expression of CD11c, a marker for pan-DCs, was not changed after the culture (FIGS. 2,3). However, all the mature DC markers including CD40, CD54, CD80, CD86, and MHC class II (I-Ad), were significantly up-regulated in the DCs grown for 40 h (FIGS. 2,3). CD8a+ DCs are the subset of DCs that can efficiently stimulate CTLs. The effect of the cytokine stimulation on CD8a+ DC population was evaluated. As shown in FIG. 4, the percentage of CD8a+ population in DCs was significantly increased during the stimulation. These results indicate that the generated cells by this method without using LPS had sufficient quality for DC-based immunization.
Secretion of IFN-γ in Response to AAH by the Splenocytes from AAH-Immunized Mice
To examine whether TH1 response was induced by the AAH immunization, the production of IFN-γ, a TH1 cytokine, in response to AAH protein was measured. Mice were immunized twice with a 2-week interval with DCs ingesting AAH or GFP-coated beads, or beads without any antigens. Two weeks after the last immunization, splenocytes were prepared from the immunized mice, cultured for 48 h in the presence of AAH, and the concentration of IFN-γ was measured in culture supernatants to examine whether splenocytes from AAH-immunized mice produce IFN-γ in response to AAH stimulation. The splenocytes from AAHimmunized mice vigorously produced IFN-γ in a dose-dependent manner (FIG. 5). Although splenocytes from three groups produced considerable amount of IFN-γ in the presence of concanavalin A (Con A), the control splenocytes produced only subtle IFN-γ when stimulated with AAH (FIG. 5). This result indicates that TH1 cells responsive to AAH were generated in the immunized mice.
Secretion of IL-4 in Response to AAH by the Splenocytes from AAH-Immunized Mice
To investigate whether humoral immunity against AAH was induced by the DCs loaded with AAH, the concentration of IL-4, a TH2 cytokine, was measured in the same samples as IFN-γ. Con A-stimulated splenocytes produced large amount of IL-4 independent of the immunization (FIG. 6). By contrast, the splenocytes prepared from the mice immunized with three types of DCs produced only trace amounts of IL-4 (FIG. 6), indicating that TH2 cells responsive to AAH were not efficiently induced by AAH immunization.
CTL Activity Against AAH-Expressing Cells in AAH-Immunized Mice
To determine whether CTLs responsive to AAH were induced in AAH-immunized mice, cytotoxicity was evaluated by using AAH-expressing SP2/0 cells as target cells. SP2/0 cells were co-cultured for 4 h with the splenocytes from the immunized mice after activated with AAH protein and IL-2. The efficiencies of target cell lyses were estimated by measuring lactate dehydrogenase activities released from dying target cells. As shown in FIG. 7, the splenocytes from the AAH-immunized mice had more CTL activity than the other control splenocytes, suggesting that AAH immunization induced AAH-responsive CTLs.
AAH-Responsive T Cells in AAH-Immunized Mice
When mice were immunized with AAH-loaded DCs, AAH-responsive TH1 cells and CTLs were induced. Antigen-specific T cell expansion is another important indicator that indicates that antigen-responsive T cells were produced.
Anti-Tumor Effect of AAH Immunization In Vivo
AAH immunization using AAH-primed DCs induces antitumor immunity. Mice are immunized with AAH-loaded DCs twice with a 2-week interval, and two weeks after the last immunization. SP2/0 cells are implanted under the skin, the growth of implanted tumor sizes is measured. Data obtained from this tumor burden model reflects the preventive effect of the immunization. To evaluate the curative effect of AAH immunization, SP2/0 cells are implanted first, then when tumors grow to 5 mm in diameter, the mice are immunized with AAH-loaded DCs, and the effect on tumor growth measured.
AAH-targeted immunotherapy also suppresses metastasis. Mice are immunized with AAH-loaded DCs followed by injection of SP2/0 cells into the tail vein. Two weeks later, the lungs are excised to count the number of nodules formed. A reduction in the amount of nodules in immunized mice compared to the amount in unimmunized mice indicates that the AAH-loaded DC immunization regimen suppresses metastasis of AAH-bearing tumors.
Vaccination with AAH-loaded mature dendritic cells dramatically reduced tumor burden (tumor volume) of an AAH-expressing tumor. The therapeutic effect of AAH vaccination on the growth of tumor in mice is shown in FIGS. 11 and 12. 5×105 BNL 1ME A.7R.1 mouse hepatoma cells were subcutaneously implanted into the flanks of 6-week-old BALB/c mice. At day 10 and 15, 5×105 mature DCs loaded with AAH or GFP, or HBSS were subcutaneously injected into the tumor-bearing mice. Tumor size was measure weekly. Tumor volumes were determined by the following formula: 0.52×(length)×(width)2. Each data point represents the mean tumor volume±standard error (n=6). Tumor volumes were found to be significantly reduced following immunization with AAH-loaded DCs. These data using an art-recognized tumor model indicate that a therapeutic method using AAH-loaded mature DCs is effective to reduce and eliminate AAH-bearing tumors in vivo. Such dendritic cell vaccines demonstrate potent anti-tumor effects against any AAH-expressing tumors.
Augmentation of Anti-Tumor Effect by Simultaneously Immunizing AAH and AFP
AFP is the only HCC-associated protein that can be antigenic to mice and humans. However, it has been shown as well that the anti-tumor effect is not sufficient for human HCC patients. However, simultaneous immunization with AAH and AFP augments the immune response to tumors expressing both AAH and AFP, like HCC.
Stable lines of AFP-expressing SP2/0 cells are made by introducing an AFP expression plasmid DNA. By using the cells, CTL activity in vitro in the mice immunized with AFP-loaded and AAH-loaded DCs is measured. Increased cytotoxicity following immunization indicates that immunization lead to an anti-tumor effect of combination AAH and AFP immunization on the growth AAH and/or AFP-bearing tumors.

## Claims

1. A method of reducing growth of an aspartyl (asparaginyl)-β-hydroxylase (AAH)-expressing tumor in a subject, comprising providing an isolated mature AAH-loaded dendritic cell and administering to said subject said isolated mature AAH-loaded dendritic cell, wherein growth of said AAH-expressing tumor is reduced after administration of said dendritic cell, and wherein said dendritic cell is primed by contact with an AAH followed by contact with a combination of cytokines comprising GM-CSF (Granulocyte-macrophage colony stimulating factor), CD40L (Cluster of Differentiation 40 ligand), and IFN-γ (interferon gamma) ex vivo, prior to said administering to said subject.

2. The method of claim 1, wherein said tumor is selected from the group consisting of liver, gastrointestinal, pancreas, breast, prostate, cervix, ovary, fallopian tube, larynx, lung, thyroid, gall bladder, kidney, bladder, and brain cancers.

3. The method of claim 1, wherein said dendritic cells are contacted with said combination of cytokines for at least 10 hours.

4. The method of claim 1, wherein said dendritic cells are contacted with said combination of cytokines for at least 40 hours.

5. The method of claim 1, wherein said subject is a human, canine, feline, equine, bovine, or porcine subject.

6. The method of claim 1, wherein said AAH-expressing tumor is a liver cancer.

7. The method of claim 6, wherein said liver cancer comprises hepatocellular carcinoma or cholangiocarcinoma.

8. A method of producing primed dendritic cells, comprising contacting isolated dendritic cells with an antigen comprising AAH (aspartyl (asparaginyl)-β-hydroxylase), and following said antigen-contacting step, contacting said dendritic cells with a combination of cytokines, said combination comprising GM-CSF (Granulocyte-macrophage colony stimulating factor), CD40L (Cluster of Differentiation 40 ligand), and IFN-γ (interferon gamma).

9. The method of claim 8, wherein said combination further comprises IL-4 (Interleukin-4).

10. The method of claim 8, wherein said antigen is in a soluble form.

11. The method of claim 8, wherein said dendritic cells are obtained from a subject by leukapheresis.

12. The method of claim 11, wherein said subject is suffering from an AAH-bearing tumor.

13. A vaccine composition comprising primed dendritic cells produced by the method of claim 8.

14. A method of inhibiting tumor growth in a mammal, comprising identifying a subject suffering from an AAH (aspartyl (asparaginyl)-β-hydroxylase)-bearing tumor, and administering to said subject autologous dendritic cells, wherein said autologous dendritic cells are primed according to the method of claim 8.

15. A method of preventing metastasis of an AAH (aspartyl (asparaginyl)-β-hydroxylase)-bearing tumor, comprising identifying a subject suffering from an AAH-bearing tumor, and administering to said subject autologous dendritic cells, wherein said autologous dendritic cells are primed according to the method of claim 8.

16. A method of producing primed dendritic cells, comprising contacting isolated dendritic cells with an antigen comprising AAH (aspartyl (asparaginyl)-β-hydroxylase) and AFP (α-fetoprotein), and following said antigen-contacting step, contacting said dendritic cells with a combination of cytokines, said combination comprising GM-CSF (Granulocyte-macrophage colony stimulating factor) and IFN-γ (interferon gamma).

17. A method of producing primed dendritic cells, comprising contacting isolated dendritic cells with an antigen comprising AAH (aspartyl (asparaginyl)-β-hydroxylase), and following said antigen-contacting step, contacting said dendritic cells with a combination of cytokines, said combination comprising GM-CSF (Granulocyte-macrophage colony stimulating factor), CD40L (Cluster of Differentiation 40 ligand) and IFN-γ (interferon gamma), and wherein said antigen is bound to a biodegradable solid support.

18. A method of producing primed dendritic cells, comprising contacting isolated dendritic cells with an antigen comprising AAH (aspartyl (asparaginyl)-β-hydroxylase), and following said antigen-contacting step, contacting said dendritic cells with a combination of cytokines, said combination comprising GM-CSF (Granulocyte-macrophage colony stimulating factor), CD40L (Cluster of Differentiation 40 ligand) and IFN-γ (interferon gamma), and wherein said antigen is bound to a solid support, wherein said solid support comprises a polystyrene bead.

19. A vaccine containing AAH-loaded mature dendritic cells for treatment of AAH-expressing tumors in a mammalian subject, wherein said dendritic cells are primed by contact with an AAH followed by contact with a combination of cytokines comprising GM-CSF (Granulocyte-macrophage colony stimulating factor), CD40L (Cluster of Differentiation 40 ligand) and IFN-γ (interferon gamma).

20. A method of reducing growth of an aspartyl (asparaginyl)-β-hydroxylase (AAH)-expressing tumor in a subject, comprising administering to said subject an isolated mature AAH-loaded dendritic cell, wherein growth of said AAH-expressing tumor is reduced after administration of said dendritic cell, and wherein said dendritic cell is activated with a combination of cytokines comprising GM-CSF and IFN-γ ex viva, prior to said administering to said subject, wherein said dendritic cell is further loaded with AFP.

21. A method of reducing growth of an aspartyl (asparaginyl)-β-hydroxylase (AAH)-expressing AAH-expressing liver tumor in a subject, comprising providing an isolated mature AAH-loaded dendritic cell and administering to said subject said isolated mature AAH-loaded dendritic cell, wherein growth of said AAH-expressing tumor is reduced after administration of said dendritic cell, and wherein said dendritic cell is primed by contact with an AAH and a combination of cytokines comprising GM-CSF (Granulocyte-macrophage colony stimulating factor), CD40L (Cluster of Differentiation 40 ligand), interleukin-4 (IL-4) and IFN-γ (interferon gamma) ex viva, prior to said administering to said subject.

22. The method of claim 21, wherein said AAH-expressing liver tumor comprises hepatocellular carcinoma or cholangiocarcinoma.