WO2002000708A2 - Vaccin - Google Patents

Vaccin Download PDF

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Publication number
WO2002000708A2
WO2002000708A2 PCT/EP2001/007079 EP0107079W WO0200708A2 WO 2002000708 A2 WO2002000708 A2 WO 2002000708A2 EP 0107079 W EP0107079 W EP 0107079W WO 0200708 A2 WO0200708 A2 WO 0200708A2
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WIPO (PCT)
Prior art keywords
protein
antigen
vaccine
prostase
expression
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PCT/EP2001/007079
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WO2002000708A3 (fr
Inventor
Teresa Elisa Virginia Cabezon Silva
Martine Marchand
Carlota Vinals Y De Bassols
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GlaxoSmithKline Biologicals SA
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SmithKline Beecham Biologicals SA
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Priority to AU2001285773A priority Critical patent/AU2001285773A1/en
Publication of WO2002000708A2 publication Critical patent/WO2002000708A2/fr
Publication of WO2002000708A3 publication Critical patent/WO2002000708A3/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6445—Kallikreins (3.4.21.34; 3.4.21.35)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/08—Drugs for disorders of the urinary system of the prostate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to protein derivatives of a protein known as prostase, a prostate-specific serine protease, to methods for their purification and manufacture, and also to pharmaceutical compositions containing such derivatives, and to their use in medicine.
  • protein derivatives find utility in cancer vaccine therapy, particularly prostate cancer vaccine therapy and diagnostic agents for prostate tumours.
  • the prostase derivatives of the invention are genetically modified prostase wherein the protein is mutated in its active site, and may further include chemically modified prostase protein wherein the antigen's disulphide bridges are reduced and the resulting thiols blocked.
  • fusion proteins comprising the mutated prostase linked to an immunological or an expression enhancer fusion partner, optionally containing blocked thiols residues are also contemplated by the present invention.
  • the present invention also provides methods for purifying the prostase derivatives and for formulating vaccines for i munotherapeutically treating prostate cancer patients and prostase-expressing tumours other than prostate tumours, prostatic hyperplasia, and prostate intraepithelilial neoplasia (PIN).
  • Prostate cancer is the most common cancer among males, with an estimated incidence of 30% in men over the age of 50. Overwhelming clinical evidence shows that human prostate cancer has the propency to metastasise to bone, and the disease appears to progress inevitably from androgen dependent to androgen refractory status, leading to increased patient mortality (Abbas F., Scardino P. "The Natural History of Clinical Prostate Carcinoma.” In Cancer (1997); 80:827-833). This prevalent disease is currently the second leading cause of cancer death among men in the US.
  • tumour-associated antigens are already known. Many of these antigens may be interesting targets for immunotherapy, but are either not fully tumour-specific or are closely related to normal proteins, and hence bear with them the risk of organ-specific auto-immunity, once targeted by a potent immune response.
  • prostate specific proteins like prostate specific antigen (PSA) and prostatic acid phosphatase (PAP), prostate-specific membrane antigen (PSMA) and prostate stem cell antigen (PSCA) have limited therapeutic potential and moreover are not always correlated with the presence of prostate cancer or with the level of metastasis (Pound C, Partin A., Eisenberg M. et al. "Natural History of Progression after PSA Elevation following Radical Prostatectomy.” In Jama (1999); 281:1591-1597) (Bostwick D., PaceUi A., Blute M. et al. "Prostate Specific Membrane Antigen Expression in Prostatic Intraepithelial Neoplasia and Adenocarcinoma.” In Cancer (1998); 82:2256-2261).
  • tumour rejection mechanisms has been recognised since several decades. Tumour antigens, though encoded by the genome of the organism and thus theoretically not recognized by the immune system through the immune tolerance phenomenon, can occasionally induce immune responses detectable in cancer patients. This is evidenced by antibodies or T cell responses to antigens expressed by the tumour (Xue BH., Zhang Y., Sosman J. et al. "Induction of Human Cytotoxic T-Lymphocytes Specific for Prostate-Specific Antigen.” In Prostate (1997); 30(2):73-78).
  • tumour antigens expressed by tumour cells can lead to complete regression of established tumours in animal models (mainly murine). It is now recognised that the expression of tumour antigens by a cell is not sufficient for induction of an immune response to these antigens. Initiation of a tumour rejection response requires a series of immune amplification phenomena dependent on the intervention of antigen presenting cells, responsible for delivery of a series of activation signals.
  • Prostase is a prostate-specific serine protease (trypsin-like), 254 amino acid- long, with a conserved serine protease catalytic triad H-D-S and a amino-terminal pre- propeptide sequence, indicating a potential secretory function (P. Nelson, Lu Gan, C. Ferguson, P. Moss, R. Gelinas, L. Hood & K. Wand, "Molecular cloning and characterisation of prostase, an androgen-regulated serine protease with prostate restricted expression, In Proc. Natl. Acad. Sci. USA (1999) 96, 3114-3119). A putative glycosylation site has been described. The predicted structure is very similar to other known serine proteases, showing that the mature polypeptide folds into a single domain. The mature protein is 224 amino acids-long, with one A2 epitope shown to be naturally processed.
  • the present invention provides mutated prostase protein derivatives and fragments and homologues thereof. Such derivatives are suitable for use in therapeutic vaccine formulations which are suitable for the treatment of a prostate tumours and prostase-expressing tumours.
  • Prostase fragments harbouring a mutation according to the invention will be of at least about 10 consecutive amino acids, preferably about 20, more preferably about 50, more preferably about 100, more preferably about 150 contiguous amino acids selected from the amino acid sequences as shown in SEQ ID N°5, SEQ ID N°6, SEQ ID N°7, SEQ ID N°8 or SEQ ID N°9. More particularly fragments will retain some functional property, preferably an immunological activity, of the larger molecule set forth in SEQ ID N°5, SEQ ID N°6, SEQ ID N°7, SEQ ID N°8 or SEQ ID N°9, and are useful in the methods described herein (e.g. in vaccine compositions, in diagnostics, etc.). In particular the fragments will be able to generate an immune response, when suitable attached to a carrier, which will recognise the protein of SEQ ID N°5, SEQ ID N°6, SEQ ID N°7, SEQ ID N°8 or SEQ ID N°9.
  • Prostase homologues will generally share substantial sequence similarity, and include isolated polypeptides comprising an amino acid sequence which has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, yet more preferably at least 95% identity, most preferably at least 97-99% identity, to that of SEQ ID N°5, SEQ ID N°6, SEQ ID NO:7, SEQ ID N°8 or SEQ ID N°9 over the entire length of SEQ ID N°5, SEQ ID N°6, SEQ ID NO:7, SEQ ID N°8 or SEQ ID N°9
  • Such polypeptides include those comprising the amino acid of SEQ ID N°5, SEQ ID N°6, SEQ ID NO:7, SEQ ID N°8 or SEQ ID N°9.
  • a mutated prostase antigen wherein the mutation occurs in the active site of the protein.
  • the prostase antigen derivative or fragments and homologues thereof carry a mutation in the active site of the protein, to reduce substantially or preferably eliminate its protease biological activity.
  • Preferred mutations involve replacing the Histidine and Aspartate catalytic residues of the serine protease.
  • prostase contains a Histidine- Alanine mutation in the active site, for example at residue 71 of prostase sequence (Ferguson, et al. (Proc. Natl. Acad. Sci. USA 1999, 96, 3114-3119).
  • Corresponding mutation in homologous proteins are expressly contemplated.
  • this mutation corresponds to position 43 in SEQ ID N°8.
  • This mutation can lead to a significant decrease in the catalytic efficiency (expressed in enzymatic specific activity) of the protease.
  • the reduction in the catalytic efficiency is at least by a factor of 10 3 , more preferably at least by a factor of 10 6 .
  • the protein which has undergone a histidine alanine mutation is hereafter referred to as * (star).
  • Preferred antigens have the amino acid sequence as set forth in SEQ ID N°l and SEQ ID N°9.
  • the mutated protein according to the invention may in one embodiment be part of a larger fusion protein, comprising the mutated prostase or fragment or homologues thereof and a heterologous protein, or part of a protein, acting as a fusion partner.
  • the protein and the fusion partner may be chemically conjugated, but are preferably expressed as recombinant fusion proteins in a heterologous expression system.
  • the fusion partner may act through a bystander helper effect linked to secretion of activation signals by a large number of T cells specific to the foreign protein or peptide, thereby enhancing the induction of immunity to the prostase component as compared to the non-fused protein.
  • the heterologous partner is selected to be recognizable by T cells in a majority of humans.
  • the invention provides a mutated prostase protein or fragment or homologues thereof linked to a fusion partner that acts as an expression enhancer.
  • the fusion partner may assist in aiding in the expression of prostase in a heterologous system, allowing increased levels to be produced in an expression system as compared to the native recombinant protein.
  • the fusion partner will be both an immunological fusion partner and an expression enhancer partner.
  • the present invention provides fusion proteins comprising a mutated tumour-specific prostase or a fragment thereof linked to a fusion partner.
  • the fusion partner is acting both as an immunological fiision partner and as an expression enhancer partner.
  • the fusion partner is the non-structural protein from influenzae virus, NSl (hemagglutinin) or fragment thereof.
  • NSl hemagglutinin
  • the N-terminal 81 amino acids are utilised, although different fragments may be used provided they include T-helper epitopes (C. hackett, D. Horowitz, M. Wysocka & S.
  • NSl 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 at higher yields than the native recombinant prostase proteins.
  • the fusion protein comprises the N-terminal 81 amino acids of NSl non structural protein fused to the 5 to 226 carboxy-terminal amino acids from mutated prostase, as set forth in SEQ ID NO: 1.
  • the mutated prostase protein, and mutated prostase-comprising fusions may in a preferred embodiment be chemically modified.
  • the prostase protein's intra- and inter-molecular disulphide bridges are reduced and the resulting thiols are blocked to prevent oxydative recoupling.
  • the derivatised thiol residues are blocked by an alkylating agent. More preferably, the alkylating agent is a carboxyalkylating agent. Most preferably the derivatised thiol residues are carboxyamidated or carboxymethylated. This treatment advantageously limits the protein aggregation and protein precipitation thereby improving the expression yield and the final purity of the protein.
  • the proteins of the present invention are expressed in an appropriate host cell, and preferably in E. coli.
  • the proteins are expressed with an affinity tag, such as for example, a histidine tail comprising between 5 to 9 and preferably six histidine residues, most preferably at least 4 histidine residues.
  • an affinity tag such as for example, a histidine tail comprising between 5 to 9 and preferably six histidine residues, most preferably at least 4 histidine residues.
  • the present invention also provides a nucleic acid encoding the proteins 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.
  • genetic constructs comprising one or more of the polynucleotides of the invention are introduced into cells in vivo. This may be achieved using any of a variety or well-known approaches.
  • One of the preferred methods for in vivo delivery of one or more nucleic acid sequences involves the use of an expression vector such as a recombinant live viral or bacterial microorganism.
  • Suitable viral expression vectors are for example poxviruses (e.g; vaccinia, fowlpox, canarypox), alphaviruses (Sindbis virus, Semliki Forest Virus, Dialoguelian Equine Encephalitis Virus), adenoviruses, adeno-associated virus, picornaviruses (poliovirus, rhinovirus), and herpesviruses (varicella zoster virus, etc).
  • poxviruses e.g; vaccinia, fowlpox, canarypox
  • alphaviruses Semliki Forest Virus, Kunststoffuelian Equine Encephalitis Virus
  • adenoviruses adeno-associated virus
  • picornaviruses poliovirus, rhinovirus
  • herpesviruses variantcella zoster virus, etc.
  • Other preferred methods for in vivo delivery of one or more nucleic acid sequences involves the use of a bacterial
  • live vaccines also form part of the invention.
  • a DNA sequence encoding the proteins of the present invention can be synthesized using standard DNA synthesis techniques, such as by enzymatic ligation as described by D.M. Roberts et al. in Biochemistry 1985, 24, 5090-5098, by chemical synthesis, by in vitro enzymatic polymerization, or by PCR technology utilising for example a heat stable polymerase, or by a combination of these techniques.
  • Enzymatic polymerisation of DNA may be carried out in vitro using a DNA polymerase such as DNA polymerase I (Klenow fragment) in an appropriate buffer containing the nucleoside triphosphates dATP, dCTP, dGTP and dTTP as required at a temperature of 10°-37°C, generally in a volume of 50 ⁇ l or less.
  • a DNA polymerase such as DNA polymerase I (Klenow fragment) in an appropriate buffer containing the nucleoside triphosphates dATP, dCTP, dGTP and dTTP as required at a temperature of 10°-37°C, generally in a volume of 50 ⁇ l or less.
  • Enzymatic ligation of DNA fragments may be carried out using a DNA ligase such as T4 DNA ligase in an appropriate buffer, such as 0.05M Tris (pH 7.4), 0.01M MgCl 2 , 0.01M dithiothreitol, ImM spermidine, ImM ATP and O.lmg/ml bovine serum albumin, at a temperature of 4°C to ambient, generally in a volume of 50ml or less.
  • a DNA ligase such as T4 DNA ligase in an appropriate buffer, such as 0.05M Tris (pH 7.4), 0.01M MgCl 2 , 0.01M dithiothreitol, ImM spermidine, ImM ATP and O.lmg/ml bovine serum albumin, at a temperature of 4°C to ambient, generally in a volume of 50ml or less.
  • the chemical synthesis of the DNA polymer or fragments may be carried out by conventional phosphotriester, phosphite or phosphoramidite chemistry, using solid phase techniques such as those described in 'Chemical and Enzymatic Synthesis of Gene Fragments - A Laboratory Manual' (ed. H.G. Gassen and A. Lang), Verlag Chemie, Weinheim (1982), or in other scientific publications, for example M.J. Gait, H.W.D. Matthes, M. Singh, B.S. Sproat, and R.C. Titmas, Nucleic Acids Research, 1982, 10, 6243; B.S. Sproat, and W. Bannwarth, Tetrahedron Letters, 1983, 24, 5771; M.D.
  • a method of producing a protein as described herein is provided a method of producing a protein as described herein.
  • the process of the invention may be performed by conventional recombinant techniques such as described in Maniatis et al, Molecular Cloning - A Laboratory Manual; Cold Spring Harbor, 1982-1989.
  • the process of the invention may preferably comprise the steps of: i) preparing a replicable or integrating expression vector capable, in a host cell, of expressing a DNA polymer comprising a nucleotide sequence that encodes the protein or an immunogenic derivative thereof; ii) transforming a host cell with said vector; ii) culruring said transformed host cell under conditions permitting expression of said DNA polymer to produce said protein; and iv) recovering said protein.
  • 'transforming' is used herein to mean the introduction of foreign DNA into a host cell. This can be achieved for example by transformation, transfection or infection with an appropriate plasmid or viral vector using e.g. conventional techniques as described in Genetic Engineering; Eds. S.M. Kingsman and A.J. Kingsman; Blackwell Scientific Publications; Oxford, England, 1988.
  • the term 'transformed' or 'transformant' will hereafter apply to the resulting host cell containing and expressing the foreign gene of interest.
  • recombinant antigens of the invention are expressed in unicellular hosts, most preferably in bacterial systems, most preferably in E. coli.
  • the recombinant strategy includes cloning a gene construct encoding a NSl fusion protein, the gene construct comprising from 5' to 3' a DNA sequence encoding NSl joined to a DNA sequence encoding the protein of interest, into an expression vector to form a DNA fragment encoding a NSl- carboxyl-terminal P703P fusion protein.
  • An affinity polyhistidine tail may be engineered at the carboxy- terminus of the fusion protein allowing for simplified purification through affinity chromatography.
  • the expression vectors are novel and also form part of the invention.
  • the replicable expression vectors may be prepared in accordance with the invention, by cleaving a vector compatible with the host cell to provide a linear DNA segment having an intact replicon, and combining said linear segment with one or more DNA molecules which, together with said linear segment encode the desired product, such as the DNA polymer encoding the protein of the invention, or derivative thereof, under ligating conditions.
  • the hybrid DNA may be pre-formed or formed during the construction of the vector, as desired.
  • the choice of vector will be determined in part by the host cell, which may be prokaryotic or eukaryotic but are preferably E. coli, yeast or CHO cells. Suitable vectors include plasmids, bacteriophages, cosmids and recombinant viruses. Expression and cloning vectors preferably contain a selectable marker such that only the host cells expressing the marker will survive under selective conditions. Selection genes include but are not limited to the one encoding protein that confer a resistance to ampicillin, tetracyclin or kanamycin. Expression vectors also contain control sequences which are compatible with the designated host. For example, expression control sequences for E.
  • promoters and ribosome binding sites include promoters and ribosome binding sites.
  • Promoter sequences may be naturally occurring, such as the ⁇ -lactamase (penicillinase) (Weissman 1981, In Interferon 3 (ed. L. Gresser), lactose (lac) (Chang et al. Nature, 1977, 198: 1056) and tryptophan (tip) (Goeddel et al. Nucl. Acids Res. 1980, 8, 4057) and lambda-derived P promoter system, hi addition, synthetic promoters which do not occur in nature also function as bacterial promoters.
  • tac synthetic hybrid promoter which is derived from sequences of the tip and lac promoters (De Boer et al, Proc. Natl Acad Sci. USA 1983, 80, 21-26). These systems are particularly suitable withE. coli.
  • Yeast compatible vectors also carry markers that allow the selection of successful transformants by conferring prototrophy to auxotrophic mutants or resistance to heavy metals on wild-type strains.
  • Control sequences for yeast vectors include promoters for glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 1968, 7, 149), PHO5 gene encoding acid phosphatase, CUPl gene, ARG3 gene, GAL genes promoters and synthetic promoter sequences.
  • Other control elements useful in yeast expression are terminators and leader sequences. The leader sequence is particularly useful since it typically encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell.
  • Suitable signal sequences can be encoded by genes for secreted yeast proteins such as the yeast invertase gene and the a-factor gene, acid phosphatase, killer toxin, the a-mating factor gene and recently the heterologous inulinase signal sequence derived from INUIA gene of Kluyveromyces marxianus.
  • yeast invertase gene and the a-factor gene such as the yeast invertase gene and the a-factor gene, acid phosphatase, killer toxin, the a-mating factor gene and recently the heterologous inulinase signal sequence derived from INUIA gene of Kluyveromyces marxianus.
  • Suitable vectors have been developed for expression in Pichia pastoris and Saccharomyces cerevisiae.
  • P. pastoris expression vectors are available based on various inducible or constitutive promoters ( Cereghino and Cregg, FEMS Microbiol. Rev. 2000,24:45-66).
  • the most commonly used P. pastoris vectors contain the very strong and tightly regulated alcohol oxidase (AOX1) promoter.
  • the vectors also contain the P. pastoris histidinol dehydrogenase (HIS4) gene for selection in his4 hosts. Secretion of foreign protein require the presence of a signal sequence and the S. cerevisiae prepro alpha mating factor leader sequence has been widly and successfully used in Pichia expression system.
  • Expression vectors are integrated into the P.
  • the preparation of the replicable expression vector may be carried out conventionally with appropriate enzymes for restriction, polymerisation and ligation of the DNA, by procedures described in, for example, Maniatis et al. cited above.
  • the recombinant host cell is prepared, in accordance with the invention, by transforming a host cell with a replicable expression vector of the invention under transforming conditions.
  • Suitable transforming conditions are conventional and are described in, for example, Maniatis et al. cited above, or "DNA Cloning" Vol. II, D.M. Glover ed., IRL Press Ltd, 1985.
  • transforming conditions depends upon the choice of the host cell to be transformed. For example, in vivo transformation using a live viral vector as the fransforming agent for the polynucleotides of the invention is described above. Bacterial transformation of a host such as E. coli may be done by direct uptake of the polynucleotides (which may be expression vectors containing the desired sequence) after the host has been treated with a solution of CaCl (Cohen et al, Proc. Nat. Acad.
  • polynucleotides include dextran mediated transfection, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) into liposomes, and direct micro-injection of the polynucleotides into nuclei.
  • the invention also extends to a host cell transformed with a nucleic acid encoding the protein of the invention or a replicable expression vector of the invention.
  • Culturing the transformed host cell under conditions permitting expression of the DNA polymer is carried out conventionally, as described in, for example, Maniatis et al and "DNA Cloning" cited above.
  • the cell is supplied with nutrient and cultured at a temperature below 50°C, preferably between 25°C and 35°C, most preferably at 30°C.
  • the incubation time may vary from a few minutes to a few hours, according to the proportion of the polypeptide in the bacterial cell, as assessed by SDS-PAGE or Western blot.
  • the product may be recovered by conventional methods according to the host cell and according to the localisation of the expression product (intracellular or secreted into the culture medium or into the cell periplasm).
  • the host cell is bacterial, such as E. coli it may, for example, be lysed physically, chemically or enzymatically and the protein product isolated from the resulting lysate.
  • the product may generally be isolated from the nutrient medium or from cell free extracts.
  • the host cell is a yeast such as Saccharomyces cerevisiae or Pichia pastoris
  • the product may generally be isolated from from lysed cells or from the culture medium, and then further purified using conventional techniques. The specificity of the expression system may be assessed by western blot using an antibody directed against the polypeptide of interest.
  • Protein isolation techniques include selective precipitation, adsorption chromatography, and affinity chromatography including a monoclonal antibody affinity column.
  • proteins of the present invention When expressed with a histidine tail (His tag), they can easily be purified by affinity chromatography using an ion metal affinity chromatography column (IMAC) column.
  • IMAC ion metal affinity chromatography column
  • a process for purifying a protein of the invention comprises treating the protein to reduce the protein's intra- and inter-molecular disulphide bonds, and blocking the thiol to prevent oxydative recoupling (referred to as "reduction/blocking step").
  • the reduction blocking step may be carried out at the end of the purification process, on the purified antigen. Preferably however the reduction/blocking step is carried out during the purification process. Accordingly therefore there is provided a process comprising treating the protein to reduce the protein's intra- and inter-molecular disulphide bonds, blocking the thiol to prevent oxydative recoupling and subjecting the protein to one or more chromatographic steps.
  • Such steps lead to an increase of approximately 10-fold in protein yield, as without such steps, the protein aggregates and precipitates, reducing the effectiveness of downstream purification. Final purity and process consistency are also improved. It is prefened to first solubilise the product in a strong chaotropic agent such as urea, guanidium hydrochloride. Zwitterionic detergents such as Empigen BB - n- dodecyl- N,N-dimethylglycine, or other detergents like for example Tween 80 (polyoxyethylene (20) sorbitan monooleate) may also be used.
  • Optimally solubilisation involves the use of both a detergent and a chaotropic agent, optionally in the presence of the reducing agent.
  • the solubilisation/reduction step is performed simultaneously, involving the use a detergent, a chaotropic agent and a reducing agent.
  • VMF Vibrating Membrane Filtration
  • the protein is solubilised utilising a combination of both a strong chaotropic agent and a detergent.
  • the detergent is in the range of 0.1% to 5%, more preferably from 0.2% to 2%, ideally from 0.5% to 1%.
  • the pH is in the range of 7 to 10, more preferably from 7.5 to 9.5, optimally between 7.5 and 9, ideally 8.5.
  • the purification involves a first step of solubilising the protein preferably utilising both a strong chaotropic agent and a detergent, secondly, filtering the product, and subsequently reducing at least one, preferably substantially all, preferably all the protein's intra- and inter-molecular disulphide bonds utilising a reducing agent such as but not limited to glutathion, and blocking the free thiol groups, and subjecting the resulting protein to one or more chromatographic steps.
  • a reducing agent such as but not limited to glutathion
  • the purification involves a first step of solubilisation/reduction using a strong chaotropic agent, a detergent and a reducing agent, secondly, filtering the product, thirdly blocking the free thiol groups, and then subjecting the resulting protein to one or more chromatographic steps.
  • the blocking agent is an alkylating agent.
  • Such blocking agents include but are not limited to alpha haloacids or alpha haloamides. More preferably the alkylating agent is a carboxyalkylating agent.
  • the carboxyalkylating agent is iodoacetic acid or iodoacetamide, which respectively results in carboxymethylation or carboxyamidation (carbamidomethylation) of the protein.
  • Other blocking agents may be used and are described in the literature (See for example, The Proteins Vol II Eds H neurath, RL Hill and C-L Boeder, Academic press 1976, or Chemical Reagents for Protein modification Vol I eds. RL Lundblad and CM Noyes, CRC Press 1985).
  • Typical examples of such other blocking agents include N-ethylmaleimide, chloroacetyl phosphate, O-methylisourea and acrylonitrile.
  • the use of the blocking agent is advantageous as it prevents the oxidation and subsequent aggregation of the prostase antigen and fusion derivatives, and ensure stability of the modified protein for downstream purification.
  • the overall benefit is an increase in the purification yield, an enhanced product purity as well as a consistency in the manufacture process.
  • One or more of these purification steps involves an ion-metal chelate affinity chromatography, preferably but not restricted to a Nickel-chelate affinity chromatography.
  • These polypeptides can be purified to high levels (greater than 80% preferably greater than 90% pure as visualised by SDS-PAGE) by undergoing further purification steps.
  • An additional purification step is a Q-Sepharose step that may be operated either before or after the IMAC column to yield highly purified protein.
  • the proteins so purified present a major single band when analysed by SDS PAGE under reducing conditions, and western blot analysis show less than 10%, preferably less than 5% host cell protein contamination.
  • the reduction/blocking treatment also advantageously leads to the eliciting of antibody response at least as good, preferably better after injection of the modified protein as compared to the unmodified one.
  • the blocking agents are selected to induce a stable covalent and ineversible derivative (eg alpha halo acids or alpha haloamides).
  • a stable covalent and ineversible derivative eg alpha halo acids or alpha haloamides.
  • other blocking agents maybe selected such that after purification the blocking agent may be removed to release the non-derivatised protein.
  • proteins of the invention having derivatised free thiol residues are new and form an aspect of the invention. hi particular, carboxyamidated or carboxymethylated derivatives are a prefened embodiment of the invention.
  • the proteins of the present invention is provided with an affinity tag, such as a polyhistidine tail or a C-LYTA tag.
  • an affinity tag such as a polyhistidine tail or a C-LYTA tag.
  • the protein is preferably subjected to affinity chromatography.
  • the affinity tag comprises a Histidine tail, fused at the carboxy-terminus of the proteins of the invention, preferably comprising between 5 to 8 histidine residues, preferably at least 4 residues, and most preferably 6 histidine residues.
  • the affinity peptide has adjacent histidine residues, preferably at least two, more preferably at least 4 residues. Most preferably the protein comprises 6 directly neighbouring histidine residues.
  • the proteins are harbouring a C- LYTA tag at their carboxy-terminus.
  • the C terminal portion of the molecule is used.
  • Lyta is derived from Streptococcus pneumoniae which synthesize an N-acetyl-L-alanine amidase, amidase LYTA, (coded by the lytA gene ⁇ Gene, 43 (1986) page 265-272 ⁇ an autolysin that specifically degrades certain bonds in the peptidoglycan backbone.
  • the C-terminal domain of the LYTA protein is responsible for the affinity to the choline or to some choline analogues such as DEAE. This property has been exploited for the development of E.coli C-LYTA expressing plasmids useful for expression of fusion proteins. Purification of hybrid proteins containing the C-LYTA fragment at its amino terminus has been described ⁇ Biotechnology: 10, (1992) page 795-798 ⁇ . As used herein a prefened embodiment utilises the repeat portion of the Lyta molecule found in the C terminal end starting at residue 178. A particularly prefened form incorporates residues 188 - 305. These preferential fusions are also new and form one aspect of the invention.
  • MAC immobilised metal ion affinity chromatography
  • the metal ion may be any suitable ion for example zinc, nickel, iron, magnesium or copper, but is preferably zinc or nickel.
  • the MAC buffer contains detergent, preferably a non-ionic detergent such as Tween 80, or a zwitterionic detergent such as Empigen BB, as this may result in lower levels of endotoxin in the final product.
  • Further chromatographic steps include for example a Q-Sepharose step that may be operated either before of after the MAC column.
  • the pH is in the range of 7.5 to 10, more preferably from 7.5 to 9.5, optimally between 8 and 9, ideally 8.5.
  • the protein thus purified present a major single band when analysed by SDS- PAGE under reducing conditions, and show less than 10%, preferably less than 5% host cell contamination as determined by Western blot analysis.
  • the proteins of the present invention are provided either soluble in a liquid form or in a lyophilised form, which is the prefened form. It is generally expected that each human dose will comprise 1 to 1000 ⁇ g of protein, and preferably 30-300 ⁇ g-
  • the present invention also provides pharmaceutical composition comprising a protein of the present invention in a pharmaceutically acceptable excipient.
  • a prefened vaccine composition comprises at least NSl-P703P*-His (SEQ ID N°l).
  • Said protein has, preferably, blocked thiol groups and is highly purified, e.g. has less than 5% host cell contamination.
  • Such vaccine may optionally contain one or more other tumour-associated antigen and derivatives.
  • suitable other associated antigen include PAP-1, PSA (prostate specific antigen), PSMA (prostate- specific membrane antigen), PSCA (Prostate Stem Cell Antigen), STEAP, and P501S (WO 98/37418).
  • Vaccine preparation is generally described in Vaccine Design ("The subunit and adjuvant approach” (eds. Powell M.F. & Newman M.J). (1995) Plenum Press New York). Encapsulation within liposomes is described by Fullerton, US Patent 4,235,877.
  • the proteins of the present invention are preferably adjuvanted in the vaccine formulation of the invention.
  • Suitable adjuvants are commercially available such as, for example, Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, MI); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ); AS-2 (SmithKline Beecham, Philadelphia, PA); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quil A.
  • Cytokines such as GM-CSF or interleukin-2, -7, or -12, and chemokines may also be used as adjuvants.
  • the adjuvant composition induces an immune response predominantly of the TH1 type.
  • High levels of Thl-type cytokines e.g., IFN- ⁇ , TNF ⁇ , IL-2 and EL- 12
  • Thl-type cytokines tend to favour the induction of cell mediated immune responses to an administered antigen.
  • the level of Thl-type cytokines will increase to a greater extent than the level of Th2-type cytokines.
  • the levels of these cytokines may be readily assessed using standard assays. For a review of the families of cytokines, see Mosmann and Coffman, Ann. Rev. Immunol. 7:145-173, 1989.
  • suitable adjuvants for use in eliciting a predominantly Thl-type response include, for example a combination of monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid A (3D-MPL) together with an aluminium salt.
  • Other known adjuvants which preferentially induce a TH1 type immune response include CpG containing oligonucleotides. The oligonucleotides are characterised in that the CpG dinucleotide is unmethylated. Such oligonucleotides are well known and are described in, for example WO 96/02555. hnmunostimulatory DNA sequences are also described, for example, by Sato et al., Science 273:352, 1996.
  • CpG- containing oligonucleotides may also be used alone or in combination with other adjuvants.
  • an enhanced system involves the combination of a CpG- containing oligonucleotide and a saponin derivative particularly the combination of CpG and QS21 as disclosed in O 00/09159 and WO 00/62800.
  • the formulation additionally comprises an oil in water emulsion and/or tocopherol.
  • Another ' prefened adjuvant is a saponin, preferably QS21 (Aquila
  • an enhanced system involves the combination of a monophosphoryl lipid A and saponin derivative, such as the combination of QS21 and 3D-MPL as described in WO 94/00153, or a less reactogenic composition where the QS21 is quenched with cholesterol, as described in WO 96/33739.
  • Other prefened formulations comprise an oil-in-water emulsion and tocopherol.
  • a particularly potent adjuvant formulation involving QS21, 3D-MPL and tocopherol in an oil-in-water emulsion is described in WO 95/17210.
  • a particularly potent adjuvant formulation involving QS21 3D-MPL & tocopherol in an oil in water emulsion is described in WO 95/17210 and is a prefened formulation.
  • prefened adjuvants include Montanide ISA 720 (Seppic, France), SAF (Chiron, California, United States), ISCOMS (CSL), MF-59 (Chiron), Detox (Ribi, Hamilton, MT), RC-529 (Corixa, Hamilton, MT) and other aminoalkyl glucosaminide 4-phosphates (AGPs).
  • prefened adjuvants include adjuvant molecules of the general formula
  • n 1-50
  • A is a bond or -C(O)-
  • R is C ⁇ - 50 alkyl or Phenyl C ⁇ - 50 alkyl.
  • One embodiment of the present invention consists of a vaccine formulation comprising a polyoxyethylene ether of general formula (I), wherein n is between 1 and 50, preferably 4-24, most preferably 9; the R component is C 1 -50, preferably C 4 - C 2 o alkyl and most preferably C 12 alkyl, and A is a bond.
  • concentration of the polyoxyethylene ethers should be in the range 0.1-20%, preferably from 0.1-10%, and most preferably in the range 0.1-1%.
  • Prefened polyoxyethylene ethers are selected from the following group: polyoxyethylene-9-lauryl ether, polyoxyethylene-9-steoryl ether, polyoxyethylene-8-steoryl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, and polyoxyethylene-23-lauryl ether.
  • Polyoxyethylene ethers such as polyoxyethylene lauryl ether are described in the Merck index (12 th edition: entry 7717). These adjuvant molecules are described in WO 99/52549.
  • the polyoxyethylene ether according to the general formula (I) above may, if desired, be combined with another adjuvant.
  • a prefened adjuvant combination is preferably with CpG as described in the pending UK patent application GB 9820956.2.
  • a vaccine comprising a mutated protein of the present invention, more preferably NS1- P703P*-His adjuvanted with a monophosphoryl lipid A or derivative thereof, QS21 and tocopherol in an oil in water emulsion.
  • the vaccine additionally comprises a saponin, more preferably QS21.
  • a saponin more preferably QS21.
  • Another particular suitable adjuvant formulation including CpG and a saponin is described in WO 00/09159 and is a prefened formulation. More preferably the saponin in that particular formulation is QS21, preferably the less reactogenic form where QS21 is quenched with cholesterol, as described in WO 96/33739.
  • the formulation additionally comprises an oil in water emulsion and tocopherol. Any of a variety of delivery vehicles may be employed within pharmaceutical compositions and vaccines to facilitate production of an antigen-specific immune response that targets tumour cells.
  • Delivery vehicles include antigen-presenting cells (APCs), such as dendritic cells, macrophages, B cells, monocytes and other cells that may be engineered to be efficient APCs.
  • APCs antigen-presenting cells
  • Such cells may, but need not, be genetically modified to increase the capacity for presenting the antigen, to improve activation and or maintenance of the T cell response, to have anti-tumour effects per se and/or to be immunologically compatible with the receiver (i.e., matched HLA haplotype).
  • APCs may generally be isolated from any of a variety of biological fluids and organs, including tumour and peri-tumoural tissues, and may be autologous, allogeneic, syngeneic or xenogeneic cells.
  • Dendritic cells are highly potent APCs (Banchereau and Steinman, Nature 392:245-251, 1998) and have been shown to be effective as a physiological adjuvant for eliciting prophylactic or therapeutic antitumour immunity (see Timmerman and Levy, Ann. Rev. Med. 50:507-529, 1999).
  • dendritic cells may be identified based on their typical shape (stellate in situ, with marked cytoplasmic processes (dendrites) visible in vitro), their ability to take up, process and present antigens with high efficiency and their ability to activate na ⁇ ve T cell responses.
  • Dendritic cells may, of course, be engineered to express specific cell-surface receptors or ligands that are not commonly found on dendritic cells in vivo or ex vivo, and such modified dendritic cells are contemplated by the present invention.
  • secreted vesicles antigen- loaded dendritic cells called exosomes
  • exosomes antigen- loaded dendritic cells
  • Dendritic cells and progenitors may be obtained from peripheral blood, bone manow, tumour-infiltrating cells, peritumoral tissues-infiltrating cells, lymph nodes, spleen, skin, umbilical cord blood or any other suitable tissue or fluid.
  • dendritic cells may be differentiated ex vivo by adding a combination of cytokines such as GM-CSF, BL-4, IL-13 and/or TNF ⁇ to cultures of monocytes harvested from peripheral blood.
  • CD34 positive cells harvested from peripheral blood, umbilical cord blood or bone manow may be differentiated into dendritic cells by adding to the culture medium combinations of GM-CSF, IL-3, TNF ⁇ , CD40 ligand, lipopolysaccharide LPS, flt3 ligand and/or other compound(s) that induce differentiation, maturation and proliferation of dendritic cells.
  • Dendritic cells are conveniently categorized as “immature” and “mature” cells, which allows a simple way to discriminate between two well characterized phenotypes. However, this nomenclature should not be construed to exclude all possible intermediate stages of differentiation. Immature dendritic cells are characterized as APC with a high capacity for antigen uptake and processing, which conelates with the high expression of Fc ⁇ receptor and mannose receptor.
  • the mature phenotype is typically characterized by a lower expression of these markers, but a high expression of cell surface molecules responsible for T cell activation such as class I and class II MHC, adhesion molecules (e.g., CD54 and CD11) and costimulatory molecules (e.g., CD40, CD80, CD86 and 4-lBB).
  • cell surface molecules responsible for T cell activation such as class I and class II MHC, adhesion molecules (e.g., CD54 and CD11) and costimulatory molecules (e.g., CD40, CD80, CD86 and 4-lBB).
  • APCs may generally be transfected with a polynucleotide encoding prostase tumour protein (or derivative thereof) such that the prostase tumour polypeptide, or an immunogenic portion thereof, is expressed on the cell surface. Such transfection may take place ex vivo, and a composition or vaccine comprising such transfected cells may then be used for therapeutic purposes, as described herein. Alternatively, a gene delivery vehicle that targets a dendritic or other antigen presenting cell may be administered to a patient, resulting in transfection that occurs in vivo.
  • In vivo and ex vivo transfection of dendritic cells may generally be performed using any methods known in the art, such as those described in WO 97/24447, or the gene gun approach described by Mahvi et al, Immunology and cell Biology 75:456-460, 1997.
  • Antigen loading of dendritic cells may be achieved by incubating dendritic cells or progenitor cells with the prostase tumour polypeptide, DNA (naked or within a plasmid vector) or RNA; or with antigen-expressing recombinant bacterium or viruses (e.g., vaccinia, fowlpox, adenovirus or lentivirus vectors).
  • 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 sterility of the formulation until use.
  • formulations may be stored as suspensions, solutions or emulsions in oily or aqueous vehicles.
  • a vaccine or pharmaceutical composition may be stored in a freeze-dried condition requiring only the addition of a sterile liquid carrier immediately prior to use.
  • the present invention also provides a process for the production of a vaccine, comprising the steps of purifying a prostase protein or a derivative thereof, by the process disclosed herein and admixing the resulting protein with a suitable adjuvant, diluent or other pharmaceutically acceptable excipient.
  • the present invention also provides a method for producing a vaccine formulation comprising mixing a protein of the present invention together 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 immunotherapeutically treating a patient suffering from prostate cancer or other prostase-associated tumours.
  • a method of treating patients susceptible to or suffering from prostate-cancer comprising administering to said patients a pharmaceutically active amount of the vaccine disclosed herein is also contemplated by the present invention.
  • Figure 1 Design of the fusion protein NSl- p703* -His expressed in E. coli
  • Figure 2 Primary structure of the fusion protein NS 1- p703* -His expressed in E. coli (SEQ ID N°1).
  • Figure 3 Coding sequence of NSl ⁇ . 81 -P703P*-His (SEQ ID N°2).
  • Figure 4 Cloning strategy to produce NSl-P703P*-His in E. coli
  • Figure 8 Characterisation of NSl-P703P*-His.
  • the reducing buffer (SB+) contains (final concentration) 7.25% v/v glycerol, 1.45% w/v SDS, 0.5 M 2-mercaptoethanol, 0.0033% w/v bromophenol blue and 58 mM Tris buffer pH 6.8.
  • the non-reducing buffer (SB-) does not contain 2-mercaptoethanol.
  • P2F stands for VMF permeate containing the antigen.
  • P2F R/C stands for VMF containing the reduced/carboxyamidated antigen.
  • FT stands for MAC flow through (lane 9) and W stands for MAC wash (lane 10).
  • Figure 10 Primary structure of the fusion protein NSl- p703 -His expressed in E. coli (SEQ ID N°3).
  • Figure 11 Coding sequence of NSl 1 . 8 ⁇ -P703P-His (SEQ ID N°4).
  • Figure 12 Primary structure of the protein p703* -His expressed in Pichia pastoris (SEQ ID N°9).
  • Figure 13 Coding sequence of P703P*-His expressed in Pichia pastoris (SEQ ID N°10).
  • EXAMPLE I Preparation of the recombinant E. coli strain expressing the fusion protein NS1- P703P*-3-His
  • the mutation of the active side was made in order to reduce substantially or preferably eliminate its proteolytical biological activity. Accordingly, the His residue at position 43 of SEQ ID N° 8, has been mutated into an Ala residue.
  • the design of the fusion protein NSl- p703* -His to be expressed in E. coli is described in figure 1.
  • This fusion contains the N-terminal (81 amino acid) of non structural protein of Influenzae virus, followed by the non processed amino acid sequence of prostate antigen (amino acids 5- 226 of p703pde5 sequence described in SEQ ID N°8 containing the mutation His->Ala of the 43 residue of the protease active site followed by the His tail.
  • the Histidine tail was added to prostase to enable versatile purification of the fusion and processed protein.
  • the length of the fusion is 313 aminoacids.
  • the primary structure of the resulting protein has the sequence described in figure 2.
  • the coding sequence conesponding to the above protein is illustrated in figure 3 and was subsequently placed under the control of ⁇ pL promoter in a E. coli expression plasmid. 2. - The E. Coli expression system
  • NSl non-structural protein from influenzae virus
  • NSl non-structural protein from influenzae virus
  • pMG 81 This plasmid utilises signals from lambda phage DNA to drive the transcription and translation of inserted foreign genes.
  • the vector contains the lambda PL promoter PL, operator OL and two utilisation sites (NutL and NutR) to relieve transcriptional polarity effects when N protein is provided (Gross et al., 1985. Mol. & Cell. Biol. 5:1015).
  • Vectors containing the PL promoter are introduced into an E. coli lysogenic host to stabilise 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 Expression. Inouya (ed) pp 1-14. Academic Press NY).
  • the lambda phage DNA directs the synthesis of the cl repressor protein which binds to the OL repressor of the vector and prevents binding of RNA polymerase to the PL promoter and thereby transcription of the inserted gene.
  • the cl gene of the expression strain AR58 contains a temperature sensitive mutation so that PL directed transcription can be regulated by temperature shift, i.e. an increase in culture temperature inactivates the repressor and synthesis of the foreign protein is initiated. This expression system allows controlled synthesis of foreign proteins especially of those that may be toxic to the cell (Shimataka & Rosenberg, 1981. Nature 292:128).
  • His protein is a derivative of the standard NTH E.coli K12 strain N99 (F- su- galK2, lacZ- thr-). It contains a defective lysogenic lambda phage (galE::TN10, 1 Kil- cI857 DHl). The Kil- phenotype prevents the shut off of host macromolecular synthesis. The cI857 mutation confers a temperature sensitive lesion to the cl repressor. The DHl deletion removes the lambda phage right operon and the hosts bio, uvr3, and chlA loci.
  • the AR58 strain was generated by transduction of N99 with a P lambda phage stock previously grown on an SA500 derivative (galE::TN10, 1 Kil- cI857 DHl).
  • the introduction of the defective lysogen into N99 was selected with tetracycline by virtue of the presence of a TN10 transposon coding for tetracyclin resistance in the adjacent galE gene.
  • N99 and SA500 are E.coli K12 strains derived from Dr. Martin Rosenberg's laboratory at the National Institutes of Health.
  • the starting materials were:
  • the cloning strategy outlined in figure 4 included the following steps:
  • PCR amplification of the p703 sequence with Ncol and Spel restriction sites The template for the PCR reaction was the cDNA plasmid received from CORIXA, the oligonucleotide sense canl39 : 5'GCG CCC ATG GTT GGG GAG GAC TGC AGC CCG 3', and the oligonucleotide antisense canl34 :
  • the antisense oligonucleotide canl41 5' CTG GAA ACA CGC TGC GGC TGA CAG 3', leading to the obtention of plasmid pRIT 14950; d) Isolation of the Ncol - Spel fragment from the plasmid pRIT 14950; e) From pMG81 plasmid, purification of NSl fragment (81aa) after digestion of the restriction sites BamHI - Ncol; f) Ligation of both fragments were ligated to the expression plasmid pRIT 14901 (pr PL long); g) Selection and characterisation of the E. coli AR58 strain transformants containing the plasmid pRIT14952 (see figure 5) expressing the NSl- p703 mutated -His fusion protein
  • the recombinant strain thus produces the NS1-P703P* His-tailed fusion protein of 313 amino acid residues long (see Figure 2), with the amino acids sequence described in H> NO:l and the coding sequence is described in ID NO:2.
  • Cells of AR58 transformed with plasmid pRIT14952 were grown in a 2 L flask containing 400 ml FEC015AA medium supplemented with kanamycin sulphate (lOOmg/L). After a 16h of incubation at 30°C and at 200 rpm, a small sample was removed from this flask for microscopic examination. 50 ml of this pre-culture was transfened into a 20-L fermentor containing 8.7 L of FEC012AF medium supplemented with kanamycin sulphate (50mg/L). The pH was adjusted to and maintained at 6.8 by addition of NH4OH (25 % v/v), and the temperature was maintained at 30°C. The aeration rate was kept constant at 20 L/min and the pO2 was regulated at 20% of saturation by feedback control of the agitation speed. The head pressure was maintained at 0.5 bar.
  • This fed-batch fermentation process is based on glycerol as a carbon source.
  • the feed solution was added at an initial rate of 0.04ml/min, and increased exponentially during the first 30 hours to limit the growth rate in order to be able to keep a minimum pO2 level of 20%.
  • the temperature of the fermentor was rapidly increased to 39.5°C in order to induce the intracellular expression of the antigen NSl-P703P*-His.
  • the feeding rate was maintained constant at 1.28 ml/min during the whole induction phase (18h). Samples of broth were taken during both growth and induction phases in order to monitor bacterial growth and antigen expression. Microbiological identification and purity tests were also realised on these materials.
  • the biomass reached an optical density of about 130, conesponding to a dry cell weight of about 50g/L.
  • the final volume was approximately 10.5 L.
  • the cells containing the antigen were directly separated from the culture medium by centrifugation at 5000g for 1 h at 4°C and the pellet was stored in plastic bags at -70°C.
  • Recombinant NSl-P703P*-His protein expressed in E. coli as inclusion bodies, was purified from cell homogenate using different steps (see figure 6). Briefly, frozen concentrated cells from fermentation harvest were thawed to +4°C before being resuspended in disruption buffer (phosphate 20 mM - NaCl 2M - EDTA 5 mM pH 7.5) to a final optical density (OD650) of 120. Two passes through a high- pressure homogeniser (1000 bars) disrupted the cells.
  • the recombinant protein, NSl-P703P*-His is produced in E. coli in the form of inclusion bodies.
  • a major issue for the set-up of the purification method was the oxidation of the recombinant protein with itself or with host cell contaminants, likely through covalent binding with disulphide bonds.
  • the process as developed aimed at reducing the massive oxidation phenomenon in order to have a highly purified product together with an acceptable global yield, while preserving the product ability to mount an effective immune response against the antigen of interest.
  • the carboxyamidated fraction was subjected to MAC (Nickel-Chelating-
  • Sepharose FF Sepharose FF, Pharmacia.
  • the column was first equilibrated with 25 mM Tris buffer pH 78.5 containing 4M urea, 0.5% (v:v) Tween 80, and 20 mM imidazole. After the sample loading, the column was washed with the same buffer. The protein was then eluted in the previous buffer with 400 mM Imidazole.
  • the conductance of the MAC-eluate was reduced to below 5 mS/cm (3.5 mS/cm) with 25 mM Tris buffer pH 8.5 containing 4M urea and 0.5 % (v:v) Tween 80.
  • the packed bed support (Q- Sepharose FF, Pharmacia) was equilibrated with the dilution buffer. After the sample loading and a washing step with the equilibration buffer, the protein was eluted with the same buffer containing 250 mM NaCl.
  • the Q-Sepharose FF-eluate was then diafiltered against the appropriate storage buffer (25 mM Tris buffer pH 8.0) in a tangential flow filtration unit equipped with a 10 Kd cut-off membrane (Omega, Filton). Ultrafiltration retentate containing NSl-P703P*-His was sterile filtered through 0.22 ⁇ m membrane.
  • the global purification yield was very high: between 2-4 g (2.5 g on average) of purified material / L of homogenate (DO120).
  • gel 1 lanes 9 and 10 show that the carboxyamidated protein when treated with a combination of 0.5% Tween 80 detergent and 4M urea chaotropic agent, shows a conect binding to the MAC column, since no protein was recovered in the flow- through (lane 9) nor in the washings (lane 10).
  • Figure 8, gel 2 shows patterns of the MAC eluate in reducing (lane 3) and non-reducing (lane 5) buffer, which is a further confirmation of the stabilisation of the oxidation. No protein of interest is detected in the consecutive elution fractions (gel 2, lanes 6-8). Other residual contaminants (endotoxin, DNA) were below the usual specification limits: respectively, 30 EU and 100 ng/100 ⁇ g protein.
  • the purified material was stable at +4°C and +37°C (1 week) although some precipitation was observed after freeze-thawing cycles. This precipitation can be avoided if the freezing is achieved in the presence of sucrose.
  • the purification process developed stepwise has allowed enhancing by a 100- fold factor the production yield of the fusion protein.
  • the final product that is being recovered harbours much lower contamination by host cell proteins, lower oxidised pattern, lower aggregation, higher stability and much more consistency from batch to batch, all features that are compatible with high-scale production for industrial applications.
  • EXAMPLE IV Vaccine preparation using NSl-P703P*-His protein
  • the vaccine used in these experiments is produced from a recombinant DNA, encoding a NSl-P703P*-His, expressed in E. coli from the strain AR58, either adjuvanted or not.
  • the formulation comprises a mixture of 3 de -O- acylated monophosphoryl lipid A (3D-MPL) and QS21 in an oil/water emulsion.
  • the adjuvant system SBAS2 has been previously described WO 95/17210.
  • 3D-MPL is an immunostimulant derived from the lipopolysaccharide (LPS) of the Gram-negative bacterium Salmonella minnesota. MPL has been deacylated and is lacking a phosphate group on the lipid A moiety. This chemical treatment dramatically reduces toxicity while preserving the immunostimulant properties (Ribi, 1986). Ribi hnmunochemistry produces and supplies MPL to SB-Biologicals. Experiments performed at Smith Kline Beecham Biologicals have shown that 3D-MPL combined with various vehicles strongly enhances both the humoral and a TH1 type of cellular immunity.
  • LPS lipopolysaccharide
  • QS21 is a natural saponin molecule extracted from the bark of the South
  • the oil/water emulsion is composed an organic phase made of of 2 oils (a tocopherol and squalene), and an aqueous phase of PBS containing Tween 80 as emulsifier.
  • the emulsion comprised 5% squalene 5% tocopherol 0.4% Tween 80 and had an average particle size of 180 nm and is known as SB62 (see WO 95/17210).
  • Tween 80 is dissolved in phosphate buffered saline (PBS) to give a 2% solution in the PBS.
  • PBS phosphate buffered saline
  • 5g of DL alpha tocopherol and 5ml of squalene are vortexed to mix thoroughly.
  • 90ml of PBS/Tween solution is added and mixed thoroughly.
  • the resulting emulsion is then passed through a syringe and finally microfluidised by using an Ml 1 OS microfluidics machine.
  • the resulting oil droplets have a size of approximately 180 nm.
  • the volumes of all compounds are adjusted to have in final: 250-50-10 ⁇ g NSl-P703P*-His, in Tris 10 mM, tween 8O 0.2%, 3.15% sucrose.
  • the vial was overfilled with by 1.25 x (reconstitution with 625 ⁇ l diluant, injection of 500 ⁇ l).
  • the adjuvant is formulated as a combination of MPL and QS21, in an oil/water emulsion.
  • Formulation composition (injection volume: lOO ⁇ l); group 1 has received P703P*-His (20 ⁇ g) formulated in a combination of MPL and QS21, in an oil/water emulsion. Group 2 has received NSl-P703P*-His (25 ⁇ g) in a combination of MPL and QS21, in an oil/water emulsion.
  • the formulations were prepared extemporaneously on the day of injection.
  • the formulations containing 3D-MPL and QS21 in an oil/water emulsion were performed as follows: P703p (20 ⁇ g) (group 2) and NSl-P703P*-His (25 ⁇ g) (group 3) were diluted in 10-fold concentrated PBS pH 6.8 and H 2 O before consecutive addition of SB62 (50 ⁇ l), MPL (20 ⁇ g), QS21 (20 ⁇ g) and 1 ⁇ g/ml thiomersal as preservative at 5 min intervals. All incubations were carried out at room temperature with agitation.
  • the non-adjuvanted formulations (Groups 4 and 5) were performed as follows: P703p (20 ⁇ g) (group 4) and NS l-P703P*-His (25 ⁇ g) (group 5) were diluted in 1.5 M NaCl and H 2 O before addition of 1 ⁇ g/ml thiomersal as preservative at 5 min intervals. All incubations were carried out at room temperature with agitation.
  • the final vaccine is obtained after reconstitution of the lyophilised NS1- P703P*-His preparation with the adjuvant or with PBS alone.
  • the adjuvant controls without antigen were prepared by replacing the protein by
  • the aim of the experiment was to characterise the immune response induced in mice by vaccination with the purified recombinant mutated NSl-p703*-His molecule produced in E coli, in the presence or the absence of an adjuvant.
  • mice Groups of 10 immunocompetent Balb/c mice, 6 to 8 weeks old mice, were vaccinated twice, intramuscularly, at 2 weeks interval with 25 ⁇ g of mutated NS1-
  • the anti P703 antibody response has been assessed in the sera of the mice 14 days after the latest vaccination. This has been done by ELISA using either carboxyamidated or non carboxyamidated NSl-P703P*-His as coating antigen. E coli extracts were used to check for the possible presence of antibodies against host contaminants.
  • P703P*-His protein alone as compared to the sera of normal control mice; 2) high antibody titers are found in animals receiving the NSl-P703P*-His carboxyamidated molecule formulated in the AS02B adjuvant; and 3) the antibodies recognise both the carboxyamidated (NSl-P703P*-His) and non carboxyamidated native form of the molecule (NSl-P703P*-His NC).
  • the isotypic profile of the NSl-p703p-His specific IgG response has also been measured. As shown in Figure 9, IgGl were detected when mice received the carboxyamidated NSl-P703P*-His protein alone, however the isotypic profile was pushed towards a TH1 response (more IgG2a) by the presence of the AS02 adjuvant.
  • the aim of the experiment was to determine whether antibodies generated in mice by vaccination with the carboxyamidated NSl-p703*-His molecule produced in E coli were cross-reacting with non carboxyamidated and carboxyamidated Pichia- produced P703P*-His (see Example VIE).
  • NSl-OspA is an unrelated antigen.
  • mice received intramuscular injection at days 0-14-28-42 of either PBS buffer or 25 ⁇ g of mutated carboxyamidated NS1-P703* C formulated or not in AS02B (25 ⁇ l SB62 / lO ⁇ g MPL / lO ⁇ g QS21) or AS01B.
  • AS01B is prepared by adding QS21 (5 ⁇ g) to small unilamellar vesicles (SUV) of dioleoyl phosphatidylcholine containing cholesterol (25 ⁇ g) (WO 96/33739) and MPL (5 ⁇ g) in the membrane.
  • SUV small unilamellar vesicles
  • MPL 5 ⁇ g
  • the anti P703 antibody response has been assessed in the sera of the mice 14 days after the latest vaccination. This has been done by ELISA using either Pichia-produced carboxyamidated P703P*-His C, Pz ' cA/ ⁇ -produced non- carboxyamidated P703P*-His NC and E coli produced carboxyamidated NS1- P703P*-His C antigen, as coating antigen.
  • the results obtained post II are shown in Table 1. They have been confirmed post IV.
  • NSl-P703P-His was prepared.
  • the amino acid and DNA sequences are depicted in SEQUENCE ID Nos. 3 and 4.
  • the strategy to express a NSl-P703P-His fusion protein in E.coli included the following steps: a) As a starting materiel, the same starting material as described in Example I (amino acids 5- 226 of p703pde5 sequence described in SEQ ID N°8); b) PCR amplification to flank the p703 unmutated sequence cloning restriction sites; c) Insertion in a PMG81 vector (promoter pL long) containing the NS 1 gene; d) Transformation of the recipient strain AR58 or AR120 e) Selection of recombinant strain.
  • the resulting protein can be purified in an analogous manner to the NS 1 -
  • P703P*-His mutated protein The primary structure of the resulting protein has the sequence described in figure 10. The coding sequence conesponding to the above protein is illustrated in figure 11.
  • Mutated P703P protein has been expressed in the yeast Pichia pastoris.
  • the P703P cDNA encodes a 254 -aa polypeptide with an amino-terminal pre-propeptide sequence indicating a potential secretory function.
  • the native P703P secretion signal sequence and the putative pro-peptide coding sequence were replaced by the Saccharomyces cerevisiae alpha pre-pro signal sequence.
  • Mutated P703P (His 4 o-» Ala 40 at the protease active site) coding sequence was fused to the Saccharomyces cerevisiae alpha prepro signal sequence. The C-terminal part of the recombinant protein was elongated by one glycine and six histidines.
  • Strain Y1786 was obtained by integrating into the yeast genome 3 to 4 copies of P703P expression cassette plus HIS4 selection gene. On medium containing methanol as carbon source, the P703P protein is efficiently secreted and accumulates into the culture medium with maximum yield after 96h of induction.
  • the starting material was the recombinant plasmid pRIT 14950 (see Figure 4).
  • This plasmid contains, inserted between Nco I and Spe I sites in the polylinker of the LITMUS 28 E.coli plasmid, the mutated P703P coding sequence.
  • the P703P coding sequence is not complete.
  • the N-terminal portion containing the signal peptide and the pro-peptide is not recovered.
  • pRIT 14950 contains the entire mature form with start at Valine 5.
  • the cloning strategy followed to construct the recombinant plasmid pRIT15043 and the integration of expression cassettes into the Pichia genome included the following steps: a) PCR amplification of the P703 sequence with primers 703P5 and 703P3. Xho l and Not I restriction sites were introduced at respectively the 5' and 3' ends of the PCR fragment.
  • the template for the PCR reaction was the pRIT14950 plasmid.
  • NRRL-Y 11430 Northern Regional Research Laboratories, Peoria, IL
  • his4 auxotrophic mutation carries the his4 auxotrophic mutation.
  • BMGY Buffered Glycerol-complex Medium
  • O.D. 26 onm O.D. 26 onm of 1-2.
  • cells were harvested and resuspended in 1/10 of the original culture volume in Buffered Methanol-complex Medium (BMMY - 1% methanol) and incubated (30°C) for 4 days. An additional 0.5% methanol was added every 24 hours.
  • the yield of secreted native P703P protein was estimated at 10 mg/liter culture supernatant (O.D.30/ml), after 4 days of induction in shake flask condition.
  • the purification scheme consists of the following sequence of steps: Culture supernatant --> filtration/0.2 ⁇ m - MAC -> IEC - UF - sterile filtration.
  • no chaotropic agent or detergent was used along the process.
  • This protocol does not include a reduction/carboxyamidation of the molecule during the purification process but this step has been added at the end of the purification process.
  • a control run has been made which did not include the reduction/carboxyamidation.
  • the purified protein either carboxyamidated or not, was used in the immunological experiment described in Example N, second experiment. Estimated purification yield is around 50 mg purified material / L culture supernatant. Solubility and stability of the purified material were good.
  • the fermentation supernatant [1 liter] was filtered through 0.2 ⁇ m filter (Millipak 100, Millipore). The filtrate was perfectly limpid.
  • the filtered supernatant was subjected to MAC ( ⁇ i-Chelating-Sepharose FF, Pharmacia).
  • the column (XK50, Pharmacia; 100 ml) was first equilibrated with PBS buffer pH 7.5. After the sample loading, the column was washed with the same buffer. The protein was then eluted in the previous buffer with a 20 CN linear gradient of increasing imidazole concentration (from 0 to 200mM). Fractions shown positive for P703P*-His were pooled after SDS-PAGE analysis.
  • the conductance of the MAC-eluate was reduced to around 4 mS/cm with 20 mM phosphate pH 7.5 buffer.
  • the packed bed support (Q-Sepharose FF - XK50 column, 60 ml, Pharmacia) was first equilibrated with 20mM phosphate pH 7.5 buffer. After the sample loading, a washing step was performed with the same buffer. The protein was then eluted with the same buffer containing a 20 CN linear gradient of increasing ⁇ aCl concentration (from 0 to 500mM). Fractions shown positive for P703P*-His were pooled after SDS- PAGE analysis. The Q-Sepharose FF-eluate was then concentrated and diafiltered against the
  • the purified antigen was diluted with an equivalent volume of 20 mM PO buffer - 8 M GuHCl - 1 % Empigen BB - 40 mM Glutathion pH 7.5 and left at room temperature in the dark under gentle agitation for 1 hour.
  • the carboxyamidation of the protein was then performed by addition of iodoacetamide up to a final concentration of 100 mM and adjustment of the pH to 7.5 with concentrated ⁇ aOH solution. The mixture was left at room temperature in the dark under gentle agitation for 30 minutes.
  • the sample was dialysed 18 hours at + 4°C against 20 mM Tris buffer - 0.2 % Tween 80 pH 8.0 and sterilised by filtration through 0.22 ⁇ m membrane.
  • the mutated protein is compatible with a product with increased safety profile, suitable for vaccine purposes.
  • fusion protein NSl-P703P*-His containing the mutated prostase when purified using the optimised process described in the invention involving a reduction/carboxyamidation treatment, can be produced at a high yield and in a less aggregated, less oxidised, more soluble and more stable form.

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Abstract

La présente invention concerne de nouvelles protéines et leur production, à partir de la famille de la prostase. En particulier, l'invention concerne une prostase génétiquement modifiée selon laquelle la protéine a subi une mutation dans son site actif. De tels antigènes peuvent être formulés pour produire des vaccins pour traiter les tumeurs de la prostate. L'invention traite également de procédés pour purifier la protéine de la prostase et ses homologues.
PCT/EP2001/007079 2000-06-27 2001-06-21 Vaccin Ceased WO2002000708A2 (fr)

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US5786148A (en) * 1996-11-05 1998-07-28 Incyte Pharmaceuticals, Inc. Polynucleotides encoding a novel prostate-specific kallikrein
US5955306A (en) * 1996-09-17 1999-09-21 Millenium Pharmaceuticals, Inc. Genes encoding proteins that interact with the tub protein
DE19805633A1 (de) * 1998-02-12 1999-08-19 Basf Ag Neue Serinprotease aus der Prostata
HUP0203035A3 (en) * 1998-07-14 2007-12-28 Corixa Corp Compositions and methods for therapy and diagnosis of prostate cancer
CA2378846A1 (fr) * 1999-07-13 2001-01-18 Smithkline Beecham Biologicals S.A. Vaccin

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