JP2003504080A - vaccine - Google Patents

Abstract

  (57) [Summary] The present invention relates to novel proteins from the prostase family and their production. In particular, the invention relates to an immunological fusion partner, such as a prostase protein or a fragment thereof fused to NS1. Such antigens can be formulated to provide a vaccine for the treatment of prostate tumor. Novel methods for purifying prostase proteins and homologs are also provided.

Description

Detailed Description of the Invention

The present invention relates to a protein known as prostase, ie a protein derivative of prostate-specific serine protease, a method for their purification and production, and also pharmaceutical compositions containing said derivative and their use in medicine. Regarding use. In particular, such derivatives find use in cancer vaccine therapy, particularly prostate cancer vaccine therapy, and diagnostic agents for prostate tumors.

[0002] In particular, the derivatives of the invention include fusion proteins comprising a prostase linked to an immunological or expression enhancer fusion partner.

The present invention relates to a method for purifying a prostase derivative, and a prostatic cancer patient, and a prostase-expressing tumor other than prostatic tumor, prostatic hyperplasia, and prostatic intraepithelial neoplasia (pr).
Also provided is a method of combining vaccines for treating ostate intraepithelial neoplasia (PIN) by immunotherapy.

Prostate cancer is the most common cancer among men with an estimated 30% incidence in men over the age of 50. Remarkable clinical evidence suggests that human prostate cancer has a tendency to metastasize to the bone, and that the disease is unavoidable from androgen depending on the androgen refractory status leading to increased patient mortality. (Abbas F., Scardino P. “The Natural His
tory of Clinical Prostate Carcinoma. ”In Cancer (1997); 80: 827-833).
This common disease is currently the second leading cause of cancer deaths among men in the United States.

Despite considerable research on therapies for the above diseases, prostate cancer remains difficult to treat. Currently, treatment is based on surgery and / or radiation therapy, but these methods are not effective in a significant percentage of cases (
Frydenberg M., Stricker P., Kaye K. “Prostate Cancer Diagnosis and Manag
ement ”The Lancet (1997); 349: 1681-1687). Several tumor-associated antigens are already known. Many of these antigens are interesting targets for immunotherapy but are completely tumor-associated. It is closely related to non-specific or normal proteins, and thus once they are targeted by a strong immune response they risk organ-specific autoimmunity.

[0006] When the autoimmune response to non-deterministic organs can be tolerated, autoimmunity to the heart, intestine, and other crucial organs could lead to unacceptable safety features. Some previously identified prostate-specific proteins, such as prostate-specific antigen (PSA) and prostate acid phosphatase (PAP), prostate-specific membrane antigen (PSMA), and prostate stem cell antigen (PSCA) are limited. And has not always been correlated with the presence of prostate cancer or the level of metastasis (Pound C., Partin A., Eisenberg M. et al. “Natural History of P
rogression after PSA Elevation following Radical Prostatectomy ”In Jama
(1999); 281: 1591-1597) (Bostwick D., Pacelli A., Blute M. et al. “Prost
ate Specific Membrane Antigen Expression in Prostatic Intraepithelial Ne
oplasia and Adenocarcinoma. ”In Cancer (1998); 82: 2256-2261).

The existence of a mechanism of tumor rejection has been recognized for decades. The tumor antigen is
Although encoded by the organism's genome and, therefore, theoretically not recognized by the immune system through the phenomenon of tolerance, it can sometimes provoke an immune response that can be detected in cancer patients. This has been demonstrated by antibodies or T cell responses to tumor expressed antigens (Xue BH., Zhang Y., Sos.
man J. et al. “Induction of Human Cytotoxic T-Lymphocytes Specific for P
rostate-Specific Antigen. ”In Prostate (1997); 30 (2): 73-78). When a relatively weak antitumor effect can be observed through the administration of antibodies that recognize cell surface markers of tumor cells. Induction of a strong T cell response to antigens expressed by tumor cells can lead to complete regression of established tumors in animal models (mainly murine).

At present, expression of tumor antigens by cells induces an immune response (in
It is recognized that it is not sufficient for the reduction. Initiation of the tumor rejection response requires a series of immune amplification events, depending on the intervening antigen presenting cells responsible for the delivery of a series of activation signals.

Prostase is a conserved serine protease-catalyzed triad HDS
And a 254 amino acid long prostate-specific protease (trypsin-like) with an amino-terminal pre-propeptide sequence that exhibits potential secretory function (P. Nelso
n, Lu Gan, C. Ferguson, P. Moss, R. Gelinas, L. Hood & K. Wand, “Molecul
ar cloning and characterisation of prostase, an androgen-regulated serin
e protease with prostate restricted expression, In Proc. Natl. Acad. Sci
USA (1999) 96, 3114-3119). Putative glycosylation sites have been described. The predicted structure closely resembles other known serine proteases, indicating that the mature polypeptide is folded into a single domain. The mature protein is 224 amino acids in length and one A2 epitope has been shown to be naturally processed.

The prostase nucleotide sequence, and the deduced polypeptide sequence and homologues,
Ferguson, et al. (Proc. Natl. Acad. Sci. USA 1999, 96, 3114-3119) and International Patent Application No. WO 98/12302 (and also the corresponding granted patent No. US).
5,955,306), WO98 / 20117 (and also correspondingly assigned patents US 5,840,871 and US 5,786,148) (
Prostate-specific kallikrein), and WO 00/04149 (P703P).

The present invention provides prostase proteins and fragments and homologues thereof (“derivatives”).
) Based prostase-protein fusion. Such derivatives are
Suitable for use in a therapeutic vaccine formulation which is suitable for the treatment of prostate tumors.

The prostase fragment of the present invention is at least about 10, preferably about 20, more preferably about 5 selected from the amino acid sequences shown in SEQ ID NO: 7 or SEQ ID NO: 8.
It will consist of 0, more preferably about 100, more preferably about 150 contiguous amino acids. More particularly, the fragment will retain some of the functional properties, preferably immunological activity, of the larger molecule set forth in SEQ ID NO: 7 or SEQ ID NO: 8,
It is then useful in the methods described herein (eg, in vaccine formulations, in diagnostics, etc.). In particular, the fragment is SEQ ID NO: 7 or SEQ ID NO: 8.
Will be able to generate an immune response when suitably attached to a carrier that will recognize the proteins of

The prostase homologues will generally share substantial sequence similarity to the sequence of SEQ ID NO: 7 or SEQ ID NO: 8 over the length of SEQ ID NO: 7 or SEQ ID NO: 8, and at least 70 %, Preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, and most preferably at least 97-99% identity of the isolated polypeptide. Will. Such polypeptides include those that include the amino acids of SEQ ID NO: 7 or SEQ ID NO: 8.

The prostase antigen derivative or fragment and homologues thereof, in a preferred embodiment,
Mutations can be carried within the active site of the protein in order to substantially reduce or preferably eliminate the biological activity of the protease. Preferred mutations include the substitution of histidine and aspartic acid catalytic residues of the serine protease. In a preferred embodiment, the prostase is P703PDE.
Residue 71 of the prostase sequence corresponding to amino acid 43 of the 5 sequence (SEQ ID NO: 8)
Containing a histidine-alanine mutation (Ferguson, et al. (Proc. Natl
Acad. Sci. USA 1999, 96, 3114-3119). The mutated protein preferably has a significant reduction in the catalytic efficiency (expressed in enzyme specific activity) of the protein as compared to the unmutated protein. Preferably, the reduction in catalytic efficiency is at least a conversion factor of 10 ³ , more preferably at least a conversion factor of 10 ⁶ . Proteins that have undergone the histidine-alanine mutation are referred to below as ^* (star).

In one aspect, the invention relates to a fusion protein comprising a tumor associated prostase or fragment or homologue thereof and a heterologous protein or portion of a protein that acts as a fusion partner. The protein and the fusion partner can be chemically linked, but are preferably expressed as a recombinant fusion protein in a heterologous expression system.

In a preferred aspect of the invention there is provided a prostase fusion protein or fragment or homologue thereof, linked to an immunological fusion partner capable of assisting the presentation of T helper epitopes. Thus, this fusion partner can work through a bystander helper effect associated with the secretion of activation signals by multiple T cells specific for foreign proteins or peptides,
Thereby enhancing the induction of immunity to the prostase component compared to the unfused protein. Preferably, the heterologous partner is chosen such that it can be recognized by T cells in the majority of humans.

In another aspect, the invention provides a prostase protein or fragment or homologue thereof, linked to a fusion partner that acts as an expression enhancer. Thus, this fusion partner aids in the expression of prostase in a heterologous system, allowing higher levels to be produced in the expression system as compared to the native recombinant protein.

Preferably, the fusion partner will be both an immunological fusion partner and an expression enhancer partner. The invention thus provides in this aspect a fusion protein comprising a tumor-specific prostase or fragment thereof linked to a fusion partner. Preferably, the fusion partner serves as both an immunological fusion partner and expression enhancer partner. Therefore,
In a preferred form of the invention said fusion partner is a non-structural protein from influenza virus, NS1 (hemagglutinin), or a fragment thereof. Typically, the N-terminal 81 amino acids are used. However, different fragments can be used as long as they contain T helper epitopes (C. Hackett,
D. Horowitz, M. Wysocka & S. Dillon, 1992, J. Gen. Virology, 73, 1339-13
43). When NS1 is the immunological fusion partner, it has the additional advantage in that it allows higher expression yields to be achieved. In particular, such fusions are expressed in higher yields than the native recombinant prostase protein.

In a preferred embodiment, the prostase component within the fusion is SEQ ID NO: 5 (
Millenium WO98 / 12302), SEQ ID NO: 6 (Incyte WO98 / 20117), SEQ ID NO: 7 (PNAS
(1999) 96, 3114-3119), and SEQ ID NO: 8 (Corixa WO00 / 04149, P703PDE5 sequence). Further, in the most preferred embodiment, the fusion protein is the N-terminal of the NS1 nonstructural protein fused to the mutated 5-226 carboxy terminal amino acid from a mutated prostase, as set forth in SEQ ID NO: 1 or SEQ ID NO: 3. Containing 81 amino acids.

The protein of the invention is expressed in a suitable host cell, preferably E. coli . In a preferred embodiment, the protein is expressed with an affinity tag, eg, a histidine tail containing 5-9, and preferably 6, and most preferably at least 4 histidine residues. They are,
For example, it is advantageous to aid purification through ionic metal affinity chromatography (IMAC).

The present invention also provides a nucleic acid encoding the protein of the present invention. Such sequences can be inserted into a suitable expression vector and used for DNA / RNA vaccination or expressed in a suitable host. In a further aspect, a genetic construct comprising one or more of the polynucleotides of the invention is introduced into a cell in vivo. This can be accomplished using any of a variety of or well known approaches. One of the preferred methods for in vivo delivery of one or more nucleic acid sequences involves the use of expression vectors, such as recombinant live virus or bacterial microorganisms. Suitable viral expression vectors include, for example, poxviruses (eg, vaccinia, fowlpox, canary pox), alphaviruses (Sindbis virus, Semliki Fores).
t) virus, Venezuelan equine encephalitis virus), adenovirus, adeno-associated virus, picornavirus (poliovirus, rhinovirus), and herpes virus (chicken pox virus, etc.). Other preferred methods for in vivo delivery of one or more nucleic acid sequences are bacterial expression vectors, eg,
Includes the use of Listeria, Salmonella, Shigella, and BCG. Inoculation with the live vector and infection in vivo will lead to expression of the antigen in vivo and induction of an immune response. The viruses and bacteria can be harmful or can be attenuated in various ways to obtain live vaccines. Such live vaccines also form part of the invention.

The DNA sequence encoding the protein of the invention may be ligated using standard DNA synthesis techniques, eg, with an enzyme as described by DM Roberts et al. In Biochemistry 1985, 24, 5090-5098. Can be synthesized by chemical synthesis, by in vitro enzymatic polymerization, or by, for example, PCR techniques utilizing thermostable polymerases, or a combination of the above techniques.
Enzymatic polymerization of DNA is carried out in vitro generally in a volume of 50 μl or less at a temperature of 10 to 37 ° C., as appropriate, with nucleoside triphosphate, dATP, d
It can be carried out using a DNA polymerase such as DNA polymerase I (Klenow fragment) in a suitable buffer containing CTP, dGTP and dTTP. Enzymatic ligation of DNA fragments is generally performed in a volume of 50 ml or less at 4 ° C to ambient temperature with a suitable buffer, eg 0.05M.
Performed with Tris (pH 7.4), 0.01M MgCl ₂ , 0.01M dithiothreitol, 1 mM spermidine, 1 mM ATP, and 0.1 mg / ml bovine serum albumin using a DNA ligase, eg, T4 ligase. You can Chemical synthesis of the above DNA polymers or fragments may be accomplished by solid phase techniques such as "Chemical and
Enzymatic Systems of Gene Fragments-A Laboratory Manual ”(ed. HG Ga
ssen and A. Lang), Verlag Chemie, Weinheim (1982), or other scientific publications,
For example, MJ Gait, HWD Matthes, M. Singh, BS Sproat, and RC Titma
s, Nucleic Acids Research, 1982, 10, 6243; BS Sproat, and W. Bannwarth
, Tetrahedron Letters, 1983, 24, 5771; MD Matteucci and MH Caruthers
, Tetrahedron Letters, 1980, 21, 719; MD Matteucci and MH Caruthers,
Journal of the American Chemical Society, 1981, 103, 3185; SP Adams e
t al., Journal of the American Chemical Society, 1983, 105, 661; ND Si
nha, J. Biernat, J. McMannus, and H. Koester, Nucleic Acids Research, 19
84, 12, 4539; and by the techniques described in HWD Matthes et al., EMBO Journal, 1984, 3, 801 by conventional phosphotriester, phosphite or phosphoramidite chemistry. You can

In a further aspect of the invention there is provided a method of making a protein as described herein. The method according to the present invention is described in, for example, Maniatis et al., Molecular Clo
ning-A Laboratory Manual; Cold Spring Harbor, 1982-1989, by conventional recombinant techniques.

In particular, the method according to the invention preferably comprises the following steps: i) a DNA polymer comprising a nucleotide sequence encoding the above protein or an immunological derivative thereof can be expressed in a host cell. Preparing a replicable or integrative expression vector; ii) transforming a host cell with the vector; iii) transforming the transformed host cell under conditions permitting expression of the DNA polymer. Culturing to produce the protein; and iv) recovering the protein.

The term “transforming” is used herein to mean the introduction of foreign DNA into a host cell. This is, for example, Genetic Engineering; Eds. SM
Kingsman and AJ Kingsman; Blackwell Scientific Publications; Oxford,
This can be accomplished using conventional techniques, such as those described in England, 1988, for example by transformation, transfection or infection with a suitable plasmid or viral vector. The term "transformed" or "transformant"
Will hereinafter be applied to the resulting host cells that contain and express the foreign gene of interest. Preferably, the recombinant antigens of the invention are expressed in a unicellular host, most preferably in a bacterial system, most preferably in E. coli.

[0026] Preferably, the recombination strategy comprises a genetic construct encoding an NS1 fusion protein (a DNA encoding NS1 linked to a DNA sequence encoding a protein of interest in the 5'to 3'direction, )) Into the expression vector to encode NS1-carboxyl terminal P703 fusion protein DN
Forming an A fragment. An affinity polyhistidine tail is engineered into the carboxy terminus of the fusion protein to allow easy purification through affinity chromatography.

[0027]   The expression vectors described above are novel and also form part of the invention.

The replicable expression vector is cleaved with a vector that is compatible with the host cell to provide a linear DNA segment with an intact replicon, and the linear segment is labeled with one or more DNAs. A molecule which, together with its linear segment, encodes the desired product, eg D, a protein according to the invention or a derivative thereof.
NA polymers may be prepared according to the invention by merging under NA conditions.

Thus, the hybrid DNA can be preformed, or formed during the construction of the vector, if desired.

The choice of vector is determined in part by the host cell, which may be prokaryotic or eukaryotic, but is preferably E. coli, yeast or CHO cells. sell. Suitable vectors include plasmids, bacteriophages, cosmids, and recombinant viruses. Expression vector and cloning
The vector preferably contains a selectable marker such that only host cells that express the marker will survive under the selection conditions. Selection genes include, but are not limited to, those encoding proteins that confer resistance to ampicillin, tetracycline or kanamycin. The expression vector also contains control sequences which are compatible with the indicated host. For example, E. Expression control sequences for E. coli, and more generally for prokaryotes, include a promoter and a ribosome binding site. The promoter sequence may be a natural, eg, β-lactamase (penicillinase) (Weis
sman 1981, In Interferon 3 (ed. L. Gresser), lactose (lac) (Chan
g et al. Nature, 1977, 198: 1056), and tryptophan (trp) (Goedde
l et al. Nucl. Acids. 1980, 8, 4057), and can be a lambda-derived P _L promoter system. In addition, non-natural synthetic promoters also serve as bacterial promoters.

This is the case, for example, with the tac synthetic hybrid promoter obtained from the sequences of the trp and lac promoters (De Boer et al., Proc. Natl Aca.
d Sci. USA 1983, 80, 21-26). The system is based on E. Especially preferred with E. coli.

Yeast-compatible vectors also carry markers that allow successful selection of transformants by conferring auxotrophic mutants with prototrophy or wild-type strains with heavy metal resistance. The control sequences for the yeast vector include promoters for glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 1968, 7, 149), PHO5 gene encoding acid phosphatase, CUP1 gene, ARG3 gene, It contains the GAL gene promoter and synthetic promoter sequences. Other control elements useful for expression in yeast are terminator and leader sequences. The leader sequence is
Especially useful. Because it typically encodes a signal peptide made from hydrophobic amino acids that direct secretion of the protein from the cell. Suitable signal sequences include genes for secreted yeast proteins, such as the yeast invertase and alpha factor genes, acid phosphatase, killer toxins, a-mating factor genes, and, more recently, Kluyveromyces marxianas. Marxianus) can be encoded by a heterologous inulinase signal sequence obtained from the INU1A gene. A suitable vector is Pichia pastoris (Pichi
a pastoris) and Saccharomyces cerevisiae (Saccharo)
has been developed for expression in Myces cerevisiae).

Different P. cerevisiae based on different inducible or constitutive promoters.
pastoris expression vector is available (Cereghino and Cregg, FEMS
Microbiol. Rev. 2000, 24: 45-66). For the production of cytoplasmic and secreted proteins, the most commonly used P. The pastoris vector contains a very strong and tightly regulated alcohol oxidase (AOX1) promoter. The vector is a P. coli vector for selection in a his4 host. pas
It also contains the toris histidinol dehydrogenase (HIS4) gene. Secretion of foreign proteins requires the presence of a signal sequence, and S. cerevi
The siae prepro alpha mating factor leader sequence has been widely and successfully used in the Pichia expression system. The expression vector is used in order to maximize the stability of the expression strain. integrated into the pastoris genome. S. cer
P. cerevisiae within sequences shared by the host genome (AOX1 or HIS4) as in E. Cleavage of the pastoris expression vector stimulates a homologous recombination event that effectively targets integration of the vector into the genomic locus.
In general, recombinant strains containing multiple integrated copies of an expression cassette are capable of producing higher amounts of heterologous protein than single copy strains. The most effective method for obtaining high copy number transformants is spheroplast.
) Technology for transformation of Pichia receptor strains (Cregg et al. 1985,
Mol. Cell. Biol. 5: 3376-3385).

Construction of a replicable expression vector is routinely carried out using the appropriate enzymes for restriction, polymerization and ligation of the DNA described above, for example by the procedure described in Maniatis et al. Cited above. Can be done.

Recombinant host cells are produced according to the invention by transforming host cells with the replicable expression vector of the invention under transformation conditions. Suitable transformation conditions are conventional, see, for example, Maniatis et al., Cited above,
Or "DNA Cloning" Vol. II, DM Glover ed., IRL Press Ltd, 1985.

The choice of transformation conditions depends on the choice of host cell to be transformed. For example, in vivo transformation using a live viral vector as a transforming agent for the polynucleotides of the invention has been previously described. A host, such as E. co
Bacterial transformation of li was performed using a solution of CaCl ₂ (Cohen et al., Proc. Nat. A.
cad. Sci., 1973, 69, 2110), or rubidium chloride (RbCl), MnC.
After treatment of the host with a solution containing a mixture of l ₂ , potassium acetate, and glycerol, and then with 3- [N-morpholino] -propane-sulfonic acid, RbCl, and glycerol, (Which can be an expression vector comprising) can be done by direct uptake of the polynucleotide. Transformation of lower eukaryotes, such as yeast cells, in culture by direct uptake is described by Hinn
en et al. (Proc. Natl. Acad. Sci. 1978, 75: 1929-1933). Mammalian cells in culture can be transformed using calcium phosphate co-precipitation of vector DNA on the cells (
Graham & Van der Eb, Virology 1978, 52, 546). Other methods for the introduction of polynucleotides into mammalian cells include dextran mediated transfection,
Includes polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotide (s) within liposomes, and direct micro-injection of polynucleotides into the nucleus.

The invention also extends to host cells transformed with a nucleic acid encoding a protein of the invention or a replicable expression vector of the invention.

Culturing the transformed host cells under conditions that permit expression of the DNA polymer is described, for example, in Maniatis et al., And “DNA Clo, cited above.
as described in "Ning", and is therefore conventionally carried out. Thus, preferably the cells are fed and below 50 ° C, preferably between 25 and
Incubation is at a temperature of 35 ° C, most preferably 30 ° C. The culture time is SDS-
It can vary from minutes to hours depending on the percentage of the polypeptide in the bacterial cells as assessed by PAGE or Western blot.

The product is recovered by conventional methods according to the host cell and according to the localization of the expression product (intracellular or secreted into the culture medium or in the periplasmic space). You can Thus, if the host cell is a bacterium, for example E. co
If it is li, it can be lysed, for example physically, chemically or enzymatically, and the protein product is isolated from the resulting lysate. If the host cell is mammalian, the product generally can be isolated from the nutrient medium or from the cell-free extract. If the host cell is a yeast, for example Saccharomyces cerevisiae or Pichia pastoris, the product can generally be isolated from the lysed cells or from the culture medium and then further using conventional techniques. It can be purified. The specificity of the expression system can be assessed by Western blot using an antibody to the polypeptide of interest.

Conventional protein isolation techniques include selective precipitation, adsorption chromatography, and affinity chromatography, including monoclonal antibody affinity columns. When the proteins according to the invention are expressed with a histidine tail (His tag), they can be easily purified by affinity chromatography using a column of ionic metal affinity chromatography column (IMAC). it can.

In a preferred embodiment of the invention, the protein according to the invention has an affinity tag, for example a polyhistidine tail.
) Is provided. In such cases, the protein after the blocking step is preferably subjected to affinity chromatography. For proteins with a polyhistidine tail, immobilized metal ion affinity chromatography (IMAC) can be performed. The metal ion can be any suitable ion, for example zinc, nickel, iron, magnesium or copper, but is preferably zinc or nickel. Preferably, the IMAC buffer is a detergent, preferably a nonionic detergent such as Tween.
n 80 ™, or a zwitterionic detergent such as Empigen BB ™. Because this can lead to lower levels of endotoxin in the final product.

Further chromatography steps include, for example, a Q-Sepharose step which can be operated either before or after the IMAC column.
Preferably, the pH is in the range of 7.5-10, more preferably 7.5-9.5, optimally between 8-9, ideally 8.5.

The proteins of the invention are provided in soluble form in liquid or lyophilized form, the latter being the preferred form. Generally, each human dose will contain 1-1000 μg of protein, and preferably 30-300 μg.

The invention also provides a pharmaceutical composition comprising a protein according to the invention in a pharmaceutically acceptable excipient. A preferred vaccine composition is at least NS1-P70
3P ^* (SEQ ID NO: 1) or NS1-P703P (SEQ ID NO: 3). The protein preferably has a blocked thiol group and is highly purified, eg having less than 5% host cell contamination. Such vaccines can optionally include one or more other tumor associated antigens and derivatives. For example, other suitable related antigens include PAP-1, PSA (prostate specific antigen), PSMA (prostate specific membrane antigen), PSCA (prostate stem cell antigen), STEAP.

Vaccine Design (“The subunit and adjuvant a
pproach ”(eds. Powell MF & Newman MJ). (1995) Plenum Press New York)
It is described inside. Encapsulation within liposomes is described by Fullerton, US Pat.
35,877.

The protein according to the invention is preferably adjuvanted in the vaccine formulation according to the invention. Suitable adjuvants are commercially available, for example from Freund's
Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit,
MI); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ); AS-2 (Smit
hkline Beecham, Philadelphia, PA): Aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphates; calcium, iron or zinc salts; insoluble suspensions of acylated tyrosine; acylated sugars; cations or anions. Derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A, and quill A. Cytokines such as GM-CSF or interleukin-2, -7, or -12 can also be used as adjuvants.

In the formulations of the present invention, the adjuvant composition induces a predominantly TH1 type immune response. High levels of Th1-type cytokines (eg IFN-γ,
TNFα, IL-2, and IL-12) tend to favor the induction of cell-mediated immune responses to administered antigens. In preferred embodiments, where the response is predominantly Th1 type, the levels of Th1 type cytokines will be increased to a greater extent than the levels of Th2 type cytokines. Levels of the cytokines can be readily assessed using standard assays. For a review of the cytokine family, see Mosmann and Coffman, Ann. Rev. Immunol. 7:14.
See 5-173, 1989.

Accordingly, suitable adjuvants used for the development of a major Th1-type response are eg aluminum salts and monophosphoryl lipid A, preferably 3
-De-O-acylated monophosphoryl lipid A (3D-MPL) combination. Other known adjuvants that predominantly induce a TH1-type immune response include CpG-containing oligonucleotides. This oligonucleotide is characterized in that the CpG dinucleotide is unmethylated. Such oligonucleotides are well known and are described, for example, in WO 96/02555. Immunostimulatory DNA sequences are also described, for example, by Sato et al., Science 273: 352, 1996. Other preferred adjuvants are saponins, preferably QS21 (Aq
uila Biopharmaceuticals Inc., Framingham, MA), which can be used alone or with other adjuvants. For example, an enhanced system is a combination of monophosphoryl lipid A and a saponin derivative, such as WO9.
A combination of QS21 and 3D-MPL as described in 4/00153 or a less reactive composition such as QS21 is quenched by cholesterol as described in WO96 / 33739. Including. Other preferred formulations include oil / water emulsions, and tocopherols. A particularly potent adjuvant formulation involving QS21,3D-MPL and tocopherol in an oil / water emulsion is described in WO 95/17210.

A particularly potent adjuvant formulation involving QS21,3D-MPL and tocopherol in an oil / water emulsion is described in WO 95/17210,
And it is a preferable compounded product.

Other preferred adjuvants are Montanide ISA 720 (Seppic, France), SAF (C
hiron, California, United States), ISCOMS (CSL), MF-59 (Chiron), Detox (
Ribi, Hamilton, MT), RC-529 (Corixa, Hamilton, MT), and other aminoalkyl glucosaminide 4-phosphates (AGPs).

Other preferred adjuvants are of the following general formula (I): HO (CH ₂ CH ₂ O) _n —A—R where n is 1 to 50 and A is a bond or —C (O). ) _-And R is C _1-50 alkyl or phenyl C _1-50 alkyl. } Adjuvant molecules represented by.

In one embodiment of the invention, n is from 1 to 50, preferably from 4 to 24, most preferably 9; R component is C _1-50 , preferably C _4- A vaccine formulation comprising a polyoxyethylene ether of general formula (I), wherein the alkyl is ₂₀ alkyl, and most preferably C ₁₄ alkyl, and A is a bond. The concentration of polyoxyethylene ether should be in the range of 0.1-20%, preferably 0.1-10%, and most preferably 0.1-1%. Preferred polyoxyethylene ethers are in the following groups: polyoxyethylene-9-lauryl ether, polyoxyethylene-9-sterol ether, polyoxyethylene-8.
-Steoryl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, and polyoxyethylene-23-
Selected from lauryl ether. Polyoxyethylene ethers, such as polyoxyethylene lauryl ether, have a Merck index (12 ^th edition: entr
y 7717). These adjuvant molecules are described in WO99 / 525.
49.

The polyoxyethylene ether according to general formula (I) above can optionally be combined with other adjuvants. For example, a preferred adjuvant combination is preferably with CpG as described in pending UK patent application GB9820956.2.

Therefore, in one aspect of the present invention, more preferably than the protein according to the present invention, in an oil / water emulsion, monophosphoryl lipid A or a derivative thereof, QS
21 and NS1-P703P ^* adjuvanted with tocopherol.

Preferably the vaccine further comprises a saponin, more preferably QS21. Other particular suitable adjuvant formulations, which include CpG and saponin, are described in WO 00/09159 and are preferred formulations. More preferably, the saponin in that particular formulation is QS21. Preferably, the formulation further comprises an oil / water emulsion and tocopherol.

Any of a variety of delivery vehicles can be used in the pharmaceutical compositions and vaccines to facilitate the generation of an antigen-specific immune response that targets tumor cells.
Delivery media include antigen presenting cells (APCs), such as dendritic cells, macrophages, B cells, monocytes, and other cells that can be genetically engineered to be efficient APCs. Such cells enhance their ability to present antigens, improve activation and / or maintenance of T cell responses, have anti-tumor effects themselves, and / or are immunologically compatible with the recipient. (Ie, matched HLA leprotype) can be, but need not be. APCs can generally be isolated from any of a variety of biological fluids and organs, including tumors and peritumoral tissues, and can be autologous, allogeneic, syngeneic, or xenogeneic cells. .

Certain preferred embodiments of the invention use dendritic cells or progenitor cells as antigen presenting cells. Dendritic cells are high and potent APCs (Banchereau and Steinman,
Nature 392: 245-251, 1998) and has been shown to be effective as a physiological adjuvant to develop prophylactic or therapeutic anti-tumor immunity (Timmerman and Le.
vy, Ann. Rev. Med. 50: 507-529, 1999). In general, dendritic cells take up and process antigens with their typical morphology (there are astrocytes in situ, with the prominent cytoplasmic processes (dendrites) found in vitro), and with high efficiency, and Can be identified based on their ability to present, as well as their ability to activate naive T cell responses. Of course, dendritic cells can be genetically engineered to express specific cell surface receptors or ligands not commonly found on dendritic cells in vivo or ex vivo, and thus modified. The generated dendritic cells are also designed according to the present invention. As an alternative to dendritic cells, secretory vesicle antigen-loaded dendritic cells (exosomes (exosomes
s)) can be used in vaccines (Zitvogel et al., Nature Me.
d. 4: 594-600, 1998).

Dendritic cells and progenitor cells are obtained from peripheral blood, bone marrow, tumor infiltrating cells, peritumoral tissue infiltrating cells, lymph nodes, spleen, skin, cord blood or any other suitable tissue or fluid. Can be done. For example, dendritic cells are
Cytokines such as GM-CSF, IL-4, IL-3, and / or TNF
The combination of α can be differentiated ex vivo by adding to monocyte cultures harvested from peripheral blood. Alternatively, CD34-positive cells harvested from peripheral blood, umbilical cord blood or bone marrow were used as a culture medium for GM-CSF, IL-3, TNFα,
Differentiating into dendritic cells by adding a combination of CD40 ligand, lipopolysaccharide LPS, flt3 ligand, and / or other compounds that induce differentiation, maturation and proliferation of dendritic cells it can.

Dendritic cells are conveniently classified as “immature” and “mature” cells, which provides a simple way of distinguishing between two well characterized phenotypes. However, this nomenclature should not be construed as excluding all possible intermediate stages of differentiation. Immature dendritic cells are characterized as APCs with high capacity for antigen uptake and processing, which are associated with Fcγ receptors and mannose
Correlate high expression of the receptor. The mature phenotype is typically characterized by low expression of the above markers, but cell surface molecules responsible for T cell activation, such as class I and class II MHC, adhesion molecules (eg CD54 and CD11). ), And high expression of costimulatory molecules (eg, CD40, CD80, CD86, and 4-1BB).

APCs are prostase oncoproteins (or derivatives thereof) such that the prostase oncoprotein, or immunogenic portion thereof, is expressed on its cell surface.
Can generally be transfected with a polynucleotide encoding Such transfection can occur ex vivo and the composition or vaccine comprising such transfected cells is then used for therapeutic, purpose, as described herein. Can be used for. Alternatively, a dendritic or other antigen presenting cell-targeting gene delivery vehicle can be administered to a patient, resulting in transfection occurring in vivo. For example, transfection of dendritic cells in vivo and ex vivo is generally performed by any method known in the art, such as those described in WO 97/24447, or Mahvi et al., Immunology a
nd cell Biology 75: 456-460, 1997, using the gene gun approach. Antigen loading of dendritic cells can be achieved by comparing dendritic or progenitor cells with prostase tumor polypeptides, DNA (naked or in a plasmid vector) or RNA; recombinant bacteria or viruses expressing the antigen (eg, vaccinia, transmissible It can be achieved by incubating with a sex epithelioma (fowlpox, adenovirus or lentivirus vector).

Vaccines and pharmaceutical compositions may be presented in unit-dose or multi-dose containers, such as sealed ampoules or vials. Such containers are preferably hermetically sealed to preserve the sterility of the formulation until use. In general, the formulations can be stored as suspensions, solutions or emulsions in oil / aqueous media. Alternatively, the vaccine or pharmaceutical composition may be stored under lyophilization conditions requiring only the addition of a sterile liquid carrier just before use.

The present invention also provides a method for producing a vaccine formulation, which comprises mixing a protein according to the present invention with a pharmaceutically acceptable excipient such as 3D-MPL.

Another aspect of the invention is the use of a protein or nucleic acid as claimed herein for the manufacture of a vaccine for the treatment of a patient with prostate cancer or other prostase associated tumor by immunotherapy. is there.

The invention will now be further described with reference to the following examples: Example I: Recombinant E. coli expressing the fusion protein NS1-P703P ^* -3-His. Preparation of E. coli strain -Protein design The expression strategy followed for the above candidates would be of the most appropriate primary structure for recombinant proteins that could have the best prediction for both good expression levels and easy purification process. Included design.

Although the chances of the recombinant protein being able to retain its protease biological activity when formulated for vaccination is, in fact, very low, mutations of its active side may result in its proteolytic degradation. This was done to substantially reduce or preferably eliminate biological activity. Therefore, the His residue at position 43 of SEQ ID NO: 8 was mutated to an Ala residue.

E. fusion protein NS1-p703 ^* -His to be expressed in E. coli
The design is shown in FIG. This fusion consists of the N-terminal (81 amino acids) of the non-structural protein of influenza virus, followed by the non-processed amino acid sequence of the prostate antigen (mutation of the 43 residues in the protease active site His-> Al).
amino acid 5 → 226 of the p703pde5 sequence described in SEQ ID NO: 8 including a),
Then includes the His tail. This histidine tail was added to prostase to allow flexible purification of the fusion and processed proteins.

The primary structure of the resulting protein has the sequence set out in FIG. The coding sequences corresponding to the above proteins are illustrated in Figure 3 and are described in E. coli
It was subsequently placed under the control of the λpL promoter in the expression plasmid.

2. E. E. coli expression system For the production of NS1, the DNA encoding the 81 amino-terminal residue of NS1 (a non-structural protein from influenza virus) was expressed in expression vector pMG81.
Cloned in. This plasmid uses signals from lambda phage DNA to drive transcription and translation of inserted foreign genes. This vector contains a lambda PL, a promoter PL, an operator OL, and two utilization sites (uti) in order to reduce the effect of transcriptional polarity when the N protein is provided.
(Lross sites) (NutL and NutR) are included (Gross et a
l., 1985. Mol. & Cell. Biol. 5: 1015). The vector containing the PL promoter was transformed into E. It is introduced into E. coli lysogenic host to stabilize the plasmid DNA.
Lysogenic host strains contain replication defective lambda phage DNA integrated into the genome (Shatzman et al., 1983; In Experimental Manipulation of Gene Expressi.
on. Inouya (ed) pp 1-14. Academic Press NY). Lambda phage DNA is
Directing the synthesis of the cI repressor protein that binds to the OL repressor of the vector and preventing the binding of RNA polymerase to the PL promoter,
And thereby prevents transcription of the inserted gene. CI of expression strain AR58
The gene contains a temperature sensitive mutation such that transcription directed by PL can be regulated by a temperature shift. That is, increasing the culture temperature inactivates the repressor, and the introduction of the foreign gene is started. This expression system allows the controlled synthesis of foreign proteins, especially those that can be toxic to the cells (Shimataka & Rosenberg, 1981. Nature 292: 128).

3. E. coli strain AR58: AR58 used for the production of NS1-P703P ^* -His protein
Lysogenic E. E. coli strains are standard NIH E. coli strains. derivatives coli K12 strain N99 (F-su-galk2, lacZ-thr -) is. It is a defective lysogenic lambda phage (galE :: TN10,1kil-cI857 DH1).
including. This kil-phenotype is a shut-off of host macromolecular synthesis.
) To prevent. The cI857 mutation confers a temperature sensitive lesion on the cI repressor. DH1 deletion is due to the right operon of lambda phage and the host bio,
Remove the uvr3 and chlA loci. The AR58 strain is a SA500 derivative (ga
1E :: TN10,1 kil-cI857 DH1) P previously grown on
Produced by transduction of N99 with lambda phage. Introduction of a defective lysogen into N99 was selected by tetracycline due to the presence of the TN10 transposon encoding tetracycline resistance within the flanking galE gene. N99 and SA500 are the National Institutes of Healt
E. H. obtained from Dr. Martin Rosenberg's laboratory. E. coli K12 strain.

[0070] 4. Construction of Vectors Designed to Express Recombinant Protein NS1-P703P ^* -His The starting materials are: 1) the cDNA received from CORIXA p703pde5 (WO00 / 04149), where its putative signal sequence, and the P703P prod. - a piece of the peptide are missing (see Fig. 1), and contains a coding sequence for prostase antigens; 2) vector pRIT14901 contains the length version of PL promoter; and 3) NS ₁ from influenza virus Plasmid PMG81, which contains the coding region.

The cloning strategy outlined in FIG. 4 comprised the following steps: a) PCR amplification of p703 sequence with NcoI and SpeI restriction sites a) Template for the PCR reaction The cDNA received from CORIXA
A plasmid, oligonucleotide sense can139: 5'GCG
CCC ATG GTT GCG GAG GAC TGC AGC CCG
3 ', and oligonucleotide antisense can134: 5'GGG AC
T AGT ACT GGC CTG GAC GGT TTT CTC 3 '
B) insertion of the amplified sequence into the commercial vector Litmus 28 (biolabs), which leads to the intermediate plasmid pRIT 14949; c) sense oligonucleotide can140: 5'CTG TCA GCC.
GCA GCG TGT TTC CAG 3 ', and antisense oligonucleotide can 141: 5' CTG GAA ACA CGC TGC G
Directed His → Ala mutagenesis of residue 43 of the p703 sequence contained in plasmid pRIT 14949 with GC TGA CAG 3 ′, which leads to the acquisition of plasmid pRIT 14950; d) the above plasmid Isolation of NcoI-SpeI fragment from pRIT 14950; e) Purification of NS1 fragment (81aa) after digestion of restriction sites BamHI-NcoI from pMG81 plasmid; f) Ligation of both fragments with expression plasmid pRIT 14901 (pr).
PL long); g) E. coli containing the plasmid pRIT 14952 (see Figure 5) expressing the NS1-p703 mutant-His fusion protein. Selection and characterization of E. coli strain AR58 transformants.

The recombinant strain thus produced the NS1-P703P ^* His tailed fusion protein with the length of 313 amino acid residues described in No. 1, the amino acid sequence of which is shown in No. 1 and the coding sequence thereof. Is described in No. 2.

Example II: Preparation of Recombinant NS1-P703P ^* -3-His Fusion Protein 1. Growth and transfer of bacterial strain B1225-NS1-P703P ^* -3-H
Expression of is AR58 cells transformed with the plasmid pRIT 14952 (strain B
1225), 400 ml of FEC0 supplemented with kanamycin sulfate (100 mg / L)
The cells were cultured in a 2 L flask containing 15AA medium. 16 at 30 ° C and 200 rpm
After a long incubation, a small sample was removed from the flask for microscopy.

50 ml of the above pre-culture was transferred into a 20-L fermentor containing 8.7 L of FEC012AF medium supplemented with kanamycin sulfate (50 mg / L). This pH is N
It was adjusted and maintained at 6.8 by the addition of H ₄ OH (25% v / v) and the temperature was maintained at 30 ° C. The aeration rate was kept constant at 20 L / min, and the pO ₂ was adjusted to 20% of saturation by feed back control of the stirring rate. Head pressure was maintained at 0.5 bar.

The fed-batch fermentation process is based on glycerol as a carbon source. The feed solution was added at a starting rate of 0.04 ml / min and increased exponentially for the first 30 hours to limit its growth rate so that a minimum pO ₂ level of 20% could be maintained. Let

After 30 hours, the fermentor temperature was rapidly raised to 39.5 ° C. in order to induce intracellular expression of the antigen NS1-P703P ^* -His. This supply rate
It was kept constant at 1.28 ml / min throughout the introduction period (18 hours).

Broth samples were taken during both the growth and induction phases to monitor bacterial growth and antigen expression. Microbiological identification and purity tests were also realized based on the above materials.

At the end of fermentation, the biomass reaches an optical density of about 130, which corresponds to a dry cell weight of about 50 g / L. The final volume was about 10.5L. Cells containing the antigen were separated directly from the culture by centrifugation at 5000g for 1 hour at 4 ° C and the pellet stored at -70 ° C in plastic bags.

2. Extraction of the above proteins: E. coli as inclusion bodies. Recombinant NS1-P703P ^* -Hi expressed in E. coli
The s protein was purified from cell homogenates using different steps (see Figure 6). Briefly, freeze-concentrated cells from the fermentation harvest were resuspended in disruption buffer (phosphate 20 mM-NaCl 2M-EDTA 5 mM pH 7.5) to a final optical density of 120 (OD650). Then, it was thawed to + 4 ° C. The cells were disrupted by passing through a high pressure homogenizer (1000 bar) twice.

Example III: Purification of the fusion protein NS1-P703P ^* -His a) Introduction As described above, the recombinant protein NS1-P703P ^* -His, in the form of inclusion bodies, was transformed into E. coli. manufactured in E. coli.
The main problem for the set-up of the purification method was probably the oxidation of the recombinant protein by itself or by host cell contaminants through covalent bonds by disulfide bonds. The developed method reduces the excessive oxidative phenomenon by having a highly purified product with an acceptable overall yield, preserving the ability of that product to mount an effective immune response against the antigen of interest. The purpose was to let.

B) Description of the above method The disrupted cell suspension was treated with Pallsep VMF (0.45 μm membrane equipped).
Vibrating Membrane Filtration) system (Pall-Filtron). The "pellet fraction" was first washed by diafiltration with 20 mM phosphate buffer pH 7.5 containing 0.5% Empigen BB detergent. The washed material is then treated with 4M guanidine hydrochloride and 20
Solubilized in the same buffer containing mM glutathione. The product was
Collected through a 45 μM filter and the permeate was treated with 200 mM iodoacetamide to prevent oxidative recoupling.

The carboxyamidated fraction was subjected to IMAC (Nickel-Chelating-Sepharose FF,
Pharmacia). The above column was first set to 4M urea, 0.5% Tween.
n 80, and 20 mM Tris buffer pH 7 containing 20 mM imidazole
． Equilibrated with 4. After loading the sample, the column was washed with the same buffer. The protein was then eluted with 400 mM imidazole in the buffer.

Before continuing the anion exchange chromatography, the electrical conductivity of the IMAC-eluent was adjusted to 20 mM T containing 4 M urea and 0.5-1.0% Tween 80.
Reduced to less than 5 mS / cm with ris buffer pH 8.5. The packed bed support (Q-Sepharose FF, Pharmacia) was equilibrated with the dilution buffer. After sample loading and washing steps with the equilibration buffer, the protein was eluted with the same buffer containing 250 mM NaCl.

Next, the Q-Sepharose FF-eluent was applied to a 10 kd cut-off membrane (Om).
Diafiltration against a suitable storage buffer (20 mM Tris buffer pH 8.0) in a tangential flow filter equipped with an ega, Filton).

The ultrafiltration retentate containing NS1-P703P ^* -His was sterile filtered through a 0.22 μm membrane.

The overall purification yield was very high: about 3-4 g of purified material (D0120) per liter of homogenate.

Example IV: Vaccine production using NS1-P703P ^* -His protein 1. Vaccine production with NS1-P703P ^* -His protein: The vaccine used in the above experiments was carried out from strains AR58 to E. coli, whether or not adjuvanted. NS1-P703P ^* -expressed in E. coli
Made from recombinant DNA, which encodes His. As an adjuvant, the formulation comprises a mixture of 3-de-O-acylated monophosphoryl lipid A (3D-MPL) and QS21 in an oil / water emulsion. This adjuvant system SBAS
2 has been previously described in WO 95/17210.

3D-MPL: is a Gram-negative bacterium, Salmonella minnesota.
minnesota) is an immunostimulant derived from lipopolysaccharide (LPS). MPL is deacylated and lacks the phosphate group on the Pyrid A component. This chemical treatment dramatically reduces toxicity while preserving its immunostimulant properties (Ribi, 1986). Ribi Immunochemistry manufactures MPL and SB-Biologic
supply to als. The experiments performed at Smith Kline Beecham Biologicals
We have shown that 3D-MPL combined with various media strongly enhances both humoral and TH1 forms of cellular immunity.

QS21: South American tree Quillaja saponaria Molina (Quillaja saponaria Mol)
Ina) is a natural saponin molecule extracted from the bark. A purification technique developed to separate individual saponins from the crude bark extract is a special saponin, QS21, which is a triterpene glycoside that exhibits stronger adjuvant activity and lower toxicity compared to its parent component. Allowed the isolation of QS21 has been shown to activate MHC class I restricted CTLs against several subunit Ags and stimulate Ag-specific lymphocyte proliferation (kensil, 1992). Aquil
a (formally Cambridge Biotech Corporation) manufactures QS21 and
S21 is supplied to SB-Biological.

Experiments performed at Smithkline Beecham Biologicals demonstrated a clear synergistic effect of the MPL and QS21 combination in the induction of both humoral and TH1-type cellular immune responses.

The oil / water emulsion consists of an organic phase made from two oils (tocopherol and squalene) and an aqueous phase of PBS containing Tween 80 ™ as an emulsifier.

The above emulsion is 5% squalene, 5% tocopherol, 0.4% Tw.
een 80, and has an average particle size of 180 nm, and SB62
(See WO 95/17210).

The experiments carried out at Smithkline Beecham Biologicals include 3D-MPL /
It was demonstrated that the addition of the above O / W emulsion to QS21 (SBAS2) further enhanced the latter's immunostimulatory properties against various subunit antigens.

2. Preparation of emulsion SB62 (2x concentrate): Tween 80 ™ is dissolved in phosphate buffered saline (PBS) to give a 2% solution in PBS. To provide 100 ml of a double concentrated emulsion, 5 g of DL alpha tocopherol and 5 ml of squalene were added,
Vortex to mix thoroughly. Add 90 ml PBS / Tween solution and mix thoroughly. The resulting emulsion is then passed through a syringe and M
Final microfluidization using a 110S Microfluidics instrument. The oil droplets obtained have a size of about 180 nm.

3. Lyophilization of NS1-P703P ^* -His: In practice, all of the compounds are placed in solution and sterilization is accomplished by filtration on a 0.2 μm membrane. Formulation was done on the day of lyophilization.

[0096]   The order of formulation is shown below:

[0097]

[Table 1]

The volume of all compounds is finally adjusted to have: Tris 10 mM, tween 80 0.2%, 3.15% sucrose,
250-50-10 μg NS1-P703P ^* -His.

The vial was overfilled with 1.25x (625 μl diluent, 500 μl injection).

A lyophilization cycle was performed over 3 days using a Lyovac GT6 lyophilizer purchased from Steris (Germany) as follows:

[0101]

[Table 2]

4. NS1-P703P ^* -His QS21 / 3D MPL oil / water (SBA
S2) Preparation of formulation: The adjuvant is formulated as a combination of MPL and QS21 in an oil / water emulsion.

1) Formulation composition (injection volume: 100 μl); Group 1 is P703P ^* -His (20) formulated in a combination of MPL and QS21 in an oil / water emulsion.
μg) was received. Group 2 received NS1-P703P ^* -His (25 μg) in a combination of MPL and QS21 in an oil / water emulsion.

[0104]   2) ingredient

[0105]

[Table 3]

[0106]   3) Combination   Formulations were prepared extemporaneously on the day of injection.

Formulation containing 3D-MPL and QS21 in an oil / water emulsion (SBAS2
Formulation B-Groups 2 and 3) was performed as follows: P703p (20 μg).
(Group 2) and NS1-P703P ^* -His (25 μg) (Group 3) were treated with SB62 (50 μl), MPL (20 μg) and QS21 (2) at intervals of 5 minutes.
0 μg), and before sequential addition of 1 μg / ml thiomersal as a preservative,
Diluted in 10 × concentrated PBS pH 6.8. All incubations were performed at room temperature with agitation.

The non-adjuvanted formulations (groups 4 and 5) were performed as follows: P703p (20 μg) (group 4) and NS1-P703P ^* -His (2).
5 μg) (group 5) were diluted in 1.5 M NaCl and H ₂ O before the addition of 1 μg / ml thiomersal as a preservative at 5 minute intervals. All incubations were performed at room temperature with agitation.

The final vaccine is obtained after reconstitution of the lyophilized NS1-P703P ^* -His preparation with adjuvant or with PBS alone.

An adjuvant control without antigen was prepared by replacing the protein with PBS.

Example V: Immunogenicity with NS1-P703P ^* -His protein 1. Immunogenicity of NS1-P703P ^* -His in Mice The purpose of this experiment was to study E. coli in the presence or absence of adjuvant. It was to characterize the immune response induced in mice by vaccination with the purified recombinant mutation NS1-p703 ^* -His produced in E. coli.

A ) -Immunization protocol: A group of 10 immunocompetent Balb / c mice, 6-8 weeks old, was treated with SBAS.
2 in 02B (50 μl SB62 / 10 μg MPL / 10 μg QS21)
With or without 5 μg of mutation NS1-P703,
The muscle was vaccinated twice at 2 week intervals.

Fourteen days after the second injection, blood was drawn and the serum was treated with anti-P703.
Tested for the presence of antibody.

B) Total IgG antibody response: Anti-P703 antibody response was assessed in the serum of the mice 14 days after the last vaccination. This is an ELI using NS1-P703P ^* as the coating antigen.
Performed by SA.

E. E. coli extract was used to check for the presence of potential antibodies to host contaminants.

C) -Results : The above results are : 1) NS1-P70 as evidenced in the serum of mice injected with NS1-P703P ^* protein alone compared to the serum of normal control mice.
3P ^* induces a higher immune response (IgG1); 2) AS02
In animals receiving the NS1-P703P ^* molecule formulated in B adjuvant,
It shows that a high antibody titer is seen.

The isotype profile of the NS1-p703p specific IgG response was also measured. Figure 8
As shown in Figure 3, IgG1 was detected when mice received the NS1-P703P ^* protein alone, whereas isotype profiles were directed to the TH1 response (more IgG2a) by the presence of AS02 adjuvant.

Example VI: 1. -NS1-P703P-His NS1-P703P-His was prepared in the same manner. The amino acid and DN
The A sequence is shown in SEQ ID NO: 3 and SEQ ID NO: 4.

Briefly, E. The strategy for expressing the NS1-P703P-His fusion protein in E. coli included the following steps: a) As starting material, the same starting material as described in Example 1 (SEQ ID NO: 8)
Amino acid 5 → 226) of the p703pde5 sequence described in 1 .; b) PCR amplification to place cloning restriction sites on both sides of the p703 non-mutated sequence; c) Insertion into the PMG81 vector (promoter pL long) containing the NS1 gene. D) transformation of the receptor strain AR58 or AR120; e) selection of recombinant strains.

The resulting protein can be purified in a similar manner to the NS1-P703P ^* -His mutein. The primary structure of the resulting protein has the sequence described in Figure 9. The coding sequence corresponding to the above protein is shown in FIG.
It shows in 0.

[0121]     References:

[0122]

[Table 4]

[Sequence list]

[Brief description of drawings]

FIG. 1 E. Design of the fusion protein NS1-p703 ^* -His expressed in E. coli.

FIG. 2 E. Primary structure of the fusion protein NS1-p703 ^* -His expressed in E. coli (SEQ ID NO: 1).

FIG. 3: Cloning sequence of NS1 _1-81 -P703P ^* -His (SEQ ID NO: 2).

FIG. 4 E. Cloning strategy for producing NS1-P703P ^* -His in E. coli.

[Figure 5]   Plasmid map of RIT 14952.

FIG. 6 E. coli NS1-P703P ^* -His fermentation process.

FIG. 7 E. coli NS1-P703P ^* -His purification process.

FIG. 8. Immunogenicity of NS1-P703P ^* adjuvanted with SBAS2.

FIG. 9 E. Primary structure of the fusion protein NS1-p703-His expressed in E. coli (SEQ ID NO: 3).

FIG. 10: Cloning sequence of NS1 _1-81- P703P-His (SEQ ID NO: 4).

[Procedure for Amendment] Submission for translation of Article 34 Amendment of Patent Cooperation Treaty

[Submission date] August 20, 2001 (2001.8.20)

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be amended] Claims

[Correction method] Change

[Contents of correction]

[Claims]

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12N 1/21 C12N 9/64 Z 5/10 15/00 ZNAA 9/64 5/00 A (31) Priority Right claim number 09 / 443,686 (32) Priority date November 18, 1999 (November 18, 1999) (33) Country of priority claim United States (US) (31) Priority claim number 09 / 483,672 (32) Priority date January 14, 2000 (January 14, 2000) (33) Priority claiming country United States (US) (31) Priority claim number 09 / 536,857 (32) Priority date 2000 March 27 (March 27, 2000) (33) Priority claiming country United States (US) (31) Priority claim number 09 / 568,100 (32) Priority date May 9, 2000 (2000.5) 9) (33) Priority claiming country United States (US) (31) Priority claiming number 09 / 570,737 (32) May 12, 2000 (May 12, 2000) (33) Priority claiming country United States (US) (31) Priority claim number 09 / 593,793 (32) Priority date June 13, 2000 (June 13, 2000) (33) United States (US) (31) Priority claim number 0015747.9 (32) Priority date June 27, 2000 (June 27, 2000) (33) Priority claiming country United Kingdom (GB) (31) Priority claiming number 09 / 605,783 (32) Priority date June 27, 2000 (June 27, 2000) (33) Priority claiming country United States (US) (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ , CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ) , UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH , CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU , SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Kabzon, Silva Teresa Belgium, Be-1330 Rique Sansart, Rue de l'Institut 89, Smith Kla N'Beacham Biologicals Societe Anonym (72) Inventor Dillon, Davin Clifford United States, Washington 98104, Theater, Suite 464, Colombia Treat 1124, Corixa Corporation F Term (reference) 4B024 AA01 AA11 BA14 BA31 CA04 CA07 DA06 EA04 GA11 HA12 4B050 CC04 DD11 GG06 LL01 LL03 4B065 AA26X AA93Y AB01 AC14 BA02 CA33 CA44 CA45 4C084 AA13 NA10 ZB262 ZC102 ZC192 4C085 AA03 BB01 BB22 DD62 EE01 EE06

Claims (18)

[Claims] 1. A prostase protein or derivative thereof bound to a fusion partner. 2. The protein of claim 1, wherein the prostase antigen is selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8. 3. The protein according to claim 1 or 2, wherein the fusion partner is an immunological fusion partner or an expression enhancer fusion partner. 4. The protein according to any one of claims 1 to 3, wherein the fusion partner is the NS1 protein from influenza or a fragment thereof. 5. The antigen of any one of claims 1-4, wherein the fusion protein further comprises an affinity tag. 6. A nucleic acid sequence encoding the protein according to any one of claims 1-5. 7. An expression vector containing the nucleic acid according to claim 6. 8. A host cell transformed with the nucleic acid sequence of claim 6 or the vector of claim 7. 9. A vaccine comprising the protein according to any one of claims 1 to 5 or the nucleic acid according to claim 6. 10. The vaccine of claim 9, further comprising an adjuvant, and / or an immunostimulatory cytokine or chemokine. 11. A vaccine according to claim 9 or 10 wherein the protein is provided in an oil / water or water / oil emulsion medium. 12. The adjuvant comprises 3D-MPL, QS21, CpG oligonucleotide, or polyethylene ether or ester.
The vaccine according to 0 or 11. 13. The vaccine according to any one of claims 9 to 12, which further comprises one or more other antigens. 14. A vaccine as claimed herein for use in medicine. 15. Use of a protein or nucleic acid as described herein for the manufacture of a vaccine for treating a patient suffering from prostate cancer or other prostase associated tumors by immunotherapy. 16. A method comprising transforming a host cell with the nucleic acid sequence of claim 6, expressing said sequence and isolating the desired product.
5. The method for producing the protein according to any one of 5 to 5. 17. A step of purifying a prostase protein or a derivative thereof according to the production method of claim 16, and the obtained protein described herein as a suitable adjuvant, diluent or other medicine. A method of making a vaccine comprising the step of mixing with an acceptable excipient. 18. A method of treating a patient susceptible to or suffering from prostate cancer, wherein said patient is administered a pharmaceutically active amount of the vaccine of any one of claims 9-14. The method, comprising: